We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.

Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs) were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These compounds were evaluated against a large spectrum of DNA and RNA viruses. Among them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against varicella-zoster virus (VZV) with EC50 values of 0.33 and 0.39 μM for wild-type and thymidine kinase deficient strains, respectively, and a selectivity index ≥200 μM. This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate analogs.

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3-H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5-97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at β-lactamase, thus protecting the antibiotic from undesirable biotransformation.

To study the influence of a linker rigidity and changes in donor-acceptor properties, three series of nucleotide analogs containing a P-X-HN-C(O)- residue (X=CH(OH)CH2, CH(OH)CH2CH2, CH2CH(OH)CH2) as a replacement for the P-CH2-O-CHR- fragment in acyclic nucleoside phosphonates, e.g., adefovir, cidofovir, were synthesized. EDC proved to provide good yields of the analogs from the respective ω-amino-1- or -2-hydroxyalkylphosphonates and nucleobase-derived acetic acids. New phosphorus-nucleobase linkers are characterized by two fragments of the restricted rotation within amide bonds and in four-atom units (P-CH(OH)-CH2-N, P-CH(OH)-CH2-C and P-CH2-CH(OH)-C) in which antiperiplanar disposition of P and N/C atoms was deduced from 1H and 13C NMR spectral data. The synthesized analogs P-X-HNC(O)-CH2B [X=CH(OH)CH2, CH(OH)CH2CH2, CH2CH(OH)CH2] appeared inactive in antiviral assays on a wide variety of DNA and RNA viruses at concentrations up to 100 μM, while two phosphonates showed cytostatic activity towards myeloid leukemia (K-562) and multiple myeloma cells (MM.1S) with IC50 of 28.8 and 40.7 μM, respectively.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.

1,3-Dipolar cycloaddition of N-methyl C-(diethoxyphosphoryl) nitrone to N3-substituted 6-bromo-2-vinyl-3H-quinazolin-4-ones gave (3-diethoxyphosphoryl) isoxazolidines substituted at C5 with quinazolinones modified at N3. All isoxazolidine cycloadducts were screened for antiviral activity against a broad spectrum of DNA and RNA viruses. Several isoxazolidines inhibited the replication of both thymidine kinase wild-type and deficient (TK⁺ and TK-) varicella-zoster virus strains at EC50 in the 5.4⁻13.6 μΜ range, as well as human cytomegalovirus (EC50 = 8.9⁻12.5 μΜ). Isoxazolidines trans-11b, trans-11c, trans-11e, trans-11f/cis-11f, trans-11g, trans-11h, and trans-11i/cis-11i exhibited moderate cytostatic activity towards the human lymphocyte cell line CEM (IC50 = 9.6⁻17 μM).

We report the synthesis and antiviral activity of a new family of non-nucleoside antivirals, derived from the indole nucleus. Modifications of this template through Mannich and Friedel-Crafts reactions, coupled with nucleophilic displacement and reductive aminations led to 23 final derivatives, which were pharmacologically tested. Tryptamine derivative 17a was found to have a selective inhibitory activity against human varicella zoster virus (VZV) replication in vitro, being inactive against a variety of other DNA and RNA viruses. A structure-activity relationship (SAR) study showed that the presence of a biphenyl ethyl moiety and the acetylation at the amino group of tryptamine are a prerequisite for anti-VZV activity. The novel compound shows the same activity against thymidine kinase (TK)-competent (TK+) and TK-deficient (TK-) VZV strains, pointing to a novel mechanism of antiviral action.

A novel series of diethyl {4-[(5-substituted-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates designed as analogues of amonafide was synthesized. All phosphonates were assessed for antiviral activity against a broad range of DNA and RNA viruses and several of them showed potency against varicella-zoster virus (VZV) [EC50 (50% effective concentration) = 27.6-91.5 μM]. Compound 16b exhibited the highest activity against a thymidine kinase-deficient (TK-) VZV strain (EC50 = 27.59 μM), while 16d was the most potent towards TK⁺ VZV (EC50 = 29.91 μM). Cytostatic properties of the compounds 14a-i-17a-i were studied on L1210, CEM, HeLa and HMEC-1 cell lines and most of them were slightly cytostatic for HeLa [IC50 (50% inhibitory concentration) = 29-130 µM] and L1210 cells [IC50 (50% inhibitory concentration) = 14-142 µM].

Hexadecyloxypropyl esters of acyclic nucleoside phosphonates containing guanine (G) or hypoxanthine (Hx) and a (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] [(S)-HPMP] or 2-(2-phosphonoethoxy)ethyl (PEE) acyclic moiety have been prepared. The activity of the prodrugs was evaluated in vitro against different virus families. Whereas ester derivatives of PEEHx and (S)-HPMPHx were antivirally inactive, monoesters of PEEG, and mono- and diesters of (S)-HPMPG showed pronounced antiviral activity against vaccinia virus and/or herpesviruses. Monoesters of (S)-HPMPG emerged as the most potent and selective derivatives against these DNA viruses. None of the compounds were inhibitory against RNA viruses and retroviruses.

A series of tricyclic penciclovir (PCV) and hydroxybutylguanine (HBG) derivatives have been prepared with enhanced lipophilicity following an efficient synthetic route. All the novel tricyclic derivatives were evaluated for inhibitory activity against herpes simplex virus 1 and 2 (HSV-1, HSV-2) and thymidine kinase deficient (ACV resistant) HSV-1. The tricyclic HBG derivatives were devoid of inhibitory activity however several of the tricyclic PCV derivatives showed promising antiviral activity, in particular 9g (R = 4-MeO-C6H4) displayed good inhibitory activity (HSV-1 EC50 1.5 μM, HSV-2 EC50 0.8 μM) and retained inhibitory activity in HSV-1 TK- cells (EC50 0.8 μM). Computational docking experiments supported the biological data observed and this preliminary study provides useful data for further development of tricyclic acyclic nucleoside derivatives with improved lipophilicity and retention of activity in HSV-1 TK deficient strains. Also, the new tricyclic derivatives were evaluated against a broad range of other DNA and RNA viruses, but were found to be inactive at subtoxic concentrations. In addition, weak to moderate cytostatic effect was observed for the new compounds.

Dipolar cycloaddition of the N-substituted C-(diethoxyphosphonyl)nitrones with N3-allyl-N1-benzylquinazoline-2,4-diones produced mixtures of diastereoisomeric 3-(diethoxyphosphonyl)isoxazolidines with a N1-benzylquinazoline-2,4-dione unit at C5. The obtained compounds were assessed for antiviral and antibacterial activities. Several compounds showed moderate inhibitory activities against VZV with EC50 values in the range of 12.63-58.48 µM. A mixture of isoxazolidines cis-20c/trans-20c (6:94) was found to be the most active against B. cereus PCM 1948, showing an MIC value 0.625 mg/mL, and also was not mutagenic up to this concentration.

A new strategy for the synthesis of N3 -benzoylated- and N3 -benzylated N1 -propargylquinazoline-2,4-diones 30a-d and 31a-d from isatoic anhydride 41 is reported. The alkynes 30a-d and 31a-d were applied in the 1,3-dipolar cycloadditions with azides 27 and 28 to synthesize acyclic 1,2,3-triazole nucleosides. The obtained alkynes and 1,2,3-triazole were evaluated for antiviral activity against a broad range of DNA and RNA viruses. The alkyne 30d showed activity against adenovirus-2 (EC50  = 8.3 μM), while compounds 37a and 37d were also active toward herpes simplex virus-1 wild-type and thymidine kinase deficient (HSV-1 TK- ) strains (EC50 values in the range of 4.6-13.8 μM). In addition, compounds 30a, 30b, 37b, and 37c exhibited activity toward varicella-zoster virus (VZV) TK+ and TK- strains (EC50  = 2.1-9.5 μM). The compound 30b proved to be the most selective against VZV and displayed marginal activity against human cytomegalovirus (HCMV). Although the compound 30a had improved anti-HCMV activity, the increase in anti-HCMV activity was accompanied by significant toxicity. Compounds 37a and 37d showed inhibitory effects toward the human T lymphocyte (CEM) cell line (IC50  = 21 ± 7 and 22 ± 1 μM, respectively), while compound 35 exhibited cytostatic activity toward HMEC-1 cells (IC50  = 28 ± 2 μM).