Article pubs.acs.org/OPRD
Process R&D of Eravacycline: The First Fully Synthetic Fluorocycline in Clinical Development Magnus Ronn,* Zhijian Zhu, Philip C. Hogan, Wu-Yan Zhang, John Niu, Christopher E. Katz, Nicholas Dunwoody, Olga Gilicky, Yonghong Deng, Diana K. Hunt, Minsheng He, Chi-Li Chen, Cuixiang Sun, Roger B. Clark, and Xiao-Yi Xiao Tetraphase Pharmaceuticals Inc., 480 Arsenal Street, Suite 110, Watertown, Massachusetts, 02472, United States ABSTRACT: Process research and development of the first fully synthetic broad spectrum 7-fluorotetracycline in clinical development is described. The process utilizes two key intermediates in a convergent approach. The key transformation is a Michael−Dieckmann reaction between a suitable substituted aromatic moiety and a key cyclohexenone derivative. Subsequent deprotection and acylation provide the desired active pharmaceutical ingredient in good overall yield.
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INTRODUCTION The use of tetracyclines, discovered half a century ago, has been widespread both for serious bacterial infections and for less serious conditions, such as acne. Naturally occurring examples of this class include chlortetracycline (1) and tetracycline (2). First-generation semisynthetic tetracyclines commonly used include doxycycline (3) and minocycline (4). Newly developed semisynthetic tetracyclines analogues are tigecycline (5) and omadacycline (6).1
containing all the functionalities within the A and B rings required for antibacterial activity. New modifications of the C and D ring of the tetracyclines are introduced by using a separate appropriately functionalized building block (vide infra). Tetraphase has continued to improve this synthetic process and discover new tetracyclines with enhanced antimicrobial profiles relative to previous generations of this class. Currently several candidates are in different stages of development. The lead compound, eravacycline (7), a broad spectrum antibiotic for serious hospital infections, has just completed phase 2 clinical studies and is active against multidrug resistant bacteria, including multidrug resistant gram-negative bacteria. Process chemistry research and development for the manufacturing of 7 in multihundred gram quantities, sufficient to supply drug substance for preclinical and early clinical development, is presented below.
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RESULTS AND DISCUSSION The Myers approach combines two key building blocks: the enone 85 and the left-hand piece (LHP) (Scheme 1). The methyl group within the LHP is deprotonated with a strong base, such as lithium diisopropylamide (LDA). Addition of the resulting benzylic anion to 8 results in a highly diastereoselective conjugate addition to the enone found within 8. The resulting ketone enolate undergoes a Dieckmann-type condensation with the phenyl ester providing a protected tetracycline derivative. Removal of the protecting groups and reductive ring opening of the isoxazole generally provides an overall high yield of the desired tetracycline analogue.6 This route allows for the production of analogues of wide structural diversity7 as well as the manufacturing of new tetracyclines using the single key intermediate 8, altering only the left-hand piece. The discovery route used to prepare 7 as well as a number of analogues is shown below (Scheme 2).8a,b The key intermediate 12 was targeted as a platform for the rapid preparation of a
Figure 1. Tetracycline antibiotics.
The widespread use of tetracyclines2 has resulted in reduced efficacy of this class of antibiotics due to increasing bacterial resistance for many of the older tetracyclines. The development of new, effective tetracyclines is desirable due to their favorable safety profile, but semisynthetic approaches have generally been limited to various substituents in the 7- and 9-positions (e.g., 5 and 6). While several totally synthetic approaches to the tetracycline antibiotics have been published, most suffer from low overall yields as well as lengthy linear sequences.3 In 2005, Myers et al. published a convergent, discovery-enabling synthetic approach to the tetracycline class of antibiotics (Scheme 1).4 This approach utilizes a single cyclohexenone-based building block © XXXX American Chemical Society
Received: January 29, 2013
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dx.doi.org/10.1021/op4000219 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 1. The Myers general approach to fully synthetic tetracycline analogues.
bromoacetyl bromide. The resulting α-bromo-amide was substituted with amines to provide the desired analogues. These analogues could be purified by preparative HPLC. This approach was advantageous for discovery purposes but was seen as suboptimal for larger scale manufacturing because of the number of steps, the use of water-soluble tetracyclines as intermediates, and the low overall yield from 8 (90%) with a HPLC purity of 98%. The possibility of using commercial LDA was investigated. Use of commercial LDA led to reaction with an inferior purity profile. We also investigated the possibility of avoiding the use of two cryogenic reactors for this step. In one reaction the anion derived from 13 generated at −70 °C was added to 8 at −20 °C. In a separate experiment a solution of 8 at −20 °C was added to a solution of the anion derived from 13 below −70 °C. In the first case the impurity profile was unacceptable, most likely due to instability of the anion derived from 13 at the higher temperature. In the second case more impurities were found, although this process may be useful with additional development. Cleavage of TBS Ether Within 14. The cleavage of the TBS group within 14 to give the tertiary alcohol 15 was initially accomplished using aqueous hydrofluoric acid (HF). The use of HF was seen as a disadvantage for larger scale manufacturing, due to safety concerns as well as its highly corrosive properties toward many materials present in the lining of reaction vessels. We investigated other reagents to effect the transformation of 14 to 15. Strong acids (trifluoroacetic acid (TFA), ptoluenesulfonic acid (TsOH), H2SO4, and HCl) and alternate sources of fluoride (HF-pyridine, tetrabutylammonium fluoride (TBAF), NH4F, and Et3N·3HF) in various solvents were
Also, the presence of excess LDA appeared to promote several unidentified side reactions. Based on these results, the procedure was modified to use a strong base for the deprotonation of 13 (LDA) and a weaker base, lithium bistrimethylsilylamide (LiHMDS), for the deprotonation of the acidic proton in the Dieckmann product necessary to drive the second stage of the Michael−Dieckmann reaction to completion. The addition of a solution of the anion derived from 13 to a solution of 8 at −70 °C provided a cleaner transformation, and moreover only 1.04 equiv of 13 was needed to consume 8 completely. Trituration of the crude product obtained after workup with methanol provided 14 in >90% yield with an HPLC purity of 98%. This procedure was verified in 150 and 200 g runs. Additional 14 could be recovered from the mother liquor (∼5%) albeit of lower purity (∼94% by HPLC). Investigation of Additives for the Deprotonation of 13. During a manufacturing campaign for clinical material, initial laboratory runs of the Michael−Dieckmann gave poor conversion of the starting materials and correspondingly lower yields. Examination of the data indicated that the anomalous results might be traced to the different lots of nbutyllithium (n-BuLi) used for the preparation of LDA solutions. One lot of n-BuLi provided a base that deprotonated 13 rapidly and quantitatively even at temperatures below −70 °C, while other lots provided incomplete deprotonation. An elegant investigation by Collum et al.10 indicated that small amounts of lithium chloride present in commercial batches of butyllithium could dramatically enhance the kinetic activity of LDA prompting us to pay particular attention to the quality of the n-BuLi as well as the possibility of using other rateenhancing additives for the deprotonation of 13. The deprotonation of 13 was investigated using methyl iodide to quench the reaction mixture followed by HPLC analysis. Three lots of n-BuLi were examined: one clear and free of suspended solids, one with some solids present, and one with a significant amount of solids. In the absence of an additive, deprotonation with LDA of the methyl group within 13 followed by quenching with methyl iodide gave the corresponding desired ethyl derivative in 39% (clear n-BuLi), 66% (some precipitate), and 100% (significant precipitate) yields, respectively, by C
dx.doi.org/10.1021/op4000219 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Figure 3. Impurities formed during the hydrogenolysis of 15.
Table 1. Scale-up of hydrogenation.
a
entry
hydrogenation timea
time for slurry in EtOH/water
final purity (HPLC %) 12:18:20:19
yield (amount)
Pd content
1 2 3 4 5
12 h 4h 7h 10 h 11 hb
2h 17 h 2h 2.5 h 4h
96.3:0.26:0.86:0.92 98.27:0.19:0.70:0.59 98.03:0.28:0.83:0.63 97.24:0.37:0.77:0.93 97.06:0.46:0.82:1.21
82.5% (25.7 g) 79.3% (36.24 g) 77.0% (155.5 g) 79.6% (186.6 g) 85.8% (349.2 g)
0.4 ppm 0.2 ppm 0.39 ppm 1.11 ppm
