Synthesis of Naringenin Amino Acid Esters as Potential CDK2 Inhibitors

Abstract

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. Among the polyhydroxylated flavones, flavopiridol (1, Figure 1) has completed phase I clinical trial where it showed antitumor effect in patients with refractory neoplasms. This encouraging result, which is consistent with the ability of 1 to inhibit proliferation of cancer cells in vitro makes polyhydroxylated flavone an interesting target for further analogue synthesis. In continuation of our studies aimed toward the development of more potent and more selective anticancer agents, we designed flavone derivatives for further biological evaluation. The flavone naringenin is widely spread in nature and easily extracted from a lot of different plants. Their protective effect against lipid peroxidation of membranes, involved in several physiological and pathological disorders, as aging, inflammation, atherosclerosis, ischemia, toxicity of oxygen and chemical substances has been largely reported. Thus, interesting biological activity as well as structural similarity with flavopiridol makes naringenin one of the most interesting candidates for designing novel CDK2 inhibitors. However, derivatives of naringenin have not been studied extensively presumably due to the synthetic difficulties. Particularly, naringenin is awkward to make derivatives due to its low solubility in water as well as in organic solvents. In view of designing naringenin derivatives, the 7-O position of naringenin (2, Figure 1) is particularly attractive because it is well known that electrophiles react with hydroxyl group preferentially at the 7-position. Also, comparison of the biological activity of 7-O-substitued naringenin with that of flavopiridol would provide interesting insights into designing novel flavonoid CDK2 inhibitors due to their subtle distinction in three dimensional structures. On the other hand, as a limiting factor of the use of flavonoids is their low water solubility, another aim of this study was to increase the solubility and dissolution rate of naringenin by conjugate formation with polar substituents. Thus, we set out to prepare various naringenin-amino acid conjugates (2, Figure 1) because amino acids substituted at the 7-O position would serve as an excellent model for our purpose: formation of the amino acid conjugate would benefit naringenin from the various substituent effect as well as increased solubility. Herein we report our efforts to synthesize the title compounds. Recently, two classes of naringenin derivatives, naringenin 7-O-oleic ester and naringenin 7-O-cetyl ether, were prepared for biological evaluation of their anti-atherogenic activity by using the mixed anhydride strategy. However, in our case, due to the epimerization problem at the amino acid αcarbon under the reaction conditions, we decided to use rather mild coupling conditions which was successfully exploited for the preparation of the amino acid esters of 5,7dihydroxy-3-phenoxychromones (3, Figure 2). Thus, a mixture of naringenin (4), N-Boc-L-alanine, and DMAP in anhydrous THF or DMF was treated with EDCI (Scheme 1) and the reaction was monitored by TLC. However, the reaction did not proceed at all presumably due to the solubility problem of the naringenin. After extensive survey of the appropriate solvent system for the coupling reaction, methylene chloride (CH2Cl2) was found to be the solvent of choice: initial slurry of naringenin and NBoc-L-alanine in anhydrous CH2Cl2 became a clear solution as the reaction proceeds, and the reaction was complete in 2