Bis(Benzofuran–1,3-N,N-heterocycle)s as Symmetric and Synthetic Drug Leads against Yellow Fever Virus

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran–1,3-imidazolidin-4-one)s and bis(benzofuran–1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC50 of <3.54 μM and SI of >15.3. The results are valuable for the development of novel antiviral drug leads against emerging diseases.


Introduction
For individuals who are severely affected by yellow fever virus (YFV) but without treatment, up to 50% of them die according to the report of the World Health Organization (WHO) [1]. Each year there are 84,000-170,000 cases, which lead to 29,000-60,000 deaths. Primarily, YFV is transmitted by a bite from an infected Aedes aegypti mosquito and that gives rise to an acute viral hemorrhagic disease [2]. Yellow fever was first identified in 1647 in Barbados, an island in the Caribbean region [3]. Since then, infected people have carried the virus to other regions by global travelling. This disease is endemic in tropical areas of Africa and Central and South America. During the 17-19th centuries, yellow fever was transported to Europe and North America. Travelers who visited yellow fever endemic regions could bring the disease to non-infected areas. Thus, this disease is difficult to eradicate because of human activities and inevitable migration. Furthermore, YFV causes large outbreaks and incurs economic disruption, retrogression, and even decimated populations.
Yellow fever can be prevented by a vaccine, which provides efficacious immunity within 30 days [1]. Nevertheless, the mass vaccination still cannot ward off the potential outbreaks due to the resurgence of infected mosquitoes in heavily populated areas. Other factors such as a decreased immunity to the infection of individuals, ongoing deforestation, increasing people s movements/urbanization, and climate change also lead this infectious benzimidazole-coumarin conjugates were reported with anti-HCV activity [50]. In 2006, Ishida and co-workers [51] disclosed the benzimidazole derivatives as inhibitors for HCV NS5B polymerase. In 2008, Sparatore et al. [15] published benzimidazole-benzotriazole conjugates that showed moderate activity against YFV.
Some synthetic compounds with a dimeric and symmetric structure have been used as clinical drugs or drug candidates. These include brilacidin (an antibiotic and a COVID-19 drug candidate) [52], cromolyn (anti-asthma and anti-inflammation) [53], daclatasvir (an FDA-approved anti-HCV drug) [54], ombitasvir (anti-HCV) [55], pyrrolobenzodiazepine dimer (SJG-136, a phase 1 trial drug candidate for anti-tumor use) [56], and suramin (an essential medicine on the WHO's list to treat African sleeping sickness and river blindness). Similar to the structural characteristics of suramin, the design and synthesis of bis(benzofuran-thiazolidinone)s and bis(benzofuran-thiazinanone)s were published in 2017 [30]. These bis-heterocycles exhibit potency against chikungunya virus with a minimum EC50 of 1.9 μM and SI~75 or higher. Herein, we report two new series of dimeric compounds with the skeletons of bis(benzofuran-imidazolidinone) and bis(benzofuranbenzimidazole) as anti-YFV agents. Their design was on the basis of the scaffold of bis(benzofuran-1,3-thiazolidin-4-one) 1 as shown in Figure 1 [30]. Accordingly, these nitrogen-containing bis-conjugates 2 and 3 were synthesized, and their antiviral activities were explored.
The structures of all new bis-conjugates 2a-k were identified according to their spectroscopic data. For instance, the exact mass of compound 2e (C 41 H 40 N 4 O 4 ) was measured as 652.3052 for the species of M + , which is very close to its theoretical value of 652.3050. The formation of two imidazolidin-4-one rings in compound 2e was supported by its 1 H NMR spectrum. The two NCHN protons resonated as a singlet at 5.60 ppm; and the two sets of diastereotopic NCH 2 C=O protons resonated at δ 3.82 ppm as a doublet with J 2 = 15.0 Hz and at δ 3.42 ppm as a doublet with J 2 = 15.0 Hz [59]. The spectrum also showed the presence of the two methylene protons in the center of this compound, which resonated at δ 4.06 ppm. Two singlets at δ 2.44 and 2.20 ppm were associated with the methyl groups from the NCH 3 in the imidazolidin-4-one and those attached to the 3,5-positions of Nphenyl group, respectively. In its 13  use of molecular sieves in Et3N and toluene at 110 °C [58]. Finally, the desired conjugates 2a-k were produced as solids in good yields (75-87%) through dehydrative cyclization. The structures of all new bis-conjugates 2a-k were identified according to their spectroscopic data. For instance, the exact mass of compound 2e (C41H40N4O4) was measured as 652.3052 for the species of M + , which is very close to its theoretical value of 652.3050. The formation of two imidazolidin-4-one rings in compound 2e was supported by its 1 H NMR spectrum. The two NCHN protons resonated as a singlet at 5.60 ppm; and the two sets of diastereotopic NCH2C=O protons resonated at δ 3.82 ppm as a doublet with J 2 = 15.0 Hz and at δ 3.42 ppm as a doublet with J 2 = 15.0 Hz [59]. The spectrum also showed the presence of the two methylene protons in the center of this compound, which resonated at δ 4.06 ppm. Two singlets at δ 2.44 and 2.20 ppm were associated with the methyl groups from the NCH3 in the imidazolidin-4-one and those attached to the 3,5-positions of N-phenyl group, respectively. In its 13 C spectrum, the NC=O carbon and the NCN carbon resonated at 170.3 ppm and 78.2 ppm, respectively. A strong absorption band at 1714 cm -1 in its IR spectrum was corresponding to the amido C=O group.

Synthesis of Bis(Benzofuran-benzimidazole) Conjugates 3 (Scheme 2) and Their Structural Identification
For establishment of the bis(benzofuran-benzimidazole) scaffold in conjugates 3, an alternative synthetic approach was taken, as illustrated in Scheme 2. The bis-ester intermediate 10 was first prepared by coupling of bissalicyaldehyde 8 [60] with ethyl bromoacetate (9) in the presence of K2CO3 (s) and dry DMF at 120 °C [61]. This annulation reaction proceeded through a nucleophilic substitution, an intramolecular aldol condensation, and then a base-catalyzed dehydration. It gave bis(benzofuran-2-carboxylate) 10 with the desired skeleton as white crystals in 76% yield. In its 1 H NMR spectrum, the peaks related to the CH2Me protons of the ester group appeared as a quartet at 4.41 ppm with J 3 = 7.2 Hz and the peaks of the CCH3 proton appeared as a triplet at 1.39 ppm with J 3 = 7.2 Hz. In its 13 C NMR spectrum, the C=O carbon resonated at 159.4 ppm.

Synthesis of Bis(Benzofuran-Benzimidazole) Conjugates 3 () and Their Structural Identification
For establishment of the bis(benzofuran-benzimidazole) scaffold in conjugates 3, an alternative synthetic approach was taken, as illustrated in Scheme 2. The bis-ester intermediate 10 was first prepared by coupling of bissalicyaldehyde 8 [60] with ethyl bromoacetate (9) in the presence of K 2 CO 3 (s) and dry DMF at 120 • C [61]. This annulation reaction proceeded through a nucleophilic substitution, an intramolecular aldol condensation, and then a base-catalyzed dehydration. It gave bis(benzofuran-2-carboxylate) 10 with the desired skeleton as white crystals in 76% yield. In its 1 H NMR spectrum, the peaks related to the CH 2 Me protons of the ester group appeared as a quartet at 4.41 ppm with J 3 = 7.2 Hz and the peaks of the CCH 3 proton appeared as a triplet at 1.39 ppm with J 3 = 7.2 Hz. In its 13 C NMR spectrum, the C=O carbon resonated at 159.4 ppm.
Saponification of bis-ester 10 was then performed by use of aqueous KOH in THF and ethanol at room temperature [62]. The desired benzofuroic acid dimer 11 was obtained in 82% yield as white solids. Then, two benzimidazole moieties were planned to build on the flanks of bis-benzofuranic acid 11, as shown in Scheme 2. Meanwhile, the N-alkylated o-phenylenediamines 14a-l were prepared from the reaction of o-phenylenediamine 12 with substituted aldehydes and ketones 13a-l. This reductive amination was achieved in 70-80% yields by use of sodium borohydride in MeOH at 0-25 • C [63]. Subsequently, coupling of these diamines 14a-l with benzofuran acid 11 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), and N-methylmorpholine (NMM) in DMF led to the desired benzofuramide dimers 15a-l in 70-83% yields. Identification of their structures is exemplified by compound 15j. One characteristic singlet at δ 4.18 ppm in its 1 H NMR spectrum showed the presence of the methylene linker (i.e., ArCH 2 Ar) in the dimer. A multiplet occurred at δ 3.57-3.51 ppm, which was related to the protons of the two CHMe 2 groups. The peaks corresponding to the twelve protons from four methyl groups were observed at δ 1.22 ppm with J 2 = 6.0 Hz. In its 13 C NMR spectrum, the peak at δ 157.0 ppm was related to the two C=O carbons of amide groups; resonances that occurred at 45.4 and 22.9 ppm were due to the CMe 2 and the CH 3 carbons, respectively. A strong absorption band at 1660 cm -1 in its IR spectrum was attributed to the carbonyl stretching vibration in the amide groups.
Finally, the formation of benzimidazole moieties in the targets 3a-l was achieved by use of acetic acid, which functioned as the catalyst and the solvent [51]. The diamides 15a-l underwent cyclization followed by acid-catalyzed dehydration at 120 • C to give benzofuranbenzimdazoles 3a-l in 80-89% yields (Scheme 2). We confirmed their structures using conjugate 3j as an example. Its exact mass was measured as 565.2599 for (C 37 H 32 N 4 O 2 H) + , which is very close to its theoretical value of 565.2603. The 1 H NMR spectrum showed a multiplet at δ 5.43-5.36 ppm and one singlet at δ 4.20 ppm, which corresponded to the two protons from two CHMe 2 groups and two protons in the methylene linker of ArCH 2 Ar, respectively. The spectrum also exhibited a doublet at δ 1.71 ppm for twelve protons related to four methyl groups from two isopropyl groups. In its 13 C NMR spectrum, resonance occurred at δ 143.6 ppm corresponding to the N=C−N carbon of the benzimidazole ring; resonances occurred at δ 49.1 and 21.5 ppm corresponding to the CMe 2 and the CH 3 carbons, respectively. There were 18 peaks, which are in agreement with the theoretical number. Saponification of bis-ester 10 was then performed by use of aqueous KOH in THF and ethanol at room temperature [62]. The desired benzofuroic acid dimer 11 was obtained in 82% yield as white solids. Then, two benzimidazole moieties were planned to build on the flanks of bis-benzofuranic acid 11, as shown in Scheme 2. Meanwhile, the Nalkylated o-phenylenediamines 14a-l were prepared from the reaction of o-phenylenediamine 12 with substituted aldehydes and ketones 13a-l. This reductive amination was achieved in 70-80% yields by use of sodium borohydride in MeOH at 0-25 °C [63]. Subsequently, coupling of these diamines 14a-l with benzofuran acid 11 with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), and Nmethylmorpholine (NMM) in DMF led to the desired benzofuramide dimers 15a-l in 70-Scheme 2. Synthesis of bis(benzofuran-benzimidazole)s 3.

Biological Activities of the New Bis-Conjugated Compounds and Their Lipophilicity
The anti-YFV activity of all new bis-conjugates and their intermediates was evaluated by use of cytopathic effect (CPE) reduction assays [10,64] on YFV strain 17D (Stamaril) in Huh-7 cells. Their biological data, including 50% effective concentration (EC 50 ) and 50% cytotoxicity concentration (CC 50 ), are summarized in Table 1. The EC 50 value is the concentration of the test compound that inhibited the virus-induced cytopathic effect on the host cell by 50%. The CC 50 value is the calculated concentration of the test compound required to reduce the metabolic activity of compound-treated cells by 50%. The selectivity index (SI = CC 50 /EC 50 ) is a ratio between cytotoxicity and antiviral activity; it reflects the therapeutic window of the test compound in the assay system. Moreover, the molecular lipophilicity (quantified as log p) of bis-conjugates with promising potency against YFV was measured by use of the "shake-flask method" [65]. The lipophilicity of chemical compounds plays a vital role in the evaluation of drug candidates [66]. In general, the log p value between -0.4 and 5.6 is considered to be an ideal range for compounds as "drug-like" candidates [67].

Essential Moieties and Substituents for Anti-YFV Activity
Some of the new compounds, including 2c, 2e, 2k, and 3j, with five-membered 1,3-N,Nheterocycles (i.e., imidazolidinone and benzimidazole) attached to bisbenzofuran showed compelling EC 50 values against YFV. Their log p values shown in Table 1 fell into an ideal range for their development as drug leads. The anti-YFV activity of 1,3-imidazolidinone 2e had better efficacy than all other compounds in the same family and benzimidazoles 3.
The substituents attached to the nitrogen atoms were found of importance to determine the anti-YFV activity. Conjugates 2a-k bearing the N-Ph moiety with a dimethyl or CF 3 substituent were more potent than others in the series. When the substituent on the phenyl groups of conjugates was changed to monomethyl, isopropyl, methoxy, or a halogen substituent, the antiviral activity of conjugates 2 decreased or abolished completely. Among benzimidazoles 3a-l, compound 3j with an isopropyl substituent on the nitrogen atom was found to be the most active one in the series. When the substituents on the nitrogen atom of conjugates 3 was changed to cycloalkyl or a substituted phenyl group, their anti-YFV activity diminished (Table 1).
The three classes of conjugated compounds 1-3 share two common features. One is all of their infrastructures possessing two benzofuran moieties linked by a methylene unit. The other is that some compounds in all of these three classes possessed antiviral activities. Nevertheless, compounds 1 had a five-membered thiazolidinone ring, some of which exhibited activity against chikungunya virus [30]. When the sulfur atoms in the two five-membered heterocyclic rings were replaced by nitrogen atoms as shown in structures 2 and 3, some showed cogent activity against YFV but not chikungunya virus.

General Information
All of the bis-conjugated compounds (i.e., 2a-k and 3a-l) were synthesized and their analytical data are presented below. The intermediates 6a-k, 10, 11, 14a-l, and 15a-l were prepared according to the reported methods. Their synthesis and analytical data are provided in the Supplementary Materials. Commercially available solvents from Mallinckrodt Chemical Co., including dichloromethane, ethyl acetate, hexanes, and tetrahydrofuran (THF), were dried and distilled by the standard procedures. Other commercially available reagents were received and used without further purification. Compounds were purified by use of gravity column chromatography with Silicycle ultra-pure silica gel with particle sizes 40-63 µM and 230-400 mesh. Analytical thin layer chromatography (TLC) was performed on precoated plates with silica gel 60 F-254. Proton NMR spectra were recorded on 400 MHz and 700 MHz spectrometers by use of chloroform-d (CDCl 3 ), dimethyl sulfoxide-d 6 (DMSOd 6 ), and methanol-d 4 (CD 3 OD) as the solvents. The chemical shifts were referenced to residual protonated solvents (δ 7.24 for chloroform, δ 2.49 for DMSO-d 6 , and δ 3.31 ppm for methanol). Carbon-13 NMR spectra were recorded on 100 MHz spectrometers by use of chloroform-d (CDCl 3 ), dimethyl sulfoxide-d 6 (DMSO-d 6 ), and methanol-d 4 (CD 3 OD) as the solvents. The chemical shifts are referenced to the center of the CDCl 3 triplet (δ 77.0 ppm), DMSO-d 6 septet (δ 39.5 ppm), and CD 3 OD septet (δ 49.0 ppm). Multiplicities are recorded by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and J, coupling constant (hertz). Infrared spectra (IR) were recorded on a Fourier transform infrared spectrometer (FT-IR). Absorption intensities are recorded by the following abbreviations: s, strong; m, medium; and w, weak. High-resolution mass spectra were measured on an electrospray ionization (ESI) or electron impact ionization (EI) mass spectrometer (MS).

Synthesis of Bis(Benzofuran-Benzimidazole)s 3 (Standard Procedure 2)
The solution of diamides 15 (1.0 equiv) in AcOH (2.5-5.0 mL) was heated to 120 • C for 4.0 h under N 2 atmosphere. The cooled reaction mixture was neutralized with cold saturated aqueous NaHCO 3 solution (10-20 mL), which was extracted with EtOAc (3 × 30 mL). The combined organic extracts were washed with water (2 × 50 mL) and then brine (2 × 50 mL), dried over MgSO 4 (s), filtered, and concentrated under reduced pressure. The residue was purified by use of silica gel column chromatography with a mixture of EtOAc and hexanes as the eluent to give bis(benzofuran-benzimidazole)s 3.

Biological Evaluation
The antiviral activity of the test compounds against YFV strain 17D (Stamaril) on Huh-7 cells was determined by use of a CPE-based assay. Their potential cytotoxic effects were evaluated in the uninfected host cells. For details to obtain the 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (CC 50 ), see the descriptions in the published papers of ours [10,64].

Study Plans of QSAR
The quantitative structure-activity relationship (QSAR) provides useful information for the design of new drugs. It involves a machine-learning process that derives the association between independent variables (molecular descriptors and structural features of compounds) and dependent variables. The 2D-QSARs of benzofuran derivatives as vasodilators were reported by Srour et al. [68]. They utilized the CODDESSA PRO program to achieve a robust model, which revealed biological activity of benzofurans. This program has been established by A. R. Katritzky, M. Karelson, and R. Petrukhin of the University of Florida, 2001-2005 [69]. The results supported the applicability of their synthesized benzofuran-based hybrids as potential vasorelaxant active agents. Similar QSAR studies to be performed on our currently synthesized bis-benzofuran derivatives could derive even more potent anti-YFV agents. We shall apply a QSAR mode first to create a relationship between chemical structures and their anti-YFV activity in a data-set of finite drug leads 2 and 3. Then QSAR models will be used to design new bis(benzofuran-1,3-imidazolidin-4-one)s and bis(benzofuran-1,3-benzimidazole)s with various N-substituents and predict their activities. The methods for QSAR analyses are essentially statistical methods. Nevertheless, the CODDESSA PRO program requires a number of descriptors in different families: 8 constitutional, 12 topological, 8 geometrical, 14 electrostatic, 40 CPSA, 7 MO related, 18 quantum chemical, and 7 thermodynamic groups. Its approach differs from the fragment (or group contribution) approach. The descriptors are computed for the system as a whole rather than from the properties of individual fragments. The anti-YFV activity of new bis-conjugates will then be expressed quantitatively as the concentration of a substance required to inhibit the virus.
On the other hand, the prediction of the partition coefficient log p is of special significance. It is an important measure used during the identification of "druglikeness" according to Lipinski s Rule of Five [70]. The log p values can be estimated by chemical fragment methods, which are related to the N-substituents to be placed in the bis-conjugates 2 and 3.
The fragmentary values to be determined will be based on the log p values shown in the last column of Table 1 for the reporting compounds. An advanced approach on fragment-based QSAR can strengthen the concept of pharmacophore similarity and will be developed by use of the data associated with the reporting bis-conjugates. This pharmacophoresimilarity-based QSAR method involves the use of topological pharmacophoric descriptors. Property prediction will assist the contribution of pharmacophore features encoded by the benzofuran, imidazolidinone, and benzimidazole moieties toward biological activity improvement [71]. These complex and useful QSAR analyses will be performed in due course.

Conclusions
We designed and synthesized two series of bis(benzofuran-1,3-N,N-heterocycle) conjugated compounds. Among 23 new conjugates, 4 (i.e., 2c, 2e, 2k, and 3j) inhibited YFV strain 17D (Stamaril) on Huh-7 cells with EC 50 values of 3.54-8.89 µM. The most appealing SI values associated with these conjugates were in the range of 6.07-15.3. Moreover, their structure-activity relationship was illustrated. Attachment of imidazolidinone or benzimidazole moieties to the bisbenzofuran nuclei greatly increased the YFV inhibition. The substituents on the N-atoms in the imidazolidinone and benzimidazole moieties influenced their anti-YFV activities. These findings provide clues for such types of dimeric compounds to be developed as antiviral agents.