Development of a Manufacturing Process for an HCV Protease

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Development of a Manufacturing Process for an HCV Protease Inhibitor Candidate Molecule Ben Littler, Michael Aizenberg, Narendra B Ambhaikar, Todd A Blythe, Timothy T Curran, Vadims Dvornikovs, Young C Jung, Valdas Jurkauskas, Elaine C Lee, Adam Robert Looker, Hoa Luong, Theodore A Martinot, David B Miller, Bobbianna J Neubert, Pieter A Otten, Peter J Rose, and Piero L Ruggiero Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op500210w • Publication Date (Web): 14 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014

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Organic Process Research & Development

Development of a Manufacturing Process for an HCV Protease Inhibitor Candidate Molecule Benjamin J. Littler,* Michael Aizenberg, Narendra B. Ambhaikar, Todd A. Blythe, Timothy T. Curran, Vadims Dvornikovs, Young C. Jung, Valdas Jurkauskas, Elaine C. Lee, Adam R. Looker, Hoa Luong, Theodore A. Martinot, David B. Miller, Bobbiana J. Neubert-Langille, Pieter A. Otten, Peter J. Rose and Piero L. Ruggiero Chemical Development, Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston Massachusetts 02210 and 11010 Torreyana Road, San Diego, California 92121 Author for correspondence: E-mail: [email protected]

Abstract

The scale-up of a prototype HCV protease inhibitor (1) from gram-scale in the laboratory to kilogram-scale in the pilot plant is described. Key features of the optimization included synthesis of bulk quantities of exomethylene proline intermediate 6, separation the diastereomers of spirocycle 2 without chromatography, isolation of the precursor to 1 to purge by-products which might raise genotoxic structural alerts, and purification of an amorphous drug substance via a crystalline acetic acid solvate.

ACS Paragon Plus Environment

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Introduction It is estimated that about 150 million people worldwide are chronically infected with the hepatitis C virus (HCV), with 3-4 million new infections every year. More than 350,000 people are thought to die every year from hepatitis C-related diseases. Following initial HCV infection approximately 80% of people exhibit no symptoms, but eventually approximately 75-85% of patients progress to a chronic infected state. An estimated 60-70% of these chronically infected people will eventually develop chronic liver diseases, such as cirrhosis or liver cancer. In 2011 two HCV protease inhibitors, telaprevir and boceprevir, were approved for treatment of hepatitis C in combination with interferon and ribavirin, affording treatment protocols that are more readily tolerated and have higher cure rates.1 Compound 1 is a prototype HCV protease inhibitor that required rapid scale-up to enable completion of toxicology and early phase clinical studies. As outlined in Figure 1 the pseudopeptide structure of compound 1 can be disconnected to spirocycle 2 and commercially available cyclohexaneacetic acid (3), N-Boc-tert-leucine (4) and chiral amine 5. Spirocycle 2 can be derived from exomethylene proline 6 and chloro-oxime 7 via a 3+2 cycloaddition.


2

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Figure 1. Retrosynthesis of Compound 1

Our colleagues in Medicinal Chemistry had prepared up to a few hundred grams of compound 1 using the chemistry shown in Scheme 1.2 Three major issues were identified as needing to be resolved before kilogram quantities of material could be produced. First, an economical synthesis of exomethylene proline 6 was required. Second, we needed replace the chromatography that was used to separate the 4:1 mixture of diastereomers produced in the 3+2 cycloaddition used to form spirocycle 2. Finally, we needed to avoid the chromatography that was used to purify compound 1.


3


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Scheme 1. Discovery Synthesis of Compound 1

In this paper we first describe the processes developed to make the first few kilograms of material in the kilo lab which enabled the project to move rapidly to the clinic. We then describe the fit-for-purpose modifications that were made to the synthesis in readiness for the pilot plant manufacture of batches of over 50 kg of compound 1. The whole synthesis development program was completed under tight timelines required to support the wider compound development program schedule.3


4

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Synthesis of Exomethylene Proline 6 As outlined in Scheme 2 the synthesis of exomethylene proline 6 has been reported on a 10 g scale without chromatography by oxidation of commercially available N-Boc-4-hydroxyproline (8) with sodium periodate and catalytic ruthenium dioxide to afford intermediate ketone 9. A Wittig reaction with the ylide derived from methyl triphenyl phosphonium bromide followed by extraction of the desired product into a basic aqueous phase and washing with Et2O to remove neutral organic by-products, such as triphenyl phosphine oxide. Acidification followed by extraction and concentration was reported to afford 6 as a foam.4

Scheme 2. Literature Preparation of Exomethylene Proline 6

Oxidation process development We wanted to avoid using ruthenium, or other metals, to oxidize 8 to 9, so a TEMPO/bleach oxidation was examined. No reaction occurred using the conventional TEMPO/bleach reaction system containing catalytic KBr and buffered with NaHCO3,5 but ketone 9 formed when KBr and NaHCO3 were omitted from the procedure. We postulate that the free carboxylate group in 8 acts as the buffer which allows bleach to survive for use in the TEMPO oxidative cycle. Catalyst stability studies were performed to determine the addition time and temperature range for the reaction. In these experiments a total of 1.0 eq of bleach was added in seven smaller portions over 24 h at fixed temperatures between 0 and 20 °C in an automated reactor while monitoring the reaction and jacket temperatures. When performed below 10 °C the reaction went


5


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to completion and the heat flow (reaction temperature minus the jacket temperature) was relatively constant for each portion added over a 24 h period, indicating that the catalyst was not being significantly deactivated over the course of the experiment. When the reaction was performed at 20 °C there was incomplete conversion to ketone 9 and the height of the heat flow spikes decreased in intensity over the course of the experiment, suggesting that the catalyst was decomposing. The data also showed that there was no heat flow between additions so the process temperature can be readily controlled by varying the rate of the bleach addition to maintain the reaction temperature between 0-5 °C. An expedient isolation procedure for isolating ketone 9 was achieved by adding the completed reaction mixture into a solution of aqueous NaHSO4 to crystallize the product. It was observed that product solid must be filtered and washed with water as quickly as possible to obtain the highest product yield and quality. Prolonged exposure of ketone 9 to acid leads to decomposition. Many batches of this oxidation were completed with isolated yields typically between 75-90% with the lower yields observed on a larger scale in the pilot plant due to the extended filtration times. In the largest single batch performed 35.0 kg N-Boc-4-hydroxyproline 8 was converted to 26.7 kg ketone 9 with a 77% yield.

Installation of the exocyclic methylene group We initially considered forming the carboxylic acid ester before the Wittig reaction to reduce the amount of base required, but decided to retain the carboxylic acid group so that we could use an aqueous work-up to remove the triphenyl phosphine oxide by-product without chromatography. The decision to isolate the carboxylic acid was further supported by the fact that our preliminary development studies found that crystalline 6 could be obtained from a slurry


6

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in i-PrOAc, whereas all attempts to crystallize the tert-butyl (10) or methyl (11) carboxylic acid ester derivatives of olefin 6 were unsuccessful. The

reported

procedures

for

preparing

olefin

6

from

ketone

9

used

both

methyltriphenylphosphonium bromide and base in at least 2-fold excess,4,6 so modification was needed to improve throughput before being suitable for kilogram scale production. The major change that we made was to reduce the amount of methyl triphenylphosphonium bromide to 1.0 equivalent. We reasoned that only an excess of base was required because the first equivalent of base would be needed to remove the carboxylic acid proton so that the second equivalent of base could generate the ylide. Gratifyingly, complete conversion of ketone 9 to olefin 6 was achieved in a trial experiment when 2.0 eq KOtBu in 2-MeTHF were added to 1.0 eq of methyl triphenylphosphonium bromide followed by treating the resulting solution with 1.0 eq of ketone 9. For the initial kilo lab runs ketone 9 was added to the reaction mixture as a solid under a blanket of nitrogen to maximize the process throughput due to the relatively low solubility of 9 in 2-MeTHF (73 g/L). For the pilot plant ketone 9 was added as a concentrated solution in NMP (500 g/L). At the end of the reaction the mixture was quenched with water and the carboxylate anion partitioned in the basic aqueous phase. A screen of solvents showed that CH2Cl2 was the most effective solvent at selectively removing both triphenylphosphine oxide and excess methyl triphenylphosphonium salts from the aqueous phase containing the desired product. CH2Cl2 also minimized the processing time because it is heavier than water which allowed multiple washes to be rapidly performed and drained from a single vessel. Olefin 6 was then isolated by acidification of the aqueous layer with citric acid followed by extraction with EtOAc. In the initial kilo lab procedure the EtOAc was removed using a rotary evaporator to afford an


7


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amorphous solid that was crystallized from only 1.5 volumes of toluene to minimize yield losses. This isolation process was used to make many batches of 6 as a pale brown crystalline solid in the kilo lab in about 70-80% yield. In the pilot plant it would not have been practical to concentrate the mixture to dryness then triturate with only 1.5 volumes of solvent, so we screened a series of inexpensive, low molecular weight amines to find a crystalline salt that could be readily isolated. Morpholine and tert-butyl amine were identified as possible crystallization partners. Morpholine was rejected because the presence of ≥1% water dissolved in EtOAc dramatically increased the solubility of 6·morpholine, whereas 6·t-BuNH2 crystallized directly from work-up solution in excellent yield and purity without requiring additional drying to remove water from the EtOAc layer after the aqueous phase was removed. The largest single batch performed in the pilot plant converted 26.0 kg ketone 9 into 30.9 kg exomethylene proline 6·t-BuNH2 with a 91% yield. The t-Bu amine salt was broken before the next step by forming a slurry in MTBE, washing with citric acid followed by azeotropic drying.

Scheme 3. Pilot Plant Synthesis of Exomethylene Proline 6

Synthesis of Chloro-oxime 7 The discovery procedure for alkylating phenol 12 used excess dimethylsulfate with K2CO3 in acetone. Methyl tosylate was selected as the alkylating agent for early development because its consumption was readily measured using a simple HPLC method. Small-scale studies showed that any unreacted phenol 12 was easily removed by basic aqueous washes, so methyl tosylate


8

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was used as the limiting reagent (0.95 equivalents). The reaction was best performed in MeCN at 60-70 °C for 16 h since the reaction rate was faster than in acetone at reflux (56 °C). The completed reaction in MeCN was quenched with water (10 L/kg of 12) and a phase split occurred so the excess phenol was simply removed in the aqueous layer. In the kilo lab methyl ether 13 was obtained as an oil after a solvent exchange from MeCN to MTBE using a rotary evaporator followed an aqueous work-up and removal of the solvent. Conversion of 13 to oxime 14 was then accomplished by adding NH2OH·HCl and NaOAc in a mixture of THF, EtOH and water. After stirring at ambient temperature for 16 h the volatile organic solvents were removed using a rotary evaporator then oxime 14 was extracted using toluene. Oxime 14 was obtained as an oil containing 5-10 wt% residual toluene. The oil partially solidified on standing, but all attempts to develop a true crystallization of 14 were unsuccessful. Fourteen kilo lab batches converted 55.9 kg of phenol 12 into 69.3 kg of oxime 14. Chloro-oxime 7 was initially prepared by a procedure that added a solution of Nchlorosuccinimide (NCS) in MeCN to a heated mixture of oxime 14 in MeCN maintaining the temperature between 45-50 °C. At the end of the reaction, the mixture was concentrated to remove MeCN and then worked up in i-PrOAc, followed by adding n-heptane to afford chlorooxime 7 as a solid. This process was scaled up to prepare 1 kg chloro-oxime 7 but differential scanning calorimetry (DSC) showed that the solid was thermally unstable above 120 °C. As a result we decided that it was safer not to isolate the solid but to telescope a solution of chlorooxime 7 in DMF directly into the 3+2 cycloaddition. In the kilo lab it was possible to add solid NCS into the reactor in small portions under a blanket of N2 while maintaining the solution of oxime 14 in 4 volumes of DMF at 33-38 °C. This allowed over 20 batches of chloro-oxime 7 in DMF to be produced up to a 4 kg scale in the kilo lab.


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Scheme 4. Kilo Lab Synthesis of Chloro-oxime 7

For the pilot plant campaign the process to make oxime 14 was further simplified to remove the need to concentrate the mixture to dryness between steps. Intermediate 13 was not isolated in the plant: once the alkylation was complete aqueous NaOH and NH2OH·HCl were added to the reaction mixture and the heating continued. Once oxime 14 had completely formed the mixture was cooled to ambient temperature and extracted with three portions of MTBE to afford a solution of oxime 14 that was telescoped directly into the cycloaddition. Using this procedure a single batch of 80 kg of phenol 12 was converted to 94 kg of oxime 14 in 98% yield and 93% AUC by HPLC. The mass of oxime 14 was estimated by quantitative HPLC analysis of the MTBE solution. Initial attempts to use the kilo lab procedure to make chloro-oxime 7 in the pilot plant demonstrated that maintaining the reaction mixture temperature between 33-38 °C during the addition of NCS proved much more difficult in the pilot plant due to the slower response that varying the jacket temperature had on the reaction mixture temperature, and the increased difficulty adding the solid NCS at a rate to ensure a consistent exotherm. We did not want the


10

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temperature to exceed 40 °C due to the thermal instability of chloro-oxime 7, but we found that the reaction rate would decrease dramatically if the reaction temperature fell below 33 °C. If a significant quantity of NCS was added with the reaction mixture below 33 °C this resulted in a strong and rapid exotherm once the mixture was heated again above 35 °C. In order to better control the exotherm we added a catalytic amount of concentrated hydrochloric acid since this has been reported to activate NCS.7 An initial small-scale reaction went to >95% conversion within 5 min of adding NCS to a mixture of oxime 14 and 0.1 equivalents of conc HCl in MTBE and DMF at 20 °C. Adding the solution of oxime 14 in MTBE to a mixture of NCS and conc HCl in DMF was equally effective and allowed the exotherm to be readily controlled. This approach looked like it might be suitable for the pilot plant but was not pursued further because an accelerating-rate calorimetry experiment on the product solution of 14 in DMF showed an exothermic event starting at 82 °C.8 Ultimately for the pilot plant we chose to implement an inherently safer process where the chloro-oxime was generated using bleach in the presence of olefin 11 so that the chlorooxime would react immediately to form cycloadduct 15.9 Heat flow measurements showed a readily controlled exotherm during the addition of bleach to an organic solution of oxime 14 and exomethylene proline 11, and no significant exotherm during the rest of the reaction. Studies indicated that 1.5 eq of NaOCl could be used to complete the reaction, but given the variability in concentration in aqueous bleach solutions, typically 2.0 eq of NaOCl were used to be certain that enough base was present to convert the chloro-oxime to the reactive dipolar nitrile oxide.


11


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Scheme 5. Pilot Plant Synthesis of Chloro-oxime 7 OH Me

OMe Me

MeOTs K2CO3

Me

Me

H2NOHHCl NaOH

MeCN H2O 93% from 12

MeCN H

O

H

12

O

13 OMe

OMe Me

Me

NaOCl

Me

Me

MTBE 20 °C H

14

N OH

Cl 7

N OH

Development of the Cycloaddition to Synthesize Spirocycle 2 A key modification that the development team made at the outset of the project was to change the protecting group on the carboxylic acid in the cycloaddition from a tert-butyl ester to a methyl ester. The medicinal chemistry team had used the tert-butyl ester protecting group in an attempt to improve the stereoselectivity of the cycloaddition by maximizing the steric bulk of the chiral element on the proline ring. Unfortunately, extensive studies by our colleagues had shown that that the stereoselectivity of the cycloaddition with the tert-butyl ester protecting group was approximately 80:20 across a wide range of aryl substituents and reaction conditions which suggested that the steric influence of the ester moiety on the cycloaddition selectivity was minimal. The extra bulk of the tert-butyl ester did improve the chromatographic separation of the diasteroemeric product mixture but this added an extra step later to the sequence in order to convert the tert-butyl ester to a more readily removed methyl ester for the end-game. We initially prepared exomethylene proline methyl ester 11 under relatively neutral conditions using a combination of EDCI [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride], DMAP and MeOH in CH2Cl2.10 A screen of solvents for the cycloaddition was performed and we were


12

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happy to find that the stereoselectivity remained at approximately 80:20 regardless of the solvent or the ester protecting group, so DMF was initially selected as the reaction solvent for the cycloaddition because it gave fewest (13% AUC by HPLC) by-products. We used the methyl ester protecting group throughout the rest of the program since this was readily removed later in the synthesis using aqueous base. We also stayed with the combination of EDCI, DMAP and MeOH to prepare the methyl ester throughout the program because the water-soluble byproducts allowed for a simple and fast work-up. In the kilo lab the esterification was performed in CH2Cl2 and methyl ester 11 was concentrated to an oil on the rotary evaporator, but for the pilot plant campaign we formed 11 in MTBE and telescoped the product solution directly into the 3+2 cycloaddition after a water wash and azeotropic drying.

Scheme 6. Initial Approach to Isolate Spirocycle 2 as a Single Diastereomer

As shown in Scheme 6 our initial approach to separate the spirocyclic diastereomers was to first remove the Boc group from 15, isolate the free base mixture of diastereomers and then form a salt that we hoped would preferentially crystallize the desired diastereomer. The Boc group was removed using methanesulfonic acid in THF,11 and after an aqueous work-up the mixture of free base spirocyclic diastereomers was treated with 12 different acids. This screen showed that maleic acid in EtOAc or i-PrOAc was able to upgrade the diastereomeric ratio from 80:20 in


13


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solution to >98:2 in the solid. This procedure was successfully demonstrated on a 200 g scale but further scale-up to 2 kg surprisingly afforded only a 94:6 mixture. Subsequent studies showed that the >98:2 selectivity was only obtained when the solid was rapidly filtered after addition of the maleic acid. Prolonged stirring led to a thermodynamic mixture that could not be raised above 95:5 without an unacceptable yield loss. Additional equilibrium solubility studies indicated that the maleic acid salt of 2 has higher thermodynamic solubility than its diastereomeric counterpart in all organic solvents screened, so a new salt with a favorable thermodynamic solubility profile was required. During the scale-up to 200 g it became clear that the aqueous work-up of the deprotection procedure using methanesulfonic acid was leading to significant losses of the desired product. Treatment of the initial Boc-protected cycloadduct product 15 with anhydrous 4 M HCl in 1,4dioxane was therefore examined as an alternative method for removing the Boc group in order to avoid an aqueous work-up. We expected to isolate a solid with an 80:20 mixture of diastereomers but remarkably we found that salt 2·HCl crystallized directly from the reaction mixture with a diasteromeric ratio of >99:1! HPLC analysis showed that almost all of the reaction by-products, including the undesired diastereomer, remained in the mother liquor and that almost all of the desired diastereomer 2 had crystallized. At this point further studies were abandoned on identifying other salts to separate the diastereomers. Approximately 30 batches of the cycloaddition, deprotection and crystallization sequence were performed in the kilo lab to convert olefin 11 and oxime 14 to supply over 100 kg of spirocycle 2·HCl. In all but two cases 2·HCl crystallized directly from the reaction mixture with diastereomer ratio of >98:2. No clear cause could be identified as to why two batches crystallized with a lower diastereomer ratio of 89:11, however a recrystallization process using MeOH and


14

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MTBE upgraded the diastereomer ratio of these two batches to >99:1 in high yield. This recrystallization process was ultimately incorporated into every kilo lab batch of 2·HCl because the initial colorless to off-white solid sometimes led to poor phase cuts and low recoveries in subsequent steps despite being >98% AUC purity. The recrystallized solid was typically colorless and >99.5% AUC purity by HPLC and gave clean phase splits in all subsequent steps. The isolated yield of 2·HCl was typically 50-55% from olefin 11 and oxime 14, which was excellent considering that the cycloaddition yield of both diasteroemers was estimated by quantitative HPLC to be 75%. The maximum possible yield of 2·HCl from 11 and 14 was approximately 60% given that there was an 80:20 mixture of diastereomers. This demonstrates just how effective the crystallization was in affording 2·HCl in excellent yield and purity from a complex mixture. For the pilot plant campaign the in situ chloro-oxime generation followed by cycloaddition reaction could be readily run in MTBE (18 h reaction time, 78:22 diasteromer ratio) or CH2Cl2 (8 h reaction time, 75:25 diastereomer ratio) at ambient temperature. MTBE was selected as the preferred solvent because the sequence could be telescoped through all steps from exomethylene proline 6·t-BuNH2 and oxime 14 through to cycloadduct 2·HCl without needing to swap solvents. Another key advantage of this procedure over the kilo lab process was that the reaction mixture after the cycloaddition was much less colored and no polymeric materials were generated, eliminating the need to recrystallize 2·HCl from MeOH and MTBE. The largest batch performed converted 33.2 kg of methyl ester olefin 11 and 25.9 kg of oxime 14 to 22.5 kg of 2·HCl in 46% yield from 11 with 99% AUC purity. The slightly lower molar yield for the pilot plant process was outweighed by the improved throughput, simplicity of operation, reduced waste and the fact that it was inherently safer.


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Scheme 7. Pilot Plant Synthesis of Spirocycle 2·HCl OMe

t-BuNH2

Me

Me

O N Boc OH 6•t-BuNH2

H

14

MeOH EDCI DMAP MTBE 92%

N OH

NaOCl MTBE OMe Me

O N Boc OMe 11

Me

+ H

7

N Cl

MTBE, 20 °C MeO

MeO

Me

Me

Me

Me

4 N HCl in dioxane

N O

46% from 11

N

O

Boc

15

OMe

78:22 mixture of diasteromers

N O ClH2N

O OMe

2•HCl >99:1 diasteromer ratio

Optimization of the Conversion of Spirocycle 2 to Compound 1 The discovery methods for converting spirocycle 2 to compound 1 proceeded in excellent yield using standard peptide coupling chemistry, so the initial goals of the development effort were to improve the process throughput and to remove the chromatography that had been used to improve the purity of compound 1. Of particular concern was the fact that no neat crystalline form of the final product had been obtained despite extensive screening, so we expected that robust processes and intermediate purifications would be needed to ensure a consistent purity profile of the drug substance 1.12


16

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Coupling of Spirocycle 2 with N-Boc-t-leucine (4) Followed by Boc-Deprotection to Afford 17·HCl Scheme 8. Pilot Plant Synthesis of Amine 17·HCl from Spirocycle 2·HCl

The coupling of spirocycle 2·HCl with N-Boc-t-leucine (4) was readily accomplished in the kilo lab using EDCI and HOBt with N-methyl morpholine (NMM) as the base and EtOAc as the solvent.13 Spirocycle 2 was used directly as its hydrochloride salt by adding an extra equivalent of NMM base (3.0 equivalents total). Robustness testing showed that the reaction could be run at 20, 30, 40 or 50 °C with no observed major side reactions, so the reaction was typically run in the kilo lab by adding NMM as the last component at 30 °C followed by stirring the resulting suspension for 12 h at 35 °C. After a standard aqueous work-up with acid and base the solvent was removed on the rotovap in the kilo lab. This afforded intermediate 16 as an oil which was directly used into the Boc-deprotection step.


17


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We initially planned to remove the Boc group from 16 using standard conditions of methanesulfonic acid in THF,11 followed by neutralization with base and an extractive workup to afford 17. The deprotection was complete within 30 min at 40 °C with no major side products detected by HPLC. The neutralization was studied with a range of bases, but as summarized in Table 1 significant quantities of a diketopiperazine impurity 18 formed during the work-up under all conditions studied.

Figure 2. Impurities generated in the conversion of Spirocycle 2 to Amine 17·HCl


18

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Table 1. Product ratio of the desired product 17 to diketopiperazine impurity 18 formed under different aqueous base work-up procedures Base

Ratio of 17 : 18 (HPLC area %)

K2CO3 (25%)

75:25

NaHCO3 (sat)

82:18

NaOH (1 M)

43:57

1N NH3 in MeOH

0:100

The original medicinal chemistry Boc-removal procedure directly precipitated the hydrochloride salt of 17 from the reaction mixture using 6 N HCl in dioxane, and diketopiperazine 18 had not been observed as a major process impurity, so we reverted back to this direct-drop process to avoid the aqueous work-up. The coupling and deprotection reactions were telescoped in the kilo lab using a rotary evaporator to swap the solvent containing intermediate 16 from EtOAc to 1,4-dioxane. Commercially available 4 N HCl in dioxane was then added and the mixture stirred at 25 °C for 12 h during which time solid started to precipitate. Heptane was added at the end of the reaction to complete the precipitation 17·HCl. This process was used to prepare a number of batches in the kilo lab and typically the product was obtained as a colorless powder in approximately 90% yield on a kilogram scale with a purity of >98% AUC. However, the following issues were identified during the kilo lab runs that meant that a new process was needed for the pilot plant: 1. One kilo lab batch of the coupling starting with 2.5 kg of spirocycle 2·HCl became so thick that it could not be mixed completely and eventually broke the pin holding the PTFE paddle onto the glass stirring rod. This led to the formation of many new by-products, with


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impurity 19 containing an additional tert-leucine residue being the most abundant. The new by-products were mostly purged after the Boc-deprotection in the crystallization of 17·HCl, but the yield of the isolated product fell to 64% and the purity was decreased from >98% to 96% AUC. 2. Diketopiperazine 18 was found to slowly form on prolonged standing of isolated 17·HCl. This would be a significant challenge when we transferred the process from the kilo lab to the plant where the longer operating cycles meant that we might have to store samples of 17·HCl for over a month. 3. If the EtOAc was not fully removed in the solvent exchange between the coupling and deprotection steps then it inhibited the precipitation of 17·HCl and afforded a sticky product containing acetamide 20 produced from the acidic hydrolysis of EtOAc. Acetamide 20 and its downstream products were found to be poorly rejected in the subsequent synthesis and purification operations, leading to contamination of the final product 1, so the formation of acetamide 20 had to be prevented.

2-Propanephosphonic acid anhydride (T3P®),14 oxalyl chloride and isobutyl chloroformate, were briefly assessed as alternative coupling reagents for the pilot plant conditions, but none was as effective as EDCI/HOBt in dipolar aprotic solvents. Notably, the coupling reaction in NMP, DMF or DMA gave greater than 97% conversion in 18-24 h at ambient temperature. DMA was ruled out as a solvent because the purity of the product in the reaction mixture was only 92% AUC compared to 97-98% AUC for DMF and NMP at 20-25 °C. In order to improve the process throughput the reactions were heated to 35 °C in DMF and NMP. Both reactions were completed


20

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within 6-8 h. However, significant degradation of the product was observed in DMF at 35 °C, so NMP was selected for the EDCI/HOBt-mediated coupling reaction. A study of the effect of reaction temperature on the product purity profile showed that it was necessary to charge NMP first, cool it to 0-5 °C, and then charge the reagents. If the reagents were charged at 20-25 °C, 1-2% AUC of impurity 19 formed via double addition of the tertleucine unit. Only 0.2-0.4% impurity 19 formed when reagents were added to NMP at 0-5 °C. It was demonstrated that the optimal amount of NMM for the coupling reaction was 3.0 equivalents with respect to 2·HCl. Slower reaction was observed when 2.5 equivalents of NMM were employed, and there was no benefit from charging 4.0 equivalents of NMM. In order to avoid the potential formation of acetamide 20 during the Boc-deprotection step the solvent in the work-up after the coupling needed to be changed from EtOAc to a more chemically inert solvent. Also a solvent with a relatively low boiling point was required in order to facilitate the solvent swap to 1,4-dioxane, so CH2Cl2 and MTBE were tested to solubilize intermediate 16 while washing the reaction with water to remove NMP. It was determined that more NMP partitioned into the aqueous phase from MTBE, so this was selected as the organic phase for the aqueous work-up of the coupling reaction for the pilot plant campaign. Prior to the pilot plant campaign we re-examined our choice of 4 N HCl in 1,4-dioxane for the Boc-deprotection since 1,4-dioxane is classified by the ICH as a Class 2 solvent with an Option 1 limit of 380 ppm. We sought to use commercially available solutions of HCl in ICH Class 3 solvents (Option 1 limit 7% of the new solvent remained in the solid. Based on these results it was deemed that removal of residual 1,4-dioxane by slurrying solid 17·HCl in other organic solvents was ineffective and that the best course of action was to plan the pilot plant processing schedule such that 17·HCl was stored under nitrogen at 5 °C and used in the next step within a week of being isolated.


22

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Four batches were scaled up in the pilot plant. The scales ranged from 2 kg to 45 kg input of 2·HCl. Stage 1 product 16 was not isolated in the plant; instead the solvent was exchanged from MTBE to 1,4-dioxane by distillation and the solution of 16 in 1,4-dioxane was used directly in Stage 2 to afford 17·HCl. Each batch of 17·HCl contained 8-11% of residual 1,4-dioxane. The uncorrected crude yields of 17·HCl were 98 to 104% over two steps from 2·HCl. The purity of the product was ≥ 98.0% AUC in each batch, as determined by HPLC.

Coupling of 17·HCl with Cyclohexaneacetic Acid (3) Followed by Hydrolysis to Afford Acid 23

Scheme 9. Pilot Plant Synthesis of Acid 23 from Amine 17·HCl CO2H

Me

MeO Me

Me N O N

HCl H2N

Me

MeO

O

O

N O

3 EDCI HOBt NMM CH2Cl2

N HN

O

OMe

O OMe

O

17•HCl Me

MeO

22

Me N O

LiOH 2-MeTHF H2O then i-PrOAc MTBE 68% from 17•HCl

N HN

O

O HO

O

23

Our main goal for developing the coupling of 17·HCl with cyclohexaneacetic acid (3) for the initial kilo lab campaign was to replace DMF with a lower boiling solvent that would provide a


23


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phase cut in an aqueous work-up. A solvent screen was performed on a gram-scale where cyclohexaneacetic acid was activated with NMM, HOBT and EDCI then 17·HCl was added. These studies showed that about 30-40% of product mixture was diketopiperazine 18 when the reaction was performed in i-PrOAc or EtOAc. Reaction in DMF showed approximately 5% diketopiperazine 18. No diketopiperazine was detected in CH2Cl2, so this was selected as the reaction solvent. Further studies showed that NMM could be added as the final reagent in CH2Cl2 without forming diketopiperazine 18, which afforded a procedure that is simpler to perform on large-scale. Using these conditions three initial batches of intermediate 22 were isolated 95-100 % yield and 93-95% AUC purity on up to a 100 g scale before a telescoped procedure to acid 23 was developed. For the kilo lab campaign the discovery hydrolysis procedure using 1N LiOH in THF was modified by switching the solvent to 2-MeTHF in order to obtain a phase cut in the aqueous work-up. The reaction went to completion overnight at 25 °C and gave a clean phase separation when the stirring was stopped. HPLC analysis showed that the carboxylate of the desired product 23 remained in the organic phase with only minor amounts in the aqueous layer. To maximize recovery, EtOAc was added to the product mixture and the aqueous phase was acidified to pH 3 with hydrochloric acid. The organic phase was isolated and concentrated to dryness on the rotovap to afford crude product 23 as an oily semi-solid. A brief screen of solvents showed that trituration of the oily semi-solid in a minimal volume of i-PrOAc (3 L/kg) at reflux followed by cooling to 5 °C afforded a crystalline solid and upgraded the material purity to greater than 98.5% AUC by HPLC without losing too much product to the mother liquors. The process was successfully transferred to the kilo lab with the solvent swap from CH2Cl2 to 2-MeTHF between the coupling and hydrolysis readily accomplished using a rotary evaporator.


24

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The two step yield from 17·HCl to 23 was typically 80-85% with 23 isolated in 98-99% AUC purity by HPLC. Prior to the pilot plant campaign alternative coupling reagents were examined to facilitate this chemical transformation, but only inferior results were obtained, especially given that the EDCI/HOBt-mediated coupling reaction in CH2Cl2 provided full conversion in about 2 h. Specifically, isobutyl chloroformate did not give full conversion and T3P gave low conversion. Development of the crystallization of 23 was a major focus for development prior to the pilot plant campaign because this offered an excellent point in the synthesis sequence to purge impurities and ensure that the final product 1 was obtained with consistent purity. The two goals for this optimization program were first to avoid the concentration to dryness when exchanging out of 2-MeTHF after the hydrolysis was completed, and second to increase the volume of the recrystallization solvent from 3 L/kg to at least 6 L/kg in order to ensure good mixing and filtration while minimizing the yield losses to the mother liquor. A solubility screen identified i-PrOAc (bp 89 °C) and t-BuOAc (bp 98 °C) for further evaluation as potential crystallization solvents with higher boiling points than CH2Cl2 (39 °C) and 2-Me-THF (79 °C). MTBE and n-heptane were selected as the anti-solvents, for further evaluation. The crystallization of acid 23 study results are summarized in Table 2.


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Table 2. Solvent system selection for the crystallization of acid 23

Entry

1

Solvent / Volume i-PrOAc / 3 vol

Initial Purity of 23,

Purity of Recrystallized 23,

% AUC

% AUC

(from 17·HCl)

60°C

94.8

98.8

48

40°C

94.8

98.0

65

50°C

94.8

99.3

62

60°C

94.8

99.1

70

Tmax

Yield, %

i-PrOAc / 3 vol 2 MTBE / 10 vol i-PrOAc / 3 vol 3

MTBE / 8.5 vol n-Heptane / 1.5 vol t-BuOAc / 5 vol

4 MTBE / 11 vol 5

t-BuOAc / 3 vol

65°C

94.8

99.0

73

6

t-BuOAc / 5 vol

65°C

93.9

98.5

75

Reflux

93.9

99.2

67

Reflux

93.9

98.8

73

Reflux

93.9

98.7

72

Reflux

93.9

98.5

75

i-PrOAc / 5 vol 7 MTBE / 11 vol t-BuOAc / 5 vol 8 MTBE / 5 vol t-BuOAc / 4 vol 9

i-PrOAc / 1 vol MTBE / 11 vol t-BuOAc / 5 vol

10 MTBE / 5 vol


26

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The best impurity rejection was obtained using i-PrOAc (5 vol) and MTBE (11 vol) at reflux (Table 1, Entry 7). From this solvent system, the purity of acid 23 was increased from 93.9% to 99.2% AUC. Crystallization employing t-BuOAc gave consistently higher isolated yields of 70% and above (Table 1, Entries 4-6 and 8-10), however, the overall purity of acid 23 was about 0.5% AUC lower when t-BuOAc was used as a crystallization solvent, so the i-PrOAc and MTBE solvent system was preferred.

Final Form Determination Three polymorphic forms were identified for compound 23 during the course of crystallization process development. The three forms exhibited different morphology and slightly different solubility but these differences were minor enough that we were able to proceed without controlling for a specific form or form composition, especially since none of the three forms was solvated. In most instances, 23 was isolated as a mixture of Forms 1 and 2. Based on slurry stability and further solid characterizations, it was found that Form 3 is the most thermodynamically stable form at room temperature.

Form 2 was found to be the most

thermodynamically stable form at elevated temperature.

Form 1 converts to Form 2 at

temperatures above 58 °C. The solvent switch during the crystallization of 22 was run at elevated temperature which explains the co-existence of Forms 1 and 2. Four batches converting 17·HCl to 23 were scaled up in the pilot plant. The scale ranged from 3 kg to 65 kg input of 17·HCl. The yields of 23 from 17·HCl ranged from 66% to 68% demonstrating the robustness of the process. Each batch of 23 contained less than 0.1% of any residual solvent after the product was dried in a filter at ambient temperature then applying


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simultaneous vacuum and nitrogen purge. The purity of the product ranged from 99.6 to 99.9% AUC by HPLC.

Coupling of Acid 23 with Amine 5 to Afford Hydroxyamide 24

Scheme 10. Pilot Plant Synthesis of Hydroxyamide 24 from Acid 23

In readiness for the kilo lab campaign a brief study to optimize the EDCI/HOBt-mediated coupling reaction showed that a 20-24 h reaction time could be obtained at 20-25 °C by treating 1.00 equiv of 23 with 1.15 equiv of HOBt, 1.15 equiv EDCI, and 1.1 equiv of 5·HCl. Raising the reaction temperature to 30-35 °C decreased the reaction time to 16 h but lowered the purity of 24 from 96.4 to 95.6% AUC by HPLC. A major by-product produced in about 1.0% AUC was identified by LCMS as pseudo-dimer 25 resulting from O-acylation of 24. Two other side products present at 0.1-0.3% were identified by LCMS as ureas 26 and 27 resulting from addition of EDCI and a subsequent rearrangement. These ureas were of particular concern since oxidation with bleach might generate small quantities of N-chloro ureas that would likely be genotoxic alert structures.


28

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Figure 3. Impurities Monitored in the Synthesis Steps to Prepare Hydroxyamide 24 and Compound 1

For the kilo lab campaign the solution of hydroxyamide 24 in CH2Cl2 after the aqueous workup was telescoped directly into the final oxidation step, but during the development work for the pilot plant campaign it became clear that we could readily isolate 24 as a solid, which reduced the risk of impurities carrying through to the drug substance and allowed the GMP steps to start at acid 23 for early phase development.


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Before the pilot plant campaign started, alternative coupling agents were screened under standard reaction conditions in CH2Cl2 with NMM or Et3N as base. Complete conversion could not be obtained with iso-butyl chloroformate, pivaloyl chloride or T3P; 5-7% of unreacted acid 23 remained when these coupling reagents were employed. Only 80% conversion was obtained when carbonyldiimidazole was used to promote the reaction. The best coupling reaction results were obtained employing EDCI/HOBt or 2-chloro-4,6dimethoxy-1,3,5-triazine (CDMT) reagents. Notably, CDMT provided desired product 24 with a crude purity of 99.0% AUC, which represented a 1.3% increase in crude purity over material obtained from the EDCI/HOBt conditions: 0.16% AUC of O-acylated impurity 25 formed when CDMT was used, compared to 1.0% AUC with EDCI/HOBt. Furthermore, the rate of the CDMT-mediated coupling reaction was higher than that with EDCI/HOBt; after 1 hour approximately 1% 23 remained with CDMT compared to 6% with EDCI/HOBt. Isolated yields of 24 following precipitation were comparable for both EDCI/HOBt and CDMT reactions: 92% and 93% respectively. The EDCI/HOBt process was used in the pilot plant because insufficient time was available to ensure that the CDMT process would perform acceptably on a kilogram scale and generate no new impurities that had not been qualified in toxicological studies. The EDCI/HOBt procedure afforded isolated solid hydroxyamide 24 in 99.7% AUC purity with less than 0.04% of ureas 26 and 27. Even when the crude coupling product was spiked with 0.5% AUC of a mixture of 26 and 27, the isolation procedure provided 24 without detectable levels (

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