Communication pubs.acs.org/OPRD
Development of a Practical Process for the Opening of Macrocyclic Cyclosporin A and Amino Acid Deletion Bernard Riss,† Arnaud Grandeury,‡ Thorsten Gut,† Manuela Seeger-Weibel,† Christian Zuercher,† Jinjing Li,† and Fabrice Gallou*,† †
Chemical and Analytical Development and ‡Pharmaceutical and Analytical Development, Novartis Pharma AG, CH-4002 Basel, Switzerland peptide.7 But the most widely encountered approach for derivatization still remains the selective opening of the macrocycle at the most readily accessible positions, namely, either the amino acid residues 1 and 11, or between 3 and 4, using the Cyclosporin A nomenclature, followed by the removal of the undesired amino acid residue(s) traditionally done via Edman degradation.8 Such an approach was disclosed in the mid 1980s and allows for robust and scalable9 protocols but suffers from a severe limitation, namely, the high demand in purification via costly methods, such as preparative chromatography. In this paper, we would like to describe our efforts to solve such process issues which led us to the development of a streamlined and cost-effective process to advanced acyclic polypeptide derivatives of Cyclosporin A.10
ABSTRACT: A practical and robust process for the derivatization of Cyclosporin A was demonstrated. The processes rely on the opening of Cyclosporin A and removal of amino acid fragments via Edman degradation, with the isolation of crystalline tetrafluoroboric salts of the corresponding acyclic polypeptides.
S
ince its discovery by isolation from fungus Tolypocladium inf latum (Beauveria nivea),1 Cyclosporin A has created tremendous fascination and received enormous interest both from the academic community and from the industry. The most significant application was made in transplantation with Cyclosporin A sold under the trade name Sandimmun that still constitutes one of the most important immunosuppressant to date.2 Continued efforts throughout the industry have led to a plethora of other derivatives in various indications such as an antiviral agent, multiple sclerosis.3 Solid-phase synthesis has been one of the strategies used in discovery mostly.4 More traditional solution phase approaches have relied on the derivatization of the more readily accessible handle 1 (amino acid residue 1 based on Cyclosporin A nomenclature as depicted in Figure 1 below), easily derivatized via either the
■
BACKGROUND The classical opening of Cyclosporin A between amino acid residue 3 (sarcosin) and 4 (methyl leucine) starts with masking of the free hydroxyl, so as to avoid isomerization ((1) in equilibrium with (1-Iso), see Figure 2 and Scheme 1) between the amide and ester forms. Not doing so leads to an equilibrium between the amide and the corresponding ester form11 that reduces the efficiency of the subsequent steps and further complicates the process. The steric hindrance around the hydroxyl group does not allow for many practical and economically viable options to mask this functionality. The acetate is therefore traditionally chosen and incorporated for industrial applications via a well-established process, which leads the desired monoacetylated product in high yields (>90% isolated yield) by direct crystallization and in purity typically around 95%. Care should nevertheless be taken of the temperature of the reaction so as not to overacetylate the polypeptide. The chemistry and process on this step were performing sufficiently well to leave this part of the process untouched. The macrocyclic peptide is then classically opened using a Meerwein salt, triethyl or trimethyloxonium tetrafluoroborate, subsequently subjected to methanolysis and acid hydrolysis. Although of limited selectivity, this step is remarkable from a complexity standpoint. Indeed, despite an excess of alkylating agent required to allow for sufficient reactivity, a remarkable selectivity for such a functionalized system is observed. The selectivity is presumably due to both steric and conformational reasons, due to the reduced steric hindrance at the sarcosine
Figure 1. Structure of Cyclosporin A (1) and its nomenclature.
hydroxyl or the olefin moieties.5 Seebach in his seminal paper on the development of chemistry on the Cyclosporin A core exemplified the strong conformational bias of the macrocyclic structure and took advantage of this feature to selectively alkylate the sarcosine amino acid residue 3.6 More recently, in collaboration with Beller, we demonstrated the direct functionalization of the macrocyclic polypeptide core via the selective reduction of the Abu fragment of the macrocyclic © XXXX American Chemical Society
Received: September 19, 2014
A
dx.doi.org/10.1021/op5003038 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Communication
Figure 2. Equilibrium amide (1)−ester (1-Iso).
Scheme 1. Original synthesis from Cyclosporin A 1−4
Scheme 2. Opening of the macrocylic structure from 2 to 3
B
dx.doi.org/10.1021/op5003038 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Communication
Table 1. Qualitative overview of salt screening for 3 screening positive hits crystallinity
HCl
TFA
H2SO4
H3PO4
pTsOH
HPF6
HBF4
32 solvents ketones, ethers, esters ++
6 solvents − +
6 solvents ethers +
6 solvents − −
6 solvents − −
30 solvents ethers, esters +
44 solvents ethers, esters +++
Table 2. Solvent screening for 3
tetrafluoroborate that differs from lot to lot with the finely dispersed powder generates variability and complexity for the process. This first opened-form intermediate (dodecapeptide) 3 can be used as such as a platform for derivatization, or amino acids can be deleted sequentially, which is classically done via Edman degradation. The major hurdle is the requirement for a laborintensive purification of the degradation product. The default method of choice is a purification via column chromatography of the trifluoroacetate salt, generated from the previous step.
residue and the transannular intramolecular interaction within the macrocycle.12 It is worth mentioning that the limited solubility of the Meerwein salt adds a number of constraints to the process. For instance, the reversible nature of the transformation (hydrolysis of the intermediate imino-ether back to the amide) creates some complications in the analytical control strategy, the purification by column chromatography generates a bottleneck in the overall process, and a high number of operations can lead to decomposition if pH is not carefully controlled during the solvent switch especially. Associated with the use of trimethyloxonium tetrafluoroborate also comes the requirement for a high solubilizing noncoordinating solvent, typically a chlorinated solvent such as dichloromethane. Besides, the quality of the trimethyloxonium
■
RESULTS With these challenges identified, we aimed at identifying a more robust process more specifically for the ring opening and C
dx.doi.org/10.1021/op5003038 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Communication
Scheme 3. Edman degradation process (from 3 to 4)
Table 3. Qualitative overview of salt screening for 4 screening positive hits crystallinity
HCl
TFA
H2SO4
H3PO4
pTsOH
HPF6
HBF4
32 solvents ketones, ethers, esters ++
6 solvents − +
6 solvents ethers +
6 solvents − −
6 solvents − −
30 solvents ethers, esters +
46 solvents ethers, esters +++
of hydantoin indeed has to be removed, along with a significant amount of carry-over impurities resulting from the nature of the original fermentation product. Purification by column chromatography was the method of choice prior to our work that allowed for obtention of material of acceptable quality of the resulting product 4 (purity >90%). We were again aiming for a chromatography-free process and were fortunate to identify rapidly an alternative to remove the hydantoin side-product. A selective liquid−liquid extraction, using the solvent already present in the process, indeed allowed us to wash away the hydantoin in a first operation. By adjusting the amount of water at completion of the reaction, the Edman degradation product indeed remained in the aqueous phase at this low pH (
