Anti-Cancer Agents in Medicinal Chemistry   Send Orders for Reprints to reprints@benthamscience.ae Anti-Cancer Agents in Medicinal Chemistry, 2017, 17, 1777-1785 1777 RESEARCH ARTICLE Design, Synthesis and Biological Evaluation of Betulinic Acid Derivatives as New Antitumor Agents for Leukemia Fernanda Waechter a , Gloria N.S. da Silva a , Júlia B. Willig b , Cristiane B. de Oliveira a , Bruna D. Vieira c , Daniela B.B. Trivella c , Aline R. Zimmer a , Andréia Buffon b , Diogo A. Pilger b and Simone C.B. Gnoatto a,* a Laboratório de Fitoquímica e Síntese Orgânica, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752, Porto Alegre, 90610-000, Brazil.; b Laboratório de Análises Bioquímicas e Citológicas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752, Porto Alegre, 90610-000, Brazil; c Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Rua Giuseppe Maximo Scolfaro, 10000, Campinas, 13083-970, Brazil Abstract: Background: Chronic myeloid leukemia (CML) is currently treated with imatinib, a Bcr-Abl inhibitor. However, resistance to this drug usually develops over time. Triptolide, a diterpenoid triepoxide, has been shown active against CML cells resistant to imatinib, acting mainly on the level of Bcr-Abl transcription inhibition. Objective: Here, we used the triterpene betulinic acid, a known proteasome inhibitor with potential antileukemic activity, as a scaffold for the generation of analogues with predicted triptolide biological activity. Method: Betulinic acid derivatives were designed based on the structure-activity relationship of triptolide and evaluated for their cytotoxic effects in CML cells, lymphocytes and human keratinocytes (HaCaT), as well as against the proteasome complex. The main modification performed on betulinic acid was fluorination at C-28 and epoxidation, both of which are responsible for enhancing activity of triptolide. A total of 10 compounds were obtained: 6 previously described and 4 novel compounds. The cytotoxic activity over a CML cell line (K562) was assessed using flow cytometry and compared to lymphocytes and HaCaT. Result: The results show that betulinic acid was the most cytotoxic compound against CML cells, showing a good selectivity index for cancer over normal cells. The most important trend for the activity in betulinic acid derivatives is the presence of a free hydroxyl group at C-3 and a carboxyl group at C-28. Results also indicated that the epoxide is important for enhancing the activity, while modification at C-28 worsens the activity. Conclusion: Proteasome inhibition assays suggest that proteasome is the main target for betulinic acid and its derivatives. Keywords: Betulinic acid, cancer chemotherapy, triptolide, chronic myeloid leukemia, fluorination, epoxidation, proteasome inhibition. 1. INTRODUCTION Chronic myeloid leukemia (CML) is mainly caused by aberrant Bcr-Abl kinases, which are responsible for 95% of the uncontrolled proliferation of myeloid cells in CML. This disease is usually treated with imatinib (Glivec ® ), a tyrosine kinase inhibitor also used for the treatment of other types of cancers. However, clinical resistance is observed in patients treated with imatinib, mainly due to mutations in the Bcr-Abl imatinib binding site. This situation leaves the patients with few options and in general, the disease progresses to blast crisis and death [1, 2]. *Address correspondence to this author at the Laboratório de Fitoquímica e Síntese Orgânica, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752, Porto Alegre, 90610-000, Brazil; Tel/Fax: +55-51-3308-5451, +55-51-3308-5313; E-mail: simone.gnoatto@ufrgs.br Recently, pre-clinical studies were carried out with triptolide (compound 1, Fig. 1A), a molecule which showed activity in vitro and in vivo against CML cells resistant to imatinib [3], offering a potential alternative therapeutic for patients. Triptolide is a diterpene present in Tripterygium wilfordii [4], a plant used for centuries in Chinese medicine to treat inflammation and auto-immune diseases [5]. Triptolide was found to reduce Bcr- Abl levels, resulting in induced mitochondrial-dependent apoptosis in either CML and CML cells containing the Bcr-Abl-T315I clini- cal mutation. It has been shown to work following a mechanism that is independent of either caspase and proteasome activation [3]. RNA polymerases I and II were identified as the main biological targets of 1 [6]. The data available for its structure-activity relation- ship [7-10] reveals that the epoxides present in 1 are important for its antitumor activity [7], while the hydroxyl group at C-14 leads to greater activity when in the β position. In addition, when the hy- droxyl group is replaced by fluorine, the activity further increases A R T I C L E H I S T O R Y Received: September 19, 2016 Revised: March 23, 2017 Accepted: March 30, 2017 DOI: 10.2174/1871521409666170412143638 1875-5992/17 $58.00+.00 © 2017 Bentham Science Publishers