New hits as phase II enzymes inducers from a focused library with heteroatom–heteroatom and Michael-acceptor motives

The increased activity of phase-II-detoxification enzymes, such as quinone reductase (QR) and glutation S-transferase (GST), correlates with protection against chemically induced carcinogenesis. Herein we studied 11 different chemotypes, pyrazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiazole, 1,3,4-oxathiazole, thienyl hydrazone, α,β-unsaturated-oxime, α,β-unsaturated-N-oxide, coumarin and α,β-unsaturated-carbonyl, as phase-II enzymes inducers in order to identify new pharmacophores with chemopreventive activity. Fifty-four compounds were analyzed on wild-type mouse-hepatoma Hepa-1c1c7 and on the aryl-hydrocarbon-nuclear-translocator (Arnt)-defective mutant BpRc1 cells. New monofunctional inducers of QR and GST were identified, the 1,2,5-oxadiazol-2-oxide (3), the 1,2,4-triazine-4-oxides (23) and (32) and the tetrahydropyrimidinones (28) and (49). It was confirmed that Nrf2 nuclear translocation is the operative molecular mechanism that allows compound (3) to exert protective effects via expression of downstream phase-II enzymes.

Cancer chemoprevention is the prevention, delay or reversal of the carcinogenesis by administration of drugs. A group of chemopreventative agents includes quinone reductase and glutation S-transferase. Herein we have studied 11 chemotypes, trying to identify new pharmacophores for chemopreventives. We found new inducers of quinone reductase and glutation S-transferase, with excellent in vitro chemopreventive indexes, the 1,2,5-oxadiazol-2-oxide (3), the 1,2,4-triazine-4-oxides (23) and (32) and the tetrahydropyrimidinones (28) and (49), confirming that Nrf2 nuclear translocation is the operative molecular mechanism that allows compound (3) to exert protection. We have therefore highlighted good candidates for further in vivo studies of cancer chemopreventive activity.
Keywords: 1,2,5-oxadiazol 2-oxide • 1,2,4-triazine 4-oxide • chemopreventive agents • phase II enzyme inducers • tetrahydropyrimidinone The process of carcinogenesis involves intricate and prolonged events launched by the cellular biomolecule damages promoted by endogenous or exogenous agents. The prevention of carcinogenesis, cancer chemoprevention, implies the avoidance, the slowing or reversing of the carcinogenic process through the use of drugs named as chemo preventive agents (ChAs). ChAs could act by suppressing the activation by metabolism of the carcinogens or blocking their formation [1]. ChAs could impede at various levels in the carcinogenesis, that is, blocking carcinogenesis initiation or suppressing cancer promotion, progression, angiogenesis, invasion and metastasis [2]. In carcinogenesis initiation, ChAs could alter the procarcinogens metabolisms, governed by phase I and phase II enzymes [3], by conjugation and excretion of the reactive and toxic metabolites [4].
On the one hand, phase I enzymes, for example, NADPH-cytochrome C reductase, Cyt b5 and Cyt P450 (CYP), participate in the reduction, oxidation or hydrolysis of  tion of carcinogenic process. On the contrary, phase II enzymes, for example, NAD(P)H:quinone reductase (QR, EC 1.6.5.2), glutathione S-transferase (GST EC 2.5.1.18) and UDP-glucuronosyltransferase [5], participate in the conjugation of xenobiotics with endogenous molecules, for example glucuronic acid and glutathione, facilitating xenobiotic excretions reducing their carcinogenic properties. QR, regarded as a phase II enzyme due to its protective functions, is induced together with other phase II enzymes and regulated by enhancers like to those that censor GST regulation [6]. Increases in phase II enzymes levels, for (1) The ChAs that induce detoxification enzymes are classified as monofunctional and bifunctional inducers. ChAs belonging to the group of monofunctional inducers (green in Figure 1A) increase selectively phase II enzymes by activation of the antioxidant response element (ARE) via the Keap1-Nrf2 system. On the other hand, the bifunctional inducers (red in Figure 1A) increase both phase I and phase II enzymes by binding to the Ah receptor and then the Ah receptor-ligand complex is translocated to the nucleus, through the Ah nuclear translocator receptor (Arnt, Figure 1A), activating the xenobiotic response element.
Herein, we describe the evaluation of selected compounds (1-15) ( Table 1) as monofunctional enzymatic inducers measuring QR phase II enzyme activity in an in vitro model using wild-type mouse hepatoma Hepa-1c1c7 and Arnt defective mutant BpRc1 cells [17,18]. Based on the results of this first analysis, we deepened the study analyzing new derivatives from those chemotypes with the best desired bioactive profile. Consequently, derivatives (16-54)  were studied in their capability to induce QR enzyme activity. From the best derivatives, since 1,2,4-triazine 4-oxide and tetrahydropyrimidinone scaffolds, studies on GST phase II enzymes activities were performed. In these studies the same cell lines, wild-type mouse hepatoma Hepa-1c1c7 and Arnt defective mutant BpRc1 cells, were used. For the most relevant derivatives the concentrations required for doubling enzymatic activity (CDs), and chemopreventive indexes (CIs, ratio between IC 50 and CDs) [16], were determined.

Results & discussion
Thirteen different chemotypes which complied with the pre-established structural requirements were New hits as phase II enzymes from a focused library Research Article selected from our chemical library, namely: pyrazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiazole and 1,3,4-oxathiazole, belonging to heteroatom-heteroatom bond containing five memberring category; thienyl hydrazone, α,β-unsaturated oxime, benzimidazole 1,3-dioxide and 1,2,4-triazine 4-oxide, belonging to α,β-unsaturated heterocarbonyl category; coumarin, 4-carboxaldehydeimidazole and tetrahydropyrimidine, belonging to α,β-unsaturated carbonyl category. The selected derivatives, (1-15) ( Figure 2), were prepared previously in our laboratory with different bioactivity [19][20][21][22][23][24][25][26][27][28]. The selected derivatives, (1-15), were tested in vitro for their capabilities to induce exclusively phase II enzymes. A model that uses two different cellular systems was employed, wild-type Hepa-1c1c7 and mutant BpRc1 cells ( Figure 1B). The increase in enzymatic activity in the mutant cell line concomitant with the increase in the wild type indicates that the compound tested is a monofunctional inducer (induces only phase II enzymes, Figure 1A). When the increase is only in the wild type, the compound is considered as a bifunctional inducer (induction of phase I and phase II enzymes, Figure 1A) [17]. According to this expected biological behavior we used as inducer descriptor the ratio of specific activity between wild and mutant cells, r H/B . When the compound has a value of r H/B near to 1, and it increases the specific enzymatic activity, it displays a monofunctional behavior. However, when the compound has a value of r H/B higher than 1, and it increases the specific enzymatic activity, it displays a bifunctional behavior. As a primary screening, QR activities were determined in both cellular models using the compounds at 10 μM (Table 1). [11] was used as control finding that it displays the typical behavior of a bifunctional inducer (Table 1), that is, high QR expression in wild-type cells with an r H/B of 3.0.
The excellent results with derivatives of the families of 1,2,4-triazine 4-oxide and tetrahydropyrimidine led us to perform a third approach, it was deepened in the study of new structural modifications on these systems. Consequently, we selected new derivatives of the 1,2,4-triazine 4-oxide system modifying substituents at the 3-and 5-position of the heterocycle and the level of oxidation (derivatives [29-36]; Tables 3 & 4) [25,34,35]. With these results we could iden-tify new 1,2,4-triazine 4-oxides with better activities than the parent compounds,  In order to complete the study, for some of the most relevant identified mono-or partially monofunctional inducers, that is,  (Table 6). The CI is defined as the ratio of the concentration which inhibited the growth of cell lines by 50% (IC 50 ) and the concentration that double QR-specific activity in the same cell line (CD). Also, in this study 4-BFV and t-butylhydroquinone (t-BHQ) were included as controls. The CIs revealed that tetrahydropyrimidine (28) was the best potential cancer ChA with clear monofunctional inducer capacity. It possessed, in both cellular models, similar values of CDs and lower cytotoxicity.   In order to complete the information about the capability of the studied compounds to induce phase II enzymes we also analyzed for the last compounds, that is,  to untreated cells, this phase II-activity in the mutant cell line without induction on the wild cells, probably extra mechanisms were operative in this cellular line that increased the GST activity.
In order to confirm that the mechanism of enzyme II induction is via ARE activation [38], we evaluated the Nrf2 nuclear translocation ability, using the 1,2,5-oxadiazole (3) as the model compound. Therefore, to further investigate effects of (3) on the Nrf2/ARE activation, we examined the subcellular location of Nrf2 in Hepa-1c1c7 cells after 1,2,5-oxadiazole treatment. Immunofluorescence analyses ( Figure 5) showed Nrf2 accumulation in the nucleus of cells upon treatment.
Lipinski's rule of five [39] serves as a guide to know the bioavailability of some studied compounds. Analysis of compounds (3), (23), (28), (32) and (49) (Figure 6) suggested that it would be orally administered fitting, in all the cases, to the rule (Table 6).

Cell culture & conditions
Mouse hepatoma Hepa-1c1c7 cell (ATCC, CRL-2026) and its mutant BpRc1 (ATCC, CRL-2217) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in α-MEM and DMEM containing 10% FBS, respectively, and grown at 37°C under a 5% CO 2-95% air atmosphere.  for 48 h, the milieu was aspirated, the cells washed twice with PBS and then detached using 0.1 ml trypsin/EDTA. Fresh milieu was added and 1.0 ml of cellular suspension was transferred into 1.5 ml tubes and centrifuged at 10.000 g for 5 min. The milieu was removed, cells rinsed once with 0.5 ml PBS and centrifuged again in the same conditions. PBS was removed and the cell pellet was suspended in 0.5 ml 25mM Tris-HCl buffer at pH 7.4. The suspension was lysed by sonication for 10 s on ice. The lysed suspension was centrifuged at 10.000 g for 5 min. The supernatant (cytosolic fraction) was used to measure QR or GST activity.

Determination of QR activity
Using 2,6-dichloroindophenol (2,6-DCIP) as the substrate, the cytosolic QR activity was measured as the decrease in absorbance at 600 nm, due to the reduction of 2,6-DCIP by QR. The assay buffer consisted of 25 mM Tris-HCl (pH 7.4), 5 μM FAD, 0.2 mM NADH, 40 μM 2,6-DCIP, 6 mg/100 ml bovine serum albumin and 10 μL/100 mL Tween-20. 5 μg of total protein for each sample was added to the cuvette containing 1 ml of assay buffer. The QR activity was determined, at room temperature, by the decrease in absorbance per min per mg of the total New hits as phase II enzymes from a focused library Research Article

Determination of GST activity
Using 1-chloro-2,4-dinitrobenzene (CDNB, 1mM) and GSH (1 mM) as the substrate, cytosolic GST activity was measured as the increase in absorbance at 340 nm, due to the formation of GS-CDNB adduct by GST. The assay buffer consisted of Na 2 HPO 4 /NaH 2 PO 4 (pH 6.5). Five μg of total protein for each sample was added to the cuvette containing 1 ml of assay buffer. GST activity was determined, at room temperature, by the increase in absorbance per min per mg of the total protein of the sample. The time of the determination was 1 min.

Cytotoxicity assay
Cells were seeded in 96-well plates (20 × 10 3 cells/well) in 100 μl final volume and were allowed to grow 24 h. After that, 100 μl of each concentration, 10.0, 50.0, 100.0, 150.0 and 200.0 μM, of the tested compounds in the culture milieu were added onto the cells and were further incubated for 48 h. Then, cells were fixed with ice-cold trichlorocetic acid for 1 h at 4°C, the plates were washed five-times in distilled water and allowed to dry in the air. Sulphorhodamine solution (SRB, 50 μl) was added to each well of the dry 96-well plates and allowed staining at room temperature for 30 min. The SRB solution and unbound dye were removed by washing the plates quickly with 1% v/v acetic acid, five-times. The washed plates were dried in the air. The bound SRB was solubilized by adding 100 μl of 10 mM unbuffered Tris Base (pH 10.5) to each well and shaking for 5 min on a shaker platform. The developed color was read in a 96-well plate reader at 492 nm. The optical density of SRB in each well is directly proportional to the cell number consequently the optical density values were plotted against concentration and the IC 50 value were determined.

Immunofluorescence staining
Hepa-1c1c7 cells were grown in 6-well plate containing glass cover slips to 60% confluence and then treated with 10 or 20 μM of compound (3) during 6 h. Untreated cells were included as a control. Culture milieu was removed, cells were fixed by adding 4% paraformaldehyde solution and incubated at room temperature for 10 min. Cells were permeabilized with PBS containing 0.1% Triton X-100. The cells were then blocked with 10% (v/v) goat serum for 45 min and incubated with mouse antibody against Nrf2 overnight at 4°C. Cover slips were washed and incubated in the dark with Alexa Fluor 594-conjugated secondary antibody (dilution 1:1000) at room temperature for 1 h. The nuclei were co-labeled with DAPI solution. Finally, the cover slips were washed and mounted on cover slides using ProLong ® Gold Antifade Reagent. The images were collected using a laser confocal microscope Leica TCS SP5 equipped with a 63× oil objective 1.4NA. Images were processed using LASAF 2.7.3v software.

Conclusion & future perspective
We reported the identification of new chemotypes, from a focused library, with ability to induce phase II enzymes. The focuses of the chemolibrary were compounds with heteroatom-heteroatom bond in five member-rings, compounds with α,β-unsaturated heterocarbonyl and compounds with α,β-unsaturated carbonyl. Among the analyzed compounds, 1,2,5-oxadiazole (3), 1,2,4-triazine (23) and (32) and tetrahydropyrimidine (28) and (49) ( Figure 6) resulted to be the great monofunctional inducers of both phase II enzymes, QR and GST. The induction property of compound (3) could be justified by its capacity to release nitric oxide, NO [40]. Recently, it has been reported that some NO-releasing compounds could act as monofunctional inducers of phase II enzyme through S-nitrosylation of 273-Cys and/or 288-Cys of Keap1 or S-guanylation of 434-Cys of Keap1, from 8-nitro-cGMP generated by NO-nitration of cGMP, and subsequent translocation to the nucleus of Nrf-2 [41][42][43][44]. Additionally, the high electrophilicity of carbon 3 of the 1,2,5-oxadiazole (3) [45,46] could promote the direct covalent bonding to Keap1 promoting its structural changes and the concomitant Nrf-2 nuclear translocation. On the other hand, compounds like (23), (32), (29) and (49) have been described as anticancer agents [25][26][27][28][34][35][36][37] that transform them as agents with potential dual use, chemopreventive and antitumor drugs. For the abovementioned points these compounds are good candidates for further in vivo studies of cancer chemopreventive activity. No writing assistance was utilized in the production of this manuscript.

Ethical conduct
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.