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The Discovery and Synthesis of the CGRP Receptor Antagonist MK-3207 Downloaded by UNIV OF FLORIDA on December 11, 2016 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

Ian M. Bell,1 Paul G. Bulger,2 and Mark McLaughlin*,2 1Department

of Discovery Chemistry, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States 2Department of Process Research & Development, Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States *E-mail: [email protected].

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and neuromodulator. Multiple lines of evidence demonstrate that CGRP plays a key role in the pathogenesis of migraine and it has become a major target for migraine drug discovery efforts. This chapter reviews the discovery of MK-3207, a novel, potent, orally acting CGRP receptor antagonist and the development of a highly efficient synthetic route that allows for large scale production of the compound.

Introduction Migraine is a common, highly disabling, neurovascular disorder that affects about 11% of adults worldwide and results in a significant burden to society in terms of lost productivity and diminished quality of life (1, 2). Migraine attacks are characterized by moderate to severe headache accompanied by other symptoms, including photophobia, phonophobia, allodynia, nausea, and vomiting (1). The duration of these attacks can be from a few hours to several days and the frequency is typically around one or two attacks per month (3). The “gold standard” agents for the acute treatment of migraine are the triptans, which are selective 5-HT1B and 5-HT1D receptor agonists. Triptans are believed to act via vasoconstriction of cranial blood vessels and by inhibition of the release of neuropeptides, including calcitonin gene-related peptide (CGRP) (4). Although triptans are effective antimigraine agents, they also cause constriction of coronary arteries and are consequently contraindicated in patients © 2016 American Chemical Society

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with cardiovascular disease and uncontrolled hypertension (5). There continues to be significant interest in the development of new and effective antimigraine drugs that are safe and that lack the cardiovascular liabilities of the triptans. Migraine is a complex disorder and the precise details of its pathogenesis are still being studied. There is, however, general agreement that a key player is CGRP, a 37-amino acid neuropeptide that is a member of the calcitonin family of peptides (6). CGRP, which is a potent vasodilator and neuromodulator, is found throughout the peripheral and central nervous systems and appears to play a role in a number of biological functions (7). Among these, its apparent involvement in cerebrovascular regulation led to the hypothesis that it could be a key player in the pathophysiology of migraine and subsequent studies have confirmed this (8). For example, it was shown that the craniovascular levels of CGRP increased significantly during migraine attacks (9). Additionally, these migraine-associated increases in CGRP concentrations appeared to return to basal levels following successful treatment of the migraine headache with sumatriptan (10). In another compelling study, it was found that intravenous infusion of CGRP induced a migraine-like headache in migraineurs (11). These lines of evidence demonstrating that CGRP was playing an important role in migraine led to speculation that a “CGRP blocker” could represent a new therapeutic approach with potential advantages over the triptans (12). The present review will focus on the discovery of one such small molecule antagonist of the CGRP receptor and on the development of novel synthetic routes that allow for large scale production of this clinical candidate, MK-3207.

CGRP and the CGRP Receptor The calcitonin family of peptides consists of calcitonin (CT), CGRP, amylin (AMY) and adrenomedullin (AM) (13, 14). The peptides share a number of features, including a cyclic structure at the N-terminus formed by a Cys-Cys disulfide bond, and a C-terminal amide group (13). There are two forms of CGRP: α-CGRP, which is produced by alternate splicing of the CT gene, and β-CGRP, which is encoded by a separate gene and differs from α-CGRP by three amino acids in humans (14). The receptors for these peptides are members of the secretin family (also known as family B or family 2) of G-protein-coupled receptors (GPCRs) (15). These family B GPCRs contain large N-terminal extracellular domains (ECDs) that are involved in binding of their peptide ligands (15). The CGRP receptor is a heterodimeric receptor, composed of the calcitonin receptor-like receptor (CLR) in association with receptor activity-modifying protein 1 (RAMP1) (16, 17). CLR can also associate with RAMP2 or RAMP3 to produce high affinity AM receptors that are designated AM1 and AM2, respectively (17). In a similar way, the calcitonin receptor (CTR) can partner with RAMP1 to produce a receptor for AMY that is designated AMY1 (17). Because CTR and CLR have significant homology, the AMY1 receptor (CTR/RAMP1) has a similar binding site to the CGRP receptor (CLR/RAMP1). 64

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The interaction of CGRP with the CGRP receptor has been studied by a number of approaches, including mutational studies and cross-linking experiments (18–20). These studies demonstrated that the ECDs of both CLR and RAMP1 are important for ligand binding and receptor activation (17). Residues in the transmembrane domains of CLR and the loops in between these transmembrane domains have also been found to play key roles in agonist binding and receptor function (21). The available data are consistent with the two-domain model for family B GPCRs described by Hoare (22). According to this model, the C-terminal region of the peptide CGRP first binds to ECDs of CLR and RAMP1. This binding event brings the N-terminal portion of CGRP into close proximity with the juxtamembrane region of CLR, allowing them to interact and produce receptor activation (22). This model neatly explains why the truncated peptide CGRP8-37, which lacks the N-terminal cyclic structure of the first seven amino acids, binds potently to the CGRP receptor but cannot activate it and acts as an antagonist (14).

Figure 1. The CGRP receptor antagonists olcegepant (1) and telcagepant (2) showing the privileged structures.

Small molecule CGRP receptor antagonists, such as olcegepant (23) (1, Figure 1) and telcagepant (24) (2), are also thought to bind to the ECDs of CLR and RAMP1 and thereby prevent binding of CGRP and receptor activation. A number of residues in the ECDs have been shown to be important for binding of small molecule receptor antagonists, including CLR Met42, RAMP1 Trp74, and RAMP1 Trp84 in the human CGRP receptor (25–28). One known exception to this antagonist binding mode is the hydroxypyridine class of antagonists, which do not seem to bind to the ECDs but appear to interact with transmembrane domain 7 in CLR (26, 29). The understanding of how small molecules antagonize the CGRP receptor was greatly enhanced by a group of researchers at Vertex, who expressed and purified a stable complex of the ECDs of CLR and RAMP1 (30). This purified ECD complex bound to small molecule CGRP receptor antagonists with high affinity but exhibited relatively weak binding to CGRP itself, consistent with the two-domain model for binding of the peptide agonist to its receptor (22). The Vertex group 65

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was able to crystallize the CLR:RAMP1 complex and solve the structures of the unliganded ectodomain as well as tertiary complexes with the small molecule antagonists olcegepant and telcagepant (31). A schematic representation of the binding of telcagepant to the CGRP ECD, as described by ter Haar et al. is shown in Figure 2, with an emphasis on the key interactions that are thought to contribute to potency (31). Telcagepant, like many small molecule CGRP receptor antagonists, has a “privileged structure” (32) that contains a terminal cyclic amide (CONH) moiety – in the case of telcagepant an azabenzimidazolone ring system that is known to be very important for CGRP receptor affinity. In the published crystal structure, this moiety makes hydrogen binding interactions with the backbone elements of CLR Thr122 (Figure 2) (31). Another residue that makes key interactions with telcagepant is CLR Trp72, which stacks against the piperidine ring of the “privileged structure” and also engages in a hydrogen bond between the tryptophan indole NH and the carbonyl oxygen of the caprolactam ring. The difluorophenyl ring in telcagepant, a group known to be crucial for binding affinity, occupies a hydrophobic pocket formed, in part, of CLR Met42, RAMP1 Trp 74, and RAMP1 Trp 84 (31). Consistent with the importance of these hydrophobic contacts, all three of these residues have been shown to be key contributors to the potency of small molecule receptor antagonists (27, 28). Overall, the complex of the CGRP receptor ECD with olcegepant is similar to that described for telcagepant and these same key residues interact with both small molecule antagonists (31).

Figure 2. Telcagepant bound to the CGRP receptor ECD. Telcagepant is shown in dark gray and the CGRP receptor ECD is shown in light gray. Key residues involved in antagonist binding are labeled. 66 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.

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Both telcagepant and olcegepant bind in an extended conformation, bridging a distance of about 18 Å between the critical hydrogen bonding interactions at CLR Thr122 and the important hydrophobic pocket formed at the CLR:RAMP1 interface (31). The crystal structures of these small molecule antagonists bound to the CGRP receptor ECD afford insight into the challenges of designing orally acting antagonists for this family B GPCR. In order to bind to this site on the protein with high affinity and block the binding of the peptide agonist, a molecule must apparently be relatively large and have multiple hydrogen bond donors and acceptors. This profile tends to be at odds with guidelines for orally bioavailable drugs, such as the Lipinski “Rule of Five” (33).

Discovery of MK-3207 Based on the significant evidence of a key role for CGRP in migraine headache, Merck initiated a program to develop orally bioavailable CGRP receptor antagonists in the early 2000s. High-throughput screening (HTS) identified the micromolar benzodiazepinone-based lead 3 (Figure 3), which was originally synthesized as a cholecystokinin receptor antagonist (34). In many ways, 3 was not an optimal lead structure for an oral drug discovery program. It only had modest binding affinity (Ki = 4.8 µM) and similar potency in a functional assay based on CGRP-stimulated production of cyclic adenosine monophosphate (cAMP) in cells (cAMP IC50 = 6 µM) (35). The combination of modest potency and large molecular size meant that it had relatively poor ligand efficiency (LE = 0.18 kcal/mol) (36). Compound 3 also possessed a significant number of hydrogen bond donors (four) and acceptors (five), which correlated with a calculated polar surface area (PSA) that predicted poor passive permeability (PSA = 147 Å2) (37). However, the structure of 3 was novel when compared with other known CGRP receptor antagonists, many of which were based on a peptidic backbone. Moreover, it was the only novel and tractable lead identified from the high-throughput screen and the team investigated the optimization of this benzodiazepinone lead. One approach to optimizing the HTS lead was based on the structural analogy between the spirohydantoin moiety in 3 and the piperidinyldihydroquinazolinone in olcegepant (1). Both contained a secondary amide hydrogen bond donor-acceptor pair embedded in a heterocycle and it was hypothesized that these rings could be key components in related “privileged structures” (32) that played a key role in binding to the GPCR. This hypothesis prompted the team to evaluate a number of piperidinyl privileged structures and led to the identification of a novel piperidinylazabenzimidazolone that provided the optimal balance of potency and chemical stability (38). Reengineering of the benzodiazepinone portion of the molecule, with the goal of improving physicochemical properties, led to caprolactam-based antagonists and ultimately to the discovery of telcagepant (2, MK-0974), the first orally bioavailable CGRP receptor antagonist to advance to the clinic (24). Thus, the initial approach to optimization of HTS lead 3 was to replace the tetralin-spirohydantoin part of the structure with a piperidinyl privileged 67

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structure. In a complementary approach, the benzodiazepinone was removed and the spirohydantoin portion of compound 3 was used as the basis of a rapid analogue screen for benzodiazepinone replacements with lower molecular weight (39). Early results indicated that the tetralin ring could be effectively replaced by an indane and led to the identification of a number of submicromolar, racemic lead structures (39). Resolution of the indanylspirohydantoin and further optimization afforded 4 (Figure 3, Ki = 21 nM; cAMP IC50 = 78 nM), in which a substituted benzimidazolone replaced the benzodiazepinone. In the simplified lead 4, the (R)-enantiomer of the spirohydantoin was preferred in terms of CGRP-R affinity and it exhibited about 200-fold higher affinity for the CGRP-R than the higher molecular weight HTS lead 3. Compound 4 exhibited a good pharmacokinetic profile in rat, dog and monkey, with low plasma clearance and good oral bioavailability (F = 29–83%) (39).

Figure 3. Spirohydantoin lead compounds and tricyclic CGRP receptor antagonists.

Although 4 was an attractive lead structure, its potency was suboptimal and incompatible with a low projected clinical dose. It was found that the 2-pyridyl ring could be replaced by a glycine substituent to provide an analogue with similar affinity for the CGRP-R. An interesting approach to potency enhancement was realized when this glycine substituent was constrained to give the tricyclic moiety found in 5 (Figure 3, Ki = 0.51 nM; cAMP IC50 = 2.4 nM) (40). This tricyclic benzimidazolone represented a 40-fold increase in CGRP-R affinity relative to the pyridyl-substituted 4. 68 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.

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Additional improvements in potency were achieved by rational modification of the spirohydantoin moiety based on its analogy to the azabenzimidazolone privileged structure in compounds like telcagepant. Specifically, replacement of the spirohydantoin in 5 with a spiroazaoxindole led to the highly potent antagonist 6 (Figure 3, Ki = 0.04 nM; cAMP IC50 = 0.28 nM), which possessed approximately 500-fold enhanced CGRP-R affinity compared with 4 (41). Unfortunately, spiroazaoxindole 6 was not orally bioavailable in preclinical species and it appeared that low passive permeability was at least partly responsible. It was known that low permeability could limit the oral absorption of related compounds and the calculated PSA of the compounds proved to be a useful guide: when PSA was greater than 130 Å2 the oral bioavailability was usually very low (39). For 6, the PSA was 149 Å2 and the aqueous solubility was poor (0.24 µg/mL at pH 7.4 for amorphous material) (42). In order to reduce the PSA, the benzimidazolone was replaced with an indoline and it was hoped that the weakly basic indoline nitrogen would afford improved solubility at acidic pH (42). These design considerations led to 7 (Figure 3, Ki = 0.35 nM; cAMP IC50 = 2.4 nM), which was orally bioavailable in rat, dog, and monkey (F = 25–49%), and possessed similar potency to telcagepant (42). Before the publication of the crystal structures determined by the Vertex team (31), conformationally constrained analogues were studied as one way to provide information on the bioactive conformation of CGRP receptor antagonists. One such example was the use of a quinoline-based central constraint as a replacement for the central amide bond in compounds such as spirohydantoin 4 (43). This modification had the added advantage that it reduced PSA and therefore might be expected to improve membrane permeability. This strategy led to quinoline 8 (Figure 3, Ki = 0.52 nM; cAMP IC50 = 2.2 nM), in which the spirohydantoin was reintroduced, in an effort to improve solubility (43). This quinoline central constraint appeared to be a good mimic of the bioactive conformation of the central amide and it also helped to impart good oral bioavailability for compound 8 in rat, dog, and monkey (F = 38–59%) (43). Not only did quinoline analogues like 8 shed light on the bioactive conformation of spiroindane-based CGRP receptor antagonists, but they also provided inspiration for the design of 9 (Figure 4, Ki = 1.9 nM), which was part of an effort to simplify the highly constrained nature of compounds like 8 to facilitate rapid exploration of SAR (44). Somewhat surprisingly, the preferred stereochemistry of the spirohydantoin 9 had switched to (S) from (R) in earlier compounds like 4. This stereochemical inversion effectively resulted from a change in the position of attachment between the amide nitrogen and the indane and this led to very different SAR for the new series. Compound 9 represented an attractive lead compound, in part because of its impressive ligand efficiency (LE = 0.34 kcal/mol), which is excellent for a CGRP receptor antagonist and compares favorably with olcegepant (1) (LE = 0.27 kcal/mol) and telcagepant (2) (LE = 0.31 kcal/mol). One area for improvement was the 370-fold selectivity of 9 for the CGRP-R over the AM2 receptor (CLR/RAMP3), which was deemed to be suboptimal (44). Initial exploration of SAR in this new series focused on improving potency and selectivity vs. the AM2 receptor. It was quickly established that fluoro 69

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substitution of the terminal benzyl group helped to address both concerns. Additionally, cyclization of the N-benzylpivalamide end group was well tolerated, affording the substituted piperidinone analogue 10 (Figure 4, Ki = 0.23 nM; cAMP IC50 = 0.91 nM) (44). Further potency enhancement was achieved by replacing the spirohydantoin with the corresponding spiroazaoxindole, in analogy with 5 and 6 (Figure 3). In the context of these piperidinones, incorporation of the spiroazaoxindole privileged structure for spirohydantoin provided a 6-fold increase in CGRP receptor affinity to give the picomolar antagonist 11 (Figure 4, Ki = 0.039 nM; cAMP IC50 = 0.16 nM) (44, 45).

Figure 4. Spiroindane CGRP receptor antagonists. Piperidinone 11 exhibited improved selectivity versus the AM2 receptor (AM2/CGRP selectivity = 4100-fold) compared with earlier analogues like 9 (44). In the cell-based functional assay, 11 had subnanomolar potency in the presence of 50% human serum (cAMP + HS IC50 = 0.35 nM). Compound 11 was found to be orally bioavailable in rat (F = 12%) and dog (F = 44%) but not in monkey (F = 0%), and this deficit was significant because it was important to fully evaluate the in vivo pharmacology of such small molecule CGRP receptor antagonists in monkeys because they exhibit significantly reduced affinity for non-primate CGRP receptors (12). In analogy with data for telcagepant, the low monkey oral bioavailability of 11 was thought to be due in part to intestinal first-pass metabolism (45). It was known that the piperidinone ring of 11 was subject to 70

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significant metabolism in vitro, so the team sought to both reduce metabolism and increase aqueous solubility by incorporating polar functionality into this ring. This strategy led to morpholinones such as 12 and piperazinones such 13 (Figure 4) (45). While morpholinone analogue 12 (Ki = 0.018 nM; cAMP IC50 = 0.32 nM) was highly potent and had good oral bioavailability in rat (F = 59%) and dog (F = 72%), it was not orally bioavailable in monkey (F = 0%) (45). Piperazinone 13 was not only potent (Ki = 0.034 nM; cAMP IC50 = 0.17 nM) but was also more soluble than the corresponding morpholinones, especially at acidic pH. The improved aqueous solubility correlated with modest oral bioavailability in monkey (F = 7%) and this observation led to a significant effort to optimize the potency, selectivity, and pharmacokinetic properties of such piperazinone analogues (45). This led to the discovery that replacement of the gem-dimethyl substituents on the piperazinone ring of 13 with a spirocyclopentyl ring provided MK-3207 (14) (Figure 4, Ki = 0.021 nM), a picomolar CGRP receptor antagonist with an excellent overall profile (45).

Preclinical Profile of MK-3207 MK-3207 was a picomolar CGRP receptor antagonist with a binding affinity (Ki = 0.021 nM) only slightly lower than the much larger olcegepant (Ki = 0.014 nM), leading to a significantly higher ligand efficiency for MK-3207 (0.35 kcal/mol) compared with olcegepant (0.27 kcal/mol). MK-3207 was also highly potent in a cell-based functional assay (cAMP IC50 = 0.12 nM) and this cell-based potency was slightly shifted in the presence of 50% human serum (cAMP + HS IC50 = 0.17 nM), suggesting that the compound was relatively free in human serum (45). The radiotracer [3H]MK-3207 was used in detailed binding studies and it was determined that the KD of MK-3207 was 60 pM and that it had a reduced off-rate (0.012 min-1) and longer dissociation half-life (59 min) compared with telcagepant (KD = 1.9 nM) (46, 47). The high in vitro potency of MK-3207 translated to an in vivo monkey pharmacodynamic model, based on capsaicin-induced dermal vasodilation (CIDV) (48). In this CIDV model, MK-3207 was found to block 90% of the capsaicin-induced increase in dermal blood flow at a plasma concentration of 7 nM (EC90 = 7 nM) (46). For telcagepant, the corresponding EC90 value in the rhesus monkey CIDV model was found to be 994 nM (49). Thus, compared to telcagepant, MK-3207 was about 40-fold more potent than in vitro and approximately 100-fold more potent in vivo. Small molecule CGRP receptor antagonists typically display pronounced species selectivity, with significantly higher affinity for the CGRP receptors of primates than for those of non-primates, and MK-3207 was no exception. Thus, while it had high affinity for rhesus monkey CGRP-R (Ki = 0.024 nM) it exhibited markedly reduced affinity for the CGRP receptors of rat (Ki = 10 nM) and dog (Ki = 10 nM) (46). MK-3207 had excellent selectivity for the human CGRP-R against the related AM1 (> 600,000-fold), AM2 (> 6,500-fold) and AMY3 (> 5,000-fold) receptors but only modest selectivity against the AMY1 receptor 71

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(30-fold). MK-3207 was screened against a panel of 169 receptors, enzymes, and transporters and was found to be greater than 50,000-fold selective for human CGRP-R versus any of these other targets (46). The preclinical pharmacokinetic profile of MK-3207 is summarized in Table 1. In rat and dog, the compound exhibited low plasma clearance and good oral bioavailability (F = 67–74%). In contrast, the plasma clearance in rhesus monkey was moderate and the oral bioavailability was relatively low at lower doses (F = 9% at 2 mg dose/kg bodyweight/day (mpk)) but improved at higher doses (F = 41% at 20 mpk) (45). These results suggest that saturable first-pass metabolism played a role in limiting the oral bioavailability in monkeys, similar to observations with the earlier compound telcagepant (50).

Table 1. Preclinical Pharmacokinetic Properties of MK-3207 Species

Dose (mpk)

Fa (%)

Clb (mL/min/kg)

Vdssb (L/kg)

IV t1/2b (h)

Rat

10 (PO); 2 (IV)

74

11

0.3

0.6

Dog

2 (PO); 0.5 (IV)

67

8.0

0.6

1.0

Monkey

2 (PO); 0.5 (IV)

9

15

1.7

1.5

Determined after dosing in 0.5% or 1% methocel vehicle. DMSO vehicle.

a

b

Determined after dosing in

As detailed in Table 1, the plasma half-life was short in preclinical species but a short half-life is quite compatible with acute treatment of migraine in the clinic. Overall, MK-3207 had an excellent preclinical profile in terms of its potency, selectivity and pharmacokinetics and it was advanced for clinical evaluation as a novel treatment for migraine with the potential for a low human dose based upon its significantly improved preclinical potency relative to telcagepant (51).

Clinical Profile of MK-3207 Consistent with results in preclinical species, MK-3207 was orally bioavailable in humans. Following oral dosing, the compound exhibited a Tmax of ~ 1–2 h and a terminal plasma half-life of ~ 9–18 h (52). The CIDV pharmacodynamic assay that was developed in rhesus monkeys could be readily translated to the clinical setting by virtue of its non-invasive nature and provided results in good agreement with the preclinical observations, with a human CIDV EC90 ≈ 14 nM (52). A detailed PK/PD analysis indicated that a 20 mg oral dose of MK-3207 should produce effective blockade of the CGRP receptor in the periphery, similar to clinically efficacious doses of telcagepant (52). 72 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.

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A placebo-controlled Phase II study evaluated seven doses of MK-3207 (from 2.5 to 200 mg) with a primary endpoint of pain freedom at 2 h (53). In this study, doses of 10 mg, 100 mg, and 200 mg were found to be superior to placebo at the primary endpoint. Based on the available data, the 200 mg dose did not appear to be more effective than any dose at or above 10 mg, and the 10 mg dose may have represented a plateau in terms of efficacy (53). This possibility is consistent with the PK/PD modeling (52). Unfortunately, although MK-3207 was generally well tolerated in this clinical trial, in extended Phase I studies there were a number of observations of delayed liver test abnormalities, typically after cessation of dosing. As a result of these findings, the development of MK-3207 was discontinued (53). The liver transaminase elevations seen for MK-3207 raised the possibility of a mechanism-related effect, since liver enzyme elevations were also observed for telcagepant. However, the pattern of clinical findings with telcagepant was quite different from that observed with MK-3207, suggesting that these effects may be compound-related rather than mechanism-based (54).

Medicinal Chemistry Synthesis of MK-3207 The synthesis of piperazinones such as MK-3207 was accomplished by amide coupling of aniline and carboxylic acid fragments described in the following two schemes. The route to aniline 25 was essentially the same as the original published synthesis (Scheme 1) (41). The 2-(trimethylsilyl)ethoxymethyl (SEM) protecting group was selected for protection of 7-azaindole (15) based on its stability under a range of conditions. This protected azaindole 16 was converted to the corresponding azaoxindole 18 using methodology described by Marfat and Carta (55). 4-Nitrophthalic acid (19) underwent standard reduction and bromination to afford 1,2-bis(bromomethyl)-4-nitrobenzene (21) in essentially quantitative yield. The key step was bis-alkylation of oxindole 18 with dibromide 21 to provide spirocycle 22 in 75% yield. This bis-alkylation employed Cs2CO3 as base, and was run at relatively low concentration in DMF to reduce the formation of oligomeric material. The nitro group in 22 was reduced under catalytic hydrogenation conditions to give the corresponding racemic aniline (±)-23, which could be separated into its individual enantiomers using chiral column chromatography. Alternatively, racemic aniline (±)-23 could be protected with a Boc group and the chiral separation conducted on carbamate (±)-24 (as shown in Scheme 1), which had improved solubility relative to the unprotected aniline. Finally, the desired enantiomer was deprotected to afford aniline (R)-25. To facilitate exploration of the piperazinone series, a practical, two-step synthesis of 6-phenylpiperazin-2-ones was developed (56). This route allowed rapid access to piperazinones such as (±)-30 (Scheme 2), albeit in moderate yield. Thus, alkylation of methyl 1-aminocyclopentanecarboxylate (27) with 3,5-difluorophenacyl bromide (26) using trisodium phosphate as base proceeded in 55% yield. The resulting aminoketone 28 was found to have poor stability 73

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and underwent oxidation at the aminoketone α-carbon to produce a symmetrical dimer. This oxidative process could be minimized by storing aminoketone 28 as the mesylate salt and by protecting it from air (45). Reductive amination of ketone 28 with glycine ethyl ester (29) and in situ cyclization of the resulting amine afforded racemic piperazinone 30 in 45% yield.

Scheme 1. Medicinal Chemistry Synthetic Route to Aniline (R)-25

To facilitate the subsequent chemistry and chromatography steps, the piperazinone was protected with a Boc group, and separation of the enantiomers provided the desired (R)-enantiomer of ester 31. Saponification of 31 led to the corresponding lithium salt 32, which was coupled with aniline 25 under standard conditions to provide, after deprotection, MK-3207 (14). 74 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.

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Scheme 2. Medicinal Chemistry Synthetic Route to MK-3207 (14) The route to aniline (R)-25 consisted of eleven total steps with an overall yield of 15% from 7-azaindole. The route from bromoacetophenone 26 to MK3207 consisted of six total steps with an overall yield of 9%. These synthetic approaches enabled the medicinal chemistry team to rapidly explore the structureactivity relationships of these piperazinone CGRP receptor antagonists. The route also allowed the synthesis of multigram quantities of MK-3207 to support early toxicological and pharmacological characterization of the compound, but it was clear that significant improvements to the chemistry would need to be made as MK-3207 advanced into development.

Process Chemistry Development Within the pharmaceutical industry, organic synthesis encompasses both medicinal chemistry and process chemistry activities. Medicinal chemists focus on the discovery of appropriate small molecules that interact with biological mechanisms in such a way as to provide a beneficial effect on a given disease state. Process chemists aim to design and develop efficient, practical and economical chemical syntheses for the drug candidates identified by medicinal chemists. The differing goals of medicinal and process chemists have a significant bearing on the nature of the synthetic chemistry strategies employed by either group of chemists. Broadly, medicinal chemists typically design flexible synthetic routes that quickly access common building blocks from which many candidate compounds 75 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.

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can be prepared, allowing structure-activity relationships to be determined and the appropriate molecular entity to be identified. Reaction yield, expense, robustness and green chemistry considerations are of secondary importance relative to speed of compound synthesis and acquisition of data from biological assays. Conversely, the industrial manufacture of drug compounds at commercial scale puts a premium on these other synthesis attributes. Consequently, process chemists seek to devise an ideal route that has maximum synthetic efficiency and is high yielding. Often this necessitates the invention of new synthetic methods, providing an excellent venue for creative chemists to conceive novel applications of existing transformations or even to develop entirely new reactions. The commercial process must be operationally safe, cost-effective and sufficiently robust to deliver active pharmaceutical ingredient (API) in consistently high quality. Furthermore, given the larger scale of operation relative to medicinal chemistry activities, minimizing the environmental impact of industrial drug production is a priority and drives the application of green chemistry principles wherever possible. Lastly, intellectual property (IP) aspects cannot be neglected when conducting commercial operations and therefore manufacturing processes need to have freedom to operate without infringing upon competitor patents. In addition to the myriad technical objectives faced by process chemists, attention must also be given to broader, cross-functional facets of development programs including project drivers and timelines. Typically, in the early stages of pre-clinical and clinical drug development, relatively small quantities of drug substance are required to support initial in vitro and in vivo toxicology studies and the speed of API delivery is normally prioritized over definition of an ideal synthesis. This being the case, it is not unusual to have an interim synthesis (“supply route”) used to support early API deliveries with a parallel (or staggered) effort to develop increasingly refined routes that are aligned with the overall program context, considering factors such as drug demand, synthesis complexity/cost and clinical timelines. In an ideal world, the best, most efficient synthesis is established as early as possible in the overall timeline but in practice it is common to observe an evolution in efficiency over the development cycle, culminating in the manufacturing route being ready as the program moves into Phase III. Excellent reviews are available that discuss chemical process development in the pharmaceutical industry at greater length (57–60). The discussion above highlights several common themes of process chemistry development across the industry. However, the details of route development, timing, and selection criteria vary, based on the unique features of individual projects and also according to the experiences and philosophies of different companies. In particular, decision-making on long-term manufacturing routes can be influenced not only by quantifiable metrics such as cost-of-goods, but also more subjective measures such as scientific elegance, novelty and broader impact in the field. The following sections in this chapter describe the process chemistry development for MK-3207. One objective is to provide instructive case studies on how and why the chemistry evolved with increasing scale and changing priorities as MK-3207 progressed into and through clinical development. Another aim 76

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is to highlight our cornerstone philosophy at Merck of implementing the best possible chemistry for our manufacturing routes and continuing to build upon a tradition of process chemistry innovation. Pursuing this goal resulted not only the identification of a highly efficient long-term route to MK-3207 but also the discovery and development of a number of synthetic methodologies; these transcended the unfortunate demise of this specific compound by subsequently impacting other projects as well as being novel scientific advances in their own right.

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MK-3207 Fragment Coupling Strategy The medicinal chemistry synthesis of MK-3207 featured the use of an amide coupling between aniline 25 and N-Boc-piperazinone 32 to complete the assembly of the molecular framework of the API (Scheme 2). This transformation was also appealing from a process chemistry perspective in that it provided a convergent approach with late-stage coupling of two fragments of approximately equal size and complexity, and also had high likelihood of being able to be developed into a robust, scalable reaction. Therefore at the outset of our work the decision was made to retain this key bond disconnection. In the forward synthetic direction, an opportunity was identified to streamline the endgame by eliminating the use of protecting groups, resulting in development of the coupling strategy illustrated in Scheme 3.

Scheme 3. Modified Fragment Coupling Strategy to Generate MK-3207 The benefit of using of unprotected piperazinone (R)-33 came at the expense of introducing a chemoselectivity challenge, as aniline (R)-25 needed to react in preference to the secondary amine in the piperazinone (R)-33. The sterically hindered nature of the latter meant that the desired coupling mode was 77 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.

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indeed feasible. However, further coupling of the initially formed MK-3207 product to another molecule of piperazinone (R)-33 resulted in the formation of a troublesome impurity 34 that was difficult to reject, thus the coupling reagents and conditions needed to be optimized. A panoply of methods exists for effecting amide couplings, many of which have been employed on scale in the pharmaceutical industry (61). It was determined that EDC-mediated coupling minimized impurity formation and generated MK-3207 in high yield. After aqueous workup the API was crystallized from EtOH and isolated as a solvated form. This provided good rejection of other impurities (including stereoisomers), and enabled recrystallization in a subsequent purification step to obtain the desired final API form. Initially this was the HCl salt, before a freebase monohydrate form was selected for early clinical development. Subsequently, a more stable anhydrate phase was discovered and was to be developed for use in the commercial tablet formulation prior to discontinuation of MK-3207. The following sections describe development of routes to the key aniline and piperazinone fragments, highlighting new chemistry that was developed along the way.

Piperazinone – Process Chemistry First-Generation Synthesis The original synthesis of the chiral piperazinone fragment of MK-3207 was racemic in nature, and utilized preparative chiral column HPLC purification in order to access enantioenriched material (Scheme 2). This approach was perfectly suited to the goal of rapid discovery of the optimal drug candidate for further development. However, upon transition into the pre-clinical development space, identification of an asymmetric approach became a high priority objective for the process chemistry team. As is typical for early development projects, speed to the clinic was also highly desirable. To this end, the team focused early efforts around modifying the discovery synthesis to introduce control of chirality while maintaining many of the already established bond formations, so as to minimize development time on the path to the first GMP delivery of MK-3207 API.

Scheme 4. First-Generation Retrosynthesis of Piperazinone (R)-33 78 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.

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Retrosynthetic analysis (Scheme 4) suggested that the piperazinone ring could be assembled via cyclization of the chiral benzylic amine 35 that would ultimately be derived from stereoselective displacement of an appropriate leaving group. Assuming the requisite leaving group would take the form of an activated alcohol (e.g., 36), ketone 28 could serve as a viable precursor to introduce the benzylic stereocenter via an asymmetric reduction process. Further disconnections to the simpler materials 26 and 37 would mirror that in the medicinal chemistry route.

Scheme 5. First Generation Synthesis of Piperazinone Acid (R)-33 The first generation process chemistry route to the chiral piperazinone acid (R)-33 began with addition of the Grignard reagent derived from 1-bromo-3,5-difluorobenzene (37, Scheme 5) to acetyl chloride in the presence of CuCl and AlCl3. The resulting 3,5-difluoroacetophenone (38) was then subjected to bromination with NBS under acidic conditions to afford 79 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.

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3,5-difluoro-α-bromoacetophenone (26). Bromination of 38 with molecular bromine resulted in the formation of around 11% of the dibromo impurity 42 (Scheme 6). This was problematic because the dibromo impurity participated in the subsequent step to form an imine 43 which reacted with another molecule of ketone 28 to generate the highly insoluble dimeric impurity 44. This impurity was first observed in the medicinal chemistry synthesis and it was difficult to remove from intermediate 28. Investigation of alternative reagents established that NBS gave a cleaner reaction profile with an acceptable level of 4% of the dibromo impurity 42.

Scheme 6. Fate of Dibromo Impurity 42: Generation of Dimer Impurity 44

Nucleophilic displacement of bromide 26 by cycloleucine 27 provided the α-aminoacetophenone derivative 28 which, as our medicinal chemistry colleagues had identified earlier, was somewhat unstable and required isolation as the more stable methanesulfonate salt (Scheme 5). Suitable conditions for the key asymmetric reduction of ketone 28 were then quickly identified using high-throughput experimentation (HTE) techniques that have been extensively developed and applied in our laboratories by our Chemocatalysis group (62–69). This transformation was performed under hydrogen in the presence of a chiral non-racemic ruthenium catalyst in MeOH/MsOH solvent mixture to deliver the aminoalcohol 39 in 60–70% enantiomeric excess (ee). In order to activate the benzylic hydroxyl group in 39 as a leaving group it was decided to form a cyclic sulfamate in a two-step process via the reaction with thionyl chloride followed by periodate oxidation. This choice of activation served to simultaneously protect the adjacent nitrogen and prevent any undesired participation from this group. The nucleophile to open cyclic sulfamate 36 was ethyl glycinate freebase (29), which must first be prepared via treatment of the commercially available hydrochloride salt 29•HCl with an appropriate base. Attempts to form the freebase process in situ using Hünig’s base were plagued by unwanted interference from soluble chloride ion, which was sufficiently nucleophilic to open the cyclic sulfamate and generate a benzyl chloride by-product 46, (Scheme 7). 80

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Scheme 7. Generation of Chloro Impurity 46 To avoid this issue, we investigated the option of forming the glycine ethyl ester free-base from the hydrochloride salt in a separate step using a two-phase aqueous Na2CO3/MTBE extraction. Unfortunately, this approach was also problematic because ethyl glycinate free-base is slightly volatile and some material was lost during the extended distillation required for azeotropic drying (water was deleterious to the cyclic sulfamate ring-opening). Further, the free-base is inherently unstable with a tendency to polymerize over time, which causes additional loss of yield. A practical solution to this dilemma that combined the most favorable attributes of aqueous and non-aqueous free-base techniques was ultimately identified. Pre-treatment of the hydrochloride salt with tetramethylguanidine (TMG) in MTBE resulted in the formation of ethyl glycine free-base in solution and concomitant precipitation of the TMG hydrochloride salt. The extremely low solubility of TMG•HCl in MTBE ensured near complete removal of the chloride ions from the solution by filtration, thereby avoiding the formation of the benzyl chloride by-product 46. The anhydrous nature of the process obviated the need for prolonged azeotropic drying. Application of this process allowed displacement of the oxygen leaving group at the benzylic center in a stereospecific fashion (inversion) to yield the intermediate 45 that spontaneously cyclized to the piperazinone ring 40 after hydrolytic cleavage of the N-sulfate residue. At this stage, the moderate enantiomeric excess stemming from the asymmetric ketone reduction was upgraded via diastereomeric salt formation using tartaric acid derivative 41 (Scheme 5). This provided material in greater than 99% ee that could be taken through the remaining chemistry with no loss of stereochemical integrity. To complete the synthesis of the piperazinone fragment, the tartrate salt was first broken and then the ethyl ester was hydrolyzed to reveal the target acid (R)-33.

Piperazinone – Process Chemistry Second-Generation Synthesis Early GMP deliveries of MK-3207 utilized the first generation synthesis of the piperazinone acid (R)-33 described above. Although this route ably supported initial animal toxicology and clinical studies, it was recognized that an alternative approach would be required in the long term. The first-generation route to piperazinone acid 33 comprised ten steps in the longest linear sequence. There were several key issues with respect to process robustness/efficiency and economics. Despite extensive study, the asymmetric hydrogenation to set the benzylic stereocenter remained only moderately effective and typically delivered 81 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.

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material with enantiomeric excess in the range of 60–70%. This relative lack of stereocontrol necessitated an upgrade via the dibenzoyltartaric acid salt in the downstream chemistry, compromising the overall process efficiency. Additionally, both the aminoketone 28 and ethyl glycinate (29) intermediates had limited chemical stability and created issues for process robustness. Furthermore, cost analysis of this route to piperazinone acid 33 identified the synthetic amino acid "cycloleucine" (27) as a major contributor. Since this reagent constitutes an integral part of the molecular structure of MK-3207, its use is essentially mandatory in any synthesis. Consequently, introduction of this component at such an early stage in a ten-step sequence was not the ideal strategy from an economic standpoint. Taking all of these factors into account, together with the Merck goal to implement the best chemistry for our commercial products, the process chemistry team set out to design a second-generation approach to the synthesis of piperazinone acid 33 that would provide higher synthetic efficiency, better stereocontrol and greater overall economy (70).

Scheme 8. Second-Generation Retrosynthesis of Piperazinone 33 Retrosynthetic analysis for the second-generation route to the piperazinone acid 33 intermediate is shown in Scheme 8. The first disconnection revealed the core piperazinone heterocycle 47 and it was anticipated that selective alkylation of the amide in the presence of the secondary amine would be achievable under appropriate conditions. A benefit of this disconnection was that the source of the acid side-chain was now a readily available α-haloacetate reagent. This would obviate a process robustness issue in the first-generation synthesis, where the unstable glycine ethyl ester freebase was used as the source of this molecular fragment. To access the core piperazinone 47, it was recognized that a cyclic sulfamate intermediate 48 with “inverted” regiochemistry from the first generation synthesis could significantly shorten the synthetic sequence and confer several additional advantages. In addition, cyclic sulfamates participate in ring-opening/ring-closing reaction sequences with bifunctional reagents (e.g., amino acids) to afford piperazinones in a single step (71, 72). Due to steric encumbrance, cycloleucine 82

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appeared to be a challenging reaction partner for this process, but in light of the overall synthetic advantage conferred by this disconnection, we deemed it worthy of investigation. Having identified the key chiral intermediate, a solution to the problem of the benzylic stereocenter was required. Asymmetric hydrogenation of unsaturated compounds represents one of the most attractive and practical options for control of functionalized stereocenters (73, 74). Consequently, cyclic imine 49 was targeted as a likely precursor of the chiral sulfamate and the team sought to derive this cyclic imine from hydroxyacetophenone 50, itself available from 1-bromo-3,5difluorobenzene 37 as the raw material. Hydroxyacetophenone Synthesis α-Hydroxyacetophenones are useful synthetic intermediates amenable to various asymmetric transformations capable of generating valuable chiral compounds (75–77). Although α-hydroxyacetophenones had featured many times in the chemical literature, at the time of this research there were only a limited number of reports directly focused on general procedures for their preparation (78–87). This is perhaps indicative of underlying stability issues associated with these intermediates. Indeed, significant stability problems were encountered during early attempts to work with intermediate 50. However, these difficulties were eventually resolved after we understood the instability of the compound towards both oxygen and neutral-to-basic pH. Thus, a straightforward and general approach to the synthesis of α-hydroxyketones using readily available reagents was developed for the synthesis of the desired compound 50 (Scheme 9) (88).

Scheme 9. Synthesis of Hydroxyacetophenone 50 The arylzinc intermediate derived from 3,5-difluorobromobenzene was acylated with α-acetoxy acetyl chloride in the presence of CuCl to produce α-acetoxy-3,5-difluoroacetophenones (51) (Scheme 9). Treatment of 51 with 5 N aqueous HCl at 40 °C in MeOH led to the formation of α-hydroxy-3,5-difluoroacetophenone (50). We noticed that the hydroxyketone 50 is relatively sensitive to oxygen in solution, presumably via facile oxidation of equilibrium concentration of the enol tautomer. For this reason, we carried out the hydrolysis of 51 with 5 N aqueous HCl under nitrogen in deoxygenated MeOH. Under these conditions the formation of polymeric degradants was minimized and the assay yield of 50 became generally good. The product 50 is isolated by direct crystallization from the reaction mixture after dilution with water. With the ready access to hydroxyacetophenone 50 secured, the team was positioned to develop the subsequent planned synthetic transformations. 83

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Cyclic Sulfamate Synthesis Cyclic sulfamates similar to 49 have been described previously in the literature. Initial laboratory investigations quickly revealed the known methods of preparation to be entirely unsuitable for large scale operation (89). The literature method (Scheme 10) relies on an in situ preparation of sulfamoyl chloride, an unstable compound that is not readily commercially available on scale. Reported procedures involve a neat reaction between N-chlorosulfonylisocyanate (CSI) and 95% aqueous formic acid, which is relatively hazardous as it releases one mole equivalent each of CO and CO2. Small scale laboratory testing revealed this reaction is highly exothermic and the mixture solidified as conversion proceeded, preventing agitation and making control of the exotherm even more problematic. In the next stage, sulfamoyl chloride was combined with the α-hydroxyacetophenone 50 in the presence of pyridine to yield the O-sulfamoyl intermediate 54, which was cyclized and dehydrated to the cyclic sulfamate via thermal treatment during workup. A significant side-reaction was the nucleophilic attack by chloride ion on intermediate 54 to yield the α-chloroacetophenone 55. Pyridine is a poor choice of base in this regard because pyridinium hydrochloride has reasonable solubility in the reaction medium and facilitates side-product formation (vide infra). In addition to having a detrimental effect on yield, chloroketone 55 is a severe lachrymator, creating handling/industrial-hygiene issues during workup.

Scheme 10. Literature Conditions for Preparation of Cyclic Sulfamate 49 This procedure was deemed unsuitable for large-scale synthesis and spurred development of a new process that was both safer and higher yielding (Scheme 11). The reaction of t-BuOH with CSI was essentially quantitative and generated N-Boc-sulfamoyl chloride (56) cleanly. Since this process is a simple addition reaction there are no gaseous by-products. Also, the reaction was conveniently carried out in 2-Me-THF, allowing for good control of the exotherm via rate of addition of reagent. Range-finding experiments indicated that over-reaction of excess t-BuOH with the initially formed N-Boc-sulfamoyl chloride was not an issue under the reaction conditions. The resulting solution of N-Boc-sulfamoyl chloride 56 had good stability, which afforded an acceptable operating window for large scale processing. 84

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Combination of 56 with the hydroxyacetophenone 50 generated negligible exotherm because there was no reaction until the subsequent addition of triethylamine; the O-sulfamoylation exotherm was then controlled via addition rate of the base. O-Sulfamoylation generates intermediates 54 and 57 that are activated towards nucleophilic displacement by chloride ion, generating the undesired α-chloroacetophenone side-product 55. The reaction temperature and age time also had a significant impact on the amount of impurity 55 formed, with greater than 90% conversion to this compound after 24 h age at room temperature using pyridine as the base. To mitigate these issues, we used triethylamine as the base and carried out the reaction at –5 to 0 °C. Under these conditions, triethylamine hydrochloride precipitated from solution, and that reduced the concentration of dissolved chloride ion to suppress the formation of 55. Cooling below –10 °C decreased the solubility of substrate 50, and thus the rate of reaction, significantly. Above 0 °C the solubility of triethylamine hydrochloride increased and the level of α-chloroacetophenone side-product 55 would accumulate over time. Under the optimal conditions, the O-sulfamoylation reaction was typically complete within 30 min and the reaction was then quenched by the addition of 0.5 M NaHSO4, maintaining the batch temperature around 0 °C. The use of 2-Me-THF as solvent allowed direct phase separation and the rejection of triethylamine hydrochloride into the lower aqueous phase. A second wash with additional 0.5 M NaHSO4 ensured negligible concentration of chloride ion in the organic phase and rendered the intermediate O-sulfamoyl compound 57 stable for continued processing at elevated temperatures.

Scheme 11. Improved Synthesis of Cyclic Sulfamate 49 Although it was possible to isolate the N-Boc-protected-O-sulfamoyl intermediate 57 via crystallization, the opportunity to continue in a through process to the desired cyclic sulfamate 49 was attractive. Accordingly, the wet 2-Me-THF solution of uncyclized intermediate 57 was treated with a catalytic quantity of p-TsOH•H2O (1 mol%) and the reaction mixture was heated at reflux to effect sequential N-Boc deprotection and cyclization/dehydration to the cyclic sulfamate 49. The N-Boc group remained largely intact until the batch 85 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.

temperature reached at least 60 °C and then cleaved smoothly under the action of catalytic acid. Over the course of several hours the conversion to cyclic sulfamate 49 reached greater than 95%, and then close to complete conversion was attained via azeotropic removal of water by distillation. The final isolation involved cooling and washing with water to remove residual inorganics (NaHSO4) from the organic phase, followed by crystallization of 49 from a mixture of 2-Me-THF and heptanes. The overall assay yield of sulfamate 49 from hydroxyketone 50 was 94% and the isolated yield was 86%.

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Asymmetric Hydrogenation of Cyclic Sulfamate Concurrent with this process development work, a literature report appeared from Zhou and coworkers on the asymmetric hydrogenation of cyclic sulfamates (89). In this paper, the levels of conversion and enantiocontrol were excellent across a variety of substrates, so we were optimistic around the use of 49. However, the optimal conditions described by Zhou had several potential drawbacks with respect to large scale operation. From an economic perspective, the use of 2,2,2-trifluoroethanol (TFE) as solvent, Pd(TFA)2 as a catalyst precursor and (S,S)-f-binaphane as the chiral ligand would all contribute to a high cost for the key asymmetric transformation in the second-generation route. Furthermore, in practical terms, the requirement for relatively high pressures of hydrogen gas (500 psig) could limit options for implementing this chemistry at vendors lacking appropriate plant equipment. To discover an improved process for this key transformation, the project team embarked upon a systematic study of the reaction conditions. Determination of the optimal conditions for this asymmetric hydrogenation was again achieved through HTE techniques. For initial screening of reaction conditions, certain aspects of the published procedure (such as the TFE solvent and the 500 psig pressure of hydrogen) were retained while the catalyst precursor and ligand were varied. A control experiment where the exact literature conditions were used was also conducted. A summary of results is shown in Table 2. The control experiment using Pd(TFA)2 and the (S,S)-f-binaphane ligand 58 gave a similar result to that published. Complete conversion was reached in all cases and excellent enantiomeric excess was also observed using Rh, Ir and Pd catalyst precursors in conjunction with several alternative commercially available phosphine ligands. The Josiphos ligand 60 was selected for further study because this was readily available on production scale as part of the process for another Merck product (Januvia®) (90). By examining the other reaction parameters (Table 3), it was established that Pd(OAc)2 could replace Pd(TFA)2 with similar performance. More significantly, in contrast to the literature report, replacement of TFE with MeOH as the reaction solvent was equally effective and conferred several process advantages such as cost reduction, alleviation of industrial hygiene concerns around TFE and the opportunity for a simple product isolation (vide infra). Also notable was the option to conduct the hydrogenation using significantly lower hydrogen pressures. 86 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.

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Table 2. Conditions for Initial Evaluation of Metal/Ligand for Asymmetric Hydrogenation of Cyclic Sulfamate 49

In the final process (Scheme 12), cyclic sulfamate 49 was dissolved in 5 volumes MeOH and subjected to 0.3 mol% catalyst loading (0.33 mol% ligand 60) under 40 psig of hydrogen at 40 °C. After complete conversion the batch was treated with carbon, filtered and the desired product was isolated via crystallization following addition of water. The isolated yield was 94% and the white solid was typically of greater than 99 wt% purity. The measured enantiomeric excess of the isolated material matched the in-process-control assay, indicating no upgrade was available via this crystallization. Subsequent crystallization of a downstream intermediate afforded the necessary upgrade in stereochemical purity (91). Piperazinone Formation Cyclic sulfamates are known to undergo reactions with amino esters that ultimately cascade to piperazinones (71, 72). For the target piperazinone 33 the amino ester required was the unnatural but commercially available "cycloleucine" methyl ester (27). Due to steric hindrance, cycloleucine is an extremely poor nucleophile. In contrast to many other amino esters, cycloleucine freebase is relatively stable for extended periods and did not polymerize to any significant extent upon storage. This lack of reactivity necessitated significant process development in order to achieve an acceptable rate of reaction with cyclic sulfamate 48. Scheme 13 illustrates the reaction conditions initially used to gain proof-of-concept for this particular piperazinone formation, as well as the subsequently optimized process. 87

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Table 3. Optimization of Catalyst Precursor and Loading, Solvent, Hydrogen Pressure and Reaction Temperature

Scheme 12. Optimized Conditions for Asymmetric Hydrogenation of 49

Cycloleucine methyl ester 27 is available commercially as the hydrochloride salt, which needed to be converted to the corresponding freebase before reaction with cyclic sulfamate 48. In our initial studies, cycloleucine freebase was extracted into toluene from aqueous potassium phosphate tribasic. Concentration of the organic phase allowed for azeotropic drying prior to reaction with the cyclic sulfamate. Heating the dry toluene solution of cycloleucine 27 and cyclic sulfamate 48 at 70 °C led to ring opening to give intermediate 61, and then 5 M aqueous HCl was added to effect the cleavage of the N-sulfate to generate 1,2-diamine 62. However, the two-phase nature of the toluene/water system made the sulfate hydrolysis very slow. Addition of an acid stable co-solvent (1,2-dimethoxyethane) was necessary to make the phases partially miscible and increase the rate of hydrolysis to a practical level. After hydrolysis, the majority of diamine 62 was present in the acidic aqueous phase (as determined by HPLC assay) while the organic phase contained some dark polymerized material, which was separated via a phase cut at this stage. Adjustment of the pH via treatment 88

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with 10 M aqueous NaOH resulted in piperazinone formation and partitioning into the freshly replaced organic phase. After phase cut and solvent switch the desired piperazinone 47 was isolated via crystallization. Although the overall yield was reasonable, a close examination of the various operations revealed several opportunities for streamlining the unit operations. The overall piperazinone process involves four distinct stages: •

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• • •

formation of cycloleucine methyl ester free base from hydrochloride 27•HCl ring opening of cyclic sulfamate (48→61) N-sulfate cleavage (61→62) pH adjustment/ring closure to form the piperazinone (62→47)

Scheme 13. Reaction Sequence for Formation of Piperazinone 47 In developing this overall process, the following aspects received close attention: • • • •

the volatility of cycloleucine methyl ester 27 during free-basing process. the dipolar aprotic solvent used to enhance nucleophilic ring opening. the potentially deleterious presence of extraneous nucleophiles (including water and certain solvents). solvent/water miscibility and pH stability across a wide range for Nsulfate cleavage and lactamization. 89

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To address the sluggish nature of the ring-opening step, we evaluated several solvents with particular focus on those likely to facilitate formation of the highly polar reaction intermediate (N-sulfate 61). Protic solvents such as MeOH and EtOH were excluded after experiments confirmed partial solvolytic opening of the cyclic sulfamate starting material 48. More surprising, but unfortunately also discouraging, were observations that typical dipolar aprotic solvents such as THF, MeCN, DMF, DMAc and NMP did not provide improved results. In fact, in the case of DMF and DMAc, these solvents were sufficiently nucleophilic to be competitive with cycloleucine 27 and consequently led to unwanted side reactions and poor reaction profiles. Following these observations attention was turned to sulfolane, a solvent that appeared a good candidate for this particular process (92). Sulfolane has one of the highest dielectric constants of any solvent and was expected to promote the initial desired ring opening to yield N-sulfate 61. Additionally, and in contrast to other common dipolar aprotic solvents, sulfolane is non-nucleophilic, which virtually eliminated non-productive solvolytic processes such as those encountered with DMF. Sulfolane is also miscible with water and stable at low pH, which helped streamline the overall piperazinone formation process. The finalized process for piperazinone formation was as follows (Scheme 13). Cycloleucine methyl ester freebase 27 was generated via partitioning of the hydrochloride salt 27•HCl between aqueous K3PO4 and MTBE followed by azeotropic drying of the MTBE layer. The relative volatility of MTBE minimized loss of the cycloleucine methyl ester free base 27 during this distillation process. Upon reaching the desired water content specification (below 100 ppm) the MTBE solution was concentrated and transferred into a solution of cyclic sulfamate 48 in sulfolane. Further distillation under reduced pressure removed MTBE from the system and the resulting highly concentrated sulfolane solution of reactants was heated to 70 °C to promote the desired ring opening. Typical conversion to intermediate 61 was greater than 95% after heating for 10 h. Addition of 5 M aqueous HCl and continued heating led to hydrolysis of the N-sulfate; the water-miscibility of sulfolane rendered this organic/aqueous system homogeneous and facilitated hydrolysis. Next, the system was adjusted to pH 10 using aqueous NaOH and heated to ensure complete lactamization. At the end of reaction, the mixture was extracted with MTBE and, after washing with brine, the majority of the sulfolane was rejected to the aqueous phase. The product piperazinone 47 was isolated via crystallization following solvent switch into n-heptane. The typical corrected isolated yield was 70% (96% ee, unchanged from the input stream of starting material 48). Given the lack of stereochemical upgrade achieved via the isolation of piperazinone 47, it appeared more attractive to through-process this intermediate into the final N-alkylation step. The crystallization after the N-alkylation step proved highly robust, consistently affording good overall purity and sufficient stereochemical upgrade, as described in the next section.

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N-Alkylation of Piperazinone Amide Chemoselective N-alkylation of the piperazinone amide was accomplished via deprotonation using NaHMDS in THF followed by treatment with ethyl bromoacetate (Scheme 14). The resulting ester 30 was hydrolyzed in situ during workup with aqueous LiOH, then acidification to pH 1 with aqueous HCl led to precipitation of the crude intermediate hydrochloride salt. Re-slurrying of this salt in water followed by treatment with one equivalent NaOAc and heating to 80 °C allowed neutralization and crystallization of the product 33 in the free form. This crystallization also afforded the necessary upgrade in enantiopurity to deliver material that matched the previously established purity specifications for this regulated API starting material (> 99 LCAP, 99.9 HPLC wt% purity, 99.7% ee). Additionally, using piperazinone acid 33 from this new route in the final amide bond formation with aniline 25 generated MK-3207 API that met the established acceptance criteria.

Scheme 14. Alkylation of Amide 47 and Hydrolysis to Generate Piperazinone Acid 33 The overall second-generation process chemistry route is illustrated in Scheme 15. The synthesis is highly enantioselective, with cascade reactions and through-processing of reaction streams figuring prominently to rapidly build up molecular complexity. The novelty and efficiency of this piperazinone route set a high bar for spirooxindole aniline (R)-25 to match, and the development efforts for this latter intermediate are described in the following section.

Spirooxindole – Process Chemistry First-Generation Synthesis As MK-3207 entered preclinical development, the problem statements for the scale-up of spirooxindole (R)-25 were analogous to those presented by the piperazinone fragment (R)-33. Spirooxindole (R)-25 is a complex synthetic target. The molecule is chiral, in this case bearing an all-carbon quaternary stereocenter that has the unique feature of being rendered stereogenic by the remote aniline substituent four bonds away. To enable initial entry to the clinic, a first GMP delivery of MK-3207 was needed on kilogram scale, representing a quantity of material two orders of magnitude greater than the total amount that had been produced up to that point. The program was on an aggressive timeline, and there 91 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.

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was only a narrow window to develop chemistry that could be scaled to deliver material that met the appropriate attribute and quality requirements.

Scheme 15. The Second-Generation Synthesis of Piperazinone Acid (R)-33 Such challenges are commonly encountered by process chemists across industry, necessitating early decisions be made on a case-by-case basis as to whether existing chemistry can be developed for initial clinical supply or if a new route should be developed. The former benefits from knowledge and experience with a proven way of making API on small scale but may encounter significant issues that slow or even prevent scale-up; the latter can yield a more efficient synthesis but at the cost of up-front commitment of time and resources at a phase of a program where industry-wide compound attrition rates are above 90% (93, 94). Successfully bridging from a discovery-focused synthesis that prizes rapid generation of diverse analogs for profiling to a scalable route for clinical supply of a specific target is one of the critical ways process chemists use their skillsets to enable programs in early development. For spirooxindole (R)-25, the initial route utilized by our medicinal chemistry colleagues offered an expedient approach to carbon-carbon bond formation with the spirocyclic ring system being generated in a single step by double alkylation of oxindole 18 with dibromide 21 (Scheme 16). This generated spirocycle 22 as a racemate, with the desired (R)-enantiomer being isolated using preparative HPLC on a chiral stationary phase. 92

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Scheme 16. Initial Synthesis of Spirooxindole (R)-25

Attracted by the good yields for the individual chemical steps and the convergent assembly of the spirocycle, the process chemistry team elected to focus on development of this route for the first GMP API delivery. Key primary objectives of this work were to: • • • •

establish robustness of the chemistry and address potential safety issues to enable successful kilogram-scale production. increase volumetric productivity of the reactions. improve product isolations. identify quick wins for yield improvement.

Additional opportunities were also identified to shorten the sequence by reducing the use of protecting groups and functional group interconversions, and potentially substitute the preparative HPLC chromatography with a more productive method for resolution of enantiomers. The modified spirooxindole route utilized for the first GMP API delivery is illustrated in Scheme 17. The commonality with the medicinal chemistry route is readily apparent, but it also features a number of developments and innovations to enable the larger-scale production. Dibromide 21 was prepared from 4-nitrophthalic acid 19 following the initial reduction/bromination sequence, but with some procedural modifications. Reduction was accomplished by addition of a THF solution of diacid 19 to 1 M BH3•THF followed by quenching with MeOH, aging with 2 M aq NaOH to break up boron complexes (95), and extraction with EtOAc. A crystallization of the resulting diol 20 from EtOAc/n-heptane was developed that eliminated the need to concentrate to dryness to obtain a solid product. For the subsequent bromination, Et2O was replaced as the reaction solvent with a safer mixture of MTBE/THF (96), and after aqueous workup the product 21 could be crystallized from EtOH/water in good overall yield. As in the initial synthesis, commercially available 7-azaindole 15 was used as the starting material for the preparation of the azaoxindole coupling partner for the dialkylation reaction. In this case, however, a one-step access to unprotected 93

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oxindole 63 was developed in collaboration with our Biocatalysis group. Mediated by chloroperoxidase in an aqueous-rich solvent system and with H2O2 as the oxidant, this direct transformation proceeded under much more environmentally benign conditions than the previous oxidation/reduction sequence and also reduced processing time, improved overall yield, and eliminated the use of the costly SEM protecting group. Critical to the success of the oxidation of 7-azaindole 15 to oxindole 63 was slow addition of H2O2 to avoid prolonged build-up of reagent in the reaction medium, as the enzyme was deactivated in the presence of excess peroxide.

Scheme 17. First Kilogram-Scale Synthesis of Spirooxindole (R)-25

The use of unprotected oxindole 63 increased the complexity of the spirooxindole formation by introducing the amide nitrogen as a competing reactive site for alkylation with dibromide 21. Use of the original Cs2CO3/DMF conditions gave an assay yield of only 21%, despite an ostensibly clean reaction profile by HPLC analysis. The formation of polymeric impurities was suspected to be the root cause of this discrepancy. Significant screening and optimization was required to ultimately arrive at the conditions shown in Scheme 17, utilizing LiOH in a mixture of THF/water at room temperature. Reaction workup was accomplished by extracting the desired product into a basic aqueous layer, with the majority of the impurities partitioning into the organic phase. Acidification then resulted in crystallization of spirooxindole 64, which was isolated in 60% yield. Conversion of racemic alkylation product 64 to final spirooxindole (R)-25 closely paralleled the initial medicinal chemistry route. Heterogeneous hydrogenation was used to reduce the aromatic nitro group to the corresponding aniline (±)-25, which was then converted to N-Boc-protected derivative 65. Initial attempts at classical resolution of intermediates in this sequence were unsuccessful, thus a chromatographic resolution was retained. Resolution was accomplished by preparative supercritical fluid chromatography (SFC) on a chiral 94

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stationary phase with reasonable recovery of the desired (R)-enantiomer. The free aniline was then liberated by acidic cleavage of the Boc group followed by neutralization to isolate spirooxindole (R)-25 as the free-base. The first-generation process chemistry route to aniline fragment (R)-25 proceeded in eight total steps (removing three steps from the original route), and was used to produce multikilogram quantities of material for the first GMP API delivery, enabling MK-3207 to rapidly advance to into Phase I clinical trials. Behind the headline numbers of step count and material quantity, this work featured a number of examples of chemistry innovation and collaborative problem-solving across the Merck process chemistry network, all conducted against a tight timeline. The successful completion of the first GMP API delivery reflected the collective efforts and accomplishments of all the team members. It also specifically exemplifies the potential for beneficial impact of applying novel enzymatic processes early in compound development. This route would also serve as the foundation for subsequent deliveries of spirooxindole (R)-25 that supported continued progression of MK-3207 through early clinical development. However, there was a critical bottleneck that needed to be addressed to enable production of this intermediate on increasing scale beyond the first GMP delivery.

Spirooxindole - Classical Resolution for Clinical Supply While the chromatographic resolution to obtain enantiomerically pure (R)-25 supported the program for the first GMP API delivery, the limitations of this methodology for larger preparation of MK-3207 were well understood. The chromatography of aniline derivative 65 was sandwiched between installation and subsequent removal of a Boc group on the aniline nitrogen (Scheme 17). This was a necessary maneuver, implemented solely for the purpose of increasing the solubility of the spirocycle enough to enable preparative-scale separation to be feasible at all. Generally poor solubility across a wide pH range was found to be one of the defining physicochemical characteristics of the rigid core spirocyclic ring system. The Boc protection/deprotection was an unfortunate sequence but considered manageable for clinical supply. The more pressing issue was that, even under the optimized conditions, the productivity and material throughput of the chromatography was still very low. The projected long cycle times and high cost (not to mention the environmental impact of large solvent volume usage) meant that it would not be practical for larger deliveries, and developing an alternative resolution protocol was viewed as a priority objective by the team. Efforts were focused on revisiting classical resolution based on diastereoselective salt formation, taking advantage of the (weakly) basic aniline and/or pyridine nitrogen atoms. Extensive salt screening was performed on the nitro compound 64 and free aniline (±)-25 intermediates in the existing synthetic sequence, as well as on a number of aniline amide derivatives (66, Figure 5). 95

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Figure 5. Substrates evaluated for classical resolution.

Following many failed attempts, there was a key breakthrough while investigating the use of di-p-toluoyl-L-tartaric acid (67) as the resolving agent with aniline (±)-25 (Scheme 18). A crucial experimental finding was that resolution was only successful when AcOH was used as the solvent. In this system the desired (R)-aniline salt preferentially formed a crystalline material that was determined to be an AcOH solvate (1:1:1 molar ratio of aniline:tartaric acid:AcOH); the undesired (S)-aniline remained primarily in solution. Other solvent systems did not exhibit this phenomenon and did not lead to efficient diastereoselective salt crystallization. Aniline salt (R)-25•DTTA•AcOH was isolated in high diastereomeric excess and in excellent recovery from the racemate. Subsequent neutralization afforded the free base (R)-25 in 96% ee, an acceptable level of stereochemical purity as the minor (S)-enantiomer could be rejected during the crystallization of the final API following coupling to the piperazinone fragment 33.

Scheme 18. Classical Resolution of Aniline (R)-25

This classical resolution eliminated the chromatography and the Boc protection/deprotection steps, and was implemented in pilot plant campaigns to produce aniline (R)-25 for further clinical supply. Persistence and attention to detail by the team members were critical to the discovery and implementation of this resolution, highlighting these as valuable traits needed for process development. 96 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.

Spirooxindole - Development of an Asymmetric Route

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Initial Route Scouting As the MK-3207 program continued to advance, our attention turned to developing chemistry to enable the production of spirooxindole (R)-25 on much larger scale to support late-stage clinical development and ultimately commercial manufacture. The route described above is relatively short and supported early clinical supply well. However, viewed through the lens of long-term manufacturing needs, our analysis was that it did not meet key criteria. This was both in direct measures such as cost, productivity, and sustainability but also, as for the piperazinone fragment 33, our high expectations of utilizing the best chemistry for our commercial products. In the route described in Scheme 17, azaindole 15 and the chloroperoxidase enzyme were expensive raw materials that were both used in the first step. The throughput of some steps was quite low—for example, the dialkylation reaction had to be run in ~ 100 volumes of solvent to maximize yield at 60%, contributing to a high overall Process Mass Intensity (PMI) (97) for the route. The overall yield was lower than desired. Ultimately, a key issue was that the synthesis was racemic. The decision was therefore made to focus on development of a new asymmetric route. 3,3′-Disubstituted oxindoles in general, and spirocyclic systems in particular, are prevalent in molecules of biological and therapeutic interest, and work across academia and industry continues to be directed towards developing methodologies for their enantioselective synthesis (98–101). Many elegant approaches have been reported, however we felt that the unique structure of spirooxindole (R)-25 presented us with an opportunity for innovation and the possibility of making our own impact in this field. Our strategy consisted of initial scouting of multiple routes in parallel, so that we may establish proof-of-principle on key transformations and make rapid decisions to focus efforts on the more promising options, and then finally select one route for full process development. Many creative ideas were generated and evaluated by the team, a selection of which is highlighted in Scheme 19. These routes feature a number of different bond disconnections and strategies for assembly of the spirocycle, but each is characterized by a unique proposal for generation of the quaternary stereocenter using asymmetric catalysis. Early no-go decisions were made on three of these routes. The reductive Heck approach was conceptualized as an extension of intramolecular Heck chemistry for the synthesis of 3,3′-disubstituted oxindoles pioneered by the Overman group (102). However, the position of the trisubstituted alkene in indene 68 was found to be challenging to control during its synthesis and prone to subsequent migration, negatively impacting the potential enantioselectivity of any cyclization. This regioisomer issue was negated in the asymmetric arylation approach by the use of saturated indane substrates 69, but in this case efficient, enantioselective Pd-mediated cyclization was thwarted by the low reactivity of this system towards generating the sterically congested quaternary center, and also the lack of discrimination between the two prochiral faces of the indane due to the remote location of the aniline substituent. 97 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.

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Scheme 19. Overview of Some Routes Evaluated for Long-Term Manufacture of Spirooxindole (R)-25

The enzymatic desymmetrization route offered a more distinct strategy for sequential assembly of the spirocyclic ring system from an acyclic precursor, but there was a lack of differentiation between the ester groups in preliminary experiments. The two approaches that were most extensively investigated were the tandem asymmetric Heck/arylation route and asymmetric phase-transfer catalysis.

Tandem Heck/Arylation Reaction The premise of this approach is illustrated in Scheme 20. It was proposed that treatment of substrate 72 with an appropriate palladium catalyst precursor and chiral ligand could lead to the formation of two carbon-carbon bonds, both the oxindole and indane rings, and the quaternary stereocenter in a single operation. This was to be achieved by combining sequential asymmetric Heck and C–H functionalization steps. The intramolecular Heck reaction is established as a powerful method for the construction of (poly)cyclic ring systems (102–105), and in recent years direct arylation has become an important part of the blossoming field of C–H activation chemistry (106). However, precedent for combination of these methodologies into a single catalytic cycle was limited (107). 98 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.

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Scheme 20. Proposed Catalytic Cycle for Tandem Pd-Mediated Reaction Cyclization precursor 83 was expeditiously prepared as shown in Scheme 21, with a key step being generation of the requisite 1,1-disubstituted alkene in a regiocontrolled manner via a Mannich reaction/decarboxylation/elimination cascade. Gratifyingly, proof-of-concept for efficient bond formation in the cascade was quickly established. Using Pd(OAc)2 and the achiral phosphine ligand P(t-Bu)3 with carbonate base in DMAc, cyclization to the desired racemic spirooxindole 84 could be effected in very high yield.

Scheme 21. Preparation and High-Yielding Cyclization of Substrate 83 Encouraged by this result, we turned our attention to the goal of developing an asymmetric variant of this transformation. Unfortunately, and despite considerable efforts with extensive screening of chiral ligands and reaction conditions, no hits were identified and ultimately it was not possible to perform 99 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.

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this chemistry in an enantioselective manner. Proposals of mechanistic rationale for this negative outcome were that a fast, but reversible, alkene insertion may be followed by a minimal discrimination between the two diastereomers of alkylpalladium(II) intermediate 76 (Scheme 20) in the subsequent C-H activation, and/or that 1,1-disubstituted alkene substrate 83 lacks the structural elements required for enantiofacial discrimination by the catalyst (1,1,2-trisubstituted alkenes are more commonly employed in asymmetric Heck reactions). The use of alternative protecting groups (or none) on the amide nitrogen did not resolve this issue. Consequently this approach was deprioritized for the long-term manufacture of spirooxindole (R)-25. Although a disappointing outcome in the specific context of the synthesis of MK-3207, this chemistry nevertheless offers a rapid entry into the complex spirooxindole architecture, and the broader scope was successfully explored as follow-up to this work (Scheme 22) (108). More recently, Zhu and co-workers have further developed Heck/C-H functionalization methodology into a cascade for the synthesis of [3,4]-fused oxindoles (109).

Scheme 22. Broader Scope of Tandem Heck/C–H Functionalization for Spirooxindole Synthesis

Phase-Transfer Catalysis The original oxindole dialkylation chemistry, first employed by our Medicinal Chemistry colleagues and subsequently modified for kilogram-scale deliveries, provided a rapid access to the spirocyclic framework in racemic form. The proposal that this chemistry could somehow be performed in an enantioselective manner (Scheme 19) was thus attractive in its simplicity; reducing it to practice was anticipated to be anything but such. We chose to focus on evaluation of asymmetric phase-transfer catalysis (PTC). Our motivation for this was driven partly by practical considerations, such as the potential to employ more environmentally benign, mild reaction conditions inherent to this organocatalytic methodology, but also by Merck’s experience and tradition in this field. It is now more than three decades since a landmark publication from our laboratories describing a synthesis of (+)-indacrinone (90, Scheme 23) employing an enantioselective methylation of indanone 87 catalyzed by the cinchonine-derived quaternary salt 88 (110, 111). This was one of the first reports of a practical method for catalytic enantioselective alkylation and helped open the door for further research in asymmetric PTC across academia 100

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and industry (112), including many additional examples from these laboratories (113–116). Today, asymmetric PTC is a burgeoning area that has seen remarkable advances in reaction scope, catalyst design, and practical application (117–119).

Scheme 23. Enantioselective PTC Alkylation in the Synthesis of (+)-indacrinone 90 The opportunity to make a novel contribution to the field with development of spirooxindole (R)-25 was therefore enticing, but this chemistry was anticipated to be challenging. At the outset of this work, only a single example of asymmetric PTC alkylation of an oxindole substrate (Scheme 24) had been reported (120) (other elegant asymmetric oxindole PTC reactions have been reported more recently) (121–126), and application to generate spirooxindoles was unprecedented. Differentiation between the two leaving groups in a bis(benzylic) electrophile 73 was not expected to be trivial (127, 128). From a practical perspective, challenges are presented by the heterogeneous nature of PTC reactions, as well gaps in detailed understanding of specific phase-transfer mechanistic pathways which often necessitate an empirical approach to reaction development.

Scheme 24. Precedent for Oxindole PTC Alkylation

Initial Results The first lesson learned from our PTC development work was that protection of the oxindole amide nitrogen was going to be required. Attempted asymmetric alkylation of the unsubstituted substrate 63 led to tarry, intractable mixtures in 101 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.

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which the starting materials were consumed but minimal amounts of the desired product 25 were formed (Scheme 25), with competing oligomerization believed to be a significant issue. That a scalable racemic alkylation of oxindole 63 using LiOH in THF/water had previously been successfully developed for the first GMP delivery underscored both the sensitivity of this chemistry to the reaction conditions and the skill of the team members in developing the earlier clinical supply route.

Scheme 25. Inauspicious First Attempts at PTC Alkylation

Hopes were initially raised with use of PMB-protected oxindole 94 (Table 4). Encouragingly, reaction with nitro-containing dibromide 21 using the original Merck catalyst 88 provided a cleaner reaction profile and measurable enantioselectivity for formation of spirooxindole (S)-64 (Table 4, entry 2). Less encouragingly, the level of enantioselectivity was mediocre. Alkylation was more rapid using the structurally distinct first- and second-generation binaphthyl-derived quaternary ammonium salt catalysts pioneered by the Maruoka group (97 (129) and 98 (130), respectively), but the enantioselectivity was not significantly higher (entries 3 and 4). The low conversion observed in the absence of catalyst (entry 1) indicated that competing background (i.e., uncatalyzed) alkylation was not a major contributing factor to the low enantioselectivities. Switching the aromatic substituent in the electrophile from nitro to methoxy resulted in marginal improvements (entries 5–7), but the enantioselectivity could not be improved further with modification of the base, solvent, or reaction temperature.

Modified PTC Strategies and Unexpected Findings A generous claim could be made that these initial experiments established proof-of-principle for an asymmetric PTC dialkylation approach. However, the results were clearly unsatisfactory. The low enantioselectivity was rationalized as resulting from either, or both, of two factors: poor regioselectivity in the first intermolecular alkylation and/or a low level of stereochemical induction imparted by the chiral catalyst during the subsequent ring-closure. Scheme 26 illustrates the multiple reaction pathways that are possible for the dialkylation. Formation 102 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.

of spirooxindole (R)-64 requires selective formation of one of the monoalkylated intermediates followed by high facial selectivity for the cyclization (solid arrow pathway). Low selectivity in the initial coupling of oxindole 94 and dibromide 21 and/or during the ring-closure leads to competing formation of (S)-64 (dotted arrows), reducing the overall enantioselectivity.

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Table 4. First Signs of Enantioselectivity in PTC Alkylation of Oxindole 94

To eliminate regioselectivity as a liability, a modified PTC strategy was proposed (Scheme 27). Alkylation of ester-substituted oxindole 101 with monohalide 102 would, by design, furnish intermediate 103 as a single benzylic regioisomer and establish the quaternary stereocenter with, ideally, high 103 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.

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selectivity. Subsequent manipulation involving intramolecular Friedel–Crafts chemistry followed by ketone reduction would then complete assembly of the spirocyclic ring system.

Scheme 26. Control of Both Intermolecular Regioselectivity and Intramolecular Facial Selectivity Is Required for High Enantioselectivity

Scheme 27. Modified Monoalkylation/Friedel–Crafts Proposal

Initial results from this approach were equally disappointing (Scheme 28): reaction of acylated oxindole 101 (which existed in the enol tautomeric form shown) with 3-nitrobenzylbromide 102 using various catalysts in the presence of hydroxide base gave the desired monoalkylated product 103 with low yield and enantioselectivity. However, it was observed that one side-product was consistently formed in significant amounts from these reactions; isolation and 104 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.

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characterization established it to be the unexpected bis-alkylated compound 105. It was subsequently confirmed that this material was formed from monoalkylated oxindole 103, presumably via a sequence of ester hydrolysis, decarboxylation, and alkylation. Evidently, the ester group in monoalkylated intermediate 103 is more prone to hydrolysis than in starting material 101, as the latter was slow to hydrolyze under the reaction conditions. The hydrolysis of intermediate 103 was promoted by the catalyst and did not occur at an appreciable rate in its absence, an observation which was recorded as a footnote at the time but which would become significant later on.

Scheme 28. Unexpected Formation of Bis-Alkylated Compound 105

At this point, we looked to turn this unexpected result to our advantage. Since dialkylation of acylated oxindole 101 was clearly feasible, we proposed that use of a bis-benzylic halide could provide an alternative entry to spirocyclic system (Scheme 29). The hope was that acylated oxindole 101 would be a more discriminating partner than the corresponding unsubstituted compound 94 in the intermolecular alkylation, resulting in improved regioselectivity for this elementary step and consequently higher enantioselectivity for the overall cascade in the presence of the chiral catalyst.

Scheme 29. Second Modified PTC Proposal

Gratifyingly this hypothesis was experimentally validated, at least to a degree, in the first experiments. As shown in Scheme 30, using catalyst 88 on screening scale the enantioselectivity for formation of (S)-spirocycles 64 and 95 was more than doubled compared to using unsubstituted oxindole 93, reaching a level of 45% ee using the methoxy-containing dibromide 94. 105 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.

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Scheme 30. Improved Spirocycle Formation Using Acylated Oxindole 101

The next round of experimentation was directed by the observation that the presence of the electron-donating methoxy group in dibromide 94 had consistently resulted in higher enantioselectivities than the corresponding electron-poor nitroanalog 21. To exploit this theme, we proposed to evaluate a stronger electrondonating aniline substituent with the use of electrophile 108.

Scheme 31. Unexpected Result in Bromination of Aniline Diol 107

Bromination of aniline diol 107 using HBr in AcOH did not afford the expected dibromide 108, but instead monobromide hydrobromide salt 109 could be isolated in good yield by crystallization from PhMe/MTBE (Scheme 31). This unexpected result was followed by two positive findings (Scheme 32): not only was monobromide 109 a viable coupling partner for the dialkylation cascade, but also the resulting spirocycle (S)-84 was generated with the highest level of enantioselectivity seen up to that point (65% ee). 106 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.

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Scheme 32. Improvement in Stereoselectivity Using Aniline Derivative 109

The former finding was more unexpected than the latter. In studying the reaction sequence further, it was shown to proceed via intermediate 110. Using a weaker base (K2CO3), the reaction stopped at this monoalkylated stage and ester hydrolysis did not occur. Intermediate 110 could be isolated, characterized, and resubjected to the reaction using a stronger, more nucleophilic base (KOH) to generate the spirocycle. The formation of intermediate 110 was rapid, and it was found that the catalyst was not required for this first alkylation step. We rationalized that the formal displacement of a hydroxyl group from compound 109 proceeded via aza-quinone methide 111. This type of reactive intermediate had previously been implicated in nucleophilic substitution of 4-hydroxymethylanilines by activated carbonyl compounds (131). Encouraged by this breakthrough, we conducted an initial screen of a small number of catalysts. The focus was on derivatives of cinchonine, largely because a modestly-sized collection of about a dozen of these had been accumulated from previous projects within the group and was thus immediately on-hand. As the number of commercially available chiral PTCs was quite limited at the time, this in-house collection served as a valuable resource for a rapid first-pass assessment of the impact of catalyst structure in this series. A selection of results is highlighted in Table 5 (132). As far as trends in catalyst structure-activity relationships could be discerned from this small data set, it appeared that an electron-deficient aromatic ring at the quaternized N-benzylic position was preferred (e.g., entries 1 and 3), as was the presence of the hydroxyl group in unprotected form (e.g., entries 4 and 5). The real breakthrough from this screen was the finding that 3,5-bis(trifluoromethyl)benzyl derivative 120 generated the spirocycle in 93% ee (entry 10). 107

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Table 5. Initial Screen Resulted in Identification of Highly Selective Catalyst 120

This result gave us the confidence to continue developing this PTC chemistry for the long-term manufacturing route, focusing on the use of aniline derivatives as the electrophile. By this stage we had also developed a better empirical understanding of the influence of the structure of the electrophilic coupling partner on the outcome of the PTC cascade. As highlighted previously, both the inter- and intramolecular steps of the cascade needed to be controlled for overall high stereoselectivity. Isolation of the monoalkylated intermediates allowed the influence of each of these steps to be characterized. As shown in Table 6, the nature of the C-4 substituent in the electrophile was found to have some influence on the facial selectivity of the intramolecular ring closure, but strongly impacted the regioselectivity of the intermolecular alkylation. Whereas the nitro-substituted compound gave a mixture of regioisomeric intermediates in the first step, formation of the ‘para’ intermediate 122 was highly selective using the electron-rich methoxy and aniline analogues, which largely translated into the overall enantioselectivity of the direct one-pot process. 108

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Table 6. Investigation of Inter- and Intramolecular Alkylation Steps

The astute reader would have noted that the studies described so far have generated the (S)-enantiomer of the spirocyclic products, i.e. the undesired enantiomer of that required for MK-3207. This was driven by our desire to establish rapid proof-of-concept on a PTC approach in our initial route-scouting efforts using the cinchonine-derived catalysts we already had in hand, with the expectation that catalyst optimization would form part of subsequent process development. That we identified 3,5-bis(trifluoromethyl)benzyl catalyst 120 during these initial efforts was a fortuitous discovery, but we were able to capitalize on this serendipity to significantly accelerate the subsequent development. We also knew that access to the desired spirooxindole (R)-84 should be provided by catalysts derived from cinchonidine (126, Figure 6), another Cinchona alkaloid described as a ‘pseudoenatiomer’ of cinchonine 124 (technically, the two are diastereomers). For both alkaloids their low cost, bulk availability, functional handles for derivatization, and generally predictable stereochemical outcomes when used for asymmetric transformations have made them popular choices in organocatalysis (133–136). These facets, combined with our assessment that we would have freedom-to-operate with these catalysts, resulted in us focusing on the cinchonidine-derived series. 109

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Figure 6. Pseudoenantiomeric cinchonine and cinchonidine scaffolds for PTC.

We quickly established that the corresponding 3,5-bis(trifluoromethyl)benzylcinchonidinum catalyst 128 indeed generated spirooxindole (R)-84 (Scheme 33), albeit with slightly diminished enantioselectivity on screening scale relative to the earlier result for (S)-84 using the cinchonine-derived analog 120. However, an issue that emerged when this chemistry was run on larger laboratory scale for the first time was that inconsistent results were observed, with the enantiomeric excess varying from 70–85% between experiments. In addition, the optimum oxindole and aniline coupling partners also needed to be defined and the downstream conversion to final MK-3207 API rigorously established. The assessment was that while the spirocyclization chemistry shown in Scheme 33 would provide a general framework for the manufacturing route, systematic study of substrates and parameters, optimization of all steps in the sequence, and detailed process characterization was going to be required. The following sections describe how each of these was successfully addressed and the manufacturing route for spirooxindole (R)-25 finalized.

Optimized Oxindole Synthesis PMB-protected oxindole 101 had served a valuable purpose to this point in discovery of the PTC cascade, but it was clear that the utility of this intermediate had run its course. Both the synthesis of substrate 101 and later removal of the PMB group from protected spirooxindole product (R)-84 were found to be challenging, and an alternative protecting group that mitigated these issues was desired. 110 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.

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Scheme 33. Generation of Spirooxindole (R)-84 Using Cinchonidinium-Derived Catalyst 128 As we had access to a supply of the parent 7-azaoxindole (63) from the early GMP deliveries of MK-3207, our first attempts involved selective protection of the amide nitrogen in this material (Scheme 34). However, this was thwarted by the formation of complex mixtures resulting from competing O- and C-alkylations. Even if it had been successful, the question of how to efficiently access 7-azaoxindole 63 from a long-term manufacturing perspective would have remained.

Scheme 34. Chemoselectivity Issues in the First Attempted Synthesis of PMB-Protected Oxindole 94 As an alternative approach we sought to construct the oxindole ring from a more readily available 2,3-disubstituted pyridine precursor, building upon an example of an anionic cyclization that had been reported by Snieckus and co-workers (137). The route used to synthesize PMB-protected compound 101, together with its unacylated progenitor 94, is illustrated in Scheme 35. Sequential N-acylation and alkylation of commercially available 2-amino3-picoline (129) gave compound 130, which upon treatment with LDA in THF underwent lateral metalation followed by cyclization to afford the desired oxindole 94 as the major product. The subsequent C-acylation could be effected in good yield using methyl cyanoformate (138), with the product being isolated as the aromatic enol tautomer. Although this was a serviceable method for obtaining gram quantities of oxindoles 94 and 101, it suffered from a liability in the formation of a significant amount of rearrangement product 133 during the anionic cyclization step. This side-product presumably resulted from competitive deprotonation at the other benzylic site in substrate 130 followed by acyl migration (formally, a 111

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[1,2]-aza-Wittig rearrangement) (139). Formation of the desired oxindole 94 was favored by higher reaction temperatures and addition of the substrate to the LDA base, but the formation of side-product 133 could not be completely suppressed.

Scheme 35. Different Chemoselectivity Issues in the Gram-Scale Synthesis of PMB-Protected Oxindoles 94 and 101

After considerable further experimentation, we settled on the use of the tertbutyl group for the oxindole protection. Somewhat underutilized as a protecting group for nitrogen in organic synthesis (140), this simple alkyl group was found to meet our criteria of efficient introduction and removal, stability during the PTC cascade, as well as being cost and (relatively) atom economical. Direct tert-butylation of 2-amino-3-picoline 129 could not be achieved due to the low nucleophilicity of the amino group, thus a high-yielding amination reaction was utilized to access compound 135 (Scheme 36). A novel one-pot anionic cascade sequence was then developed to generate the acylated oxindole. Treatment of compound 135 with n-HexLi followed by methyl chloroformate resulted in conversion to carbamate 136; subsequent treatment with n-HexLi followed by further charges of i-Pr2NH and n-HexLi (to generate LDA in situ) resulted in cyclization to Li-enolate 137 which, in turn, was acylated upon the final addition of a further equivalent of methyl chloroformate. The acylated oxindole 139 was formed in 69% solution assay yield for the overall sequence. tert-Butyl intermediate 136 could not undergo competing rearrangement as was observed for PMB-protected analog 130, however control of the reaction conditions was required to minimize formation of dimeric impurity 140 which was difficult to reject. It was found that addition of a substoichiometric amount of n-HexLi to the solution of carbamate 136 and aging for 30 min prior to addition of the subsequent reagents suppressed the formation of dimer 140 by consuming any excess methyl chloroformate still present before the cyclization. 112

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Scheme 36. Synthesis of Tert-Butyl-Protected Oxindole 139

While evaluating acid/base aqueous workup procedures for the isolation of product 139, the serendipitous finding was made that the corresponding potassium salt 138 could be efficiently partitioned into the organic phase with rejection of impurities to aqueous layers, and then crystallized from THF/n-heptane with good recovery and purity. A subsequent salt break (dissolution of potassium salt 138 in MeOH, acidification with AcOH, addition of water antisolvent, and filtration) enabled isolation of 139 in the free form.

Optimized Chloride Synthesis The first synthesis of aniline bromide electrophile 109 is illustrated in Scheme 37. Beginning with 4-bromophthalic acid (141), esterification followed by palladium-catalyzed amination gave aniline 143. The implementation of the esterification step was primarily to improve substrate solubility; attempted amination of the diacid starting material resulted in poor conversion even at high catalyst/ligand loadings. Reduction of diester 143 to diol 107 was accomplished using LiAlH4. Conversion to monobromide 109 then proceeded as described previously. Chloride electrophile 144 could be formed from diol 107 by treatment with SOCl2. Analogously to the corresponding bromide 109, electrophile 144 was isolated as the monobenzylic halide. A small, but nonetheless significant, improvement in enantioselectivity (typically, 2–3% ee) was observed when chloride 144 was used in place of bromide 109 for the PTC spirocyclization cascade, therefore it was selected for further development. 113

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Scheme 37. First Route to Aniline Electrophiles 109 and 144

Scheme 38. Improved Route to Chloride Electrophile 144

An issue with the first synthesis of chloride 144 was that 4-bromophthalic acid (141) was relatively expensive, and a more cost-effective alternative was needed for long-term manufacture. The route that was subsequently developed is shown in Scheme 38. We returned to 4-nitrophthalic acid (19) as a cheap, commercially 114 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.

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available starting material. Conversion to aniline diester 145 was achieved via a one-pot protocol. Fischer esterification using a small excess of methanesulfonic acid in MeOH (141, 142) at 80 °C for 16 h was followed by cooling, addition of 10% Pd/C (3 wt% relative to diacid 19) and hydrogenation below 40 °C at 60 psig of hydrogen. At the end of reaction, the solution was filtered to remove the catalyst, the bulk of the MeOH was removed and the solvent switched to EtOAc for the basic aqueous workup. Finally, crystallization from PhMe/n-heptane gave the product 145 in 84% overall yield. Dibenzylation was accomplished using BnCl, K2CO3, and a catalytic amount of KI as promoter in DMAc at 90 °C (143). This was a heterogeneous reaction and the particle size of the K2CO3 base was important. Competitive acylation of the aniline by the DMAc solvent would result in significant levels of side-products 147 and 148 when granular K2CO3 was used; this liability was avoided using a powdered form of the base. After an organic/aqueous workup using MTBE, the product stream was switched into THF for use directly in the next reduction step. We used the commercially available solution of LiAlH4(1 M in THF) for the reduction of diol 110 to avoid challenges of handling the solid reducing agent on scale. Thus, LiAlH4 solution was added slowly to the diester solution while maintaining the temperature below 5 °C. Reduction was complete within a few hours, and then the reaction was worked up using a modified Fieser protocol. Excess hydride reagent was first quenched by the slow addition of solution of 20% v/v water in THF below 10–15 °C (144), followed by the addition of 15% aq NaOH and then finally water. The resulting slurry was then filtered through a bed of cellulose as a filter aid, and the filtrate was concentrated and the product was crystallized from toluene/n-heptane. Diol 107 was isolated in 87% overall yield for the two steps and in greater than 97% purity. Chlorination was accomplished by charging diol 110 in portions to a solution of SOCl2 in MeCN below 20 °C. Once the starting material had been fully consumed, the reaction mixture was diluted with MTBE. Seeding of the solution resulted in crystallization of the monochloride HCl salt 144. Alternative combinations of solvents for reaction and crystallization were evaluated, but MeCN/MTBE provided the best reaction profile and crystallization attributes. The product was isolated in 90% yield corrected for the 94% weight purity; the mass balance was primarily residual solvents together with small amounts of dimeric impurities.

Optimization of PTC Chemistry and Deprotection We set out on our development of the PTC spirocyclization reaction with a number of goals in mind, namely to: • •

define critical parameters and address reproducibility issues seen in early lab-scale reactions. optimize the yield, enantioselectivity and practicality of the process. · establish a robust product isolation protocol. 115

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The path to establishing reproducibility was guided by piecing together a number of observations from earlier experiments: •



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a catalyst was not necessary for the formation of the monoalkylated intermediate but was required for the subsequent ester hydrolysis to occur at an appreciable rate under the reaction conditions. the enantioselectivity of the spirocyclization was consistently higher starting from isolated monoalkylated intermediate than in the one-pot double-alkylation process. the enantioselectivity was also highly dependent on the structure of the catalyst.

Thus, based on the these goals and guidelines, we arrived at the protocol shown in Scheme 39. Oxindole 139 and chloride 144 were added to a heterogeneous mixture of KOH, water and toluene at 8–12 °C and allowed to react in the absence of catalyst. Once formation of intermediate 149 was complete (in less than 1 h), catalyst 128 (5 mol%) was charged and agitation continued for a further 12–14 h to generate the spirooxindole. A number of parameters were important for the efficiency and reproducibility of this process, including reagent stoichiometry and order of addition, base and solvent, concentration, temperature, and agitation rate. Each of these was extensively investigated and optimized.

Scheme 39. Optimized PTC Chemistry

To achieve the highest enantioselectivity, it was critical that chloride 144 be almost completely consumed prior to introduction of the catalyst. As an explanation for the variability in performance when the catalyst was present in the mixture from the beginning, we believed that the electrophile would competitively alkylate the catalyst (e.g., on the hydroxy group) to generate a new species that was still catalytically active but afforded much lower enantioselectivity. 116 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.

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In practice, control of residual chloride 144 was achieved by employing a slight excess of oxindole 139 and establishing an in-process control of less than 1% chloride 144 remaining before charging the catalyst 128. Sampling and analysis of the reaction was non-trivial given the heterogeneous nature of the mixture and the limited stability of chloride 144 in solution, highlighting the skill of our analytical colleagues in establishing a reliable method. We were also able to reduce the loading of catalyst 128 to 5 mol% without impacting the enantioselectivity (145). Evaluation of organic solvent and base systems confirmed that toluene and aqueous KOH were the optimum combination, used in a 2.6:1 (v/v) ratio. The yield and enantioselectivity increased with the concentration of the base solution. For convenience, commercially available 50 wt% KOH in water was initially used for lab-scale development, but it was noted that the overall reaction generated molar equivalents of water, thus the concentration of KOH decreased slightly over the course of the reaction. By using a slurry of 55 wt% KOH in water (prepared by charging solid KOH to the commercial 50 wt% solution prior to addition of the other reagents), the concentration of KOH remained above the saturation limit in water (~51 wt% at the reaction temperature) throughout the sequence, maximizing the enantioselectivity. The reaction mixture was therefore comprised of a triphasic system of KOH(s), water, and organic phases (146). The enantioselectivity was also dependent on the total reaction volume, increasing at greater dilution. On laboratory scale, spirooxindole (R)-84 was formed in 90% ee at a reaction volume of 40 mL/g oxindole starting material 139, but the selectivity dropped to 85% ee at 20 mL/g. Analogous dependence on concentration has previously been reported for asymmetric PTC reactions (114). To balance overall yield against material throughput, a reaction volume of 40 mL/g oxindole 139 was selected for scale-up. Similarly, a reaction temperature of 8–12 °C was selected as the best compromise between two competing trends as the temperature was lowered: for the intrinsic enantioselectivity of the spirocyclization to increase, but at the expense of increasing viscosity of impeding agitation of the thick, heterogeneous mixture. Effective agitation and mixing of the heterogeneous mixture was critical to maximizing reaction performance. On kilo-lab scale using a simple U-shaped paddle stirrer in a 100 L cylindrical vessel, (R)-84 was obtained in 88% ee. But in a pilot plant setting with more efficient agitation (using either a retreat-curve impeller or Rushton disk turbine), higher enantioselectivities of up to 93% ee were achieved. At the end of reaction, an aqueous workup afforded crude spirocycle (R)84 as a solution in toluene in 89–92% assay yield. Product isolation was greatly facilitated by the discovery of a crystalline toluene solvate that afforded upgrade of both chemical and enantiopurity. Concentration of the crude product solution to reach supersaturation and trigger crystallization was followed by addition of MeOH as antisolvent to reduce liquor losses and then filtration. In the first kilo-lab demonstration of this chemistry, spirocycle (R)-84 was obtained in 77% yield and 95% ee. Upgrade of stereochemical purity to the required level could be achieved in crystallizations of subsequent steps to the MK-3207 API. Further development of the isolation of spirocycle (R)-84, in particular definition of the minimum amount of toluene required to maintain the 117

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solvate form during the crystallization, filtration and cake washes (147), enabled the product to be isolated in 83% yield and greater than 99% ee in a later pilot plant campaign. Two impurities observed at low levels during early lab scale experiments were the novel pyran-containing spirocycle 150 (the configuration at the chiral center was not determined) and the ‘aniline dimer’ 151 (Figure 7). Pyran 150 was proposed to arise from enolate oxidation via adventitious intrusion of oxygen into the reaction mixture (148), and its formation was easily eliminated using standard inertion techniques. Aniline dimer 151, shown to arise by alkylation of monoalkylated intermediate 149 with a second molecule of chloride 144, was more problematic as it was only partially rejected during the crystallization of spirocycle (R)-84. The downstream fate of aniline dimer 151 was to react in subsequent steps to generate a new impurity in the API that had not been qualified in toxicological studies. The control strategy implemented was to restrict the level of formation of aniline dimer 151 to no more than 0.6% by the end of the PTC reaction. This was achieved in practice by controlling the stoichiometry of oxindole 139 and chloride 144 starting materials to limit the amount of the latter remaining after formation of intermediate 149.

Figure 7. Impurities 150 and 151 formed during the PTC reaction.

A through-process was developed for the two-stage removal of the protecting groups from spirocycle (R)-84 to give free aniline (R)-25 in excellent yield and purity (Scheme 40). The tert-butyl group was first cleaved from the oxindole nitrogen using excess methanesulfonic acid and a small volume of toluene at 90 °C. The main role of the toluene was to sequester the liberated tert-butyl cation and prevent undesired alkylation of the aniline core. The residual toluene present in the solvate form of starting material (R)-84•PhMe also served this purpose to a degree, but we found that using additional PhMe as co-solvent improved the scavenging efficiency. After cooling to ambient temperature, the solution of oxindole (R)-152 was diluted with MeOH and hydrogenated at 60 psig of H2 in the presence of catalytic Pd/C. Once hydrogenolysis of the benzyl groups was complete, the mixture was filtered and a workup performed in which the product (R)-25 was extracted into the aqueous layer as the corresponding MsOH salt. Adjustment of pH to 6–8 using aq. NaOH resulted in crystallization of free aniline (R)-25. 118

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Scheme 40. Deprotection To Generate Free Aniline (R)-25

Spirooxindole Route Summary The overall route that was developed for the long-term manufacture of (R)-spirooxindole (R)-25 is shown in Scheme 41 (149). The synthesis comprised ten steps, and features at its heart a novel PTC-mediated reaction to generate the spirocyclic ring system and quaternary stereocenter in high yield and enantioselectivity. The overall yield was 40%, representing a four-fold improvement over the earlier classical resolution route. One of the goals of this chapter section on the spirooxindole has been to describe the evolution of the chemistry from the medicinal chemistry synthesis that enabled discovery of MK-3207, through the first generation process chemistry route for speed to the clinic, to the manufacturing route for practical, economical long-term supply. Beyond the specific chemistry details, however, another aim has been to use this story to convey more general lessons learned from our experiences as well as some of our broader thoughts on chemical process development. The first is that effective teamwork and communication are essential to deliver on a project of any complexity. The successes described above reflect the collective efforts of dozens of outstanding colleagues and collaboration between many different groups. A second key theme is the impact of bringing innovation to process development, and being willing to take risks in developing new chemistry to tackle the most challenging synthetic problems. From the enzymatic oxidation used in the first GMP delivery to the Heck/C–H functionalization chemistry and then the PTC spirocyclization, this spirooxindole work exemplified: • • • •

the development of novel reactions. new extensions of established reactions. cascade processes to rapidly assemble molecular complexity. the enabling ability of asymmetric catalysis and HTE.

A third lesson is that persistence, attention to detail, mechanistic understanding, and the ability to take advantage of opportunities presented by serendipity are all important traits in process development. The optimized PTC chemistry to prepare (R)-25 (Scheme 41) ended up being quite different to original 119 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.

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proposal at the outset of long-term route scouting (Scheme 25). The connection between the two was neither intuitively obvious nor direct, but instead resulted from several rounds of analysis and strategy revision based on unanticipated findings.

Scheme 41. Summary of New Route to Spirooxindole (R)-25 Finally, the development of novel methodologies and capabilities to solve challenges on one project can have broader benefits, as is described below. Development of a PTC Library and Its Application Reflecting back on the development of the PTC spirocyclization chemistry, there was an element of good fortune in 3,5-bis(trifluoromethyl)benzylcinchoninium bromide 120 (Table 5) being present among the small collection of catalysts we had in hand at the time of initial screening, and that we were thus able to identify a high-performing catalyst so quickly. Establishing catalyst structure-activity relationships for any given PTC reaction has been largely empirical and qualitative to date, although more quantitative approaches to catalyst design have begun to be developed (150, 151). In addition, the number of commercially available phase-transfer catalysts is fairly limited. To address these capability gaps and improve the odds of success on future projects, we sought to generate a much larger library of catalysts that would be available for colleagues to use in-house. In its initial form, this library consisted of derivatives of the cinchonine and cinchonidine alkaloids quaternized on the quinuclidine nitrogen with a diverse 120

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set of commercially available benzyl halides. Over 100 of these catalysts were prepared in each pseudoenantiomeric series (Figure 8). The goal was for this library to enable efficient screening and hit generation for various transformations using HTE techniques. The following two examples highlight some of the applications and new discoveries that this library has enabled so far.

Figure 8. A library of cinchonine- and cinchonidine-derived catalysts. One of the early applications was in our studies towards developing a cost-effective synthesis of vinylcyclopropane 156 (Scheme 42), a structural motif present in several NS3/4A protease inhibitors, including grazoprevir (157) (152, 153), for the treatment of hepatitis C virus (154, 155). A number of synthetic routes to this valuable building block had been reported (156), including racemic dialkylation of a glycine imine derivative to generate the cyclopropane followed by enzymatic resolution (157). In our hands, screening of this cyclopropanation reaction with the set of cinchonidinium derivatives under PTC conditions resulted in the rapid identification of catalyst 155 as a promising lead that could serve as a starting point for further development (158, 159).

Scheme 42. Early Demonstration of Utility of the PTC Library Screening for Synthesis of Vinylcyclopropane 156 The second example serves as another case study of the value of capitalizing on serendipity in research. MK-8825 (158, Figure 9) was identified as another CGRP receptor antagonist candidate (160) following MK-3207, and 121 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.

process chemistry was engaged in developing a scalable synthesis of the core spirooxindole component 159.

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Figure 9. MK-8825 (158) and spirooxindole 159. The direct one-pot PTC dialkylation cascade developed for the related MK3207 spirooxindole proved less effective when applied to this new system. The team evaluated a number of other routes, including the stepwise approach shown in Scheme 43 in which alternative chemistry was used to access intermediate 160, but asymmetric PTC was still envisaged as being used to effect the critical intramolecular ring closure.

Scheme 43. Initial Screening of Intramolecular Cyclization of Oxindole 160 and Discovery of ‘Bis-Quat’ Catalyst 163 Screening of the cinchonidinium-derived catalysts generated an initial data set comprised largely of incomplete conversions and poor-to-middling enantioselectivities. However, catalyst 162 was a unique outlier in furnishing spirocycle 161 in high yield and 92% ee. Encouraged by this promising result the team began laboratory-scale development, at which point the performance of the cyclization promptly dropped (Scheme 43). The team noted that different batches of catalyst 162 were used between screening and scale-up, and closer inspection indicated that the newer batch of catalyst prepared for the scale-up studies was of 122 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.

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higher purity than the screening material that had been prepared earlier as part of the library generation. After further excellent detective work, doubly-quaternized species 163 (Scheme 43) was identified as the true actor responsible for the high performance of the screening catalyst batch, despite being present only as a low-level impurity in this material. The remarkable catalytic efficiency of compound 163 was underscored in the optimized cyclization conditions (Scheme 44) which required only 0.3 mol% catalyst loading (161), an unprecedentedly low level for Cinchona alkaloid-based phase-transfer catalysis.

Scheme 44. Optimized Spirocyclization Using Doubly-Quaternized Catalyst 163

Diligent investigation of some initially confounding results therefore led to the serendipitous discovery of a novel class of doubly-quaternized Cinchona alkaloid derivatives that we believe will have broader utility in asymmetric PTC. As an example, development of an efficient intramolecular aza-Michael PTC reaction for the enantioselective synthesis of the clinical drug candidate letermovir (167, Scheme 45) has recently been reported by these laboratories (162).

Scheme 45. Application of ‘Bis-Quat’ PTC in the Asymmetric Synthesis of letermovir 167 123 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 advances in PTC chemistry described in this chapter, from development of the MK-3207 spirooxindole cascade to the discovery of a new catalyst class, build upon Merck’s legacy in this field and offer exciting new directions for the future.

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Conclusions The discovery of MK-3207, an orally acting, picomolar CGRP receptor antagonist with the potential for a low clinical dose, together with the development of scalable methods for its production, were significant achievements. Unfortunately, there were a number of liver enzyme abnormalities observed in the clinic after dosing with the compound and its development was discontinued after the work described in this chapter had been completed. Nonetheless, the preclinical and clinical studies with MK-3207 demonstrated that it is possible to achieve CGRP receptor blockade with a low oral dose of a suitable antagonist. Moreover, the challenge of synthesizing such a complex molecule on large scale led to novel approaches and scientific discoveries that highlight the impact of process chemistry in a complex multidisciplinary area and the importance of driving innovation in chemistry research.

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