Esters of betulin and betulinic acid with amino acids have improved water solubility and are selectively cytotoxic toward cancer cells Malgorzata Drag-Zalesinska a , Julita Kulbacka a , Jolanta Saczko a , Teresa Wysocka a , Maciej Zabel a,b , Pawel Surowiak a,c , Marcin Drag d, * a University School of Medicine, ul. Chalubinskiego 6a, 50-356 Wroclaw, Poland b Department of Histology and Embryology, Medical University of Poznan, Poland c Lower Silesian Oncology Centre, pl. Hirszfelda 12, 53-413 Wroclaw, Poland d Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland article info Article history: Received 27 April 2009 Revised 7 June 2009 Accepted 11 June 2009 Available online 14 June 2009 Keywords: Betulin, Betulinic acid Cytotoxicity Apoptosis abstract Betulin and betulinic acid are naturally occurring pentacyclic triterpenes showing cytotoxicity towards a number of cancer cell lines. Unfortunately they are practically insoluble in aqueous media and therefore their overall absorption index is not satisfactory. We have modified structures of both compounds by simple transformation to mono- and disubstituted esters of L-amino acids. This allowed us to achieve bet- ter water solubility without loss of the observed earlier anticancer properties. Comet assay on various cancer cell lines demonstrate that these compounds act via an apoptotic mechanism. Ó 2009 Elsevier Ltd. All rights reserved. Natural compounds to treat various types of cancer have re- cently attracted considerable interest due to their versatile biolog- ical properties and usually broad safety window during administration. One such group of compounds are triterpenes with betulinic acid being the best documented example demonstrating cytotoxicity toward several cancer cell lines. 1 This feature of betu- linic acid originally demonstrated by Pisha et al. on melanoma cells makes this compound an interesting drug candidate. Betulinic acid, for example, is currently undergoing phase II clinical trials for the treatment of melanoma. 2 Unfortunately, betulinic acid and its metabolic precursor betu- lin, that also harbors anticancer properties, are not very much sol- uble in aqueous media and therefore their overall absorption index is not satisfactory enough. However, their versatile molecular structure makes them ideal candidates for modifications using medicinal chemistry approaches. To date several reports have been published demonstrating that either simple or advanced modifica- tions may be performed without loss of the desired biological properties. There are two types of possible modifications of betulin and betulinic acid. 3 First, the hydroxy groups could be transformed into more soluble derivatives. 4,5 While, the second approach involves changing the triterpenes core rings, mostly by various substitu- tions or other types of chemical modifications. 6,7 In the approach presented in this manuscript, we decided to transform betulin and betulinic acid into their respective esters of amino acids. In the patent literature there is one example of a glycine ester of betulinic acid showing some cytotoxicity toward melanoma cells, but no further studies were performed within this group of compounds. 8 We assumed that modifications of betulin into diester derivatives should especially benefit the solubility index of these compounds and even if esterases cleave the ester 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.06.046 * Corresponding author. E-mail address: marcin.drag@pwr.wroc.pl (M. Drag). H O H H O O NH 3 + R POSSIBLE ESTERASE CLEAVAGE SITE O + H 3 N R POSSIBLE ESTERASE CLEAVAGE SITE R = side chain of the amino acid C-3 C-28 Figure 1. Esters of betulin with amino acids and possible cleavage sites by esterases. Bioorganic & Medicinal Chemistry Letters 19 (2009) 4814–4817 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl