ARTICLE pubs.acs.org/OPRD
Synthesis, Chromatographic Purification, and Isolation of EpothiloneFolic Acid Conjugate BMS-753493 Soong-Hoon Kim,*,†,( Nuria de Mas,*,‡,b Luca Parlanti,‡,0 Olav K. Lyngberg,‡ Guido Str€ohlein,z Zhenrong Guo,‡,9 Konstantinos Dambalas,‡ Victor W. Rosso,‡ Bing-Shiou Yang,‡,4 Kevin P. Girard,‡,2 Zerene A. Manaloto,‡,O Germano D’Arasmo,|| Riccardo E. Frigerio,|| Wei Wang,§ Xujin Lu,§ Mark S. Bolgar,§ Madhushree Gokhale,^ and Ajit B. Thakur^ †
Oncology Discovery Chemistry, Bristol-Myers Squibb Company, Route 206 & Province Line Road, Princeton, New Jersey 08543, United States Process Research and Development, §Analytical Research and Development, ^Biopharmaceutics; Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, New Jersey 08901, United States z ChromaCon AG, Technoparkstrasse 1, 8005 Zurich, Switzerland NerPharMa DS, Nerviano Medical Sciences Group, Via Pasteur 10, 20014 Nerviano, Milan, Italy
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bS Supporting Information ABSTRACT: We describe the synthesis, chromatographic purification, and isolation of the epothilonefolic acid conjugate BMS753493, an investigational new drug candidate for the treatment of cancer. The main challenges for process development were the instability of BMS-753493 in aqueous solution, the design and optimization of the preparative chromatography, and the removal of phosphate salts and water from the purified material. The operating conditions of the batch chromatographic purification were optimized using a column adsorption model. The free-salt active pharmaceutical ingredient was isolated via the precipitation of its zwitterion following a careful determination of the isolation parameters that controlled thermal and pH-related decomposition. This process enabled the manufacturing of several batches (1030 g) of cGMP quality BMS-753493.
’ INTRODUCTION The folate receptor (FR) is a cell surface receptor that is highly expressed in tumor tissues of epithelial origin while minimally expressed in normal tissues.1 The FR binds folic acid and its conjugates tightly (dissociation constant Kd < 109 M). Receptor endocytosis, dissociation, and release of the conjugate inside the cell have been demonstrated in preclinical in vitro and in vivo models. Studies with cytotoxic folic acid conjugates in cell lines and tumor models are consistent with the selective targeting of cells that overexpress the FR.2 This differential tissue selectivity suggests a potential for increased therapeutic index and reduced toxicity. BMS-753493 (1), an epothilonefolic acid conjugate, shows preclinical efficacy consistent with the selective delivery of the cytotoxic epothilone into tissues that overexpress the FR and is an investigational new drug (IND) candidate for the treatment of cancer.3 Epothilones are cytotoxics with proven clinical efficacy.4 Other groups are pursuing similar FR-targeted strategies using different cytotoxics (e.g., vinblastinefolate).5 The dominant structural feature in compound 1 (molecular weight 1570 Da) is the highly polar peptide fragment, which has a major influence in its physicochemical properties. Additionally, 1 contains functional groups that are pH (lactone, chiral centers, carbonate, and aziridine), UV (folate), and chemically (disulfide bond) sensitive. The compound contains multiple ionizable groups, including four carboxylic acids (pKa 3.0, 4.3, 4.4, 5.9), an aziridine (pKa 6.6), and a guanidine (calculated pKa ≈ 13.8), with an isoelectric point (pI) in the range of 3.54.5. r 2011 American Chemical Society
The manufacturing of peptide-based active pharmaceutical ingredients (API) nearly always involves a chromatographic purification step 6 as peptides are difficult to crystallize. In addition, the aqueous solubility of charged peptides limits the number of processing options for isolation. Understanding the transition from reaction to chromatography, developing a robust chromatographic purification, and defining the processing ranges that maintained API stability were regarded as the important issues for the development of an early-stage synthesis process of BMS-753493. We here describe a synthesis process that addresses these issues.
’ RESULTS AND DISCUSSION Form Selection. During the discovery process, 1 was isolated after batch reverse-phase high-performance liquid chromatography (HPLC) as a lyophile containing sodium phosphate salts.7 A large variation in API content (3181 wt %) as well as in the levels of sodium phosphate salts and water in the individual lyophile batches occurred on scale, presumably because of the variable amount of API injected per column volume (load) during the chromatographic purification and the hygroscopicity of the lyophiles. Before undertaking process optimization, API form evaluation studies were initiated to identify the form that offered minimal Received: February 1, 2011 Published: May 03, 2011 797
dx.doi.org/10.1021/op200023g | Org. Process Res. Dev. 2011, 15, 797–809
Organic Process Research & Development
ARTICLE
disulfide 3 (Scheme 1). The reaction in THF/aqueous NaHCO3 was heterogeneous, and the impure API solids were separated using centrifugation. THF was removed by distillation, and the resulting solution was passed through a 1-μm filter and purified by preparative reverse-phase HPLC (NaH2PO4/Na2HPO4 and acetonitrile). Acetonitrile was removed from the API fractions by distillation, and the solution was lyophilized. A second chromatography with water/acetonitrile to remove the phosphate salts was followed by yet a second lyophilization to isolate 1 as a lyophile.9 Such a process possessed several challenges. Solids were removed from the reaction mixture by centrifugation because filtration was unacceptably slow. Only the supernatant was processed further; however, API remained in the pellet, reducing the yield. The poor thermal stability of the API required avoiding the high-temperature distillation of water (2% API degradation at 30 C in 6 h in the concentration range 1.25.9 g/L). A second chromatography was required to obtain the salt-free final API solution. In addition, 1 is a potent cytotoxic agent that was handled using the highest level of engineering controls in specialized containment scale-up facilities, underscoring the need for a more streamlined process.10 Strategically, we viewed the IND toxicology campaign (5 g, 3 mmol) as an opportunity to define an alternate synthesis strategy with process controls, to generate process knowledge, and to define processing ranges that maintained API stability. The first cGMP campaign (>10 g of 1) was subsequently used to define the scalability limits of this new synthesis process with stricter requirements for purity. Process Development. To initiate our studies, the relationship between stoichiometry and reaction outcome (product distribution, relative reaction rates) was examined (Table 2). The reaction followed a simple profile: excess of reagent 3 consumed its counterpart 2 and vice versa, and no significant side products were observed. A small amount of oxidative dimerization of peptide 2 (
