Synthesis of some oxazolo[4,5-d ]pyrimidine derivatives and evaluation of their antiviral activity and cytotoxicity

oxazolo[4,5-d ]pyrimidine derivatives were synthesized and their some physicochemical and ADMET properties were analyzed by in silico prediction. Antiviral activity against DNA viruses (human cytomegalovirus, varicella-zoster virus, herpes simplex virus and BK virus) and cytotoxicity, including control experiments, were evaluated in vitro . Some of the compounds synthesized were characterized by low toxicity; however, they did not exhibit favorable antiviral effects


Introduction
Oxazole and pyrimidine scaffolds, as well as their fused heterocycles, are well-known structures utilized in the design of bioactive molecules in drug discovery.A significant number of studies have been carried out on the biological properties of oxazolo [5,4-d]pyrimidines, uncovering their activities as receptor modulators and enzyme inhibitors. 1 However, it seems that oxazolo [4,5-d]pyrimidines have only been synthesized, and that there is little information in the literature concerning their bioactivity. 2Among these bioactivity reports, inhibition of fatty acid amide hydrolase and monoglyceride lipase 3 by these compounds have been described.5][6] Since oxazoleand pyrimidine-based heterocycles, including oxazolo [5,4-d]pyrimidines, have demonstrated antiviral effects, [7][8][9] the synthesis of new compounds that contain oxazolo [4,5-d]pyrimidine fragment was of interest to us and therefore we decided to synthesize them and to evaluate their antiviral activity and toxicity.
In this paper, new oxazolo [4,5-d]pyrimidine derivatives were synthesized, characterized in silico on druglike properties and tested in vitro for cell cytotoxicity and efficacy against some viral opportunistic infections.

Investigation of molecular properties and drug-like parameters
Drug-like parameters (Table 1), as calculated by Molinspiration software (http://www.molinspiration.com),indicated that only four of the 15 synthesized compounds are in compliance with Lipinski rule of five. 11Twothirds of the derivatives have molecular weights lower than 500 Da.Accordig to the predictive software, only compounds 5, 6, 10, and 13 are expected to have logP values lower than 5, and the other oxazolo [4,5d]pyrimidine derivatives are more lipophilic.All of the compounds were predicted to have H-bond accepting abilities in the range of 5-8, and less than half of them can be characterized by one H-bond donating group.The topological polar surface area prediction values (from 64 to 94) were found to be in the acceptable range.Pharmacokinetic properties (Table 1 and Table S1) predicted by the pkCSM online server 12 suggested that all the oxazolo [4,5-d]pyrimidine derivatives could potentially have good human intestinal absorption, and compound 1 can also have a good possibility in terms of blood-brain barrier permeability.

Antiviral and Cytotoxicity Assays
Activity against human cytomegalovirus (HCMV), varicella-zoster virus (VZV), herpes simplex virus (HSV-1) and BK virus (BKV) was assessed via in vitro antiviral assays.The data obtained in the study, along with positive controls for each virus, are given in Table 2.Only compound 3 showed activity against BKV with EC50 of 3.51 μM and CC50 of 23.53 μM (selectivity index SI50 is 7), and none of the other compounds demonstrated favorable antiviral activity.At the same time, CC50 values indicated a high degree of cytotoxicity of many of the tested compounds.It should also be noted, that in silico ADMET profile showed that the compounds could potentially inhibit hERG II and exhibit hepatotoxicity (Table S1).Giving the in vitro cytotoxicity results, we analyzed the possible link between the toxicity of the tested compounds and their lipophilicity.Cytotoxic compounds 1-4, 9, and 15 have calculated logP values from 5.51 to 6.72, while the compounds with low toxicity (CC50 >150 μM), 5, 6, 8, 10, 13, are characterized by logP values from 3.65 to 5.24.This indicates that high lipophilicity of the oxazolo [4,5-d]pyrimidine derivatives may cause their high toxicity, probably due to higher permeability through the membrane and binding to intracellular targets of both infected and non-infected cells.

Conclusions
New oxazolo [4,5-d]pyrimidine derivatives were synthesized and their antiviral activities against four DNA viruses were evaluated in vitro.Some of physicochemical and pharmacological properties of the compounds were predicted in silico.Biologically relevant antiviral assays demonstrated a lack of antiviral effects of the compounds, with cytotoxicity being a major limitation in many of them.This could be due to the high predicted logP values for the compounds and is consistent with predicted good human intestinal absorption.Therefore, the main task of further functionalization of the oxazolo [4,5-d]pyrimidine framework is to reduce their toxicity and increase the selectivity.

1-15
b nOHNH -Number of OH and NH groups as H-bond donors.c nON -Number of O and N atoms as H-bond acceptors.d nrotb -Number of rotatable bonds.e TPSA -Topological polar surface area.