Design, Synthesis, Anti-Varicella-Zoster and Antimicrobial Activity of (Isoxazolidin-3-yl)Phosphonate Conjugates of N1-Functionalised Quinazoline-2,4-Diones

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.

In the vast majority of natural pyrimidine nucleosides, e.g., uridine and 2′-deoxythymidine and their synthetic analogues, furanose ring or its mimetic is linked to the N1 of a pyrimidine moiety; however, analogues with nucleobase attached to sugar via the N3 atom of pyrimidine have been also obtained. Among them, nucleotide analogues 10 and 11 ( Figure 3) show even higher antiviral activity than the respective N1-isomeric nucleotides [16,17]. Recently, we achieved the synthesis of isoxazolidine nucleotide analogues 12a-c with functionalized quinazoline-2,4-dione as a nucleobase replacer, and their promising antivaricella-zoster virus (VZV) activity has been recognized (EC50 = 3.0-5.1 µM) [18]. Based on the observed biological properties of various quinazoline-2,4-dione derivatives, such as 12, and in continuation of our studies on isoxazolidine analogues of nucleosides, a new series of compounds of the general formula 13 with quinazoline-2,4-dione linked via the N3 atom to the isoxazolidine moiety was synthesized to evaluate the biological activity. The route to construct the designed compound 13 relies on the 1,3-dipolar cycloaddition of N-substituted C-(diethoxyphosphonyl)nitrones 14-15 [19] with selected N3-allylated quinazoline-2,4-dione 16 functionalized at N3 with the respective benzyl group (Scheme 1). Recently, we achieved the synthesis of isoxazolidine nucleotide analogues 12a-c with functionalized quinazoline-2,4-dione as a nucleobase replacer, and their promising antivaricella-zoster virus (VZV) activity has been recognized (EC 50 = 3.0-5.1 µM) [18]. Based on the observed biological properties of various quinazoline-2,4-dione derivatives, such as 12, and in continuation of our studies on isoxazolidine analogues of nucleosides, a new series of compounds of the general formula 13 with quinazoline-2,4-dione linked via the N3 atom to the isoxazolidine moiety was synthesized to evaluate the biological activity. The route to construct the designed compound 13 relies on the 1,3-dipolar cycloaddition of N-substituted C-(diethoxyphosphonyl)nitrones 14-15 [19] with selected N3-allylated quinazoline-2,4-dione 16 functionalized at N3 with the respective benzyl group (Scheme 1).

Chemistry
The 1,3-dipolar cycloadditions of the respective nitrone 22 (R = Me) or 23 (R = Bn) with the selected N 3 -allyl-N 1 -benzylquinazoline-2,4-diones 24a-d were carried out at 60 °C in toluene. In all cases, the regiospecific formation of the diastereoisomeric mixtures of 3,5-disubstituted isoxazolidines trans-20 and cis-20 or trans-21 and cis-21 was observed, with the trans-isomer predominating (Scheme 3, Table 1). In the case of isoxazolidines 20a and 20c as well as 21a-d trans/cis ratios, diastereoisomers were determined based on analyses of 31 P NMR spectra of crude products, since two well-separated signals were observed. For diastereoisomeric pairs of isoxazolidines 20b and 20d, trans/cis ratios were calculated from 1 H NMR spectra of crude reaction mixtures by comparison of diagnostic resonances of CH3-N protons in the isoxazolidine ring. The diastereoselectivity values (d.e.) ranged from 70 to 84%. The mixtures of the respective cycloadducts were subjected to purification on silica gel columns; however, attempts to isolate pure diastereoisomers were fruitless and, in each case, only mixtures of diastereoisomers isoxazolidines trans-20 and cis-20 or trans-21 and cis-21 were isolated.  Table 1). In the case of isoxazolidines 20a and 20c as well as 21a-d trans/cis ratios, diastereoisomers were determined based on analyses of 31 P NMR spectra of crude products, since two well-separated signals were observed. For diastereoisomeric pairs of isoxazolidines 20b and 20d, trans/cis ratios were calculated from 1 H NMR spectra of crude reaction mixtures by comparison of diagnostic resonances of CH 3 -N protons in the isoxazolidine ring. The diastereoselectivity values (d.e.) ranged from 70 to 84%. The mixtures of the respective cycloadducts were subjected to purification on silica gel columns; however, attempts to isolate pure diastereoisomers were fruitless and, in each case, only mixtures of diastereoisomers isoxazolidines trans-20 and cis-20 or trans-21 and cis-21 were isolated.    The relative configurations of the isoxazolidine cycloadducts trans-20 and cis-20 or trans-21 and cis-21 were established by taking in account our previous studies on the stereochemistry of cycloaddition of N-substituted C-diethyoxyphosphonylated nitrones 22 and 23 with allylated derivatives of various (hetero)aromatic compounds [21,22], including N3-substituted N1-allylquinazoline-2,4-diones [18]. Since modification of the substituents in quinazoline-2,4-dione moiety, including the relocation of substituents at N1 and N3, has no influence on the stereochemical outcome of the cycloaddition of nitrones 22 and 23 to Nallylquinazoline-2,4-dione dipolarophiles, configurations of all major isoxazolidines 20 and 21 were assigned trans, while minor isomers were assigned cis, by analogy to previously established configurations of transand cis-isoxazolidines 12a-c [18]. -10000), mycophenolic acid, zanamivir, amantadine, and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the effective concentration required to reduce virus plaque formation (VZV, HCMV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses).
Since mutagenic compounds can be capable of inducing cancer [28], we decided to evaluate the mutagenic potential of the most active agent, i.e., the inseparable mixture of compounds cis-20c/trans-20c (6:94) ( Table 3) using the Ames mutagenicity assay (the bacterial reverse mutation test). The study was performed using a standard microplate Ames MPF TM Penta I kit (Xenometrix, Allschwil, Switzerland), compliant with the OECD guideline 417 [29] and the International Organization for Standardization [30,31]. This test is a widely accepted short-term bacterial assay for the identification of substances that can produce genetic damage that leads to gene mutations. The bacterial strains used in this assay have various mutations that inactivate a gene involved in the synthesis of essential amino acids, either histidine (Salmonella typhimurium, TA98, TA100, TA1535 and TA1537 strains) or tryptophan (Escherichia coli, WP2uvrA[pKM101 strain), so they can only grow in the culture medium that is supplemented with that amino acid. The mutagenic potential of the sample was assessed after metabolic activation in the presence of Aroclor 1254-induced rat liver S9 (S9 Cofactor kit, Xenometrix, Allschwil, Switzerland). When the bacteria are exposed to a mutagen, mutations occur that may restore or reverse the ability of the bacteria to synthesize the amino acid and to continue growing once the limited amount of amino acid in the liquid medium is depleted. The following mutagenic compounds were used as positive controls: 2-aminoanthracene (for S. typhimurium TA98, TA100, TA1535 and TA1537) and 2-aminofluorene (for E.coli strain WP2 uvrA[pKM101]). As the negative control, 50% DMSO was used (solvent for the tested compound). Based on the obtained results, the tested agent, the inseparable mixture of compounds cis-20c/trans-20c (6:94), should be considered as not mutagenic in the tested species of bacteria at the concentration up to 0.625 mg/mL.

Materials and Methods
3.1. General Information 1 H, 13 C, and 31 P NMR spectra were taken in CDCl 3 on the Bruker Avance III spectrometers (600 MHz) with TMS as internal standard at 600, 151, and 243 MHz, respectively. IR spectra were measured on an Infinity MI-60 FT-IR spectrometer. Melting points were determined on a Boetius apparatus and were uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of this faculty on Perkin-Elmer PE 2400 CHNS analyzer. The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh), analytical TLC, and Merck TLC plastic sheets silica gel 60 F 254 . Nmethyl-and N-benzyl-C-(diethoxyphosphonyl)nitrones 14 and 15 were obtained according to procedures in the literature [19]. 1 H-, 13 C-and 31 P-NMR spectra of all new synthesized compounds are provided in Supplementary Materials.

Antibacterial Activity Assays
The antimicrobial tests were performed using reference strains of microbia from the American Type Culture Collection (ATCC), including E. faecalis ATCC 29212, S. aureus ATCC 2593, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and two fungal strains, C. albicans ATCC 10241 and A. brasiliensis ATCC 16404. From the Polish Collection of Microorganisms (PCM), B. cereus PCM 1948 was used. The antimicrobial activity of the compounds was assessed according to their minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The MIC and MBC were expressed in mg/mL. Antibacterial and antifungal activities were determined using the broth microdilution method in a liquid medium according to The European Committee on Antimicrobial Susceptibility (EUCAST) recommendations. The Mueller-Hinton liquid medium (pH~7.2) (BioMerieux, Marcy L Etoile, France) was used for bacteria. Liquid medium RPMI-1640 (pH~7.2) (Sigma, Darmstadt, Germany) was used for the fungal strains. Each tested compound was dissolved in 10 mg/mL in sterile water. Two-fold series dilutions of the different compounds in the growth medium were performed in the 96-well sterile microtiter plates (Kartell Labware, Noviglio, Italy). Inocula were freshly prepared and standardized as microbial suspensions (McFarland scale) containing 10 8 colony forming units (cfu/mL), added at a volume of 10 µL, to the wells of the microtiter plate together with the serial dilutions of the compounds in the growth medium. After 24 h of incubation at 37 • C, microbial growth was evaluated spectrophotometrically at 595 nm using a Microplate reader 680 (BioRad, Hercules, CA, USA). The lowest concentration of the tested compounds resulting in total growth inhibition was taken as the MIC value. To determine the MBC, 10 µL of the culture were collected from each well, where no visible growth of microorganisms was recorded and plated onto the surface of Brain Heart Infusion Agar (BioMerieux, Marcy L Etoile, France). The cultures were incubated for 24 h at 37 • C. An absence of microbial growth indicated bactericidal activity by the tested compounds. Plates with A. brasiliensis were incubated at 37 • C for three days. The tests were performed in two independent experiments. Amikacin (Sigma, Darmstadt, Germany) and fluconazole (Sigma) were used as antimicrobial standards.

Microbial Mutagenicity Assay-The Ames Test
Mutagenicity was determined using a standard microplate AMES MPF TM PENTA I kit according to the manufacturer's instructions (Xenometrix, Allschwil, Switzerland) [29]. Bacteria were exposed to 25 µL of the tested compound (0.625 mg/mL) as well as positive and negative controls for 90 min in a medium-containing sufficient histidine (S. typhimurium) or tryptophan (E. coli) to support approximately two cell divisions. After exposure, the cultures were diluted in pH indicator medium lacking histidine or tryptophan and aliquoted into 48 wells of a 384-well plate. Within two days, cells that had undergone reversion to amino acid prototrophy grew. Bacterial metabolism reduces the pH of the medium, changing the color of that well. The number of wells containing revertant colonies was counted for each dose and compared to a solvent (negative) control. Each dose was performed in triplicate to allow for statistical analysis of the data. A dose-dependent increase in the number of revertant colonies upon exposure to the test sample relative to the solvent control indicates that the sample is mutagenic in the Ames MPF assay. The mutagenic potential of the sample was assessed after metabolic activation in the presence of Aroclor 1254-induced rat liver S9 (S9 Cofactor kit, Xenometrix, Allschwil, Switzerland). The following mutagenic compounds were used as positive controls: 2-aminoanthracene (for S. typhimurium TA98, TA100, TA1535 and TA1537) and 2-aminofluorene (for E.coli strain WP2 uvrA[pKM101]). As the negative control, 50% DMSO was used.

Acknowledgments:
The authors wish to express their gratitude to Leentje Persoons, Brecht Dirix, and Wim Werckx for their excellent technical assistance.