Design, Synthesis, and Biological Evaluation of Benzimidazole Derivatives as Potential Lassa Virus Inhibitors

Abstract

The Lassa virus (LASV) causes Lassa fever, a highly infectious and lethal agent of acute viral hemorrhagic fever. At present, there are still no effective treatments available, creating an urgent need to develop novel therapeutics. Some benzimidazole compounds targeting the arenavirus envelope glycoprotein complex (GPC) are promising inhibitors of LASV. In this study, we synthesized two series of LASV inhibitors based on the benzimidazole structure. Lentiviral pseudotypes bearing the LASV GPC were established to identify virus entry inhibitors. Surface plasmon resonance (SPR) was further used to verify the binding activities of the potential compounds. Compounds 7d−Z, 7h−Z, 13c, 13d, and 13f showed relatively excellent antiviral activities with IC₅₀ values ranging from 7.58 to 15.46 nM and their SI values above 1251. These five representative compounds exhibited stronger binding affinity with low equilibrium dissociation constants (K_D < 8.25 × 10⁻⁷ M) in SPR study. The compound 7h−Z displayed the most potent antiviral activity (IC₅₀ = 7.58 nM) with a relatively high SI value (2496), which could be further studied as a lead compound. The structure–activity relationship indicated that the compounds with lipophilic and spatially larger substituents might possess higher antiviral activity and a much larger safety margin. This study will provide some good guidance for the development of highly active compounds with a novel skeleton against LASV.

1. Introduction

The Lassa virus (LASV), which belongs to the Mammarenavirus of the Arenaviridae family, causes severe viral hemorrhagic fever (HF) in humans and represents a serious public health problem. It has been reported that 58 million people are at risk of contracting LASV, with an estimated 100,000 to 300,000 cases and 5000 deaths per year in West Africa [1]. However, these numbers are likely to be underestimated due to the lack of appropriate diagnosis in poor areas such as rural areas and the non-specific febrile symptoms of Lassa fever [2]. Hospitalized LASV HF patients have a mortality rate of 15–20%, and survivors often suffer permanent damage to their bilateral hearing after surviving the infection [3,4,5,6]. Currently, there are still no licensed vaccines or drugs to prevent and treat LASV infection. According to the Centers for Disease Control and Prevention (CDC), LASV is considered a Category A pathogen due to its high mortality rates and limited therapeutic options [7]. The World Health Organization (WHO) has made the disease the top priority of research and development [8].

As viral invasion is a crucial step in the viral life cycle, the related protein promises one of the most important targets for antiviral drug design. During LASV entry, the glycoprotein complex (GPC) plays a crucial role. GPC is first synthesized as a single polypeptide and then cleaved by signal peptidase and subtilisin/kexin-isoenzyme-1/site-1 proteases (SKI-1/S1P) into three segments: the receptor-binding subunit GP1, the membrane fusion subunit GP2, and the stable signal peptide (SSP) [9,10]. Following the interaction of GP1 with the cellular receptor α-cystine (α-DG) and lysosome-associated membrane protein 1 (LAMP1), viral endocytosis occurs, and GP2 is subjected to a pH-dependent conformational rearrangement, prompting the fusion of viral and endosomal membranes [8,9,11,12,13]. By interacting with GP2 subunits, SSP promotes GPC-mediated membrane fusion at pH and promotes GPC maturation [14,15]. As a category A reagent, LASV requires Biosafety Level 4 (BSL-4) containment, which presents significant obstacles and safety challenges to anti-infective drug discovery for LASV [16]. Therefore, the surrogate assays of LASV GPC incorporation into lentiviral pseudotypes as an antiviral drug screen method is an accepted alternative [17,18,19].

Currently, the therapeutic strategies for Lassa fever are limited to the off-label use of broad-spectrum antiviral ribavirin, which is usually used in the early stages of the disease [20,21]. As we know, drug repurposing is a promising strategy to identify new uses from approved or experimental drugs, which has also been used in searching for LASV inhibitors [22]. Favipiravir (T−705), an RNA polymerase inhibitor, gave better results than ribavirin in treating LASV infection [23,24]. Losmapimod, a drug used clinically in cardiovascular disease and chronic obstructive pulmonary disease, was identified as an inhibitor of LASV infection [25]. Lacidipine, an anti-hypertension drug, was screened from an FDA-approved drug library as a compound with anti-LASV activity [26]. The antifungal isavuconazole was demonstrated to inhibit the entry of LASV by targeting the stable signal peptide-GP2 subunit interface of LASV GP [27]. However, the antiviral activities of these old drugs are not very satisfactory. Benzimidazole derivatives display a variety of biological activities in many diseases through various mechanisms, such as antiviral [28,29], antimicrobial [30,31], antiproliferative [32,33], and anticancer activities [34,35]. Benzimidazole derivatives are special lead compounds for antiviral drugs, attracting interest from many chemists [18,36,37,38,39]. LHF−535 and ST−193, derivatives of benzimidazoles, are being investigated as promising anti-LASV entry inhibitors (Figure 1) [8,40]. As a small-molecule compound targeting the GPC of LASV, LHF−535 has broad-spectrum activity against different lineages of LASV and related arenaviruses causing HF disease in South America [39]. ST−193 with a benzimidazole scaffold also showed potent inhibition of lentiviral pseudotyped viruses expressing an enveloped GPC assay against LASV [18]. Currently, these small molecules are still in the early stage of drug development, and there are no clinically specific therapeutic agents for LASV. Therefore, it is an urgent task to discover new antiviral agents against LASV.

In this study, inspired by these facts, we designed and synthesized two new series of benzimidazole derivatives with high antiviral activities using LHF−535 and ST−193 as lead structures (Figure 1). An in vitro infection model of LASV pseudovirus containing firefly luciferase (Fluc) gene and enhanced green fluorescent protein (EGFP) gene was established and used to evaluate the antiviral activities of the target compounds. In addition, we analyzed the binding activities of the target compounds to GP2 protein using the SPR technique and calculated their kinetic parameters. We determined five compounds (7d−Z, 7h−Z, 13c, 13d, and 13f) that inhibit LASV entry, exhibit excellent antiviral activities and low cytotoxicity, as well as better binding affinity to target proteins. 7h−Z displayed optimal antiviral activity (IC₅₀ = 7.58 nM), and SI value is almost twice that of LHF−535.

2. Results

2.1. Chemistry

2.1.1. Synthesis of 5-vinyl-benzo[d]imidazole Derivatives 7a−7h(E/Z)

The series I of the target compounds was designed by introducing different substituents R₁ into the benzimidazole scaffold. The preparation of 5-vinyl-benzo[d]imidazole derivatives 7a−7h(E/Z) has been accomplished as described in Scheme 1 [41,42]. Initially, compound 3 was synthesized by the substitution reaction using commercially available 4-fluoro-3-nitrobenzonitrile (1) and 4-isopropoxyaniline (2) as starting materials in the presence of triethylamine. Using tin (II) chloride dihydrate as a reductant, compound 3 was reduced to produce compound 4. Then, compound 4 was treated with formamidine acetate and subjected to a cycloaddition reaction to give compound 5. Subsequently, the cyano group was reduced by adding Raney Nickel in the presence of 75% formic acid to obtain compound 6. Finally, the aldehyde group of compound 6 reacted with suitable Wittig reagents to give the title compounds 7a−7h (Z/E). In the reaction, n-BuLi was used as a strong base to obtain the Wittig reagents, which then reacted with aldehydes at room temperature to form the corresponding cis-trans isomers. Products with cis structures usually have higher yields.

2.1.2. Synthesis of 5-amino-benzo[d]imidazole Derivatives 13a−13j

The reaction route of compounds 13a−13j was outlined in Scheme 2 [43]. Firstly, 1-fluoro-2, 4-dinitrobenzene (8) was reacted with 4-methoxyaniline (9) under the presence of cesium carbonate in THF to obtain compound 10. Subsequently, compound 10 was reduced to obtain compound 11, using sodium hydrosulfite as the catalytic reagent for hydrogenation. Compound 11 underwent a cycloaddition reaction in the presence of hydrochloric acid (4.0 M) and formic acid to obtain compound 12. Lastly, the amino group of compound 12 underwent dehydration condensation and reduction reactions with suitable aromatic aldehydes to give compounds 13a−13j.

The synthesis methods are simple and feasible and can be used as a general method for synthesizing a series of bioactive benzimidazole derivatives. The structures of these synthesized compounds were further confirmed by NMR. The data were collected in the Supplemental Materials Figures S1–S60.

2.2. Biochemical Assays

The LASV is classified as a category A agent, and BSL-4 facilities are required to conduct experiments with live viruses, which restricts the use of live viruses for experiments. In this study, we used HIV-1 pseudotyped viruses bearing the GPC of LASV to establish a validated cell-based pseudovirus infection model (LASVpv) to evaluate the pharmacological activities of the compounds. In this model, VSV pseudotyped lentivirus (VSVpv) constructed with the G protein of the vesicular stomatitis virus was used as a specific control. Based on the pseudoviral model, the antiviral activities of the compounds were determined in HEK-293 cells. LHF−535 was used as a positive drug to validate the reliability of the model. The results showed that the positive drug did not exhibit an antiviral effect against VSVpv infection in the experimental concentration range (Figure 2B). However, the inhibition of LASVpv infection for the positive drug was concentration-dependent (Figure 2A) with an IC₅₀ value of 3.04 nM (Figure 3), which corresponded to the data in the reported literature [39]. Therefore, the model can be used to evaluate the antiviral activities of title compounds. All synthesized compounds exhibit low nanomolar IC₅₀ activities against LASVpv, as shown in Table 1 and Table 2. The concentration–response curves of the representative compounds and the positive drug to LASVpv are shown in Figure 3. The IC₅₀ values of 7d−Z, 7h−Z, 13c, 13d, and 13f were 13.56 nM, 7.58 nM, 15.46 nM, 13.81 nM, and 11.87 nM, respectively, which were closer to the IC₅₀ of positive drugs (3.04 nM), and, notably, they had higher SI values.

2.3. Initial Inhibitory and Structural Analysis of Benzimidazole Derivatives

Initially, exploring the structure–activity relationships (SAR) of the R₁ showed that R₁ with electron-withdrawing groups might reduce the activities of compounds against LASVpv. For example, compounds substituted with 4-(difluoromethoxy)phenyl group (7e−Z), 4-(trifluoromethoxy)phenyl (7f−Z), and trifluoromethyl phenyl (7g−Z) showed a 9 to 11 fold reduction in activities against LASVpv compared to hydroxyisopropyl phenyl. Several other lipophilic groups retained low nanomolar activities of the compounds (7a−Z, 7d−Z, and 7h−Z, IC₅₀ ≤ 15 nM) except the spatially small substituent 4-methylphenyl (7c−Z, IC₅₀ = 36.89 nM). Among them, compound 7h−Z, with a more lipophilic naphthyl group, was more active than compounds with other substituents. Compounds with cis configuration showed submicromolar IC₅₀ values, while the compounds with trans configuration showed relatively low activities efficiency (IC₅₀ > 50.0 nM). This result is consistent with previous studies [41].

For 5-amino-benzo[d]imidazole derivatives 13a−13j, the replacement of isopropyl (in ST−193) with electron-withdrawing groups of dichloromethyl (13i) and trifluoromethyl (13j) resulted in a decrease in activity. The substitution of R₂ by the more lipophilic tert-butylphenyl group (13−f) increased potency. Compound 13g, which has a less lipophilic 4-methoxyphenyl group, reduced the activity against LASVpv, except for compound 13c (IC₅₀ = 15.47 nM), which has a spatially larger substituent. However, compound 13d with large lipophilic 2,4,5-trimethoxyphenyl showed a relative increase in antiviral activity. The introduction of polar hydroxyphenyl (13a, 13b, 13e, 13h) led to a significant decrease in activities against LASVpv. Especially, the substitution of 3-hydroxyphenyl (13h, IC₅₀ > 100 nM) led to the loss of activity.

2.4. SPR-Based Binding Assay for Compounds to GP2

Surface plasmon resonance (SPR) is a powerful tool for monitoring interactions between small molecules and target proteins. This interaction was characterized by determining kinetic parameters and affinities [44]. The association rate constant (ka), the dissociation rate constant (k_d), and the equilibrium dissociation constant (K_D) can be determined using SPR-based binding assays. Commonly, active compounds bind to ligand proteins with K_D values ranging from 10⁻⁷ to 10⁻⁴ M [45]. Lower K_D values indicate stronger binding.

To our knowledge, the evaluation binding affinity of different small compounds towards LASV GP2 using SPR is reported for the first time in our study. Immobilization of GP2 on the SPR sensor surface is achieved by amino coupling, resulting in a stable surface with an immobilization level of approximately 15,783 response units (RU). The binding parameters of title compounds were determined with the chip at concentrations between 0.19 μM and 50.00 μM. Based on Biacore software’s 1:1 binding fitting model, the binding kinetic was analyzed. The K_D values of all the tested title compounds binding to GP2 are shown in Table 3. For 7d−Z, 7h−Z, 13c, 13d, and 13f, they are nearly within the same quantitative grade compared with LHF−535 (The positive drug), and for other analytes, their K_D values (>10^–6 M) are higher. The five representative compounds and the positive drug bound to GP2 with a clear association and dissociation phase (Figure 4). These compounds exhibit distinctive fast-binding and slow-dissociation curves, resulting in a significant and dose-dependent increase in RU. Representative compounds exhibited strong binding activities to GP2 with dissociation constants K_D < 8.25 × 10⁻⁷ M. These results indicated that compounds 7d−Z, 7h−Z, 13c, 13d, and 13f had a long duration of drug efficacy and excellent inhibitory potency, which is consistent with the results of LASVpv inhibition assays shown in Table 1 and Table 2.

3. Materials and Methods

3.1. Chemistry

3.1.1. Chemicals and Instruments

All solvents and chemicals were purchased from commercial suppliers and used without further purification if not indicated. Melting points (Mp) were determined in capillary tubes on a Jiahang melting point JH70L apparatus. The progress of all reactions was routinely monitored by TLC silica gel glass plates (UV wavelength: 254 and 365 nm). The synthesized products were further purified by a Biotage^@Selekt automated flash purification system. Proton (¹H) NMR spectra were obtained at 600 MHz in Bruker AVANCE 600 spectrometer using DMSO-d6 or CDCl₃ as the solvent. Carbon (¹³C) NMR spectra were recorded at 151 MHz using the same instrument and solvent conditions. Chemical shifts are marked as parts per million (ppm). High-resolution mass spectroscopy (HRMS) data were analyzed in Agilent 1290II-6460.

3.1.2. Synthesis of 5-vinyl-benzo[d]imidazole Derivatives

4-((4-isopropoxyphenyl)amino)-3-nitrobenzonitrile(3). 4-fluoro-3-nitrobenzonitrile 1 (20.00 g, 0.12 mol), 4-isopropoxyaniline 2 (22.76 g, 0.15 mol), and triethylamine (19.46 mL, 0.14 mol) were combined in acetonitrile (100 mL). The mixture was stirred at 90 °C for 24 h. The organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by slurrying with tert-butylmethylether and filtered to afford compound 3 (35.08 g, 98%) as a red solid. Mp 130–132 °C. ¹H NMR (600 MHz, Chloroform-d): δ 9.71 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 9.1, 2.2 Hz, 1H), 7.16 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 9.1 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 4.61–4.52 (m, 1H), 1.37 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.38, 146.91, 137.26, 132.12, 131.71, 129.01, 127.56, 117.94, 117.16, 116.93, 99.47, 70.43, 22.07. HRMS (ESI) m/z: (M + H)⁺ calcd for C₁₆H₁₅N₃O₃ 298.1113; found 298.1187.

3-amino-4-((4-isopropoxyphenyl)amino)benzonitrile (4). A solution of compound 3 (24.00 g, 0.08 mol) in EtOAc (500 mL) was heated to 50 °C, and tin (II) chloride dihydrate (63.75 g 0.28 mol) was added in portions. The mixture was heated for 3 h at 60–62 °C. The reaction mixture was cooled to room temperature, and the pH was adjusted to alkaline by adding aqueous sodium bicarbonate, and this mixture was stirred for 1 h. The reaction mixture was filtered through a pad of Celite. The filtrate was washed with water and EtOAc. The organic phase was dried over anhydrous sodium sulfate overnight and concentrated; the residue product was purified using an automated chromatography system to afford compound 4 as a brownish-red solid (20.50, 95%). Mp 146−147 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.05 (dd, J = 8.3, 2.0 Hz, 1H), 7.02 (s, 1H), 6.97 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.8 Hz, 2H), 5.53 (s, 1H), 4.55–4.45 (m, 1H), 4.07–3.28 (m, 2H), 1.34 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 154.50, 139.27, 135.21, 134.03, 125.78, 122.92, 120.11, 120.09, 117.32, 115.24, 102.85, 70.66, 22.24. HRMS (ESI) m/z: (M + H)⁺ calcd for C₁₆H₁₇N₃O 268.1372; found 268.1446.

1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (5). A mixture of compound 4 (20.00 g, 0.07 mol), formamidine acetate (10.13 g, 0.10 mol), and ethanol (500 mL) was stirred at 88 °C for 9 h. After the mixed solution was cooled to room temperature and stirred overnight, the solvent was removed under reduced pressure; the residue was diluted with water, stirred for 30 min, and filtered. Then, the residue was purified by slurrying with methanol and filtered to afford compound 5 (20.13 g, 97%) as an orange solid. Mp 193−195 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.22 (s, 1H), 8.21 (d, J = 1.7 Hz, 1H), 7.57 (dd, J = 8.4, 1.5 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.39–7.35 (d, J = 8.9 Hz, 2H), 7.07 (d, J = 8.9 Hz, 2H), 4.67–4.60 (m, 1H), 1.40 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 158.59, 145.02, 143.02, 137.01, 127.53, 127.13, 126.10, 125.65, 119.71, 117.16, 111.84, 106.37, 70.67, 22.07. HRMS (ESI) m/z: (M + H)⁺ calcd for C₁₇H₁₅N₃O 278.1215; found 278.1292.

1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole-5-carbaldehyde (6). The mixture of compound 5 (8.40 g, 0.03 mol) and Raney Ni (8.40 g) in 75% formic acid (168 mL) was stirred at 100 °C for 1–2 h. The reaction mixture was filtered through a pad of Celite. The filtrate was washed with water and EtOAc. The organic phase was separated and dried over anhydrous sodium sulfate overnight. It was then filtered and concentrated; the residue was purified using an automated chromatography system to afford compound 6 (8.15 g, 96%) as a white solid. Mp 130–131 °C. ¹H NMR (600 MHz, Chloroform-d): δ 10.09 (s, 1H), 8.34 (d, J = 1.5 Hz, 1H), 8.17 (s, 1H), 7.89 (dd, J = 8.4, 1.5 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 4.67–4.58 (m, 1H), 1.39 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 191.99, 158.39, 144.88, 143.75, 138.58, 132.26, 128.01, 126.03, 124.89, 123.99, 117.11, 111.27, 70.64, 22.07. HRMS (ESI) m/z: (M + H)⁺ calcd for C₁₇H₁₆N₂O₂ 281.1212; found 281.1287.

3.1.3. General Procedure for Synthesis of Compounds 7a−7h(E/Z)

The corresponding Wittig reagents (1.00 eq.) were added to a stirring solution of tetrahydrofuran (5.00 mL) at −80 °C. Sodium butyllithium (2.50 M in hexanes, 1.05 eq.) was added and stirred for 1 h. Subsequently, compound 6 (1.00 eq.) was added, and the mixed solution was warmed to room temperature slowly. After 12 h, the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography to obtain the products 7a−7h(E/Z).

(Z)-5-(4-(tert-butyl)styryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole (7a−Z) [41]. Compound 7a−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-(tert-butyl)benzyl)triphenylphosphonium. Compound 7a−Z was obtained as a light yellow solid in 45% yield. Mp 172–174 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.06 (s, 1H), 7.84 (s, 1H), 7.42 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.28–7.23 (m, 4H), 7.07 (d, J = 8.9 Hz, 2H), 6.74 (d, J = 12.3 Hz, 1H), 6.60 (d, J = 12.2 Hz, 1H), 4.64 (p, J = 6.1 Hz, 1H), 1.43 (d, J = 6.1 Hz, 6H), 1.32 (s, 9H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.85, 150.64, 144.46, 143.28, 134.91, 133.96, 132.84, 128.88, 128.50, 127.62, 126.25, 125.73, 122.38, 118.52, 116.99, 110.63, 70.55, 34.74, 31.44, 22.12. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₈H₃₀N₂O 411.2358; found 411.2422.

(E)-5-(4-(tert-butyl)styryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole (7a−E) [41]. Compound 7a−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-(tert-butyl)benzyl)triphenylphosphonium. Compound 7a−E was obtained as a white solid in a yield of 23%. Mp 161–162 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.06 (s, 1H), 7.97 (s, 1H), 7.47 (ddd, J = 59.1, 21.1, 8.9 Hz, 8H), 7.25 (d, J = 16.1 Hz, 1H), 7.14 (d, J = 16.2 Hz, 1H), 7.06 (d, J = 8.5 Hz, 2H), 4.63 (p, J = 6.5 Hz, 1H), 1.41 (d, J = 6.3 Hz, 6H), 1.35 (s, 9H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.79, 150.14, 144.06, 143.01, 134.46, 133.43, 132.46, 129.92, 129.50, 128.94, 128.72, 125.70, 125.26, 125.04, 120.69, 116.96, 110.14, 70.54, 34.67, 31.42, 22.12. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₈H₃₀N₂O 411.2358; found 411.2420.

(Z)-4-(2-(1-(4-isopropoxyphenyl)-1H-benzo[d]imidazol-5-yl) vinyl) benzonitrile (7b−Z) [41]. Compound 7b−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-cyanobenzyl) triphenylphosphonium. Compound 7b−Z was obtained as a yellow solid in a yield of 36%. Mp 165–166 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.03 (s, 1H), 7.71 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.4, 1.7 Hz, 1H), 7.04 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 12.2 Hz, 1H), 6.58 (d, J = 12.1 Hz, 1H), 4.65–4.57 (m, 1H), 1.39 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.90, 144.12, 143.37, 142.47, 133.91, 133.76, 132.16, 130.97, 129.69, 128.62, 127.75, 125.69, 124.76, 120.92, 119.11, 116.97, 110.50, 110.40, 70.53, 22.07. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁N₃O 380.1685; found 380.1747.

(E)-4-(2-(1-(4-isopropoxyphenyl)-1H-benzo[d]imidazol-5-yl) vinyl) benzonitrile (7b−E) [41]. Compound 7b−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-cyanobenzyl) triphenylphosphonium. Compound 7b−E was obtained as a yellow solid in a yield of 20%. Mp 161–162 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.07 (s, 1H), 7.99 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.41–7.34 (m, 3H), 7.11 (d, J = 16.2 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 4.68–4.59 (m, 1H), 1.40 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.97, 144.40, 143.66, 142.21, 134.70, 133.03, 132.59, 131.49, 128.59, 126.83, 125.74, 125.72, 122.67, 119.33, 119.26, 117.00, 110.91, 110.31, 70.55, 22.09. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁N₃O 380.1685; found 380.1747.

(Z)-1-(4-isopropoxyphenyl)-5-(4-methylstyryl)-1H-benzo[d]imidazole (7c−Z). Compound 7c−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-methylbenzyl)triphenylphosphonium. Compound 7c−Z was obtained as a yellow solid in a yield of 25%. Mp 168–169 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.01 (s, 1H), 7.77 (s, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.18 (d, J = 7.9 Hz, 2H), 7.07–6.94 (m, 4H), 6.71 (d, J = 12.2 Hz, 1H), 6.58 (d, J = 12.2 Hz, 1H), 4.66–4.57 (m, 1H), 2.30 (s, 3H), 1.39 (d, J = 6.2 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.75, 144.06, 142.97, 136.83, 134.54, 133.39, 132.30, 129.95, 129.59, 129.06, 128.90, 125.64, 125.04, 120.77, 116.93, 110.06, 70.51, 22.09, 21.34. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₄N₂O 369.1889; found 369.1955.

(E)-1-(4-isopropoxyphenyl)-5-(4-methylstyryl)-1H-benzo[d]imidazole (7c−E). Compound 7c−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-methylbenzyl)triphenylphosphonium. Compound 7c−E was obtained as a yellow solid in a yield of 15%. Mp 168–170 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.04 (s, 1H), 7.96 (s, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.49–7.36 (m, 5H), 7.23 (d, J = 16.3 Hz, 1H), 7.18 (d, J = 7.9 Hz, 2H), 7.12 (d, J = 16.3 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.67–4.57 (m, 1H), 2.37 (s, 3H), 1.40 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.84, 144.47, 143.26, 137.35, 134.88, 133.95, 132.80, 129.51, 128.87, 128.25, 127.72, 126.43, 125.70, 122.35, 118.50, 116.98, 110.63, 70.54, 22.11, 21.38. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₄N₂O 369.1889; found 369.1953.

(Z)-5-(4-ethoxystyryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole (7d−Z). Compound 7d−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with 4-ethoxybenzyl)triphenylphosphonium. Compound 7d−Z was obtained as a yellow solid in a yield of 50%. Mp 161–162 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.05 (s, 1H), 7.82 (s, 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 6.9 Hz, 2H), 7.06 (d, J = 6.9 Hz, 2H), 6.77 (d, J = 6.7 Hz, 2H), 6.69 (d, J = 12.1 Hz, 1H), 6.58 (d, J = 12.2 Hz, 1H), 4.64 (dtq, J = 9.2, 5.8, 3.2, 2.6 Hz, 1H), 4.02 (dt, J = 7.3, 2.4 Hz, 2H), 1.42 (dt, J = 5.0, 2.5 Hz, 9H). ¹³C NMR (151 MHz, Chloroform-d): δ 158.08, 157.72, 144.06, 142.92, 133.30, 132.45, 130.21, 129.75, 129.24, 129.04, 128.88, 125.60, 124.99, 120.66, 116.91, 114.25, 110.07, 70.48, 63.38, 22.07, 14.94. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₆H₂₆N₂O₂, 399.1994; found 399.2057.

(E)-5-(4-ethoxystyryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole(7d−E). Compound 7d−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with 4-ethoxybenzyl)triphenylphosphonium. Compound 7d−E was obtained as a yellow solid in a yield of 30%. Mp 152–153 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.04 (s, 1H), 7.94 (s, 1H), 7.59–7.36 (m, 6H), 7.18–7.00 (m, 4H), 6.89 (d, J = 6.2 Hz, 2H), 4.67–4.56 (m, 1H), 4.10–4.01 (m, 2H), 1.51–1.35 (m, 9H). ¹³C NMR (151 MHz, Chloroform-d): δ 158.62, 157.81, 144.44, 143.20, 133.79, 132.98, 130.33, 128.88, 127.69, 127.38, 127.05, 125.68, 122.26, 118.24, 116.97, 114.79, 110.60, 70.53, 63.60, 22.10, 14.98. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₆H₂₆N₂O₂ 399.1994; found 399.2060.

(Z)-5-(4-(difluoromethoxy)styryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole (7e−Z). Compound 7e−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-(difluoromethoxy)benzyl)triphenylphosphonium. Compound 7e−Z was obtained as a yellow solid in a yield of 30%. Mp 173–174 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.02 (s, 1H), 7.74 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8.9 Hz, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.7 Hz, 2H), 7.18 (dd, J = 8.4, 1.7 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 12.1 Hz, 1H), 6.56 (d, J = 12.1 Hz, 1H), 6.48 (t, J = 74.1 Hz, 1H), 4.65–4.56 (m, 1H), 1.39 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.83, 150.20 (t, J = 2.7 Hz), 144.09, 143.14, 134.75, 133.57, 131.75, 131.06, 130.50, 128.79, 128.31, 125.67, 124.90, 120.77, 119.20, 116.96, 116.08 (t, J = 259.0 Hz), 110.29, 70.53, 22.08. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₂F₂N₂O₂ 421.1649; found 421.1712.

(E)-5-(4-(difluoromethoxy)styryl)-1-(4-isopropoxyphenyl)-1H-benzo[d]imidazole (7e−E). Compound 7e−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (4-(difluoromethoxy)benzyl)triphenylphosphonium. Compound 7e−E was obtained as a yellow solid in a yield of 26%. Mp 173–174 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.05 (s, 1H), 7.96 (s, 1H), 7.56–7.48 (m, 3H), 7.43 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.21 (d, J = 16.3 Hz, 1H), 7.14–7.07 (m, 3H), 7.05 (d, J = 8.8 Hz, 2H), 6.52 (t, J = 74.0 Hz, 1H), 4.67–4.58 (m, 1H), 1.40 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.87, 150.46 (t, J = 2.7 Hz), 144.48, 143.40, 135.12, 134.17, 132.29, 129.59, 128.78, 127.76, 126.37, 125.70, 122.39, 119.84, 118.70, 116.98, 116.08 (t, J = 260.0 Hz), 110.72, 70.54, 22.10. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₂F₂N₂O₂ 421.1649; found 421.1713.

(Z)-1-(4-isopropoxyphenyl)-5-(4-(trifluoromethoxy)styryl)-1H-benzo[d]imidazole (7f−Z). Compound 7f−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with triphenyl(4-(trifluoromethoxy)benzyl)phosphonium. Compound 7f−Z was obtained as a yellow solid in a yield of 30%. Mp 161–162 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.03 (s, 1H), 7.75 (d, J = 1.7 Hz, 1H), 7.37 (d, J = 8.9 Hz, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 7.18 (dd, J = 8.5, 1.7 Hz, 1H), 7.07–7.00 (m, 4H), 6.80 (d, J = 12.2 Hz, 1H), 6.56 (d, J = 12.1 Hz, 1H), 4.65–4.56 (m, 1H), 1.39 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.87, 148.10 (q, J = 1.5 Hz), 144.12, 143.20, 136.18, 133.66, 131.62, 131.57, 130.42, 128.79, 128.08, 125.70, 124.84, 120.85, 120.76, 120.57 (q, J = 257.2 Hz), 116.98, 110.35, 70.55, 22.10. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁F₃N₂O₂ 439.1555; found 439.1619.

(E)-1-(4-isopropoxyphenyl)-5-(4-(trifluoromethoxy)styryl)-1H-benzo[d]imidazole (7f−E). Compound 7f−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with triphenyl(4-(trifluoromethoxy)benzyl)phosphonium. Compound 7f−E was obtained as a yellow solid in a yield of 10%. Mp 160–162 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.05 (s, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.52 (dd, J = 8.5, 1.7 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 7.25–7.19 (m, 3H), 7.11 (d, J = 16.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.66–4.57 (m, 1H), 1.40 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.93, 148.41, 144.44, 143.45, 136.49, 134.28, 132.17, 130.30, 128.76, 127.65, 126.17, 125.74, 122.47, 121.33, 120.63 (q, J = 257.2 Hz), 118.81, 117.01, 110.77, 70.56, 22.10. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁F₃N₂O₂ 439.1555; found 439.1619.

(Z)-1-(4-isopropoxyphenyl)-5-(4-(trifluoromethyl)styryl)-1H-benzo[d]imidazole (7g−Z). Compound 7g−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with triphenyl(4-(trifluoromethyl)benzyl)phosphonium. Compound 7g−Z was obtained as a yellow solid in a yield of 35%. Mp 165–166 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.03 (s, 1H), 7.74 (s, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.39–7.33 (m, 4H), 7.30 (dd, J = 8.5 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.06–7.00 (m, 2H), 6.86 (d, J = 12.2 Hz, 1H), 6.60 (d, J = 12.2 Hz, 1H), 4.66–4.55 (m, 1H), 1.41–1.35 (m, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.87, 144.10, 143.25, 141.25, 133.76, 132.72, 131.33, 129.27, 128.90 (q, J = 32.0 Hz), 128.72, 128.09, 125.68, 125.29 (q, J = 3.9 Hz), 124.85, 124.29 (q, J = 272.3 Hz), 120.89, 116.97, 110.39, 70.53, 22.07. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁F₃N₂O 423.1606; found 423.1667.

(E)-1-(4-isopropoxyphenyl)-5-(4-(trifluoromethyl)styryl)-1H-benzo[d]imidazole (7g−E). Compound 7g−E was synthesized according to the general procedure, replacing the corresponding Wittig reagents with tri-phenyl(4-(trifluoromethyl)benzyl)phosphonium. Compound 7g−E was obtained as a yellow solid in a yield of 20%. Mp 166–167 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.06 (s, 1H), 7.99 (s, 1H), 7.69–7.57 (m, 4H), 7.53 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 5.2 Hz, 1H), 7.39 (d, J = 5.2 Hz, 2H), 7.35 (d, J = 16.1 Hz, 1H), 7.15 (d, J = 16.2 Hz, 1H), 7.06 (d, J = 5.4 Hz, 2H), 4.66–4.57 (m, 1H), 1.44–1.35 (m, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.97, 144.44, 143.55, 141.18, 134.50, 131.89, 131.81, 129.08 (q, J = 32.0 Hz), 128.71, 126.57, 126.18, 125.76, 125.73, 124.41 (q, J = 271.5 Hz), 122.61, 119.10, 117.02, 110.84, 70.58, 22.11. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₁F₃N₂O 423.1606; found 423.1668.

(Z)-1-(4-isopropoxyphenyl)-5-(2-(naphthalene-2-yl)vinyl)-1H-benzo[d]imidazole (7h−Z). Compound 7h−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (naphthalen-2-ylmethyl)triphenylphosphonium. Compound 7h−Z was obtained as a brown solid in a yield of 26%. Mp 170–171 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.02 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.74–7.69 (m, 1H), 7.73–7.69 (m, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.43–7.40 (m, 2H), 7.39–7.35 (m, 3H), 7.28–7.22 (m, 2H), 7.02 (d, J = 8.9 Hz, 2H), 6.84 (d, J = 12.1 Hz, 1H), 6.78 (d, J = 12.2 Hz, 1H), 4.65–4.56 (m, 1H), 1.38 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.79, 143.99, 143.07, 135.23, 133.65, 133.53, 132.67, 132.16, 131.02, 129.56, 128.83, 128.11, 128.02, 127.73, 127.65, 127.10, 126.06, 125.91, 125.65, 125.12, 120.99, 116.95, 110.13, 70.52, 22.09. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₈H₂₄N₂O 405.1889; found 405.1955.

(E)-1-(4-isopropoxyphenyl)-5-(2-(naphthalen-2-yl)vinyl)-1H-benzo[d]imidazole (7h−E). Compound 7h−Z was synthesized according to the general procedure, replacing the corresponding Wittig reagents with (naphthalen-2-ylmethyl)triphenylphosphonium. Compound 7h−E was obtained as a yellow solid in a yield of 15%. Mp 172–173 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.06 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.85–7.80 (m, 3H), 7.79 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48–7.38 (m, 6H), 7.31 (d, J = 16.2 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 4.68–4.59 (m, 1H), 1.41 (d, J = 6.1 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d): δ 157.86, 144.46, 143.34, 135.17, 134.12, 133.88, 133.05, 132.63, 129.60, 128.81, 128.40, 128.08, 127.84, 127.81, 126.49, 126.41, 125.88, 125.72, 123.65, 122.48, 118.69, 116.98, 110.73, 70.54, 22.11. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₈H₂₄N₂O 405.1889; found 405.1951.

3.1.4. General Procedure for Synthesis of Compounds 13a−13j

To a mixture of compound 12 (1.00 eq.) and relatively aromatic aldehyde (R₂-CHO, 1.00 eq.) in 1, 2-dichloroethane (3 mL) was added the Na(AcO)₃BH (1.50 eq.) and stirred at room temperature for 6 h. After the mixture was stirred for 6 h, TLC monitors showed the complete consumption of compound 12. Following cooling, the mixture was divided between EtOAc and H₂O, and the layers were separated, with the aqueous layer extracted with EtOAc and the organic layer washed with H₂O. The organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain the products 13a−13j.

2-methoxy-5-(((1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)amino)methyl)phenol (13a). Compound 13a was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with isovanillin. Compound 13a was obtained as a white solid in a yield of 87%. Mp 182.1–183.3 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.892 (s, 1H), 7.372 (t, 1H, J1 = 4.8, J2 = 1.8), 7.357 (t, 1H, J1 = 2.4, J2 = 3.0), 7.228 (d, 1H, J = 10.4) 7.034 (s, 2H), 7.019 (t, 1H, J1 = 2.4, J2 = 3.6), 6.978 (d, 1H, J = 1.8), 6.876 (m, 1H), 6.805 (dd, 1H, J = 10.4), 6.689 (dd, 1H) 5.666 (s, 1H), 4.274 (s, 2H), 3.852 (m, 6H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.72, 146.89, 146.82, 145.65, 145.44, 142.60, 133.41, 129.78, 126.11, 125.15, 118.27, 115.52, 115.02, 112.92, 112.61, 110.91, 100.66, 56.13, 55.95, 47.28, 40.35, 40.21, 40.07, 39.94, 39.80, 39.66, 39.52. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₂H₂₁N₃O₃ 376.1656; found 376.1654.

2-methoxy-4-(((1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)amino)methyl)phenol (13b). Compound 13b was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with vanillin. Compound 13b was obtained as a yellow solid in a yield of 91%. Mp 157.6–159.0 °C. ¹H NMR (600 MHz, Chloroform-d): δ7.908 (s, 1H), 7.374 (t, 1H, J1 = J2 = 2.4), 7.367 (t, 1H, J1 = 2.4, J2 = 3.6), 7.227 (d, 1H, J = 7.8), 7.066 (d, 1H), 7. 039 (t, 1H, J = 2.4), 7. 027 (t, 1H, J = 1.8), 6.932 (d, 1H, J = 1.8), 6.891 (dd, 1H, J = 8.4), 6.874 (d, 1H, J = 7.8), 6.703 (dd, 1H, J = 6.6), 5.598 (s, 1H), 4.283 (s, 2H), 3.868 (d, 6H, J = 3.0). ¹³C NMR (151 MHz, DMSO-d6): δ 158.71, 147.98, 145.74, 145.66, 145.44, 142.61, 131.50, 129.79, 126.16, 125.15, 120.12, 115.63, 115.53, 112.99, 112.05, 110.88, 100.78, 56.01, 55.96, 47.74. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₂H₂₁N₃O₃ 376.1656; found 376.1652.

N-(2, 4-dimethoxybenzyl)-1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-amine (13c). Compound 13c was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 2, 4-dimethoxybenzaldehyde. Compound 13c was obtained as a yellow solid in a yield of 88%. Mp 143.1–145.2 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.517 (s, 1H), 7.385–7.412 (m, 3H), 7.112 (s, 1H), 7.097 (s, 1H), 7.063 (s, 1H), 6.924 (d, 1H, J = 7.8), 6.845 (s, 1H), 6.829 (s, 1H), 6.815 (s, 1H), 6.776 (d, 1H, J = 3.0), 4.377 (s, 2H), 3.908 (s, 3H), 3.897 (s, 3H), 3.733 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 160.58, 141.36, 139.40, 131.50, 131.06, 129.73, 129.53, 127.99, 126.88, 126.87, 115.86, 115.68, 113.83, 56.48, 56.19, 45.82. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₃H₂₃N₃O₃ 390.1812; found 390.1810.

1-(4-methoxyphenyl)-N-(2, 4,5-trimethoxybenzyl)-1H-benzo[d]imidazol-5-amine (13d). Compound 13d was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 2, 4,5-trimethoxybenzaldehyde. Compound 13d was obtained as an orange solid in a yield of 88%. Mp 167.8–169.8 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.894 (s, 1H), 7.374 (t, 1H, J1 = 3.6, J2 = 1.8), 7.360 (t, 1H, J1 = 1.8, J2 = 3.6), 7.226 (d, 1H, J = 8.4), 7.094 (d, 1H, J = 1.8), 7. 033 (t, 1H, J1 = 3.6, J2 = 1.8), 7. 019 (t, 1H, J1 = 1.8, J2 = 3.6), 6.920 (s, 1H), 6.716 (dd, 1H, J = 6.6), 6.536 (s, 1H), 4.304 (s, 2H), 3.870 (s, 3H), 3.860 (s, 3H), 3.839 (s, 3H), 3.779 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 203.62, 158.72, 156.07, 151.76, 148.86, 145.73, 145.46, 142.98, 142.64, 125.15, 119.33, 115.53, 114.17, 112.89, 110.93, 100.64, 98.73, 56.92, 56.68, 56.34, 55.96, 42.08. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₄H₂₅N₃O₄ 420.1918; found 420.1920.

2, 6-dimethoxy-4-(((1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)amino)methyl)phenol (13e). Compound 13e was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with Syringaldehyde. Compound 13e was obtained as an orange solid in a yield of 95%. Mp 115.7–118.1 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.917 (d, 1H, J = 3.6), 7.377 (t, 1H, J1 = J2 = 1.8), 7.366 (t, 1H, J1 = 1.8, J2 = 3.6), 7.244 (d, 1H, J = 6.0), 7.068 (d, 1H, J = 2.4), 7. 037 (t, 1H, J1 = 1.8, J2 = 1.2), 7. 025 (t, 1H, J1 = 2.4, J2 = 3.0), 6.708 (dd, 1H, J = 6.0), 6.651 (s, 2H), 6.482 (s, 1H),5.407 (d, 1H), 4.528 (s, 1H), 4.284 (s, 2H), 3.864 (s, 9H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.72, 148.50, 148.41, 134.63, 125.79, 125.15, 115.62, 115.53, 113.03, 110.90, 105.28, 105.04, 100.85, 56.41, 55.96, 48.21. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₃H₂₃N₃O₄ 406.1761; found 406.1762.

N-(4-(tert-butyl)benzyl)-1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-amine (13f). Compound 13f was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with p-t-butylbenzaldehyde. Compound 13f was obtained as a yellow solid in a yield of 95%. Mp 175.8–176.9 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.897 (s, 1H), 7.326–7.3776 (m, 6H), 7.240 (d, 1H, J = 9.0), 7.054 (d, 1H, J = 2.4), 7. 036 (t, 1H, J = 2.4), 7. 025 (t, 1H, J = 2.4), 6.706 (dd, 1H, J = 6.6), 4.343 (s, 2H), 3.862 (s, 3H), 1.303 (s, 9H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.73, 149.33, 145.61, 145.46, 142.63, 137.81, 129.78, 127.49, 126.17, 125.46, 125.16, 115.53, 112.94, 110.97, 100.62, 55.96, 47.34, 34.60, 31.67. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₅H₂₇N₃O 386.2227; found 386.2330.

N-(4-methoxybenzyl)-1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-amine (13g). Compound 13g was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 4-methoxybenzaldehyde. Compound 13g was obtained as a yellow solid in a yield of 90%. Mp 128.0–129.3 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.897 (s, 1H), 7.374 (t, 1H, J1 = 3.0, J2 = 2.4), 7.359 (t, 1H, J1 = 2.4, J2 = 3.6), 7.325 (d, 1H, J = 3.0), 7.311 (d, 1H, J = 3.0), 7.235 (d, 1H, J = 8.4), 7. 054 (d, 1H, J = 2.4), 7.035 (t, 1H, J1 = 3.0, J2 = 2.4), 7.020 (t, 1H, J1 = 2.4, J2 = 3.6), 6.871 (t, 1H, J1 = 3.0, J2 = 1.8), 6.857 (t, 1H, J1 = 1.8, J2 = 3.0), 6.695 (dd, 1H, J = 6.6), 4.303 (s, 2H), 3.861 (s, 3H), 3.785 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.73, 158.50, 145.57, 145.46, 142.62, 132.63, 129.78, 128.88, 126.18, 125.15, 115.53, 114.14, 112.98, 110.93, 100.76, 55.96, 55.47, 47.13. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₂H₂₁N₃O₂ 360.1707; found 360.1705.

3-(((1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)amino)methyl)phenol (13h). Compound 13h was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 3-hydroxybenzaldehyde. Compound 13h was obtained as a yellow solid in a yield of 91%. Mp 182.9–184.3 °C. ¹H NMR (600 MHz, Chloroform-d): ¹H NMR (600 MHz, Chloroform-d): δ 7.926 (s, 1H), 7.365 (t, 1H, J1 = 3.6, J2 = 2.4), 7.350 (t, 1H, J1 = 2.4, J2 = 3.6), 7.226 (d, 1H, J = 8.4), 7.185 (t, 1H, J1 = J2 =7.8), 7. 035 (t, 1H, J1 = J2 = 2.4), 7. 020 (t, 1H, J1 = 3.6, J2 = 2.4), 6.928 (d, 1H, J = 7.2), 6.781 (dd, 1H, J = 6.0), 4.295 (s, 2H), 3.826 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.73, 157.88, 145.60, 145.44, 142.64, 142.51, 129.77, 129.67, 126.16, 125.17, 118.23, 115.53, 114.34, 113.93, 112.86, 110.96, 100.62, 55.96, 47.62. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₁H₁₉N₃O₂ 346.1550; found 346.1547.

N-(3, 4-dichlorobenzyl)-1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-amine (13i). Compound 13i was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 3, 4-dichlorobenzaldehyde. Compound 13i was obtained as a yellow solid in a yield of 89%. Mp 134.1–135.8 °C. ¹H NMR (600 MHz, Chloroform-d): δ 8.926 (s, 1H), 7.455 (t, 1H, J1 = 3.0, J2 = 1.8), 7.444 (d, 1H, J = 1.8), 7.435 (d, 1H, J = 1.2), 7.365 (d, 1H, J = 8.4), 7.265 (d, 1H, J = 9.0), 7. 220 (dd, 1H, J = 7.2), 7.096 (t, 1H, J1 = J2 = 3.6), 7.081 (t, 1H, J1 = 2.4, J2 = 3.0), 6.897 (d, 1H, J = 9.6), 4.356 (s, 2H), 3.878 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 158.76, 146.23, 145.43, 145.11, 142.77, 129.72, 128.28, 127.82, 127.62, 126.38, 125.79, 125.59, 125.21, 115.53, 112.87, 111.14, 100.75, 55.96, 47.11. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₁H₁₇C_l2N₃O 398.0821; found 398.0824.

1-(4-methoxyphenyl)-N-(4-(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-5-amine (13j). Compound 13j was synthesized according to the general procedure, replacing the corresponding aromatic aldehyde with 4-(trifluoromethyl)benzaldehyde. Compound 13j was obtained as a white solid in a yield of 90%. Mp 198.3–200.5 °C. ¹H NMR (600 MHz, Chloroform-d): δ 7.895 (s, 1H), 7.557 (d, 1H, J = 8.4), 7.518 (d, 1H, J = 7.8), 7.365 (t, 1H, J1 = J2 = 2.4), 7.350 (t, 1H, J1 = 2.4, J2 = 3.0), 7.245 (d, 1H, J = 8.4), 7.034 (t, 1H, J1 = 3.6, J2 = 1.8), 7.020 (t, 1H, J1 = 1.8, J2 = 3.6), 6.974 (d, 1H, J = 1.8), 6.695 (dd, 1H, J = 6.6), 4.463 (s, 2H), 4.160 (s, 1H), 3.860 (s, 3H). ¹³C NMR (151 MHz, DMSO-d6): δ 160.62, 153.55, 151.59, 133.35, 126.92, 126.84, 116.42, 115.69, 115.22, 113.81, 112.29, 112.05, 56.20, 55.74. HRMS (ESI) m/z: (M + H)⁺ calcd for C₂₂H₁₈F₃N₃O 398.1475; found 398.1480.

3.2. Determining the Antiviral Activities of Compounds

3.2.1. Cells, Viruses, and Compounds

HEK-293 cells (ATCC, CRL-1573) and 293T cells (ATCC, CRL-3216) were cultured under 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium with 10% heat inactivated fetal bovine serum (FBS). LASV pseudotyped virus (LASVpv) based on HIV-1 expression system with enhanced green fluorescent protein (EGFP) and firefly luciferase (Luc) encoded in LASV GPC (Josiah strain) were produced by VectorBuilder (Vector Builder Inc., Guangzhou, China). The titers of LASVpv were 8 × 10⁸ TU/mL. Benzimidazole Compounds disclosed herein were synthesized as described in our research.

3.2.2. Cytotoxicity Evaluation

The title compounds were dissolved by DMSO into 20mM solution as stock solution and were stored at −80 °C until being used. The working solutions of the 8 different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 μM) for the test compounds were serially diluted in the culture medium. HEK-293 cells were inoculated in clear 96-well plates at an approximate density of 2 × 10⁴ cells/well and cultured for 24 h at 37 °C with 5% CO₂. Then, the working solution of the test compounds was added to each well. PBS was used to wash the dead cells after the plates had been cultured for 4 days.

In this study, the Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of cells, and the optical density (OD) data were used to estimate the survival rate of the cells. The absorbance was measured at 450 nm with a microplate reader. CC₅₀ value (50% cytotoxicity Concentration) was calculated using log (inhibitor) vs. response–variable slope in GraphPad Prism 8 software (San Diego, CA, USA).

3.2.3. LASVpv Infections and Inhibition Assays

To test antiviral activities, HEK-293 cells were cultured in opaque white 96-well plates. The assay medium consisted of phenol red-free Dulbecco’s Modified Eagle’s Medium, 2% FBS, 25 mM HEPES, 4.5 g/L D-glucose, and L-glutamine. Cell density and MOI (Multiplicity of Infection) were optimized for the antiviral activity assay in preliminary studies. HEK-293 cells at different densities (5 × 10³ to 3 × 10⁴ cells/well) were infected at MOI from 0.5 to 10. 0. The optimized cell density (2 × 10⁴ cells per well) and the dose for LASVpv (MOI = 5) were selected for our assay. A negative control was the addition of only culture medium containing 0.5% DMSO; a positive control was adding virus without compound.

HEK-293 cells were cultured at a density of 2 × 10⁴ cells per well in opaque white 96-well plates. After incubating overnight, cells were treated with compound solutions of different concentrations (50, 5, 0.5, 0.05, 0.005, 0.0005, 0.00005, 0.000005 μM). Subsequently, HEK-293 cells were infected with LASVpv, and firefly luciferase activity was assayed at 48 h post-infection.

Luciferase signals were detected with Luciferase Assay System (Promega, Madison, WI, USA). A SpectraMax i3X microplate reader (Molecular Devices, San Jose, CA, USA) was used to quantify luminescence using a 1 s read time per well. Visualization and counting of EGFP-positive cells were carried out using the FLoid^® Cell Imaging Station. IC₅₀ value (50% growth inhibitory concentration) was calculated also using log (inhibitor) vs. response–variable slope in GraphPad Prism 8 software.

3.3. Surface Plasmon Resonance (SPR) Studies

Analyses of SPR interactions were performed on a Biacore T200 optical biosensor (GE Healthcare Life Sciences, Chicago, IL, USA). Data were analyzed using BiacoreT200 evaluation software ver 3.2.1 (Chicago, IL, USA). The curves were plotted using Origin 2022 software (Paris, France). The expressing vector pET-22b-His-GST-GP2 was constructed, and GP2 protein was produced by Abiocenter Biotechnology Co., Ltd. (Beijing, China). The phosphate-buffered saline composed of 0.2 M phosphate buffer, 27 mM KCl, 27 mM KCl, 1.37 M NaCl, and 0.5% surfactant P20 were used as the base buffer. Diluted the base buffer 10 times and added 5% DMSO when pH was adjusted to 7.4 as running buffer. The running buffers were used as blank injections, and solvents were used to correct for volume effects.

3.3.1. Ligand Protein Immobilization

The ligand protein GP2 was immobilized on a CM5 chip by standard amine coupling. Using a 1:1 mixture of d 100 mM N-hydroxysuccinimide (NHS) and 100 mM Nethyl-N′-(dimethylaminopropyl)-carbodiimide (EDC), the flow cell surface was activated for 400 s at a flow rate of 10 μL/min. Then, GP2 (50 μg/mL) dissolved in 0.1 M sodium acetate buffer pH 4.0 was immobilized on a CM5 chip for 600 s until the desired immobilization level of 15,783 RU. Finally, the activated carboxyl groups remaining on the surface were blocked by a 400 s injection of methanolamine (pH 8.5).

3.3.2. Screening and Kinetic Analysis

All the binding experiments were performed at 25 °C at a continuous flow rate 10 μL/min with 20 mM phosphate-buffered saline. Compounds were dissolved with DMSO to make 20 mM stock solution and then diluted with the prepared PBS-P⁺ running buffer to fit the concentration (50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 μM) for the assay. Analytes solutions with a series of increasing concentrations (0.19–50 µM) were applied to channel F1 in SPR-binding buffer at a 10 µL/min flow rate at 25 °C. For reference purposes, one of the flow channels F2 was left untouched (immobilized blankly). The contact and dissociation times were kept at 90 s, and data were double-referenced with both reference cell RU values and zero concentration (5% DMSO) signals.

All equilibrium dissociation constant (K_D) analyses were performed using Biacore T200 evaluation software version 3.2.1, and the data were fitted to a 1:1 binding.

4. Conclusions

Viral hemorrhagic fever caused by LASV is an infectious disease that seriously threatens human health. No specific anti-LASV drug is currently licensed for clinical use. GPC, the glycoprotein complex of LASV, plays a key role in its invasion process and can be used as a target for anti-LASV drugs. Research on envelope glycoprotein inhibitors could help to discover effective therapeutic agents against LASV.

In this study, we prepared two series of novel benzimidazole derivatives targeted at the LASV GPC. Their antiviral activities were tested through pseudovirus bioassays and SPR technical analysis, in which some compounds showed submicromolar anti-LASV activities. Preliminary structure–activity relationship studies found that although 5-amino-benzo[d]imidazole derivatives have lower antiviral activities than series I compounds, their cytotoxicity is also lower in HEK-293 cells, resulting in a higher SI and better safety profile. The presence of subamine bonds in series II compounds generally leads to their higher stability relative to the carbon–carbon double bonds in series I compounds. Substituents with lipophilicity and those that are spatially larger, such as naphthylphenyl, ethoxyphenyl, tert-butylphenyl, 3, 6-dimethoxyphenyl, and 2, 4, 5-trimethoxyphenyl, resulted in the better inhibitory activities of the compounds against LASV, indicating that the antiviral activity is closely related to the lipophilic nature of the para-substituted phenyl group. Additionally, the R₁ site in series I and the R₂ site in series II can accommodate larger substituents, which indicates that the group with larger spatial site resistance is a better choice at this position. As a result, potent antiviral compounds were identified. In particular, the IC₅₀ values of 7d−Z, 7h−Z, 13c, 13d, and 13f showed activities comparable to the positive control compound, LHF−535, against LASVpv. However, the cytotoxicities of five representative compounds was investigated on the human embryonic kidney cell line (HEK-293), and the results showed that these compounds had lower toxicities in this study. The SI values of these compounds (1251, 2494, 4798, 9761, and 8602, respectively) were all higher than LHF−535 (1208). Among them, it is worth pointing out that compound 7h−Z, with a large conjugated system by introducing naphthalenyl groups, showed the best antiviral activity (IC₅₀ = 7.58 nM) with a 2-fold increase in SI, which can be further investigated as a lead compound. Moreover, in the SPR study, we further determined the kinetic parameters of the title compounds. These five representative compounds showed low k_d values (<0.03 s⁻¹) and low K_D (<8.25 × 10⁻⁷ M), which expose their long residence time (t = 1/K_d) on their targets, slower dissociation behavior, and strong binding affinity. In conclusion, introducing lipophilicity and spatially larger substituents was very important for prolonging the residence time and enhancing the binding affinities of the target compound on its target; this may give a larger safety margin. Further investigations are underway to modify the benzimidazole derivatives, aiming to improve the antiviral activity against LASV.

To design and synthesize compounds with potent activities against LASV, the influence of substituents needs to be fully considered. This paper provides some references for the design and synthesis of highly active compounds against LASV. In the future, we will further optimize the molecular structure to obtain excellent candidates for clinical use.

Author Contributions

Conceptualization, L.X. and Y.S.; methodology, J.C., L.X. and Z.B.; investigation, J.C. and L.X.; resources, Y.S.; data curation, J.C., L.X., D.Z. and L.Z.; writing—original draft preparation, J.C.; writing—review and editing, L.X., B.W. and Y.S.; supervision, Y.S.; project administration, L.X.; funding acquisition, Y.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We are grateful to Lin Li for his helpful suggestion.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 7a−7h(Z/E) and 13a−13j are available from the authors.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures, Schemes and Tables

Figure 1. The structure of LHF−535 and ST−193 and two kinds of substituted benzimidazole structures.

Scheme 1. Synthesis of 7a−7h Z/E (Z: cis structure, E: trans structure). Reagents and conditions: (i) Et3N, CH3CN, 90 °C; (ii) SnCl2·2H2O, EtOAc, 50 °C; (iii) Formamidine acetate, EtOH, 88 °C; (iv) Raney Nickel, formic acid, 100 °C; (v) Wittig reagents, n-BuLi, THF, −80 °C.

Scheme 2. Synthesis of derivatives 13a−13j. Reagents and conditions: (i) Cesium carbonate, THF, 48 °C; (ii) Sodium hydrosulfite, EtOH, rt; (iii) 4.0 M hydrochloric acid, formic acid, 100 °C; (iv) aromatic aldehydes, DCM, Na(AcO)3BH, rt.

Figure 2. Expression of EGFP in pseudovirus-infected HEK-293 cells. (A) The positive drug LHF−535 inhibited LASVpv infection in a concentration-dependent manner. (B) The positive drug LHF−535 shows little or no effect on pseudotyped viruses expressing the VSVpv.

Figure 3. Concentration–response curves of representative compounds 7d−Z, 7h−Z, 13c, 13d, 13f, and LHF−535 to LASVpv.

Figure 4. Binding sensory plots of representative compounds (A) 7d−Z, (B) 7h−Z, (C) 13c, (D) 13d, (E) 13f, and (F) LHF−535 interacting with immobilized GP2.

Table 1

Inhibition of LASVpv pseudotyped viruses by synthetic compounds 7a−7h(Z/E).

Core	[Image omitted. Please see PDF.]
Compd.	R1	CC50 (μM) b	IC50 (nM) a	SI (CC50/IC50) c
LHF−535	[Image omitted. Please see PDF.]	3.68	3.04	1208
7a−Z	[Image omitted. Please see PDF.]	3.01	3.91	770
7a−E	[Image omitted. Please see PDF.]	>100	>50	-
7b−Z	[Image omitted. Please see PDF.]	23.58	>50	49
7b−E	[Image omitted. Please see PDF.]	>100	>50	-
7c−Z	[Image omitted. Please see PDF.]	20.93	36.89	567
7c−E	[Image omitted. Please see PDF.]	>100	>50	-
7d−Z	[Image omitted. Please see PDF.]	16.97	13.56	1251
7d−E	[Image omitted. Please see PDF.]	>100	>50	-
7e−Z	[Image omitted. Please see PDF.]	17.88	34.39	520
7e−E	[Image omitted. Please see PDF.]	>100	>50	-
7f−Z	[Image omitted. Please see PDF.]	9.47	33.89	279
7f−E	[Image omitted. Please see PDF.]	>100	>50	-
7g−Z	[Image omitted. Please see PDF.]	24.38	29.09	838
7g−E	[Image omitted. Please see PDF.]	>100	>50	-
7h−Z	[Image omitted. Please see PDF.]	18.92	7.58	2494
7h−E	[Image omitted. Please see PDF.]	>100	>50	-

^a Activity is provided as IC₅₀ (nM). ^b Cytotoxicity in HEK-293 cells is shown as CC₅₀ (μM). ^c The selection index is given as SI.

Table 2

Inhibition of LASVpv pseudotyped viruses by synthetic compounds 13a−13j.

Core	[Image omitted. Please see PDF.]
Compd.	R2	CC50 (μM) b	IC50 (nM) a	SI (CC50/IC50) c
ST−193	[Image omitted. Please see PDF.]	56.29	44.49	1265
13a	[Image omitted. Please see PDF.]	62.60	47.58	1316
13b	[Image omitted. Please see PDF.]	87.28	65.75	1327
13c	[Image omitted. Please see PDF.]	74.18	15.46	4798
13d	[Image omitted. Please see PDF.]	134.80	13.81	9761
13e	[Image omitted. Please see PDF.]	88.78	50.49	1758
13f	[Image omitted. Please see PDF.]	102.10	11.87	8602
13g	[Image omitted. Please see PDF.]	99.96	55.39	1805
13h	[Image omitted. Please see PDF.]	40.34	>100	152
13i	[Image omitted. Please see PDF.]	81.73	53.15	1538
13j	[Image omitted. Please see PDF.]	108.80	63.78	1706

^a Activity is provided as IC₅₀ (nM). ^b Cytotoxicity in HEK-293 cells is shown as CC₅₀ (μM). ^c The selection index is given as SI.

Table 3

The SPR data for the binding of active compounds.

Compd.	ka (M−1s−1)	kd (s−1)	KD (M)
7a−Z	3.28 × 105	0.04	1.46 × 10−7
7b−Z	3908	0.28	7.34 × 10−5
7c−Z	6.56 × 104	0.07	1.18 × 10−6
7d−Z	8.65 × 104	0.02	3.07 × 10−7
7e−Z	4.34 × 104	0.08	1.98 × 10−6
7f−Z	1.56 × 104	0.03	2.19 × 10−6
7g−Z	5.01	0.13	2.70 × 10−6
7h−Z	2.94 × 105	0.03	1.29 × 10−7
13a	1.32 × 104	0.03	2.78 × 10−6
13b	5.23 × 104	0.20	3.93 × 10−6
13c	3.95 × 104	0.03	8.25 × 10−7
13d	5.67 × 104	0.03	6.56 × 10−7
13e	5.03 × 104	0.22	4.39 × 10−6
13f	1.78 × 105	0.03	1.73 × 10−7
13g	6.59 × 104	0.30	4.69 × 10−6
13h	1.19 × 104	0.42	3.60 × 10−5
13i	1.94 × 104	0.49	2.54 × 10−5
13j	8532	0.04	5.49 × 10−6
ST−193	8.32 × 104	0.01	2.00 × 10−7
LHF−535	3.04 × 105	0.03	1.14 × 10−7

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28041579/s1, Figures S1–S60: ¹H and ¹³C NMR spectra of intermediates and final compounds.

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