4A protease inhibitor for HCV

Tetrahedron 75 (2019) 4271e4286

Contents lists available at ScienceDirect

Tetrahedron journal homepage: www.elsevier.com/locate/tet

Process development of ABT-450 e A first generation NS3/4A protease inhibitor for HCV Daniel D. Caspi, Russell D. Cink, Dean Clyne, Moiz Diwan, Kenneth M. Engstrom,  G. Napolitano, Timothy Grieme, Jianzhang Mei, Robert W. Miller, Clifford Mitchell, Jose Nandkishor Nere, Matthew M. Ravn*, Ahmad Sheikh, Seble Wagaw, Hongqiang Zhang AbbVie Inc., 1 North Waukegan Road, North Chicago, IL 60064, United States

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 April 2019 Received in revised form 27 May 2019 Accepted 29 May 2019 Available online 5 June 2019

ABT-450 (8), a potent hepatitis C (HCV) NS3/4A protease inhibitor, was approved as part of AbbVie's first generation HCV treatment for the United States in December 2014. A series of process optimizations were developed over six years to support the program starting with recycling of a previous protease inhibitor candidate through route development and final process. This discussion will focus on optimization of the final six steps starting from dipeptide 12 and amino acid 13 and highlights the use of a large scale ring closing metathesis (RCM), reactive crystallizations for isolation of intermediates, and detailed process understanding of the final sulfonamide coupling. The process provides ABT-450 (8) in 72% overall yield for the final 6 steps. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Ring closing metathesis Macrocycle Acyl sulfonamide HCV protease ABT-450 Process optimization

1. Introduction

intermediate for initial deliveries followed by subsequent development of a specific synthesis for ABT-450 (8).

The hepatitis C virus (HCV) is a blood borne disease estimated to affect between 71 and 185 million people worldwide (Fig. 1) [1]. Persons infected with HCV can remain asymptomatic for decades; if left untreated HCV can lead to liver failure, liver cancer and death. In December 2006, Abbott Laboratories, which later split to become AbbVie, started a collaborative effort with Enanta Pharmaceuticals focused on the development of NS3/4A HCV protease inhibitors. The collaboration led to the development of ABT-450 (8) as a component of AbbVie's first generation treatment for HCV [2]. Subsequently, second generation therapies [3] have been developed to address the key issue of pan-genotypic activity [4], resulting in multiple treatment options that represent a curative therapy for all major HCV genotypes [5]. Process development of ABT-450 (8) involved two routes and multiple iterations for each step to streamline the synthesis. Initial deliveries were expedited by recycling a previous protease inhibitor candidate as an

* Corresponding author. E-mail address: [email protected] (M.M. Ravn). https://doi.org/10.1016/j.tet.2019.05.064 0040-4020/© 2019 Elsevier Ltd. All rights reserved.

Fig. 1. 2015 WHO estimate of Hepatitis C (HCV) prevalence [1].

4272

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

The predecessor candidate to ABT-450 (8) was an analogous macrocyclic compound ABT-515 (1) which had been developed through initial scale-up campaigns [6]. Toxicology studies identified risks which precluded it from continuing in development. Continued efforts identified ABT-450 (8) as the next candidate for development, and to accelerate generation of initial supplies, efforts to utilize the on hand ABT-515 (1) resulted in a recovery route to prepare ABT-450 (8). This recovery route enabled a manufacture of the first in human (FIH) supplies six months ahead of timelines utilizing the final route synthesis. The recovery route from ABT-515 (1) started with reductive removal of the oxime to give alcohol 3 followed by installation of the phenanthridine heterocycle to give 5 (Scheme 1). Subsequent removal of the Boc protecting group and installation of the pyrazine amide provided ABT-450 (8). This synthesis provided rapid access to ABT-450 (8) and was executed twice at kilogram scale to support initial toxicology and phase I clinical supply. Longer term, an efficient synthesis of ABT-450 (8) was required to support the continued clinical trials and commercial supplies. The retrosynthetic analysis for the route to ABT-450 (8), as shown in Scheme 2, would utilize a final coupling of sulfonamide 9 and the Step 5 acid 10. Step 5 acid 10 would be formed via ring closing metathesis, deprotection and hydrolysis of the step 2 protected diene 11, which would be prepared from dipeptide fragment 12 [7] and amino acid 13 [7]. The synthetic disconnections were based on published work for a similar macrocyclic ring target [8]. In that work, the addition of a protecting group on the prolamide position (below Boc protection for diene 11) provided substantial rate enhancements to the RCM closure and better control of dimeric impurity formation. Building on this work, the shown retrosynthesis provided an efficient route for ABT-450 (8) (Schemes 2 and 3). 2. Results and discussion 2.1. ABT-515 recovery route Prior to the toxicology findings, ABT-515 (1) was a lead candidate which had been prepared at kilogram scale [6]. Utilization of this material as an advanced intermediate to supply GLP toxicology and phase I clinical studies of ABT-450 (8) shortened the development timeline by approximately 6 months. Recovery of ABT-515 (1) started with a reductive removal of the oxime to afford macrocyclic alcohol 3 (Scheme 1). Reduction of the oxime was conducted at 30
C by portionwise addition of zinc powder (3.4 equivalents) to 1 in acetic acid to control the reaction temperature (adiabatic temperature increase of 84
C). Additionally, the portionwise addition provided better suspension of the zinc which was required for complete conversion. After reaction, the excess zinc and solid salts were removed by dilution with IPAc and filtration. A series of washes with water and dilute phosphoric acid served to remove the 9-fluorenylamine byproduct (2) (>99%) and the majority of the acetic acid (23 w/w% relative to 3 after washes) with a loss of 0.8% product to the aqueous washes. The washed organic layer was solvent exchanged to a toluene solution (chase distilled with 25 vol of toluene, 11 vol toluene target endpoint for distillation) and crystallized by addition of n-heptane (target toluene:n-heptane of 1:1 v/v, 2% product loss to filtrate) to afford macrocyclic alcohol 3 (98.6 area% purity by HPLC, 89% yield). Alkylation of the macrocyclic alcohol 3 with chlorophenanthridine 4 required the use of strong base (Na or K alkoxides, NaO-tPent utilized) in polar aprotic solvents to afford Boc intermediate 5. The primary impurities formed were resultant of dimerization via loss of the Boc protecting group affording dimers 14 and 15, or the des-Boc freebase 6 (Fig. 2). These impurities were minimized by

Fig. 2. Recovery route - Impurities from step 2 alkylation.

maintaining the reaction temperature at 0
C during addition of base to 3 and 4. N-methyl pyrrolidine (NMP) was utilized as the reaction solvent as N,N-dimethylformamide (DMF) formed significant levels of urea 16 as an impurity. Isolation by crystallization was evaluated but impurities were not well rejected in this step. As such, a solution of Boc intermediate 5 was carried forward to the next step (94.5 area% purity, 96% yield by assay). Removal of the Boc group from 5 proved more challenging than initially considered. The freebase of amine 6 was unstable, necessitating isolation as a salt. However, the use of acids such as HCl and p-toluenesulfonic acid (TsOH) was not possible due to precipitation of starting material as the phenanthridine salts resulting in incomplete reaction. The use of trifluoroacetic acid (TFA) as the acid for deprotection provided a homogenous reaction that affected the desired transformation with low levels of hydrolysis to form phenanthridinone 18 (typically < 5 mol%) and trifluoroacetylation impurity 17 (typically 5 area%), which could be removed in subsequent crystallizations (Fig. 3). Isolation of the product was accomplished by an inverse addition of the reaction mixture (IPAc/ TFA) to a solution of HCl in IPA/MeOH that allowed for isolation as the bis HCl salt (purity 91.5 area%, 83% yield). The methyl pyrazine 7 was installed as the final step by addition of the amine salt 6 to a preactivated solution of pyrazine acid 7 and hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU)/diisopropylethylamine (DIPEA) in DMF at less than 5
C. The product was isolated by aqueous extraction into IPAc using only water and brine washes as the use of stronger acids (e.g. aqueous HCl) resulted in precipitation of the product, presumably as the phenanthridine salt. Losses to the aqueous washes were typically very low (<0.1%). For initial deliveries, ABT-450 (8) was isolated as a hydrate crystal form I [9] from a mixture of IPAc/water/EtOH (target 2/1/17 v/v/v) via a slow cooling from 70
C to 0
C. If required, the material was recrystallized a second time to control impurities to levels suitable for the studies (purity 98.8 area% after first crystallization, 99.6% after second crystallization, total losses to liquors 16%, 83% yield). Polymorph screening of ABT-450 (8) identified a number of solvates and hydrates, but the above process reproducibly provided

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

4273

Scheme 1. Recovery route for ABT-450 (8) utilizing ABT-515 (1).

control of other physical characteristics such as particle size and agglomeration was not required.

2.2. Final route to ABT-450 (8)

Fig. 3. Recovery route - Impurities from Step 3 Boc removal.

the fit for purpose hydrate form I [9]. Form I completely dehydrates at approximately 120
C and the dehydrated phase maintains its crystallinity until melting at approximately 210
C. At this phase of development a liquid formulation was to be utilized, and therefore

2.2.1. Dipeptide 12 and amino acid 13 Dipeptide 12 and amino acid 13, the synthesis of which has been described [7], were selected as significant fragments for the convergent synthesis of ABT-450 (8). As starting materials for the planned regulatory synthesis, significant understanding of the impurities present in these materials and how they translated to impurities in ABT-450 (8) was essential. Impurities in dipeptide 12 were well tolerated by the process and did not translate to impurities in ABT-450 (8) (Fig. 4). The predominant stereoisomers of the four chiral centers and the

Scheme 2. Retrosynthetic analysis of the final route to ABT-450 (8).

4274

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Scheme 3. Final route to ABT-450 (8).

tripeptide homologs (two proline or vinylcyclopropyl amino acids) were well removed (reduced to < 0.10 area%) by the synthetic process when controlled to appropriate levels (0.2e0.4 area%) in dipeptide 12. Conversely, not all impurities in amino acid 13 were removed by the synthetic process (Fig. 5). Several impurities were poorly rejected and generated corresponding daughter impurities in ABT-450 (8) (Fig. 6). For this reason they are controlled to appropriate levels in amino acid 13 so that ABT-450 (8) is of suitable purity.

Fig. 4. Impurities of dipeptide 12 rejected by the ABT-450 (8) process (controlled 0.2 to 0.4 area%).

2.2.2. Ring closing metathesis (steps 1e3) The first three steps of the ABT-450 (8) process are conducted as a single process stream with steps 1 and 2 carried forward as solutions in toluene after extractive workups, then Step 3 Macrocycle 20 is isolated at Step 3 after extractive workup, carbon treatment and crystallization. Step 1 diene 19 could be isolated by crystallization but doing so provided no additional benefit to downstream processing (Scheme 4). The Step 1 coupling involves reaction of amino acid 13 with dipeptide 12. Initial conditions screened provided adequate conversion however significant amounts of epimerization (1e8%) of the amino acid 13 stereocenter was observed under a variety of

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

4275

Fig. 6. Impurities of amino acid 13 which are poorly rejected in the ABT-450 (8) process.

Fig. 5. Impurities of amino acid 13 rejected by the ABT-450 (8) process (controlled 0.2 to 1.0 area%).

conditions examined, including HATU/Et3N/DMF with or without CuCl2, HATU/2,6-lutidine/DMF, 2-chloro-4,6-dimethoxy-triazine (CDMT)/N-methylmorpholine/THF and EDAC/HONB/DMF with or without collidine, 2,6-lutidine, Et3N. Further screening identified T3P/DIPEA as a suitable coupling agent which gave significantly less epimerization of the amino acid (<0.5%). Additionally, the phosphate byproduct of T3P was easily removed by the aqueous washes [10,11]. The reaction was conducted in a mixture of NMP/toluene with DIPEA (3.5 equivalents) and a small excess of T3P (1.25 equivalents, 50 w/w% solution in toluene) added at 0
C followed by reaction at room temperature. Under these conditions, conversions were generally >99% using a small excess of amino acid 13 (1.05 equivalents). Aqueous workup (acid/base/water, < 1% loss to washes) in toluene provided a solution of Step 1 diene 19 after concentration. The step 2 Boc protection, as mentioned in the introduction, was modeled after process and mechanistic papers from BoehringerIngelheim for RCM reactions of the same macrocyclic core [8]. In those studies, installation of a protecting group on the prolamide position provided lower levels of dimeric impurities and allowed the RCM to be conducted at much higher concentrations. As discussed below, that work translated well to the synthesis of ABT-450 (8) and a similar strategy was utilized. For the protection of Step 1 diene 19, conditions similar to those reported were used. A catalytic

amount of DMAP (20 mol%) and an excess of Boc anhydride (1.65 equivalents) in toluene at ambient temperature provided robust conversion (>99%) and less than 1% each of two bis-Boc impurities as N- and O- protection of the pyrazine amide at 20
C). After reaction completion, consumption of excess Boc-anhydride was required as Boc anhydride can persist through the workup and concentration to form additional bis-Boc impurities. To consume the excess Boc anhydride, the reaction was quenched by the addition of a small amount of water to saturate the toluene reaction mixture followed by mixing for 2 h to ensure complete quench before continuing to the workup. The mixing time was based on ReactIR data following the consumption of Boc-anhydride under the reaction conditions (Fig. 7). Aqueous washes were conducted at 35
C for improved phase cuts removed DMAP, which can poison the subsequent RCM reaction (<1% loss to washes). After concentration. The toluene solution of step 2 protected diene 11 was used

Fig. 7. ReactIR trend at 1050-1100 cm1 depicting (Boc)2O concentration.

4276

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Scheme 4. ABT-450 (8) steps 1-3.

directly for the Step 3 RCM. During the development of ABT-515 (1), considerable study was conducted using unprotected diene 37 and protected diene 39 which were informative to the development of the ABT-450 (8) RCM (Scheme 5). This work extends the published RCM strategy [8] by demonstration of benzoate as an alternative protecting group and extension to alternate heterocycles. In addition, the studies on unprotected diene 37 identified reactivity of product in the catalytic cycle as a key issue precluding the use of fed batch concepts as a solution. The initial manufacture of ABT-515 (1) utilized the RCM of unprotected diene 37 which suffered from several disadvantages. This RCM required high dilution (120 L/kg) and use of HoveydaGrubbs Type I [12] catalysts to control dimeric impurities to an acceptable levels (<5 area%). Grubbs Type I [13] catalysts (bisphosphine) were not considered based on potential isomerization of the cyclopropane reported on similar systems [14]. The use of higher temperatures or Hoveyda-Grubbs type II [15] catalysts resulted in unacceptably high levels of dimers. The initial supplies of ABT-515 (1) utilized in the recovery route were prepared as shown below (Scheme 5) using dichloromethane (120 L/kg) and a Hoveyda-Grubbs Type I catalyst (Zhan C [16], 3 mol%) which provided acceptable intermediate (4% dimers). As part of this development, fed batch processes, which are commonly used to limit byproduct formation in RCM reactions [17], with slow addition of diene and/or catalyst under reduced volume were evaluated but only showed minor improvements in dimer formation. As this was somewhat surprising, a deuterium labeled D8/D0 crossover experiment (Scheme 6) starting with an equimolar mixture of D8 diene and D0 product was conducted under the same reaction conditions to evaluate the relative rates of starting material and product reaction to form dimers. The initial dimers formed were analyzed by LC/MS and showed an almost equal amount of D16 dimer (expected dimerization of two molecules of D8 diene) and D8 dimer (unexpected dimerization of D8 diene with D0 product). This indicated

Scheme 5. RCM development for ABT-515 (1).

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

4277

Scheme 6. Crossover RCM reaction showing product participation in dimer formation.

that the relative dimerization rates of the starting material and product were similar which explained the limitations of fed batch application to this reaction. In preparation for future deliveries of ABT-515 (1), additional development identified the benzamide protected diene 39 as a suitable RCM substrate which allowed the utilization of HoveydaGrubbs type II catalyst (Zhan B, 0.2 mol%) at higher concentration (13 L/kg, 9X reduction in volume) while still providing acceptable levels of dimers. Interestingly, in contrast to the identified reactivity of product 38 under the reaction conditions, protected product 40 does not seem to be reactive as additional catalyst charges after reaction completion do not result in further dimer formation. The benzamide protection was selected as ABT-515 (1) contains a Boc group as part of the final product and selective deprotection of a bis-Boc protected macrocycle would have been difficult. While this modification was never performed on scale due to the discontinuation of ABT-515, the reaction understanding gained on these substrates informed the RCM reaction development for ABT-450 (8). For the Step 3 RCM of ABT-450, development focused exclusively on the step 2 diene 11. Initial studies were conducted in EtOAc but later switched to toluene to align the reaction and isolation solvents for the Step 1/2/3 process. Catalyst loading could be lowered to <1 mol% with successful runs at 0.25 mol% and modest improvements to dimeric impurities using 0.7 mol% (Table 1, Run 1 and 2). Due to concerns about trace contaminants causing catalyst deactivation with further reductions in catalyst [18], the final process utilized catalyst loads in the range of 0.44e0.6 mol% to balance conversion with conservation of catalyst (cost and control of ruthenium downstream). The use of the step 2 diene 11 allowed for higher reaction concentrations utilizing a total final reaction volume of 13 L/kg which balances dimer formation

and throughput. Fed batch processing offered some advantage compared to the batch process where dimer production was reduced from 5% to 2% (Table 1 Run 4 and 5). As the catalyst was found to deactivate over time under the reaction conditions, the final process included adding the diene as a solution in toluene over 4 h and the catalyst as a solution in toluene over 5 h to ensure the presence of fresh catalyst throughout the reaction process. Of interest for the fed batch process, additional charges of catalyst towards the end of the reaction (5e10% starting material remaining) did not produce significant changes in the dimer levels, suggesting that the prolamide protected product 20 reacts slowly with the catalyst. On scale, use of a nitrogen sparge to remove the byproduct ethylene was needed for robust conversion [18]. This was conducted as shown in Fig. 8 where nitrogen was introduced in a high mixing zone with a ½ inch dip tube and flow at 11 SCFM in a 500 gal reactor. The Step 3 ring closing metathesis generates several key impurities in the ABT-450 (8) process as shown in Fig. 9. Several key observations aided the control of each impurity: 1. M- 14 41 levels do not vary based on the Step 3 processing conditions (temperature, solvent, catalyst) and are derived from impurities in the Step 1 starting material (Fig. 6). Control of these impurities in amino acid 12 is particularly important as the M-14 impurity is not removed by downstream processing. 2. The amount of trans macrocycle 42 formed is controlled by reaction temperature with levels of 0.8 area% at 40
C/1.0 area% at 65
C and 1.2 area% at 80
C. As reaction conversions were not robust at 40
C, the reaction temperature was increased to 65
C to balance conversion with trans impurity 42 formation. This impurity is typically reduced by 50% in the Step 3 crystallization and further reduced to about 25% in the Steps 4 and 5 isolations.

Table 1 Lab scale evaluation of parameters in Step 3 RCM. Run

1 2 3 4 5 6

Toluene (L/kg) Reaction

Diene

Catalyst

9 9 9 9 11 7

2 2 2 2

2 2 2 2 2 1

[1] Batch mode process, addition time for catalyst only.

Addition time (h)

Catalyst (mol%)

Temperature (
C)

Assay Yield (%)

Starting material (conversion, %)

Dimers (area%)

4 4 4 4 41 31

0.7 0.25 0.7 0.5 0.5 1.0

60 60 80 80 80 65

90 88 90 90 86 76

>99 >99 >99 >99 >99 >99

2.0 2.8 1.7 1.7 5.3 9.8

4278

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Fig. 8. Lab and plant scale RCM reactions (fed batch).

3. The dimer impurities are initially present in an “open” form, but cyclize as the reaction proceeds to provide two major dimer isomers 43 and 44 determined to be the “head to tail” trans/ trans and “head to tail” trans/cis isomers. These dimeric

Fig. 9. Key impurities from Step 3.

impurities, typically 2e3 area% in the reaction, are not rejected in the Step 3 crystallization but are reduced by steps 4 and 5 isolations to 0.3 area%. In contrast to many metal catalyzed reactions where metal removal constitutes a difficult part of the processing, the control of Step 3 Macrocycle 20 color was more relevant to downstream processing. Removal of ruthenium by subsequent steps provided good controls where Step 3 Macrocycle 20 containing elevated ruthenium of 175 ppm (versus nominal of <40 ppm) was reduced to < 20 ppm by Step 5 Acid 10. In contrast, strongly colored solutions carried downstream resulted in undesirable color prompting a need for control in Step 3 where processing would deliver light yellow to clear reaction solutions. Workup and control of ruthenium and color has been accomplished by two processes. The first process involved quench of the reaction mixture with imidazole [19] (2 x catalyst equivalents) to deactivate the catalyst followed by absorption on to Grade F4 Filterol (50 w/w% relative to diene, BASF Catalysts LLC), a chemically treated clay filter aid. This was very effective in removing ruthenium and color from the reaction solutions. However, attempts to utilize Filterol as a packed bed did not give consistent results due to uneven flow and breakthrough of the packed bed. To overcome this, the process was run by slurry treatment in the reactor which required extensive cleaning to remove the residual Filterol. As shown in Table 2, screening identified silica gel and carbon as alternate absorption agents. However, the appropriate equipment to utilize silica gel was not available at the time for the process and carbon treatment required an impractical loading of 10% (Fig. 10). Because of this, an aqueous workup based on published literature using 2-mercaptonicotinic acid [20] quench (4 mol%, 40
C for 2 h) followed by a basic aqueous wash was developed. This aqueous workup provided substantial ruthenium and color removal to give an amber product solution of Step 3 Macrocycle 20 after workup with 60e90 ppm ruthenium (versus 20, theoretical starting 540 ppm based on initial catalyst charge). A subsequent carbon treatment (AKS6 Pall Carbon, 2.5 w/w% carbon) served to further reduce color and ruthenium (30e50 ppm) and provided consistent characteristics for

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

4279

Table 2 Initial solid phase treatment screening of Step 3 with imidazole quench. Treatment

W/w% Loading Range

% Product Loss Over Range

Minimum W/w% Loading for Color Removal

Ruthenium in 20 at Minimum Loading

Silica Neutral Alumina Basic Alumina F1/F4 Filterol Carbons

15e30% 12.5e25% 12.5e25% 10e50% 2.5e10%

2e4% 0e1% 1e2% 0e1% 0e4%

15% 25% >25% 50% 10%

<20 ppm N.D. N.D <20 ppm 45 ppm

Fig. 10. Examples of imidazole quench reaction solutions treated with various solid phase agents after overnight mixing. Weight percent treatment is with respect to the Step 3 product. All vials are approximately 8 w/w% 20 in toluene as reaction mixtures.

downstream steps (Fig. 11). Step 3 macrocycle 20 was isolated from the toluene reaction solution after workup and carbon treatment by crystallization from toluene:heptanes. The crystallization was conducted by concentration of the solution to approximately 3 vol of toluene (approximately 28 w/w% Step 3 macrocycle 20 in toluene) followed by slow addition of heptanes (9 vol) at 60
C and cooling to 20
C (filtrate level 3e8 mg/mL by HPLC, typically 5e10% losses to liquors).

Fig. 11. Picture showing from left to right: crude toluene solution after 2mercaptonicotinic acid workup, after treatment with Pall AKS1 or after treatment with Pall AKS6 carbon (2.5 w/w%).

2.2.3. Boc deprotection (step 4) The Boc removal involves a reactive crystallization of the HCl salt of Step 4 macrocycle 21 (Scheme 7). The reaction is conducted in a mixture of toluene and acetonitrile (1:1 v/v, 10 vol total) which provided good reaction profile and appropriate solubility characteristics to achieve a reactive crystallization. Screening of reaction conditions identified the use of acetonitrile with HCl as providing

Scheme 7. Step 4 reaction scheme.

4280

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Fig. 12. Micrograph of Step 4 macrocycle 21 at 100X. Comparison of unseeded and seeded particle presentation.

the best impurity profile for the reaction but resulted in high levels of product in the liquors even after partial removal of HCl via sparging after reaction completion. To enable direct isolation, toluene was added as a co-solvent which reduced the product solubility while maintaining the desired reaction profile. HCl solution was freshly prepared by gassing anhydrous HCl into acetonitrile (titrated to determine molarity, targeted 9 to 11 w/w%, 6.5 equivalents HCl charged for reaction). The reaction was conducted by mixing at 20
C for 1 h, during which time approximately 10% conversion occurred, followed by addition of product seeds to enable crystallization as the reaction progressed. Without seeding, the product salt self-nucleates as the reaction proceeds, but resulted in very fine needles which were difficult to filter (Fig. 12). After seeding the reaction was warmed slowly (8
C/h) to 60
C and held until reaction completion (<0.5% Step 3 macrocycle 20, typically about 5 h) then cooled to 20
C and filtered (filtrate level 2e4 mg/ mL by HPLC, 2e4% loss to liquors).

2.2.4. Hydrolysis (step 5) The Step 5 hydrolysis was conducted with aqueous alkoxide as base to neutralize the HCl salt and saponify the ester (Scheme 8). Initial reaction conditions utilized THF/EtOH/water as the reaction solvent at 65
C. Workup was conducted with 2-MeTHF and aqueous phosphoric acid/aqueous brine washes at 35
C to improve phase cuts. 2-MeTHF was required as the solubility characteristics of the free acid were very low in other typical workup solvents (Table 3). The organic extracts were concentrated and chase distilled with EtOH to remove THF/2-MeTHF/water (THF and 2MeTHF < 1 w/w% as they greatly increase the solubility and losses in the crystallization). Step 5 acid 10 was isolated by crystallization from EtOH by dissolving at 70
C, seeding at 65
C and then isolation after cooling to 0
C (typically 5% loss to liquors). In order to streamline the process, a direct crystallization of Step 5 acid 10 from the reaction mixture was developed. However, the sodium carboxylate salt of 10 was unreactive in the step 6 acyl

imidazolide formation [21]. This required that the direct crystallization of Step 5 acid 10 ensure that the free acid was obtained with <0.1 w/w% sodium (corresponding to 3 mol% carboxylate salt) to give an acceptable reaction conversion profile in the next step. The phenanthridine ring of ABT-450 (8) was susceptible to hydrolysis in the presence of strong acids. For this reason, a process was initially developed using HOAc in MeOH/water to crystallize Step 5 acid 10 directly from the reaction mixture. Based on the charge of NaOH used for the hydrolysis, a minimum of 3.5 equivalents of HOAc was required to neutralize the sodium salt. Solubility profiles demonstrated that the addition of additional HOAc (3.5e5 equivalents) provided decreased solubility in a range of solvent mixtures and provided acceptable impurity profiles but high residual sodium levels (0.2e0.4%) which negatively impacted downstream acyl imidazolide conversion (Fig. 13). An estimated pKa equilibrium based on the initial experiments at 3.5 and 5 equivalents HOAc predicted that further increase of HOAc equivalents would reduce the level of sodium salt in the Step 5 acid. This was demonstrated as shown in Fig. 14 where increasing to 10 to 25 equivalents HOAc provided acceptable sodium levels (<0.l%). Based on this, direct crystallization could be accomplished by addition of HOAc (25 equivalents) at 60
C to the reaction mixture (MeOH/water 8.5/1.5 vol) followed by seeding and cooling (loss to liquors 5.5%, purity > 99 area%) that contained < 0.006% remaining sodium carboxylate salt. The final process used formic acid in place of acetic acid; the lower pKa of formic acid relative to HOAc allowed lower equivalents to drive the desired crystallization. In this final process, the hydrolysis was conducted using NaOH

Table 3 Solubility of Step 5 acid 10. Solvent

mg/mL

THF 2-MeTHF EtOH IPAc MTBE MeCN

>100 36 9.7 1.5 1.1 0.9

Fig. 13. Solubility data (after 3 h of equilibration) for Step 5 acid 10 as a function temperature in various MeOH (10 vol)/H2O ratios as a function of AcOH equivalents.

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Fig. 14. Residual sodium in Step 5 acid 10 as a function of equivalents of AcOH.

Scheme 8. Step 5 reaction scheme.

(charge based on HCl content in ester starting material, 2.6 equivalents over HCl content) in aqueous methanol followed by neutralization with formic acid (3.4 equivalents total, 2.6 equivalents to neutralize base and 0.8 equivalents excess to drive crystallization) at 67
C followed by cooling with seeding (filtrate level 3 mg/mL by HPLC, typically 3% loss to liquors)(see Scheme 8).

2.2.5. Acyl sulfonamide formation (step 6) Formation of the acylsulfonamide bond of ABT-450 (8) is accomplished in Step 6 via a two-step reaction sequence. First, the Step 5 acid 10 was reacted with CDI (1.4 equivalents) at 0
C to form the acyl imidazolide. Reaction conversion is determined by HPLC analysis of a sample of the reaction mixture quenched into 0.1 M DBU in MeOH and measurement of the remaining carboxylic acid versus the methyl ester (typical conversion > 99%). Cyclopropylsulfonamide (2.0 equivalents) and DBU (1.5 equivalents) were then added followed by heating to 40
C to complete the acyl sulfonamide formation (typical conversion > 99%). Interestingly, the acyl imidazolide was demonstrated to convert to an azlactone intermediate upon exposure to DBU as shown in Scheme 9 which prompted concerns of epimerization at the proline stereocenter. Fortunately, even under stressed reaction conditions with excess DBU (3 equivalents) at elevated temperature (80
C) and extended hold (32 h) only 0.87% isomer was observed which is fully removed by the Step 6 crystallization (<0.03%). After reaction completion, ABT-450 (8) is isolated by an aqueous workup followed by crystallization from a mixture of IPAc, IPA, and water.

4281

Multiple variables impact the Step 6 acyl imidazolide formation. First, water hydrolyzes both CDI and the acyl imidazolide. To control water entering the process, the NMP solution of Step 5 acid 10 (typically contains 2e4% water from the Step 5 acid 10) was azeodried using IPAc to < 0.10% water before addition of CDI. Second, CDI itself was found to have variable sensitivity to atmospheric moisture depending on its physical characteristics (particle size and chloride content) and handling (exposure and storage) [22]. Finally, incomplete acyl imidazolide formation can be caused by sodium carboxylate salt in the Step 5 acid 10 which is much less reactive towards CDI [21]. Once formation of the acyl imidazolide is complete, a strong amine base is required for acyl sulfonamide formation to occur at a reasonable rate. While DBU was the ideal choice for this particular acyl sulfonamide formation, its use presented two challenges. First, any water introduced during acyl sulfonamide formation regenerated Step 5 acid 10. Because DBU is hygroscopic, it was dried before use by treatment with CDI (0.2 equivalents) as a reactive drying agent. Second, the acyl sulfonamide formation rate was significantly impacted by carbon dioxide remaining from the acyl imidazolide formation step, presumably through sequestration of DBU [23]. A nitrogen sparge applied during acyl imidazolide formation sufficiently removes carbon dioxide prior to DBU addition. In summary, the step 6 reaction requires a substantial number of controls to ensure robust performance including:  Control of residual sodium carboxylate salt in Step 5 acid 10 which prevents activation of the carboxylate  Azeodrying of the Step 5 acid 10 NMP/IPAc solution to remove water before use which consumes CDI  Control of CDI equivalents to ensure sufficient conversion to acyl imidazolide without an excess of CDI present to consume cyclopropylsulfonamide 9  Control of water in DBU by reactive drying using CDI to avoid hydrolysis of activated acid  Removal of carbon dioxide generated in the acyl imidazolide formation step by nitrogen sparging to avoid sequestration of DBU ABT-450 (8) was isolated by aqueous workup followed by crystallization. Dilution of the reaction mixture with IPAc followed by a 2 M H3PO4 wash and brine washes served to remove imidazole, DBU, and unreacted cyclopropylsulfonamide. Conducting these washes at 35
C dramatically improved layer separations. ABT-450 was crystallized from IPAc/IPA/water to afford ABT-450 (8) in 91% yield. A complete discussion of the crystallization process including thermodynamic polymorph stability in the ternary system and controls relevant to physical properties has been published [24]. Form II, isolated from IPAc/IPA/water, was identified as the most suitable form for development. Form II completely dehydrates to an amorphous state around 110
C making it ideally suited for the commercial formulation process, wherein amorphous solid dispersion is prepared via hot-melt extrusion based formulation process [25]. Form II crystallizes in the triclinic space group P1 with one molecule of ABT-450 (8) and 2 molecules of water in the asymmetric unit (see Fig. 15). The two molecules of water occupy ca. 7.2% (75 Å3) of the unit cell volume, highlighted in light blue, and participate in two different hydrogen bonding motifs. When the relative humidity (RH) is increased beyond 40% the form II unit cell starts accommodating up to a full molecule of water at 95% (RH). The higher hydration state also crystallizes in the P1 space group with two molecules of ABT-450 (8) in the unit cell, five fully occupied water molecule sites, two partially occupied water sites, and a doubling of the unit cell volume. The hydrogen bonding scheme is much more complex at the higher

4282

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Scheme 9. Step 6 reaction scheme.

enable efficient isolations, and a mechanistically complex acylsulfonamide formation as the final step. This process was developed through the course of four years and has been utilized for commercial manufacture since its launch in December of 2014. 4. Experimental section

Fig. 15. Ball-and-stick representation of ABT-450 (8) Form II highlighting the hydrogen bonding motifs of the two water molecules O8 and O9. Hydrogens on each water molecule could not be located in the difference map and are therefore not included. O8 acts as a hydrogen bond donor and acceptor, bridging two ABT-450 molecules at N3/O8 and O2/O8 distances of 2.99 Å and 2.94 Å, respectively. O9 acts as a hydrogen bond donor to the sulfonamide group at an O9/O5 distance of 3.00 Å.

hydrate state, however, the same functional groups are involved. Water occupies a greater volume of the unit cell compared to the dihydrate (138 Å3 versus 75 Å3), however the fraction of the unit cell volume that water occupies remains relatively constant (6.9% versus 7.2%). 3. Conclusion Process development of ABT-450 (8) enabled the development and commercialization of a complex macrocycle which features an RCM reaction on greater than 200 kg scale, process streamlining of reaction and workup solvents and reactive crystallizations to

General Information: All reagents and solvents were purchased from commercial vendors and used without further purification. 1H NMR spectra were recorded on either a 400 or 700 MHz spectrometer and chemical shifts (d) are referenced to either TMS or the NMR solvent. 13C NMR spectra were obtained at either 101 or 176 MHz and referenced to the NMR solvent. HPLC samples were analyzed using an Agilent 1100 or 1200 system equipped with a UV-DAD detector. Numerous HPLC methods were developed for the analysis of the reactions and products. An example of a typical method as follows: Ascentis Express C8 or C18 column, 10 or 15 cm  4.6 mm, and 2.7 mm using acetonitrile and either 0.1% H3PO4 or 0.1% HClO4 with a gradient from 10% acetonitrile to 90% acetonitrile over 10e15 min. UV detection typically at 205 or 210 nm. Reported conversion and purity values were generally obtained using HPLC. Potency was generally obtained using HPLC by comparison to are reference material. LC/MS (ESI positive) samples were analyzed using similar methods with the exception of using 0.1% formic acid in both the aqueous and the acetonitrile mobile phases. Macrocyclic alcohol 3: A solution of ABT-515 1 (5.0 kg, 6.5 mol) and HOAc (20.3 L) was maintained at 30
C as zinc dust was charged in three portions (1.5 kg total, 22.5 mol, 3.5 equiv) each time allowing the temperature to return to 30
C before the subsequent charge. After 15 min (>99% conversion by HPLC) the slurry was cooled to 23
C, diluted with IPAc (8.5 kg) and filtered through celite/hyflo using IPAc (2  8.5 kg) as a rinse. The combined organics were washed with water (57.7 kg) and the aqueous layer was back extracted with IPAc (26.0 kg). The

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

combined organics were washed with water (31.5 kg), 1 M phosphoric acid (31.0 kg) and water (31.5 kg). The organic layer was concentrated to approximately 37 L and then chase distilled with toluene (105 kg) maintaining a constant volume during which time product partially crystallized. The slurry was warmed to 50
C at which time most solids had dissolved, maintained for 3 h then cooled over 5 h to 20
C and maintained at 20
C for 5 h. n-Heptane (25.5 kg) was added over 1 h followed by mixing for 30 min (filtrate level 0.3 mg/mL by HPLC) then filtered and washed with n-heptane (3  5 kg). The solids were dried under reduced pressure at 40
C for 2 days to give macrocyclic alcohol 3 as a white solid (3.4 kg, 98.4 w/w% by assay, 98.6 area% purity, 89% potency adjusted yield, < 10 ppm zinc). 1H NMR (400 MHz, Chloroform-d) d 10.34 (s, 1H), 7.17 (s, 1H), 5.70 (q, J ¼ 8.7 Hz, 1H), 5.15 (d, J ¼ 7.1 Hz, 1H), 4.98 (t, J ¼ 9.3 Hz, 1H), 4.60 (s, 1H), 4.52 (t, J ¼ 8.1 Hz, 1H), 4.26 (td, J ¼ 7.4, 3.8 Hz, 1H), 4.11 (d, J ¼ 11.0 Hz, 1H), 3.72 (dd, J ¼ 10.9, 3.3 Hz, 1H), 2.91 (tt, J ¼ 8.0, 5.0 Hz, 1H), 2.50e2.46 (m, 1H), 2.33e2.22 (m, 3H), 1.94e1.89 (m, 2H), 1.88e1.77 (m, 2H), 1.59e1.44 (m, 7H), 1.42 (s, 9H), 1.33e1.29 (m, 2H), 1.19e1.04 (m, 2H), 0.98e0.86 (m, 1H). Amine salt 6: Macrocyclic alcohol 3 (4.2 kg, 7.3 mol) and 6chlorophenantiridine 4 (1.6 kg, 7.3 mol, 1.0 equiv) were dissolved in NMP (13.1 kg, anhydrous) by warming to 40
C. The solution was cooled to 5
C and maintained below 10
C as a solution of NaO-tBu (2.3 kg, 20.0 mol, 2.7 equiv) in NMP (11.0 kg, anhydrous) was slowly added to give a deep purple solution. After 20 h at 0
C, the mixture had converted to a thick yellow syrup. The reaction was warmed and held for 12 h at 20
C (<1 area% alcohol 3 remaining by HPLC). The reaction was maintained below 23
C while water (30 kg) was added. The quenched reaction solution was extracted twice with IPAc (2  27 kg). The combined organic layers were diluted with additional IPAc (27 kg) then washed three times with 10% KH2PO4 (3  44 kg) and 20% NaCl (48 kg). The organic layer was concentrated under reduced pressure to approximately 15 L then diluted with IPAc (65 kg) and filtered to provide Boc intermediate 5 as a solution in IPAc (91.8 kg solution, 5.65 w/w%, 5.2 kg 5, 94.5 area % purity, 96% potency adjusted yield). 1H NMR (400 MHz, DMSO‑d6) d 11.08 (s, 1H), 8.91 (s, 1H), 8.73 (d, J ¼ 8.3 Hz, 1H), 8.63 (dd, J ¼ 8.3, 1.3 Hz, 1H), 8.34 (d, J ¼ 7.7 Hz, 1H), 7.90 (t, J ¼ 7.0 Hz, 1H), 7.83 (dd, J ¼ 8.2, 1.3 Hz, 1H), 7.71e7.58 (m, 2H), 7.53 (t, J ¼ 6.9 Hz, 1H), 5.94 (s, 1H), 5.60 (q, J ¼ 8.8 Hz, 1H), 5.08 (t, J ¼ 9.4 Hz, 1H), 4.70 (d, J ¼ 11.4 Hz, 1H), 4.51 (dd, J ¼ 10.0, 6.9 Hz, 1H), 4.07e3.97 (m, 1H), 3.93 (d, J ¼ 10.9 Hz, 1H), 2.95e2.85 (m, 1H), 2.73 (dd, J ¼ 14.0, 6.9 Hz, 1H), 2.65 (d, J ¼ 12.2 Hz, 1H), 2.42 (ddd, J ¼ 14.8, 10.8, 4.4 Hz, 1H), 2.34 (q, J ¼ 8.6 Hz, 1H), 1.81e1.65 (m, 2H), 1.63e1.51 (m, 2H), 1.37 (d, J ¼ 13.8 Hz, 4H), 1.23 (s, 1H), 1.21e1.15 (m, 1H), 1.13 (s, 9H), 1.11e0.92 (m, 5H), 0.89e0.81 (m, 1H). 13C NMR (101 MHz, DMSO) d 177.0, 172.9, 169.8, 157.9, 155.8, 143.2, 135.2, 132.3, 129.8, 128.3, 126.5, 125.6, 125.4, 123.6, 123.3, 122.9, 120.3, 78.9, 76.3, 60.0, 54.2, 53.2, 44.3, 36.1, 33.0, 32.6, 32.0, 31.1, 29.7, 29.3, 28.5, 28.1, 27.4, 24.1, 23.5, 22.8, 15.4, 7.2, 7.2. Most of the Boc intermediate 5 solution (5.1 kg content, 6.8 mol) was concentrated to 18.7 kg solution then trifluoroacetic acid (7.4 kg, 5 L, 65 mol, 9.6 equiv) was charged with an IPAc (4.4 kg) rinse, and the reaction was heated to 70
C. After 18 h, the reaction was cooled to 20
C (>99% conversion by HPLC, 91% assay yield). In a separate reactor, HCl/IPA (4 M, 4.2 L, 17 mol, 2.5 equiv) and MeOH (15.8 kg) were combined and maintained at 37
C as a portion (10%) of the above reaction mixture was added over 10 min resulting in a thin suspension. The suspension was aged for 15 min then the remainder of the reaction solution was added over 1 h. The slurry was cooled to 20
C over 1 h, filtered and rinsed with IPAc. The solids were dried under reduced pressure at 50
C to give amine salt 6 as a white solid (4.48 kg, 81.5 w/w% freebase by assay,

4283

91.5 area% purity, 83% potency adjusted yield). 1H NMR (400 MHz, DMSO‑d6) d 11.17 (s, 1H), 9.20 (s, 1H), 8.77 (d, J ¼ 8.4 Hz, 1H), 8.65 (dd, J ¼ 8.2, 1.2 Hz, 1H), 8.46e8.22 (m, 4H), 7.95 (ddd, J ¼ 8.4, 7.1, 1.4 Hz, 1H), 7.85 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.75 (ddd, J ¼ 8.1, 7.1, 1.0 Hz, 1H), 7.68 (ddd, J ¼ 8.2, 7.1, 1.4 Hz, 1H), 7.56 (ddd, J ¼ 8.3, 7.1, 1.3 Hz, 1H), 6.03 (s, 1H), 5.62 (q, J ¼ 8.4 Hz, 1H), 5.13 (t, J ¼ 9.5 Hz, 1H), 4.59 (dd, J ¼ 9.0, 7.4 Hz, 1H), 4.28 (d, J ¼ 11.7 Hz, 1H), 4.23e4.10 (m, 2H), 2.92 (tt, J ¼ 7.8, 4.9 Hz, 1H), 2.74 (dd, J ¼ 14.0, 7.2 Hz, 1H), 2.55 (ddd, J ¼ 14.2, 9.5, 4.8 Hz, 1H), 2.23 (q, J ¼ 8.9 Hz, 1H), 1.95e1.81 (m, 1H), 1.81e1.71 (m, 2H), 1.63 (dd, J ¼ 8.2, 5.1 Hz, 1H), 1.57 (dd, J ¼ 9.4, 5.0 Hz, 1H), 1.53e1.34 (m, 4H), 1.30e1.14 (m, 2H), 1.14e0.95 (m, 4H). ABT-450 (8): To ensure accurate equiv, amine salt 6 was dissolved in DMF and analyzed for freebase content. The prepared solution contained amine 6 (3.0 kg, 24.5 w/w%, 4.6 mol) and DMF (approximately 9.0 kg). In a separate reactor, pyrazine acid 7 (0.77 kg, 5.6 mol, 1.2 equiv) and HATU (2.12 kg, 5.6 mol, 1.2 equiv) were dissolved in DMF (8.5 kg) and cooled to 0
C. The reaction was maintained below 10
C as DIPEA (2.16 kg, 16.7 mol, 3.6 equiv) was slowly added. After 30 min amine 6 in DMF was slowly added maintaining the temperature below 20
C. After 15 min (>99% conversion by HPLC), the reaction was diluted with IPAc (26.2 kg) and water (30 kg) maintaining the temperature below 25
C. The layers were separated and the aqueous layer was extracted with IPAc (10.5 kg). The combined organic layers in addition to an IPAc rinse (2.6 kg) to aid transfers were washed with water (30 kg), 5% NaCl (31 kg) and 10% NaCl (32 kg) and concentrated to approximately 7 L under reduced pressure (52.1 w/w% solution in IPAc, approximately 1.25 vol IPAc). The organic solution was diluted with water (3.0 kg) and EtOH (42.6 kg) then heated and maintained at 75
C as IPAc (4.4 kg) was added in portions until a homogenous solution was observed. The solution was cooled to 70
C, seeded, then cooled to 10
C over 24 h (filtrate level 5.1 mg/mL by HPLC). The slurry was filtered and washed with EtOH (2  4.7 kg). For the second crystallization, the wetcake was suspended in IPAc (5.2 kg), water (3.0 kg) and EtOH (40.2 kg) and heated to 75
C to dissolve. The solution was cooled to 70
C, seeded, then cooled to 10
C over 40 h (filtrate level 2.1 mg/mL by HPLC). The slurry was filtered and washed with EtOH (2  4.7 kg). The solids were dried at 50
C under reduced pressure to give ABT-450 (8) as a white solid (3.0 kg, 97.1 w/w% by assay, 99.5 area% purity, 83% potency adjusted yield). Analytical data for 8 is tabulated below. Amino acid 13: 1H NMR (700 MHz, DMSO‑d6) d 12.84 (s, 1H), 9.05 (d, J ¼ 1.6 Hz, 1H), 8.72 (d, J ¼ 8.1 Hz, 1H), 8.64 (d, J ¼ 1.6 Hz, 1H), 5.76 (ddt, J ¼ 16.9, 10.2, 6.7 Hz, 1H), 4.96 (dq, J ¼ 17.2, 1.7 Hz, 1H), 4.90 (ddt, J ¼ 10.2, 2.4, 1.3 Hz, 1H), 4.45 (td, J ¼ 8.2, 5.2 Hz, 1H), 2.60 (s, 3H), 1.98 (qd, J ¼ 6.7, 3.3 Hz, 2H), 1.85 (pd, J ¼ 8.0, 3.7 Hz, 2H), 1.36e1.22 (m, 6H). 13C NMR (176 MHz, DMSO) d 173.2, 162.9, 157.2, 142.9, 142.5, 141.6, 138.7, 114.6, 52.0, 33.0, 30.7, 28.1, 28.0, 25.2, 21.4. HRMS calcd C15H21N3O3 [MþH]þ: 292.1656, Found 292.1664. Melting point 97e98
C. Dipeptide 12: 1H NMR (700 MHz, DMSO‑d6) d 8.72 (dd, J ¼ 8.2, 1.1 Hz, 1H), 8.68 (s, 1H), 8.61 (dd, J ¼ 8.3, 1.5 Hz, 1H), 8.35 (dd, J ¼ 8.1, 1.4 Hz, 1H), 7.91 (ddd, J ¼ 8.3, 7.0, 1.4 Hz, 1H), 7.79 (dd, J ¼ 8.2, 1.3 Hz, 1H), 7.73 (ddd, J ¼ 8.1, 7.0, 1.1 Hz, 1H), 7.65 (ddd, J ¼ 8.2, 7.0, 1.4 Hz, 1H), 7.52 (ddd, J ¼ 8.2, 7.0, 1.4 Hz, 1H), 5.79 (ddt, J ¼ 6.6, 4.5, 2.0 Hz, 1H), 5.65 (ddd, J ¼ 17.2, 10.4, 8.9 Hz, 1H), 5.30 (dd, J ¼ 17.1, 2.1 Hz, 1H), 5.10 (dd, J ¼ 10.3, 2.0 Hz, 1H), 4.10 (dq, J ¼ 10.9, 7.1 Hz, 1H), 4.04 (dq, J ¼ 10.9, 7.1 Hz, 1H), 3.89 (t, J ¼ 7.9 Hz, 1H), 3.36e3.30 (m, 2H), 3.21 (dt, J ¼ 12.6, 1.7 Hz, 1H), 2.50 (p, J ¼ 1.9 Hz, 1H), 2.39e2.33 (m, 1H), 2.31e2.20 (m, 2H), 1.68 (dd, J ¼ 8.0, 5.1 Hz, 1H), 1.34 (dd, J ¼ 9.4, 5.2 Hz, 1H), 1.18 (t, J ¼ 7.1 Hz, 3H). 13C NMR (176 MHz, DMSO) d 175.0, 170.0, 157.7, 142.6, 134.4, 134.3, 131.6, 129.1, 127.8, 127.6, 124.9, 124.8, 122.8, 122.5, 122.0, 119.5, 117.7, 77.2, 60.8, 59.8, 52.9, 37.4, 32.1, 22.6, 14.2. HRMS calcd C26H27N3O4 [MþH]þ: 446.2075,

4284

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

Found 446.2086. Melting point 158e159
C. Step 1 diene 19 and step 2 protected diene 11: Amino acid 13 (82.3 kg, 282 mol, 1.05 equiv), dipeptide 12 (120.0 kg, 269 mmol) were dissolved in NMP (245 kg), toluene (209 kg) and DIPEA (122 kg, 944 mol, 3.5 equiv) was added. The solution was cooled to 0
C and maintained below 5
C as T3P in toluene (50 w/w% solution, 215.2 kg solution, 338 mol, 1.25 equiv) was added with a toluene (17 kg) rinse. After 3 h (>99% conversion by HPLC) the reaction was diluted with toluene (414 kg) and washed with 5% NaCl in 1 M H3PO4 (727 kg) followed by 5% NaHCO3 (711 kg) and 5% NaCl (711 kg) at 40
C for improved phase cuts. The organic layer was concentrated under reduced pressure to approximately 670 L to give Step 1 diene 19 as a solution in toluene (98.3 area% purity, Kf < 0.10% water). 1H NMR (rotomers observed) (700 MHz, DMSO‑d6) d 8.69 (d, J ¼ 1.5 Hz, 1H), 8.68 (d, J ¼ 8.4 Hz, 1H), 8.64e8.57 (m, 1H), 8.43 (d, J ¼ 1.5 Hz, 1H), 8.21 (dd, J ¼ 8.2, 1.4 Hz, 1H), 7.87 (ddd, J ¼ 8.3, 7.0, 1.4 Hz, 1H), 7.81 (dd, J ¼ 8.2, 1.3 Hz, 1H), 7.67 (ddd, J ¼ 8.2, 6.9, 1.3 Hz, 1H), 7.63 (td, J ¼ 7.4, 7.0, 1.0 Hz, 1H), 7.54 (tt, J ¼ 7.0, 1.3 Hz, 1H), 6.01 (tt, J ¼ 4.1, 1.8 Hz, 1H), 5.80 (ddd, J ¼ 15.4, 9.5, 7.7 Hz, 1H), 5.79e5.70 (m, 2H), 5.31 (t, J ¼ 8.2 Hz, 1H), 5.17 (dd, J ¼ 17.2, 2.0 Hz, 1H), 5.10 (dd, J ¼ 10.2, 2.1 Hz, 1H), 4.95 (dt, J ¼ 17.0, 1.9 Hz, 1H), 4.92e4.88 (m, 1H), 4.72 (q, J ¼ 6.9 Hz, 1H), 4.44 (d, J ¼ 11.7 Hz, 1H), 4.17e3.97 (m, 3H), 2.91e2.81 (m, 1H), 2.53 (s, 3H), 2.43e2.34 (m, 1H), 1.98 (q, J ¼ 6.8 Hz, 2H), 1.82 (dd, J ¼ 8.9, 5.7 Hz, 1H), 1.76e1.66 (m, 1H), 1.64 (dd, J ¼ 9.8, 5.8 Hz, 1H), 1.38 (s, 7H), 1.37 (s, 2H), 1.15 (t, J ¼ 7.1 Hz, 2H). 13C NMR (176 MHz, DMSO) d 176.0, 174.3, 173.2, 170.4, 170.4, 169.7, 169.1, 168.9, 168.7, 168.6, 162.2, 162.2, 161.8, 161.5, 157.4, 157.1, 157.1, 157.0, 156.9, 156.9, 152.3, 152.3, 152.2, 152.0, 151.5, 143.0, 142.9, 142.6, 142.6, 142.2, 142.2, 142.1, 142.0, 142.0, 141.2, 141.1, 141.0, 141.0, 138.6, 138.6, 138.5, 137.2, 134.3, 134.2, 134.1, 133.8, 133.5, 133.5, 133.3, 131.7, 131.4, 131.4, 129.1, 128.9, 128.9, 128.8, 128.1, 127.8, 127.7, 127.6, 127.5, 127.4, 125.2, 125.0, 124.8, 124.8, 124.4, 124.4, 124.1, 124.0, 122.7, 122.6, 122.3, 122.3, 122.0, 121.9, 119.0, 119.0, 118.9, 118.8, 118.1, 117.9, 117.8, 117.7, 114.5, 114.5, 114.4, 84.2, 83.7, 83.3, 83.0, 80.1, 74.5, 74.3, 72.4, 72.3, 61.1, 61.1, 60.9, 60.8, 60.4, 59.9, 59.4, 53.0, 52.4, 52.2, 52.0, 50.3, 50.3, 49.4, 44.0, 43.9, 43.8, 43.7, 39.8, 39.7, 39.6, 39.5, 39.4, 39.2, 39.1, 37.1, 36.3, 36.2, 35.9, 34.8, 34.7, 34.1, 33.1, 32.9, 32.9, 32.9, 32.9, 32.8, 31.0, 30.8, 28.4, 28.2, 28.0, 28.0, 27.6, 27.6, 27.4, 27.3, 27.2, 25.7, 24.7, 24.6, 24.4, 24.2, 21.3, 21.3, 21.0, 14.0, 13.9. HRMS calcd C41H46N6O6 [MþH]þ: 719.3552, Found 719.3564. Melting point 64e67
C. The Step 1 diene 19 solution was combined with DMAP (6.9 kg, 56 mol, 0.2 equiv) and diluted with toluene to approximately 1075 L. The solution was cooled to 10
C and Boc-anhydride (70% solution in toluene, 141 kg solution, 452 mol, 1.68 equiv) was added then the reaction was warmed to 20
C. After 16 h (>99% conversion by HPLC) the reaction was diluted with water (97 kg) and mixed for 2 h to consume excess Boc-anhydride. The reaction was then diluted with toluene (492 kg) and washed with 10% KH2PO4 (808 kg) and water (775 kg) at 35
C for improved phase cuts. The organic layer was concentrated under reduced pressure to approximately 665 L then transferred with a toluene rinse (38 kg) to drums for storage to give step 2 protected diene 11 as a solution in toluene (664 kg solution, 33.6 w/w%, 223 kg protected diene 11, 98.1 area% purity, calculates to 101% yield thus 100% yield assumed for combined steps). Data on a concentrated sample: 1H NMR (rotomers present) (700 MHz, DMSO‑d6) d 9.07e9.04 (m, 2H), 8.76 (d, J ¼ 8.4 Hz, 1H), 8.74 (s, 3H), 8.68e8.61 (m, 8H), 8.58 (dd, J ¼ 8.3, 1.4 Hz, 3H), 8.53 (d, J ¼ 8.1 Hz, 3H), 8.45 (d, J ¼ 8.0 Hz, 1H), 8.36 (d, J ¼ 1.4 Hz, 3H), 8.23 (dd, J ¼ 8.1, 1.3 Hz, 1H), 8.19 (dd, J ¼ 8.2, 1.4 Hz, 3H), 7.94 (ddd, J ¼ 8.3, 7.0, 1.4 Hz, 1H), 7.89e7.78 (m, 7H), 7.75e7.65 (m, 5H), 7.62 (ddd, J ¼ 8.1, 6.9, 1.1 Hz, 3H), 7.59e7.54 (m, 1H), 7.57e7.52 (m, 3H), 7.18e7.11 (m, 1H), 5.95 (dp, J ¼ 4.5, 2.1 Hz, 3H), 5.89 (dp, J ¼ 4.9, 2.4 Hz, 1H), 5.75 (ddt, J ¼ 16.9, 10.2, 6.6 Hz, 3H),

5.71e5.63 (m, 5H), 5.26 (dd, J ¼ 17.2, 1.8 Hz, 3H), 5.16e5.09 (m, 4H), 5.03 (dd, J ¼ 10.3, 2.1 Hz, 1H), 4.96 (dq, J ¼ 17.2, 1.8 Hz, 3H), 4.92e4.83 (m, 5H), 4.79e4.71 (m, 4H), 4.62e4.55 (m, 4H), 4.31 (dt, J ¼ 12.0, 1.7 Hz, 3H), 4.17 (dd, J ¼ 11.8, 4.2 Hz, 3H), 4.14e4.05 (m, 5H), 4.07e4.01 (m, 4H), 3.95 (dd, J ¼ 13.0, 4.5 Hz, 1H), 2.89e2.82 (m, 1H), 2.69 (s, 1H), 2.62 (ddq, J ¼ 12.9, 6.7, 2.4 Hz, 3H), 2.59 (s, 3H), 2.58e2.54 (m, 1H), 2.53 (s, 9H), 2.38 (ddd, J ¼ 13.6, 8.6, 4.9 Hz, 3H), 2.30e2.23 (m, 2H), 2.18e2.09 (m, 3H), 2.00e1.94 (m, 6H), 1.87 (ddt, J ¼ 18.4, 14.6, 6.8 Hz, 6H), 1.80e1.75 (m, 1H), 1.75e1.69 (m, 2H), 1.71e1.66 (m, 1H), 1.69 (s, 2H), 1.66 (dd, J ¼ 7.9, 5.1 Hz, 3H), 1.60 (dd, J ¼ 8.0, 4.9 Hz, 1H), 1.45 (dd, J ¼ 9.4, 5.0 Hz, 1H), 1.36e1.29 (m, 8H), 1.30e1.27 (m, 1H), 1.29e1.26 (m, 1H), 1.27 (s, 2H), 1.19 (t, J ¼ 7.1 Hz, 11H), 1.18 (s, 1H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR (176 MHz, DMSO) d 174.2, 172.3, 172.1, 170.9, 170.4, 170.3, 162.7, 162.2, 157.9, 157.6, 157.5, 157.4, 143.5, 143.1, 142.7, 142.7, 142.5, 141.7, 141.5, 139.2, 139.1, 137.8, 134.9, 134.8, 134.8, 134.7, 132.2, 131.9, 129.7, 129.5, 129.4, 128.7, 128.3, 128.1, 127.9, 127.9, 125.8, 125.5, 125.3, 124.9, 124.6, 123.3, 123.2, 123.1, 122.8, 122.5, 122.4, 119.5, 119.5, 117.8, 117.8, 115.1, 115.1, 74.9, 73.2, 61.3, 61.2, 59.4, 58.7, 53.1, 53.1, 51.1, 50.7, 48.9, 40.4, 40.3, 40.1, 40.0, 39.9, 39.8, 39.7, 37.5, 35.3, 33.6, 33.5, 33.4, 33.1, 33.1, 31.3, 30.6, 29.5, 28.9, 28.8, 28.7, 28.5, 25.0, 24.9, 22.7, 22.7, 21.9, 21.8, 21.5, 17.7, 14.5. HRMS calcd C46H54N6O8 [MþH]þ: 819.4076, Found 819.4098. Melting point 48e51
C. Step 3 Macrocycle 20: To the main reactor, two ½ inch dip tubes were installed near the impeller, one for nitrogen sparge and one for diene solution addition (note e catalyst solution was added from a top port to minimize heating before entering the reaction). The catalyst solution was prepared by dissolving Zhan 1B catalyst (862 g, 1.2 mol, 0.44 mol%) with toluene (190 kg) followed by purging with nitrogen for 10 min to give a green solution. The Step 2 protected diene 11 (664 kg solution, 269 mol) was prepared by dilution with toluene (93 kg) to an approximate toluene content of 3 L/kg. The reaction vessel was prepared by charging toluene (1322 kg) and heating to 65
C with a continuous nitrogen sparge at a rate of 12 SCFM (maintained throughout reaction). While maintaining 65
C, the diene solution and 90% of the catalyst solution were added over 4 h with a toluene rinse (30 kg). The remaining catalyst was then charged over 1 h. After 2 h (conversion > 99% by HPLC), the nitrogen sparge was stopped, the reaction was cooled to 60
C and then quenched with a solution of 2-mercaptonicotinic acid (1.7 kg, 11 mol, 4 mol%) and DIPEA (1.5 kg, 11 mol, 4 mol%) in MeOH (2.7 kg) and toluene (1.5 kg). After 3 h, the reaction was concentrated under reduced pressure to approximately 2000 L (560 L distillate collected), cooled to 40
C and extracted with 1% NaHCO3 (667 kg) and water (665 kg) at 40
C to improve phase separation. The organic layer was transferred through a carbon filter (Pall AKS6 filter, 5.2 kg carbon, 2.3 w/w% relative to 11) with a toluene rinse (76 kg). The treated organic layer was concentrated under reduced pressure to approximately 672 L (1450 L distillate collected) then heated to 80
C to dissolve all solids and maintained at 80
C as heptanes (1376 kg) was slowly added over 3 h during which time solids were observed. The slurry was cooled to 20
C over 3 h then mixed for 15 h (filtrate level 6 mg/mL by HPLC). The slurry was centrifuged and washed with 3:1 v/v heptanes:toluene (600 kg total) in portions. The solids were utilized as the wetcake (230.8 kg wetcake, solution assay in Step 4 185 kg Step 3 macrocycle 20, 87% potency adjusted yield for 3 steps, 97.1 area% purity, 0.5 area% trans 42, 1.8 area% RCM dimers 43/44, 36 ppm ruthenium) for the subsequent step as the HCl stoichiometry was in excess and drying was not required. 1H NMR (700 MHz, Chloroform-d) d 9.03 (s, 1H), 8.49 (d, J ¼ 8.3 Hz, 1H), 8.42 (d, J ¼ 8.2 Hz, 1H), 8.36 (d, J ¼ 7.9 Hz, 1H), 8.32 (s, 1H), 8.29 (d, J ¼ 8.1 Hz, 1H), 7.87 (d, J ¼ 8.1 Hz, 1H), 7.77 (t, J ¼ 7.7 Hz, 1H), 7.63 (t, J ¼ 7.7 Hz, 1H), 7.56 (t, J ¼ 7.5 Hz, 1H), 7.49 (t, J ¼ 7.5 Hz, 1H), 6.15e6.10 (m, 1H), 5.64 (t, J ¼ 7.7 Hz, 1H), 5.57 (td,

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

J ¼ 10.3, 5.8 Hz, 1H), 5.47 (t, J ¼ 10.2 Hz, 1H), 4.93 (td, J ¼ 8.5, 2.6 Hz, 1H), 4.43 (d, J ¼ 11.7 Hz, 1H), 4.33 (dd, J ¼ 11.6, 4.8 Hz, 1H), 4.20 (dq, J ¼ 44.6, 10.8, 7.1 Hz, 1H), 4.13 (dq, J ¼ 10.8, 7.1 Hz, 1H), 3.03e2.96 (m, 1H), 2.60 (s, 3H), 2.59e2.52 (m, 2H), 2.26 (dq, J ¼ 13.9, 8.6 Hz, 1H), 2.10 (ddd, J ¼ 12.8, 8.6, 4.3 Hz, 2H), 1.87 (dd, J ¼ 9.1, 5.7 Hz, 1H), 1.83e1.74 (m, 1H), 1.69 (dd, J ¼ 10.0, 5.6 Hz, 1H), 1.61e1.47 (m, 4H), 1.45 (s, 9H), 1.43e1.37 (m, 1H), 1.34e1.27 (m, 1H), 1.25 (t, J ¼ 7.2 Hz, 3H). 13C NMR (176 MHz, CDCl3) d 177.6, 170.8, 170.1, 162.6, 157.7, 157.1, 152.4, 143.2, 143.0, 142.5, 141.7, 135.0, 133.8, 131.1, 128.9, 128.2, 127.3, 125.2, 124.8, 124.8, 122.7, 122.2, 121.9, 112.0, 83.7, 74.3, 61.4, 61.2, 54.0, 50.5, 45.0, 35.6, 31.3, 31.0, 28.0, 27.6, 27.2, 26.7, 26.2, 21.9, 21.5, 14.4. HRMS calcd C44H50N6O8 [MþH]þ: 791.3763, Found 791.3788. Melting point 180e182
C. Step 4 Macrocycle 21: An HCl solution was prepared by gassing anhydrous HCl (56.6 kg, 1550 mol, 6.6 equiv) into acetonitrile (520 kg) then maintained below 10
C (titrated as 9.6 w/w% HCl compared to expected 9.8 w/w%). A solution of Step 3 macrocycle 20 (230.8 kg wetcake, solution assay 185 kg 20, 234 mol) was dissolved in toluene (819 kg) and acetonitrile (149 kg) then maintained below 0
C as the HCl solution was transferred over 3 h using acetonitrile (74 kg) as a rinse. The reaction was warmed to 20
C, seeds (4.8 kg) were charged and warming was continued to 60
C at approximately 8
C per hour. After 3 h (>99% conversion by HPLC), the slurry was cooled to 20
C over 1.5 h, filtered and washed with 1:1 v/v toluene:acetonitrile (939 kg). The solids were dried in a hastelloy drier under reduced pressure at 60
C to give Step 4 macrocycle 21 as a white solid (178 kg subtracting for seed charge, 98.2 area% purity, 87.6 w/w% potency by assay, 8.1% chloride as HCl by IC, 0.8% water, < 10 ppm ruthenium, 246 ppm acetamide, 96% potency adjusted yield). 1H NMR (700 MHz, DMSO‑d6) d 8.83 (s, 1H), 8.75 (d, J ¼ 1.3 Hz, 1H), 8.70 (d, J ¼ 8.4 Hz, 1H), 8.60 (d, J ¼ 7.6 Hz, 1H), 8.52 (s, 1H), 8.42 (d, J ¼ 7.1 Hz, 1H), 8.26 (d, J ¼ 8.8 Hz, 1H), 7.87 (t, J ¼ 7.6 Hz, 1H), 7.83 (d, J ¼ 8.1 Hz, 1H), 7.65 (dt, J ¼ 16.1, 7.5 Hz, 2H), 7.53 (t, J ¼ 8.1 Hz, 1H), 7.24e7.21 (m, 1H), 6.02 (s, 1H), 5.50 (d, J ¼ 7.9 Hz, 1H), 5.33 (t, J ¼ 9.9 Hz, 1H), 4.70 (td, J ¼ 8.2, 2.8 Hz, 2H), 4.66 (t, J ¼ 7.6 Hz, 1H), 4.36 (d, J ¼ 11.6 Hz, 1H), 4.14 (dd, J ¼ 11.4, 4.5 Hz, 1H), 4.04 (dq, J ¼ 11.1, 7.1 Hz, 1H), 4.00 (dq, J ¼ 11.1, 7.1 Hz, 1H), 2.57e2.54 (m, 1H), 2.53 (s, 3H), 2.50e2.45 (m, 1H), 2.27e2.22 (m, 1H), 2.17 (q, J ¼ 9.0 Hz, 1H), 2.01e1.89 (m, 2H), 1.67 (t, J ¼ 11.0 Hz, 1H), 1.55 (dd, J ¼ 9.4, 4.9 Hz, 1H), 1.48 (dd, J ¼ 8.5, 4.9 Hz, 1H), 1.43e1.33 (m, 2H), 1.31e1.18 (m, 4H), 1.16 (t, J ¼ 7.1 Hz, 3H). 13C NMR (176 MHz, DMSO) d 172.9, 170.4, 170.2, 162.0, 157.4, 157.2, 143.0, 142.3, 142.1, 141.3, 134.4, 132.7, 131.7, 129.1, 127.8, 127.5, 126.3, 125.0, 124.7, 122.8, 122.5, 122.1, 119.3, 75.0, 60.5, 58.4, 52.8, 50.6, 34.1, 30.7, 30.4, 27.6, 26.9, 26.0, 23.6, 21.4, 21.4, 14.2. HRMS calcd C39H42N6O6 (freebase) [MþH]þ: 691.3239, Found 691.3270. Melting point 165e167
C. Step 5 acid 10: A solution of sodium hydroxide (36.5 kg, 98.4% potency, 898 mol, 4.4 equiv, net 2.6 equiv after calculation of HCl content in Step 4 macrocycle) in water (49 kg) and methanol (927 kg) was maintained at 55
C as Step 4 macrocycle 21 (161.2 kg, 87.0 w/w% potency, 8.0% chloride, 203 mol) was added in portions (note inverse addition avoids clumping of ester freebase during addition). After addition, the reaction was heated to 65
C and additional water (157 kg) was added. After 1 h (>99% conversion by HPLC), formic acid (33.4 kg, 96% potency, 697 mol, 3.4 equiv, 0.8 equiv excess over sodium hydroxide charge) was added followed by a slurry of seeds (1.3 kg) in MeOH (9 kg) and water (2 kg). The slurry was maintained at 65
C for 1 h, cooled to 20
C over 6 h then filtered in portions using MeOH:water (7:1 v/v, 275 kg total) followed by MeOH (135 kg total to remove water) as a rinse (2.7% loss to filtrate). The solids were dried under reduced pressure at 85
C to give Step 5 acid 10 as a white solid (130.3 kg after removing seeds, 99.3 area% purity, 98.1 w/w% potency, 95% potency adjusted yield). 1 H NMR (700 MHz, Chloroform-d) d 8.99 (d, J ¼ 1.2 Hz, 1H), 8.36 (d,

4285

J ¼ 7.5 Hz, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 8.32 (s, 1H), 8.29 (d, J ¼ 7.8 Hz, 1H), 8.16 (d, J ¼ 7.9 Hz, 1H), 7.80 (d, J ¼ 8.1 Hz, 1H), 7.65 (t, J ¼ 7.7 Hz, 1H), 7.57e7.52 (m, 2H), 7.46 (t, J ¼ 7.5 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 6.00 (s, 1H), 5.57 (q, J ¼ 10.0 Hz, 1H), 5.26 (t, J ¼ 9.6 Hz, 1H), 4.89 (dt, J ¼ 7.8, 4.0 Hz, 1H), 4.86 (dt, J ¼ 8.0, 6.3 Hz, 1H), 4.26 (d, J ¼ 11.7 Hz, 1H), 4.16 (dd, J ¼ 11.7, 5.0 Hz, 1H), 2.86 (dt, J ¼ 14.0, 5.8 Hz, 1H), 2.57 (s, 3H), 2.53 (ddd, J ¼ 12.6, 8.2, 3.0 Hz, 1H), 2.25 (q, J ¼ 8.9 Hz, 1H), 2.23e2.11 (m, 2H), 2.07e2.00 (m, 1H), 1.82 (dd, J ¼ 8.1, 5.6 Hz, 1H), 1.76 (t, J ¼ 12.5 Hz, 1H), 1.65 (dd, J ¼ 9.6, 5.5 Hz, 1H), 1.51e1.38 (m, 2H), 1.37e1.34 (m, 2H), 1.26e1.22 (m, 1H). 13C NMR (176 MHz, CDCl3) d 174.2, 172.8, 172.0, 162.7, 157.4, 157.3, 143.0, 142.8, 141.4, 134.8, 134.7, 131.0, 128.9, 128.1, 127.2, 125.4, 124.9, 124.8, 122.5, 122.1, 121.8, 119.6, 74.4, 58.8, 53.3, 50.8, 41.7, 33.6, 31.5, 29.5, 27.9, 26.6, 25.9, 23.0, 22.4, 21.8. HRMS calcd C37H38N6O6 [MþH]þ: 663.2926, Found 663.2943. Melting point 185e187
C. ABT-450 (8): The Step 5 acid 10 (127.6 kg, 98.1 w/w% potency, 189 mol) and cycloproylsulfonamide (45.5 kg, 375 mol, 2.0 equiv) were dissolved in NMP (347 kg) and IPAc (543 kg) and concentrated under reduced pressure to approximately 400 L followed by continued chase distillation with additional IPAc (518 kg) to a final approximate volume of 350 L (Kf < 0.1%). The chase distilled solution was cooled to 0
C, carbonyl diimidazole (CDI) (43.5 kg, 268 mol, 1.4 equiv) was charged maintaining the temperature below 25
C then the activated acid solution was maintained at 0
C with a nitrogen sparge (10 SCFM) applied. In a separate reactor, CDI (3.1 kg, 19.1 mol, 0.1 equiv) in IPAc (54 kg) was maintained below 30
C as 1,8-diazabicycle (5.4.0) undec-7-ene (DBU) (42.3 kg, 278 mol, 1.5 equiv) was charged to prepare the dried DBU solution. After 1 h (derivatization test for conversion by MeOH/DBU quench of activated acid sample was >99.9% by HPLC), the dried DBU solution was charged to the reaction maintaining the temperature below 40
C with an IPAc rinse (54 kg) then the reaction was maintained at 40
C. After 8 h (conversion 99.3% by HPLC), a supplemental charge of DBU (3.0 kg, 19.7 mol, 0.1 equiv) was added to continue the reaction completion maintained at 40
C. After an additional 1 h (conversion remained 99.3% by HPLC), the reaction was cooled to 20
C, diluted with IPAc (953 kg) then washed at 35
C with 2 M H3PO4 (817 kg). The organic layer was diluted with IPAc (434 kg) then washed at 35
C with 5% NaCl (2  770 kg). The organic layer was warmed to 60
C (to avoid precipitation of 8) and transferred through a carbon filter (Pall AKS6 filter, 5.2 kg carbon, 4.1 w/w% relative to 10) with an IPAc rinse (434 kg). The solution of 8 was concentrated under reduced pressure to approximately 430 L. In a crystallization reactor containing a solution of IPAc/IPA/ water (1/2/2 v/v/v, 427 kg) circulating through a high speed rotorstator based wet mill, a simultaneous addition of 8 solution and aqueous IPA (1:1 v/v) was performed at 2.66 wt basis addition rate ratio (1 kg solution of 8 per 2.66 kg aqueous IPA added per unit time). A small portion of 8 solution (13 kg) and aqueous IPA (34 kg) was added to saturate the crystallization reactor with ABT-450 (8). The crystallization was seeded (Form II 8, 2.7 kg). Then while circulating the seed slurry through the high speed rotor-stator based wet mill, a simultaneous addition of the remaining solution of 8 and aqueous IPA was completed. The slurry was filtered (estimated 5% loss to filtrate) and washed with IPAc/IPA/water (1/2/ 2 v/v/v, 379 kg). The solids were dried under reduced pressure at 68
C with humidified nitrogen to remove residual solvents while maintaining the desired crystal form (Form II) to give ABT-450 (8) as a white solid (139.6 kg, 99.8 area% purity, 99.6 w/w% potency (anhydrous, 5.6% water), 94.0% w/w potency (calculated), 91% potency adjusted yield). 1H NMR (700 MHz, DMSO‑d6) d 11.09 (s, 1H), 8.91 (s, 1H), 8.73 (d, J ¼ 8.5 Hz, 1H), 8.66 (d, J ¼ 1.2 Hz, 1H), 8.64 (d, J ¼ 7.5 Hz, 1H), 8.54 (d, J ¼ 1.0 Hz, 1H), 8.50 (d, J ¼ 6.7 Hz, 1H), 8.32 (dd, J ¼ 8.1, 1.4 Hz, 1H), 7.90 (ddd, J ¼ 8.3, 7.4, 1.2 Hz, 1H), 7.86 (dd,

4286

D.D. Caspi et al. / Tetrahedron 75 (2019) 4271e4286

J ¼ 8.1, 1.0 Hz, 1H), 7.68 (ddd, J ¼ 8.3, 7.2, 1.2 Hz, 1H), 7.64 (t, J ¼ 7.5 Hz, 1H), 7.55 (ddd, J ¼ 8.2, 7.4, 1.2 Hz, 1H), 6.04 (s, 1H), 5.62 (q, J ¼ 8.3 Hz, 1H), 5.12 (t, J ¼ 9.5 Hz, 1H), 4.61 (d, J ¼ 11.5 Hz, 1H), 4.59e4.53 (m, 2H), 4.09 (dd, J ¼ 11.4, 3.7 Hz, 1H), 2.93 (tt, J ¼ 7.9, 4.9 Hz, 1H), 2.73 (dd, J ¼ 13.9, 7.1 Hz, 1H), 2.59e2.57 (m, 1H), 2.55 (s, 3H), 2.52e2.47 (m, 2H), 2.33 (q, J ¼ 8.7 Hz, 1H), 1.98 (q, J ¼ 9.7 Hz, 1H), 1.84e1.76 (m, 1H), 1.59 (dd, J ¼ 8.2, 5.1 Hz, 1H), 1.58e1.57 (m, 1H), 1.56 (dd, J ¼ 9.4, 4.8 Hz, 1H), 1.49e1.33 (m, 4H), 1.27e1.19 (m, 2H), 1.15e1.08 (m, 1H), 1.08e1.03 (m, 1H), 1.03e0.98 (m, 1H). 13C NMR (176 MHz, DMSO) d 176.0, 171.0, 169.3, 162.4, 157.3, 157.1, 142.9, 142.4, 142.2, 141.3, 134.4, 134.2, 131.6, 129.1, 127.6, 127.5, 125.6, 124.9, 124.7, 122.8, 122.4, 122.1, 119.4, 75.0, 59.0, 53.1, 50.9, 42.7, 34.7, 31.3, 30.7, 30.1, 27.0, 27.0, 26.0, 22.7, 21.4, 21.4, 5.8, 5.7. HRMS calcd C40H43N7O7S [MþH]þ: 766.3018, Found 766.3043. Melting point 169e172
C. Disclosures This study was sponsored and funded by AbbVie Inc. AbbVie contributed to the design, participated in the collection, analysis, interpretation of data, writing, reviewing, and approving the final publication. All authors are employees of AbbVie Inc. and may own AbbVie stock. Acknowledgments The authors would like to thank Evelina Kim for helpful discussions, Carmina Presto for obtaining the NMR data, Collin Morris and Rodger Henry for support of the X-ray structure and Shiyue Zhou for conducting the HRMS analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tet.2019.05.064. References [1] (a) World Health Organization, Global Hepatitis Report 2017, WHO, Geneva, 2017; (b) M. Martinello, B. Hajarizadeh, J. Grebely, G.J. Dore, V. Gail, G.V. Matthews, Nat. Rev. Gastroenterol. Hepatol. 15 (2018) 412. https://doi.org/10.1038/ s41575-018-0026-5; (c) J.P. Messina, I. Humphreys, A. Flaxman, A. Brown, G.S. Cooke, O.G. Pybus, E. Barnes, Hepatology 61 (2015) 77. https://doi.org/10.1002/hep.27259; (d) B. Hajarizadeh, J. Grebely, G. Dore, J. Nat. Rev. Gastroenterol. Hepatol. 10 (2013) 553. https://doi.org/10.1038/nrgastro.2013.107. [2] (a) J.J. Feld, K.V. Kowdley, E. Coakley, S. Sigal, D.R. Nelson, D. Crawford, O. Weiland, H. Aguilar, J. Xiong, T. Pilot-Matias, B. DaSilva-Tillmann, L. Larsen, T. Podsadecki, B.N. Bernstein, Engl. J. Med. 370 (2014) 159. https://doi.org/10. 1056/NEJMoa1315722; (b) E.D. Deeks, Drugs 75 (2015) 1027. https://doi.org/10.1007/s40265-0150412-z.

[3] Y.N. Lamb, Drugs 77 (2017) 1797. https://doi.org/10.1007/s40265-017-0817y. [4] S. Zeuzem, G.R. Foster, S. Wang, A. Asatryan, E. Gane, J.J. Feld, T. Asselah, re, P.J. Ruane, H. Wedemeyer, S. Pol, R. Flisiak, F. Poordad, W.M. Bourlie L. Chuang, C.A. Stedman, S. Flamm, P. Kwo, G.J. Dore, G. Sepulveda-Arzola, S.K. Roberts, R. Soto-Malave, K. Kaita, M. Puoti, J. Vierling, E. Tam, H.E. Vargas, R. Bruck, F. Fuster, S.-W. Paik, F. Felizarta, J. Kort, B. Fu, R. Liu, T.I. Ng, T. PilotMatias, C.-W. Lin, R. Trinh, F.J.N. Mensa, Engl. J. Med. 378 (2018) 354. https:// doi.org/10.1056/NEJMoa1702417. [5] EASL Recommendations on Treatment of Hepatitis C, J. Hepatol. 69 (2018) 461, 2018, https://doi.org/10.1016/J.JHEP.2018.03.026. [6] M.M. Ravn, “Development of a Highly Efficient Process to a Macrocyclic HCV Protease Inhibitor” Poster at 239th ACS National Meeting, March 2010. San Francisco. [7] Y. Ku, K.F. McDaniel, H.-J. Chen, J.P. Shanley, D.J. Kempf, D.J. Grampovnik, Y. Sun, D. Liu, Y. Gai, Y.S. Or, S.H. Wagaw, K.M. Engstrom, T. Grieme, A. Sheikh, J. Mei, PCT Int. Appl. (2010). WO 2010/030359 A2. [8] (a) C. Shu, X. Zeng, M.-H. Hao, X. Wei, N.K. Yee, C.A. Busacca, Z. Han, V. Farina, C.H. Senanayke, Org. Lett. 10 (2008) 1303e1306, https://doi.org/10.1021/ ol800183x; (b) V. Farina, C. Shu, X. Zeng, X. Wei, Z. Han, N.K. Yee, C.H. Senanayake, Org. Process Res. Dev. 13 (2009) 250e254. https://doi.org/10.1021/op800225f. [9] P.J. Brackemeyer, M. Diwan, Y. Gong, A. Pal, A.Y. Sheikh, S. Wagaw, G.J. Zhang, PCT Int. Appl. (2015). WO 2015/084953 A1. [10] Basavaprabhu, T.M. Vishwanatha, N.R. Panguluri, V.V. Sureshbabu, Synthesis 45 (2013) 1569e1601. https://doi.org/10.1055/s-0033-1338989. [11] T3P was analyzed for “acetyl” content (acetic acid and acetic anhydride) by GC to control acetylation of dipeptide 13 as an impurity [12] J.S. Kingsbury, J.P.A. Harrity, P.J. Bonitatebus, A.H. Hoveyda, J. Am. Chem. Soc. 121 (1999) 791e799. https://doi.org/10.1021/ja983222u. [13] S.T. Nguyen, L.K. Johnson, R.H. Grubbs, J.W. Ziller, J. Am. Chem. Soc. 114 (1992) 3974e3975. https://doi.org/10.1021/ja00036a053. [14] X. Zeng, X. Wei, V. Farina, E. Napolitano, Y. Xu, L. Zhang, N. Haddad, N.K. Yee, N. Grinberg, S. Shen, C.H. Senanayake, J. Org. Chem. 71 (2006) 8864. https:// doi.org/10.1021/jo061587o. [15] S.B. Garber, J.S. Kingsbury, B.L. Gray, A.H. Hoveyda, J. Am. Chem. Soc. 122 (2000) 8168e8179. https://doi.org/10.1021/ja001179g. [16] Zhan, Z.J. US Pat. (2007) US 2007/0043180 A1. [17] M. Yu, S. Lou, F.G. Bobes, Org. Process Res. Dev. 22 (2018) 918e946. https:// doi.org/10.1021/acs.oprd.8b00093. [18] T. Nicola, M. Brenner, K. Donsbach, P. Kreye, Org. Process Res. Dev. 9 (2005) 513e515. https://doi.org/10.1021/op0580015. [19] Brenner, M.; Meineck, S.; Wirth, T. US Pat. (2007) US 7183374 B2. [20] N.K. Yee, V. Farina, I.N. Houpis, N. Haddad, R.P. Frutos, R. Gallow, X. Wang, X. Wei, R.D. Simpson, X. Feng, V. Fuchs, Y. Xu, J. Tan, L. Zhang, J. Xu, L.L. SmithKeenan, J. Vitous, M.D. Ridges, E.M. Spinelli, M. Johnson, K. Donsbach, T. Nicola, M. Brenner, E. Winter, P. Kreye, W. Samstang, J. Org. Chem. 71 (2006) 7133e7145. https://doi.org/10.1021/jo060285j. [21] K.M. Engstrom, Org. Process Res. Dev. 22 (2018) 1294e1297. https://doi.org/ 10.1021/acs.oprd.8b00121. [22] K.M. Engstrom, A. Sheikh, R. Ho, R.M. Miller, Org. Process Res. Dev. 18 (2014) 488e494. https://doi.org/10.1021/op400281h. [23] D.J. Heldebrant, P.G. Jessop, C.A. Thomas, C.A. Eckert, C.L. Liotta, J. Org. Chem. 70 (2005) 5335e5338. https://doi.org/10.1021/jo0503759. [24] N.K. Nere, M. Diwan, A.M. Czyzewski, J.C. Marek, H. Sinha, Case studies on crystallization scale-up”, in: Chemical Engineering in the Pharmaceutical Industry: Active Pharmaceutical Ingredients, second ed., John Wiley and Sons, 2019, pp. 617e634. David J. am Ende and Mary Tanya am Ende. [25] A.Y. Sheikh, M. Diwan, A. Mattei, R. Ho, T.B. Borchardt, G. Danzer, N. Ding, X. Xu, Applications of thermodynamics towards pharmaceutical problem solving, in: Chemical Engineering in the Pharmaceutical Industry: Active Pharmaceutical Ingredients, second ed., John Wiley and Sons, 2019, pp. 419e438. David J. am Ende and Mary Tanya am Ende.