Mass spectrometry based identification of geometric isomers during metabolic stability study of a new cytotoxic sulfonamide derivatives supported by quantitative structure-retention relationships

Abstract

A set of 15 new sulphonamide derivatives, presenting antitumor activity have been subjected to a metabolic stability study. The results showed that besides products of biotransformation, some additional peaks occurred in chromatograms. Tandem mass spectrometry revealed the same mass and fragmentation pathway, suggesting that geometric isomerization occurred. Thus, to support this hypothesis, quantitative structure-retention relationships were applied. Human liver microsomes were used as an in vitro model of metabolism. The biotransformation reactions were tracked by liquid chromatography assay and additionally, fragmentation mass spectra were recorded. In silico molecular modeling at a semi-empirical level was conducted as a starting point for molecular descriptor calculations. A quantitative structure-retention relationship model was built applying multiple linear regression based on selected three-dimensional descriptors. The studied compounds revealed high metabolic stability, with a tendency to form hydroxylated biotransformation products. However, significant chemical instability in conditions simulating human body fluids was noticed. According to literature and MS data geometrical isomerization was suggested. The developed in sillico model was able to describe the relationship between the geometry of isomer pairs and their chromatographic retention properties, thus it supported the hypothesis that the observed pairs of peaks are most likely geometric isomers. However, extensive structural investigations are needed to fully identify isomers' geometry. An effort to describe MS fragmentation pathways of novel chemical structures is often not enough to propose structures of potent metabolites and products of other chemical reactions that can be observed in compound solutions at early drug discovery studies. The results indicate that the relatively non-expensive and not time- and labor-consuming in sillico approach could be a good supportive tool assisting the identification of cis-trans isomers based on retention data. This methodology can be helpful during the structural identification of biotransformation and degradation products of new chemical entities--potential new drugs.

Figure 1. Chemical structures of the 15 studied compounds.

Figure 2. Definition of the torsion angle in cis and trans isomers of the studied benzensulfonamide derivatives.

Figure 3. Exemplary liquid chromatography results: (A) Extracted ion chromatogram of water dissolved compounds 3 and 4; proposed peak assignment is presented, *compound 3 injected immediately after dissolution.

(B) Extracted ion chromatograms of compound 13 and its oxidized biotransformation products after incubation in the presence of HLM and NADPH, proposed peak assignment is presented. Experimental details in the text.

Figure 4. Common ions present in MS/MS fragmentation spectra of compound 14 and its three main biotransformation products.

M1 – hydroxylated metabolite, M2 – debrominated metabolite, M3 – metabolite both hydroxylated and debromianted.

Figure 5. MS/MS fragmentation spectrum of protonated compound 14 (m/z 471, A) and metabolite M1 (m/z 587, B) along with proposed structures of formed fragment ions.

Figure 6. MS/MS fragmentation spectrum of protonated M2 (m/z 493, A) and M3 (m/z 509) metabolite of compound 14 along with proposed structures of formed fragment ions.

Figure 7. Proposed MS/MS fragmentation pathway of compound 14.

Figure 8. 3-dimentional structures of compound 1 isomers obtained after optimization in the HyperChem software.

Figure 9. Correlation between experimental log k_w values and those predicted by the developed model.