Insight into the mechanism of three component condensation leading to aminomethylenebisphosphonates Ewa Da ˛ browska a , Agnieszka Burzyn ´ ska a , Artur Mucha a , Ewa Matczak-Jon b , Wanda Sawka-Dobrowolska c , Lukasz Berlicki a , Paweł Kafarski a, * a Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze _ ze Wyspian ´skiego 27, 50-370 Wrocław, Poland b Department of Inorganic and Structural Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze _ ze Wyspian ´skiego 27, 50-370 Wrocław, Poland c Department of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland article info Article history: Received 8 May 2009 Received in revised form 7 July 2009 Accepted 10 July 2009 Available online 16 July 2009 The contribution is dedicated to Prof. Roman Tyka on his 85th anniversary Keywords: Aminomethylenebisphosphonates Reaction mechanism Organophosphorus chemistry abstract Three-component reaction of a primary amine, diethyl phosphite and triethyl orthoformate followed by acid hydrolysis of the adduct provides N-substituted aminomethylenebisphosphonic acids in good yields. Being extremely versatile, it is commonly utilized for preparation of compounds possessing potential antiosteoporotic, antibacterial, anticancer, antiparasitic or herbicidal activity. However, the mechanism of the reaction remains unknown. p-Nitroaniline has been found an interesting tool to shed light on this matter. Its use allowed to separate and identify four intermediates, both non-phosphorus and phospho- rus containing, and subsequently suggest the mechanism of the whole process. The acquired knowledge was helpful in explanation the route and the final product structure obtained for more complex reaction proceeding with the use of 4-aminopyridine. Additional alkylation of the pyridine nitrogen atom, leading to unexpected N-(1-alkylpyridinium-4-amino)methylenebisphosphonic acids was unambiguously proved. Ó 2009 Elsevier B.V. All rights reserved. 1. Introduction Bisphosphonic acids are hydrolytically stable analogues of pyro- phosphate characterized by a common P–C–P fragment, in which the oxygen-to-phosphorus bonds are replaced by the carbon-to- phosphorus bonds. Bisphosphonates have been employed as ther- apeutic agents for treatment of bone disorders, hypocalcaemia of malignancy and osteoporosis for over two decades [1]. Despite this, the molecular mode of their action is still not clear and attracts considerable attention [2]. Recently, a promising drug delivery sys- tem using bisphosphonate moiety for targeting osseous tissues was also proposed [3]. Additionally, bisphosphonates have been found to have antibacterial [4] and anticancer [5] properties and to stimulate cd T cells of immune system, drawing interest in can- cer immunotherapy [6]. A subclass of bisphosphonates, derivatives of aminomethylene- bisphosphonic acid, has been also described to exhibit promising antiparasitic [7] and herbicidal activities [8]. It is worth to note that a representative of the latest generation of antiosteoporotic drugs – cycloheptylaminomethylenebisphosphonate, discovered in the early 90s [9] and commercialized as Incadronate, belongs to such modified bisphosphonic acids. The simplest procedure for preparation of amino- methylenebisphosphonates relies on three-component reaction between a primary or secondary amine, triethyl orthoformate and diethyl phosphite, followed by acid hydrolysis (Scheme 1) [10]. Although this straightforward procedure affords desired products of a wide structural variety, the detailed route leading to the target structure is not recognized yet. The yield is usually satisfactory, however it happens unpredictably low. Moreover, unexpected by-products are obtained in those particular cases. In this paper we wish to present the results of our studies on elucidation of the mechanism of the title three component conden- sation. The overall reaction route has been proposed on the basis of the structure of isolated intermediates and conversion reactions observed between them. These findings supported elucidation of the structure of unusual alkylated products obtained upon forma- tion of pyridinylaminomethylenebisphosphonates. 2. Results and discussion 2.1. Reaction with p-nitroaniline and identification of intermediates The analysis of the general mechanism of amino- methylenebisphosphonates formation in the condensation of dial- kyl phosphite, trialkyl orthoformate and amine was performed using p-nitroaniline as the nucleophilic component. This compound was 0022-328X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jorganchem.2009.07.025 * Corresponding author. Tel.: +48 71 320 36 82; fax: +48 71 328 40 64. E-mail address: pawel.kafarski@pwr.wroc.pl (P. Kafarski). Journal of Organometallic Chemistry 694 (2009) 3806–3813 Contents lists available at ScienceDirect Journal of Organometallic Chemistry journal homepage: www.elsevier.com/locate/jorganchem