Synthesis of Novel Nitrogen-Containing Heterocycle Bromophenols and Their Interaction with Keap1 Protein by Molecular Docking

We previously reported 5,2’-dibromo-2,4’,5’-trihydroxydiphenylmethanoe (LM49), a bromophenol analogue that shows strong protection from oxidative stress injury owing to its superior anti-inflammatory, antioxidant, and anti-apoptotic properties. A series of novel nitrogen-containing heterocycle bromophenols were herein synthesized by introducing substituted piperidine, piperazine, and imidazole to modify 2-position of the lead compound LM49. By further evaluating their cytoprotective activity against H2O2 induced injury in EA.hy926 cells, 14 target bromophenols showed moderate-to-potent activity with EC50 values in the range of 0.9–6.3 μM, which were stronger than that of quercetin (EC50: 18.0 μM), a positive reference compound. Of these, the most potent compound 22b is a piperazine bromophenol with an EC50 value of 0.9 μM equivalent to the LM49. Molecular docking studies were subsequently performed to deduce the affinity and binding mode of derived halophenols toward the Keap1 Kelch domain, the docking results exhibited that the small molecule 22b is well accommodated by the bound region of Keap1-Kelch and Nrf2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. The above facts suggest that 22b is a promising pharmacological candidate for further cardiovascular drug development. Moreover, the targeting Keap1-Nrf2 protein-protein interaction may be an emerging strategy for halophenols to selectively and effectively activate Nrf2 triggering downstream protective genes defending against injury.


Introduction
The vascular endothelium is the major barrier for a cardiovascular system fighting against oxidative stress injury and inflammation [1]. In recent years, various novel skeleton halophenols derived from natural marine algaes and their derivatives obtained by structural optimization have been discovered showing the excellent vascular endothelial protective properties [2][3][4][5][6][7][8][9][10][11][12]. In support of these growing interests, we expanded upon the continuing structural optimization and mechanistic investigation on halophenols for finding a candidate compound. As a fact, we have reported the plentiful synthesis of a series of diphenylketone, diphenylmethane and phenyl furan-2-yl ketone halophenols, and their protective activity against H 2 O 2 induced injury in human  [11,13]. Moreover, we indeed found a "hit" compound, 5,4', (Figure 1) possessing an EC 50 value of 0.4 µM with the strong vascular endothelium protective ability [11], as evidenced being attributed to its anti-apoptotic, antioxidant, and anti-inflammatory abilities by further mechanical study [1,14]. To our knowledge, nitrogen-containing heterocycle derivatives have been reported to exist in many natural products and applied in many fields such as medicines and chemical products [15]. The existence of a heterocyclic unit in numerous natural products often plays an essential role in their biological activities [16][17][18][19][20][21][22]. Moreover, it helps the dosage form design of drugs such as those being prepared for injection. Based on these considerations, we focused on introducing substituted piperidine, piperazine and imidazole to modify the 2-position of lead compound LM49 to synthesize a series of analogues aiming to find the promising pharmacological candidates for further cardiovascular drug development.
Molecules 2017, 22, 2142 2 of 21 2,4', (Figure 1) possessing an EC50 value of 0.4 μM with the strong vascular endothelium protective ability [11], as evidenced being attributed to its anti-apoptotic, antioxidant, and anti-inflammatory abilities by further mechanical study [1,14]. To our knowledge, nitrogen-containing heterocycle derivatives have been reported to exist in many natural products and applied in many fields such as medicines and chemical products [15]. The existence of a heterocyclic unit in numerous natural products often plays an essential role in their biological activities [16][17][18][19][20][21][22]. Moreover, it helps the dosage form design of drugs such as those being prepared for injection. Based on these considerations, we focused on introducing substituted piperidine, piperazine and imidazole to modify the 2-position of lead compound LM49 to synthesize a series of analogues aiming to find the promising pharmacological candidates for further cardiovascular drug development. In addition, sustained oxidative stress and elevated redox state are the major causes of the development of chronic inflammation related cardiovascular diseases such as atherosclerosis and diabetes. The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE pathway plays a key role in the endogenous antioxidant system. Under basal conditions, the antioxidant transcription factor Nrf2 is bound to Keap1 protein and targets proteasomal degradation in the cytoplasm. In response to cellular injury, Nrf2 dissociates from Keap1 and activates the transcription of protective genes, defending against injury [1,23]. In our recent study, we reported that diphenylketone halophenols can protect vascular endothelial cells against the oxidative stress injury and inflammation by the activation of Nrf2 up-regulating heme oxygenase-1 (HO-1) protein expression [1], which prompted us to investigate the influence of halophenols on the Keap1-Nrf2 protein-protein interaction (PPI). Inspired by the above, we herein investigated the action mode and mechanism of halophenols interacting with the Keap1 by molecular docking.

Chemistry
In this paper, 36 new target bromophenols were prepared by Friedel-Crafts acylation, aromatic bromination, radical substitution, nitrogen-containing heterocyclic nucleophilic substitution, and demethylation reaction according to the preparation route described in Scheme 1. All structures of target compounds were confirmed by ESI-MS, 1 H-NMR, and 13 C-NMR spectrum. The obtained active bromophrnols were further characterized by IR and HR-MS spectra.
The intermediate 1 was prepared from 5-bromo-2-methyl benzoic acid with anhydrous SOCl2 dropped little N,N-dimethyl formamide (DMF) via acylating chlorination, then reacted with 1,2dimethoxybenzene catalyzed by AlCl3 to yield intermediate 2. Aluminum chloride is an effective and cheap Lewis acid catalyst and is widely used in Friedel-Crafts acylation. Bromination reaction of intermediate 2 was quickly conducted with bromine to obtain the important compound 3 in acetic acid solvent at room temperature by electrophilic substitution in benzene ring, and was subsequently reacted with N-bromosuccinimide (NBS) to gain the key intermediate 4 in anhydrous CH2Cl2 using benzoyl peroxide (BPO) as the catalyst by free radical substitution. In this process, sunlight was beneficial to accelerate reaction velocity and shorten reaction time [24]. Compound 4 was treated with substituted piperidine, piperazine or imidazole in the presence of anhydrous Na2CO3 to prepare important intermediates 5a-40a. Then, 5a-40a were demethylated with BBr3 as the demethylation reagent in anhydrous CH2Cl2 at −78 °C to obtain target bromophenols 5b-40b in moderate to high yields. In addition, sustained oxidative stress and elevated redox state are the major causes of the development of chronic inflammation related cardiovascular diseases such as atherosclerosis and diabetes. The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE pathway plays a key role in the endogenous antioxidant system. Under basal conditions, the antioxidant transcription factor Nrf2 is bound to Keap1 protein and targets proteasomal degradation in the cytoplasm. In response to cellular injury, Nrf2 dissociates from Keap1 and activates the transcription of protective genes, defending against injury [1,23]. In our recent study, we reported that diphenylketone halophenols can protect vascular endothelial cells against the oxidative stress injury and inflammation by the activation of Nrf2 up-regulating heme oxygenase-1 (HO-1) protein expression [1], which prompted us to investigate the influence of halophenols on the Keap1-Nrf2 protein-protein interaction (PPI). Inspired by the above, we herein investigated the action mode and mechanism of halophenols interacting with the Keap1 by molecular docking.

Chemistry
In this paper, 36 new target bromophenols were prepared by Friedel-Crafts acylation, aromatic bromination, radical substitution, nitrogen-containing heterocyclic nucleophilic substitution, and demethylation reaction according to the preparation route described in Scheme 1. All structures of target compounds were confirmed by ESI-MS, 1 H-NMR, and 13 C-NMR spectrum. The obtained active bromophrnols were further characterized by IR and HR-MS spectra.

Biological Evaluation
To assess the cytoprotective activity of all synthesized target compounds 5b-40b compared to important intermediates 5a-40a against H 2 O 2 induced injury in endothelial-derived EA.hy926 cells by MTT assay, we first conducted the preliminary screening to test their cytoprotective rates at a concentration of 10 µM. If the protective rates of tested compounds were higher than 45% then their EC 50 (50% effective concentration) values were determined by examining cell viability at different concentrations of 0.3125, 0.625, 1. 25, 2.5, 5, 10 µM, as presented in Tables 1-3, the values are the average of at least three independent experiments. Quercetin was used as a positive reference standard. The activity data showed that 14 target bromophenols 11b-14b, 16b, 21b, 22b, 24b-26b, 35b-38b and 15 key intermediates 5a, 10a, 14a, 15a, 17a, 21a, 24a, 27a-32a, 39a, 40a exhibited moderate-to-potent activity with EC 50 values in the range of 0.9-7.4 µM, which were stronger than that of quercetin (EC 50 : 18.0 µM). The most promising bromophenol derivative 22b showed the highest activity with an EC 50 value of 0.9 µM, which was almost identical to that of the lead compound LM49 (EC 50 : 0.7 µM). Due to the presence of a piperazine ring, compound 22b suggests the preferably potential druggability in comparison with LM49.
a EC50 values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
a EC50 values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
a EC50 values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.
a EC50 values were an average of three separate determinations. b Used as a lead compound. c Used as a positive control.

Structure-Activity Relationships
Based on the data of cytoprotective activity listed in the Tables 1-3, the preliminary structure-activity relationships (SARs) of novel bromophenols analogues could be summarized. Target  compound 11b-13b, 16b, 21b, 22b, 24b-26b, 35b-38b with two hydroxyl groups displayed better activity than their corresponding intermediates 11a-13a, 16a, 21a, 22a, 24a-26a, 35a-38a. These findings revealed that the presence of hetercycles and hydroxyl groups contributes to the increase of their anti-oxidative stress abilities, which is consistent with our previously presented results [11].
In 26 piperidine analogues (Table 1), five bromophenol derivatves 11b-14b, 16b with EC 50 values of 1. 4-6. 1 µM and five intermediates 5a, 10a, 14a, 15a, 17a with EC 50 values of 1.7-5.2 µM exhibited moderate-excellent activity. Bromophenol derivatives 5b-8b with no substituted groups or only a single methyl group that existed in the ortho-, metaor para-position of piperidine, showed no activity. Two isomers 13b and 9b with two methyl groups in the orthoor meta-position of nitrogen atom, respectively, displayed significantly different activity, bromophenol 13b demonstrated higher activity with an EC 50 value 5.4 µM than compound 9b. Compound 14b with an EC 50 value 6.1 µM, all hydrogen atoms in the ortho-position of nitrogen atom replaced by methyl group, exhibited nearly the same activity to compound 13b, moreover, corresponding intermediate 14a possessed more potent activity with an EC 50 value 1.9 µM than bromophenol derivative 14b, which indicated that the presence of methyl groups in two ortho-positions of nitrogen atom favored for the activity. In addition, compounds 15b and 17b are isomers with a hydroxymethyl group in the orthoor para-position of nitrogen atom, no activity was observed. However, their corresponding intermediates 15a and 17a showed better activity with an EC 50 value of 1.7 µM and 5.2 µM, respectively. Bromophenol derivative 11b, the meta-position of nitrogen atom replaced by withdrawing group ethoxycarbonyl, demonstrated excellent activity with an EC 50 value 1.4 µM compared with compound 7b and 9b substituted by donating group methyl.
Among 28 piperazine analogues (Table 2), five bromophenol derivatives 21b, 22b, 24b-26b showed moderate-potent activity with EC 50 values in the range of 0.9-6.3 µM, seven key intermediates 21a, 24a, 27a-31a exhibited middle activity with EC 50 values of 2.2-7.4 µM. Target compound 22b showed the most potent protective activity with an EC 50 value 0.9 µM, which was comparable to the lead compound LM49 (EC 50 = 0.7 µM). Replacement of 4-position of piperazine by methyl, isopropyl or diphenylmethyl group, bromophenol derivatives 24b-26b displayed moderate-superior activity with EC 50 values of 6.3, 2.2 and 1.5 µM, respectively. To bromophenol derivatives 18b, 20b, 23b, and 29b-30b, 4-position hydrogen of piperazine was replaced by acyl-, nitro-or fluro-substituted phenyl group, their activity was disappeared. Conversely, compound 21b, with a methoxyl group on the 2-position of piperazine, showed better activity. Evidently, the electron withdrawing effect on the benzene ring exerted a negative effect on the activity. The above results suggest that the electronic effect and steric hindrance effect at the 4-position of piperazine play a pivotal role to the cytoprotective activity of bromophenols.
In 18 prepared imidazole analogues (Table 3), three intermediates 32a, 39a and 40a showed moderate activity with EC 50 values of 1.9 µM, 4.0 µM, and 3.2 µM, respectively. Target bromophenol derivatives 35b-38b, replaced by ethyl, isopropyl or phenyl group on the 2-position of imidazole, showed excellent activity with EC 50 values of 1.3-1.6 µM. For bromophenol derivatives 32b, 33b, 34b and 39b, with no substituent or one to two methyl groups on the imidazole, no activity was observed. From these, we can conclude that the substitution groups such as ethyl, isopropyl and phenyl existed in the 2-position of imidazole and contributed to the activity improvement. Clearly, the protective activity of imidazole bromophenols is ascribed to the electron donating effect of alkyl groups.

Molecular Docking Study
Nrf2 contains multiple basic residues and possesses a tight four-residue β-hairpin conformation comprising of the residues Asp-77, Glu-78, Glu-79, Thr-80, Gly-81. In particular, Glu-79 is one of the critical functional residues in the interaction of Keap1 protein and Nrf2, the side chain of which Molecules 2017, 22, 2142 7 of 22 is wedged between Arg-415 and Arg-508. The unique feature of Arg-415, adopting an unusual left-handed helical conformation (58 • , 49 • ), may cause the potential interaction of Arg-415 with Glu-79. When the ligand occupies the region closed to Arg-415, this may result in the change of the rotational isomer of Arg-415 and may also affect the electrostatic interaction. When the ligand enters the bound region of Keap1-Kelch and Nrf2, it may influence the nature of the residue Arg-415 in the active site, causing a series of changes in the electrostatic force and the acting force to weaken the interaction with Glu-79, and then bringing the dissociation of Nrf2 into the nucleus, completing the task of protein expression [23][24][25][26][27].
In the current study, the most potent compound 22b was employed to investigate the binding modes of derived halophenols to the kelch domain of keap1 protein by molecular docking experiment. As can be seen from the left side of Figure 2, the closer to the brown it was, the higher lipotropy or hydrophobicity it showed. Conversely, the nearer to the blue it was, the stronger hydrophily it exhibited. The cavity of the receptor presented with a brown color, which suggested strong hydrophobicity. The benzene ring, a hydrophobic group, approached the inside of the cavity. In parallel, the hydrophile groups hydroxyls and carbonyls closed to the hydrophile area of the receptor. The docking results (Figure 2) showed that a small molecule 22b was well accommodated in the active pocket of the receptor and entered the bound region of Keap1-Kelch and Nrf2, and also exhibited excellent interaction via hydrogen bonds and hydrophobic interaction. Figure 3 showed that the amino acids in the distance of 5A from small molecule included Ser-602, Arg-415 and Gln-530. The hydroxyl group of 22b was 1.82A distant from the Ser-602 residue. The carbonyl group linked to the two benzene rings was 1.87A and 2.09A away from Arg-415, respectively. The distance between the carbonyl group on the piperazine ring and the Gln-530 was 1.98A. The presence of multiple hydrogen bonds together with hydrophobic interaction contributed to the enhancement of affinity and stability between the ligand and receptor. handed helical conformation (58°, 49°), may cause the potential interaction of Arg-415 with Glu-79. When the ligand occupies the region closed to Arg-415, this may result in the change of the rotational isomer of Arg-415 and may also affect the electrostatic interaction. When the ligand enters the bound region of Keap1-Kelch and Nrf2, it may influence the nature of the residue Arg-415 in the active site, causing a series of changes in the electrostatic force and the acting force to weaken the interaction with Glu-79, and then bringing the dissociation of Nrf2 into the nucleus, completing the task of protein expression [23][24][25][26][27].
In the current study, the most potent compound 22b was employed to investigate the binding modes of derived halophenols to the kelch domain of keap1 protein by molecular docking experiment. As can be seen from the left side of Figure 2, the closer to the brown it was, the higher lipotropy or hydrophobicity it showed. Conversely, the nearer to the blue it was, the stronger hydrophily it exhibited. The cavity of the receptor presented with a brown color, which suggested strong hydrophobicity. The benzene ring, a hydrophobic group, approached the inside of the cavity. In parallel, the hydrophile groups hydroxyls and carbonyls closed to the hydrophile area of the receptor. The docking results (Figure 2) showed that a small molecule 22b was well accommodated in the active pocket of the receptor and entered the bound region of Keap1-Kelch and Nrf2, and also exhibited excellent interaction via hydrogen bonds and hydrophobic interaction. Figure 3 showed that the amino acids in the distance of 5A from small molecule included Ser-602, Arg-415 and Gln-530. The hydroxyl group of 22b was 1.82A distant from the Ser-602 residue. The carbonyl group linked to the two benzene rings was 1.87A and 2.09A away from Arg-415, respectively. The distance between the carbonyl group on the piperazine ring and the Gln-530 was 1.98A. The presence of multiple hydrogen bonds together with hydrophobic interaction contributed to the enhancement of affinity and stability between the ligand and receptor.

Chemistry
The main reagents including 5-bromo-2-methyl benzoic acid, dimethoxybenzene, substituted piperdine, piperazine and imidazole were purchased from J & K Chemical Technology. Other chemical reagents and solvents were commercially available unless otherwise indicated. Dichloromethane was distilled from calcium hydride.
Melting points were taken on a micromelting point apparatus, which were uncorrected. The IR spectra of the compounds were recorded using a Thermo Scientific Nicolet iS 50 Fourier transform IR (FTIR) spectrometer. The 1 H-and 13 C-NMR spectra were recorded with a Bruker-AV 600 spectrometer in CDCl3 or DMSO-d6 with TMS as reference. Chemical shifts (δ values) and coupling constants (J values) were given in ppm and Hz, respectively. ESI mass spectra were obtained on an API QTRAP 3200 MS spectrometer, and HR-MS were recorded on a Bruker Daltonics Apex IV 70e FTICR-MS (Varian 7.0T). 4 5-Bromo-2-methyl benzoic acid (4.5 g, 7.0 mmol) was dissolved in 24 mL dried SOCl2 with a few drops DMF, the mixture was refluxed for 7 h. The solvent was evaporated under reduced pressure to give compound 1 as a transparent liquid. Dimethoxybenzene 4.5 mL (35.4 mmol) was added to 30 mL dried CH2Cl2 and stirred at 0 °C. Next, anhydrous AlCl3 (3.0 g, 22.7 mmol) was added portionwise. The obtained compound 1 was then added to the solution, which was allowed to warm to room temperature and stirred for 3 h and quenched with 30 mL distilled water. The organic phase was separated, washed with 30 mL water and dried over anhydrous Na2SO4, and then concentrated via

Chemistry
The main reagents including 5-bromo-2-methyl benzoic acid, dimethoxybenzene, substituted piperdine, piperazine and imidazole were purchased from J & K Chemical Technology. Other chemical reagents and solvents were commercially available unless otherwise indicated. Dichloromethane was distilled from calcium hydride.
Melting points were taken on a micromelting point apparatus, which were uncorrected. The IR spectra of the compounds were recorded using a Thermo Scientific Nicolet iS 50 Fourier transform IR (FTIR) spectrometer. The 1 H-and 13 C-NMR spectra were recorded with a Bruker-AV 600 spectrometer in CDCl 3 or DMSO-d 6 with TMS as reference. Chemical shifts (δ values) and coupling constants (J values) were given in ppm and Hz, respectively. ESI mass spectra were obtained on an API QTRAP 3200 MS spectrometer, and HR-MS were recorded on a Bruker Daltonics Apex IV 70e FTICR-MS (Varian 7.0T). 4 5-Bromo-2-methyl benzoic acid (4.5 g, 7.0 mmol) was dissolved in 24 mL dried SOCl 2 with a few drops DMF, the mixture was refluxed for 7 h. The solvent was evaporated under reduced pressure to give compound 1 as a transparent liquid. Dimethoxybenzene 4.5 mL (35.4 mmol) was added to 30 mL dried CH 2 Cl 2 and stirred at 0 • C. Next, anhydrous AlCl 3 (3.0 g, 22.7 mmol) was added portion-wise. The obtained compound 1 was then added to the solution, which was allowed to warm to room temperature and stirred for 3 h and quenched with 30 mL distilled water. The organic phase was separated, washed with 30 mL water and dried over anhydrous Na 2 SO 4 , and then concentrated via rotary evaporation. The crude product was purified by silica gel chromatography with ethyl acetate-petroleum ether (v/v, 1/8) as the eluent to afford compound 2. The product was recrystallized from methanol to give a white powder in 63% total yield. m. Compound 2 2.3 g (5.5 mmol) was dissolved in the mixed solvent of 30 mL acetic acid and 8 mL dichloromethane. Next the bromine 2 mL was added to the mixture. The reaction process was monitored by thin layer chromatography (TLC). After being stirred for 0.5 h at room temperature, the mixture was slowly poured into 50 mL strong ammonia water and then cooled to room temperature. The mixture was extracted twice with CH 2 Cl 2 (2 × 30 mL). The combined organics were washed to neutral with water, dried over anhydrous Na 2 SO 4 , and then concentrated via rotary evaporation. The crude product was purified by silica gel chromatography with ethyl acetate-petroleum ether (v/v, 1/16) as the eluent to gain 2.

General Procedure for the Synthesis of Intermediate Compounds 5a-40a
Compound 4 0.2 g (0.41 mmol) and 25 µL piperdine (0.82 mmol) was added to the 1.0 mL dried CH 2 Cl 2 . Anhydrous Na 2 CO 3 20 mg was then added to the mixture, which was stirred for 12 h. The mixture was washed with the distilled water, the organic phase was separated and dried over anhydrous Na 2 SO 4 , and then concentrated viarotary evaporation. The crude product was purified by silica gel chromatography with petroleum ether-acetone-strong ammonia water (v/v/v, 8/1/0.1) as the eluent to gain 0.18 g yellow solid compound 5a in 90% yield.
Compounds 6a-40a were also obtained from intermediate 4 in a similar manner as for the preparation of 5a in 70-93% yield. Note that the preparation of compound 13a and 14a was requested for the circumstance of heating and refluxing. BBr 3 solution (BBr 3 /CH 2 Cl 2 , v/v, 1/9) 1.5 mL was dropwise added to a cooled (−78 • C) solution of 0.259 g (0.52 mmol) compound 5a in 5 mL dried CH 2 Cl 2 . The mixture was allowed to warm to room temperature and stirred for 2 h, and poured into 30 mL ice-water. The precipitate was filtered, washed with a little distilled water and dried CH 2 Cl 2 , respectively, and dried in a vacuum drying oven to obtain 0.183 g yellow solid compound 5b in 75% yield. The total yield of target compound 5b was 18.8%.