Molecular Dynamic Study and Synthesis of 1H-benzo[ d ]imidazole-5-carboxamide Derivatives as Inhibitors for Yellow Fever and Zika Virus Replication (cid:10)

Flaviviridae family comprises the flavivirus genotype that represents a significant world health problem as it includes the Yellow fever virus (YFV) and Zika virus (ZIKV) which are responsible for large outbreaks and for which novel therapies are in urgent demand. The benzimidazole scaffold has been widely reported for its antiviral activity, and hence a new series of 1H-benzo[ d ]imidazole-5-carboxamide derivatives (VIIa-x, VIIIa-h & IXa, b) was designed, synthesized, and biologically evaluated for their antiviral activity. Five Compounds ( VIId , VIIe , VIIh , VIIn , and VIIt) showed antiviral activity against YFV in the low micromolar range using the human hepatoma Huh-7 cells and Vero cells. One compound (VIId) exhibited activity on both YFV (EC 50 = 1.7±0.8 µM) and ZIKV (EC 50 = 4.5±2.1 µM). Molecular docking and molecular dynamics simulation studies were conducted to understand the SAR of newly synthesized compounds, to explore the potential target of compound VIId, and to investigate the possible binding mode to its target. (cid:10)


INTRODUCTION
The Flaviviridae family comprises a large number of genera representing a major cause of public health problems, mainly the Hepacivirus genus, the Flavivirus genus, and the Pestivirus genus Flaviviruses began to spread about 75,000 years ago due to human migration from Africa [4]. According to the world health organization (WHO), 200,000 new YFV cases occur annually with outbreaks recorded in 2016-2017 in several countries in Africa and the Americas [5-7]. As for ZIKV, an estimated 440,000-1,300,000 cases were recorded in late 2015 during the Brazilian outbreak, and as a result of an increase in ZIKV-associated microcephaly cases in 2016, the WHO declared ZIKV a Public Health Emergency of International Concern [8,9].
Despite the availability of a highly efficacious YFV vaccine (17D vaccine) the recent large YFV outbreaks clearly show that antivirals against YFV and all flaviviruses are desperately needed. Aiming to decrease the viremia during early infection and block the viral replication and growth, various viral proteins are targeted [10]. The flaviviral proteins are classified into structural and non-structural proteins. The structural proteins include the capsid protein (C), the envelope protein (E), and the membrane protein (M) [11][12][13]. The non-structural proteins include: NS1, NS2A, NS2B, NS3 (serine protease/ helicase enzyme), NS4A, NS4B, NS5 (methyltransferase/ polymerase enzyme) [14 -19].
Various benzimidazole-based compounds were reported to be active leads in the search for new antiviral agents acting against Flaviviridae family members especially yellow fever virus and HCV (Fig. 1) [32][33][34][35].

Fig.1. Benzimidazole-based reported anti-Flaviviridae compounds
Herein, a variety of 1H-benzo[d]imidazole-5carboxamide derivatives (VIIa-x, VIIIa-h & IXa, b) were synthesized and biologically evaluated for their anti-YFV activity. Besides, molecular modeling studies (docking and molecular dynamics) of compound VIId which exhibited antiviral activity against both YFV (on Huh-7 and VeroA cells) and ZIKV were conducted to gain insight into its antiviral mechanism. These studies suggested that VIId may be a ZIKV polymerase inhibitor.

Fig. 2. Benzimidazole-based compounds showing activity against Flaviviridae family
The first series (2-naphthyl benzimidazoles) evaluated the effect of different substituents at positions 5 and 6 of the benzimidazole ring, nearly 3 compounds (1-3) showed activity on YFV.
As for the third series, (styrylbenzimidazoles), the phenyl ring was separated from the benzimidazole nucleus by an ethylene spacer, and compounds with various substituents at position 5, 6 of the benzimidazole nucleus and the phenyl ring were tested. All compounds from this series showed no activity against YFV. Compound 5 displayed the highest antiviral activity against BVDV among all active derivatives.
The fourth series (5-acetyl-2arylbenzimidazoles) showed the best anti-Flaviviridae activity with about 9 compounds showing antiviral activity ranging between 0.8-8 µM on YFV, BVDV, and HCV representing the three genera of Flaviviridae family (Flavivirus, Pestivirus, and Hepacivirus) respectively. SAR studies within the reported compounds demonstrated that: 1) 5-Acetyl group improved the anti-BVDV and anti-YFV activity due to its electronegativity.
3) Cyclohexyl substituent introduction at position 1 improved the anti-YFV activity better than the unsubstituted derivative and that was obvious in compounds 8 and 9.
Based on the SAR study of the previously reported compounds, in the present study, we were able to design, synthesize and biologically evaluate novel series of benzimidazole derivatives targeting the Flaviviridae family (especially YFV and ZIKV).
Our structure-based design strategy was based on:

1)
Keeping the benzimidazole nucleus which is beneficial for anti-viral activity.

2)
Keeping the cyclohexyl ring in the N1 position.

3)
Exploration of various substituents on the 2-phenyl ring compared to the unsubstituted derivatives.

4)
Exploration of various substituents on 5 positions of benzimidazole ring.
Accordingly, three series of compounds were designed based on the modification of the lead compound 4, which possesses high potency to be synthesized and biologically evaluated against YFV and ZIKV as shown in (Fig. 3).

Chemistry
Starting materials and reagents were purchased from Sigma-Aldrich (USA), Loba Chemie (India), and Alfa-Aesar organics and used without further purification. Solvents were purchased from Fisher Scientific and Sigma-Aldrich and utilized after drying. Reactions were monitored by analytical TLC, performed on silica gel 60 F 254 packed on Aluminum sheets, purchased from Merck, visualized under U.V. light (254 nm). For flash chromatography, Merck silica gel 60 was used with a particle size (230-400 mesh). Melting points were measured on the Stuart Scientific apparatus. 1 HNMR spectra were recorded in δ scale given in ppm on a Bruker 400 MHz and referred to TMS as an internal reference and 13 CNMR spectra were recorded on a Bruker 101 MHz at the Center of Drug Discovery and Research Development, Ain Shams University. EI-MS spectra and Elemental analyses were performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University.

General procedure to prepare Intermediates I-III
The starting compound (4-chlorobenzoic acid) was purchased from Sigma-Aldrich (USA) and used as it is. 4-Chlorobenzoic acid (6.24 g, 40 mmol) was dissolved in 20 mL concentrated sulfuric acid portion-wise in an ice bath at 0 °C, then 20 mL of concentrated nitric acid was added dropwise for 30 minutes at 0 °C. The reaction mixture was left to stir at room temperature for 3 h, then poured dropwise on ice to give a white precipitate. The precipitate was filtered and washed with excess water to get rid of excess acids and left to dry to give the titled compound I as a white powder. To a solution of compound 1 (6 g, 29.8 mmol) in 48 mL ethanol, 6 mL of concentrated sulfuric acid was added dropwise while in an ice bath at 0 °C. Then the reaction mixture was stirred at 70 °C for 24 h. After the reaction was completed, it was poured on ice/water to give an off-white precipitate. The precipitate was neutralized with sodium carbonate then filtered and washed with excess water and left to dry. The product was crystallized from ethanol to give the titled compound II as an off-white powder. For the preparation of compound III, cyclohexylamine (9.35 mL, 93.3 mmol, 3 equivalents) and triethylamine (9.3 g) were added to a solution of compound II (7.14 g, 31.1 mmol, 1 equivalent) in DMSO (23 mL). The reaction mixture was then stirred at 90 °C for 4 h. After completion of the reaction, the reaction mixture was poured dropwise on ice with vigorous stirring to give a yellow precipitate. The precipitate was filtered and washed with water and diethyl ether, then left to dry and then crystallized from n-hexane to yield the titled compound III as yellow solid. (39)(40)(41)

General procedure to prepare Intermediate IV
A solution of Ethyl 4-(cyclohexyl amino)-3nitrobenzoate III (2 g, 6.8 mmol) and 10% Pd/C (0.2 g) in ethyl acetate (100 mL) were placed in a parr bottle and the bottle was placed in the parr hydrogenator apparatus, flushed 3 times with hydrogen and filled with hydrogen (40 psi

General procedure to prepare compounds (VIIa-x)
A suspension of the appropriate acid derivative VIa-e (3.14 mmol, 1 equivalent) in dry DCM (15 mL) was cooled to 0 °C in an ice bath and thionyl chloride (5 mL, 4 equivalent) was added dropwise with stirring, the reaction mixture was stirred under reflux for 2-4 h. The solvent was then evaporated under vacuum giving brownish solid of the respective acid chloride that was used directly without further purification.

Biological Evaluation
The antiviral activity against both YFV and ZIKV was determined using a CPE-based assay. For YFV assay using Huh-7 cells, cells were seeded in 96-well plates at a density 5.5 × 103 cells/well in 100 µL culture medium: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 2% HEPES. Cells could adhere overnight in a CO 2 incubator at 37 °C. The next day, compounds were serially diluted in the culture medium. 100 µl/well of YFV diluted in assay medium (DMEM supplemented with 2% FCS, 1% NEAA, and 2% HEPES) was added to the 96-well plates, after which plates were incubated for 4 days at 37 °C. For YFV assays using Vero cells, cells were seeded at a density of 2 × 104 cells/well in 100 µL culture media (MEM Rega3, 10% FBS, 2 mM L-glutamine, and 0.075% sodium bicarbonate) in 96-well plates. The next day, the culture medium was removed and 100 µL/well of assay medium (same as culture medium except that 10% FBS was replaced by 2% FBS) containing serial dilution of the compounds was added to the 96-well plates followed by an incubation period of 7 days. A potential toxic effect on the host cells was tested in parallel assays using the same protocol except that virus infection was omitted. In both antiviral and metabolic assays, the colorimetric readout was performed by using the MTS/PMS method, as described previously. [44] CPE-based assays for ZIKV were performed as described previously [45]. The 50% effective concentration (EC 50 ; the compound concentration that is required to protect 50% of the cells from virus-induced CPE) and the 50% cytotoxic concentration (CC 50 ; the concentration that reduces the number of viable cells by 50%) were determined using logarithmic interpolation, as described previously [44].

Molecular Docking
Molecular docking was performed using AutoDock Vina version 1.1.2 [46]. First, the protein (PDB ID: 5WZ3) was prepared using Discovery Studio 2.5 protein preparation protocol, and missing loops were added. Both protein and ligand files were converted to AutoDock's PDBQT format. The XYZ coordinates were chosen to be 24.1, 72.6, and 130.1, respectively. An equal length of X, Y, and Z axes was chosen to be 15 angstroms. A random seed number and standard exhaustiveness were used. Nine poses were generated where the best pose showed a binding affinity of -5.6 kcal/mol.

Molecular Dynamics Simulations
Molecular dynamics simulations were performed using Sybyl-X molecular modeling software. The simulation length was adjusted to 500 ps. The time step was set to 2 fs. An NTV ensemble was used with a temperature adjusted to 310 K. Boltzmann distribution was used to calculate the initial velocities and a random seed was used. At the end of the simulation, the total energy vs. time plot was generated to visualize energy changes. Molecular graphics of the docked poses and molecular dynamics were generated using Maestro visualizer.

Chemistry
The designed compounds were synthesized according to the chemical pathways outlined in schemes 1 and 2. Finally, the reductive cyclization method was used in the presence of sodium dithionite as a reducing agent for the nitro group followed by cyclization using the appropriate aldehyde in a one-pot reaction [42] to afford the ethyl 1- via catalytic hydrogenation using palladium on activated charcoal (Pd/C) and hydrogenator [43] to give Ethyl 3-amino-4-(cyclohexylamine) benzoate (IV) followed by its condensation with 4-nitro benzaldehyde in the presence of sodium acetate in ethanol [47] to give the ethyl 1- The preparation of the targeted 1-cyclohexyl- Eventually, synthesis of amides directly from esters and amines has been utilized for the preparation of the final two derivatives (IX a, b) using Na metal in absolute ethanol to prepare sodium ethoxide which was added immediately to compound Ve and the corresponding amines.  Compounds Va and VIa bearing the ester and acid moieties respectively showed no YFV inhibition with EC 50 >100 µm.
So, modification of the 5-carboxylate ester and 5-carboxylic acid groups into the amide group potentiated the anti-YFV activity.
Compounds VIIa-j with unsubstituted 2phenyl showed the best anti-YFV activity with 5 compounds (out of 10 compounds) exhibiting EC 50 values below 3.5 µM.
Compounds VIIIe-h with the 2-(4hydroxyphenyl) group were the most potent compounds with YFV EC 50 values of below 3µM.
Further investigation among the previously obtained results revealed: In compounds (VIIa-j) (unsubstituted 2phenyl) substitutions on the amidic N with phenyl ring bearing electron-withdrawing group as nitro, chloro, and fluoro groups at the para position in compounds VIId, VIIe and VIIh, respectively, showed very good anti-YFV activity (huh-7 cells) with YFV EC 50 values of 1.7±0.8, 2.4 ± 0.7 and 3.5±1.6 µM, respectively. Cyclic aliphatic cyclohexyl substitution in compound VIIa also showed the same good activity with YFV (huh-7 cells) EC 50 value of 2.5 ±1.3 µM. The other aromatic substitutions as phenyl, benzyl, and phenethyl in compounds VIIb, VIIc, and VIIf, respectively, and the aliphatic substitution, isobutyl and butyl chains in compounds VIIg and VIIj, respectively, showed poor anti-YFV activity.
In compounds (VIIk-p) (2-(4methoxyphenyl) group), substitutions on the amidic N with phenyl ring bearing the nitro electron-withdrawing group at the para position in compound VIIn and the aliphatic cyclohexyl substitution in compound VIIk showed very good anti-YFV activity with YFV EC 50 values of 2.1±0.6 and 3.0±1.4 µM, respectively. The other aromatic substitutions as benzyl and phenyl in compounds VIIm and VIIl, respectively, showed poor anti-YFV activity. While the aliphatic isobutyl and cyclopropyl substituents in compounds VIIo and VIIp, respectively, showed no anti-YFV activity.
In compounds (VIIIa-d), compound VIIIc with the 6-Chlorobenzo[d]thiazol substitution showed poor anti-YFV activity, and compounds VIIIa, VIIIb and VIIId showed no anti-YFV activity Compounds (VIIIe-h) with the 2-(4hydroxyphenyl) group were the most active compounds. Both para-substitution on the Nsubstituted phenyl ring with the nitro electronwithdrawing group in compound VIIIh and the cyclic aliphatic cyclohexyl substitution in compound VIIIe showed very good anti-YFV activity with YFV EC 50 values of 1.3±0.2 and 1.6±0.2 µM, aromatic substitutions as benzyl and phenyl in compounds VIIIg and VIIIf, respectively also showed very good anti-YFV activity with YFV EC 50 values of 1.6±0.7 and 3.0±1.7 µM, respectively.
In an attempt to elucidate a SAR study from the previously discussed results, it was clear that the nature of the amidic-N substitution contributes the most to the biological activity, the phenyl ring (substituted with electronwithdrawing groups on its para-position) and the cyclohexyl ring were the most active ones. The variations in the 2-position substituents did not influence the activity much except for the 4-OH phenyl substitution.

Molecular Modelling
To understand the interesting SAR of our compound series, we conducted different molecular modeling experiments. The goal of our experiments was to explore the potential target of compound VIId which showed very good activity against YFV (Huh-7 and Vero cells) and also against ZIKV VeroE6 cells and to investigate the possible binding mode to its target.
As has been mentioned, the design rationale relied on retaining the benzimidazole scaffold and exploring various substituents on the phenyl ring to improve activity against members of the Flaviviridae family. An important observation is that the synthesized compound series has a high structural similarity with CMF, a potent HCV NS5b inhibitor reported by Di Marco et al. [51].
The structures show scaffold similarity differing only in one extra nitrogen atom within the benzimidazole nucleus of compound VIId. Both CMF and compound VIId contain phenyl and cyclohexyl on the fused bicyclic nucleus. The major differences between both compounds are an extra morpholine amide found in the CMF structure, and compound VIId contains an amide substituent on the benzimidazole nucleus instead of the carboxylic acid found in CMF. Fig. 4. shows the field alignment of key features of CMF and compound VIId. From the previous observations, and due to high structural conservancy among the proteins of the Flaviviridae family, the Zika virus NS5 polymerase was predicted to be a potential target of compound VIId [52,53]. We conducted several molecular modeling experiments to predict the potential binding mode of compound VIId to Zika virus NS5 polymerase. It is worth mentioning that no co-crystal structure of the Zika virus NS5 polymerase and an inhibitor had been determined yet.

Molecular Docking
Due to the high similarity between CMF and compound VIId, we anticipated the co-crystal structure between CMF and HCV NS5b (PDB ID: 2BRK) as a good starting point to explore the potential target and its binding to compound VIId [51] Fig. 5 shows the 2D interactions between CMF and HCV NS5b.

Fig. 5. 2D representation of the interactions between CMF and HCV NS5b (PDB ID: 2BRK)
By examining this crystal structure, it is observed that CMF binds to an allosteric site in the thumb domain of HCV NS5b, this binding relies mainly on Van der Waal's forces with hydrophobic residues. The only charge interaction found is between Arg503 and the carboxylate of CMF. The morpholine amide of CMF does not contribute to binding but protrudes towards the solvent (Fig. 5). molecular docking was utilized to study the possible binding mode of compound VIId to Zika virus NS5 polymerase. Zika virus NS5 crystal structure was prepared (PDB ID: 5WZ3) and docking of compound VIId was performed using AutoDock Vina molecular docking software. The best binding pose showed a binding affinity of -5.6 kcal/mol. Compound VIId occupied a groove within the thumb domain of the protein where the phenyl and cyclohexyl substituents showed hydrophobic contacts with residues at the tip of the priming loop. The nitrotolyl moiety showed pi-pi stacking with Tyr885 as shown in (Fig. 6).
This binding mode gives insight into key features that improve the binding and activity of the tested compound. Pi-pi stacking with Tyr885 is strengthened with electron-withdrawing groups as the p-nitro group found in compound VIId. Compound binding depends mainly on hydrophobic contacts with the side chains of Val787, Asp810, Met883, Asp884, and Tyr885. This is also observed to be the driving force for the binding of CMF with HCV NS5b [51].
This binding mode also suggests a possible mechanism of action for compound VIId by allosterically influencing the geometry of the binding site. It was observed that VIId forms hydrophobic contact with Val787 and Asp810. These two residues are at the tip of the priming loop of the protein which extends from Val785 to Asp810 (Fig. 6). The dynamics of this loop is crucial for the activity of the NS5 protein as it regulates the allosteric positioning of the 3' terminus of the RNA template at the active site. [52] Binding of VIId to the tip of the priming loop could lead to a dynamic cascade which leads to disruption of the priming loop and active site deformity. To test this hypothesis, we conducted molecular dynamics simulations. VIId bound to it using Sybyl-X molecular modeling software. The simulation showed the stabilization of total energy after 500 ps (Fig. 7).
The priming loop was shown to have been moved significantly throughout the simulation showing an RSMD of 9.59 when compared to its conformation in the protein crystal structure (Fig.   8). This significant motility of the priming loop upon binding of VIId could explain the inhibitory mechanism of VIId. SAR study among the compounds showed that modification of the 5-carboxylate ester and 5-carboxylic acid groups into amide group potentiated the anti-YFV activity, Variation of 2position substituents between phenyl, 4-methoxy phenyl, 2-methoxy phenyl, 4-hydroxy phenyl, and 4-nitro phenyl didn't influence too much in the anti-YFV activity except 4-hydroxy phenyl derivatives (VIIIe-h) which demonstrated very good activity. Variation of 5-position carboxamide derivatives had a bigger influence on the anti-YFV activity. The variation of the anti-YFV activity of the carboxamide derivatives depends on the nature of the N-substitution.