Synthesis and Biological Evaluation of Analogues of AKT (Protein Kinase B) Inhibitor-IV

Abstract

Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl₄-catalyzed cyclization of 1,2-arylenediamines with α,β-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells, and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

Introduction

In higher eukaryotes, the phosphatidylinositol-3-kinase (PI3K) pathway regulates a number of critical physiological functions including vesicular trafficking, cellular growth, survival, and proliferation. When activated, receptor tyrosine kinases, such as the epidermal growth factor receptor, recruit PI3K to the plasma membrane, resulting in the conversion of membrane-bound phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3,4,5-triphosphate. This lipid second messenger in turn recruits the serine-threonine kinase AKT (also known as protein kinase B) to the plasma membrane by binding the pleckstrin homology (PH) domain of this enzyme. At the plasma membrane, AKT is fully activated upon phosphorylation by 3-phosphoinositide-dependent kinase (PDK1) and the rapamycin-insensitive mammalian target of rapamycin complex (mTORC2). Phosphorylation of substrates by activated AKT subsequently results in a cascade of downstream signaling events. Mutations in PI3K that result in constitutive growth-factor-independent activation of AKT are frequently observed in human cancers.^{1, 2}

In addition to its established contribution to cancer progression, the PI3K/AKT pathway also plays key roles in the infection and replication of certain viral pathogens. This pathway is hijacked to promote host cell survival in cells infected by Influenza A virus³ and HIV-1,⁴ it plays a critical role in the cellular entry of Ebola virus,⁵ and it has been hypothesized in some studies,^{6, 7} but not others,⁸ to be involved in the replication of nonsegmented negative stranded RNA viruses such as parainfluenza virus 5 (PIV5). Among the numerous small molecules known to block the PI3K/AKT pathway, many inhibit AKT through interactions with the ATP-binding site, allosteric sites, or the PH domain.⁹ Other inhibitors of this pathway bind PI3K, other upstream components, kinases between PI3K and AKT, or downstream proteins.¹⁰ AKT inhibitor-IV (1, also known as ChemBridge 5233705, Figure 1) was initially proposed to target the ATP binding site of unknown kinases upstream of AKT but downstream of PI3K.¹¹ In 786-O cells, 1 blocks AKT-mediated nuclear export of the transcription factor FOXO1a (IC₅₀ = 625 nM)¹¹ and cell proliferation (IC₅₀ < 1.25 μM).^12–15 More recent studies indicate that a low (1 μM) concentration of 1 inhibits phosphorylation of substrates downstream of AKT, but the antiviral effects observed at this concentration do not result from direct inhibition of kinase activity of the AKT1 or AKT2 subtypes or other known kinases of the PI3K pathway.⁸ Consequently, the mechanism of antiviral and antiproliferative activities of 1 remains poorly understood, and molecular probes that facilitate the identification of specific protein targets of this compound are needed.

Figure 1.

Structure of AKT inhibitor IV (1).

To better define the structural basis of its antiviral/antiproliferative activities, we report here a new synthetic approach that allowed preparation of 1 and 21 related analogues. This route is based on a ZrCl₄-mediated cyclization reaction of substituted N-aryl 1,2-arylenediamines with α,β-unsaturated aldehydes. We evaluated the in vitro antiviral activity of these compounds against PIV5 in infected HeLa cells, examined the cytotoxic effects of the most potent compounds against HeLa cells and normal human bronchial/tracheal epithelial (NHBE) cells, and assessed effects on replication of the intracellular bacterium M. fortuitum in HeLa cells. These studies identified two analogues with greater biological activity than the parent compound and provide strategies for structural modification that may be valuable for future target identification studies.

Results and discussion

Benzimidazole represents an important scaffold in natural products and is considered a privileged structure in medicinal chemistry.^16–18 Although numerous routes to benzimidazoles have been reported,¹⁹ an efficient route for preparation of the commercially available benzimidazole derivative AKT inhibitor-IV (1) has not been previously described. For the synthesis of simpler benzimidazoles, unconjugated aldehydes can often be condensed with 1,2-phenylenediamines to generate benzimidazoline intermediates^20–22 that can be further oxidized with mild oxidants such as potassium peroxymonosulfate,²⁰ MnO₂,²³ and DDQ,²⁴ to afford the desired products. However, the use of this approach with sensitive α,β-unsaturated aldehyde substrates as building blocks for the preparation of more complex benzimidazole derivatives such as 1 is known^{20, 23, 24} to be problematic. To develop an improved route to these more sensitive derivatives, we initially synthesized the novel 1,2-arylenediamine 7 (Scheme 1) in three steps from 4-chloro-3-nitrobenzaldehyde (2). Compound 2 was condensed with 2-aminothiophenol (3) to generate benzothiazole 4 using conditions described by Mortimer for similar substrates.²⁵ Treatment of 4 with freshly distilled aniline (5) afforded 6, which was reduced with hydrazine in the presence of Pd(0)²⁶ to yield 7. However, when 7 and the known²⁷ α,β-unsaturated-γ-amino aldehyde 10 were subjected to well-precedented conditions for generating benzimidazolines, such as refluxing in ethanol, the reaction was extremely sluggish. Moreover, subsequent addition of potassium peroxymonosulfate,²⁰ MnO₂,²³ or DDQ,²⁴ resulted in decomposition, and complex reaction mixtures were obtained. To provide better methodology for the preparation of benzimidazoles using α,β-unsaturated aldehyde coupling partners, we alternatively executed the cyclization and oxidation steps separately. We found that in the presence of 3Å molecular sieves, the benzimidazoline intermediate 11 was generated as a major byproduct in refluxing ethanol, and MnO₂ was sufficiently mild to afford 12, but unfortunately this reaction generated only low yields of the desired product 12. To improve this outcome, we examined the use of Lewis acids such as ZrOCl₂, ZrCl₄, CuSO₄, and FeCl₃ previously reported^28–31 for the preparation of benzimidazoles, benzothiazoles, and purines. By screening a variety of Lewis acids, we found that addition of 0.5 equivalents of ZrCl₄ to 7 and 10 in refluxing ethanol afforded 11, which could be oxidized in situ with MnO₂ to afford 12 in a remarkably high 75% yield. As shown in Scheme 1, alkylation of 12 with excess ethyl iodide followed by purification by flash column chromatography afforded AKT inhibitor-IV (1). Analogues 13-32, shown in Figure 2, were prepared by this zirconium-mediated cyclization route or similar methods as illustrated in Schemes 2 and 3.

Scheme 1.

Synthesis of AKT inhibitor-IV (1).

Figure 2.

Structures of analogues 12 to 32. For 13 to 16 and 18 to 31, compounds were isolated as ammonium iodides.

Scheme 2.

Synthesis of 13-16 and 18-31.

Scheme 3.

Synthesis of 17 and 32.

To examine the antiviral activity of compounds 12-32, we constructed a recombinant parainfluenza virus 5 minigenome system (rPIV5-RL). As shown in Figure 3, this system is analogous to a previously reported³² recombinant PIV5 that expresses GFP, but we replaced the gene encoding GFP with renilla luciferase (RL), which was inserted into the viral genome between the HN and L genes. To validate the utility of rPIV5-RL for testing compounds related to 1, we quantified luciferase activity in infected HeLa cells, a human cervical carcinoma line suitable for virus replication, as a function of multiplicity of infection (MOI). The strong correlation of MOI with luciferase activity, as shown in Figure 3, confirmed the utility of rPIV5-RL for evaluation of antiviral activity of synthetic compounds. Assessment of antiviral activity initially involved infection of HeLa cells with rPIV5-RL at 1 MOI followed by treatment with compounds 1 and 12-32 at 1 μM to generate singleton data points. As shown in Figure 4, compounds comparable in activity to 1 at this concentration were further studied at 0.5 μM as singletons. Dose-response curves against rPIV5-RL and cytotoxic effects towards HeLa cells were used to generate IC₅₀ values for 1 and the three most potent analogues (29-31, Figure 5). Importantly, two compounds were identified (30, 31) that exhibit greater antiviral activity than the parent compound 1. However, the cytotoxicity of all of these compounds towards HeLa cancer cells, assessed by quantifying cellular ATP using a firefly luciferase-based assay, generally paralleled their antiviral activity. Interestingly, 6-fold (for 1) to >20-fold (for 31) higher concentrations of these compounds were required for comparable cytotoxic effects against NHBE cells, suggesting the possibility of inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. Control experiments examining inhibition of both renilla luciferase and firefly luciferase present in cell extracts confirmed that the observed biological activity does not result from inhibition of either these reporter enzymes (data provided in the supporting information). To further confirm that the biological activity of analogues 29-31 does not relate to non-specific detergent-like action, we evaluated the effect of these compounds on replication of intracellular M. fortuitum bacteria in infected HeLa cells. Whereas the non-ionic detergent Triton X-100 (1%) reduced bacterial survival by 18-fold compared with DMSO vehicle alone, no deleterious effects of 1 or 29-31 (0.5 μM) on bacterial replication was observed. Together, these results suggest that 1 and the potent analogues 29-31 selectively inhibit one or more mammalian host proteins necessary for both proliferation of HeLa cervical carcinoma cells and replication of PIV5.

Figure 3.

Panel A: Design of recombinant PIV5 expressing renilla luciferase. The gene encoding luciferase was inserted into the PIV5 genome between the HN and L genes. Panel B: Validation of rPIV5-RL by luciferase assay of infected HeLa cells. The activity of the highest dose of infection, 34 infectious viruses per cell, was set as 100%. Error bars represent standard errors of the mean.

Figure 4.

Initial evaluation of compounds 1 and 12-32 against rPIV5-RL at one day post infection. All compounds were tested at 1 μM. Compounds comparable in activity to 1 were further examined at 0.5 μM (concentrations (μM) shown in brackets). Error bars represent standard errors of the mean.

Figure 5.

Panels A-C: Dose-dependent effects of compounds 1 and 29-31 on renilla luciferase expressed by rPIV5-RL in HeLa cells (Panel A), on viability of HeLa cells after 24 h (Panel B), and on viability of NHBE cells after 40 h (Panel C). Viability of mammalian cells was measured by quantification of cellular ATP with firefly luciferase. Panel D: Examination of effects of compounds (0.5 μM) compared with Triton X-100 (1%) on replication of intracellular M. fortuitum bacteria in HeLa cells. Data are in triplicate or larger numbers of measurements. Standard errors of the mean are ≤ 20%.

How AKT inhibitor-IV (1) affects the PI3K/AKT pathway is not well understood. In studies of the relatively high concentration of 10 μM, 1 was originally proposed by Silver¹¹ to target the ATP binding site of unknown kinases upstream of AKT but downstream of PI3K. However, a more recent investigation⁸ by Connor of cells treated with 1 μM of 1 indicates that this compound exhibits potent antiviral and antiproliferative effects through a unique mechanism that does not involve inhibition of the AKT1 or AKT2 subtypes or inhibition of other known kinases within the PI3K/AKT pathway. At 1 μM, 1 is known to promote hyperphosphorylation of AKT in multiple mammalian cell lines, a process that requires PI3K activity, and could potentially involve activation of the PDK1 kinase directly upstream of AKT.⁸ Additionally, in a high-throughput screening assay against 84 kinases, 1 (1 μM) showed no inhibitory activity against 82 of these enzymes, and only slight inhibition of the AGC kinases SGK1 and MKK1 was observed.⁸ However, treatment of cells with 1 is known to decrease phosphorylation of AKT substrates such as 4E-BP-1, suggesting that inhibition occurs downstream of AKT kinase, possibly implicating mTORC2 as a target.⁸ Inhibition or activation of other aspects of normal cellular function outside of the PI3K/AKT pathway may also play key roles in the potent biological activities of 1.⁸

In conclusion, we synthesized the potent antiviral/antiproliferative agent AKT inhibitor-IV (1) by developing a zirconium-mediated condensation approach that allows coupling of the sensitive α,β-unsaturated aldehydes with the novel 1,2-arylenediamine 7. This methodology allowed the preparation of a collection of 21 analogues. Evaluation of these analogues identified two compounds (30, 31) with greater activity than 1 against recombinant PIV5 expressing luciferase in HeLa cells. Although these compounds were found to exhibit potent cytotoxic activity against HeLa carcinoma cells, this toxicity was 6-fold to >20-fold lower against normal NHBE cells, and no effect against replication of intracellular M. fortuitum bacteria was observed, suggesting the possibility of selective inhibition of unidentified host protein(s) involved in proliferation of cancer cells and PIV5. The ability to substitute AKT inhibitor-IV (1) with long side chains at the N3 position without deleteriously affecting its antiviral and cytotoxic activity may be useful for future affinity chromatography³³ or related methods directed at identification of protein target(s) of AKT inhibitor-IV (1).

Experimental Section

General

Chemical reagents and solvents were obtained from Acros, Aldrich, EMD Biosciences and Combi-Blocks. Commercial grade reagents were used without further purification unless otherwise noted. Compounds 10²⁷ and 33³⁴ were prepared using previously described methods. Compounds 35 and 36 were prepared by converting 4-chloro-3-nitro-benzoic acid to the acid chloride with thionyl chloride followed by reaction with methanol and aniline, respectively. Anhydrous solvents were obtained after passage through a drying column of a solvent purification system from GlassContour (Laguna Beach, CA). All reactions were performed under an atmosphere of dry nitrogen. Reactions were monitored by analytical thin-layer chromatography on plates coated with 0.25 mm silica gel 60 F254 (EMD Chemicals). TLC plates were visualized by UV irradiation (254 nm) or stained with 20% phosphomolybdic acid in ethanol. Flash column chromatography employed silica gel (ICN SiliTech, 32–63 μm). Melting points were measured with a Thomas Hoover capillary melting point apparatus and are uncorrected. Infrared spectra were obtained with a Perkin Elmer 1600 Series FTIR. NMR spectra were obtained with Bruker CDPX-300 and AV-400 instruments with chemical shifts reported in parts per million (ppm, δ). High-resolution mass spectra were obtained from the proteomics and mass spectrometry core facility at Penn State University, University Park, PA or the mass spectrometry facility at the University of Kansas. Peaks are reported as m/z. Analytical purity of compounds was determined by analytical reverse-phase HPLC with a PRP-1 (polystyrene-divinylbenzene) reverse-phase column (4.1 × 250 mm, 7 μm; Hamilton) running a gradient of 10% to 99.9% CH₃CN in nanopure water (containing 0.1% TFA) over 20 min at a flow rate of 0.8 mL/min. All compounds subjected to biological assays were ≥98% pure by analytical reverse phase HPLC.

2-(4-Chloro-3-nitro-phenyl)-benzothiazole (4)

To a solution of compound 2 (5.5 g, 30 mmol) in anhydrous ethanol (250 mL) was added 2-aminothiophenol (3, 3.3 mL, 30 mmol). The reaction was refluxed for 16 h and cooled to 22 °C. A white precipitate was collected by filtration with a fritted funnel, and the solid crude product was washed with cold ethanol (10 mL × 3). Recrystallization from toluene afforded the product (6.26 g, 72%) as an off white solid; mp 163–164 °C; R_f = 0.51 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (s, 1H), 8.35 (d, 1H, J = 8.4 Hz), 8.21 (d, 1H, J = 8.0 Hz), 8.12 (d, 1H, J = 8.0 Hz), 7.96 (d, 1H, J = 8.4 Hz), 7.58 (dd, 1H, J = 8.4 Hz), 7.52 (dd, 1H, J = 8.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.3, 153.7, 148.6, 135.4, 133.4, 133.2, 132.3, 127.8, 127.6, 126.8, 124.1, 123.8, 123.2; IR (film) ν max 1604, 1566, 1538, 1456, 1430, 1339, 1127, 1050, 990, 887, 835, 764, 730 cm⁻¹; HRMS (ESI-) m/z 288.9831 (M-H⁻, C₁₃H₆ClN₂O₂S requires 288.9839).

(4-Benzothiazol-2-yl-2-nitrophenyl)phenylamine (6)

To a solution of compound 4 (2.9 g, 10 mmol) in DMSO (50 mL) was added freshly distilled aniline (5, 4.6 mL, 50 mmol). The reaction was heated to 70 °C and stirred for 8 h. The reaction was cooled to 22 °C and poured into water (200 mL). The aqueous phase was extracted with diethyl ether (100 mL × 4). The organic extracts were combined and washed with saturated aqueous NaCl (200 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (CH₂Cl₂ eluent) to afford the product (2.46 g, 71%) as a orange solid; mp 160–162 °C; R_f = 0.56 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 1H), 8.86 (d, 1H, J = 2.1 Hz), 8.07 (dd, 1H, J₁ = 2.1 Hz, J₂ = 9.0 Hz), 8.00 (d, 1H, J = 8.1 Hz), 7.86 (d, 1H, J = 9 Hz), 7.49-7.44 (m, 3H), 7.36-7.25 (m, 5H); ¹³C NMR (75 MHz, CDCl3) δ 165.6, 156.6, 144.5, 137.8, 134.7, 134.0, 132.7, 129.9 (× 2), 126.5, 126.4, 126.0, 125.2, 124.8 (× 2), 123.3, 123.0, 121,6, 116.4; IR (film) ν max 3333, 1624, 1594, 1567, 1531, 1496, 1353, 1266, 1210, 753 cm⁻¹; HRMS (ESI+) m/z 348.0833 (M+H⁺, C₁₉H₁₄N₃O₂S requires 348.0807).

4-Benzothiazol-2-yl-N¹-phenylbenzene-1,2-diamine (7)

To a slurry of compound 6 (1.74 g, 5 mmol) in ethanol (50 mL) was added palladium on carbon (10%, 530 mg, 0.5 mmol) and anhydrous hydrazine (0.5 mL, 16 mmol). The reaction was heated to 80 °C and refluxed for 30 min. The reaction was cooled to 22 °C and filtered through a fritted funnel. The solid Pd/C was washed with ethanol (10 mL), and the combined filtrate was concentrated in vacuo to afford the crude product. Recrystallization from CH₂Cl₂/hexane afforded the product (1.52 g, 95%) as a light yellow solid; mp 154–156 °C; R_f = 0.13 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, DMSO-d₆) δ 8.06 (d, 1H, J = 7.5 Hz), 7.97 (d, 1H, J = 7.7 Hz), 7.55 (d, 1H, J = 2.0 Hz), 7.52-7.46 (m, 1H) 7.44 (s, 1H) 7.41-7.36 (m, 1H) 7.29-7.19 (m, 4H), 7.01 (d, 2H, J = 8 Hz) 6.83 (t, 1H, J = 7.3 Hz), 5.17 (s, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 168.0, 153.8, 143.7, 140.2, 134.1, 132.3, 129.1 (× 2), 126.4, 126.3, 124.8, 122.2, 122.1, 119.7, 118.4, 117.0 (× 2), 116.4, 113.2; IR (film) ν max 3360, 3037, 1595, 1496, 1434, 1313, 1194, 1000, 810, 749, 728, 693 cm⁻¹; HRMS (ESI+) m/z 318.1066 (M+H⁺, C₁₉H₁₆N₃S requires 318.1065).

[(E)-2-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)ethenyl]methyl phenylamine (12)

To a solution of compound 7 (64 mg, 0.2 mmol) and 10 (32 mg, 0.2 mmol) in anhydrous ethanol (15 mL) was added ZrCl₄ (24 mg, 0.1 mmol). The reaction was heated to 80 °C and refluxed for 30 min. When the starting material was consumed as evidenced by TLC, MnO₂ (70 mg, 0.8 mmol) was added and the reaction was stirred at room temperature (22 °C) for 5 min. The reaction was cooled and filtered through a fritted funnel. The solid MnO₂ was washed with ethanol (10 mL), and the combined filtrate was concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 4:1) afforded the product (68 mg, 75%) as a yellow solid; mp 215–217 °C; R_f = 0.13 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.34 (s, 1H), 8.31 (d, 1H, J = 0.9 Hz), 8.05 (d, 1H, J = 6.0 Hz), 7.99 (dd, 1H, J₁ = 1.2 Hz, J₂ = 6.3 Hz), 7.89 (d, 1H, J = 5.8 Hz), 7.64-7.44 (m, 5H), 7.34 (dd, 3H, J₁ = 5.6 Hz, J₂ = 6.0 Hz), 7.18 (d, 2H, J = 5.8 Hz), 7.13 (d, 1H, J = 6.3 Hz), 7.08 (t, 1H, J = 5.5 Hz), 5.22 (d, 1H, J = 9.9 Hz), 3.19 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 169.8, 155.9, 154.8, 147.0, 143.2 (× 2), 139.1, 136.2, 135.5, 130.4 (× 2), 129.8 (× 2), 129.2, 128.7, 128.0 (× 2), 126.4, 125.1, 123.9, 121.9, 121.3, 119.8, 117.7, 114.9, 111.7, 109.9 87.5, 36.5; IR (film) ν max 3059, 2944, 2901, 1627, 1594, 1492, 1464, 1435, 1347, 1298, 1270, 1127, 978, 910, 758, 730, 696 cm⁻¹; HRMS (ESI+) m/z 459.1663 (M+H⁺, C₂₉H₂₃N₄S requires 459.1643).

5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-3H-benzo-imidazol-1-ium, iodide (1)

A slurry of 12 (35 mg, 0.076 mmol) in ethyl iodide (76, 3 mL) was heated to 75 °C and refluxed for 24 h. The solvent was removed in vacuo. The crude product was purified by flash column chromatography (CH₂Cl₂/MeOH, 20:1) to afford the product (41 mg, 88%) as a yellow solid; mp 165–167 °C; R_f = 0.18 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.43 (s, 1H), 8.10-8.00 (m, 3H), 7.83-7.69 (m, 5H), 7.53-7.14 (m, 7H), 6.74 (d, J = 6.5 Hz, 2H), 5.52 (d, J = 12.3 Hz, 1H), 4.61 (q, J = 7.1 Hz, 2H), 3.41 (s, 3H), 1.61 (t, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 167.2, 154.1, 152.1, 150.7 (× 2), 135.9, 135.3, 135.0, 132.1, 131.9 (× 2), 131.5, 131.2, 129.7 (× 2), 128.1 (× 2), 127.0, 126.3, 126.0, 125.5, 123.0, 122.1, 120.4 (× 2), 112.0 (× 2), 109.6, 78.2, 40.3, 12.9; IR (film) ν max 3290, 3058, 2924, 1688, 1618, 1507, 1537, 1494, 1465, 1371, 1308, 1203, 1132, 1029, 800, 763, 697 cm⁻¹; HRMS (ESI+) m/z 487.1949, (M+H⁺, C₃₁H₂₇N₄S requires 487.1956).

(4-Benzooxazol-2-yl-2-nitrophenyl)phenylamine (40)

2-(4-Chloro-3-nitro-phenyl)benzooxazole (33, 1.2 g, 4.5 mmol) and freshly distilled aniline (1.8 mL, 20 mmol) were used to synthesize 40 using the procedure described for preparation of 6. Column chromatography (CH₂Cl₂) afforded the product (2.24 g, 84%) as a orange solid, mp 164–166 °C; R_f = 0.15 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 9.82 (s, 1H), 9.09 (s, 1H), 8.20 (d, J = 9.0 Hz, 1H), 7.73 (m, 1H), 7.57 (m, 1H), 7.52-7.47 (m, 2H), 7.36-7.29 (m, 5H); ¹³C NMR (75 MHz, CDCl₃) δ 161.3, 150.6, 144.9, 142.0, 137.6, 134.0, 132.6, 130.0 (× 2), 126.7, 126.4, 125.1, 125.0 (× 2), 124.7, 119.7, 116.4 (× 2), 110.5; IR (film) ν max 3339, 1628, 1596, 1507, 1452, 1352, 1264, 1242, 1152, 1077 cm⁻¹; HRMS (ESI+) m/z 332.1035 (M+H⁺, C₁₉H₁₄N₃O₃ requires 332.1035).

(2-Nitrophenyl)phenylamine (41)

1-Chloro-2-nitrobenzene (34, 1.57 g, 10 mmol) and freshly distilled aniline (2.7 mL, 30 mmol) were used to synthesize 41 using the procedure described for preparation of 6. Column chromatography (CH₂Cl₂) afforded the product (1.58 g, 74%) as a orange solid, mp 73–75 °C; R_f = 0.18 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 9.50 (s, br, 1H), 8.20 (dd, J₁ = 7.6 Hz, J₂ = 1.4 Hz, 1H), 7.46-7.21 (m, 7H) 6.78 (m, 1H); ¹³C NMR (75 MHz, CDC_l3) δ 143.1, 138.7, 135.7, 133.2, 129.7, 126.7, 125.6, 124.4, 117.5, 116.0; IR (film) ν max 3389, 1630, 1591, 1576, 1508, 1452, 1415, 1351, 1262, 1241, 1153, 1076 cm⁻¹. HRMS (ESI+) m/z 215.0822 (M+H⁺, C₁₂H₁₁N₂O₂ requires 215.0821).

3-Nitro-4-phenylaminobenzoic acid methyl ester (42)

4-Chloro-3-nitrobenzoic acid methyl ester (35, 2.15 g, 10 mmol) and freshly distilled aniline (2.7 mL, 30 mmol) were used to synthesize 42 using the procedure described for preparation of 6. Column chromatography (CH₂Cl₂) afforded the product (2.37 g, 87%) as a yellow solid, mp 121–123 °C; R_f = 0.48 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, br, 1H), 8.93 (d, J = 2.0 Hz, 1H), 7.97 (dd, J₁ = 8.9 Hz, J₂ = 2.1 Hz, 1H), 7.48 (dd, J₁ = J₂ = 7.6 Hz, 2H), 7.32 (m, J₁ = 8.9 Hz, J₂ = 2.1 Hz, 3H), 7.17 (d, J = 9.0 Hz, 1H), 3.92 (s, 3H); ¹³C NMR (100 MHz, CDC_l3) δ 165.4, 146.0, 137.6, 135.9, 132.2, 129.8 (× 2), 129.3, 126.8, 125.1 (× 2), 119.2, 115.6; IR (film) ν max 3337, 2951, 1718, 1624, 1596, 1567, 1524, 1497, 1436, 1354, 1312, 1281, 1212, 1151, 1119, 1071, 987, 918, 756, 692 cm⁻¹; HRMS (ESI+) m/z 273.0851 (M+H⁺, C₁₄H₁₃N₂O₄ requires 273.0875).

3-Nitro-N-phenyl-4-phenylaminobenzamide (43)

4-Chloro-3-nitro-N-phenylbenzamide (2.2 g, 8 mmol) and freshly distilled aniline (3.7 mL, 40 mmol) were used to synthesize 43 using the procedure described for preparation of 6. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the product (2.24 g, 84%) as a orange solid, mp 212–215 °C; R_f = 0.16 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 9.81 (s, 1H), 8.74 (d, J = 1.5 Hz, 1H), 7.98 (dd, J₁ = 1.3 Hz, J₂ = 6.7 Hz, 1H), 7.79 (s, 1H), 7.66 (d, J = 5.9 Hz, 2H), 7.50 (t, J = 5.9 Hz, 2H), 7.43 (t, J = 5.6 Hz, 2H), 7.34-7.27 (m, 3H), 7.19 (t, J = 5.6 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 165.8, 144.1, 139.8, 139.6, 131.9, 129.0 (× 2), 128.8, 128.5 (× 2), 123.1, 120.1 (× 2), 119.3, 118.3, 116.5 (× 2), 116.3, 114.7; IR (film) ν max 3331, 1648, 1623, 1596, 1440, 1319, 1267, 1214, 1152, 753, 690 cm⁻¹; HRMS (ESI+) m/z 334.1181 (M+H⁺, C₁₉H₁₆N₃O₃ requires 334.1192).

4-Benzooxazol-2-yl-N¹-phenylbenzene-1,2-diamine (47)

Compound 40 (900 mg, 2.7 mmol) was treated with anhydrous hydrazine (0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (758 mg, 93%) as a yellow solid; R_f = 0.53 (hexane/ethyl acetate, 1:1); mp 142–143 °C; ¹H NMR (300 MHz, CDCl₃) δ 7,79-7.69 (m, 3H), 7.60-7.57 (m, 1 H), 7.37-7,29 (m, 5H), 7.01-6.98 (m, 3H), 5,60 (s, 1H), 3.65 (s, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 163.4, 150.6, 142.9, 142.2, 138.7, 134.2, 129.4 (× 2), 124.6, 124.4, 121.6, 121.1, 119.7, 119.7, 119.5, 117.6 (× 2), 115.6, 110.5; IR (film) ν max 3364, 3048, 1614, 1594, 1557, 1497, 1454, 1313, 1244, 744 cm⁻¹; HRMS (ESI+) m/z 302.1302 (M+H⁺, C₁₉H₁₆N₃O requires 302.1293).

N-Phenylbenzene-1,2-diamine (48)

Compound 41 (1.07 g, 5.0 mmol) was treated with anhydrous hydrazine (0.75 mL, 24 mmol) and Pd/C (530 mg, 0.5 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the product (840 mg, 91%) as a yellow solid; R_f = 0.65 (hexane/ethyl acetate, 1:1); mp 77–79 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.19 (dd, J₁ = 7.6 Hz, J₂ = 8.5 Hz, 2H), 7.10 (dd, J₁ = 7.9 Hz, J₂ = 1.5 Hz, 1H), 7.00 (dd, J₁ = J₂ = 7.2 Hz, 1H), 6.81-6.71 (m, 5H), 5,15 (s, br, 1H), 3.70 (s, br, 2H); ¹³C NMR (75 MHz, CDCl₃) δ145.3, 141.9, 129.2 (× 2), 128.5, 125.6, 124.9, 119.2, 119.0, 116.1, 115.1 (× 2); IR (film) ν max 3445, 3388, 3360, 3048, 1604, 1594, 1502, 1461, 1323, 1265, 1254 cm⁻¹; HRMS (ESI+) m/z 185.1075 (M+H⁺, C₁₂H₁₂N₂ requires 185.1073).

3-Amino-4-phenylaminobenzoic acid methyl ester (49)

Compound 42 (816 mg, 3.0 mmol) was treated with anhydrous hydrazine (0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (675 mg, 93%) as a white solid; R_f = 0.45 (hexane/ethyl acetate, 1:1); mp 89–90 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 7.39 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.24 (dd, J₁ = J₂ = 7.6 Hz, 2H), 7.15 (dd, J₁ = 2.0 Hz, J₂ = 7.6 Hz, 1H), 7.07 (d, J₁ = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 2H), 6.85 (dd, J = 7.6 Hz, 1H), 3.77 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.0, 143.6, 139.2, 134.5, 129.6 (× 2), 122.5, 120.6, 119.1, 118.1 (× 2), 116.8, 116.1, 52.0; IR (film) ν max 3368, 3049, 2949, 1698, 1594, 1518, 1497, 1447, 1311, 1252, 1112, 1079, 993, 887, 763, 749, 695 cm⁻¹; HRMS (ESI+) m/z 243.1136 (M+H⁺, C₁₄H₁₅N₂O₂ requires 243.1134).

3-Amino-N-phenyl-4-phenylaminobenzamide (50)

Compound 43 (1.0 g, 3.0 mmol) was treated with anhydrous hydrazine (0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (875 mg, 96%) as a white solid; R_f = 0.53 (hexane/ethyl acetate, 1:1); mp 176–178 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.76 (d, J = 7.6 Hz, 2H), 7.34-7.29 (m, 4H), 7.23-7.10 (m, 4H), 7.05 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.6 Hz, 2H), 6.79 (t, J = 7.2 Hz, 1H), 5.0 (s, 2H), 7.19 (t, J = 5.6 Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 165.8, 144.1, 139.8, 139.6, 131.9, 129.1 (× 2), 128.8, 128.5 (× 2), 123.1, 120.1 (× 2), 119.3, 118.3, 116.5 (× 2), 116.4, 114.7; IR (film) ν max 3324, 1643, 1594, 1497, 1434, 1316, 1241, 886, 774, 691 cm⁻¹; HRMS (ESI+) m/z 304.1457 (M+H⁺, C₁₉H₁₈N₃O requires 304.1450).

[(E)-2-(5-Benzooxazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)ethenyl]methylphenylamine (61)

Compound 47 (60 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 61 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (48 mg, 54%) as a light yellow solid; mp 194–196 °C; R_f = 0.54 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.55 (d, J = 1.1 Hz, 1H), 8.35 (d, J = 13.2 Hz, 1H), 8.09 (dd, J₁ = 1.5 Hz, J₂ = 8.4 Hz, 1H), 7.78 (m, 1H), 7.68-7.54 (m, 4 H), 7.51-7.49 (m, 2H), 7.39-7.31 (m, 4H), 7.21-7.02 (m, 4H), 5.36 (d, J = 13.2 Hz, 1H), 3.15 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 164.3, 155.6, 150.8, 146.6, 142.8, 142.4, 140.0, 135.7, 130.0 (× 2), 129.3 (× 3), 128.9, 127.6 (× 2), 124.4, 124.3, 123.5, 121.4, 121.4, 119.6, 119.4 (× 2), 117.1, 110.4, 109.5, 87.0, 36.1; IR (film) ν max 3060, 1627, 1594, 1582, 1492, 1453, 1347, 1297, 1244, 1127, 746, 696 cm⁻¹; HRMS (ESI+) m/z 443.1875 (M+H⁺, C₂₉H₂₃N₄O requires 443.1872).

Methyl-phenyl-[(E)-2-(1-phenyl-1H-benzoimidazol-2-yl)ethenyl]amine (62)

N-Phenylbenzene-1,2-diamine (48, 37 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 62 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (39 mg, 61%) as a yellow solid; mp 118–121 °C; R_f = 0.54 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.30 (d, J = 13.3 Hz, 1H), 7.71(d, J = 7.9 Hz, 1H), 7.64-7.46 (m, 5H), 7.34 (t, J = 7.9 Hz, 2H), 7.25-7.05 (m, 6H), 5.26 (d, J = 13.3 Hz, 1H), 3.20 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 153.8, 146.7, 142.0, 136.5, 136.3, 129.8 (× 2), 129.3 (× 2), 128.5, 127.6 (× 2), 123.1, 122.4, 121.1, 119.2 (× 2), 117.6, 109.2 (× 2), 87.6, 35.9; IR (film) ν max 3060, 1629, 1594, 1492, 1454, 1347, 1301, 1286, 1267, 1127, 757, 695 cm⁻¹; HRMS (ESI+) m/z 326.1651 (M+H⁺, C₂₂H₂₀N₃ requires 326.1657).

2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-benzoimidazole-5-carboxylic acid methyl ester (63)

3-Amino-4-phenylaminobenzoic acid methyl ester (49, 48 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 63 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (50 mg, 65%) as a light yellow solid; mp 169–170 °C; R_f = 0.64 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J = 1.3 Hz, 1H), 8.28 (d, J = 13.3 Hz, 1H), 7.82 (dd, J₁ = 1.5 Hz, J₂ = 8.4 Hz, 1H), 7.63-7.53 (m, 3H), 7.45-7.43 (m, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 7.07 (m, 2H), 5.20 (d, J = 13.3 Hz, 1H), 3.93 (s, 3H), 3.17 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 167.9, 155.7, 146.6, 143.3, 142.7, 139.9, 135.8, 130.0 (× 2), 129.3 (× 2), 128.8, 127.5 (× 2), 124.4, 123.5, 123.0, 119.6, 119.3 (× 2), 108.6, 87.1, 52.0, 36.1; IR (film) ν max 1712, 1628, 1492, 1438, 1348, 1297, 1224, 1127, 1085, 751, 696 cm⁻¹; HRMS (ESI+) m/z 384.1731 (M+H⁺, C₂₄H₂₂N₃O₂ requires 384.1712).

2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-benzoimidazole-5-carboxylic acid phenylamide (64)

Compound 50 (61 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 64 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (50 mg, 56%) as a yellow solid; mp 121–123 °C; R_f = 0.50 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.28 (d, J = 17.3 Hz, 1H), 8.12(s, 1H), 7.72-7.07 (m, 18H), 5.21 (d, J = 13.4 Hz, 1H), 3.16 (s, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 169.3, 158.3, 157.5, 148.0, 144.5, 143.7, 140.1, 140.0, 136.8, 131.4 (× 2), 130.8, 130.6 (× 2), 130.5, 129.8 (× 2), 128.7, 125.4, 124.8, 122.6, 122.4 (× 2), 120.3 (× 2), 117.4, 110.2, 87.1, 36.3; IR (film) ν max 3290, 3059, 1625, 1595, 1540, 1498, 1433, 1316, 1297, 1127, 751 cm⁻¹; HRMS (ESI+) m/z 445.2021 (M+H⁺, C₂₉H₂₅N₄O requires 445.2028).

5-Benzooxazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, iodide (16)

Compound 61 (35 mg, 0.079 mmol) and ethyl iodide (3 mL) were used to synthesize 16 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (40 mg, 86%) as a yellow solid, mp 181–184 °C; R_f = 0.22 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, CDCl₃) δ 8.75 (s, 1H), 8.44 (d, J = 12.2 Hz, 1H), 7.71-7.56 (m, 7H), 7.31-7.03 (m, 7H), 6.52 (m, 2H), 5.41 (d, J = 11.6 Hz, 1H), 4.50 (q, J = 6.7 Hz, 2H), 3.17 (s, 3H), 1.50 (t, J = 6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 163.4, 153.3, 152.2, 151.8, 142.9, 137.3, 136.0, 132.9 (× 3), 132.6, 130.7 (× 3), 129.2 (× 3), 127.3, 127.0, 126.3, 126.2, 125.4, 120.8, 113.1, 111.9, 111.3, 79.1, 41.1, 36.9, 13.9; IR (film) ν max 3425, 1681, 1617, 1586, 1537, 1494, 1453, 1365, 1298, 1197, 1136, 761, 697 cm⁻¹; HRMS (ESI+) m/z 471.2168 (M⁺, C₃₁H₂₇N₄O requires 471.2185).

1-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenyl-3H-benzoimidazol-1-ium, iodide (15)

Compound 62 (30 mg, 0.092 mmol) and ethyl iodide (3 mL) were used to synthesize 15 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (37 mg, 85%) as a yellow solid; mp 43–45 °C; R_f = 0.28 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 7.83-7.77 (m, 4H), 7.69-7.66(m, 2H), 7.52 (t, J = 7.2, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.31-7.08 (m, 5H), 6.74 (d, J = 7.9 Hz, 2H), 5.56 (d, J = 11.3 Hz, 1H), 4.58 (d, J = 7.3 Hz, 2H), 3.41 (s, 3H), 1.58 (t, J = 7.3 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 152.1, 151.6, 151.5, 147.6, 136.8, 135.5, 133.2 (× 2), 132.9, 132.7, 131.1 (× 2), 129.7 (× 2), 127.4, 127.3, 127.2, 121.8, 112.9, 112.8, 79.5, 41.6, 37.5, 14.4; IR (film) ν max 3360, 1688, 1623, 1585, 1533, 1494, 1361, 1310, 1201, 1177, 1129, 759, 697 cm⁻¹; HRMS (ESI+) m/z 354.1949 (M⁺, C₂₄H₂₄N₃ requires 354.1970).

1-Ethyl-6-methoxycarbonyl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenyl-3H-benzoimi-dazol-1-ium, iodide (14)

Compound 63 (30 mg, 0.078 mmol) and ethyl iodide (3 mL) were used to synthesize 14 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (39 mg, 93%) as a yellow solid, mp 43–46 °C; R_f = 0.25 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, DMSO-d₆) δ 8.47 (s, 1H), 8.02 (dd, J₁ = 8.6 Hz, J₂ = 1.2 Hz, 1H), 7.84-8.63 (m, 5H), 7.29 (t, J = 8.1 Hz, 2H), 7.21-7.14 (m, 3H), 6.70 (d, J = 7.9 Hz, 2H), 5.76 (d, J = 13.5 Hz, 1H), 4.70 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.41 (s, 3H), 1.46 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 165.6, 152.0, 149.5, 145.2, 136.6, 134.5, 131.5 (× 2), 131.1, 130.9, 129.5 (× 3), 128.0 (× 2), 126.5, 126.4, 125.6, 119.6, 112.7, 111.3, 79.1, 52.5, 39.4, 36.4, 13.7; IR (film) ν max 3412, 1716, 1692, 1622, 1588, 1538, 1621, 1588, 1538, 1494, 1463, 1373, 1312, 1290, 1266, 1198, 1134, 764, 698 cm⁻¹; HRMS (ESI+) m/z 412.2000 (M⁺, C₂₆H₂₆N₃O₂ requires 412.2028).

3-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-5-phenylcarbamoyl-3H-benzoimidazol-1-ium, iodide (18)

Compound 64 (35 mg, 0.079 mmol) and ethyl iodide (3 mL) were used to synthesize 18 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (40 mg, 84%) as a yellow solid, mp 285–287 °C (decomp.); R_f = 0.44 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, DMSO-d₆) δ 10.4 (s, 1H), 8.52(s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.82 (m, 7H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.24-7.15 (m, 4H), 6.73 (d, J = 7.6 Hz, 2H), 5.77 (d, J = 12.8 Hz), 4.69 (q, J = 6.5 Hz, 2H), 3.43 (s, 3H), 1.53 (t, J = 6.2 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 164.8, 152.0, 149.7, 145.6, 139.3, 135.8, 134.9, 132.2, 131.9 (× 2), 131.4, 131.1, 129.9 (× 2), 129.1 (× 2), 128.4 (× 2), 125.9, 125.7, 124.4, 121.0 (× 2), 119.9 (× 2), 111.6, 111.3, 79.6, 40.9, 36.8, 14.2; IR (film) ν max 3260, 1662, 1620, 1587, 1525, 1493, 1463, 1440, 1369, 1306, 1251, 1128, 1209, 758 cm⁻¹; HRMS (ESI+) m/z 473.2335 (M⁺, C₃₁H₂₉N₄O requires 473.2341).

2-(1,2-Diphenyl-1H-benzoimidazol-5-yl)benzothiazole (68)

Compound 7 (64 mg, 0.2 mmol) and benzaldehyde (20 mL, 0.2 mmol) were used to synthesize 68 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (42 mg, 52%) as a light yellow solid; mp 184–186 °C; R_f = 0.28 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.54 (d, J = 1.3 Hz, 1H), 8.14 (dd, J₁ = 1.7 Hz, J₂ = 8.6 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.60-7.43 (m, 6H), 7.38-7.29 (m, 7H); ¹³C NMR (75 MHz, CDCl₃) δ168.8, 154.3, 153.9, 143.2, 139.2, 136.6, 135.1, 130.0 (× 2), 129.8, 129.5 (× 2), 129.0, 128.9, 128.4 (× 2), 127.3 (× 2), 126.6, 126.2, 123.0, 122.9 (× 2), 121.5, 119.7, 111.0 cm⁻¹; HRMS (ESI+) m/z 404.1249 (M+H⁺, C₂₆H₁₈N₃S requires 404.1221).

2-(2-Phenethyl-1-phenyl-1H-benzoimidazol-5-yl)benzothiazole (71)

Compound 7 (64 mg, 0.2 mmol) and 3-phenylpropionaldehyde (26 mL, 0.2 mmol) were used to synthesize 71 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 3:1) afforded the product (41 mg, 48%) as a light yellow solid; mp 56–58 °C; R_f = 0.10 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.48 (s, 1H), 8.07 (dd, 2H, J₁ = 3.2 Hz, J₂ = 4.8 Hz), 7.91 (d, 1H, J = 7.9 Hz), 7.55-7.31 (m, 5H), 7.25-7.14 (m, 6H), 7.08 (d, 2H, J = 7.0 Hz), 3.21 (t, 4H, J = 6.5 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.9, 156.0, 154.3, 142.8, 140.6, 138.4, 135.3, 135.1, 130.0 (× 2), 129.3, 128.5 (× 2), 128.4 (× 2), 127.3 (× 2), 126.3, 126.2, 124.9, 123.0 (× 2), 122.4, 121.5, 119.1, 110.6, 34.0, 29.8; IR (film) ν max 3052, 3028, 2921, 2862, 1596, 1514, 1498, 1462, 1434, 1393, 1314, 1274, 758, 728, 699 cm⁻¹; HRMS (ESI+) m/z 432.1542 (M+H⁺, C₂₈H₂₂N₃S requires 432.1534).

2-[2-((E)-2-Furan-2-yl-ethenyl)-1-phenyl-1H-benzoimidazol-5-yl]benzothiazole (69)

To a solution of compound 7 (64 mg, 0.2 mmol) and trans-3-(2-furyl)acrolein (24 mg, 0.2 mmol) in ethanol (15 mL) was added ZrCl₄ (24 mg, 0.1 mmol). The reaction was heated to 80 °C and refluxed for 30 min. The reaction was cooled and poured into aqueous ammonium hydroxide (5%, 50 mL). The product was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over anhydrous Na₂SO₄, and concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (69 mg, 82%) as a yellow solid; mp 97–99 °C; R_f = 0.18 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 8.47 (d, 1H, J = 1.1 Hz), 8.11 (dd, 1H, J₁ = 1.5 Hz, J₂ = 8.5 Hz), 8.07 (d, 1H, J = 7.9 Hz), 7.91 (d, 1H, J = 7.8 Hz), 7.83 (d, 1H, J = 15.7 Hz), 7.68-7.58 (m, 3H), 7.50-7.46 (m, 3H), 7.40-7.37 (m, 2H), 7.26 (d, 1H, J = 8.5 Hz), 6.71 (d, 1H, J = 15.7 Hz), 6.57 (d, 1H, J = 3.3 Hz), 6.45 (dd, 1H, J₁ = 1.8 Hz, J₂ = 3.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 168.7, 154.3, 152.3, 152.0, 144.2, 138.4, 135.2, 134.9, 130.3 (× 2), 130.1, 129.5, 127.7 (× 2), 127.4, 125.7, 123.1, 123.0, 122.9 (× 2), 118.8, 118.7, 113.5, 112.5, 112.3, 110.9, 110.7; IR (film) ν max 3284, 3060, 2931, 2860, 1652, 1608, 1595, 1498, 1434, 1380, 1314, 1273, 1224, 1016, 757, 695 cm⁻¹; HRMS (ESI+) m/z 420.1166 (M+H⁺, C₂₆H₁₈N₃OS requires 420.1171).

2-(1-Phenyl-2(E)-styryl-1H-benzoimidazol-5-yl)benzothiazole (70)

Compound 7 (64 mg, 0.2 mmol) and trans-cinnamaldehyde (26 mL, 0.2 mmol) were used to synthesize 70 using the procedure described for preparation of 69. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (73 mg, 85%) as a yellow solid; mp 218–220 °C; R_f = 0.20 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.48 (d, 1H, J = 1.4 Hz), 8.11 (dd, 1H, J₁ = 1.6 Hz, J₂ = 8.5 Hz), 8.07 (d, 1H, J = 8.1 Hz), 8.01 (d, 1H, J = 16.0 Hz), 7.90 (d, 1H, J = 7.6 Hz), 7.66-7.55 (m, 3H), 7.50-7.45 (m, 5H), 7.37-7.32 (m, 5H), 6.85 (d, 1H, J = 16.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.8, 154.3, 152.4, 143.4, 138.5, 138.1, 135.8, 130.14 (× 2), 129.3 (× 2), 129.2 (× 2), 128.8 (× 2), 127.4, 126.6 (× 2), 126.2, 123.0 (× 2), 122.7, 121.5, 119.2 (× 2), 113.5, 110.7 (× 2); IR (film) ν max 3059, 2217, 1633, 1596, 1499, 1433, 1386, 1340,1274, 1216, 909, 756, 729 cm⁻¹; HRMS (ESI+) m/z 430.1393 (M+H⁺, C₂₈H₂₀N₃S requires 430.1378).

2-{2-[(E)-2-(4-Methoxyphenyl)ethenyl]-1-phenyl-1H-benzoimidazol-5-yl} benzothiazole (72)

Compound 7 (64 mg, 0.2 mmol) and trans-4-methoxycinnamaldehyde (33 mg, 0.2 mmol) were used to synthesize 72 using the procedure described for preparation of 69. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (79 mg, 86%) as a yellow solid; mp 177–179 °C; R_f = 0.17 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.46 (d, 1H, J = 1.4 Hz), 8.10 (dd, 1H, J₁ = 1.6 Hz, J₂ = 8.6 Hz), 8.07 (d, 1H, J = 7.2 Hz), 7.98 (d, 1H, J = 16.0 Hz), 7.91 (d, 1H, J = 7.9 Hz), 7.66-7.59 (m, 3H), 7.49-7.34 (m, 6H), 7.25 (d, 1H, J = 8.5 Hz), 6.88 (d, 2H, J = 8.7 Hz), 6.69 (d, 1H, J = 16.0 Hz), 3.81 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 168.9, 160.6, 154.3, 152.9, 143.3, 138.5, 137.9, 135.2, 135.1, 130.2 (× 2), 129.3, 129.1 (× 2), 129.0, 128.6 (× 2), 127.5, 126.2, 124.9, 123.0, 122.5, 121.5, 118.9, 114.3 (× 2), 111.0, 110.5, 55.3; IR (film) ν max 3061, 2954, 2929, 2837, 1603, 1514, 1500, 1434, 1304, 1254, 1173, 759 cm⁻¹; HRMS (ESI+) m/z 460.1473 (M+H⁺, C₂₉H₂₂N₃OS requires 460.1484).

2-(2-Nona-1(E),3(E)-dienyl-1-phenyl-1H-benzoimidazol-5-yl)benzothiazole (73)

Compound 7 (64 mg, 0.2 mmol) and trans,trans-2,4-decadienal (35 mL, 0.2 mmol) were used to synthesize 73 using the procedure described for preparation of 69. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the product (63 mg, 67%) as a yellow glassy solid; R_f = 0.40 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.43 (d, J = 1.5 Hz, 1H), 8.07 (m, 2H), 7.89 (d, J = 7.9 Hz, 1H), 7.64-7.56 (m, 4H), 7.50-7.33 (m, 4H), 7.23 (d, J = 19.0 Hz, 1H), 6.24-6.04 (m, 3H), 2.18-2.11 (m, 2H), 1.47-1.23 (m, 6H), 0.88 (t, J = 6.9 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 168.9, 154.4, 152.9, 143.5, 142.0, 139.1, 138.5, 135.3, 135.1, 130.0 (× 2), 129.5, 129.2, 129.0 (× 2), 127.5, 126.2, 124.8, 123.0, 122.4, 121.5, 119.0, 114.4, 110.4, 33.0, 31.4, 28.6, 22.5, 14.0; IR (film) ν max 3048, 2925, 2855, 1638, 1614, 1596, 1498, 1455, 1434, 1388, 1314, 1291, 993, 757 cm⁻¹; HRMS (ESI+) m/z 450.2025 (M+H⁺, C₂₉H₂₈N₃S requires 450.2004).

2-(1-Phenyl-1H-benzoimidazol-5-yl)benzothiazole (74)

To a solution of 7 (130 mg, 0.4 mmol) and 10 (65 mg, 0.4 mmol) in anhydrous ethanol (15 mL) was added activated 3Å molecular sieves (0.5 g). The reaction was heated to 80 °C and refluxed for 48 h. The reaction was cooled and filtered through a fritted funnel. The molecular sieves were washed with ethanol (10 mL), and the combined filtrate was concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 2:1) afforded the product (20 mg, 31%) as a white solid; mp 138–140 °C; R_f = 0.17 (hexane/ethyl acetate, 1:1); ¹H NMR (400 MHz, CDCl₃) δ 8.54 (d, J = 1.1 Hz, 1H), 8.15 (s, 1H), 8.14 (dd, J₁ = 1.4 Hz, J₂ = 9.9 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.58-7.54 (m, 3H), 7.49-7.44 (m, 4H), 7.34 (t, J = 7.3 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 168.4, 154.2, 144.3, 143.7, 135.8, 135.5, 135.0, 130.2 (× 2), 128.8, 128.4, 126.9, 126.2, 124.9, 124.0 (× 2), 123.2, 123.0, 121.5, 120.3, 111.0; IR (film) ν max 3382, 3064, 2954, 2360, 1617, 1599, 1509, 1461, 1435, 1314, 1284, 1240, 1216, 1167, 750, 719, 692 cm⁻¹; HRMS (ESI+) m/z 328.0897 (M+H⁺, C₂₀H₁₄N₃S requires 328.0908).

3-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)chromen-2-one (75)

To coumarin-3-carboxylic acid (40 mg, 0.2 mmol) in a round bottom flask (25 mL) fitted with a condenser was added thionyl chloride (2 mL, 10 mmol). The reaction was heated to 80 °C and refluxed for 2 h. Excess thionyl chloride was removed by distillation, and the residue was further dried by applying high vacuum for 30 min. The residue was dissolved in anhydrous toluene (5 mL), compound 7 (64 mg, 0.2 mmol) was added, and the reaction was stirred at 22 °C for 30 min. The reaction was subsequently heated to 110 °C and refluxed for 12 h. The reaction was cooled and concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (36 mg, 38%) as a off white solid; mp 164–166 °C; R_f = 0.20 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J = 1.4 Hz, 1H), 8.40 (s, 1H), 8.21 (dd, J₁ = 1.6 Hz, J₂ = 8.5 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.64-7.35 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 168.5, 157.8, 154.5, 154.3, 149.0, 146.4, 142.9, 138.6, 136.1, 135.1, 133.1, 129.8 (× 2), 129.2, 128.9, 128.8, 126.2 (× 2), 126.1,125.0, 124.9, 123.7, 124.9, 123.7, 123.0, 121.6, 119.9, 119.3, 118.5, 116.8, 111.2; IR (film) ν max 3060, 2966, 1735, 1608, 1499, 1456, 1434, 1386, 1325, 1282, 1242, 1215, 756 cm⁻¹; HRMS (ESI+) m/z 472.1113 (M+H⁺, C₂₉H₁₈N₃O₂S requires 472.1120).

5-Benzothiazol-2-yl-3-ethyl-1,2-diphenyl-3H-benzoimidazol-1-ium, iodide (19)

Compound 68 (30 mg, 0.074 mmol) and ethyl iodide (3 mL) were used to synthesize 19 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (37 mg, 90%) as a white solid; mp 72–74 °C; R_f = 0.33 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.88 (s, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.09 (t, J = 8.2 Hz, 2H), 7.73-7.47 (m, 13H), 4.65 (q, J = 7.1 Hz, 2H), 1.64 (t, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 166.6, 154.1, 152.5, 135.6, 135.0, 133.4, 133.3, 132.9, 132.0, 131.2, 130.7 (× 2), 130.5 (× 2), 129.7 (× 2), 127.7 (× 2), 127.4, 127.1, 126.3, 123.3, 122.2, 121.4, 114.6, 112.2, 42.6, 14.0; IR (film) ν max 3425, 3060, 2990, 1778, 1737, 1688, 1502, 1454, 1434, 1198, 1151, 1138, 760, 703 cm⁻¹; HRMS (ESI+) m/z 432.1518 (M⁺, C₂₈H₂₂N₃S requires 432.1534).

5-Benzothiazol-2-yl-3-ethyl-2-phenethyl-1-phenyl-3H-benzoimidazol-1-ium, iodide (22)

Compound 71 (35 mg, 0.08 mmol) and ethyl iodide (3 mL) were used to synthesize 22 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (44 mg, 93%) as a white solid; mp 131–133 °C; R_f = 0.30 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.73 (s, 1H), 8.30 (dd, J₁ = 1.4 Hz, J₂ = 8.6 Hz, 1H), 8.04 (t, J = 8.1 Hz, 2H), 7.83-7.72 (m, 3H), 7.58-7.44 (m, 5H), 7.24-7.21 (m, 3H), 6.99-6.96 (m, 2H), 4.70 (q, J = 7.2 Hz, 2H), 3.52 (t, J = 7.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 1.65 (t, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 167.5, 155.8, 155.0, 139.4, 136.6, 135.8, 133.9, 133.2, 132.9, 132.5, 132.0 (× 2), 130.2 (× 2), 129.4 (× 2), 128.5 (× 2), 128.4, 128.1, 128.0, 127.2, 124.2, 123.1, 115.1, 112.8, 42.9, 33.8, 27.8, 14.9; IR (film) ν max 3416, 3064, 1733, 1688, 1506, 1471, 1455, 1199, 1135, 762, 701 cm⁻¹; HRMS (ESI+) m/z 460.1833 (M⁺, C₃₀H₂₆N₃S requires 460.1847).

5-Benzothiazol-2-yl-3-ethyl-2-((E)-2-furan-2-yl-ethenyl)-1-phenyl-3H-benzo imidazol-1-ium, iodide (20)

Compound 69 (30 mg, 0.07 mmol) and ethyl iodide (3 mL) were used to synthesize 20 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (35 mg, 88%) as a yellow solid; mp 83–85 °C; R_f = 0.30 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.60 (d, 1H, J = 1.0 Hz), 8.19 (dd, J₁ = 1.5 Hz, J₂ = 8.7 Hz, 1H), 7.95 (t, J = 7.3 Hz, 2H), 7.72-7.62 (m, 6H), 7.47-7.35 (m, 3H), 6.85 (d, J = 1.4 Hz, 2H), 6.73 (d, J = 3.5 Hz, 1H), 6.51 (q, J = 1.8 Hz, 1H), 4.73 (q, J = 7.3 Hz, 2H), 1.61 (t, J = 7.3 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.0, 155.5, 152.0, 150.4, 149.0, 136.9, 136.8, 134.8, 134.7, 134.3, 133.6, 133.4, 132.7, 131.8, 129.2 (× 2), 128.5 (× 2), 127.6, 120.2, 115.1, 114.9, 112.7, 104.4, 43.2, 15.2; IR (film) ν max 3429, 3063, 2342, 1688, 1626, 1447, 1199, 1128, 762 cm⁻¹; HRMS (ESI+) m/z 448.1454 (M⁺, C₂₈H₂₂N₃OS requires 448.1484).

5-Benzothiazol-2-yl-3-ethyl-1-phenyl-2(E)-styryl-3H-benzoimidazol-1-ium, iodide (21)

Compound 70 (35 mg, 0.08 mmol) and ethyl iodide (3 mL) were used to synthesize 21 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (43 mg, 92%) as a light yellow solid; mp 199–202 °C; R_f = 0.33 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.76 (d, J = 0.9 Hz, 1H), 8.34 (dd, J₁ = 1.5 Hz, J₂ = 8.7 Hz, 1H), 8.06 (t, J = 7.7 Hz, 2H), 7.82-7.76 (m, 6H), 7.57-7.42 (m, 9H), 7.21 (q, J = 7.2 Hz, 2H), 1.73 (t, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 166.6, 154.1, 149.5, 148.9, 135.6, 135.4, 134.1, 133.6, 133.0, 132.1, 132.0, 131.8, 131.2 (× 2), 129.4 (× 2), 128.5 (× 2), 127.8 (× 2), 127.2, 127.1, 126.3, 123.3, 122.2, 114.0, 111.6, 106.6, 42.0, 13.9; IR (film) ν max 3416, 3061, 2988, 1688, 1632, 1503, 1470, 1441, 1199, 1135, 760 cm⁻¹; HRMS (ESI+) m/z 458.1684 (M⁺, C₃₀H₂₄N₃S requires 458.1691).

5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(4-methoxyphenyl)ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, iodide (23)

Compound 72 (35 mg, 0.076 mmol) and ethyl iodide (3 mL) were used to synthesize 23 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (42 mg, 90%) as a yellow solid; mp 188–192 °C; R_f = 0.33 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.51 (d, J = 1.0 Hz, 1H), 8.09 (d, J = 1.4 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 7.68-7.66 (m, 5H), 7.60-7.33 (m, 5H), 6.90 (d, J = 3.1 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.67 (q, J = 7.2 Hz, 2H), 3.68 (s, 3H), 1.55(t, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 170.4, 167.2, 157.8, 153.4, 152.3, 139.2, 139.0, 137.4, 136.4, 135.7, 135.5, 135.0 (× 2), 134.4 (× 2), 131.6 (× 2), 130.9, 130.8, 130.7, 130.0 126.9, 125.9, 118.5 (× 2), 117.4, 115.0, 107.0, 58.8, 46.2, 17.6; IR (film) ν max 3404, 3064, 1688, 1628, 1599, 1573, 1520, 1470, 1256, 1199, 1176, 1116, 763 cm⁻¹; HRMS (ESI+) m/z 488.1770 (M⁺, C₃₁H₂₆N₃OS requires 488.1797).

5-Benzothiazol-2-yl-3-ethyl-2-nona-1(E),3(E)-dienyl-1-phenyl-3H-benzoimidazol-1-ium, iodide (24)

Compound 73 (30 mg, 0.067 mmol) and ethyl iodide (3 mL) were used to synthesize 24 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (36 mg, 89%) as a yellow solid; mp 66–68 °C; R_f = 0.33 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.68 (d, J = 1.0 Hz, 1H), 8.26 (dd, J₁ = 1.5 Hz, J₂ = 8.7 Hz, 1H), 8.04 (t, J = 6.4 Hz, 2H), 7.81-7.30 (m, 9H), 6.73-6.62 (m, 1H), 6.42-6.34 (m, 1H), 6.11-6.01 (m, 1H), 4.73 (q, J = 7.2 Hz, 2H), 2.22-2.16 (m, 2H), 1.65 (t, J = 7.2 Hz, 3H), 1.44-1.29 (m, 6H), 0.89 (t, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 166.7, 154.1, 149.6, 149.4, 135.6, 135.4, 134.0, 133.5, 132.8, 132.0, 131.9, 131.2 (× 2), 129.6, 127.8 (× 2), 127.1, 127.0, 126.2, 123.2, 122.2, 113.7, 111.3, 107.4, 41.7, 33.2, 31.5, 28.3, 22.5, 13.8, 13.6; IR (film) ν max 3416, 3051, 2950, 2923, 2862, 1687, 1632, 1613, 1501, 1469, 1198, 1166, 1126, 762 cm⁻¹; HRMS (ESI+) m/z 478.2298 (M⁺, C₃₁H₃₂N₃S requires 478.2317).

5-Benzothiazol-2-yl-3-ethyl-1-phenyl-3H-benzoimidazol-1-ium, iodide (13)

Compound 74 (35 mg, 0.11 mmol) and ethyl iodide (3 mL) were used to synthesize 13 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (49 mg, 93%) as a white solid; mp 122–124 °C; R_f = 0.30 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 9.95 (s, 1H), 8.71 (d, J = 1.0 Hz, 1H), 8.34 (dd, J₁ = 1.5 Hz, J₂ = 8.8 Hz, 1H), 7.98 (t, J = 7.1 Hz, 2H), 7.87 (d, J = 8.8 Hz, 1H), 7.76-7.68 (m, 5H), 7.49-7.32 (m, 2H), 4.70 (q, J = 7.3 Hz, 2H), 1.72 (t, J = 7.3 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 167.4, 155.1, 144.4, 136.6, 134.7, 134.5, 134.2, 133.5, 132.2, 131.8 (× 2), 128.3, 128.1, 127.3, 126.3 (× 2), 124.3, 123.1, 115.7, 113.4, 44.4, 14.5; IR (film) ν max 3434, 3131, 3064, 1780, 1736, 1686, 1561, 1499, 1478, 1446, 1416, 1314, 1200, 1143, 758, 706 cm⁻¹; HRMS (ESI+) m/z 356.1200 (M⁺, C₂₂H₁₈N₃S requires 356.1221).

5-Benzothiazol-2-yl-3-ethyl-2-(2-oxo-2H-chromen-3-yl)-1-phenyl-3H-benzoimidazol-1-ium, iodide (25)

Compound 75 (20 mg, 0.042 mmol) and ethyl iodide (3 mL) were used to synthesize 25 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (21 mg, 81%) as a light yellow solid; mp 206–208 °C; R_f = 0.25 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, DMSO-d₆) δ 8.88 (d, J = 1.0 Hz, 1H), 8.63 (s, 1H), 8.43 (dd, J₁ = 1.5 Hz, J₂ = 8.8 Hz, 1H), 8.06 (dd, J₁ = 1.0 Hz, J₂ = 8.1 Hz, 2H), 7.78-7.38 (m, 12H), 4.76 (q, J = 7.2 Hz, 2H), 1.60 (t, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 166.7, 158.2, 155.8, 154.7, 153.5, 147.2, 136.7, 136.2, 135.5, 134.1, 133.0, 132.5, 132.4, 131.4 (× 2), 131.3, 128.4, 128.0 (× 2), 127.9, 127.0, 123.1, 118.3, 117.8, 115.6, 113.3, 110.8, 43.7, 15.0; IR (film) ν max 3412, 3060, 1725, 1687, 1609, 1576, 1503, 1437, 1255, 1201, 1174, 1128, 761 cm⁻¹; HRMS (ESI+) m/z 500.1440 (M⁺, C₃₁H₂₂N₃O₂S requires 500.1433).

(4-Benzothiazol-2-yl-2-nitrophenyl)cyclohexylamine (44)

To a solution of 4 (580 mg, 2.0 mmol) in DMSO (5 mL) was added cyclohexylamine (1.16 mL, 10.0 mmol). The reaction was stirred at 22 °C for 48 h. The reaction was poured into aqueous HCl (1.0 M, 50 mL), and the product was extracted with diethyl ether (50 mL × 3). The combined organic phase was washed with saturated aqueous NaCl (100 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Column chromatography (CH₂Cl₂) afforded the product (609 mg, 87%) as a orange solid, mp 138–140 °C; R_f = 0.47 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.78 (s, 1H), 8.28 (d, J = 7.2 Hz,, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.45 (dd, J₁ = 7.4 Hz, J₂ = 7.9 Hz, 1H), 7.36 (dd, J₁ = 7.6 Hz, J₂ = 7.6 Hz, 1H), 6.92 (d, J = 9.1 Hz, 1H), 3.56 (m, 1H), 2.05 (m, 2H), 1.81 (m, 2H), 1.69-1.21 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 166.1, 154.0, 145.8, 135.1, 134.6, 134.3, 131.2, 126.5, 126.3, 124.9, 122.7, 121.5, 120.8, 114.7, 51.3, 32.6 (× 2), 25.5, 24.5 (× 2); IR (film) ν max 3556, 2931, 2854, 1624, 1567, 1534, 1488, 1436, 1361, 1266, 1217, 1155, 757 cm⁻¹; HRMS (ESI+) m/z 354.1280 (M+H⁺, C₁₉H₂₀N₃O₂S requires 354.1276).

(4-Benzothiazol-2-yl-2-nitrophenyl)hexylamine (45)

Compound 4 (580 mg, 2.0 mmol) and n-hexylamine (1.3 mL, 10.0 mmol) were used to synthesize 45 using the procedure described for preparation of the synthesis of 44. The reaction was stirred at 22 °C for 48 h. The reaction was poured into aqueous HCl (1.0 M, 50 mL), and the product was extracted with diethyl ether (50 mL × 3). The combined organic phase was washed with saturated aqueous NaCl (100 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel with CH₂Cl₂ to afford the product (625 mg, 88%) as a orange solid, mp 98–100 °C; R_f = 0.50 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 8.78 (t, J = 2.0 Hz, 1H), 8.28 (s, br, 1H), 8.15 (d, J = 6.9 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.45 (dd, J₁ = 7.2 Hz, J₂ = 8.1 Hz, 1H), 7.34 (dd, J₁ = 7.3 Hz, J₂ = 7.8 Hz, 1H), 6.89 (dd, J₁ = 2.4 Hz, J₂ = 9.1 Hz, 1H), 3.31 (m, 2H), 1.73 (m, 2H), 1.45 (m, 2H), 1.35 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 166.0, 154.0, 146.7, 134.6, 134.4, 131.3, 126.3, 126.2, 125.0, 122.8, 121.5, 121.1, 114.3, 43.3, 31.4, 28.8, 26.7, 22.5, 14.0; IR (film) ν max 3372, 2955, 2931, 2858, 1626, 1566, 1488, 1360, 1269, 1214, 1159, 758 cm⁻¹; HRMS (ESI+) m/z 356.1413 (M+H⁺, C₁₉H₂₂N₃O₂S, requires 356.1433).

(4-Benzothiazol-2-yl-2-nitrophenyl)-(4-methoxyphenyl)amine (46)

Compound 4 (580 mg, 2.0 mmol) and p-anisidine (1.23 g, 10.0 mmol) was used to synthesize 46 using the procedure described for preparation of the synthesis of 44. Column chromatography (CH₂Cl₂) afforded the product (560 mg, 74%) as a orange solid, mp 188–190 °C; R_f = 0.40 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 9.65 (s, 1H), 8.84 (s, 1H), 8.02 (m, 2H), 7.85 (d, J = 9.0 Hz, 1H), 7.48-6.96 (m, 7H), 3.85 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 165.7, 158.4, 154.0, 145.7, 134.7, 134.0, 132.1, 130.3, 127.2 (× 2), 126.4, 126.0, 125.1, 122.9, 122.7, 121.6, 116.3, 115.1 (× 2); IR (film) ν max 3337, 1626, 1593, 1568, 1510, 1486, 1352, 1247, 1208, 1147, 753 cm⁻¹; HRMS (ESI+) m/z 378.0919 (M+H⁺, C₂₀H₁₆N₃O₃S requires 378.0912).

4-Benzothiazol-2-yl-N¹-cyclohexylbenzene-1,2-diamine (51)

Compound 44 (354 mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the product (291 mg, 90%) as a yellow solid, mp 168–170 °C; R_f = 0.37 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.08 (d, J = 7.9 Hz,, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.51 (m, 2H), 7.42 (dd, J₁ = 7.7 Hz, J₂ = 7.7 Hz, 1H), 7.29 (dd, J₁ = 7.6 Hz, J₂ = 7.6 Hz, 1H), 6.65 (d, J = 8.7 Hz, 1H), 3.48 (br, 3H), 3.32 (m, 1H), 2.08 (m, 2H), 1.78 (m, 2H), 1.66 (m, 1H), 1.29 (m, 5H); ¹³C NMR (75 MHz, CDCl₃) δ 169.1, 154.3, 140.6, 134.6, 133.1, 126.0, 124.2, 122.5, 121.9, 121.3, 115.9, 110.6, 51.4, 33.3 (× 2), 25.9, 24.9 (× 2); IR (film) ν max 3369, 3215, 3050, 2930, 2862, 1604, 1462, 1431, 1300, 1256, 1147, 756 cm⁻¹; HRMS (ESI+) m/z 324.1506 (M+H⁺, C₁₉H₂₂N₃S requires 324.1534).

4-Benzothiazol-2-yl-N¹-hexylbenzene-1,2-diamine (53)

Compound 45 (356 mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol) according to the procedure described for 7. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the product (282 mg, 87%) as a yellow solid, mp 151–153 °C; R_f = 0.40 (hexane/ethyl acetate, 3:1); ¹H NMR (300 MHz, CDCl₃) δ 7.88 (d, J = 8.1 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.53 (m, 1H), 7.42 (dd, J₁ = 7.3 Hz, J₂ = 7.8 Hz, 1H), 7.29 (dd, J₁ = 7.3 Hz, J₂ = 7.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 3.77 (br, 1H) 3.38 (br, 2H), 3.14 (t, J =7.1 Hz, 2H), 1.65 (m, 2H), 1.39 (m, 2H), 1.32 (m, 4H), 0.90 (t, J = 6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 169.0, 154.3, 141.7, 134.6, 133.2, 126.0, 124.3, 122.9, 122.3, 121.8, 121.4, 115.4, 110.2, 43.9, 31.6, 29.4, 26.9, 22.6, 14.0; IR (film) ν max 3379.8, 3212.9, 3018.9, 2952.1, 2916.7, 2848.5, 1647.9, 1594.3, 1459.8, 1438.6, 1364.1, 1307.4, 1215.5, 1160.7 769.8, 753.6, 726.3 cm⁻¹; HRMS (ESI+) m/z 326.1666 (M+H⁺, C₁₉H₂₄N₃S, requires 326.1691).

4-Benzothiazol-2-yl-N¹-cyclohexylbenzene-1,2-diamine (53)

Compound 46 (378 mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol) using the procedure described for preparation of 7. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (295 mg, 85%) as a yellow solid, mp 155–157 °C; R_f = 0.33 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.47-7.30 (m, 3H), 7.04 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 5.40 (s, 1H), 3.79 (s, 3H), 3.68 (br, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 168.4, 155.1, 154.2, 137.3, 136.2, 135.7, 134.8, 127.1, 126.1, 124.6, 122.6, 121.5, 121.0 (× 2), 120.2, 117.3, 115.3, 114.8 (× 2), 55.6; IR (film) ν max 3361, 3260, 3058, 3003, 2953, 2833, 1600, 1509, 1477, 1437, 1311, 1243, 1034, 822, 758 cm⁻¹; HRMS (ESI+) m/z 348.1156 (M+H⁺, C₂₀H₁₈N₃OS requires 348.1156).

[(E)-2-(5-Benzothiazol-2-yl-1-cyclohexyl-1H-benzoimidazol-2-yl)ethenyl] methylphenylamine (65)

Compound 51 (65 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 65 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (64 mg, 69%) as a yellow solid; mp 114–116 °C; R_f = 0.53 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.30 (d, J = 13.1 Hz,, 1H), 8.25 (d, J = 1.5 Hz,, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.95 (dd, J₁ = 8.1 Hz, J₂ = 1.5 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.45 (dd, J₁ = J₂ = 7.7 Hz, 1H), 7.36-7.20 (m, 5H), 7.06 (t, J = 7.3 Hz, 1H), 5.46 (d, J = 13.2 Hz, 1H), 4.26 (m, 1H), 3.39 (s, 3H), 2.25 (m, 2H), 2.02 (m, 4H), 1.84 (m, 1H), 1.45 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 169.6, 155.1, 154.4, 146.8, 144.2, 143.1, 136.5, 135.1, 129.4 (× 2), 127.2, 126.0, 124.6, 123.4, 122.8, 121.5, 119.8, 119.5 (× 2), 117.6, 111.2, 87.0, 56.0, 36.4, 30.1 (× 2), 26.2 (× 2), 25.4; IR (film) ν max 3060, 2931, 2849, 1625, 1593, 1490, 1466, 1436, 1301, 1127, 755 cm⁻¹; HRMS (ESI+) m/z 465.2129 (M+H⁺, C₂₉H₂₉N₄S requires 465.2113).

[(E)-2-(5-Benzothiazol-2-yl-1-hexyl-1H-benzoimidazol-2-yl)ethenyl]methyl phenylamine (66)

Compound 52 (65 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 66 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the product (66 mg, 71%) as a yellow solid; mp 60–62 °C; R_f = 0.53 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.34 (d, J = 13.1 Hz, 1H), 8.24 (d, J = 1.5 Hz, 1H), 8.03 (m, 2H) 7.87 (d, J = 7.9 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.38-7.23 (m, 6H), 7.09 (t, J = 8.2 Hz, 1H), 5.36 (d, J = 13.1 Hz, 1H), 4.10 (t, J = 7.1 Hz, 2H) 3.37 (s, 3H), 1.82 (m, 2H), 1.33 (m, 6H), 0.90 (t, J = 6.9 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 169.7, 155.2, 154.4, 146.7, 143.7, 142.9, 137.8, 135.1, 129.4 (× 2), 127.6, 126.0, 124.6, 123.5, 122.8, 121.5, 120.2, 119.5 (× 2), 117.4, 108.7, 86.3, 43.3, 36.4, 31.4, 29.8, 22.5, 14.0; IR (film) ν max 3059, 2953, 2928, 2856, 1628, 1593, 1491, 1467, 1437, 1325, 1302, 1265, 1127, 756 cm⁻¹; HRMS (ESI+) m/z 467.2257 (M+H⁺, C₂₉H₃₁N₄S requires 467.2269).

{(E)-2-[5-Benzothiazol-2-yl-1-(4-methoxyphenyl)-1H-benzoimidazol-2-yl]ethenyl}methylphenylamine (67)

Compound 53 (70 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl₄ (24 mg, 0.1 mmol) were used to synthesize 67 using the procedure described for preparation of 12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (70 mg, 72%) as a yellow solid; mp 182–184 °C; R_f = 0.53 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.31-8.27 (m, 2H), 8.04 (d, J = 8.2 Hz,, 1H), 7.96 (dd, J₁ = 8.3 Hz, J₂ = 1.6 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.45-7.30 (m, 6H), 7.17-6.92 (m, 6H), 5.18 (d, J = 13.3 Hz, 1H), 3.90 (s, 3H), 3.16 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 169.5, 159.7, 155.9, 154.4, 146.6, 143.8, 142.4, 139.2, 135.1, 129.3 (× 2), 128.8 (× 2), 128.3, 128.1, 126.0, 124.6, 123.4, 122.8, 121.5, 120.7, 119.2 (× 2), 117.7, 115.1 (× 2), 109.5, 87.3, 55.6, 36.0; IR (film) ν max 3060, 2007, 2934, 2836, 1627, 1592, 1514, 1491, 1465, 1435, 1296, 1251, 1127, 755 cm⁻¹; HRMS (ESI+) m/z 489.1754 (M+H⁺, C₃₀H₂₅N₄OS requires 489.1749).

5-Benzothiazol-2-yl-1-cyclohexyl-3-ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-3Hbenzoimidazol-1-ium, iodide (26)

Compound 65 (30 mg, 0.065 mmol) and ethyl iodide (3 mL) were used to synthesize 26 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (36 mg, 90%) as a yellow solid; mp 112–114 °C; R_f = 0.28 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.40 (s, 1H), 8.11 (m, 2H), 7.98 (m, 2H), 7.66 (dd, J₁ = 7.3 Hz, J₂ = 6.3 Hz, 1H), 7.52-7.31 (m, 6H), 7.23 (dd, J₁ = J₂ = 7.3 Hz, 1H), 5.50 (d, J = 13.5 Hz, 1H), 4.63 (m, 1H), 4.49 (q, J = 7.2 Hz, 2H), 3.53 (s, 3H), 2.35 (m, 2H), 2.02 (m, 4H), 1.78 (m 1H), 1.54 (t, J = 7.2 Hz, 3H), 1.56-1.37 (m, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.6, 155.5, 154.4, 154.3 (× 2), 151.3 (× 2), 148.0, 136.8, 134.5, 134.0, 132.3, 131.4 (× 2), 128.4, 127.5, 126.3, 124.4, 123.5, 122.4, 116.8, 111.9, 79.4, 61.6, 43.1, 38.3, 32.0 (× 2), 27.3 (× 2), 26.3, 14.8; IR (film) ν max 3342 (br), 3060, 2931, 2948, 1688, 1616, 1588, 1528, 1494, 1461, 1363, 1305, 1199, 1172, 1130, 761 cm⁻¹; HRMS (ESI+) m/z 493.2415 (M⁺, C₃₁H₃₃N₄S requires 493.2426).

5-Benzothiazol-2-yl-3-ethyl-1-hexyl-2-[(E)-2-(methylphenylamino)ethenyl]-3H-benzoimidazol-1-ium, iodide (27)

Compound 66 (30 mg, 0.064 mmol) and ethyl iodide (3 mL) were used to synthesize 27 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (36 mg, 91%) as a yellow solid; mp 128–131 °C; R_f = 0.25 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.33 (d, J = 1.1 Hz, 1H), 8.13 (m, 1H), 7.98 (m, 2H), 7.80 (m, 2H), 7.49-7.17 (m, 7H), 5.45 (d, J = 13.2 Hz, 1H), 4.50 (q, J = 7.3 Hz, 2H) 4.32 (t, J = 7.7 Hz, 2H), 3.53 (s, 3H), 1.87 (m, 2H), 1.53 (t, J = 7.2 Hz, 1H) 1.33 (m, 2H), 1.27 (m, 4H), 0.83 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.7, 155.5, 153.5, 151.4, 136.8, 135.6, 133.8, 132.5, 131.5 (× 3), 128.4, 127.9, 127.4, 126.7, 124.4, 123.5, 122.9, 122.8, 113.9, 111.4, 79.8, 47.5, 42.7, 32.9 (× 2), 30.2, 27.8, 24.0, 14.7, 14.6; IR (film) ν max 3412.8, 3060.3, 2942.7, 2919.2, 2860.5, 1689.5, 1620.5, 1587.7, 1535.6, 1493.0, 1357.9, 1199.8, 1130.2, 760.2 cm⁻¹; HRMS (ESI+) m/z 495.2572 (M⁺, C₃₁H₃₅N₄S requires 495.2582).

5-Benzothiazol-2-yl-3-ethyl-1-(4-methoxyphenyl)-2-[(E)-2-(methylphenylamino)ethenyl]-3Hbenzoimidazol-1-ium, iodide (28)

Compound 67 (35 mg, 0.072 mmol) and ethyl iodide (3 mL) were used to synthesize 28 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 20:1) afforded the product (42 mg, 92%) as a yellow solid; mp 174-176 °C; R_f = 0.22 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.34 (s, 1H), 7.92 (m, 2H), 7.48 (d, J = 8.8 Hz, 2H), 7.41-7.03 (m, 9H), 6.70 (d, J = 7.5 Hz, 2H), 5.36 (d, J = 14.0 Hz, 1H), 4.47 (q, J = 6.9 Hz, 2H), 3.85 (s, 3H), 3.30 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.6, 163.7, 155.5, 153.5, 151.9, 147.6, 137.6, 136.7, 133.3, 132.4, 131.1 (× 2), 130.8 (× 2), 128.4, 127.8, 127.4, 124.4, 123.5, 121.8, 118.3 (× 2), 113.4, 110.9, 79.8, 57.0, 41.7, 37.3, 14.4; IR (film) ν max 3412 (br), 3050, 2932, 2840, 1682, 1613, 1588, 1504, 1410, 1369, 1256, 1201, 1126, 762 cm⁻¹; HRMS (ESI+) m/z 517.2045 (M⁺, C₃₂H₂₉N₄OS requires 517.2062).

5-Benzothiazol-2-yl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenethyl-1-phenyl-3Hbenzoimidazol-1-ium, iodide (29)

Compound 12 (30 mg, 0.066 mmol) and (2-iodoethyl)benzene (3 mL) were used to synthesize 29 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 30:1) afford the product (43 mg, 87%) as a yellow solid; mp 83–85 °C; R_f = 0.41 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.31 (d, J = 1.3 Hz, 1H), 8.18-8.03 (m, 3H), 7.78-7.75 (m, 3H), 7.62-7.42 (m, 4H), 7.31-7.02 (m, 9H), 6.83 (d, J = 13.5 Hz, 1H), 6.65 (br, 2H), 5.03-4.84 (m, 5H), 4.52 (m, 2H), 3.23 (s, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.5, 155.5, 154.1, 151.4, 139.2, 136.9, 136.8, 136.3, 133.5, 133.3 (× 2), 132.9, 132.7, 131.1(× 2), 130.9 (× 2), 130.4 (× 2), 129.4 (× 2), 128.9, 128.4, 127.7, 127. 4, 126.9, 124.4, 123.5, 121.6, 113.4, 111.6, 79.8, 48.0, 37.0, 35.9; IR (film) ν max 3428, 3061, 2943, 2860, 1689, 1616, 1586, 1535, 1494, 1464, 1370, 1307, 1200, 1167, 1127, 799, 760, 697 cm⁻¹; HRMS (ESI+) m/z 563.2261 (M⁺, C₃₇H₃₁N₄S requires 563.2269).

5-Benzthiazol-2-yl-3-hexyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, iodide (30)

Compound 12 (35 mg, 0.076 mmol) and 1-iodo-hexane (3 mL) were used to synthesize 30 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 30:1) afforded 30 (47 mg, 92%) as a yellow solid, mp 86–88 °C; R_f = 0.37 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.40 (d, J = 1.1 Hz, 1H), 8.06-7.95 (m, 3H), 7.78-7.74 (m, 3H), 7.67-7.64 (m, 2H), 7.48-7.38 (m, 2H), 7.26 (m, 2H), 7.23-7.12 (m, 3H), 6.72 (br, 2H), 5.47 (d, J = 11.9 Hz, 1H), 4.50 (m, 2H), 3.36 (s, 3H), 1.95 (m, 2H), 1.51 (m, 2H), 1.36 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.6, 155.5, 153.8, 152.1, 137.2, 136.7, 136.4, 134.0, 133.3 (× 2), 133.0, 132.6, 131.1 (× 2), 129.6 (× 2), 128.4, 127.4, 126.9, 124.4, 123.5, 121.9, 113.5, 111.2, 79.9, 46.5, 36.8, 33.0, 30.0, 27.9, 24.1, 14.8; IR (film) ν max 3331, 3060, 2943, 2931, 2849, 1688, 1617, 1586, 1537, 1494, 1464, 1369, 1310, 1120, 1167, 1127, 799, 761, 697 cm⁻¹; HRMS (ESI+) m/z 543.2548 (M⁺, C₃₅H₃₅N₄S requires 543.2582).

5-Benzothiazol-2-yl-3-dodecyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, iodide (31)

Compound 12 (30 mg, 0.066 mmol) and 1-iodo-dodecane (3 mL) were used to synthesize 31 using the procedure described for preparation of 1. Flash column chromatography (CH₂Cl₂/MeOH, 30:1) afforded the product (43 mg, 87%) as a yellow glassy solid; R_f = 0.44 (CH₂Cl₂/MeOH, 10:1); ¹H NMR (300 MHz, MeOH-d₄) δ 8.42 (s, 1H), 8.07-7.95 (m, 3H), 7.79-7.64 (m, 5H), 7.51-7.36 (m, 2H), 7.27-7.15 (m, 5H), 6.69 (br, 2H), 5.53 (d, J = 12.3 Hz, 1H), 4.52 (m, 2H), 3.36 (s, 3H), 1.97 (m, 2H), 1.50-1.00 (m, 20H), 0.78 (t, J = 6.1 Hz, 3H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.3, 155.1, 153.4, 151.7, 136.8, 136.4, 136.1, 133.6, 132.9 (× 2), 132.6, 132.3, 130.1 (× 2), 129.2 (× 2), 128.0, 127.1, 126.6, 124.0, 123.1, 121.4, 113.1, 110.9, 79.5, 46.1, 37.1, 33.0, 30.8, 30.7, 30.6, 30.5, 30.3, 29.5, 27.7, 23.7, 14.4; IR (film) ν max 3425, 3060, 2919, 2849, 1689, 1620, 1586, 1537, 1494, 1465, 1370, 1310, 1200, 1131, 799, 762, 697 cm⁻¹; HRMS (ESI+) m/z 627.3523 (M⁺, C₄₁H₄₇N₄S requires 627.3521).

2-(3-Chloro-4-nitrophenyl)benzooxazole (82)

To a slurry of 3-chloro-4-nitro-benzoic acid (80, 1.0 g, 5.0 mmol) in DMF (15 mL) was added HATU (1.9 g, 5.0 mmol) and DIEA (1.7 mL, 10 mmol). The reaction was stirred at 22 °C for 5 min prior to addition of 2-aminophenol (81, 550 mg, 5.0 mmol). The reaction was stirred at 22 °C for 4 h followed by addition of saturated aqueous NaCl (150 mL). The aqueous phase was extracted with diethyl ether (100 mL × 3). The organic phases were combined and washed with saturated aqueous NaCl (100 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. To the slurry of resulting residue in xylene (30 mL) was added p-toluenesulfonic acid (3.8 g, 20 mmol). The reaction was refluxed for 1 h, cooled, and poured into aqueous NaOH (1.0 M, 100 mL). The aqueous phase was extracted with diethyl ether (100 mL × 3). The organic phases were combined and washed with saturated aqueous NaCl (100 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 10:1) afforded the product (1.02 g, 71%) as a off white solid; mp 162–164 °C; R_f = 0.58 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 8.40 (d, J = 1.7 Hz, 1H), 8.23 (dd, J₁ = 1.7 Hz, J₂ = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 1H), 7.79 (m, 1H), 7.61 (m, 1H), 7.41 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 159.4, 151.0, 149.0, 141.7, 131.8, 130.6, 128.0, 126.6, 126.2 (× 2), 125.4, 120.7, 111.0; IR (film) ν max 3093, 1610, 1580, 1547, 1523, 1468, 1446, 1331, 1067, 762, 750 cm⁻¹; HRMS (ESI+) m/z 275.0238 (M+H⁺, C₁₃H₈N₂O₃Cl requires 275.0224).

(5-Benzooxazol-2-yl-2-nitrophenyl)ethylamine (85)

To a slurry of 82 (200 mg, 0.73 mmol) in DMSO (2 mL) was added ethylamine (83, 2.0 M in THF, 4 mL). The reaction was stirred at 22 °C for 48 h. The reaction was poured into aqueous HCl (1.0 M, 30 mL). The aqueous phase was extracted with diethyl ether (50 mL × 3). The organic phases were combined and washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Recrystallization of the crude product from CH₂Cl₂/MeOH afforded the product (190 mg, 92%) as a orange solid; mp 168–170 °C; R_f = 0.55 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 8.9 Hz, 1H), 8.01 (br, 1H), 7.80 (m, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.59 (m, 1H), 7.46 (d, J₁ = 1.6 Hz, J₂ = 8.9 Hz, 1H), 7.39 (m, 2H), 3.48 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 161.2, 150.9, 145,3, 141,9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.1, 113.6,112.8, 110.8, 38.0, 14.4; IR (film) ν max 3379, 2969, 2912, 2850, 1627, 1621, 1580, 1553, 1523, 1490, 1402, 1339, 1320, 1281, 1215, 1188, 1048, 743 cm⁻¹; HRMS (ESI+) m/z 284.1036 (M+H⁺, C₁₅H₁₄N₃O₃ requires 284.1035).

(5-Benzooxazol-2-yl-2-nitrophenyl)hexylamine (84)

Compound 82 (200 mg, 0.73 mmol) was added to hexylamine (38, 1.0 mL, 7.7 mmol) in DMSO/THF (2 mL/2 mL) and stirred at 22 °C for 48 h. The reaction was worked up as described for preparation of 85. Recrystallization of the crude product from CH₂Cl₂/MeOH afforded the product (233 mg, 94%) as a orange solid; mp 134–135 °C; R_f = 0.68 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 8.9 Hz, 1H), 8.09 (br, 1H), 7.81 (m, 1H), 7.72 (d, J = 0.9 Hz, 1H), 7.60 (m, 1H), 7.46 (d, J₁ = 0.9 Hz, J₂ = 8.9 Hz, 1H), 7.40 (m, 2H), 3.42 (q, J = 5.2 Hz, 2H), 1.80 (m, 2H), 1.49 (m, 2H), 1.38 (m, 4H), 0.93 (t, J = 5.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 161.3, 150.9, 145.4, 141.9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.5, 113.5, 112.9, 110.8, 43.3, 31.5, 28.9, 26.7, 22.6, 14.0; IR (film) ν max 3368, 3077, 2950, 2924, 2857, 1630, 1622, 1582, 1556, 1523, 1491, 1470, 1451, 1407, 1318, 1284, 1260, 1244, 1211, 1191, 1180,1050, 760, 744 cm⁻¹; HRMS (ESI+) m/z 340.1646 (M+H⁺, C₁₉H₂₂N₃O₃ requires 340.1661).

4-Benzooxazol-2-yl-N²-ethylbenzene-1,2-diamine (87)

Compound 85 (160 mg, 0.56 mmol) was treated with anhydrous hydrazine (0.1 mL, 3.2 mmol) and Pd/C (10%, 64 mg, 0.06 mmol) using the procedure described for preparation of 7. Recrystallization from CH₂Cl₂/hexane afforded the product (132 mg, 93%) as a light yellow solid; mp 172–173 °C; R_f = 0.30 (hexane/ethyl acetate, 1:1); ¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (br, 2H), 7.36-7.18 (m, 4H), 6.66 (d, J = 7.6 Hz, 1H), 5.44 (s, 2H), 4.70 (br, 1H), 3.15 (m, 2H), 1.27 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.7, 150.4, 142.7, 140.4, 135.8, 124.7, 124.4, 119.1, 118.3, 114.7, 113.3, 110.7, 108.0, 38.4, 14.8; IR (film) ν max 3429, 3379, 3335, 3225, 2973, 2956, 2923, 2852, 1643, 1607, 1586, 1495, 1451, 1036, 1278, 1240, 1150, 1053, 856, 793, 759, 746 cm⁻¹; HRMS (ESI+) m/z 254.1287 (M+H⁺, C₁₅H₁₆N₃O requires 254.1293).

4-Benzooxazol-2-yl-N²-hexylbenzene-1,2-diamine (86)

Compound 84 (160 mg, 0.47 mmol) was treated with anhydrous hydrazine (0.1 mL, 3.2 mmol) and Pd/C (10%, 53 mg, 0.05 mmol) using the procedure described for preparation of 7. Recrystallization from CH₂Cl₂/hexane afforded the product (136 mg, 94%) as a light yellow solid; mp 126–127 °C; R_f = 0.24 (hexane/ethyl acetate, 3:1); ¹H NMR (400 MHz, CDCl₃) δ 7.72 (m, 2H), 7.62 (dd, J₁ = 1.8 Hz, J₂ = 8.0 Hz, 1H), 7.53 (m, 2H), 7.28 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 3.6 (br, 3H), 3.20 (t, J = 7.2 Hz, 2H), 1.69 (m, 2H), 1.44 (m, 2H), 1.34 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.3,150.6, 142.4, 138.5, 137.5,124.2 (× 2), 119.3 (× 2), 118.7, 115.4, 110.8, 110.2, 44.4, 31.7, 29.7, 27.0, 22.6, 14.1; IR (film) ν max 3410, 3360, 3220, 2950, 2927, 2846, 1646, 1591, 1582, 1557, 1505, 1484, 1455, 1443, 1318, 1292, 1158, 955, 851, 797, 758, 743 cm⁻¹; HRMS (ESI+) m/z 310.1899 (M+H⁺, C₁₉H₂₄N₃O requires 310.1919).

[(E)-2-(6-Benzooxazol-2-yl-1-ethyl-1H-benzoimidazol-2-yl)ethenyl]methyl phenylamine (17)

To a solution of 87 (51 mg, 0.2 mmol) and 10 (32 mg, 0.2 mmol) in ethanol (10 mL), ZrOCl₂·8H₂O (32 mg, 0.1 mmol) was added. The reaction was stirred at 22 °C for 30 min. The reaction was heated to 80 °C and refluxed for 5 min before the addition of MnO₂ (86 mg, 1.0 mmol). After 10 min, the solution was cooled to 22 °C and filtered through a fritted funnel. The MnO₂ was washed with ethanol (10 mL). The combined filtrate was concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate, 3:1) afforded the product (53 mg, 68%) as a yellow solid; mp 171–173 °C; R_f = 0.15 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J = 13.0 Hz, 1H), 8.12 (m, 2H),7.72 (m, 2H), 7.58 (m, 1H), 7.39-7.22 (m, 6H), 7.12 (t, J = 6.7 Hz, 1H), 5.38 (d, J = 13.0 Hz, 1H), 4.23 (q, J = 6.7 Hz, 2H), 3.38 (s, 3H), 1.47 (t, J = 6.7 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 164.5, 156.0, 150.8, 146.8, 146.6, 143.5 (× 2), 142.5, 135.3, 129.4(× 2), 124.4, 124.3, 123.7, 121.9, 119.7 (× 2), 119.4, 119.0, 117.6, 110.3, 107.8, 85.9, 38.0, 36.5, 15.0; IR (film) ν max 3379, 3060, 2978, 2934, 1628, 1594, 1558, 1491, 1453, 1410, 1347, 1326, 1291, 1242, 1128, 809, 747 cm⁻¹; HRMS (ESI+) m/z 395.1849 (M+H⁺, C₂₅H₂₃N₄O requires 395.1872).

[(E)-2-(6-Benzooxazol-2-yl-1-hexyl-1H-benzoimidazol-2-yl)ethenyl]methylphenylamine (32)

To a solution of compound 86 (62 mg, 0.2 mmol) and 10 (32 mg, 0.2 mmol) in ethanol (10 mL), was added ZrOCl₂·8H₂O (32 mg, 0.1 mmol) to synthesize 32 using the procedure described for preparation of 17. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product (58 mg, 64%) as a yellow solid; mp 88–89 °C; R_f = 0.40 (hexane/ethyl acetate, 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 13.1 Hz, 1H), 8.11-8.09 (m, 2H), 7.76 (m, 1H), 7.67 (dd, J₁ = 0.9 Hz, J₂ = 8.0 Hz, 1H), 7.56 (m, 1H), 7.38-7.30 (m, 4H), 7.23-7.21 (m, 2H), 7.10 (m, 1H), 5.37 (d, J = 13.1 Hz, 1H), 4.15 (t, J = 7.4 Hz, 2H), 3.37 (s, 3H), 1.85 (m, 2H), 1.35 (m, 6H), 0.89 (t, J = 7.4 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 164.5, 156.3, 150.8, 146.6, 143.4, 142.5, 135.8, 129.4 (× 2), 124.4, 124.3, 123.7, 121.9, 119.7 (× 3), 119.4, 119.0, 117.6, 110.3, 108.0, 86.2, 43.3, 36.5, 31.5, 29.8, 26.6, 22.6, 14.0; IR (film) ν max 3357, 3060, 2950, 2929, 2853, 1628, 1594, 1559, 1491, 1452, 1409, 1326, 1294, 1268, 1243, 1128, 1001, 823, 746, 695 cm⁻¹; HRMS (ESI+) m/z 451.2472 (M+H⁺, C₂₉H₃₁N₄O requires 451.2498).

Biological assays

To construct the PIV5 strain used for antiviral assays, recombinant PIV5 encoding GFP between the viral genes HN and L³² was modified by replacement of GFP in the PIV5 genome with renilla luciferase. Recombinant PIV5 expressing renilla luciferase (rPIV5-RL) was confirmed by RT-PCR sequencing and functional assays. To examine whether luciferase activity of infected cells reflects the rate of viral infection, a serial dilution (2 ✕) of rPIV5-RL virus was used to infect HeLa cells grown on a 96-well plate. At 1 day post infection (dpi), the cells were lysed and assayed for renilla luciferase activity (commercial detection kit from Promega Inc., Madison, WI). As shown in Figure 3, at 0.06 to 8 MOI (multiplicity of infectivity, corresponding to the number of infectious viruses per cell), the luciferase activity directly correlated with the amount of virus applied, indicating that rPIV5-RL can be used to quantitatively assess viral replication. To test the effects of compounds on replication of rPIV5-RL, HeLa cells in 96-well plates were infected with 1 MOI of rPIV5-RL. The cells were incubated with compounds, collected at 1 day post infection, and assayed for renilla luciferase activity.

To examine the toxicity of compounds to mammalian cells, cellular viability was quantified after 24 h (HeLa cells) or 40 h (NHBE cells) with a firefly luciferase-based CellTiter-Glo luminescent cell viability assay (Promega Inc., Madison, WI). Normal human bronchial epithelial (NHBE) cells were cultured in an air-liquid interface system as previously described.³⁵ Briefly, NHBE cells (passage-2, 2×10⁴ cells/cm², Clonetics, San Diego, CA) were cultured in Transwell-clear culture inserts (24.5 mm, 0.45 μm pore size; Costar, Cambridge, MA) thin-coated with rat tail collagen, type I (Collaborative Res., Bedford, MA). Cells were cultured submerged for the first 5 to 7 days. After this period, the apical medium was removed to create the air-liquid interface, and cells were fed medium on their basal surface only. For an additional 28 days, the apical surface of the cells was exposed to a humidified 95% air/5% CO₂ environment. NHBE cells were cultured for a total of 35 days.

Cultures of Mycobacterium fortuitum, a generous gift of Dr. Fred Quinn, were maintained at 37 °C in 7H9 broth supplemented with OADC and 0.05% glycerol. The assay in infected HeLa cells was based on a related survival assay for M. tuberculosis.³⁶ HeLa cells were seeded in 24 well plates at 5 × 10⁵ cells per well in EMEM containing 10% FBS (HeLa medium) overnight in a CO₂ (5%) incubator at 37 °C. M. fortuitum was washed with uptake buffer (1 part Basal Uptake Buffer (4.5 mg/mL glucose, 5 mg/mL BSA, 0.1 mg/mL CaCl₂, 0.1 mg/mL MgCl₂, 1 mg/mL gelatin in PBS): 4 parts Wash Buffer (5% FBS in DMEM)). After the wells had been washed to remove antibiotics, M. fortuitum in uptake buffer was added to the wells containing HeLa cells (MOI = 5). After infection for 24 h, the medium was removed, the wells were washed with warm DMEM, and this DMEM was replaced with HeLa medium containing compounds 1 (0.5 μM), 29 (0.5 μM), 30 (0.5 μM), 31 (0.5 μM), TritonX-100 (1%), or DMSO (0.1%, vehicle control). After an additional 24 h, the medium was removed and the cells were lysed, diluted, and plated for determination of colony forming units (CFU) on 7H11 agar plates.

Abbreviations

AKT

protein kinase B

AGC

cAMP-dependent, cGMP-dependent and protein kinase C

FOXO

forkhead box-O

NHBE

normal human bronchial epithelial

PIV5

parainfluenza virus 5

PI3K

phosphatidylinositol-3-kinase

PH

pleckstrin homology

RL

renilla luciferase

MOI

multiplicity of infection

CFU

colony forming units