1 ′ -Homocarbocyclic Nucleoside Analogs with an Optically Active Substituted Bicyclo[2.2.1]Heptane Scaffold †

: An optically active bicyclo[2.2.0]heptane fragment was introduced in the molecule of new 1 ′ -homonucleosides on a 2- 6-chloro-amino-purine scaffold to obtain 6-substituted carbocyclicnu-cleozide analogs as antiviral compounds. The synthesis was realized by a Mitsunobu reaction of the base with the corresponding bicyclo[2.2.0]heptane intermediate, and then the nucleoside analogs were obtained by substitution of the 6-chlorime with selected pharmaceutically accepted amines. A molecular docking study of the compounds on influenza, HSV and low active coronavirus was realized. Experimental screening of the compounds on the same viruses is being developed and soon will be finished.


Introduction
Nucleosides are a recognized class of antiviral and anticancer drugs. The resistance acquired in time and the toxicity are the main factors that motivated the discovery of new more active and selective analogs. The modifications were realized on the nucleobase and/or on the sugar moiety. With guanine and 2-amino-6-substituted purine as nucleobase, recognized carbocyclic nucleoside drugs or active compounds studied in different clinical phases, like carbovir and its prodrug abacavir (with 6-cyclopropylamino substituent), entecavir, lobucavir and cyclohexenyl G, and also recognized acyclic nucleosides like acyclovir, ganciclovir, penciclovir and their valine esters valaciclovir, valganciclovir and famciclovir became recognized as milestone compounds in the treatment of antiviral and anticancer diseases [1,2].

Results
Synthesis of the new 1′-homocarbanucleosides with an optically active bicyclo[2.2.1]heptane fragment as sugar moiety and guanine, 6-O-alkyl-guanine, 2,6-diaminopurine and 2-amino-6-N-substitutedpurine as nucleobase started from diol 2, was obtained crystallized by NaBH4 reduction of the keto-compound 1 [3]. The unprotected diol 2 was used in the Mitsunobu reaction with 2-amino-6-chloropurine, taking into account that the primary alcohol will react more quickly than the secondary one. Indeed, the primary alcohol reacted selectively to give the key nucleoside intermediate 5G isolated by simple crystallization in 71.3% yield (Scheme 1). For comparison, 6-chloropurine reacted with diol 2 in the same Mitsunobu reaction in 67.6% yield [4,5].
The following 1′-homocarbocyclic nucleosides were synthesized by substitution of the chlorine atom with ammonia, with primary or secondary pharmacological amines, with methoxide or ethoxide or by substitution with hydroxyl (acid hydrolysis) to the guanine, all in good yield [6]. Twenty-four new compounds were obtained, fully characterized and used for antiviral screening against influenza, herpes simplex virus and low active coronavirus.
A molecular docking study was realized using CLC Drug Discovery Workbench Software, on 24 compounds to obtain accurate predictions about the structure and interactions of the studied compounds in complex with a protein/enzyme receptor to evaluate the biological activity.
Docking evaluation against herpes simplex type-1 thymidine kinase: docking studies were performed to achieve accurate predictions on the optimized conformations for both ligand and protein target to form a stable complex. All ligands were docked on the crystal structure of the herpes simplex type-1 thymidine kinase (PDB ID: 2KI5). The docking pose of the co-crystallized AC2 interacting with amino acid residues of the active site is shown in Figure 3. The oseltamivir and ribavirin were taken as reference ligands to compare the docking results of all the studied compounds. The docking studies revealed that the 5jG compound has the best docking score −66.42 (RMSD: 0.55) (Table 1, Figure 4).   The docking pose of the 5jG compound is shown in Figure 5. After analyzing the data obtained from the docking study, it was observed that all the compounds were placed in the same binding site of 2KI5 as the co-crystallized ( Figure 6). Docking evaluation against wild-type influenza N2 neuraminidase: docking studies were performed to achieve accurate predictions on the optimized conformations for both ligand and protein target to form a stable complex. All ligands were docked on the crystal structure of the wild-type influenza N2 neuraminidase (PDB ID: 4H52). The docking pose of the co-crystallized FSI A 508 interacting with amino acid residues of the active site is shown in Figure 7. Oseltamivir and ribavirin were taken as reference ligands to compare the docking results of all the studied compounds. The docking studies revealed that the 5kG compound has the best docking score −67.57 (RMSD: 0.58) (Table 1, Figure 4). The docking pose of the 5kG compound is shown in Figure 8.  After analyzing the data obtained from the docking study, it was observed that all the compounds were placed in the same binding site of 4H52 as the co-crystallized (Figure 9). Docking evaluation against SARS coronavirus main protease: docking studies were performed to achieve accurate predictions on the optimized conformations for both ligand and protein target to form a stable complex. All ligands were docked on the crystal structure of the SARS coronavirus main protease (PDB ID: 3TNT). The docking pose of the cocrystallized G85A 501 interacting with amino acid residues of the active site is shown in Figure 10. Oseltamivir and ribavirin were taken as reference ligands to compare the docking results of all the studied compounds. The docking studies revealed that the 5jjG compound has the best docking score −81.11 (RMSD: 1.90) (Table 1, Figure 4). The docking pose of the 5jjG compound is shown in Figure 11.  After analyzing the data obtained from the docking study, it was observed that all the compounds were placed in the same binding site of 3TNT as the co-crystallized ( Figure 12). Important molecular properties-molecular weight, flexible bonds, the number of hydrogen bond donors, the number of hydrogen bond acceptors and log P-were calculated (Table 2). These parameters can predict if a molecule possesses properties that might turn it into an orally active drug, according to Lipinski's rule of five. The number of violations of the Lipinski rules allows one to evaluate drug likeness for a molecule. According to the data presented in Table 2, only the 5lG compound failed with respect to the Lipinski rules (Lipinski violation is 1, hydrogen bond donors >5). The compounds were screened against influenza virus and two compounds, 5fG and 5gG, had SI of 25 and 23 (IC50 = 8 μM and 12.8 μM) and two SI of 10 (5kG, IC50 = 24 μM and 5sG, IC50 = 29 μM). The screening of the compounds against HSV and low active coronavirus is being developed and soon will be finished.
In conclusion, a number of 24 new 1′-homonucleoside with a 2-amino-6-substituted purine as nucleobase and an optically active bicyclo[2.2.0]heptane scaffold were synthesized, and a molecular docking study on three viruses and an experimental screening of the compounds against influenza virus were realized.

Conflicts of Interest:
The authors declare no conflict of interest.