Synthesis and characterization of impurities of an anticonvulsant drug, Pregabalin

During the process development of Pregabalin 1 , a known anticonvulsant drug, six potential impurities were identified in the final crude material ranging from 0.01 to 0.15% by LCMS. All six impurities were subsequently synthesized and characterized by IR, MS and NMR spectral data. Four of the six related substances are known as 4-isobutylpyrrolidin-2-one 6 , 3-isobutylglutaric acid 2 , ( R )-(-)-3-carbamoylmethyl-5-methylhexanoic acid 5 and ( R )-(-)-3- aminomethyl-5-methylhexanoic acid 8 , whilst ( S )-3-aminomethyl-5-methylhexanoic acid isobutyl ester 9 and ( S )-3-aminomethyl-5-methylhexanoic acid isopropyl ester 10 are new compounds reported for the first time in our process. The present work describes the formation, synthesis and characterization of these impurities.


Introduction
Pregabalin 1 ((S)-(+)-3-aminomethyl-5-methylhexanoic acid) is a novel and potent anticonvulsant agent for the treatment of epilepsy and pain. 1 It has also been found to be more active than Gabapentin in preclinical models of epilepsy. 2It has more potent and robust activity in various models of epilepsy, neuropathic pain and anxiety. 3he presence of impurities in an active pharmaceutical ingredient (API) can have a significant impact on the quality and safety of the drug products.Therefore, it is necessary to study the impurity profile of the API to be used in the manufacturing of drug substance.International Conference on Harmonization (ICH) guidelines recommended identifying and characterizing all impurities that are present at a level of 0.10% or more. 4,5As Pregabalin 1 is an important drug substance and to obtain information on product profile, a comprehensive study was undertaken on the impurities generated during the process development (Scheme 1). 6 Scheme 1. Reported synthetic scheme for Pregabalin.

Results and Discussion
During the process development of Pregabalin 1, HPLC analysis of crude Pregabalin revealed six impurities ranging from 0.01-0.15%.According to ICH (International Conference on Harmonization) guidelines the amount of acceptable level for known and unknown impurities in a final drug candidate must be less than 0.15% and 0.10% respectively.In order to meet the stringent regulatory requirements, the impurities needed to be identified and characterized.Hence, samples of Pregabalin 1 were initially analyzed by LCMS to provide parent ions at m/z 142, 187, 188, 216, 202 for five related impurities and at m/z 160 for the enantiomer of Pregabalin 1, and thus provide a basis for initial identification.To confirm their proposed structures and complete their characterization, all six substances were individually synthesized and characterized by their respective IR, MS, and NMR spectral data.The structure of these related substances were assigned as 4-isobutylpyrrolidin-2-one 6, 3-isobutylglutaric acid 2, (R)-(-)-3-carbamoylmethyl-5-methylhexanoic acid 5, (S)-3-aminomethyl-5-methylhexanoic acid isobutyl ester 9, (S)-3-aminomethyl-5-methylhexanoic acid isopropyl ester 10 and (R)-(-)-3aminomethyl-5-methylhexanoic acid 8 respectively.

Formation of related compounds
The substance 6 was formed during Hofmann reaction of (R)-(-)-3-carbamoylmethyl-5methylhexanoic acid 5 in the presence of bromine and sodium hydroxide.8a The related compound 6 was synthesized by the reaction of (R)-(-)-3-carbamoylmethyl-5-methylhexanoic acid 5 with excess of sodium hydroxide and bromine at elevated temperature (Scheme 2).The mass spectrum displayed a protonated molecular ion at m/z 142 and the NMR spectrum showed a peak at δ 7.44 ppm corresponding to cyclic amide -NH proton.Based on the spectral data the structure was confirmed as 4-isobutylpyrrolidin-2-one 6.  Related substance 2, a potential impurity which is base hydrolyzed product during Hofmann reaction, 8b was synthesized by the reaction of (R)-(-)-3-carbamoylmethyl-5-methylhexanoic acid 5 with excess of sodium hydroxide at elevated temperature (Scheme 3).The mass spectrum of compound 2, displayed a negative molecular ion at m/z 187 whilst in the IR spectrum a sharp band appeared at 1575 cm -1 corresponding to an aliphatic acid carbonyl and the spectral data is consistent with the structure of 3-isobutylglutaric acid 2. (R)-(-)-3-Carbamoylmethyl-5-methylhexanoic acid 5 is one of the key intermediate in the synthesis of Pregabalin 1.It was prepared by the resolution of racemic 3-carbamoylmethyl-5methylhexanoic acid 4 with (R)-1-phenylethylamine in the presence of chloroform and ethanol (Scheme 4).The mass spectrum of the substance 5, showed a protonated molecular ion at m/z 188.The IR spectrum showed a sharp band at 1712 cm -1 corresponding to acid carbonyl and at 1644 cm -1 corresponding to amide carbonyl.The NMR spectrum displayed a peak at δ 6.76 and δ 7.43 ppm corresponding to amide -NH2 protons.Based on the spectral data, the structure was confirmed as (R)-(-)-3-carbamoylmethyl-5-methylhexanoic acid 5. Any traces of (S)-(+)-3-carbamoylmethyl-5-methylhexanoic acid 7 that is present in (R)-(-)-3carbamoylmethyl-5-methylhexanoic acid 5 converts to enantiomer of Pregabalin i.e., (R)-(-)-3aminomethyl-5-methylhexanoic acid 8 during the Hofmann reaction with bromine and sodium hydroxide.Substance 8 was synthesized by the resolution of racemic amide 4 with (S)-1phenylethylamine in chloroform and ethanol followed Hofmann reaction (Scheme 5).The mass spectrum of the substance 8 displayed a protonated molecular ion at m/z 160.The IR spectrum showed a sharp band at 1644 cm -1 corresponding to acid carbonyl and the specific optical rotation of [α]D 25 -10.7°confirms the structure of (R)-(-)-3-aminomethyl-5methylhexanoic acid 8. Hofmann reaction of substance 5 with bromine and sodium hydroxide affords Pregabalin 1.However, as a side reaction isobutanol which is used as solvent for the extraction, reacts with 1 and leads to the formation of isobutyl ester of Pregabalin.This impurity was quantitatively synthesized in the form of hydrochloride salt by the reaction of Pregabalin 1 with thionyl chloride in the presence of isobutanol (Scheme 6).The mass spectrum of the substance 9 displayed a protonated molecular ion at m/z 216 and a sharp band at 1727 cm -1 was observed in the IR spectrum which was attributed to ester C=O stretching.The NMR spectrum exhibited 12 protons at δ 0.83-0.90ppm corresponding to four methyl groups and a doublet at δ 3.8 ppm corresponding to two protons of methylene group adjacent to oxygen atom.Based on the spectral data, the structure of this impurity is assigned as (S)-3-aminomethyl-5-methylhexanoic acid isobutyl ester hydrochloride 9.

Scheme 6
During Hofmann reaction, isopropanol which is used as a solvent for isolation reacts with 1, as a side reaction and leads to the formation of isopropyl ester of Pregabalin.This impurity was synthesized in the form of hydrochloride salt by the esterification of Pregabalin 1 with thionyl chloride in the presence of isopropanol (Scheme 7).The mass spectrum of the substance 10 displayed a protonated molecular ion at m/z 202 and a sharp band at 1727 cm -1 was observed in the IR spectrum which was attributed to ester C=O stretching.The NMR spectrum displayed a doublet at δ 1.2 ppm corresponding to two methyl groups and a multiplet at δ 4.86-4.95ppm corresponding to -CH proton of isopropyl ester.Based on the spectral data the structure of this impurity is assigned as (S)-3-aminomethyl-5-methylhexanoic acid isopropyl ester hydrochloride 10.

Figure 2 .
Figure 2. LC-MS Spectrum of pregabalin and its related substances.