N-Terpenyl Benzisoselenazolones — Evaluation of the Particular Structure-Bioactivity Relationship †

Different activity of compounds with the same molecular formula but possessing alternative bonding arrangement or orientation of atoms in space is a very important issue when constructing molecules that can selectively interact with certain domains of the biological target. Herein, we present the synthesis of the new chiral benzisoselenazol-3(2H)-ones substituted on the nitrogen atom with three monoterpene moieties: p-menthane, pinane and carane. All derivatives were obtained by the reaction of an appropriate amine with 2-(chloroseleno)benzoyl chloride. The applied terpenyl amines were first synthetized by a multistep procedure starting from the corresponding alcohol (p-menthane system) or alkene (pinene and carene systems). Finally, all compounds were tested as antioxidants and antiproliferative agents on breast cancer MCF-7 and human promyelocytic leukemia cell line HL-60. The correlation between the structure of the obtained organoselenium compounds, representing examples of various isomers including enantiomers, diastereoisomers and regioisomers, and their bio-activity was thoroughly evaluated.


Introduction
When selecting new drug candidates several characteristics have to be considered e.g., therapeutic potential, toxicity, bioavailability, metabolism and selectivity towards specific targets. The binding affinity between the molecule and the active site of the particular protein/enzyme is determined by their structural compatibility. As biochemical reactions are often highly stereoselective, a properly fitted 3-dimensional structure of the drug candidate is an important feature for the selective molecular interaction [1,2]. The goal of this research was to evaluate the influence of a chiral substituent on the bio-activity of new organoselenium derivatives and recognize the structural motifs that increase the selective drug-target bonding. The designed compounds are a combination of two building blocks: (1) a benzisoselenazol-3(2H)-one core, that is essential for the GPx-like activity and oxidative stress prevention and (2) an optically active monoterpene substituent that was constructed using terpenyl amines obtained from three different monoterpene systems: pmenthane 1-3, carane 4, 5 and pinane 6-8 [3]. All synthetized compounds were tested as antioxidants and anticancer agents. The activitystructure correlation was evaluated and several proactive features were selected.

Results and Discussion
First, a series of the new N-substituted benzisoselenazol-3(2H)-ones was synthetized by our previously reported multistep procedure starting from anthranilic acid 9 which was transformed to 2-(chloroseleno)benzoyl chloride 10 and further converted to the final product through the reaction with an appropriate terpenyl amine 1-8 (Scheme 2) [4].

Scheme 2. Synthesis of benzisoselenazol-3(2H)-ones 11-18.
Next, the ability to reduce peroxides was tested by an NMR assay where dithiol (DTT red ) is transformed to a disulphide (DTT ox ) by a selenocatalyst first oxidized in the presence of hydrogen peroxide ( Figure 1) [5]. The rate of the reaction was measured based on changes in the 1 H NMR spectra (Table 1).  The highest antioxidant activity was observed for all derivatives from the pinane system: enantiomers 16/17 and regioisomer 18, the total substrate conversion was observed after 30 and 60 min, respectively. These results correlate with our previous observation that the bulky bicyclic bornane skeleton also enhances the antioxidant capacity of benzisoselenazol-3(2H)-one core and reduces the DTT red oxidation time to 5 min [4].
Next, the antiproliferative capacity was measured by a cell viability assay (MTT) on breast cancer MCF-7 and human promyelocytic leukemia cell line HL-60 (  The N-isopinocampheyl-1,2-benzisoselenazol-3(2H)-one 16 exhibited the highest antiproliferative potential with IC50 of 7.1 ± 0.4 µM (HL-60 cell line). Attachment of the isoselenazolone ring to the C10 carbon decreased the activity, what was highly emphasized in the results obtained for HL-60 cells with the increase of IC50 from 7.1 ± 0.4 to 250 ± 24.7 µM.
For MCF-7 cell line the lowest IC50 was measured for N-menthyl-1,2-benzisoselenazol-3(2H)-one 13 with the value of 11.9 ± 0.2 µM. We have also observed a different activity for both enantiomeric pairs derived from p-menthane and pinane. The diversity was more apparent in the case of compounds 11 and 12, where one enantiomer was far more active with IC50 values 12.4 ± 0.4 µM and 85.5 ± 4.0 µM, respectively. Compounds 11 and 13, with good antiproliferative capacity, have the same configurations on C1 and C4 carbon atoms, on the contrary to less active enantiomer 12. The stereochemistry of C2, attached to the isoselenazolone ring, seems not to influence the reactivity. Moreover, the molecule 13 possesses the same structural motif that was present in the structure of other active N-alkyl benzisoselenazolones 19 and 20-an incorporated 2-methylbutyl chain (Scheme 3) [7]. In the case of carane regioisomers 14 and 15 the IC50 was lower when the N-Cterpene bond was more hindered by the methyl group and the cyclopropane bridge. The overall activity decreased when the compounds were tested on HL-60 cell line.

Conclusions
In this paper we have synthetized a series of N-terpenyl benzisoselenazol-3(2H)-ones derived from three monoterpene systems: p-menthane, carane and pinane. The procedure was based on the reaction of 2-(chloroseleno)benzoyl chloride with an appropriate terpene monocyclic or bicyclic amine. The obtained 8 chiral organoselenium compounds included enantiomers, epimers and regioisomers were further tested as antioxidants and anticancer agents. The obtained results and deducted structure-activity evaluation enabled to propose that: (1) the more bulky the terpenyl substituent the higher the antioxidant potential; (2) the antiproliferative activity differs significantly for both enantiomeric pairs derived from p-menthane and pinane; (3) the configuration of C1 and C4 carbon in the p-menthane skeleton can be essential for the improvement of the bio-activity, (4) the presence of an incorporated 2-methylbutyl chain can increase the anticancer capacity; (5) the more hindered the N-Cterpene bond the higher the antiproliferative potential.