Process Development and Scale-up of Fully Synthetic Tetracycline TP

Dec 28, 2015 - Wu-Yan Zhang, Cuixiang Sun, Diana Hunt, Minsheng He, Yonghong Deng, Zhijian Zhu, Chi-Li Chen, Christopher E. Katz, John Niu, Philip C. ...
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Process Development and Scale-up of Fully Synthetic Tetracycline TP-2758: A Potent Antibacterial Agent with Excellent Oral Bioavailability Wu-Yan Zhang,* Cuixiang Sun, Diana Hunt, Minsheng He, Yonghong Deng, Zhijian Zhu, Chi-Li Chen, Christopher E. Katz, John Niu, Philip C. Hogan, Xiao-Yi Xiao, Nicholas Dunwoody, and Magnus Ronn Tetraphase Pharmaceuticals Inc, 480 Arsenal Street, Suite 110, Watertown, Massachusetts 02472, United States ABSTRACT: Process research and development of the fully synthetic broad spectrum tetracycline TP-2758, with a chiral pyrrolidine side chain at the C-8 position, is described. The process utilizes two key intermediates, 7 and 10, in a convergent approach that allows for manufacturing of sufficient quantities of API to supply preclinical and early clinical development. The pyrrolidine moiety was introduced into the left-hand piece (LHP) 10 with high enantioselectivity using Ellman’s sulfinamide chemistry, and the absolute configuration was confirmed by X-ray crystal structure analysis.



INTRODUCTION Using our fully synthetic tetracycline technology,1 Tetraphase has synthesized more than 3000 novel, highly active tetracycline analogs, including our lead clinical candidate eravacycline (1), which is currently undergoing phase 3 clinical studies (Figure 1).2

modest yield (41%), even at gram scale. The product of the Michael−Dieckmann reaction (8), which is obtained as a ∼1:1 diastereomeric mixture, required extensive flash chromatography to isolate the desired diastereomer, a process considered costly at large scale. Large scale manufacturing required a more robust and economical approach. For an efficient synthesis, we planned to prepare the LHP as a fully built single enantiomer 10 (Scheme 2), which should undergo Michael−Dieckmann reaction with enone 7,5 followed by deprotection steps to produce desired tetracycline 2. Stereoselective introduction of the chiral pyrrolidine moiety within LHP 10 is envisioned from bromide 9. Research and Manufacture of Intermediate 9. To manufacture material for GLP toxicity studies and a phase 1 clinical trial, several routes for intermediate 9 were investigated simultaneously using commercially available starting materials. The main goal for the compound synthesis was to make the route easily accessible and robust for early manufacturing and to minimize the cost of goods. Three major preliminary process research routes explored are described below. Synthesis of Intermediate 9 Starting with 2,5Dimethoxybenzoic Acid. Outlined in Scheme 3 is one of the initial routes we investigated using 2,5-dimethoxybenzoic acid (11) as the starting material. Regioselective methylation of acid 11 was achieved by modifying a procedure from the eravacycline program at Tetraphase.6 The key transformation in this route is the regioselective bromination at the C-4 position. After extensive investigations, we concluded that bromination of the C-4 position could only be achieved under the directing effect of a 3-hydroxy group. Owing to the electron withdrawing phenyl ester group, the C-6 methoxy within compound 13 could be selectively demethylated but not the C-3 one. Thereby both methoxy groups were cleaved with BBr3 to provide compound 14. Attempts to selectively protect the C-6 hydroxyl group with methyl, acetyl, benzyl, and TBS groups respectively failed. Finally, it was found that the C-3 hydroxyl group can be

Figure 1. Tetracyclines developed at Tetraphase.

TP-2758 (2) is another new tetracycline analog with novel substitutions at C-7 and C-8, which are difficult to install by semisynthetic methodologies. TP-2758 demonstrated potent activity against tetracycline-resistant isolates of Gram-negative bacteria including Enterobacteriaceae strains containing extendedspectrum β-lactamases, Proteus and Acinetobacter, but not Pseudomonas spp. TP-2758 also retains significant Gram-positive coverage. TP-2758 exhibited excellent oral and IV efficacy in multiple murine infection models, including pyelonephritis models with either uropathogenic E. coli or ESBL-producing K. pneumoniae and lung infection models with either tetracycline-resistant S. aureus or S. pneumoniae. The favorable IV/oral pharmacokinetic profile of TP-2758 further supports its candidacy for development as an IV/oral antibiotic for the empiric treatment of serious infections with a high incidence of Gram-negative pathogens.3 The CMC group was tasked to supply hundreds of grams of TP-2758 for toxicity studies and phase I clinical trials.



RESULTS AND DISCUSSION The discovery route used to prepare TP-2758 as well as a number of other analogs is shown in Scheme 1.4 The racemic left-hand piece (LHP) 6 was synthesized in 13 steps from commercially available but expensive starting material 3, including one reaction to install the methoxy group (from 4 to 5) with © 2015 American Chemical Society

Received: December 8, 2015 Published: December 28, 2015 284

DOI: 10.1021/acs.oprd.5b00404 Org. Process Res. Dev. 2016, 20, 284−296

Organic Process Research & Development

Article

Scheme 1. Discovery Route to TP-2758

Scheme 2. Retrosynthetic Analysis of TP-2758

Scheme 3. Synthesis of Intermediate 9 Starting with 2,5-Dimethoxybenzoic Acid

such as i-PrMgCl·LiCl or i-PrMgCl were not as regioselective as MeLi. However, the formation of acetonide 19 suffered from low yield and low throughput while the phenyl ester 23 formation required a large excess of TFA and TFAA, rendering this route less attractive. Further optimization of these two steps is required if this route were to be used for future production. Research and Manufacturing of Intermediate 9 Starting with 1,4-Dimethoxybenzene. Intermediate 9 was manufactured in kilogram scale according to the route shown in Scheme 5, starting from cheap and readily available 1,4dimethoxybenzene. In this 6-step sequence, the yields in all the steps are above 90% except for one bottleneck reaction, the methylation of phenyl ester 27 to produce compound 28. The methylation reactions of the corresponding acid 26, methyl, and tert-butyl ester substrates were also investigated. None of these proved to be a better substrate than phenyl ester 27; therefore, phenyl ester 27 was investigated thoroughly (Table 1). For all experiments the anion was generated by addition of phenyl ester 27 to a freshly prepared base solution at or below −70 °C. In general, the major issues causing low yield are low

selectively protected using Boc2O to give compound 15 along with ∼7% bis-Boc byproduct and ∼7% starting material. Protection of the C-6 hydroxyl group as a benzyl ether followed by Boc removal gave compound 16. Regioselective bromination of compound 16 with bromine in the presence of NaOAc gave the desired bromide 17, which was then methylated to provide intermediate 9. The protecting group manipulations made this 8-step route laborious, and a few steps required chromatographic purifications. In addition, the starting material, 2,5-dimethoxybenzoic acid, is much more expensive than the 1,4-dimethoxybenzene in Scheme 5 (vide infra). The cost and length of the route led to discontinuation of its optimization. Synthesis of Intermediate 9 Starting with 2,5Dihydroxybenzoic Acid. An approach outlined in Scheme 4 using commercially available 2,5-dihydroxybenzoic acid 18 as starting material was explored simultaneously with the above route. The key step in this approach is the regioselective methylation of compound 20 to produce compound 21, which was achieved using 1 equiv of MeLi followed by quenching of the resulting aryllithium with iodomethane. Other reagents 285

DOI: 10.1021/acs.oprd.5b00404 Org. Process Res. Dev. 2016, 20, 284−296

Organic Process Research & Development

Article

Scheme 4. Synthesis of Intermediate 9 from 2,5-Dihydroxybenzoic Acid

Scheme 5. Manufacture of Intermediate 9 from 1,4-Dimethoxybenzene

Table 1. Summary of Methylation Reactions entry

Base (1.05 equiv)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

LDA LDA LDA LDA LDA LDA LDA LDA LDA LDA LDA LTMP LiNEt2 LiNCy2 LiNsBu2 LiNtBuiPr

17

LiNsBu2

Additivesa TMEDA TMEDA TMEDA TMEDA TMEDA

Solvent

Conditionsb −60 °C −40 °C −20 °C 0 °C −78 °C −40 °C −78 °C −40 °C −40 °C −40 °C −40 °C −40 °C −40 °C −40 °C −40 °C −78 °C

TMEDA/Et3N·HCl TMEDA/Et3N·HCl TMEDA/Et3N·HCl

THF THF THF THF THF THF THF THF THF 2-MeTHF THF THF THF THF THF THF

A, A, A, A, B, A, B, A, A, A, A, A, A, A, A, B,

TMEDA/Et3N·HCl

THF

A, −40 °C

MgCl2 Et3N·HCl TMEDA/Et3N·HCl TMEDA/Et3N·HCl TMEDA/Et3N·HCl TMEDA

MeI (equiv) 6 6 6 6 6 6 6 6 6 6 1.5 6 6 6 6 6 3

28/27/PhOH (by 1H NMR) 1:0.51:0.81 1:0.22:0.38 1:0.35:0.50 1:0.37:0.48 1:0.24:0.59 Mostly SM 27 1:0.18:0.44 1:0.19:0.16 1:0.43:1.4 1:0.63:0.87 1:0.77:1.45 mostly amide 1:0.15:0.16 1:0:0.25 1:1.63:1.55 (0.57 undesired methylated regioisomer) 1:0.05:0.37

Yield of 28 (isolated)