Fragmentation of diamide derivatives of 3,4-ethylenedioxythiophene Ivana Stolić 1 , Igor Bratoš 2 , Goran Kovačević 3 and Miroslav Bajić 1 * 1 Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia 2 TAPI Research & Development, PLIVA Croatia Ltd., Prilaz baruna Filipovića 25, 10000 Zagreb, Croatia 3 Ruđer Bošković Institute, P.O.B. 180 HR-10002 Zagreb, Croatia The sequential product ion (MS n ) fragmentation of four symmetric diamide derivatives of 3,4-ethylenedioxythiophene were characterized using ion trap mass spectrometry with electrospray ionization and their fragmentation patterns were studied. The experimental data consists of mass spectra obtained by tandem mass spectrometry, and calculations were obtained by the M06-2X/6-31 G (d,p) method. Investigated compounds represent building blocks in synthesis of compounds used in different areas of chemistry and industry such as in medicinal chemistry, as potential anticancer and anticonvulsant agents, in organic chemistry as linkers for solid-phase synthesis, and in the synthesis of a variety of materials in polymer chemistry. We present herein the investigation of the fragmentation pathway of protonated diamide derivatives of 3,4-ethylenedioxythiophene that involves the identification of fragments, influence of proton transfer on direction of fragmentation and mechanisms of reactions by which the fragmentation process occurs. Data obtained from product ion spectra of these protonated compounds and density functional theory (DFT) calculations indicate that the fragmentation process takes place via four main reactions: amido-iminol proton transfer, reverse cycloaddition, cleavage of the amide bond, and isocyanic acid elimination. The 3,4-ethylenedioxythiophene-2,5-dicarboxamide was observed as an intermediate in the fragmentation of its alkyl derivatives. To our knowledge, this work brings the first correct description of the mechanism of elimination of isocyanic acid. Copyright © 2012 John Wiley & Sons, Ltd. 3,4-Ethylenedioxythiophene (EDOT) is an interesting com- pound, particularly its derivatives and varieties of bondings it is involved in. [1] It is an important motif in the formation of chains and, more interestingly, bridges in polymers and in varieties of composite materials. [2–4] The polymer chemistry of the derivatives of EDOT has led to some interesting practical applications, including antistatic treatment of plastics and electrode material for solid electrolyte capacitors. [5–8] Recently, the derivatives of EDOT have been recognized as precursors or intermediates for the synthesis of a variety of potential biologically active compounds, arylamidine being one of them. [9–11] Arylamidine is one of the most investigated compounds in anticancer treatment; [12,13] however, the com- pounds in this class display numerous serious side effects. [13] Therefore, the main focus of present research is to modify the structures of potential anticancer compounds from this class by reducing the systematic side effects without losing their biological activity. One of these strategies involves incorpora- tion of the robust 3,4-ethylenedioxythiophene moiety as a linker between two arylamidines, instead of an unstable alkyl chain or mono-heteroaromatic ring. [14] Characterization of unknown metabolites in biological sam- ples is one of the most challenging tasks in metabolomic research. [15–17] Metabolism studies are essential in the drug discovery and development process and are important to support the safety studies and the clinical development of drug candidates. [18,19] Several methods have been applied in the anal- ysis of drugs and their metabolites, such as radioimmunoassay (RIA), liquid chromatography (LC), and many more, but among them mass spectrometry (MS) is the most widespread method for detection of metabolites and fragments produced by degra- dation of organic compounds. [20–23] The method is very sensitive and selective, thus enabling fast analysis of biologically active compounds. As part of our ongoing project of diarylamidine derivatives of 3,4-ethylenedioxythiophene as potential biologically active compounds, the fragmentation pathways of synthesized amide derivatives will provide additional information about the fragmentation of more complex compounds which in their structure exhibit a diamide-3,4-ethylenedioxythiophene part as the conserved structural core. To date, chemical transformations of 3,4-ethylenedioxythio- phene have resulted in the synthesis of three series of amidine derivatives whose anticancer activity depends on their chemical structure. Metabolite identification of molecules from this class is crucial to the drug discovery process because it can be used to investigate the metabolites that are likely to be formed in vivo as well the influence of chemical structure on formation of pharmacologically active or toxic metabolites. Early identifi- cation of potential metabolic liabilities provides a better perspec- tive in the design of the synthetic route with the aim to optimize the pharmacokinetic and safety profiles of newly synthesized drug candidates of this class of compounds. Thus, as part of our ongoing project on the use of electro- spray ionization tandem mass spectrometry (ESI-MS/MS) in the structural elucidation of potential anticancer agents, * Correspondence to: M. Bajić, Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia. E-mail: mbajic@vef.hr Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 1023–1031 Research Article Received: 16 November 2011 Revised: 9 February 2012 Accepted: 10 February 2012 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2012, 26, 1023–1031 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6196 1023