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Chapter 12

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Telaprevir: From Drug Discovery to the Manufacture of Drug Substance Gerald J. Tanoury,*,1 Stephen Eastham,2 Cristian L. Harrison,1 Benjamin J. Littler,1 Piero L. Ruggiero,1 Zhifeng Ye,2 and Anne-Laure Grillo 1Process

Chemistry, 50 Northern Avenue, Boston, Massachusetts 02210, United States 2Technical Operations, and 50 Northern Avenue, Boston, Massachusetts 02210, United States 3Chemistry Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210 *E-mail: [email protected].

This chapter describes the discovery of telaprevir as a HCV NS3•4A protease inhibitor and development of a process for commercial manufacture of the drug substance. The first section of the chapter covers the drug discovery efforts, covering the SAR that identified the essential structural requirements for the protease inhibitor and the final optimization that led to the discovery of telaprevir. The remainder of the chapter describes, in detail, the efforts required to develop a commercial manufacturing process for telaprevir, a tetrapeptide possessing a pyrazine cap on the P4 residue and a cyclopropyl amide on the P1 residue. The development efforts were divided into several stages: process development for the commercial manufacture of the P1 and P2 residues (aminoalcohol 20 and bicycloproline ester 12, respectively), amine deprotections (debenzylation) and amide couplings, and TEMPO-mediated oxidation of hydroxyamide 19 to give telaprevir. Although discussion of the development of 20 and 12 is brief, detailed descriptions of the development activities for the remaining stages of the commercial process are provided. The final commercial process © 2016 American Chemical Society

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

provided >60 metric tons of drug substance over 150 batches, with a total impurity level of ≤ 0.03%, providing telaprevir in ≥99.97% purity.


Introduction Infection with hepatitis C virus (HCV) represents a major medical burden worldwide, with over 185 million people affected by the disease (1). Following the acute infection phase, 80% of individuals progress to a chronic infection stage, associated with significant morbidity and mortality. Long-term HCV infection has been shown to lead to liver disease, such as fibrosis, cirrhosis, and hepatocellular carcinoma (2, 3). Extrahepatic manifestations of HCV infection are also common and burdensome (4). The hepatitis C virus was identified as the causative agent of non-A and non-B hepatitis in 1989 (5, 6). The first approved therapy consisted of a weekly administration of interferon-alpha (IFN-α) for 48 weeks, and achieved a sustained viral response (SVR) in 12-15% of patients (7). In 1991, ribavirin (RBV) was introduced (8). Its administration in combination with pegylated interferon-alpha (PegIFN-α) resulted in SVR rates of about 50% across HCV genotypes (9). This combination of PegIFN-α and RBV remained the standard of care therapy until 2011, when the first direct-acting antiviral therapies (DAAs), boceprevir and telaprevir, were approved to be used in combination with pegIFN-α and RBV (10–13). Since then, more than 30 DAAs and host-targeting agents (HTAs) have been investigated in the clinic, and all-oral therapy combinations are now available that can achieve SVR rates greater than 90% (14, 15). The introduction of DAAs achieved a paradigm shift in HCV treatment, and was made possible by key discoveries that furthered our knowledge of HCV. These included molecular virology advances that uncovered the HCV replication cycle, and identified druggable targets for drug discovery (14). Additionally, the introduction of the HCV subgenomic replicon assay in 1999, an in vitro viral replication system that allowed the evaluation of the cellular activity of investigational compounds, further bolstered drug discovery efforts (16). The hepatitis C virus is a single-stranded flaviviridae virus. Following infection, the virus circulates in the blood as a complex with lipoproteins, and enters the hepatocyte through interaction with a number of cell-surface receptors and subsequent endocytosis (17). The positive-stranded viral RNA is released into the cytosol and translated by cellular ribosomes into a single 3,000-amino acid polyprotein chain that contains all the structural and non-structural viral proteins required for replication. Cleavage of this polyprotein chain by cellular and viral proteases generates structural proteins Core, E1 and E2, as well non-structural proteins p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The non-structural proteins assemble into the multi-protein replicase complex that is responsible for the production of progeny of HCV RNA. Final steps in the HCV lifecycle are the packaging and maturation of HCV RNA into infectious virus that is eventually released into the bloodstream (18, 19). 282 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The non-structural proteins NS3, NS4A, NS5A and NS5B are all essential to HCV RNA replication, and have been the focus of intense efforts aimed at discovering small molecule inhibitors of the HCV lifecycle (20). A number of small molecule DAAs targeting these proteins have been approved or are in development for HCV treatment (15). Telaprevir, approved by the FDA in 2011 (12, 13), targets the non-structural protein NS3•4A. NS3 is a bi-functional protein comprising a catalytic serine protease domain in its N-terminal portion and a helicase domain in its C-terminal portion. NS3 associates tightly and non-covalently with NS4A to form the NS3•4A complex. This complex is responsible for the cleavage of the viral polyprotein between NS3 and NS4A, NS4A and NS4B, NS4B and NS5A, and NS5A and NS5B (21–23). NS3•4A also plays a role in helping HCV evade the immune system through its cleavage of host innate proteins MAVS and TRIF, eventually suppressing interferon synthesis (24). Thus NS3•4A is an attractive target for therapeutic intervention. This chapter describes the discovery of the NS3•4A inhibitor telaprevir, as well as its chemical development and manufacture.

The Discovery of Telaprevir Publication of the crystal structure of the HCV NS3•4A protease in 1996 sparked intense research efforts in pharmaceutical companies toward the identification of inhibitors of this enzyme for therapeutic intervention (25, 26). As of today, more than a dozen of HCV NS3•4A protease inhibitors have entered the clinic, with a few being approved for the treatment of HCV. These are part of multi-drug regimens designed to suppress the emergence of resistance (14, 15). However, early on in the discovery process, the identification of a therapeutically useful inhibitor of this enzyme presented several challenges: •

• • •

while enzyme assays were quickly developed to drive medicinal chemistry efforts, there was initially no cellular assay available to establish compound activity in a cellular environment. there was no existing animal model to evaluate inhibitor efficacy in vivo and establish PK/PD relationships. crystal structures of the enzyme revealed a flat hydrophobic active site presenting few pockets where binding affinity could be gained. early high throughput screens to identify inhibitor starting points yielded no hits.

Hence the Vertex approach to enable the discovery of an HCV protease inhibitor relied on: •

developing and implementing enzymatic and cellular assays to drive compound optimization. Early on, the subgenomic replicon assay developed by Bartenschlager et al. was implemented (16). This assay 283



•

was later supplemented by an infectious HCV replication assay in primary human hepatocytes and by an HCV protease mouse model that allowed the measurement of protease activity in the liver, thus enabling PK/PD evaluation (27). using a natural substrate of the enzyme as a starting point and utilizing structure-based drug design iteratively as an integral part of the optimization cycle.

An additional element of the strategy included the profiling of compounds in rodents early in the assay cascade to assess their exposure at the intended site of action—the liver. This approach was justified by the observation that upon liver transplantation, HCV infected patients underwent a dramatic drop in HCV RNA levels, suggesting that the liver was the primary site of HCV replication. Vertex Pharmaceuticals entered the HCV drug discovery field in 1997 in collaboration with Eli Lilly. Joint research efforts over the next three years resulted in the discovery of telaprevir, which has been published (28–30). A summary is presented in this chapter. Due to the lack of hits from high throughput screening efforts, decapeptide 1 (Scheme 1, Ki = 0.89 uM), derived from the natural NS5A-NS5B substrate of the NS3•4A protease, served at the starting point for inhibitor optimization. This decapeptide spanned the S6 to S4′ sites of the enzyme binding site. Early work focused on identifying the optimal inhibitor size that would provide enough binding affinity, with the goal of achieving a molecular weight range in-line with a drug-like profile. Truncation was also supported by the observation that the NS3•4A protease was inhibited by its own cleavage products (31, 32). Truncation studies showed that removal of the P4′ amino acid resulted in significant loss of activity, but that additional truncations at P2′ and P3′ had little additional negative effect on enzyme affinity. Also, removal of the acidic residues at P5 and P6 resulted in significant loss of activity. Furthermore, removal of the P3 and P4 hydrophobic residues was also deleterious to binding to the enzyme, suggesting that productive hydrophobic interactions between enzyme and inhibitor at these sites could be found (33). Taken together these data supported pursuing inhibitors spanning up to S4 on the non-prime side. To compensate for the resulting loss in affinity, a covalent reversible inhibitor containing an electrophilic warhead at the C-terminus was pursued—an approach supported by a number of publications in the literature (34). Reversible and covalent inhibitors of serine proteases have been shown to be 10- to 1,000-fold more potent than non-covalent inhibitors, and aldehydes are the simplest functionality that can serve as electrophilic moieties. Hence, aldehyde warhead incorporation and optimization of the P2 substituent to a proline-based residue, yielded inhibitor 2 (Scheme 1) (35). Replacement of the P5 and P6 residues with a heteroaryl cap yielded tetrapeptide aldehyde 3 (Ki = 12 uM) as a starting point for further optimization (35). X-ray analyses of aldehyde-containing HCV NS3•4A inhibitors show that the C-terminal electrophilic warhead undergoes nucleophilic attack by Ser139, resulting in the formation of a covalent, reversible bond between the protease and the inhibitor. The resulting tetrahedral complex is stabilized through additional 284



ionic interactions with two additional residues, His57 and Asp81, which together with Ser139 form the catalytic triad (29).

Scheme 1. From Decapeptide Substrate 1 to Tetrapeptide Aldehyde 3 Both sequential and parallel explorations were conducted to optimize tetrapeptide aldehyde 3. The aldehyde warhead served as an excellent tool for the parallel synthesis of a number of inhibitors with variations at P1 and P2 (36). P1 Residue The S1 specificity pocket of the HCV NS3•4A protease is defined by the side chains of Leu135, Phe154 and Ala157, and is responsible for selectivity versus the clotting cascade of serine proteases such as thrombin. The consensus sequence for substrates resulting in trans cleavage all have a cysteine at P1, a nucleophilic residue incompatible with an electrophilic warhead. Examination of SAR at this position showed a preference for small hydrophobic side chains such as ethyl, propyl and trifluoroethyl. Polar atoms such as oxygen and disubstitution alpha to the aldehyde were not tolerated. P2 Residue The S2 pocket had been recognized as important for inhibitor recognition and potency, and incorporation of bulky P2 substituents had been shown to result in increased affinity. These findings repeated for this series of tetrapeptide aldehydes. 285


For example, replacement of the benzyl ether in 3 with a tetrahydroisoquinolyl carbamate (4) led to a gain of about 13-fold due to optimized contacts of the inhibitor in the S2 pocket.


From Aldehydes to α-Ketoamides While the aldehyde warhead was a great tool to explore SAR at various subsites of the molecule, the moiety was easily oxidized in vivo and hence needed to be replaced. Investigation of a number of warheads such as carboxylic acids, trifluoromethyl ketones, chloromethyl ketones, etc., all commonly used in serine protease inhibitors, met with failure. A key discovery was the finding that the aldehyde could be replaced with an α-ketoamide, which resulted in up to 40-fold improvements in binding affinity (37). X-ray structure analysis of α-ketoamides such as 5 (Scheme 2) showed an unexpected arrangement of the tetrahedral intermediate, where the oxyanion hole defined by Ser139 and Gly137 was occupied by the non-electrophilic carbonyl of the warhead, and the negatively charged oxygen was pointing out toward solvent (37). An additional ten-fold improvement in potency could be gained via the introduction of a carboxylic acid moiety on the prime side (compound 6, Scheme 2). However, the charged nature of this compound and related analogs prevented entry into the cell resulting in loss of cell potency in the replicon assay.

Scheme 2. Comparison of Aldehydes and Ketoamide Warheads 286 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


P2: From 4-Hydroxyprolines to 3-Alkylprolines α-Ketoamides based on 4-hydroxyproline at P2, such as 5 and 7 (Schemes 2 and 3), showed good potency against the enzyme. However, due to their high molecular weights, they were generally associated with poor metabolic stability and poor exposure in the liver—the target organ. A key advance toward telaprevir was the replacement of the 4-hydroxyproline residue at P2 with a 3-alkylproline residue. This finding originated with the structure-based design hypothesis that a proline-based P2 bearing a 1- to 4-carbon substituent at the 3-position on the α face could result in displacement of a putative water molecule in the crystal structure of compound 7 bound to HCV NS3•4A, potentially resulting in improved binding affinity (38). Compound 8 was prepared and showed a Ki of 1.4 µM. Subsequent crystal structure examination disproved the water molecule displacement hypothesis, yet the reasonable activity of 7 led to further exploration of P2 proline substituents at the 3-position (38). Further exploration of 3-alkylproline-based inhibitors was also supported by their improved PK profile and liver exposure. Final Optimization to Telaprevir Key compounds that led to the synthesis of telaprevir are shown in Scheme 3. Extension of the 3-methylproline substituent in 8 (Ki = 1.4 µM) to ethyl, and matrix exploration of substituents at P1′, P3 and P4, led to compounds with improved potencies, 9 (Ki = 0.22 µM) and 10 (Ki = 0.15 µM). The observed potency improvement in 9 led to the synthesis of bicyclic ketone 11 (Ki = 0.040 µM). Finally, reduction of the ketone moiety to the bicyclic carbocycle, coupled with the incorporation of optimal residues at P1′, P3 and P4 eventually led to telaprevir (Scheme 3) (39). Telaprevir was selected for advancement based on its potency and its good liver exposure in preclinical animal species (27–29).

Development of a Chemical Process for the Manufacture of Telaprevir Telaprevir is a polypeptide composed of four amino acid residues and a pyrazine cap. Scheme 4 shows a retrosynthetic disconnection. Retrosynthetic analysis by amide bond cleavages reveals residues B – E and the pyrazine carboxylic acid A as appropriate building blocks for the construction of telaprevir. Pyrazine carboxylic acid A, N-protected cyclohexylglycine B and N-protected t-butyl glycine C are available commercially, but D and E require development of a commercial process. Scheme 5 depicts the processing steps to manufacture telaprevir from the residues shown in Scheme 4. Apart from the manufacture of D and E, the process can be divided into four sections: • •

amide coupling and Cbz-deprotection to provide H-t-Leu dipeptide 14. a second amide coupling and Cbz-deprotection to provide H-Chg tripeptide 16. 287


•


•

capping the N-terminus of H-Chg tripeptide 16 with pyrazine carboxylic acid with subsequent t-butyl ester cleavage to give P-cap acid 18. amide coupling of P-cap acid 18 with 20 followed by oxidation of the hydroxyamide 19 to provide telaprevir.

Scheme 3. Final Optimization Toward Telaprevir

This section of the chapter will discuss the activities required to develop the manufacturing route in Scheme 5, as well as a brief discussion of the processes to manufacture bicycloproline ester 12 and 20 (40–43).

Manufacture of Bicycloproline Ester 12 The initial manufacture of the key starting material 12 began with the reaction sequence shown in Scheme 6. Conversion of t-butyl glycine to the chiral imine with the unnatural enantiomer of camphor gave imine ester 21. A stereoselective Michael addition of imine ester 21 to cyclopentenyl ester 22 proceeded in low yield but gave Michael adduct 23 in high diastereomeric and chemical purities. Deprotection with hydroxylamine gave lactam 24, which was protected and then reduced with borane to give Z-protected bicycloproline ester 25 in high 288 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


enantiomeric purity. Removal of the Cbz group and crystallization as the oxalate salt provided bicycloproline ester 12 in 10 steps with high enantiomeric and chemical purities, and manageable yield. While this process was fit-for-purpose to manufacture up to 100 kg, we had tremendous interest in developing an alternative route to lower the cost of manufacture and to reduce material sourcing risks, especially for the unnatural enantiomer of camphor.

Scheme 4. Retrosynthetic Analysis of Telaprevir

Scheme 5. Processing Route for the Manufacture of Telaprevir 289 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

The new process (Scheme 7) utilized a stereoselective lithiation/carboxylation sequence (44). In a fashion similar to Beak’s stereoselective α-lithiation of Boc-protected amines (45–47), our in-house-developed achiral ligand dipropylbispidine (DPBP) (see Scheme 7) induced excellent diastereoselectivity to provide the exo isomer in 95:5 diastereoselectivity. Direct, same-pot resolution with (S)-THNA (see Scheme 7) and recrystallization provided 27 (S)-THNA salt in >99.5:0.5 e.r. and d.r. Further processing of 27 (S)-THNA salt required the following:


1. 2. 3.

conversion to the t-butyl ester with Boc2O, DMAP, and t-BuOH. chemoselective removal of the Boc group with MsOH/THF. crystallization of 12 as the oxalate salt.

The process was accomplished in an overall yield of 27% from Boc-protected 3-azabicyclo[3.3.0]octane 26 (based on total molar charge of 26), and was used for commercial manufacture of metric tons of bicycloproline ester 12.

Scheme 6. Initial Process for Bicycloproline Ester 12

Manufacture of Aminoalcohol 20 The development of a process for manufacture of aminoalcohol 20 used an epoxidation and subsequent nitrogen nucleophilic ring-opening protocol to establish the correct relative stereochemistry (Schemes 8 and 9). Racemic epoxidation of trans-2-hexenoic acid was performed with Oxone™ to give 290 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


racemic trans-epoxide 28, which was converted to the corresponding racemic epoxy amide 29 (Scheme 8). Enantioselective epoxidation of hexenoic amide 30 was accomplished the Shibasaki protocol (48) to give trans-epoxyamide 31 in high yield and high enantiomeric purity. Ring-opening of epoxy amide 29 with benzylamine followed by deprotection gave racemic aminoalcohol rac-20 (Scheme 9), which was subsequently resolved to give aminoalcohol 20 in high enantiomeric purity. Using the identical ring-opening/deprotection strategy for trans-epoxyamide 31 gave aminoalcohol 20 in equally high enantiomeric purity, without the loss to a resolution process.

Scheme 7. Commercial Route to Bicycloproline Ester 12

Scheme 8. Synthetic Routes to trans-Epoxy Amides 29 and 31 291 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 9. Synthetic Routes to Aminoalcohol 20

Development of a Process for the Manufacture of Dipeptide H-t-Leu Dipeptide 14 from Bicycloproline 12 The first stage for the manufacture of the polypeptide chain of telaprevir comprises the coupling of Cbz-protected tert-leucine (Z-t-Leu-OH) with 12 to afford Z-t-Leu dipeptide 13, and subsequent hydrogenolysis of the Cbz group to give H-t-Leu dipeptide 14. Cbz-protected tert-leucine was obtained commercially as the dicyclohexylamine (DCHA) salt, and 12 was obtained as the oxalate salt by custom commercial manufacturing, vide supra. In this section the development of the commercial manufacturing process shown in Scheme 10 is described. The first operations for development were the salt breaks to afford solutions of 12 and Z-t-Leu-OH in the same solvent. Next, amide coupling to manufacture Z-t-Leu dipeptide 13 was improved with a particular focus on identifying the best way to remove the excess Z-t-Leu-OBt ester from the reaction mixture. Finally the conditions for the Cbz removal and isolation of dipeptide H-t-Leu dipeptide 14 were screened which ultimately led to a decision to telescope a solution of H-t-Leu dipeptide 14 into the next stage rather than isolate H-t-Leu dipeptide 14 as a solid. 292



Scheme 10. Commercial Manufacturing Process for H-t-Leu Dipeptide 14

Optimization of 12 Oxalate Salt Break A DoE study was setup to optimize the amount of K2CO3 and water used in the 12 oxalate salt break. Choosing the factors shown in Table 1 with their corresponding ranges, and setting a limit of 1% loss of 12 to the aqueous layer gave an acceptable operating range. In this study, conditions at 3 equivalents K2CO3 and 3 volumes of water minimized the loss of 12, but precipitated a high level of salts that complicated the phase separation. However, increasing the amount of water to four volumes kept all salts in solution with minimal loss of 12 to the aqueous phase. A water wash was used to ensure removal of dissolved base in the isopropyl acetate (i-PrOAc) organic phase. 293 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Table 1. Factor Ranges in DoE Study of 12 Oxalate Salt Break K2CO3 (equiv)

Water (vol)

Time

% 12 in aqueous layer

1

5

15 min

11.9

3

3

3h

0.3

1

3

20 min

5.1

3

5

1h

2.2

In the course of investigating the stability of the i-PrOAc solution of 12, the N-acetyl impurity 32 formed when stored at room temperature for several days. Figure 1 shows the rate of growth of 32 in an i-PrOAc solution of 12 at various temperatures. Based on these results, recommendations were made to store the i-PrOAc solution of 12 at room temperature for less than two days, or at lower temperature for a longer time, if necessary.

Figure 1. Stability of 12 in i-PrOAc.

Optimization of the Coupling Work-up Process In the previous procedures, histamine•2HCl as well as a 25% K2CO3 solution were utilized to remove the excess Z-t-Leu-OBt ester from the reaction mixture after the formation of Z-t-Leu dipeptide 13. Histamine is efficient, but considering its toxicity (49), a replacement was needed for commercial manufacturing. Potassium carbonate is inexpensive and nontoxic. However, we noticed that increased levels of impurities in the manufacturing of Z-t-Leu dipeptide 13 were related to the incomplete quenching of the activated ester by the 25% K2CO3 wash. This was also complicated by poor detection of the activated ester by HPLC analysis, which was solved by derivatization with benzylamine. 294 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


A series of nitrogen-based nucleophiles was screened to quench the excess activated ester in the reaction solution. The combination of an amine base, Et3N or N-methylmorpholine (NMM), and amino acids, L-glycine, L-lysine, or L-cysteine, were examined. Based on efficiency of the reaction (complete conversion of HOBt-activated ester Z-t-Leu-OBt) and the time required for completion and commercial availability, L-lysine (1 equiv) with NMM (2 equiv) was chosen as the quench reagent combination. The mono-Lys adduct 33 (Scheme 11) was completely removed from the organic phase after the K2CO3 and HCl aqueous solution washes, and bis-Lys adduct (34) typically remained at 0.1 - 0.5 %.

Scheme 11. Amide Products from L-lysine/NMM Quench

Optimization of CBz-removal from Z-t-Leu Dipeptide 13 To Manufacture H-t-Leu Dipeptide 14 Catalyst Loading The legacy process for the hydrogenation step used a 10 wt% loading of Pd(OH)2/C as catalyst. A lower catalyst loading was investigated in order to improve the process efficiency on a production scale. The study, ranging from 10 wt% to 1 wt% catalyst loading, was complicated by the deleterious effect of residual acetyl impurity 32 on the reaction efficiency. With typical levels of acetyl impurity 32 at ~0.5% in the plant, experiments demonstrated 8 wt% catalyst loading was optimal. Unfortunately, lower loadings did not provide an efficient reaction.

Stability of H-t-Leu Dipeptide 14 After completion of the hydrogenolysis, and work-up, the process required a solvent switch from i-PrOAc to n-heptane. The stability of H-t-Leu dipeptide 14 in i-PrOAc at various temperatures was investigated (Figure 2). Two impurities, diketopiperidine 35 and N-acetyl dipeptide 36 (Scheme 12) were formed when the H-t-Leu dipeptide 14/i-PrOAc solution was stored at various temperatures for a certain period. To limit formation of these impurities, a solvent swap temperature of ≤ 40 °C was chosen. 295 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 12. Impurities from Decomposition of H-t-Leu Dipeptide 14 in i-PrOAc

Figure 2. Stability of H-t-Leu Dipeptide 14 in i-PrOAc Solution at Various Temperature. In summary, a scalable and robust isolation process for H-t-Leu dipeptide 14 was developed. H-t-Leu dipeptide 14 was taken forward to the next step as a solution in i-PrOAc.

Development of Manufacturing Process for GMP Starting Material H-Chg Tripeptide 16 from H-t-Leu Dipeptide 14 Scheme 13 shows the process for the conversion of H-t-Leu dipeptide 14 to H-Chg tripeptide 16. However, the legacy process used DMF as solvent for the amide coupling (H-t-Leu dipeptide 14 to Z-Chg tripeptide 15). The optimization of this sequence therefore started with adjusting the protocol for charging the amide coupling reagents to the reactor because H-t-Leu dipeptide 14 was now being introduced as a solution in i-PrOAc rather than a solid. Other key developments included an improved workup procedure to completely quench the activated esters after the amide coupling, and development of the crystallization 296 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of H-Chg tripeptide 16. A number of stability, and spike and purge experiments were also performed to verify that tripeptide H-Chg tripeptide 16 could be obtained with a consistent purity profile because it is the designated GMP starting material for telaprevir.

Scheme 13. Conversion of H-t-Leu Dipeptide 14 to H-ChgTripeptide 16

Optimization of the Process to Synthesize Z-Chg Tripeptide 15 Optimization of the EDCI/HOBt Coupling Optimization studies began with a screen of various conditions and equivalents of reagents, resulting in the following procedure (Scheme 13) : 1. 2.

3. 4.

A reactor is charged with EDCI•HCl (1.05 equiv), HOBt•H2O (1.05 equiv), and NMP (2.5 vol). This suspension is cooled to 0 °C. A solution of Z-Chg-OH (1.05 equiv) in NMP (3.0 vol) is then added to the cooled suspension, maintaining the internal temperature at 0 ± 5 °C. After stirring for 1.5 h, the reaction mixture is treated with a solution of H-t-Leu dipeptide 14 (1.0 equiv) in i-PrOAc (10 vol), at 0 ± 5 °C. Once the addition is complete, the mixture is warmed to 20 ± 5 °C, after which the reaction is stirred for 5 - 17 h. 297


Compound Z-Chg tripeptide 15 is stable to the reaction conditions if left overnight, and complete conversion was obtained in these instances.

Development of a New Method for Quenching Z-Chg-OBt


In light of the impurity issues associated with activated ester Z-t-Leu-OBt, a derivatization method for Z-Chg-OBt was developed using benzylamine to generate the corresponding benzyl amide 37 (Scheme 14). This provided a means to accurately analyze the amount of Z-Chg-OBt remaining at reaction completion.

Scheme 14. Derivatized Product from Z-Chg-OBt With an improved analytical method in hand, a more efficient quench of the Z-Chg-OBt was investigated to avoid generation of the Chg-dimer impurity 38 from reaction with the deprotected amine H-Chg tripeptide 16 (Scheme 15).

Scheme 15. Impurity Resulting from Incomplete Quench of Z-Chg-OBt The original process used histamine in order to quench any unreacted activated esters of Z-Chg-OH, rendering them as water-soluble histamine amides. However, due to health issues surrounding histamine (49) as well as incomplete quenching of the activated ester, another work-up procedure was sought. A variety of reagents was examined. Screening experiments were carried out as follows: the coupling of Z-Chg-OBt and H-t-Leu dipeptide 14 to form Z-Chg tripeptide 15 was performed, followed by treatment of the reaction mixture with an amine base (Et3N or NMM) and/or an amino acid. The most desirable trapping combination would rapidly generate the desired amide, which would be stable to hydrolysis and readily be removed in the subsequent aqueous washes. L-Lysine and L-cysteine were capable of efficiently trapping the activated ester, but emulsions formed during basic, acidic, and neutral aqueous washes. However, 298


glycine (1.0 equiv) with NMM (2.0 equiv) efficiently trapped Z-Chg-OBt as glycyl amide 39 (Scheme 16) and no epimerization of Z-Chg tripeptide 15 was observed. An additional water wash was incorporated in order to avoid a thick residue forming on the walls of the vessel.


Scheme 16. Glycine Adduct of Z-Chg-OBt Optimization of the workup procedure showed that after a single wash with 5% K2CO3, a second wash with 25% K2CO3 was needed to effectively quench Z-Chg-OBt, and a subsequent acid wash (1N HCl) was required to remove the urea by-product formed from the EDCI reagent, as well as any unreacted H-t-Leu dipeptide 14 and other basic impurities. A final water wash was included in order to remove any excess acid present before beginning the hydrogenolysis of the Cbz group. Hydrogenolysis proceeded to completion in less than 35 min rather than the previously typical 1.5 h, suggesting that the additional water wash may provide an opportunity to further reduce the catalyst loading from 5 wt % Pd(OH)2/C. Final isolation and purification of H-Chg tripeptide 16 was achieved by solvent exchange to n-heptane and crystallization. The robustness of the crystallization process was verified by confirming the ability to purge intermediates Z-t-Leu dipeptide 13, H-t-Leu dipeptide 14, Z-Chg tripeptide 15, and impurities 40-44 (Scheme 17) from a spiked crude i-PrOAc solution of H-Chg tripeptide 16. Intermediate Z-Chg tripeptide 15 and ethyl ester 44 were the only two compounds that were not completely purged in the spike/purge studies (0.08 and 0.15%, respectively). Implementing the solvent exchange and n-heptane crystallization process during manufacture provided H-Chg tripeptide 16 in 85% recovery and 100% purity. Development of a Process for the Manufacture of P-cap Acid 18 From H-Chg Tripeptide 16 Chemical Development of the Conversion of H-Chg Tripeptide 16 to P-cap Acid 18 The initial process for converting H-Chg tripeptide 16 to P-cap acid 18 is a two-step process which required precipitation of the desired product by addition of water to a DMF solution (Scheme 18). The major disadvantage of this approach was that P-cap ester 17 filtered poorly in the plant and was very time-consuming to dry. So, the initial focus of the commercial process development was to telescope the steps directly from H-Chg tripeptide 16 to P-cap acid 18. 299 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 17. Structures of Spiked Impurities for Purging Studies of H-Chg Tripeptide 16

Scheme 18. Original Process for Conversion of H-Chg Tripeptide 16 to P-cap Acid 18 300 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Once the most process-friendly set of reaction conditions (reagents, solvents, catalysts) were established for the formation of P-cap acid 18 from H-Chg tripeptide 16, the limits of the process were defined. In particular, a process characterization was performed and the acceptable limits of impurities were established. In order to evaluate the impact and safe limits of impurities in the starting material or product, a series of spike and purge experiments were performed. Impurities were spiked at the start of the process for P-cap acid 18. The spiked and residual impurities were tracked throughout the normal operation of the process and are described in the subsections below.

Solvent and Coupling Reagent Selection A qualitative solvent screen for the conversion of H-Chg tripeptide 16 and P-cap acid 18 in solvents of medium to high polarity which were likely to be compatible with the reaction conditions was performed initially. P-cap acid 18 had low solubility in most of the solvents examined except for, which provided good solubility and was expected to be inert to strong acid. However, due to the high solubility of P-cap ester 17 in CH2Cl2, and the low likelihood of crystallization, development of a telescoped process from H-Chg tripeptide 16 to P-cap acid 18 was pursued. The solubility studies also identified toluene as the preferred solvent for crystallization of P-cap acid 18. The next area of development was the activation of the pyrazine carboxylic acid as its acid chloride. We prepared a sample of the pure acid chloride by sublimation to afford a colorless solid, but discoloration occurred after a week. This prompted the decision to generate the acid chloride in situ and use it immediately. The initial screening studies for the telescoped acid chloride formation and coupling indicated that oxalyl chloride gave a consistently homogenous solution, the best conversion and the least discoloration. A solvent-dependent impurity was seen in the reactions with oxalyl chloride, with CH2Cl2 showing minimal impurity formation. Attempts with EDCI/HOBt or CDI as the coupling agent produced results inferior to oxalyl chloride. A final study to examine the preferred amine base showed NMM to be superior to i-Pr2NEt. Optimization of the reaction stoichiometry was pursued next. An initial screen showed that 1.4 equivalents of pyrazine carboxylic acid activated with 1.2 equivalents of oxalyl chloride in the presence of 5.0 equivalents of NMM produced the cleanest conversion to P-cap ester 17. Subsequent development of the reaction showed formation of the acid chloride was rapid (

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