Synthesis and Characterization of Impurities in the Production Process of Lopinavir

Lopinavir is an antiretroviral drug used for the inhibition of HIV protease. Four related substances of lopinavir were observed during the manufacturing process of lopinavir in the laboratory and they were identified. The present work describes the origin, synthesis, characterization, and control of these related substances.

The presence of impurities in an Active Pharmaceutical Ingredient (API) will influence the quality and safety of the drug product. In the regulatory guidelines of the International Conference on Harmonization (ICH), it is recommended that impurities amounting to more than 0.1% [1] should be identified and characterized. Impurities are required in pure form to check the analytical performance characteristics such as specificity, linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), robustness, system suitability testing, and relative retention factor [2].
During the process development of lopinavir 1 in our laboratory, we observed the formation of four substances that are structurally related to lopinavir. These unknown related substances were identified, monitored, and their structures were tentatively assigned on the basis of their fragmentation patterns in LC-MS [3]. In the present work, the identified related substances of lopinavir were synthesized and characterized by various spectroscopic techniques and further confirmed by co-injection studies using qualitative HPLC analysis. A few references [3][4][5][6][7][8][9] were found in the literature for related substances of lopinavir and its metabolites.

Fig. 1.
Possible synthetic pathways for lopinavir 1 and its related substances (7)(8)(9)(10) Phenols have a tendency to polymerize during storage; it is therefore necessary to study the dimers of phenols. We observed a dimer (13) at a level of ~0.3% in 2,6-dimethylphenol used in the laboratory and it was subsequently reacted to give lopinavir dimer 7. Lopinavir dimer 7 was independently prepared by the dimerization of 2,6-dimethylphenol to give compound 13. Compound 13 was acylated with chloroacetic acid to give compound 14.
Compound 14 was condensed with compound 4 to produce lopinavir dimer 7 (as shown in Scheme 3).

Lopinavir Carboxymethyl Analog 8
During the acylation of 2,6-dimethylphenol with chloroacetic acid (as shown in Scheme 2), the formation of ~0.2% of compound 19 was observed and was reacted in the subsequent steps to give lopinavir carboxymethyl analog 8.

Lopinavir Diacylated Impurity 10
Lopinavir diacylated impurity 10 was formed due to the acylation on the nitrogen presented in the pyrimidine ring of lopinavir with 2,6-dimethylphenoxy acetic acid during the synthesis of lopinavir. It was independently prepared by the condensation of compound 1 with compound 12. Related substances 8-10 were eliminated during the purification of lopinavir 1. Related substance 7 originated due to the presence of dimer 13 in 2,6-dimethylphenol. It was controlled by keeping the limit in the specification of 2,6-dimethylphenol raw material.

(Carboxymethoxy)-3,5-dimethylphenyl]acetic acid (19):
A mixture of (4-acetyl-2,6-dimethylphenoxy)acetic acid (17, 25 g, 112 mmol) and sulfur powder (5.4 g, 168 mmol) in morpholine (24.52 g, 281 mmol) was heated to 130°C and stirred for 12 h at 130°C. The reaction mass was cooled to 20-25°C and we added 10% w/w ethanolic sodium hydroxide solution (150 g, 375 mmol). Again, the mass temperature was raised to 85 °C and stirred for 10 h at 85°C. Thereafter, the reaction mass was concentrated at 85°C under reduced pressure, the obtained residue was dissolved in DM water (250 mL) and washed with ethyl acetate (250 mL). The aqueous layer was adjusted to pH 5.0 with concentrated hydrochloric acid (24 mL) at 20-25°C and again washed with ethyl acetate (125 mL) at 20-25°C. After that, the aqueous layer was adjusted to pH 1.0 with concentrated hydrochloric acid (12 mL) at 20-25°C and we extracted the product with dichloromethane (4X 100 mL). The combined dichloromethane layer was concentrated below 40°C under reduced pressure to obtain a gummy solid, which was crystallized from ethyl acetate and hexane to obtain 15 g (46%) of pure [4-