Investigating the Stability of Six Phenolic TMZ Ester Analogues, Incubated in the Presence of Porcine Liver Esterase and Monitored by HPLC

Abstract

Previous published data from our group showed the encouraging in vitro activities of six phenolic temozolomide (TMZ) ester analogues (ES8-ES12 and ES14) with up to a five-fold increase in potency compared to TMZ against glioblastoma multiform cell lines and TMZ-resistant O⁶-methylguanine-DNA methyl transferase (MGMT)-positive primary cells. This study investigated the stabilities of the six phenolic TMZ ester analogues in the presence of porcine liver esterase (PLE) as a hydrolytic enzyme, using high-performance liquid chromatography (HPLC), monitored by a diode-array detector (DAD). Determining the rates of hydrolysis of the esters provided a useful insight into the feasibility of progressing them to the next phase of drug development. Fifty percent of TMZ esters consisting of para nitro, chloro, phenyl and tolyl groups (ES9, ES10, ES12 and ES14) were hydrolysed within the first 4.2 min of PLE exposure, while the TMZ esters consisting of para methoxy and nitrile groups (ES8 and ES11) demonstrated increased stability, with 50% hydrolysis achieved in 7.3 and 13.7 min, respectively. In conclusion, the survival of these phenolic TMZ esters on route to the target site of a brain tumor would be a challenge, mainly due to the undesirable rapid rate of hydrolysis. These findings therefore pose a question regarding the effectiveness of these esters in an in vivo setting.

Keywords: diode-array detector (DAD); glioblastoma multiform (GBM); high-performance liquid chromatography (HPLC); hydrolysis; phenolic TMZ esters; porcine liver esterase (PLE); stability; temozolomide (TMZ).

Figure 1

Structures of temozolomide (TMZ) and phenolic TMZ esters assessed for esterase-mediated stability.

Scheme 1

Esterase-mediated degradation of phenolic TMZ esters.

Figure 2

Comparative calibration curves for six TMZ ester analogues, highlighting the relationship between the concentration and the peak area (PA). Relative regression equations and R² values are shown in Table 2.

Figure 3

Comparative calibration graphs for the corresponding six alcohols, highlighting the relationship between the concentration and the PA. Relative regression equations and R² values are shown in Table 2.

Figure 4

Shows an example of how the LLOQ and the LLOD were determined. Chromatogram (A) was included in order to provide a comparison and identify the peak responses at low concentrations. Chromatogram (B), with TMZ acid (320 nM) and ES8 (207 nM) with retentions times of 2.3 and 9.7 min, respectively, was estimated to be the LLOQ for both TMZ acid and ES8. Chromatogram (C) represented a one-in-two dilution of the LLOQ. Chromatogram (D), with TMZ acid and ES8 at the concentrations of 80 and 52 nM, respectively, was estimated to be the LLOD. Chromatogram (E) represented a one-in-two dilution of the LLOD concentration and clearly indicated the absence of both the TMZ and ES8 analytes. The analysis was carried out at a wavelength of 325 nm.

Figure 5

A series of chromatograms showing the monitored degradation of ES11 into its corresponding TMZ acid and 4-Hydroxybenzonitrile. Each series of chromatograms showed a different wavelength, allowing all analytes to be visualised. (A) Breakdown of ES11 and the formation of 4-Hydroxybenzonitrile (250 nm). (B) Breakdown of ES11 and the formation of TMZ acid (325 nm).