Design, Synthesis, and Anticancer Activity of Novel Trimethoxyphenyl-Derived Chalcone-Benzimidazolium Salts.

A series of novel trimethoxyphenyl-derived chalcone-benzimidazolium salts were synthesized. The biological properties of the compounds were screened in vitro against five different human tumor cell lines. The results suggest that the 5,6-dimethyl-benzimidazole or 2-methyl-benzimidazole ring as well as the 2-naphthylmethyl, 4-methylbenzyl, or 2-naphthylacyl substituent at position-3 of the benzimidazole ring was important to the cytotoxic activity. Notably, (E)-5,6-dimethyl-3-(naphthalen-2-ylmethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium bromide (7f) was more selective to HL-60, MCF-7, and SW-480 cell lines with IC50 values 8.0-, 11.1-, and 5.8-fold lower than DDP. Studies of the antitumor mechanism of action showed that compound 7f could induce cell-cycle G1 phase arrest and apoptosis in SMMC-7721 cells.

Introduction

Chalcones are one of the most important structural scaffolds in natural products.[1] The chalcone family exhibits a wide range of remarkable biological activities,[2−12] for example, anti-inflammatory,[3] antihistaminic,[4] hypnotic,[5] antispasmodic,[6] antioxidant,[7] antiobesity,[8] antiprotozoal,[9] and antitumor activities.[10−12] Chalcones are excellent anticancer scaffolds and have attracted much attention in the anticancer domain. The molecular mechanism of some chalcone-based anticancer compounds has been studied at the cellular level over the past two decades.[1]Figure shows that chalcones 1a and 1b are potent inhibitors of tubulin polymerization.[13,14] The IC50 value of compound 1a against the P388 murine leukemia cell line is 2.6 nM, and compound 1b has an IC50 of 0.21 nM against the K562 human chronic myelogenous leukemia cell line. Their phosphates are under preclinical evaluation for cancer as prodrugs.[11]

Figure 1

Representative structures of chalcone, imidazolium salts, and trimethoxyphenyl-derived compounds.

Imidazole and their derivatives have attracted much interest due to their broad and interesting biological and pharmacological activities,[15−20] especially antitumor activity.[21−23] For instance, Lepidiline A and B are isolated from the roots of Lepidium meyenii and show potent cytotoxic activity against four human cancer cell lines, that is, bladder carcinoma, pancreatic adenocarcinoma, breast carcinoma, and ovarian carcinoma.[24] In our previous work, a library of novel imidazolium salts was synthesized, and their antitumor activities were evaluated.[25−29] Many compounds showed potential antitumor activity, such as NTIB.[25] Studies on molecular targets and docking calculations suggested that these imidazolium salt hybrids may be mammalian target of rapamycin (mTOR) signaling inhibitors.[25−27]

In addition, the trimethoxyphenyl group is a well-known fragment in some natural tubulin polymerization inhibitors,[30−32] such as CA-4,[30] colchicine,[31] and podophyllotoxin[32] (Figure ). The 3,4,5-trimethoxyphenyl group can be linked to another aromatic system and is the only well-defined pharmacophore for the inhibition of tubulin polymerization.[33]

Currently, combination chemotherapy, that is, using two or more than two drugs with different molecular mechanisms or targets in treatment, is one of the most important methods to treat cancer.[34] However, drug combination regimens always increase hospitalizations, hematological toxicities, and other side effects.[35] Molecular hybridization, involving the rational design of new chemical entities by the fusion of two or more than two drugs, has been one of the most potent strategies for drug discovery during the past decades.[36−40] Therefore, the design of a single anticancer drug that combines two or more than two pharmacophores with different molecular mechanisms in one molecule is a very important assignment for pharmaceutical synthetic scientists. Considering the anticancer activities of chalcone-derived compounds, naturally occurring trimethoxyphenyl-derived compounds, and the potent cytotoxic activities of natural and synthetic imidazolium salts, we synthesized a number of novel hybrid compounds with chalcone units (shown in red in Figure ), trimethoxyphenyl groups (shown red in Figure ), and N-benzyl- or phenacyl-substituted imidazole moieties (shown in blue in Figure ). Although imidazothiazole-chalcone derivatives were synthesized and found to exhibit potent anticancer effect,[11] to the best of our knowledge, no reports concerning antitumor activity of trimethoxyphenyl-derived chalcone-imidazolium salts have yet been reported.

Here, we describe our efforts toward the synthesis and cytotoxic activity evaluation of novel trimethoxyphenyl-derived chalcone-imidazolium salts with the ultimate aim of developing potent antitumor agents. The resulting compounds were evaluated for their cytotoxic activity toward five different human tumor cell lines.

Results and Discussion

Chemistry

The 3,4,5-trimethoxybenzaldehyde 2 and 4′-hydroxyacetophenone 3 were selected as the starting materials for the syntheses of a series of trimethoxyphenyl-derived chalcone-imidazolium salts (7a–j, 8a–j, and 9a–j, Scheme ). Scheme shows that the trimethoxyphenyl-derived (E)-chalcone 4 was synthesized via a NaOH-catalyzed Claisen–Schmidt condensation between 4′-hydroxyacetophenone and 3,4,5-trimethoxybenzaldehyde in 66% yield. The (E)-chalcone 4 was alkylated with 1,3-dibromopropane in the presence of NaH to afford compound 5 in 63% yield. The respective chalcone-imidazoles 6a–c were obtained in the presence of K2CO3 in acetone with 66–75% yield. Finally, trimethoxyphenyl-derived chalcone-imidazolium salts (7a–j, 8a–j, and 9a–j) were prepared with excellent yield (75–91%) by treating chalcone–imidazole hybrid 6 with various alkyl halides in acetone or toluene at 80 °C. The detailed structures and yields of trimethoxyphenyl-derived chalcone-imidazolium salts are shown in Table .

Scheme 1

Syntheses of Hybrid Compounds 6a–c, 7a–j, 8a–j, and 9a–j

Table 1

Structures and Yields of Hybrid Compounds 6a–c, 7a–j, 8a–j, and 9a–j

entry	compd	benzimidazole ring	R2	molecular formula	yields (%)
1	6a	5,6-dimethyl-benzimidazole		C30H32N2O5	66
2	6b	2-methyl-benzimidazole		C29H30N2O5	75
3	6c	benzimidazole		C28H28N2O5	75
4	7a	5,6-dimethyl-benzimidazole	4-methoxyphenacyl	C39H41BrN2O7	87
5	7b	5,6-dimethyl-benzimidazole	phenacyl	C38H39BrN2O6	84
6	7c	5,6-dimethyl-benzimidazole	4-bromophenacyl	C38H38Br2N2O6	85
7	7d	5,6-dimethyl-benzimidazole	4-bromobenzyl	C37H38Br2N2O5	82
8	7e	5,6-dimethyl-benzimidazole	2-bromobenzyl	C37H38Br2N2O5	79
9	7f	5,6-dimethyl-benzimidazole	naphthylmethyl	C41H41BrN2O5	82
10	7g	5,6-dimethyl-benzimidazole	benzyl	C37H39BrN2O5	80
11	7h	5,6-dimethyl-benzimidazole	4-nitrobenzyl	C37H38BrN3O7	90
12	7i	5,6-dimethyl-benzimidazole	naphthylacyl	C42H41BrN2O6	85
13	7j	5,6-dimethyl-benzimidazole	4-methylbenzyl	C38H41BrN2O5	89
14	8a	2-methyl-benzimidazole	4-methoxyphenacyl	C38H39BrN2O7	84
15	8b	2-methyl-benzimidazole	phenacyl	C37H37BrN2O6	91
16	8c	2-methyl-benzimidazole	4-bromophenacyl	C37H36Br2N2O6	85
17	8d	2-methyl-benzimidazole	4-bromobenzyl	C36H36Br2N2O5	90
18	8e	2-methyl-benzimidazole	2-bromobenzyl	C36H36Br2N2O5	79
19	8f	2-methyl-benzimidazole	naphthylmethyl	C40H39BrN2O5	86
20	8g	2-methyl-benzimidazole	benzyl	C36H37BrN2O5	82
21	8h	2-methyl-benzimidazole	4-nitrobenzyl	C36H36BrN3O7	91
22	8i	2-methyl-benzimidazole	naphthylacyl	C41H39BrN2O6	86
23	8j	2-methyl-benzimidazole	4-methylbenzyl	C37H39BrN2O5	89
24	9a	benzimidazole	4-methoxyphenacyl	C37H37BrN2O7	79
25	9b	benzimidazole	phenacyl	C36H34Br2N2O6	81
26	9c	benzimidazole	4-bromophenacyl	C36H34Br2N2O6	83
27	9d	benzimidazole	4-bromobenzyl	C35H34Br2N2O5	88
28	9e	benzimidazole	2-bromobenzyl	C35H34Br2N2O5	84
29	9f	benzimidazole	naphthylmethyl	C39H37BrN2O5	75
30	9g	benzimidazole	benzyl	C35H35BrN2O5	80
31	9h	benzimidazole	4-nitrobenzyl	C35H34BrN3O7	75
32	9i	benzimidazole	naphthylacyl	C40H37BrN2O6	81
33	9j	benzimidazole	4-methylbenzyl	C36H37BrN2O5	88

To verify the structures of the chalcone-imidazolium salt derivatives, imidazolium salt 8d was selected as a representative compound and characterized by X-ray crystallography (CCDC 1589720) as shown in Figure .

Figure 2

X-ray crystal structure of compound 8d.

Cytotoxic Activity and Structure–Activity Relationship (SAR)

The cytotoxic potential of all newly synthesized hybrid compounds was evaluated in vitro against a panel of human tumor cell lines according to procedures described in the literature.[41,42] The panel consisted of leukemia (HL-60), myeloid liver carcinoma (SMMC-7721), lung carcinoma (A549), breast carcinoma (MCF-7), and colon carcinoma (SW480). Cisplatin (DDP) was used as the reference drug. The results are summarized in Table (here, the IC50 value is defined as the concentration corresponding to 50% growth inhibition).

Table 2

Cytotoxic Activities of Hybrid Compounds in Vitroa (IC50, μMb)

entry	compd	HL-60	SMMC-7721	A-549	MCF-7	SW480
1	6a	1.95	3.90	5.87	4.84	5.10
2	6b	1.08	2.50	2.28	3.56	2.90
3	6c	1.69	2.16	2.19	3.48	2.95
4	7a	1.18	7.51	>20	2.90	6.85
5	7b	>20	>20	>20	>20	>20
6	7c	>20	>20	>20	>20	>20
7	7d	2.23	7.23	12.68	5.49	8.10
8	7e	1.93	6.57	13.32	4.38	7.69
9	7f	0.83	6.35	7.97	1.57	2.92
10	7g	4.19	7.16	12.26	5.44	7.88
11	7h	>20	>20	>20	>20	>20
12	7i	1.16	7.55	>20	4.03	7.79
13	7j	0.59	5.92	8.15	2.83	7.31
14	8a	2.00	5.64	11.18	2.49	4.68
15	8b	4.73	5.28	11.38	2.42	5.01
16	8c	1.21	5.89	11.75	3.07	5.51
17	8d	6.82	5.44	>20	4.82	6.21
18	8e	1.42	4.71	11.60	3.50	4.15
19	8f	1.18	5.02	6.70	4.78	6.48
20	8g	3.25	6.55	8.82	3.64	4.94
21	8h	>20	>20	>20	>20	>20
22	8i	1.14	4.76	4.74	1.25	1.63
23	8j	1.19	6.24	9.25	3.11	4.64
24	9a	>20	>20	>20	>20	>20
25	9b	>20	>20	>20	>20	>20
26	9c	>20	>20	>20	>20	>20
27	9d	7.04	7.62	10.98	5.91	7.20
28	9e	6.29	11.57	14.97	4.89	6.57
29	9f	1.00	7.40	9.56	5.96	7.93
30	9g	6.58	7.48	8.28	6.41	7.93
31	9h	>20	>20	>20	>20	>20
32	9i	6.58	8.82	10.18	5.25	10.64
33	9j	1.54	7.48	7.53	5.36	7.63
34	DDP	2.11	11.27	6.94	17.43	17.05

Data represent the mean values of three independent determinations.

Cytotoxicity as IC50 for each cell line; this is the concentration of compound that reduced the optical density of treated cells by 50% with respect to untreated cells using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay.

Table shows that the structures of the chalcone-imidazoles have a significant effect on the cytotoxic activity. Chalcone-imidazoles 6a–c showed good activity for HL-60, SMMC-7721, A-549, MCF-7, and SW480 cell lines at a concentration of 20 μM. The IC50 value was 1.08–5.87 μM on the study of five human tumor cell lines, and this was more active than DDP. More interestingly, chalcone-imidazolium salts (7a–j, 8a–j, and 9a–j) exhibited a degree of cytotoxic activity or higher cytotoxic activity. The difference in cytotoxicity between neutral compounds and imidazolium salts may be due to changes in molecular structure, charge distribution, and water solubility.[43]

In terms of the substituents with different positions of benzimidazole rings, the imidazolium salts 7a, 7d–g, 7i, and 7j having two methyl groups at 5,6-position of the benzimidazole ring showed strong cytotoxic activity against five cell lines. In particular, compounds 7f and 7j showed the best cytotoxic activity against leukemia cell lines (HL-60 and IC50 values of 0.83 and 0.59 μM, respectively). Simultaneously, the benzimidazolium salts 8a–c, 8e–g, 8i, and 8j having the methyl group at 2-position of the benzimidazole ring exhibited moderate cytotoxic activity to the five cell lines. For example, the benzimidazolium salts 8a, 8b, 8c, and 8i have IC50 values of 2.49, 2.42, 3.07, and 1.25 μM, respectively. These showed much higher inhibitory activity in vitro than DDP for breast cancer (MCF-7). However, most of the benzimidazolium salts without any substituent at the benzimidazole ring showed lower inhibitory in vitro activity than those with benzimidazolium salts at the 2-position or 5,6-position of the benzimidazole ring.

In terms of the substituents with different positions of imidazolium salts, the imidazolium salts 7b, 7c, 7h, 8h, 9a–c, and 9h have a formyl substituent and show a lack of activity against five tumor cell lines. Compounds 7d, 7e, 7g, 8d, 8e, 8g, 9d, 9e, and 9f have a benzyl substituent at the 3-position of the benzimidazole ring and are more effective than the above-mentioned formyl-substituted imidazolium salt derivatives. In particular, compounds 7f, 8f, 9f, 7i, 8i, and 9i have a naphthylmethyl or 4-methylbenzyl substituent at the 3-position of the imidazole ring and exhibit similar or higher cytotoxic activity in vitro compared with DDP. Interestingly, the hybrid compounds 7f and 7j with naphthylmethyl and 4-methylbenzyl substituents at the 3-position of 5,6-dimethyl-benzimidazole were the most potent derivatives against the five human tumor cell lines with an IC50 value of 0.59–8.15 μM; they were more active than DDP. We note that the heterozygous compound 7f selectively treated leukemia (HL-60, IC50 value of 0.83 μM), breast cancer (MCF-7, IC50 value of 1.57 μM), and colon cancer (SW480, IC50 value of 2.92 μM) with 8.0-fold, 11.1-fold, and 5.8-fold lower concentrations than DDP, respectively.

This finding suggests that the existence of the 5,6-dimethylbenzimidazole or 2-methyl-benzimidazole ring as well as substitution of the imidazolyl-3-position with a naphthylmethyl or benzyl group was important for antitumor activity. In general, the structure–activity relationship (SAR) results are summarized in Scheme .

Scheme 2

Structure Analysis of Trimethoxyphenyl-Derived Chalcone-Benzimidazolium Salts

Apoptosis

SMMC-7721 cells were exposed to increasing concentrations of compound 7f, and cell apoptosis was determined with annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labeled cell cytometry. Figure shows that the apoptotic cell rates were 14.95 ± 0.89 and 97.43 ± 5.96% after treatment of SMMC-7721 cells with compound 7f at 5 and 10 μM for 48 h, respectively. These were statistically significantly different from the control (2.35 ± 0.22%).

Figure 3

Compound 7f caused significant apoptosis of SMMC-7721 cells. (A) Cells were treated with 5 and 10 μM compound 7f for 48 h. Cell apoptosis was determined by the annexin V–FITC/PI double-staining assay. (B) Quantification of cell apoptosis.

Cell-Cycle Analysis

The results of cell-cycle analysis on SMMC-7721 cells treated with compound 7f are presented in Figure . Compared with the control cells, the percentage of cells in the G1 phase increased during incubation with compound 7f in a dose-dependent manner. Compound 7f treatment caused 83.14 ± 0.48% cells in the G0/G1 phase versus controls showing 62.20 ± 0.68%. In contrast, the fractions of cells in the S and G2/M phases decreased slightly from 14.09 ± 0.17 to 9.40 ± 0.23% and 2.57 ± 0.39 to 4.17 ± 0.19%, respectively. These data showed that compound 7f may induce G1 phase arrest in the cell cycle (Figure ).

Figure 4

Effects of compound 7f on SMMC-7721 cell-cycle progression. (A) Cells were treated with 2.5 and 5 μM of compound 7f for 24 h. Cell cycle was determined by PI staining and cell cytometry. (B) Percentage of cells in different phases was quantified. At least three independent experiments were performed, and data from one representative experiment is shown. Data represents the mean ± standard deviation (S.D.) of three independent experiments.

Docking Analysis

In our previous work, we found that tailored imidazolium salts might be potential inhibitors of mammalian target of rapamycin (mTOR) signaling.[25−27] To rationalize the observed SARs for this series of compounds, we attempted to dock hybrid 7f with some crystal structure of proteins in this signaling pathway, for example, mTORC1, mTORC2, and PI3K, using Autodock 4.2. Although compound 7f could not dock with mTORC1 or mTORC2, it could dock nicely with PI3Kγ (PDB code 3PRZ). Figure shows that hybrid 7f is predicted to engage a hydrogen bond with ASN498 using phenol oxygen and a hydrogen bond with GLY970 using methoxyl oxygen. The results also show that hybrid 7f can foster van der Waals interactions with the gap bounded by ASN971, LYS875, GLY489, SLR488, ASP500, ASN498, PHE497, THR1043, MET1039, GLN1041, PRO1040, GLN840, and GLY970. These favorable interactions contribute to a good docking score (AutoDock binding energy is −7.47 kcal/mol) and an excellent inhibitory activity. These interesting findings are useful for further studies.

Figure 5

Model of compound 7f docked into PI3Kγ.

Conclusions

Compounds 7f, 7j, 8f, 8i, and 8j, with a 5,6-dimethyl-benzimidazole or 2-methyl-benzimidazole ring, and 2-naphthylmethyl substituent, 4-methylbenzyl or 2-naphthylacyl substituent at position-3 of the benzimidazole ring, were found to be the most potent derivatives. Notably, compounds 8f and 8i were the most potent derivative against five human tumor cell lines with IC50 values below 6.70 μM. These values showed more selectivity toward HL-60 and MCF-7 cell lines. Compound 7f was more selective to HL-60, MCF-7, and SW-480 cell lines with IC50 values that were 8.0-fold, 11.1-fold, and 5.8-fold lower than DDP, respectively. Studies of the antitumor mechanism of action showed that compound 7f could induce cell-cycle G1 phase arrest and apoptosis in SMMC-7721 cells. The trimethoxyphenyl-derived chalcone-benzimidazolium salts 7f, 7j, 8f, 8i, and 8j are promising leads for further structural modifications guided by our SARs.

Experimental Section

General Methods

Melting points were obtained on an XT-4 melting-point apparatus and were uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz. Carbon-13 nuclear magnetic resonance (13C NMR) was recorded on a Bruker Avance 400 spectrometer at 100 MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane for all of the recorded NMR spectra. High-resolution mass spectra were taken on an AB QSTAR Pulsar mass spectrometer. Silica gel (200–300 mesh) for column chromatography and silica GF254 for thin-layer chromatography (TLC) were produced by Merck KGaA (Germany). All of the air- or moisture-sensitive reactions were conducted under an argon atmosphere. Starting materials and reagents used in the reactions were obtained commercially from Acros, Sigma-Aldrich, and Fluka and were used without purification unless otherwise indicated.

Synthesis of Compound 4

A 100 mL flask was charged with 3,4,5-trimethoxybenzaldehyde 2 (8.22 g, 42.00 mmol) and a solution of sodium hydroxide (8.40 g, 210.00 mmol) in a 4:1 (v/v) mixture of ethanol/H2O (200 mL). The resulting mixture was stirred at room temperature for 5 min. The 4′-hydroxyacetophenone 3 (6.86 g, 50.40 mmol) was then added to the reaction, and the resulting mixture was stirred at room temperature for 13 days. The reaction was quenched with HCl (2 N, 150 mL), and the resulting suspension was filtered through a Büchner funnel. The solid cake was washed with water (100 mL) and ether (20 mL). Compound 4 (8.72 g) was afforded as a yellow powder in 66% yield. IR (KBr) νmax (cm–1): 3856, 3133, 2345, 1646, 1606, 1500, 1422, 1285, 1213, 1127, 1035, 825, 733, 550. 1H NMR [400 MHz, dimethyl sulfoxide (DMSO)], δ (ppm): 10.47 (1H, brs), 8.13 (2H, d, J = 8.4 Hz), 7.91 (1H, d, J = 15.6 Hz), 7.68 (1H, d, J = 15.6 Hz), 7.25 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 3.90 (6H, s), 3.75 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.09, 162.17, 153.12, 143.25, 139.48, 131.22, 130.48, 129.22, 121.28, 115.36, 106.34, 60.15, 56.13. High-resolution mass spectrometry (HRMS) [electrospray ionization-time-of-flight (ESI-TOF)] m/z calcd for C18H19O5 [M + H]+ 315.1227, found 315.1227.

Synthesis of Compound 5

Compound 4 (4.95 g, 15.75 mmol) was dissolved in dimethylformamide (120 mL) in a 250 mL round-bottom flask. The NaH (0.40 g, 16.60 mmol) was added under N2 protection and ice bath. After stirring for 15 min, the mixture was slowly warmed to room temperature. After stirring for 30 min, 1,3-dibromopropane (8.00 mL, 78.73 mmol) was added in one portion. The reaction was then monitored by TLC using ethyl acetate/petroleum ether (1:2 v/v) as the solvent system. After the reaction was complete, the reaction was quenched with HCl (2 N, 10 mL), and the residue was diluted with EtOAc (300 mL). The organic layer was washed with water and brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C/ethyl acetate = 4:1) to afford compound 5 (4.34 g) as a yellow solid in 63% yield. IR (KBr) νmax (cm–1): 3384, 2331, 1656, 1601, 1503, 1452, 1416, 1314, 1280, 1247, 1122, 1006, 838, 742, 601, 558. 1H NMR (400 MHz, CDCl3), δ (ppm): 8.04 (2H, d, J = 8.8 Hz), 7.72 (1H, d, J = 15.6 Hz), 7.42 (1H, d, = 15.6 Hz), 6.99 (2H, d, J = 8.8 Hz), 6.86 (2H, s), 4.20 (2H, t, J = 6.0 Hz), 3.93 (6H, s), 3.90 (3H, s), 3.62 (2H, t, J = 6.0 Hz), 2.39–2.33 (2H, m). 13C NMR (100 MHz, CDCl3), δ (ppm): 188.80, 162.62, 153.59, 144.36, 140.38, 131.44, 130.96, 130.68, 121.32, 114.43, 105.68, 65.64, 61.14, 56.35, 32.24, 29.86. HRMS (ESI-TOF) m/z calcd for C21H24BrO5 [M + H]+ 435.0802, found 435.0803.

General Procedure for the Synthesis of Compounds 6a–c

A mixture of the previous bromide 5 (2 mmol), K2CO3 (12 mmol), and substituted imidazole (3 mmol) was stirred in acetone (20 mL) at 60 °C in a tube for 12–24 h. After cooling to room temperature, the solvent was concentrated and the residue was diluted with EtOAc (30 mL). The organic layer was washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C/ethyl acetate = 1:2) to give the yellow solid compounds 6a–c in 66–75% yield.

(E)-1-(4-(3-(5,6-Dimethyl-1H-benzo[d]imidazol-1-yl)propoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6a)

Yellow powder, m.p.: 89–90 °C, yield 66%. IR (KBr) νmax (cm–1): 3445, 2360, 2025, 1656, 1604, 1448, 1320, 1256, 1123, 1068, 862, 566. 1H NMR (400 MHz, CDCl3), δ (ppm): 8.18 (1H, s), 8.00 (2H, d, J = 8.8 Hz), 7.70 (1H, d, J = 15.6 Hz), 7.58 (1H, s), 7.38 (1H, d, J = 15.6 Hz), 7.17 (1H, s), 6.93 (2H, d, J = 8.8 Hz), 6.85 (2H, s), 4.47 (2H, t, J = 6.4 Hz), 3.99 (2H, t, J = 6.4 Hz), 3.92 (6H, s), 3.89 (3H, s), 2.38 (2H, t, J = 6.4 Hz), 2.35 (3H, s), 2.29 (3H, s). 13C NMR (100 MHz, CDCl3), δ (ppm): 188.74, 162.20, 153.57, 144.41, 142.01, 140.39, 140.30, 133.14, 132.23, 131.90, 131.59, 130.97, 130. 61, 121.22, 119.67, 114.35, 110.13, 105.68, 64.29, 61.12, 56.34, 41.83, 29.45, 20.63, 20.36. HRMS (ESI-TOF) m/z calcd for C30H33N2O5 [M + H]+ 501.2311, found 501.2386.

(E)-1-(4-(3-(2-Methyl-1H-benzo[d]imidazol-1-yl)propoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6b)

Yellow powder, m.p.: 139–140 °C, yield 75%. IR (KBr) νmax (cm–1): 3443, 2288, 1667, 1608, 1508, 1456, 1322, 1254, 1129, 1068, 865, 737, 543. 1H NMR (400 MHz, DMSO), δ (ppm): 8.17 (2H, d, J = 8.4 Hz), 7.89 (1H, d, J = 15.6 Hz), 7.66 (1H, d, J = 15.6 Hz), 7.53–7.48 (2H, m), 7.22 (2H, s), 7.14–7.12 (2H, m), 7.07 (2H, d, J = 8.8 Hz), 4.39 (2H, t, J = 6.8 Hz), 4.06 (2H, t, J = 5.6 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.51 (3H, s), 2.23 (2H, t, J = 6.1 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.79, 162.65, 153.58, 144.15, 142.74, 140.08, 135.51, 131.42, 131.15, 130.83, 121.91, 121.66, 118.65, 114.87, 110.16, 106.93, 65.29, 60.61, 56.61, 40.41, 28.98, 13.78. HRMS (ESI-TOF) m/z calcd for C29H31N2O5 [M + H]+ 487.2155, found 487.2225.

(E)-1-(4-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6c)

Yellow powder, m.p.: 171–172 °C, yield 75%. IR (KBr) νmax (cm–1): 3440, 2323, 1646, 1601, 1501, 1459, 1328, 1285, 1259, 1126, 1068, 864, 822, 743, 544. 1H NMR (400 MHz, DMSO), δ (ppm): 8.23 (1H, s), 8.16 (2H, d, J = 8.8 Hz), 7.88 (1H, d, J = 15.2 Hz), 7.68–7.61 (3H, m), 7.23–7.07 (4H, m), 7.05 (2H, d, J = 8.8 Hz), 4.46 (2H, t, J = 6.8 Hz), 4.06 (2H, t, J = 5.8 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.32–2.29 (2H, m). 13C NMR (100 MHz, DMSO), δ (ppm): 187.80, 162.74, 153.58, 144.57, 144.14, 143.91, 140.10, 134.30, 131.37, 131.12, 130.83, 122.75, 121.92, 121.68, 119.93, 114.89, 110.75, 106.94, 65.54, 60.61, 56.62, 41.52, 29.39. HRMS (ESI-TOF) m/z calcd for C28H29N2O5 [M + H]+ 473.1993, found 473.2074.

General Procedure for the Syntheses of Chalcone-Imidazolium Salts 7a–j, 8a–j, and 9a–j

A mixture of chalcone–imidazole hybrid 6a–c (1 mmol) and phenacyl or alkyl halide (1.2 mmol) was stirred in acetone (10 mL) or toluene (10 mL) at 80 °C in a tube. A yellow or white solid was formed. After completion of the reaction as indicated by TLC, the precipitate was filtered through a small pad of Celite and washed with ethyl acetate (5 × 20 mL) and then dried to give the title compound 7a–j, 8a–j, and 9a–j in 75–91% yield.

(E)-3-(2-(4-Methoxyphenyl)-2-oxoethyl)-5,6-dimethyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7a)

Yellow powder, m.p.: 183–184 °C, yield 87%. IR (KBr) νmax (cm–1): 3448, 2849, 2027,1671, 1460,1266, 1122, 1067, 995, 951, 862, 544,51. 1H NMR (400 MHz, DMSO), δ (ppm): 9.69 (1H, s), 8.21 (2H, d, J = 8.4 Hz), 8.14 (2H, d, J = 8.6 Hz), 7.96 (1H, d, J = 15.4 Hz), 7.94 (1H, s), 7.88 (1H, s), 7.71 (1H, d, J = 15.4 Hz), 7.28 (2H, s), 7.23 (2H, d, J = 8.4 Hz), 7.07 (2H, d, J = 8.8 Hz), 6.34 (2H, s), 4.83 (2H, brs), 4.26 (2H, brs), 3.94 (3H, s), 3.91 (6H, s), 3.76 (3H, s), 2.50 (2H, d, J = 5.1 Hz), 2.40 (6H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 189.91, 187.70, 164.67, 162.48, 153.56, 144.23, 142.79, 140.00, 136.90, 136.76, 131.36, 131.10, 130.94, 130.81, 129.80, 127.00, 121.53, 114.83, 114.77, 106.88, 65.44, 60.61, 56.59, 53.12, 44.65, 28.70, 20.41, 20.37. HRMS (ESI-TOF) m/z calcd for C39H41N2O7 [M – Br]+ 649.2914, found 649.2909.

(E)-5,6-Dimethyl-3-(2-oxo-2-phenylethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7b)

Yellow powder, m.p.: 177–178 °C, yield 84%. IR (KBr) νmax (cm–1): 3449, 2380, 2027, 1656, 1460, 1267, 1068, 994, 952, 861, 546, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 9.69 (1H, s), 8.22–8.16 (4H, m), 7.94–7.91 (3H,m), 7.84 (1H, t, J = 6.8 Hz), 7.73–7.71 (3H, m), 7.27 (2H, s), 7.07 (2H, d, J = 8.0 Hz), 6.40 (2H, s), 4.83 (2H, brs), 4.27 (2H, brs), 3.91 (6H, s), 3.76 (3H, s), 2.50 (2H, brs), 2.39 (6H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 191.68, 187.75, 162.47, 153.58, 144.18, 142.76, 140.14, 136.93, 136.80, 135.07, 134.22, 131.34, 131.18, 130.95, 130.81, 129.84, 129.53, 128.89, 121.62, 114.86, 113.91, 113.61, 106.98, 65.47, 60.62, 56.65, 53.54, 44.70, 28.73, 20.41, 20.35. HRMS (ESI-TOF) m/z calcd for C38H39N2O6 [M – Br]+ 619.2808, found 619.2801.

(E)-3-(2-(4-Bromophenyl)-2-oxoethyl)-5,6-dimethyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7c)

Yellow powder, m.p.: 183–184 °C, yield 85%. IR (KBr) νmax (cm–1): 3451, 2445, 2027, 1658, 1603, 1462, 1122, 1068, 997, 951, 861, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.61 (1H, s), 8.17 (2H, d, J = 8.8 Hz), 8.05 (2H, d, J = 8.8 Hz), 7.91–7.89 (4H, m), 7.86 (1H, s), 7.67 (1H, d, J = 15.2 Hz), 7.23 (2H, s), 7.02 (2H, d, J = 8.8 Hz), 6.33 (2H, s), 4.78 (2H, t, J = 6.4 Hz), 4.21 (2H, t, J = 5.2 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.44–2.43 (2H, m), 2.34 (6H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 191.11, 187.75, 162.47, 153.58, 144.18, 142.73, 140.13, 136.92, 136.81, 133.28, 132.61, 131.36, 131.17, 130.86, 129.82, 129.19, 121.63, 114.85, 113.94, 113.63, 106.98, 65.49, 60.62, 56.66, 53.57, 44.72, 28.73, 20.42, 20.35. HRMS (ESI-TOF) m/z calcd for C38H38BrN2O6 [M – Br]+ 697.1913, found 697.1908.

(E)-3-(4-Bromobenzyl)-5,6-dimethyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7d)

Yellow powder, m.p.: 200–201 °C, yield 82%. IR (KBr) νmax (cm–1): 3453, 2361, 2027, 1656, 1602, 1463, 1265, 1128, 1068, 952, 861, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.90 (1H, s), 8.16 (2H, d, J = 8.4 Hz), 7.90 (1H, d, J = 15.6 Hz), 7.88 (1H, s), 7.78 (1H, s), 7.67 (1H, d, J = 15.6 Hz), 7.58 (2H, d, J = 8.0 Hz), 7.45 (2H, d, J = 8.4 Hz), 7.23 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 5.70 (2H, s), 4.69 (2H, t, J = 6.4 Hz), 4.20 (2H, t, J = 5.2 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.46–2.43 (2H, m), 2.35 (3H, s), 2.32 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.73, 162.45, 153.58, 144.18, 141.96, 140.11, 136.94, 136.91, 133.96, 132.32, 131.37, 131.16, 130.95, 130.82, 130.37, 129.79, 122.48, 121.62, 114.79, 113.83, 113.66, 106.97, 65.57, 60.62, 56.65, 49.45, 44.71, 28.50, 20.47, 20.35. HRMS (ESI-TOF) m/z calcd for C37H38BrN2O5 [M – Br]+ 669.1964, found 669.1959.

(E)-3-(2-Bromobenzyl)-5,6-dimethyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7e)

Yellow powder, m.p.: 122–123 °C, yield 79%. IR (KBr) νmax (cm–1): 3450, 2361, 2341, 2027, 1655, 1603, 1460, 1160, 1068, 952, 861, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.97 (1H, s), 8.22 (2H, d, J = 8.8 Hz), 7.95 (1H, d, J = 15.6 Hz), 7.93 (1H, s), 7.83 (1H, s), 7.71 (1H, d, J = 15.5 Hz), 7.63 (2H, d, J = 8.4 Hz), 7.52–7.50 (2H, m), 7.27 (2H, s), 7.00 (2H, d, J = 8.8 Hz), 5.76 (2H, s), 4.74 (2H, t, J = 6.6 Hz), 4.26 (2H, t, J = 5.5 Hz), 3.92 (6H, s), 3.76 (3H, s), 2.39 (2H, t, J = 5.8 Hz), 2.40 (3H, s), 2.37 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.74, 162.45, 153.58, 144.20, 142.36, 140.11, 137.47, 137.06, 133.74, 133.17, 131.39, 131.37, 130.93, 130.66, 130.10, 128.90, 123.45, 121.61, 114.79, 113.90, 113.65, 106.97, 65.43, 60.62, 56.65, 50.74, 44.67, 28.63, 20.48, 20.35. HRMS (ESI-TOF) m/z calcd for C37H38BrN2O5 [M – Br]+ 669.1964, found 669.1955.

(E)-5,6-Dimethyl-3-(naphthalen-2-ylmethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7f)

Yellow powder, m.p.: 166–167 °C, yield 82%. IR (KBr) νmax (cm–1): 3450, 2344, 2027, 1657, 1605, 1564, 1461, 1313, 1272, 1218, 1160, 1068, 952, 861, 548. 1H NMR (400 MHz, DMSO), δ (ppm): 9.93 (1H, s), 8.14 (2H, d, J = 8.4 Hz), 8.06 (1H, s), 7.95–7.84 (6H, m), 7.68 (1H, d, J = 15.6 Hz), 7.56 (3H, brs), 7.23 (2H, s), 6.96 (2H, d, J = 8.0 Hz), 5.89 (2H, s), 4.71 (2H, brs), 4.22 (2H, brs), 3.88 (6H, s), 3.73 (3H, s), 2.47 (2H, brs), 2.35 (3H, s), 2.33 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.74, 162.45, 153.59, 144.19, 141.98, 140.14, 136.92, 136.88, 133.17, 131.96, 131.35, 131.17, 130.81, 130.42, 130.00, 129.24, 128.33, 128.14, 127.86, 127.18, 126.04, 121.63, 114.77, 113.82, 113.73, 106.98, 65.61, 60.62, 56.65, 50.38, 44.75, 28.52, 20.47, 20.34. HRMS (ESI-TOF) m/z calcd for C41H41N2O5 [M – Br]+ 641.3015, found 641.3008.

(E)-3-Benzyl-5,6-dimethyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7g)

Yellow powder, m.p.: 114–115 °C, yield 80%. IR (KBr) νmax (cm–1): 3449, 2362, 1656, 1601, 1564, 1459, 1162, 1068, 953, 862, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.91 (1H, s), 8.16 (2H, d, J = 8.0 Hz), 7.89 (1H, d, J = 15.2 Hz), 7.87 (1H, s), 7.78 (1H, s), 7.67 (1H, d, J = 15.2 Hz), 7.46 (2H, brs), 7.39–7.36 (3H, m), 7.23 (2H, s), 6.96 (2H, d, J = 8.4 Hz), 5.71 (2H, s), 4.69 (2H, brs), 4.20 (2H, brs), 3.87 (6H, s), 3.71 (3H, s), 2.45 (2H, brs), 2.35 (3H, s), 2.32 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.72, 162.45, 153.57, 144.24, 141.87, 140.07, 136.92, 136.87, 134.59, 131.37, 131.14, 130.80, 130.38, 129.84, 129.44, 129.12, 128.57, 121.56, 114.77, 113.79, 113.69, 106.92, 65.54, 60.62, 56.62, 50.14, 44.68, 28.51, 20.48, 20.36. HRMS (ESI-TOF) m/z calcd for C37H39N2O5 [M – Br]+ 591.2859, found 591.2850.

(E)-5,6-Dimethyl-3-(4-nitrobenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7h)

Yellow powder, m.p.: 203–204 °C, yield 90%. IR (KBr) νmax (cm–1): 3450, 2345, 2027, 1656, 1603, 1461, 1351, 1160, 1068, 952, 861, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.93 (1H, s), 8.28 (1H, d, J = 8.4 Hz), 8.22 (2H, d, J = 8.4 Hz), 8.15 (2H, d, J = 8.4 Hz), 7.90 (1H, s), 7.76–7.70 (4H, m), 7.23 (2H, s), 6.97 (2H, d, J = 8.8 Hz), 5.90 (2H, s), 4.70 (2H, t, J = 6.4 Hz), 4.21 (2H, t, J = 5.2 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.46 (2H, t, J = 6.4 Hz), 2.34 (3H, s), 2.33 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.66, 162.45, 153.55, 148.01, 144.22, 142.84, 141.90, 140.01, 139.84, 137.42, 137.05, 136.15, 131.37, 131.12, 130.00, 129.79, 129.46, 129.35, 124.50, 121.50, 114.32, 106.88, 60.61, 56.60, 49.58, 44.76, 28.43, 20.47, 20.37. HRMS (ESI-TOF) m/z calcd for C37H38N3O7 [M – Br]+ 636.2710, found 636.2703.

(E)-5,6-Dimethyl-3-(2-(naphthalen-2-yl)-2-oxoethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7i)

Yellow powder, m.p.: 169–170 °C, yield 85%. IR (KBr) νmax (cm-1): 3449, 2342, 1667, 1604, 1460, 1260, 1165, 1123, 1068, 1003, 953, 862, 745, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.72 (1H, s), 8.94 (1H, s), 8.25–8.09 (6H, m), 7.92 (3H, brs), 7.77–7.70 (3H, m), 7.23 (2H, s), 7.04 (2H, d, J = 8.0 Hz), 6.50 (2H, s), 4.82 (2H, brs), 4.25 (2H, brs), 3.88 (6H, s), 3.73 (3H, s), 2.51 (2H, brs), 2.37 (6H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 191.60, 187.76, 162.48, 153.58, 144.19, 142.83, 140.14, 136.96, 136.82, 136.05, 132.51, 131.53, 131.36, 131.18, 131.00, 130.82, 130.18, 129.87, 129.19, 128.37, 127.89, 123.82, 121.63, 114.87, 113.93, 113.65, 106.98, 65.50, 60.62, 56.65, 53.59, 44.74, 28.72, 20.43, 20.37. HRMS (ESI-TOF) m/z calcd for C42H41N2O6 [M – Br]+ 669.2965, found 669.2961.

(E)-5,6-Dimethyl-3-(4-methylbenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (7j)

Yellow powder, m.p.: 111–112 °C, yield 89%. IR (KBr) νmax (cm–1): 3448, 2361, 1655, 1460, 1267, 1068, 952, 862, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.83 (1H, s), 8.15 (2H, d, J = 8.4 Hz), 7.90 (2H, d, J = 15.6 Hz), 7.86 (1H, s), 7.77 (1H, s), 7.67 (1H, d, J = 15.6 Hz), 7.35 (2H, d, J = 8.0 Hz), 7.23 (2H, s), 7.17 (2H, d, J = 7.6 Hz), 6.94 (2H, d, J = 8.4 Hz), 5.63 (2H, s), 4.67 (2H, t, J = 6.4 Hz), 4.18 (2H, brs), 3.86 (6H, s), 3.71 (3H, s), 2.43 (2H, t, J = 11.5 Hz), 2.35 (3H, s), 2.32 (3H, s), 2.26 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.75, 162.44, 153.58, 144.18, 141.74, 140.13, 138.54, 136.87, 136.80, 131.53, 131.36, 131.15, 130.81, 130.37, 129.94, 128.67, 121.63, 114.78, 113.87, 113.74, 106.99, 65.59, 60.62, 56.67, 50.08, 44.69, 28.50, 21.16, 20.47. HRMS (ESI-TOF) m/z calcd for C38H41N2O5 [M – Br]+ 605.3015, found 605.3008.

(E)-3-(2-(4-Methoxyphenyl)-2-oxoethyl)-2-methyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8a)

Yellow powder, m.p.: 190–191 °C, yield 84%. IR (KBr) νmax (cm–1): 3447, 2343, 1656, 1603, 1421, 1245, 1067, 952, 862, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 8.17 (1H, s), 8.15-8.12 (4H, s), 7.99 (1H, d, J = 8.8 Hz), 7.89 (1H, d, J = 15.6 Hz), 7.66 (1H, d, J = 15.2 Hz), 7.60–7.58 (2H, m), 7.23 (2H, s), 7.19 (2H, d, J = 8.8 Hz), 7.02 (2H, d, J = 8.8 Hz), 6.40 (2H, s), 4.82 (2H, t, J = 6.4 Hz), 4.22 (2H, t, J = 5.2 Hz), 3.90 (3H, s), 3.87 (6H, s), 3.71 (3H, s), 2.86 (3H, s), 2.40 (2H, t, J = 5.6 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 189.78, 187.76, 164.81, 162.38, 153.58, 153.49, 144.19, 140.13, 132.01, 131.68, 131.38, 131.22, 130.82, 127.05, 126.69, 126.63, 121.63, 114.82, 114.70, 113.56, 113.48, 106.99, 65.28, 60.62, 56.65, 56.32, 51.94, 43.14, 28.21, 10.90. HRMS (ESI-TOF) m/z calcd for C38H39N2O7 [M – Br]+ 635.2757, found 635.2750.

(E)-2-Methyl-3-(2-oxo-2-phenylethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8b)

Yellow powder, m.p.: 128–129 °C, yield 91%. IR (KBr) νmax (cm–1): 3445, 2343, 2027, 1656, 1385, 1266, 1121, 1067, 1009, 953, 863, 545, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 8.17–8.15 (4H, m), 8.10 (1H, d, J = 8.8 Hz), 8.01 (1H, d, J = 8.8 Hz), 7.89 (1H, d, J = 15.6 Hz), 7.80–7.79 (1H, m), 7.69–7.59 (5H, m), 7.22 (2H, s), 7.02 (2H, d, J = 8.8 Hz), 6.45 (2H, s), 4.82 (2H, t, J = 6.4 Hz), 4.22 (2H, t, J = 5.2 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.87 (3H, s), 2.40 (2H, t, J = 6.4 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 191.62, 187.75, 162.38, 153.58, 144.19, 140.13, 135.15, 134.20, 131.97, 131.38, 131.24, 130.81, 129.42, 129.21, 126.71, 121.61, 114.81, 113.62, 106.98, 65.29, 60.62, 56.64, 52.33, 43.17, 28.20, 10.87. HRMS (ESI-TOF) m/z calcd for C37H37N2O6 [M – Br]+ 605.2652, found 605.2647.

(E)-3-(2-(4-Bromophenyl)-2-oxoethyl)-2-methyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8c)

Yellow powder, m.p.: 162–163 °C, yield 85%. IR (KBr) νmax (cm–1): 3445, 2342, 1654, 1602, 1424, 1263, 1158, 1068, 953, 861, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 8.17 (2H, d, J = 8.8 Hz), 8.08 (2H, d, J = 8.8 Hz), 8.01(1H, d, J = 8.8 Hz), 7.93–7.90 (4H, m), 7.67 (1H, d, J = 15.6 Hz), 7.60–7.58 (2H, m), 7.23 (2H, s), 7.02 (2H, d, J = 8.8 Hz), 6.45 (2H, s), 4.82 (2H, t, J = 6.4 Hz), 4.21 (2H, t, J = 5.6 Hz), 3.86 (6H, s), 3.70 (3H, s), 2.87 (3H, s), 2.39 (2H, t, J = 5.8 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 191.06, 187.72, 162.38, 153.55, 153.50, 144.23, 140.00, 133.22, 132.48, 131.91, 131.41, 131.18, 130.82, 129.29, 126.69, 121.54, 114.79, 113.66, 113.48, 106.89, 65.27, 60.61, 56.60, 52.37, 43.17, 28.17, 10.90. HRMS (ESI-TOF) m/z calcd for C37H36Br2N2O6 [M – Br]+ 683.1757, found 683.1750.

(E)-3-(4-Bromobenzyl)-2-methyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8d)

Yellow powder, m.p.: 191–192 °C, yield 90%. IR (KBr) νmax (cm–1): 3449, 2344, 1657, 1605, 1468, 1251, 1123, 1067, 994, 952, 862, 770, 546, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 8.15 (2H, d, J = 8.4 Hz), 8.08 (1H, d, J = 4.8 Hz), 7.92–7.87 (2H, m), 7.67 (1H, d, J = 15.6 Hz), 7.59–7.57 (4H, m), 7.32 (2H, d, J = 7.6 Hz), 7.23 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 5.80 (2H, s), 4.73 (2H, t, J = 6.0 Hz), 4.21 (2H, t, J = 5.6 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.95 (3H, s), 2.38 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.72, 162.36, 153.57, 152.74, 144.20, 140.10, 134.12, 132.31, 131.54, 131.40, 131.35, 131.19, 130.81, 130.14, 126.76, 126.68, 122.08, 114.76, 113.58, 106.95, 65.35, 60.62, 56.63, 56.13, 47.90, 43.08, 28.12, 11.26. HRMS (ESI-TOF) m/z calcd for C36H36BrN2O5 [M – Br]+ 655.1808, found 655.1804.

(E)-3-(2-Bromobenzyl)-2-methyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8e)

Yellow powder, m.p.: 220–221 °C, yield 79%. IR (KBr) νmax (cm–1): 3449, 2342, 1658, 1462, 1067, 992, 952, 862, 739, 546. 1H NMR (400 MHz, DMSO), δ (ppm): 8.17 (2H, d, J = 8.4 Hz), 8.12 (1H, d, J = 8.0 Hz), 7.90 (1H, d, J = 15.6 Hz), 7.78–7.76 (1H, m), 7.71 (2H, t, J = 15.6 Hz), 7.65 (1H, d, J = 15.2 Hz), 7.62–7.58 (1H, m), 7.32 (2H, t, J = 4.4 Hz), 7.23 (2H, s), 7.02 (2H, d, J = 8.4 Hz), 6.97 (1H, brs), 5.81 (2H, s), 4.78 (2H, t, J = 6.4 Hz), 4.21 (2H, t, J = 5.2 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.91 (3H, s), 2.40 (2H, t, J = 5.6 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.76, 162.41, 153.58, 153.25, 144.21, 140.13, 133.72, 133.25, 131.57, 131.42, 131.23, 130.85, 128.85, 126.89, 126.76, 122.42, 121.62, 114.84, 113.74, 113.49, 107.51, 106.98, 65.19, 60.62, 56.65, 49.32, 43.09, 28.22, 11.36. HRMS (ESI-TOF) m/z calcd for C36H36BrN2O5 [M – Br]+ 655.1808, found 655.1802.

(E)-2-Methyl-3-(naphthalen-2-ylmethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8f)

Yellow powder, m.p.: 231–232 °C, yield 86%. IR (KBr) νmax (cm–1): 3447, 1655, 1601, 1462, 1262, 1124, 1067, 998, 952, 863, 743, 545, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 8.11 (3H, d, J = 8.8 Hz), 8.02–8.00 (1H, m), 7.92 (3H, d, J = 8.8 Hz), 7.88–7.86 (2H, m), 7.67 (1H, d, J = 15.6 Hz), 7.60 (2H, d, J = 8.8 Hz), 7.53 (2H, d, J = 8.8 Hz), 7.48 (1H, d, J = 8.4 Hz), 7.24 (2H, s), 6.94 (2H, d, J = 8.4 Hz), 6.00 (2H, s), 4.78 (2H, t, J = 6.4 Hz), 4.22 (2H, t, J = 5.6 Hz), 3.87 (6H, s), 3.72 (3H, s), 3.02 (3H, s), 2.41 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.72, 162.36, 153.56, 152.74, 144.22, 140.00, 133.17, 132.99, 132.21, 131.57, 131.40, 131.11, 130.82, 129.25, 128.27, 128.12, 127.17, 127.06, 126.89, 126.74, 126.69, 126.65, 125.54, 121.55, 114.71, 113.69, 113.59, 106.87, 65.39, 60.62, 56.61, 48.77, 43.13, 28.13, 11.35. HRMS (ESI-TOF) m/z calcd for C40H39N2O5 [M – Br]+ 627.2859, found 627.2852.

(E)-3-Benzyl-2-methyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8g)

Yellow powder, m.p.: 125–126 °C, yield 82%. IR (KBr) νmax (cm–1): 3448, 2345, 1655, 1460, 1246, 1122, 1067, 997, 951, 863, 743, 545, 515. 1H NMR (400 MHz, DMSO), δ (ppm): 8.16 (2H, d, J = 8.4 Hz), 8.08 (1H, d, J = 6.0 Hz), 7.94–7.89 (2H, m), 7.67 (1H, d, J = 15.6 Hz), 7.60–7.58 (2H, m), 7.37 (5H, brs), 7.23 (2H, s), 6.96 (2H, d, J = 8.8 Hz), 5.84 (2H, s), 4.75 (2H, t, J = 6.4 Hz), 4.22 (2H, t, J = 4.8 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.99 (3H, s), 2.39 (2H, t, J = 5.2 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.76, 162.38, 153.58, 152.61, 144.19, 140.12, 134.68, 131.56, 131.42, 131.18, 130.82, 129.44, 128.81, 127.84, 126.69, 126.64, 121.64, 114.76, 113.67, 113.57, 106.98, 65.41, 60.62, 56.66, 48.57, 43.12, 28.17, 11.31. HRMS (ESI-TOF) m/z calcd for C36H37N2O5 [M – Br]+ 577.2702, found 577.2698.

(E)-2-Methyl-3-(4-nitrobenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8h)

Yellow powder, m.p.: 205–206 °C, yield 91%. IR (KBr) νmax (cm–1): 3443, 2860, 2340, 2027, 1659, 1606, 1522, 1472, 1415, 1346, 1251, 1214, 1121, 1066, 1003, 951, 863, 741, 542, 515. 1H NMR (400 MHz, DMSO), δ (ppm): 8.23 (2H, d, J = 8.8 Hz), 8.16 (2H, d, J = 8.8 Hz), 8.12 (1H, d, J = 8.0 Hz), 7.92 (1H, s), 7.91 (1H, d, J = 15.6 Hz), 7.68 (1H, d, J = 15.6 Hz), 7.63–7.61 (4H, m), 7.23 (2H, s), 6.99 (2H, d, J = 8.8 Hz), 6.02 (2H, s), 4.77 (2H, t, J = 6.8 Hz), 4.23 (2H, t, J = 5.6 Hz), 3.88 (6H, s), 3.71 (3H, s), 2.96 (3H, s), 2.41 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.72, 162.38, 153.57, 153.05, 147.80, 144.19, 142.12, 140.11, 131.60, 131.39, 131.19, 130.81, 129.02, 127.12, 126.86, 126.78, 124.41, 121.58, 114.81, 113.67, 113.49, 106.96, 65.31, 60.61, 56.64, 47.91, 43.13, 28.15, 11.33. HRMS (ESI-TOF) m/z calcd for C36H36N3O7 [M – Br]+ 622.2553, found 622.2547.

(E)-2-Methyl-3-(2-(naphthalen-2-yl)-2-oxoethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8i)

Yellow powder, m.p.: 195–196 °C, yield 86%. IR (KBr) νmax (cm–1): 3450, 2345, 1689, 1654, 1601, 1461, 1259, 1121, 1067, 993, 954, 864, 821, 770, 614, 540, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 8.97 (1H, s), 8.23–8.07 (8H, m), 7.91 (1H, d, J = 15.6 Hz), 7.78–7.71 (2H, m), 7.67 (1H, d, J = 15.6 Hz), 7.62–7.61 (1H, m), 7.23 (2H, s), 7.04 (2H, d, J = 8.4 Hz), 6.60 (2H, s), 4.85 (2H, t, J = 6.0 Hz), 4.23 (2H, brs), 3.87 (6H, s), 3.71 (3H, s), 2.91 (3H, s), 2.42 (2H, t, J = 5.2 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 191.54, 187.76, 162.40, 153.58, 153.55, 152.82, 144.20, 140.12, 136.10, 132.44, 132.02, 131.83, 131.53, 131.40, 131.27, 131.21, 130.82, 130.16, 129.91, 129.05, 128.38, 127.91, 126.69, 124.01, 121.62, 114.82, 113.64, 113.52, 107.51, 106.98, 65.32, 60.62, 56.64, 52.39, 43.21, 28.21, 10.94. HRMS (ESI-TOF) m/z calcd for C41H39N2O6 [M – Br]+ 655.2808, found 655.2803.

(E)-2-Methyl-3-(4-methylbenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (8j)

Yellow powder, m.p.: 191–192 °C, yield 89%. IR (KBr) νmax (cm–1): 3448, 1652, 1603, 1468, 1261, 1121, 1068, 995, 864, 814, 751, 538, 514. 1H NMR (400 MHz, DMSO), δ (ppm): 8.14 (2H, d, J = 8.8 Hz), 8.07 (1H, d, J = 9.2 Hz), 7.95–7.93 (1H, m), 7.90 (1H, d, J = 15.6 Hz), 7.67 (1H, d, J = 15.6 Hz), 7.60–7.57 (2H, m), 7.25–7.18 (4H, s), 7.17 (2H, d, J = 8.4 Hz), 6.92 (2H, d, J = 8.8 Hz), 5.76 (2H, s), 4.73 (2H, t, J = 6.4 Hz), 4.19 (2H, t, J = 5.6 Hz), 3.86 (6H, s), 3.70 (3H, s), 2.95 (3H, s), 2.37 (2H, t, J = 6.0 Hz). 2.26 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.71, 162.34, 153.56, 152.46, 144.24, 140.00, 138.23, 131.68, 131.49, 131.42, 131.37, 131.11, 130.81, 129.97, 127.88, 126.67, 126.61, 121.52, 114.71, 113.69, 113.54, 106.87, 65.37, 60.61, 56.60, 48.33, 43.08, 28.07, 21.13, 11.23. HRMS (ESI-TOF) m/z calcd for C37H39N2O5 [M – Br]+ 591.2859, found 591.2856.

(E)-3-(2-(4-Methoxyphenyl)-2-oxoethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9a)

Yellow powder, m.p.: 139–140 °C, yield 79%. IR (KBr) νmax (cm–1): 3453, 2360, 2340, 2027, 1655, 1459, 1159, 1070, 953, 860, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 9.85 (1H, s), 8.18 (2H, d, J = 8.8 Hz), 8.17–8.16 (1H, m), 8.11 (2H, d, J = 8.8 Hz), 8.08–8.06 (1H, m), 7.91 (1H, d, J = 15.2 Hz), 7.68–7.65 (3H, m), 7.23 (2H, s), 7.18 (2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 9.2 Hz), 6.39 (2H, s), 4.85 (2H, t, J = 6.8 Hz), 4.25 (2H, t, J = 5.6 Hz), 3.89 (3H, s), 3.86 (6H, s), 3.71 (3H, s), 2.47 (2H, t, J = 6.4 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 189.89, 187.74, 164.70, 162.48, 153.57, 144.19, 144.06, 140.06, 132.47, 131.39, 131.37, 131.13, 130.82, 127.22, 127.06, 127.02, 121.60, 114.80, 114.44, 114.15, 106.94, 65.59, 60.61, 56.63, 56.27, 53.28, 44.88, 28.70. HRMS (ESI-TOF) m/z calcd for C37H37N2O7 [M – Br]+ 621.2601, found 621.2597.

(E)-3-(2-Oxo-2-phenylethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9b)

Yellow powder, m.p.: 148–149 °C, yield 81%. IR (KBr) νmax (cm–1): 3450, 2360, 2027, 1657, 1601, 1460, 1069, 993, 952, 861, 755, 546. 1H NMR (400 MHz, DMSO), δ (ppm): 9.91 (1H, s), 8.20–8.12 (6H, m), 7.94 (1H, d, J = 15.6 Hz), 7.81 (1H, t, J = 7.4 Hz), 7.70–7.66 (5H, m), 7.25 (2H, s), 7.02 (2H, d, J = 8.8 Hz), 6.49 (2H, s), 4.88 (2H, t, J = 6.4 Hz), 4.27 (2H, t, J = 5.6 Hz), 3.88 (6H, s), 3.72 (3H, s), 2.48 (2H, d, J = 6.0 Hz), 13C NMR (100 MHz, DMSO), δ (ppm): 191.72, 187.75, 162.49, 153.57, 144.19, 144.01, 140.06, 135.08, 134.21, 132.46, 131.39, 131.36, 131.13, 130.84, 129.53, 128.95, 127.23, 127.08, 121.63, 114.83, 114.52, 114.18, 106.95, 65.61, 60.61, 56.65, 53.77, 44.91, 31.17, 28.73. HRMS (ESI-TOF) m/z calcd for C36H35BrN2O6 [M – Br]+ 591.2495, found 591.2488.

(E)-3-(2-(4-Bromophenyl)-2-oxoethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9c)

Yellow powder, m.p.: 209–210 °C, yield 83%. IR (KBr) νmax (cm–1): 3449, 2362, 1655, 1459, 1068, 951, 861, 614, 591, 545. 1H NMR (400 MHz, DMSO), δ (ppm): 9.85 (1H, s), 8.17–8.16 (3H, m), 8.08–8.06 (3H, m), 7.91 (3H, brs), 7.69–7.65 (3H, m), 7.23 (2H, s), 7.00 (2H, d, J = 8.0 Hz), 6.44 (2H, s), 4.86 (2H, brs), 4.25 (2H, brs), 3.87 (6H, s), 3.71 (3H, s), 2.47 (2H, brs). 13C NMR (100 MHz, DMSO), δ (ppm): 191.11, 187.73, 162.47, 153.56, 144.20, 143.97, 140.04, 133.26, 132.62, 132.42, 131.38, 131.12, 130.89, 129.23, 127.24, 127.10, 121.60, 114.81, 114.52, 114.18, 106.93, 65.61, 60.61, 56.63, 53.71, 44.92, 28.70. HRMS (ESI-TOF) m/z calcd for C36H34BrN2O6 [M – Br]+ 669.1600, found 669.1595.

(E)-3-(4-Bromobenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9d)

Yellow powder, m.p.: 122–123 °C, yield 88%. IR (KBr) νmax (cm–1): 3449, 2344, 2027, 1655, 1460, 1068, 996, 953, 861, 546. 1H NMR (400 MHz, DMSO), δ (ppm): 10.04 (1H, s), 8.17–8.15 (1H, m), 8.15 (2H, d, J = 8.8 Hz), 7.98 (1H, d, J = 8.4 Hz), 7.90 (1H, d, J = 15.2 Hz), 7.68 (1H, d, J = 7.2 Hz), 7.65 (2H, s), 7.59 (2H, d, J = 8.4 Hz), 7.48 (2H, d, J = 8.4 Hz), 7.23 (2H, s), 6.92 (2H, d, J = 9.2 Hz), 5.77 (2H, s), 4.74 (2H, t, J = 6.8 Hz), 4.23 (2H, t, J = 5.6 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.46 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.69, 162.44, 153.56, 144.21, 143.23, 140.04, 133.77, 132.33, 131.94, 131.38, 131.27, 131.13, 131.09, 130.81, 127.17, 122.57, 121.57, 114.74, 114.35, 114.28, 106.92, 65.66, 60.61, 56.62, 49.64, 44.88, 28.46. HRMS (ESI-TOF) m/z calcd for C35H34BrN2O5 [M – Br]+ 641.1651, found 641.1647.

(E)-3-(2-Bromobenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9e)

Yellow powder, m.p.: 104–105 °C, yield 84%. IR (KBr) νmax (cm–1): 3406, 2326, 1654, 1603, 1502, 1419, 1253, 1010, 822, 744, 549. 1H NMR (400 MHz, DMSO), δ (ppm): 9.93 (1H, s), 8.19–8.16 (1H, m), 8.15 (2H, d, J = 8.8 Hz), 7.96–7.92 (2H, m), 7.87 (1H, d, J = 8.8 Hz), 7.74 (1H, d, J = 7.6 Hz), 7.69–7.68 (2H, m), 7.41 (2H, s), 7.37 (1H, d, J = 7.6 Hz), 7.23 (2H, s), 6.94 (2H, d, J = 8.8 Hz), 5.84 (2H, s), 4.77 (2H, t, J = 6.4 Hz), 4.21 (2H, t, J = 5.6 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.45 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.70, 162.45, 153.57, 152.82, 144.22, 143.68, 140.05, 133.78, 133.01, 131.84, 131.50, 131.37, 131.24, 130.81, 128.90, 127.37, 127.25, 123.59, 121.56, 114.74, 114.46, 114.27, 106.92, 65.55, 60.61, 56.62, 50.92, 44.86, 28.59. HRMS (ESI-TOF) m/z calcd for C35H34BrN2O5 [M – Br]+ 641.1651, found 641.1645.

(E)-3-(Naphthalen-2-ylmethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9f)

Yellow powder, m.p.: 99–100 °C, yield 75%. IR (KBr) νmax (cm–1): 3450, 2345, 1655, 1460, 1160, 1068, 996, 953, 861, 744, 547. 1H NMR (400 MHz, DMSO), δ (ppm): 10.06 (1H, s), 8.13–8.10 (4H, m), 8.03 (1H, d, J = 8.8 Hz), 7.94–7.86 (4H, m), 7.69–7.64 (3H, m), 7.58–7.54 (3H, m), 7.23 (2H, s), 6.92 (2H, d, J = 8.4 Hz), 5.94 (2H, s), 4.76 (2H, t, J = 6.8 Hz), 4.24 (2H, t, J = 4.8 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.47 (2H, t, J = 6.4 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.75, 162.45, 153.58, 144.18, 143.24, 140.67, 133.39, 133.19, 132.61, 131.78, 131.46, 130.83, 129.25, 128.34, 128.12, 127.98, 127.18, 126.46, 126.15, 125.90, 125.72, 124.75, 121.65, 114.74, 114.37, 106.98, 65.73, 60.62, 56.66, 50.60, 44.93, 28.50. HRMS (ESI-TOF) m/z calcd for C39H37N2O5 [M – Br]+ 613.2702, found 613.2695.

(E)-3-Benzyl-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9g)

Yellow powder, m.p.: 200–201 °C, yield 80%. IR (KBr) νmax (cm–1): 3448, 2344, 1654, 1460, 1265, 1121, 1067, 998, 952, 862, 749, 545. 1H NMR (400 MHz, DMSO), δ (ppm): 10.08 (1H, s), 8.15 (3H, d, J = 8.4 Hz), 7.99 (1H, d, J = 5.2 Hz), 7.90 (1H, d, J = 15.2 Hz), 7.66 (1H, d, J = 15.2 Hz), 7.65 (2H, s), 7.50 (2H, d, J = 6.4 Hz), 7.39–7.37 (3H, m), 7.23 (2H, s), 6.93 (2H, d, J = 8.4 Hz), 5.79 (2H, s), 4.76 (2H, t, J = 6.4 Hz), 4.24 (2H, t, J = 5.2 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.47 (2H, t, J = 6.0 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 187.77, 162.46, 153.58, 144.18, 143.19, 142.38, 142.25, 141.08, 140.12, 134.44, 131.96, 131.53, 131.39, 130.83, 129.48, 129.41, 128.81, 127.27, 127.12, 121.67, 114.92, 114.76, 114.53, 114.34, 106.99, 65.73, 60.62, 56.68, 50.51, 44.89, 28.53. HRMS (ESI-TOF) m/z calcd for C35H35N2O5 [M – Br]+ 563.2540, found 563.2431.

(E)-3-(4-Nitrobenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9h)

Yellow powder, m.p.: 153–154 °C, yield 75%. IR (KBr) νmax (cm–1): 3448, 2844, 1654, 1460, 1265, 1121, 1067, 998, 952, 862, 545, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 10.12 (1H, s), 8.22 (2H, d, J = 8.4 Hz), 8.15 (2H, d, J = 8.8 Hz), 7.95 (1H, d, J = 8.0 Hz), 7.89 (1H, d, J = 15.6 Hz), 7.75 (2H, d, J = 8.8 Hz), 7.68–7.65 (4H, m), 7.23 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 5.98 (2H, s), 4.77 (2H, t, J = 6.4 Hz), 4.25 (2H, t, J = 5.2 Hz), 3.87 (6H, s), 3.71 (3H, s), 2.50 (2H, brs). 13C NMR (100 MHz, DMSO), δ (ppm): 187.72, 162.45, 153.57, 148.08, 144.15, 141.68, 140.10, 131.96, 131.54, 131.37, 130.83, 130.08, 127.51, 127.24, 126.57, 124.45, 121.62, 114.92, 114.50, 106.97, 65.69, 60.61, 56.67, 49.80, 44.98, 28.46. HRMS (ESI-TOF) m/z calcd for C35H34N3O7 [M – Br]+ 608.2397, found 608.2392.

(E)-3-(2-(Naphthalen-2-yl)-2-oxoethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9i)

Yellow powder, m.p.: 158–160 °C, yield 81%. IR (KBr) νmax (cm–1): 3449, 2343, 2027, 1686, 1600, 1461, 1260, 1123, 1068, 994, 952, 862, 749, 544, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 10.04 (1H, s), 8.15 (4H, d, J = 8.4 Hz), 7.98 (1H, d, J = 4.8 Hz), 7.90 (1H, d, J = 15.6 Hz), 7.69–7.65 (4H, m), 7.59 (3H, d, J = 8.0 Hz), 7.48 (2H, d, J = 8.0 Hz), 7.23 (2H, s), 6.92 (2H, d, J = 7.6 Hz), 5.77 (2H, s), 4.74 (2H, t, J = 6.8 Hz), 4.23 (2H, t, J = 5.2 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.46 (2H, t, J = 6.1 Hz). 13C NMR (100 MHz, DMSO), δ (ppm): 191.97, 191.58, 162.49, 153.58, 144.08, 143.53, 141.09, 140.11, 136.06, 132.53, 132.20, 131.62, 131.41, 130.90, 130.19, 129.85, 129.19, 128.37, 127.90, 127.29, 126.59, 123.83, 115.75, 114.93, 114.53, 113.85, 106.98, 65.64, 60.62, 56.67, 53.41, 44.96, 28.71. HRMS (ESI-TOF) m/z calcd for C40H37BrN2O6 [M – Br]+ 641.2652, found 641.2648.

(E)-3-(4-Methylbenzyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium Bromide (9j)

Yellow powder, m.p.: 100–101 °C, yield 88%. IR (KBr) νmax (cm–1): 3448, 2343, 1655, 1602, 1460, 1264, 1121, 1067, 995, 863, 747, 541, 516. 1H NMR (400 MHz, DMSO), δ (ppm): 10.01 (1H, s), 8.16–8.13 (3H, m), 7.97 (1H, d, J = 3.6 Hz), 7.89 (1H, d, J = 15.2 Hz), 7.66 (1H, d, J = 15.2 Hz), 7.65 (2H, s), 7.38 (2H, d, J = 7.6 Hz), 7.23 (2H, s), 7.17 (2H, d, J = 7.6 Hz), 6.91 (2H, d, J = 8.4 Hz), 5.71 (2H, s), 4.75 (2H, t, J = 6.4 Hz), 4.22 (2H, t, J = 5.2 Hz), 3.86 (6H, s), 3.71 (3H, s), 2.46 (2H, t, J = 6.0 Hz), 2.26 (3H, s). 13C NMR (100 MHz, DMSO), δ (ppm): 187.75, 162.44, 153.58, 144.18, 143.01, 140.11, 138.69, 138.62, 131.95, 131.37, 131.13, 130.82, 130.01, 129.95, 128.83, 127.20, 127.10, 121.64, 114.74, 114.38, 106.98, 65.72, 60.62, 56.66, 50.19, 44.90, 28.48, 21.16. HRMS (ESI-TOF) m/z calcd for C36H37BrN2O5 [M – Br]+ 577.2702, found 577.2698.

Cytotoxicity Assay

The assay was performed in five cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW480). Cells were cultured at 37 °C under a humidified atmosphere of 5% CO2 in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal serum and dispersed in replicate 96-well plates. Chalcone-benzimidazolium hybrids were then added. After 48 h exposure to the compounds, cell viability was determined by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cytotoxicity assay by measuring the absorbance at 570 nm with a microplate spectrophotometer. Each test was performed in triplicate.

Flow Cytometric Evaluation of Apoptosis

To analyze the cells for apoptosis, cells were plated and allowed to adhere overnight. The SMMC-7721 cells were treated with 0, 5, or 10 μM 7f for 0–48 h. Cells treated with vehicle alone (100% ethanol) were used as a control. Cells were seeded in 6-well plates at a density of 1.2 × 106 cells/well. After treatment, cells were then collected and washed twice with cold phosphate-buffered saline (PBS) and resuspended in 100 μL of a binding buffer containing annexin V–FITC and propidium iodine (PI). This step was followed by incubation with FITC-conjugated annexin V and propidium iodide (PI) for 15 min at room temperature in the dark. The fluorescence intensity was measured using the FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). The percentages of cells in different phases were quantified, and at least three independent experiments were performed. The data of one representative experiment are shown. The data represent the mean ± S.D. of three independent experiments.

Cell-Cycle Assay

To analyze the DNA content by flow cytometry, cells were collected and washed twice with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS and then stained with a 50 μg/mL propidium iodide (PI) solution containing 50 μg/mL RNase A for 30 min at room temperature. Fluorescence intensity was analyzed by the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The percentage of cells distributed in the different phases of the cell cycle was obtained using ModFIT LT 2.0.

Molecular Modeling

Chalcone-benzimidazolium hybrid 7f was docked into PI3Kγ, a crystal obtained from the complex (PDB code: 3PRZ) of PI3Kγ with 4-amino-2-methyl-N-(1H-pyrazol-3-yl)quinazoline-8-carboxamide by means of AutoDock 4.2. A grid of 126, 126, and 126 points was constructed, the x, y, and z directions of which were centered on 32.02, −0.082, and 25.843. A grid spacing of 0.736 Å and a distance-dependent function of the dielectric constant for the energetic map calculations were used in this modeling. The Lamarckian genetic algorithm was selected as docking simulations of the compounds. A protocol with an initial population of 150 randomly placed individuals, a mutation rate of 0.02, an elitism value of 1, a crossover rate of 0.8, and a maximum number of 250 million energy evaluations was used in the calculations. Fifty independent docking runs were carried out between PI3Kγ and compound 7f. The resulting conformations, differing by 1.6 Å in positional root-mean-square deviation, were clustered by selecting the most populated cluster with the best energy in all of the cases.[44]