Tetraphase Pharmaceuticals Achieves Process-Scale Synthesis of New Tetracycline Derivative - Pharmaceutical Technology
Tetraphase Pharmaceuticals Achieves Process-Scale Synthesis of New Tetracycline Derivative
Synthesis of tetracycline derivatives with novel substituents has been challenging. Using an approach based on a Michael-Dieckmann reaction, new compounds with enhanced antibiotic properties are now being prepared on a large scale.


Pharmaceutical Sciences, Manufacturing & Marketplace Report

Tetracyclines comprise a group of antibiotics that are recognized as safe and effective and are thus commonly used to treat serious bacterial infections and other less severe conditions such as acne. Unfortunately, because tetracyclines are commonly used, many bacteria have developed resistance to the older versions of these drugs. Recent efforts have thus been directed at developing new tetracycline derivatives.

Scientists at Tetraphase Pharmaceuticals are overcoming this barrier by implementing a new synthetic route first reported by Myers in 2005 (1). This approach involves the coupling of a cyclohexenone intermediate that contains the key tetracycline functionalities with a second functionalized aromatic intermediate via a Michael-Dieckmann reaction, thus enabling the incorporation of a variety of different substituents at various positions in the tetracycline skeleton. Using this methodology, Magnus Ronn, vice-president of CMC at Tetraphase Pharmaceuticals and his colleagues at the company recently reported the successful preparation of eravacycline, a fully synthetic broad spectrum 7-fluorotetracycline in clinical development, in multihundred gram quantities (2). A summary of their work is presented below.

A single key intermediate
The advantage of this approach to the synthesis of tetracycline analogues is that a single key intermediate can be used to access a wide range of substituted tetracycline active pharmaceutical ingredients (APIs),” says Ronn. This key intermediate is a tricyclic cyclohexenone with three chiral centers  (the synthesis of this compound was reported previously (3)).  The enone is reacted with a suitably functionalized phenol bearing an ortho-carboxyphenyl group and a meta-methyl substituent. Other functionalities are included as needed to produce the desired tetracycline analogue.

This aromatic compound, referred to by the researchers as the lefthand piece (LHP), is deprotonated with a strong base to form a benzylic anion, which then undergoes diastereoselective 1,4-conjugate (Michael) addition to the enone moiety when added to the cyclohexenone. The ketone enolate that forms from this step undergoes a Dieckmann-type condensation with the phenyl ester to produce the protected tetracycline compound. To obtain the desired tetracycline analogue, this intermediate is subjected to subsequent silyl-ether cleavage and hydrogenolysis of the benzyl protecting groups with concomitant reductive ring opening of the isoxazole (2).

A suitable LHP
The LHP selected for the preparation of eravacycline is a benzyl-protected phenol with a fluorine atom and a dibenzylamine substituent. It was prepared from a commercially available starting material in seven steps, the synthesis of which will be published in the future (2).

The key MichaelDieckmann reaction
One of the hurdles that the researchers had to overcome in developing the large-scale synthesis of eravacycline was the sensitivity of the Michael−Dieckmann transformation to the reaction conditions, according to Ronn. Not only the order of addition, but the strength of the base was important for the two different deprotonation steps (2). Thus, the researchers reported that it was necessary to first deprotonate the LHP (1.04 equivalents of LHP is used) with lithium diisopropylamide (LDA, 1.13 equivalents) and then add the generated anion to a solution of the cyclohexenone and the weaker base lithium bistrimethylsilylamide (LiHMDS) at -70 °C. The desired adduct was isolated after workup and trituration with methanol in > 90% yield a 98% purity (using high-performance liquid chromatography), even on the 200-g scale (2).

Because both the deprotonation and the Michael−Dieckmann reaction should be performed at -70°C, two cryogenic reactors are required. The researchers reported that attempts to eliminate one of those reactions by raising the temperature of the cyclohexenone solution to -20 °C led to increased production of impurities (2).

Final steps
To obtain eravacycline, the first step after the Michael-Dieckmann reaction involved cleavage of the tert-butyl silyl (TBS) protecting group. Despite the issues associated with using hydrofluoric acid in commercial manufacturing, the researchers reported that this reagent gave better results than other investigated alternatives and it was thus selected for scale-up (2).

Reductive ring opening of the isoxazoline group and removal of the four benzyl groups using palladium on carbon(Pd/C)/hydrogen to give the 9-amino-7-fluoro-sancycline required extensive investigation by the researchers (2). A mixed solvent system of tetrahydrofuran (THF) in methanol (1:3) was required because of solubility issues. An acid additive was also needed to improve the rate of the hydrogenation reaction, but epimerization at the C-4 position and reduction of undesired groups led to the formation of impurities, including one that was very difficult to separate from the desired product. The reaction was optimized using concentrated aqueous hydrochloric acid (HCl) because it is a stable reagent with a reliable concentration. The palladium on carbon was removed using Celite, and residual palladium was eliminated with the metal scavenger (SiliaBond DMT, Silicycle). The desired hydrochloride salt was precipitated from water/ethanol in approximately 80% yield and high purity (< 2% of the undesired impurities), even on a large scale (2).

Next, the hydrochloride salt of the fully deprotected penultimate intermediate was coupled with the desired side chain to prepare eravacycline. The reaction was carried out in acetonitrile and water. To achieve complete conversion, several charges of the acid chloride were necessary, and it was also found that adjustment of the pH from approximately 3 to approximately 7 after the second charge aided the complete dissolution of the starting material, allowing the reaction to go to completion. After the completion of the coupling, the pH of the reaction solution was brought to pH 6.8 to ensure hydrolysis of any over-acylated compounds to the desired tetracycline product.

Eravacycline was extracted using dichloromethane at pH 7.4. As an added benefit, the researchers found that the undesired C-4 epimer was partly removed in the aqueous layer and when the dichloromethane solution was dried with sodium sulfate prior to evaporation, thus increasing the purity of the tetracycline product (2). Finally, the bis-hydrochloride salt of eravacycline was prepared using an ethanol−methanol mixture containing an excess of hydrogen chloride and precipitated with addition of ethyl acetate.

“While some of the steps presented challenges, this overall route to eravacycline has enabled the production of sufficient quantities of the API for clinical testing. This tetracycline derivative has completed Phase II clinical studies and has been shown to be active against multidrug resistant bacteria and is therefore a candidate as a broad spectrum antibiotic for serious hospital infections. We are continuing to improve the process for future larger-scale manufacturing and are also developing an isolation procedure that will be suitable for commercial production of eravacycline,” Ronn notes.

References

  1. M.G. Charest et al. Science 308 (5720) 395-398 (2005).
  2. M. Ronn et al., Org. Process Res. Dev. 17 (5) 838-845 (2013).
  3. J. D. Brubaker and A. G. Myers, Org. Lett. 9 (18) 3523-3525 (2007).
Source: Pharmaceutical Sciences, Manufacturing & Marketplace Report,
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