Characterization of DNA Interstrand Cross-Linking Agents by Liquid Chromatograhy-Mass Spectrometry

Abstract

This study is to characterize novel reactive oxygen species (ROS)-activated nitrogen mustard analogues and UV-activated alkylating agents by Liquid Chromatography-Mass Spectrometry (LC-MS). First, we optimized the instrument conditions to successfully ionize a few novel binapthalene analogues and methoxybenzene analogues by MS. The highest signal intensity for compounds 3-10 was observed when atmospheric pressure chemical ionization (APCI) with an optimized corona needle position of 5mm or 10 mm was used, which led to successful analysis of compounds 3-10 by MS. Second, we determined pharmacokinetic properties of a drug compound (FAN-NM-CH3), including animal study, assay development, optimization of MS/MS instrumental parameter and the development of a calibration model, and finally quantification of FAN-NM-CH3 in different tissues, including blood, liver, and brain by LC-MS/MS. The results indicated that the methyl analogue FAN-NM-CH3 showed a t1/2 of 8.84 min that is two times of the parent compound CWB-20145 with a t1/2 of 4.92 min. The results suggested that introduction of an alkyl group (CH3) greatly increases in vivo duration, which is an important guide for further modification. The rate of elimination for the parent compound CWB-20145 in the blood is two times faster (Erate = 0.141 min−1) than that of FAN-NM-CH3 (Erate = 0.078 min−1). FAN-NM-CH3 showed an area under the curve (AUC) of 16.25 µg•min/mL, which is significantly higher than that of CWB-20145 (10.88 µg•min/mL). Further in vitro microsomal stability studies revealed that FAN-NM-CH3 is significantly more stable in human (t1/2 = 77.06 min) than in mouse (t1/2 = 33.0 min). The human microsomal stability of FAN-NM-CH3 supports its design as a human therapeutic.