Herpesviruses are widespread among humans and may cause many diseases. The primary infection is usually followed by a lifelong latency of the virus and its reactivation usually occurs during immunosuppression of the host. Infection with varicella-zoster virus (VZV) results in varicella (chickenpox) which usually takes a mild course in children but may be more severe in adults. Later on, after establishing latency in neural tissues, the virus can reactivate causing herpes zoster (shingles) which is often accompanied by neuralgic pain and can lead to post-herpetic neuralgia (PHN) as well as other complications such as loss of vision (zoster ophthalmicus) [1, 2] .
In most cases immunocompetent patients infected by herpesviruses do not require antiviral therapy. However, reactivation of the virus is of significant concern in immunocompromised individuals, e.g. recipients of solid-organ and hematopoietic stem cell transplant, patients under aggressive chemotherapy or individuals with acquired immunodeficiency syndrome (AIDS). Under these circumstances, efficient antiviral drugs are of crucial importance. Effective treatments of herpesviridae species, including herpes simplex virus (HSV), VZV and human cytomegalovirus (HCMV) [3, 4] are available but they are hampered by emergence of drug resistance and significant drug toxicities for some anti-herpesvirus agents (such as ganciclovir, foscavir and cidofovir). Four compounds are currently licensed for the treatment of VZV infections, namely acyclovir, valaciclovir, famciclovir and brivudin [5, 6] . Regrettably, AIDS patients often do not respond well to acyclovir therapy or other antiviral drugs due to the emergence of thymidine kinase-deficient or thymidine kinase-altered mutations of VZV [7, 8] . Therefore, the extensive search for new anti-VZV agents with superior efficacy compared to currently approved drugs is of high importance.
Numerous structurally diversified compounds have already been synthesized and tested as new potential anti-VZV agents including bicyclic nucleoside analogues, non-nucleoside DNA polymerase inhibitors and N-(a-methylbenzyl)-N-arylthiourea analogues ( Fig. 1) [9e18].
On the other hand, the antiviral activity of several 1,3disubstituted quinazoline-2,4-diones (Fig. 2 ) has been discovered in recent years [19] . A 3-benzylquinazolin-2,4-dione derivative 1 was reported to posses the anti-HIV-1 activity in MT-4 cells and inhibited the recombinant RT in vitro [20] . A quinazolinone-2,4dione 2 was a potent inhibitor of RSV-induced cytopathic effect (EC 50 ¼ 2.14 mM) [21] . Several other analogues, namely 3e5, proved to be very active toward Respiratory Syncytial Virus (RSV) [21] . Recently, the N3-benzoylquinazolinonedione moiety was successfully incorporated as a nucleobase mimetic into the 1,2,3-triazole analogues of nucleotides 6 [22] and 7 [23] . While the compound 6 showed a moderate activity against both herpes simplex viruses (HSV-1 and HSV-2) (EC 50 ¼ 17 mM) as well as feline herpes virus (EC 50 ¼ 24 mM), its dihydroxylated derivative (1R,2S)-7 proved to be even more potent (EC 50 ¼ 2.9, 4 and 4 mM toward HSV-1, HSV-2 and feline herpes virus, respectively), while the enantiomer (1S,2S)-16 was inactive [23] . From several functionalized quinazoline-2,4diones studied as allosteric inhibitors of the NS5B polymerase compounds 8e10 exhibited the highest affinity to the enzyme [24] . On the basis of these observations one may conclude that for the antiviral activity of quinazoline-2,4-diones substitution at N3 with aryl, benzyl or benzoyl groups is beneficial.
Recently, we successfully accomplished the syntheses of homonucleoside analogues 11 which proved inactive against a broad spectrum of DNA and RNA viruses while some of them appeared slightly cytostatic toward several cancerous cell lines [25] . However, later on they were additionally screened for inhibition of VZV and HCMV replication and two compounds 11a (B ¼ N-benzoyluracil) and 12a (R ¼ benzoyl, R 0 ¼ methyl) showed noticeable activity toward VZV (Table 3) . Based on this discovery we designed a new series of analogues (Scheme 1) installing at N3 of the quinazoline-2,4-dione skeleton either substituted benzoyl groups (compounds 2 and 3) or substituted benzyl residues (compounds 14 and 15).
As previously reported [25] , N 1 -allyl-N 3 -benzoylquinazoline-2,4-dione 18a was obtained in three steps in 20% overall yield starting from quinazoline-2,4-dione employing bis-N 1 ,N 3 -benzoylation with benzoyl chloride followed by the selective N 1 -debenzoylation and subsequent allylation. However, this procedure appeared tedious and the N 1 -debenzylation step was the least effective. For this reason another strategy for the syntheses of N 1allyl-N 3 -benzoylquinazoline-2,4-diones 18a-18d was designed which relied on N-allylation of the commercially available isatoic anhydride 20 followed by a subsequent condensation of compound 21 with urea [26, 27] and concluded with N 3 -benzoylation of the resulted N-allylquinazoline-2,4-dione 22 with selected benzoyl chlorides (Scheme 2). Moreover, under these circumstances compounds 19a-d could be obtained in one step by benzylation of allylquinazoline-2,4-dione 22 (Scheme 2).
1,3-Dipolar cycloadditions of nitrones 16 (R 0 ¼ Me) or 7 (R 0 ¼ Bn) with the respective N 1 -allyl-N 3 -benzoylquinazoline-2,4-diones 18a-d were carried out at 60 C in toluene or toluene-ethanol mixtures as solvents and afforded mixtures of diastereoisomeric isoxazolidines trans-12 and cis-12 or trans-13 and cis-13 (Scheme 3, Table 1 ) with the trans-isomer predominating. The cis/trans ratios of diastereoisomeric products were determined on the basis of the 31 P NMR spectral data. The reactions proceeded with moderate diastereoselectivities (d.e. 28e60%) and with good to excellent overall yields. The isolation of pure isomers was successfully accomplished chromatographically for major isomers trans-12a, trans-12b, trans-12c, trans-12d, trans-13c and trans-13d but also for minor isomers cis-12a, cis-12d and cis-13a.
To eliminate rigidity within the substituted quinazoline-2,4dione moiety benzoyl substituents at N3 were replaced by the functionalized benzyl residues. 1,3-Dipolar cycloadditions of nitrones 16 (R 0 ¼ Me) or 7 (R 0 ¼ Bn) with the respective N 1 -allyl-N 3benzylquinazoline-2,4-diones 19a-d were carried out under conditions already described for compounds 18. Diastereoisomeric cycloadducts trans-14 and cis-14 or trans-15 and cis-15 (Scheme 4, Table 2 ) were formed in good to excellent overall yields and with moderate diastereoselectivities (d.e. 28e60%) which were slightly higher for reactions of the nitrone 7 (R 0 ¼ Bn). Chromatographic isolation of pure isomers was achieved for trans-14a, cis-14b, trans-14b, trans-14c and trans-14d. 
The pure isomers of quinazoline-2,4-dione -conjugates [trans-11a, cis-11a, trans-12a, cis-12a, trans-13a, cis-13a, trans-12b, trans-12c, trans-13c, trans-12d, cis-12d, trans-13d, trans-14a, trans-14b, cis-14b, trans-15b, trans-14c, trans-14d] and the respective mixtures of cis/trans isomers [cis-12b/trans-12b (87:13), trans-13b/cis-13b (90:10), cis-2c/trans-2c (94:6), cis-13c/trans-13c (86:14), cis-13d/trans-13d (85:15), cis-14a/trans-14a (75:25), trans-15a/cis-15a (90:10), cis-15a/trans-15a (50:50), cis-15b/trans-15b (87:13), cis-14c/trans-14c (97:3), trans-15c/cis-15c (90:10), cis-15c/trans-15c (80:20), cis-14d/trans-14d (75:25), trans-15d/cis-15d (95:5), cis-15d/trans-15d (75:52)] were screened as inhibitors of a wide variety of DNA and RNA viruses using the following cell-based assays: mycophenolic acid, Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses). Several synthesized quinazoline-2,4-diones inhibited the replication of both TK þ and TK À VZV strains (Table 3) . A 95:5 trans-15d/ cis-15d mixture and a 87:13 cis-15b/trans-15b mixture emerged as the most active derivatives with EC 50 's of, respectively, 4.7 mM and 3 mM (VZV TK þ strain) and of 3.6 mM and 5.1 mM (VZV TK À strain).
These two quinazoline-2,4-diones were 4e6-fold less active against the TK þ virus but proved to be 10e14-fold more active against the TK e strain when compared to the reference drug acyclovir. These data clearly indicate that these novel derivatives do not require activation by the viral TK. Although these quinazoline-2,4-diones did not significantly altered the morphology of cells in the antiviral assays, they showed considerable cytostatic activity (in the same range as the antiviral activity).
Compounds trans-13c, a 86:14 cis-13c/trans-13c mixture, trans-13d, and a 85:15 cis-13d/trans-13d mixture inhibited both VZV TK þ and TK e viruses with EC 50 's in the range of 6.0e8.5 mM. Compounds trans-12a, cis-12a, cis-13a, trans-12d and a 50:50 cis-15a/trans-15a mixture also proved active against VZV TK þ strain (EC 50 's of 6.9e7.5 mM) and VZV TK À strain (EC 50 's of 10e14 mM), slightly exceeding in potency against VZV TK À strains the reference drugs acyclovir and brivudin (EC 50 ¼ 50.3 mM and 22.7, respectively).
However, majority of the studied compounds exhibited significant cytotoxicity and the 95:5 trans-15d/cis-15d mixture reduced cell growth (CC 50 ) at concentration as low as 6.6 mM which was almost two orders of magnitude lower than that for acyclovir (CC 50 ¼ 440 mM).
Among the investigated quinazoline-2,4-diones, the N3benzoylated compounds (cis-and trans-12/13) were found inactive toward both human cytomegalovirus (HCMV) strains. On the other hand, isoxazolidine phosphonates having benzyl substituents both at N3 of the quinazoline-2,4-dione skeleton and at N2 of the isoxazolidine ring (cis-and trans-15) showed weak antiviral activity with EC 50 in the range of !3e!14.5 mM (Table 4 ).
structure-activity relationship observations revealed a lack of significant differences in activity of cis vs. trans isoxazolidines and higher potency of isoxazolidines carrying Nbenzyl substituents in comparison with their N-methyl counterparts especially well pronounced for the N 3 -benzylquinazoline-2,4diones 15. For the active compounds the introduction of a fluorine atom into the benzene ring either in benzyl or benzoyl residues did not improve their efficacy. While the quinazoline-2,4-diones substituted at N3 with benzoyl and benzyl moieties were found effective against VZV, only those carrying substituted benzyl components proved active toward HMCV.
All synthesized isoxazolidine phosphonates were also subjected to antiviral screening with other viruses, but only compound trans-12d appeared slightly active against other herpesviruses, adenovirus-2 and human Coronavirus ( Table 5 ).
The 50% cytostatic inhibitory concentration (IC 50 ) causing a 50% decrease in cell proliferation was determined against murine leukemia L1210, human lymphocyte CEM, human cervix carcinoma HeLa and immortalized human dermal microvacsular endothelial cells (HMEC-1) ( Table 6 ). Among all tested compounds only quinazoline-2,4-diones trans-15/cis-15 having benzyl substituents at N3 in the quinazolinone core and the benzyl group at N2 of the isoxazolidine unit showed significant cytostatic activity toward the tested cell lines. For the CEM cell line, these derivatives were as active as the reference drug 5-fluorouracil. It was noticed that the replacement of the benzyl component within the isoxazolidine moiety for the methyl group (trans-15/cis-15 vs. the respective trans-14/cis-14) resulted in decrease in potency by roughly an order of magnitude.
Several series of {5-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl) methyl-2-methylisoxazolidin-3-yl})phosphonates (cis-12/trans-12 and cis-14/trans-14) and {5-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl-2-benzylisoxazolidin-3-yl})phosphonates (cis-13/ trans-13 and cis-15/trans-15) modified at N3 in the quinazoline-2,4dione moiety have been obtained by the 1,3-dipolar cycloaddition of N-substituted (C-diethoxyphosphoryl)nitrones 16 (R¼Me) and 17 (R¼Bn) and the respective N 1 -allylated quinazoline-2,4-diones substituted at N3 with benzoyl (compounds 18) or benzyl groups (compounds 19).
The synthesized isoxazolidine phosphonates were evaluated against a variety of DNA and RNA viruses and several derivatives appeared to be active against varicella-zoster virus and human cytomegalovirus. Among all tested compounds, a 95:5 trans-15d/ cis-15d (95:5) (EC 50 ¼ 3.0 mM) and a cis-15b/trans-15b (87:13) (EC 50 ¼ 4.7 mM) showed the highest activity toward TK þ VZV strain.
The potency of these derivatives was 4e6 fold lower than that of acyclovir, used as reference drug.
On the other hand, compounds trans-13c, mixture cis-13c/trans-13c (86:14), trans-13d, and a 85:15 mixture of cis-13d/trans-13d exhibited potency not only against TK þ VZV strain but also toward TK À VZV strain and their anti-TK À VZV activity was significantly higher than that of the reference drugs acyclovir and brivudin (EC 50 ¼ 50.3 and 22.7 mM, respectively). The isoxazolidine phosphonates cis-15a-d/trans-15a-d having benzyl substituents both at N3 of the quinazoline-2,4-dione skeleton and at N2 of the isoxazolidine ring (cis-and trans-5) showed some activity toward human cytomegalovirus (EC 50 in the range of !3 to !14.5 mM). Table 2 Cycloadditions of the nitrones 16 or 17 and N 1 -allyl-N 3 -benzylquinazoline-2,4-diones 19a-d.
a Yield of the pure isomer. b Yield of the pure mixture of cis-and trans-isomers.
The quinazoline-2,4-diones endowed with anti-VZV and anti-HCMV activity did not alter the morphology of cells used in the antiviral assays up to a concentration of 100 mM. However, these derivatives showed considerable cytostatic activity. Cytostatic activity of the obtained compounds was also evaluated on L1210, CEM, HeLa and HMEC-1 cells. Among all tested compounds, only quinazoline-2,4-diones (trans-15/cis-15) bearing benzyl substituents at N3 in the quinazolinone core and the benzyl group at N2 of the isoxazolidine unit showed significant (IC 50 ¼ 10e98 mM) activity toward tested cell lines.
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 are 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 (70e230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F 254 . N-methyl-and N-benzyl-C-(diethoxyphosphoryl)nitrones 16 and 17 were obtained according to the literature procedures [28] .
To a solution of 1-allyl-1H-benzo[d] [1, 3] oxazine-2,4-dione 21 (0.500 g, 2.46 mmol) in DMF (10 mL) urea (0.221 g, 3.69 mmol) was added. The reaction mixture was stirred for 5 h, the solvent was removed in vacuo and the residue was crystallized from ethanol to give pure 22 as a yellowish amorphous solid, m.p. ¼ 218e219 C. To a solution of N-allylquinazoline-2,4-dione 22 (1.00 mmol) in acetonitrile (10 mL) triethylamine was added (3.00 mmol) followed by the respective benzoyl chloride (2.20 mmol). The mixture was stirred at room temperature for 72 h. The solvent was removed in vacuo, the residue was dissolved in methylene chloride (10 mL) and washed with water (3 Â 10 mL). The organic layer was dried (MgSO 4 ), concentrated and purified by column chromatography Table 6 The inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa) and immortalized human dermal microvascular endothelial cells (HMEC-1). 
To a solution of N-allylquinazoline-2,4-dione 22 (1.00 mmol) in acetonitrile (15 mL) potassium hydroxide (3.00 mmol) was added followed by the respective benzyl chloride (1.10 mmol). The reaction mixture was stirred at 105 C for 4 h. The solvent was removed in vacuo, the residue was dissolved in methylene chloride (10 mL) and washed with water (3 Â 10 mL). The organic layer was dried (MgSO 4 ), concentrated and the crude product was purified by column chromatography with methylene chloride-hexane mixture (7:3, v/v) and further crystallized from a chloroform-petroleum ether mixture.
An amorphous solid, m. 
The compounds were evaluated against different herpesviruses, including herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK e ) HSV-1 KOS strain resistant to ACV (ACV r ), herpes simplex virus type 2 (HSV-2) strain G, varicellazoster virus (VZV) strain Oka, TK e VZV strain 07-1, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as feline herpes virus (FHV), the poxvirus vaccinia virus (Lederle strain), para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, respiratory syncytial virus (RSV), feline coronovirus (FIPV) and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and influenza B virus (B/HK/5/72) and human immune deficiency virus (5HVV-1 and HIV-2). The antiviral assays, other than HIV, were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey kidney cells (Vero), human epithelial cervix carcinoma cells (HeLa), Crandell-Rees feline kidney cells (CRFK), or Madin Darby canine kidney cells (MDCK) . Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation (VZV) was recorded as soon as it reached completion in the control virusinfected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 or compound concentration required to reduce virus-induced cytopathicity or viral plaque formation by 50%. Cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that caused a microscopically detectable alteration of cell morphology.
Murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa) and immortalized human dermal microvascular endothelial cells (HMEC-1) were suspended at 300,000e500,000 cells/mL of culture medium, and 100 mL of a cell suspension was added to 100 mL of an appropriate dilution of the test compounds in 200 mL-wells of 96-well microtiter plates. After incubation at 37 C for two (L1210), three (CEM) or four (HeLa) days, the cell number was determined using a Coulter counter. The IC50 was defined as the compound concentration required to inhibit cell proliferation by 50%.