Antiviral Profiling of C-18- or C-19-Functionalized Semisynthetic Abietane Diterpenoids

Viral infections affect several million patients annually. Although hundreds of viruses are known to be pathogenic, only a few can be treated in the clinic with available antiviral drugs. Naturally based pharmacotherapy may be a proper alternative for treating viral diseases. Several natural and semisynthetic abietane-type diterpenoids have shown important antiviral activities. In this study, a biological evaluation of a number of either C-18- or C-19-functionalized known semisynthetic abietanes against Zika virus, Dengue virus, Herpes virus simplextype 1, and Chikungunya virus are reported. Semisynthetic abietane ferruginol and its analogue 18-(phthalimid-2-yl)ferruginol displayed broad-spectrum antiviral properties. The scale-up synthesis of this analogue has been optimized for further studies and development. This molecule displayed an EC50 between 5.0 and 10.0 μM against Colombian Zika virus strains and EC50 = 9.8 μM against Chikungunya virus. Knowing that this ferruginol analogue is also active against Dengue virus type 2 (EC50 = 1.4 μM, DENV-2), we can conclude that this compound is a promising broad-spectrum antiviral agent paving the way for the development of novel antivirals.

Adapted from Malkowsky and co-workers. 1 A solution of compound 27 from previous step (24.9 g, ca. 42 mmol) in DCM (300 mL) was cooled in an ice-bath and AcCl (10.45 mL, 11.53 g, 147 mmol, 3.5 equiv.) was added followed by AlCl3 (16.8 g, 126 mmol, 3.0 equiv.). The reaction mixture became from yellowish to dark brown and stirred for 20 min. Then, the ice-bath was S4 removed and the reaction was stirred for 29 h at rt. After this time, the resulting brownish-red solution was cooled in an ice-bath and quenched dropwise in a beaker with saturated aq. NaHCO3 (100 mL) (Gas evolution!). The mixture was poured onto saturated aq. NaHCO3 (200 mL) in a 1L separation funnel and the phases were separated. Care must be taken with continuous opening of the tap of the funnel to avoid CO2 pressure. The aqueous phase was extracted with DCM (2 × 100 mL). The combined organic phases were washed with H2O (100 mL) and brine (50 mL), dried (MgSO4) under stirring overnight. Next day, the extract was filtered and concentrated to give 29.2 g of yellowish brown semi-solid which was crystallized with EtOH (90 mL) overnight. The resulting greenish solid was filtered off under vacuum and washed with cold EtOH (60 mL) and dried under vacuum to give 16.1 g of acetyl derivative 28 as a pale greenish solid (84%, two steps), which had 1 H and 13 C NMR data in agreement with those reported. 1 From the mother liquor were recovered, after chromatography on silica eluting with n-hexane-EtOAc (8:2), additional 4.8 g of product as a yellow solid. 1
Adapted from Malkowsky and co-workers. 1 Compound 28 (20.7 g, 45.7 mmol) and metachloroperbenzoic acid (MCPBA, 27.3 g, 118.9 mmol, 2.6 equiv.) were dissolved in DCM (125 mL) and cooled in an ice-bath. Then, trifluoroacetic acid (3.5 mL, 5.2 g, 45.7 mmol, 1.0 equiv.) was added dropwise and the mixture was stirred for 20 min. before allowed to warm to rt and stirring continued for 23h. Next day, the reaction mixture was diluted with DCM (80 mL) and quenched with 10% aq. Na2SO3 (120 mL). Phases were separated in a 1L separation funnel and the aqueous phase was extracted with DCM (60 mL). The combined organic phases were washed with H2O (100 mL + 10 mL of brine), 50% saturated aq. NaHCO3 (2 × 125 mL), and brine (100 mL), dried over MgSO4 under stirring for 30 min., filtered and concentrated to give a crude of 23 g as a pale oil. The crude was chromatographed on silica eluting with n-hexane-EtOAc (7:3) to give 16.9 g (80%) of acetate 29 as a yellow foam, which had 1 H and 13 C NMR data in agreement with those reported: 1   The combined organic phases were dried over MgSO4 under stirring overnight. Next day, it was filtered and concentrated to give a crude of 9.6 g as a yellow solid. The crude was chromatographed on silica eluting with n-hexane-EtOAc (7:3) to give 8.94 g (90%) of phenol 6 as a yellow foam, which had 1 H and 13

Reagents and compounds
Dulbecco

End-point titration technique (EPTT).
The technique EPTT (Vlietinck et al., 1995), 4  h for Zika _459148 and CHIK virus; fixation and staining were performed, as previously described for the plaque forming unit (PFU) assay. Viral plaque counting was performed in 3 independent assays to determine the inhibition of plaque formation. The effective concentration (EC50) was defined as the concentration that reduces viral plaques by 50%, and graphically determined from the corresponding concentration-response curve.
The antiviral effect of the most effective compound por Zika was confirmed with other viral strain (ZIKA/Col, isolated from serum from a patient infected during the 2015 epidemic in Colombia 5 S13 by quantification of infectious viral particles in supernatants; and genome and viral protein in monolayers obtained from cells treated in a post-infection stage evaluation assay with that strain. 6

Quantification of infectious viral particles by plaque assay
Serial dilutions of the supernatants were inoculated for 2 h on monolayers of Vero cells (1.2 × 10 4 /well). Afterwards, the viral inoculum was removed, and 1.5% carboxymethylcellulose (Sigma-Aldrich) was added. The monolayers were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with crystal violet after 6 days of incubation. The plaques were then counted and the results were expressed as plaque-forming units per milliliter (PFU/mL). 7

Quantification of intracellular viral genome by qPCR
RNA was extracted from the infected and treated monolayers following the manufacturer's instructions (Zymo Quick-RNA Viral Kit). The qRT-PCR was performed from 500 ng of RNA using the qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (QuantaBio) using previously described primers (1086 and 1162c with a 1107-FAB probe) that targeted a region of the envelope protein gen. 5 The samples were amplified in a QuantStudio 3 thermocycler (Thermo Scientific®) and the thermal profile used was 1 cycle at 50 °C for 2 min, 1 cycle at 95°C for 2 min, 40 cycles at 95°C for 15 s and 60°C for 1 min. For the absolute quantification of the genomic copies, we used standard curves of plasmids previously constructed. 8

Intracellular quantification of viral protein by Cell-ELISA
The monolayers were fixed with paraformaldehyde 4% and then permeabilized with Triton X-100

III) Molecular Docking Methodology
Molecular docking is a computational tool in which the affinity energies in a protein-ligand complex are measured. 10 This in silico study is used for a virtual screening of a list of molecules S15 with possible therapeutic effects thus facilitating the identification of active principles in proteins.
Three compounds ferruginol (5), 13 compound 6 1,14 and methyl 14-hydroxy-dehydroabietate (24) 15 ( Figure S1) were evaluated against cellular targets in post-infection stage with inhibitors reported in the literature, to compare results, such as Rhoa (5c4m) 16 and its inhibitor rhosin, 17 β-tubulin subunit (1JFF) 18 and its inhibitor taxol, 19 G-actin (3EKS) 20 and its inhibitor cythochalasin, 21 PI3KY (1E7U) and its inhibitor wortmannin, 22 Arp2/3 complex (3UKR) and its inhibitor CK-666, 23 Cdc42 (1KI1) 24 and its inhibitor ZCL278 25 and the last cellular target RAC1 (3TH1) 26 and its inhibitor NCS23766. 27 The above cellular targets are responsible for the translation of viral proteins and cytoskeleton rearrangement. For dengue virus the following non-structural proteins NS5 rdrp (3VWS) 28 and its inhibitor NITD-107, 29 NS5 methyltransferase (3P97) and its inhibitor SAH analogue, 30 NS3 helicase (2BMF) 31 without reported inhibitor and NS3 serine protease (3U1I) 32 and its inhibitor Bz-NKKR-H. 33 For Zika virus, the following non-structural proteins NS5 rdrp (5WZ3) 34 and its inhibitor PSI-7409, 35 NS5 methyltransferase (5M5B) 36 and its inhibitor 36A, 37 NS3 helicase (5JPS) 38 and its inhibitor GTPγ-S 39 and last NS3 serine protease (5H4I) 40 and its inhibitor Benzimidazol-1-γlmetanol 41 were selected. For chikungunya virus, the protease nsP2 (3TRK) with its inhibitor FT-0679525 42 was studied and for herpes virus type 1 ( HHV-1) TK (4IVP) and its inhibitor acyclovir. 43 All binding sites depend on the chemical characteristics and are widely explained in the literature for each viral nonstructural protein and cellular target. A screening of binding pockets was performed and the corroborated ones with high binding energies S16 were selected. The procedure followed for the treatment of the viral protein was the identification of the active sites by means of a study of the bibliography, treatment of the viral proteins in the UCSF chimera 1.12 software 44 eliminating inactive chains, solvents and cofactors that affect the selectivity of the docking process. Later, using autodock tools 1.5.7 software, we added to the viral protein the type of atom, polar hydrogens and the gasteiger charges and finally, it was changed the format from .pdb to .pdbqt. Using the avogadro software 45 the pH is set to 7.4 and the energy is reduced to a minimum, thus guaranteeing a 3D structure suitable for molecular docking. The ligand is then treated using autodock tools 1.5.7 where polar hydrogens, rotatable bonds and the torsion point are added, and finally converted to .pdbqt format. With autodock vina 1.12 software, 46 the docking was performed and the affinity energies between the studied ligands ( Fig. S1) are measured and the results are tabulated in Table S1. -7.5 -6.8 -7.1 ZCL278 (-6.5) Rac1 (3th5) -6.9 -6.3 -6.0 NCS23766 (-6.6) Figure S1. Studied molecules by molecular docking.