Article pubs.acs.org/OPRD
Chromatography- and Lyophilization-Free Synthesis of a PeptideLinker Conjugate Javier Magano,*,† Brandon Bock,‡ John Brennan,† Douglas Farrand,‡ Michael Lovdahl,‡ Mark T. Maloney,† Durgesh Nadkarni,§ Wendy K. Oliver,‡ Mark J. Pozzo,∥ John J. Teixeira,† Jian Wang,‡ John Rizzo,⊥ and David Tumelty⊥ †
Chemical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States ‡ Analytical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States § Biotherapeutics Pharmaceutical Sciences, Pfizer Worldwide Research & Development, 401 North Middletown Road, Pearl River, New York 10965, United States ∥ Biotherapeutics Pharmaceutical Sciences, Pfizer Worldwide Research & Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017, United States ⊥ CovX Research, 9381 Judicial Drive, San Diego, California 92121, United States S Supporting Information *
ABSTRACT: An optimized and scalable process to manufacture peptide−linker conjugate 1 is reported that avoids the chromatographic purification and lyophilization that are typically required for the isolation of this type of compound. An operationally simple protocol has been developed that couples the peptide to the linker in DMF followed by precipitation with MeCN. A scalable synthesis of the linker is also described which features the N-acylation of 2-azetidinone promoted by 1propanephosphonic acid anhydride (T3P). The number of operations during the second step of the synthesis (nitrobenzene reduction to aniline) has been simplified by telescoping the aniline into the next step (reaction with diglycolic anhydride to form an acid), thus avoiding an additional isolation. Finally, two efficient activation methods for the acid have been developed by means of the corresponding pentafluorophenyl (PFP) and p-nitrophenyl (PNP) esters.
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INTRODUCTION Biotechnology-based pharmaceuticals, or biopharmaceuticals, have seen a considerable expansion in recent years. “Bioconjugation”, which refers to the covalent derivatization of biomolecules, can be employed to bring together two or more molecules to form a complex that displays the combined properties of each one of its individual components.1 This technique has been applied to stabilize substances in blood, for the protection of substances prone to enzymatic degradation, and for decreasing the immunogenicity of polypeptides, among others.2 An interesting application is in drug delivery systems, such as in some oncology treatments, where the drug is attached to a carrier by means of a linker or spacer that can bind to its specific receptor contained on the cancer cell. As a result, the therapeutic molecule can be directed to the desired site of action.3 Both monoclonal antibodies4 and synthetic polymers5 have been used as carriers. Conjugate 1 (Figure 1) is an advanced intermediate in the synthesis of antibody drug bioconjugate 5, currently under evaluation at Pfizer as part of an oncology program.6 The preparation of 5 involves two steps (Scheme 1): (a) activated linker 2 undergoes reaction with a lysine residue on 22-mer peptide 3, which is the therapeutic portion of the molecule, to form peptide−linker conjugate 1 via a new amide bond; (b) peptide−linker conjugate 1 is then subjected to a second coupling with a lysine residue on monoclonal antibody 4 to © XXXX American Chemical Society
form drug substance 5 via azetidinone ring-opening and creation of a β-alanine functionality.
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MEDICINAL CHEMISTRY SYNTHESIS OF 1 The original synthesis of 1 is shown in Scheme 2.6a,7 Acid 6 and N-hydroxysuccinimide (7) underwent reaction with N,N′diisopropylcarbodiimide (DIC) as coupling reagent to afford N-hydroxysuccinimido ester 8 in quantitative yield. Crude 8 was immediately used in the subsequent reaction with peptide 3 and N-methylmorpholine (NMM) as base in DMF to provide conjugate 1, which was then subjected to extensive reversephase chromatographic purification followed by lyophilization to afford a 40% yield of purified 1. The lyophilized fractions were reconstituted8 in MeCN/H2O 1:1 (v/v) to generate a homogeneous lot of intermediate 1. After filtration of some insoluble material,9 the filtrates were subjected to a second lyophilization to generate 1 in 12% yield. This lengthy and both energy- and solvent-intensive process was employed for the generation of small batches of material (up to 50 g), but it quickly became clear that its implementation to manufacture even larger quantities of 1 would be impractical due to the very low throughput. In addition, peptide 3 was only available at a Received: October 20, 2013
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dx.doi.org/10.1021/op4002998 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
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Figure 1. Structure of peptide−linker conjugate 1.
Scheme 1. Preparation of peptide−linker−antibody bioconjugate 5
to provide crude acid chloride 10. In a separate flask, 2azetidinone (11) was deprotonated at −70 °C with n-BuLi to generate Li salt 12 which, without isolation, underwent reaction with acid chloride 10 at 0 °C to give intermediate 13 in 91% yield. The nitro group on 13 was reduced using catalytic hydrogenation with 10% Pd/C to give aniline HCl salt 14·HCl in 68% yield. The last step involved the reaction between 14 and diglycolic anhydride (15) using Hunig’s base to provide acid 6 in 83% yield. During preliminary experiments to determine the scalability of this route, it was found that the yield for the coupling between acid chloride 10 and azetidinone 11 was not reproducible and dropped to 40−55% when the reaction was run on 200-g scale. As a result, a search for alternative
considerable expense from a commercial source,10 and the very low overall yield would have made this method difficult to implement on scale for cost reasons. When the need for larger amounts of antibody drug bioconjugate 5 for clinical trials arose, our group was requested to develop an optimized synthesis of 1 capable of producing multihundred gram quantities under current good manufacturing process (cGMP) conditions in a cost-effective manner.11
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PROCESS DEVELOPMENT FOR THE SYNTHESIS OF LINKER 6 We first addressed the preparation of the linker portion of the molecule. The Medicinal Chemistry synthesis of acid 6 is shown in Scheme 3.12 Acid 9 was treated with Cl2SO at reflux B
dx.doi.org/10.1021/op4002998 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
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Scheme 2. Medicinal Chemistry peptide−linker conjugation conditions
Scheme 3. Medicinal Chemistry synthesis of linker acid 6a
A major breakthrough took place when 1-propanephosphonic acid anhydride (T3P) was investigated.16 This coupling reagent is commercially available as a 50 wt % solution in EtOAc,17 and the reaction is usually carried out at ambient temperature. Initial experiments showed that the addition of T3P solution to a mixture of acid 9 and 2-azetidinone (11) in DMF and pyridine as base gave desired product 13 in 20−45% yield. A base (pyridine, DBU, DIPEA) and solvent (THF, EtOAc, DMF, MeCN) screen revealed that consistent yields around 40% could be obtained when the reaction was performed with 3 equiv of DIPEA, 1.2 equiv of 2-azetidinone, and 1.2 equiv of T3P in MeCN at 20 °C for 18 h. Further optimization led to the use of 5 equiv of DIPEA, 1.5 equiv of 2azetidinone, and 1.5 equiv of T3P in MeCN at 20 °C for 20 h, which provided coupling product 13 in 55−65% yield (Scheme 4). Clear advantages of this method are the operational simplicity (slow addition of the 50 wt % T3P solution in EtOAc to a mixture containing the two coupling partners and base in MeCN), mild reaction conditions, and yield reproducibility. Upon reaction completion, the solvent was removed under vacuum, and the residue was redissolved in i-PrOAc. The Scheme 4. Optimized synthesis of linker acid 6a
Reagents and conditions. (a) Cl2SO, reflux. (b) n-BuLi, −70 °C, THF. (c) 0 °C, 91% (from 9). (d) H2, 10% Pd/C, MeOH, HCl, 40 °C, 68%. (e) DIPEA, CH2Cl2, rt, 83%. a
conditions was carried out with the additional goal of avoiding running the process at cryogenic temperature. A literature search disclosed that the N-acylation of 2azetidinone has been accomplished through the use of acid chlorides13 or anhydrides.14 After further exploring the first approach, we noted that the preparation of acid chloride 10 using oxalyl chloride and the subsequent reaction with lithium salt 12 prepared via deprotonation with LiHMDS at −30 to −60 °C led to intermediate 13 but in low yield and purity. Alternatively, the activation of acid 9 with CDI was readily achieved, but the subsequent coupling with 2-azetidinone or its anion failed in solvents such as CH2Cl2, THF, or EtOAc, most likely due to the low nucleophilicity of this substrate. Finally, KF on alumina was employed on the basis of some literature reports15 to promote a slow deprotonation of the azetidinone, but low conversion to 13 was observed over a 3-day period.
a
Reagents and conditions. (a) T3P (50% in EtOAc), DIPEA, MeCN, 20 °C, 20 h, 60−68%. (b) H2 (15 psig), 10% Pd/C (10 wt %), THF, 25 °C, 1 h. (c) THF, 22 °C, 15 min, 80% (2 steps).
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dx.doi.org/10.1021/op4002998 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
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solvent ratio of 9:1 i-PrOAc/THF was employed. The telescoping of the THF solution of free base allowed for a much simpler process and saved a considerable amount of time in the plant. Since this linker is also employed in another project that has stricter residual solvent specifications (99% chemical purity that met residual solvent specifications. This procedure delivered significantly smaller particles (∼30 μm) than the original THF/i-PrOAc crystallization (100−200 μm), and the smaller particle size may explain why less solvent was trapped in the crystals.
organic phase was washed with aqueous citric acid, and after a solvent switch to 2-propanol, intermediate 13 precipitated from solution in high purity (≥98% area %). Before using this material in the next nitro-reduction step, a Darco G-60 treatment in EtOAc was performed to remove some color and trace impurities present in acid 9.18,19 A further benefit of this carbon treatment was that the level of impurity 16 (Figure 2), resulting from the opening of the azetidinone ring by the newly formed amino group in the subsequent nitro reduction step, was kept below 0.3%.
Figure 2. Impurities produced during the nitro reduction step.
The nitro reduction step was originally carried out in MeOH, but due to the formation of considerable amounts of methyl ester impurity 17 (Figure 2), the Medicinal Chemistry group switched to dioxane to prevent its formation. Since this solvent is a known carcinogen and undesirable on scale, we decided to search for a safer alternative. Catalytic hydrogenation (15 psig) with 10% Pd/C (10 wt %) in THF gave a fast reaction (
