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Article

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

1
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
2
Laboratory of Virology and Chemotherapy, Rega Institute, Department of Microbiology, Immunology and Transplantation, KU Leuven, B-3000 Leuven, Belgium
3
Department of Pharmaceutical Microbiology and Microbiological Diagnostics, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(19), 6526; https://doi.org/10.3390/molecules27196526
Received: 14 September 2022 / Revised: 26 September 2022 / Accepted: 28 September 2022 / Published: 2 October 2022

Abstract

:
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.

Graphical Abstract

1. Introduction

Quinazoline-2,4-diones belong to an important class of nitrogen-containing heterocyclic compounds with a wide spectrum of biological activities, including anticancer [1,2,3,4,5], antihypertensive [6], hypoglycemic [7] and anticonvulsant [8] activities, among others. Considerable attention has been focused on studies of antimicrobial and antiviral activities of 1,3-substituted quinazoline-2,4-diones, as some of them have promising biological activity (Figure 1). For example, N1-methylquinazoline-2,4-diones 1a–e exhibited high antibacterial properties toward MRSA and B. subtilis and were slightly active against S. aureus [9], whereas compounds 2a–b appeared to be suitable gyrase inhibitors that are active toward multidrug-resistance Gram-positive bacterial strains [10]. On the other hand, substituted quinazoline-2,4-dione derivatives 3a–d displayed potent inhibitory activity against respiratory syncytial virus (RSV) (EC50 = 0.7–2.2 µM) [11], while compound 4 showed antiviral activity against HIV-1 and inhibited the recombinant RT in vitro [12], and N1-propargylquinazoline-2,4-dione 5 proved to be active towards adenovirus-2 (EC50 = 8.3 µM) [13].
Compounds with promising antiviral activity have been found as nucleoside and nucleotide analogues (Figure 2). For instance, 1,2,3-triazoles 6a–b [13] and their phosphonylated analogues 7–8 [1,14] functionalized with quinazoline-2,4-dione moiety, both designed as acyclic nucleoside analogues, appeared to be active against herpes simplex viruses (HSV-1 and HSV-2) (EC50 values in the range of 2.9–17 µM, 4–11 µM towards HSV-1 and HSV-2, respectively) [1,13,14]. Additionally, compound 6a inhibited the replication of varicella-zoster virus (VZV) at EC50 = 6.8–8.2, while derivatives 7 and 8 were also active toward feline herpes viruses (EC50 = 4, 24 µM for 7 and 8, respectively) [1,13,14]. Quinazoline-2,4-dione-containing nucleosides 9a–b showed antiviral activity against influenza virus (H1N1) (IC50 = 30–42 µM) [15].
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 anti-varicella-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).

2. Results and Discussion

2.1. Chemistry

The respective N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d were synthesized from the commercially available isatoic anhydride 25 (Scheme 2). Benzylation of 25 with the selected benzyl bromide [20], followed by the reaction of the resulted compounds 26a–d with urea, led to the formation of the respective N1-benzylquinazoline-2,4-diones 27a–d. Subsequent allylation of derivatives 27a–d with allyl bromide produced compounds 24a–d.
The 1,3-dipolar cycloadditions of the respective nitrone 22 (R = Me) or 23 (R = Bn) with the selected N3-allyl-N1-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 31P 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 1H 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.
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 N-allylquinazoline-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 trans- and cis-isoxazolidines 12a–c [18].

2.2. Antiviral and Antimicrobial Evaluation

2.2.1. Antiviral Activity

All synthesized alkenes 24a–d and the respective diastereoisomeric mixtures of isoxazolidines cis-20a/trans-20a (10:90), cis-20b/trans-20b (8:92), cis-20c/trans-20c (6:94), cis-20d/trans-20d (10:90), cis-21a/trans-21a (10:90), cis-21b/trans-21b (15:85), cis-21c/trans-21c (10:90), and cis-21d/trans-21d (15:85) were tested for inhibitory activity of a wide variety of DNA and RNA viruses, using the following cell-based assays: (a) human embryonic lung (HEL) cells: herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), herpes simplex virus-1 (TK KOS ACVr), cytomegalovirus (AD-169 strain, Davis strain), varicella-zoster virus (TK+ VZV OKA strain and TK VZV 07-1 strain), vaccinia virus, adenovirus-2, human coronavirus (229E); (b) HeLa cell cultures: vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus; (c) Vero cell cultures: para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, yellow fever virus; and (d) Madin–Darby canine kidney (MDCK) cell cultures: influenza A virus (H1N1 and H3N2 types), influenza B virus. Ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, alovudine, UDA, ribavirin, dextran sulfate (molecular weight 10000, DS.-10000), mycophenolic acid, zanamivir, amantadine, and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC50: the effective concentration required to reduce virus plaque formation (VZV, HCMV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses).
The synthesized compounds only showed some antiviral activity toward varicella-zoster virus, including both TK+ and TK VZV strains (Table 2). Among the series of tested compounds, isoxazolidines cis-20a–d/trans-20a–d and 21a–d/trans-21a–d showed higher inhibitory activity against VZV than the respective alkenes 24a–d. Isoxazolidiens cis-21b/trans-21b (15:85) and cis-21a/trans-21a (10:90) exhibited the highest activity against TK+ VZV OKA strain (EC50 = 12.63 µM and EC50 = 14.5 µM, respectively); however, this antiviral activity was lower than that of reference drug acyclovir. Moreover, preliminary structure–activity relationship observations revealed the slightly higher activity of compounds with benzyl group at nitrogen in the isoxazolidine ring in comparison to their N-methyl analogues (cis-21/trans-21 vs. analogous cis-20/trans-20).

2.2.2. Antimicrobial Activity

Due to the increasing resistance of bacteria to antibiotics, new compounds with antibacterial properties are being searched as alternatives to antibiotics. For this reason, the biological screening was expanded to evaluate the antimicrobial activity of alkenes 24a–d and diastereoisomeric mixtures of isoxazolidines (cis-20a/trans-20a (10:90), cis-20b/trans-20b (8:92), cis-20c/trans-20c (6:94), cis-20d/trans-20d (10:90), cis-21a/trans-21a (10:90), cis-21b/trans-21b (15:85), cis-21c/trans-21c (10:90), and cis-21d/trans-21d (15:85)) towards selected bacterial strains (E. faecalis ATCC 29212, S. aureus ATCC 2593, B. cereus PCM 1948, E. coli ATCC 25922, and P. aeruginosa ATCC 27853) and two fungal strains (C. albicans ATCC 10241 and A. brasiliensis ATCC 16404). Previous studies have shown that compounds containing a substituted quinazoline-2,4-dione moiety exhibit promising activity against Gram-negative and Gram-positive bacteria and fungi [9,23,24,25]. The antimicrobial activity was expressed as the MIC, minimal inhibitory concentrations, and the MBC, minimal bactericidal concentrations. Among all tested isoxazolidines, compounds cis-20c/trans-20c were found to be the most active against B. cereus PCM 1948 (MIC = 0.625 mg/mL). B. cereus is a Gram-positive spore-forming bacterium commonly found in the environment and can contaminate food. B. cereus bacteria can multiply rapidly at room temperature and produce toxins that can cause food poisoning of the diarrheal and vomiting type. B. cereus is also associated with infections of the eye, respiratory tract, and wounds [26,27]. Alkene 24c and isoxazolidines cis-20a/trans-20a, cis-21c/trans-21c and cis-21d/trans-21d showed noticeable activity against E. coli ATCC 25922 (MIC = 1.25 mg/mL). None of the synthesized compounds exhibited antifungal activity against tested strains (Table 3).
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 MPFTM 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.

3. Materials and Methods

3.1. General Information

1H, 13C, and 31P NMR spectra were taken in CDCl3 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 F254. N-methyl- and N-benzyl-C-(diethoxyphosphonyl)nitrones 14 and 15 were obtained according to procedures in the literature [19].
1H-, 13C- and 31P-NMR spectra of all new synthesized compounds are provided in Supplementary Materials.

3.2. General Procedure for Benzylation of 2H-Benzo[d][1,3]Oxazine-2,4-Diones 25

Sodium hydride (1.10 mmol) was added under argon atmosphere to a solution of 2H-benzo[d][1,3]oxazine-2,4-dione 25 (1.00 mmol) in anhydrous DMF (3 mL) and stirred at room temperature for 1 h. The respective benzyl bromide (1.10 mmol) was added and the reaction mixture was stirred for 18 h. The reaction mixture was poured onto ice water (20 mL). The suspension was filtered, washed with water (3 × 10 mL), and dried and crystallized from a chloroform-petroleum ether mixture to produce 26a–d.
1-Benzyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (26a). According to the general procedure from 2H-benzo[d][1,3]oxazine-2,4(1H)-dione 25 (1.00 g, 6.13 mmol), sodium hydride (0.162 g, 6.74 mmol), and benzyl bromide (0.838 mL, 6.74 mmol), 1-benzyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 26a (1.14 g, 73%) was obtained as an amorphous solid. M.p. 141–143 °C (lit. m.p. 140–142 °C) [20]. IR (KBr, cm–1) νmax: 3054, 2924, 1780, 1719, 1604, 1453, 1320, 1242, 1027, 758, 683. 1H NMR (600 MHz, CDCl3): δ = 8.19 (dd, J = 7.8 Hz, J = 1.4 Hz, 1H), 7.67–7.65 (m, 1H), 7.40–7.38 (m, 2H), 7.34–7.32 (m, 3H), 7.29 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 5.34 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 158.32 (C(O)), 148.48 (C(O)), 141.45, 137.17, 134.44, 130.87, 129.16, 128.15, 126.62, 124,15, 114.73, 111.90, 48.55. Anal. calcd. for C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.10; H, 4.16; N; 5.62.
1-(2-Fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (26b). According to the general procedure from 2H-benzo[d][1,3]oxazine-2,4(1H)-dione 25 (1.00 g, 6.13 mmol), sodium hydride (0.162 g, 6.74 mmol), and 2-fluorobenzyl bromide (0.838 mL, 6.74 mmol), 1-(2-fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 26b (1.16 g, 70%) was obtained as an amorphous solid, m.p. 149–151.5 °C (lit. m.p. 151–154 °C) [20]. IR (KBr, cm–1) νmax: 3052, 2932, 1787, 1720, 1585, 1456, 1331, 1228, 1034, 788, 681. 1H NMR (600 MHz, CDCl3): δ = 8.20 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.71–7.68 (m, 1H), 7.34–7.27 (m, 3H), 7.17–7.12 (m, 3H), 5.40 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 160.27 (d, 1J(CF) = 246.4 Hz, C2’), 158.17 (C(O)), 148.53 (C(O)), 141.10, 137.35, 130.95, 129.99 (d, 3J(CCCF) = 7.9 Hz, C6’), 128.43 (d, 3J(CCCF) = 3.7 Hz, C4’), 124.92 (d, 4J(CCCCF) = 3.9 Hz, C5’), 124.30, 121.57 (d, 2J(CCF) = 13.4 Hz, C3’), 115.86, 115.79 (d, 2J(CCF) = 21.2 Hz, C1’), 114.26 (d, 5J(CNCCCF) = 2.2 Hz), 111.85, 42.11 (d, 3J(CCCF) = 4.9 Hz). Anal. calcd. for C15H10FNO3: C, 66.42; H, 3.72; N, 5.16. Found: C, 66.71; H, 3.50; N, 5.29.
1-(3-Fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (26c). According to the general procedure from 2H-benzo[d][1,3]oxazine-2,4(1H)-dione 25 (1.00 g, 6.13 mmol), sodium hydride (0.162 g, 6.74 mmol), and 3-fluorobenzyl bromide (0.838 mL, 6.74 mmol), 1-(3-fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 26c (1.18 g, 71%) was obtained as an amorphous solid. M.p. 131–133 °C (lit. m.p.133–136 °C) [20]. IR (KBr, cm–1) νmax: 3058, 2926, 1786, 1723, 1592, 1451, 1321, 1251, 1029, 791, 681. 1H NMR (600 MHz, CDCl3): δ = 8.22 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.70–7.67 (m, 1H), 7.38–7.36 (m, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.05–7.02 (m, 2H), 5.33 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 163.24 (d, 1J(CF) = 247.8 Hz, C3), 158.09 (C(O)), 148.40 (C(O)), 141.23, 137.00 (d, 3J(CCCF) = 7.1 Hz, C5), 131.04, 130.87 (d, 3J(CCCF) = 8.3 Hz, C1), 124.34, 122.19 (d, 4J(CCCCF) = 2.8 Hz, C6), 115.23 (d, 2J(CCF) = 21.2 Hz, C2), 114.46, 113.75 (d, 2J(CCF) = 22.6 Hz, C4), 111.91, 48.06. Anal. calcd. for C15H10FNO3: C, 66.42; H, 3.72; N, 5.16. Found: C, 66.72; H, 3.56; N, 5.27.
1-(4-Fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (26d). According to the general procedure from 2H-benzo[d][1,3]oxazine-2,4(1H)-dione 25 (1.00 g, 6.13 mmol), sodium hydride (0.162 g, 6.74 mmol), and 4-fluorobenzyl bromide (0.838 mL, 6.74 mmol), 1-(4-fluorobenzyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 26d (1.29 g, 78%) was obtained as an amorphous solid. M.p. 141–143 °C (lit. m.p. 143–145 °C) [20]. IR (KBr, cm–1) νmax: 3022, 2924, 1702, 1680, 1607, 1485, 1324, 1221, 1018, 759, 686. 1H NMR (600 MHz, CDCl3): δ = 8.22 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.70–7.67 (m, 1H), 7.38–7.36 (m, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.05–7.02 (m, 2H), 5.33 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 162.46 (d, 1J(CF) = 247.4 Hz, C4), 158.21 (C(O)), 148.46 (C(O)), 141.24, 137.27, 131.10, 130.21 (d, 4J(CCCCF) = 3.2 Hz, C1), 128.56 (d, 3J(CCCF) = 8.5 Hz, C2, C6), 121.31, 116.18 (d, 2J(CCF) = 21.9 Hz, C3, C5), 114.52, 46.57. Anal. calcd. for C15H10FNO3: C, 66.42; H, 3.72; N, 5.16. Found: C, 66.61; H, 3.51; N, 5.27.

3.3. General Procedure for the Synthesis of 1-Benzylquinazoline-2,4-Diones 26a–d

Urea (1.50 mmol) was added to a solution of the respective 1-benzylbenzo[d][1,3]oxazine-2,4-dione 26a–d (1.00 mmol) in anhydrous DMF (10 mL) and the mixture was heated under reflux for 5 h. The solvent was removed in vacuo and the residue was crystallized from ethanol to produce the corresponding 27a–d.
1-Benzylquinazoline-2,4-dione (27a). According to the general procedure from 1-benzylbenzo[d][1,3]oxazine-2,4-dione 26a (0.500 g, 1.97 mmol) and urea (0.178 g, 2.96 mmol), 1-benzylquinazoline-2,4-dione 27a (0.238 g, 48%) was obtained as a white amorphous solid, m.p. 218–220 °C. IR (KBr, cm–1) νmax: 3170, 2923, 1704, 1605, 1480, 1311, 1151, 1020, 856, 722. 1H NMR (600 MHz, CDCl3): δ = 8.70 (s, 1H, NH), 8.25 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.60–7.58 (m, 1H), 7.38–7.36 (m, 2H), 7.32–7.29 (m, 3H), 7.26 (t, J = 7.9 Hz, 1H), 7.16 (t, J = 8.5 Hz, 1H), 5.39 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 161.84 (C(O)), 150.72 (C(O)), 141.11, 135.56, 135.39, 129.04, 128.81, 127.78, 126.50, 123.32, 116.13, 114.95, 46.58. Anal. calcd. for C15H11FN2O2: C, 71.42; H, 4.79; N, 11.10. Found: C, 71.12; H, 4.54; N, 10.87.
1-(2-Fluorobenzyl)quinazoline-2,4-dione (27b). According to the general procedure from 1-(2-fluorobenzyl)benzo[d][1,3]oxazine-2,4-dione 26b (0.500 g, 1.84 mmol) and urea (0.166 g, 2.76 mmol), 1-(2-fluorobenzyl)quinazoline-2,4-dione 27b (0.253 g, 49%) was obtained as a white amorphous solid, m.p. = 228–230 °C. IR (KBr, cm–1) νmax: 3177, 2922, 1701, 1606, 1457, 1371, 1230, 1020, 852, 749. 1H NMR (600 MHz, CDCl3): δ = 8.42 (s, 1H, NH), 8.25 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.64–7.61 (m, 1H), 7.32–7.27 (m, 2H), 7.19–7.13 (m, 3H), 7.11–7.09 (m, 1H), 5.44 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 161.49 (C(O)), 160.31 (d, 1J(CF) = 245.7 Hz, C2’), 150.58 (C(O)), 140.79, 135.72, 129.53 (d, 3J(CCCF) = 8.0 Hz, C6’), 128.87, 128.18 (d, 3J(CCCF) = 3.9 Hz, C4’), 124.76 (d, 4J(CCCCF) = 3.9 Hz, C5’), 123.47, 122.46 (d, 2J(CCF) = 14.2 Hz, C3’), 116.11, 115.65 (d, 2J(CCF) = 21.7 Hz, C1’), 114.47 (d, 5J(CNCCCF) =1.7 Hz), 40.18 (d, 3J(CCCF) = 5.4 Hz). Anal. calcd. for C15H11FN2O2: C, 66.66; H, 4.10; N, 10.37. Found: C, 66.55; H, 3.90; N, 10.60.
1-(3-Fluorobenzyl)quinazoline-2,4-dione (27c). According to the general procedure from 1-(3-fluorobenzyl)benzo[d][1,3]oxazine-2,4-dione 26c (0.500 g, 1.84 mmol) and urea (0.166 g, 2.76 mmol), 1-(3-fluorobenzyl)quinazoline-2,4-dione 27c (0.225 g, 45%) was obtained as a white amorphous solid, m.p. = 227–229 °C. IR (KBr, cm–1) νmax: 3176, 2964, 1691, 1607, 1439, 1317, 1244, 1020, 939, 738. 1H NMR (600 MHz, CDCl3): δ = 8.65 (s, 1H, NH), 8.25 (dd, J = 7.9Hz, J = 1.5 Hz, 1H), 7.63–7.60 (m, 1H), 7.36–7.34 (m, 1H), 7.33–7.27 (m, 1H), 7.12 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 7.02–6.99 (m, 2H), 5.37 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 163.25 (d, 1J(CF) = 247.7 Hz, C3), 161.46 (C(O)), 150.47 (C(O)), 140.91, 138.03 (d, 3J(CCCF) = 6.8 Hz, C5), 135.61, 130.68 (d, 3J(CCCF) = 7.9 Hz, C1), 128.96, 123.50, 122.07 (d, 4J(CCCCF) = 2.6 Hz, C6), 116.17, 114.85 (d, 2J(CCF) = 21.0 Hz, C2), 114.66, 113.61 (d, 2J(CCF) = 22.1 Hz, C4), 45.05 (d, 4J(CCCCF) = 1.6 Hz). Anal. calcd. for C15H11FN2O2 × 0.5 H2O: C, 64.51; H, 4.33; N, 10.03. Found: C, 64.41; H, 4.03; N, 10.31.
1-(4-Fluorobenzyl)quinazoline-2,4-dione (27d). According to the general procedure from 1-(4-fluorobenzyl)benzo[d][1,3]oxazine-2,4-dione 26d (0.500 g, 1.84 mmol) and urea (0.166 g, 2.76 mmol), 1-(4-fluorobenzyl)quinazoline-2,4-dione 27d (0.250 g, 50%) was obtained as a white amorphous solid, m.p. = 214–215 °C. IR (KBr, cm–1) νmax: 3153, 3020, 2922, 1679, 1606, 1437, 1321, 1224, 1018, 920, 759. 1H NMR (600 MHz, CDCl3): δ = 9.08 (s, 1H, NH), 8.26 (dd, J = 7.9Hz, J = 1.5 Hz, 1H), 7.62–7.60 (m, 1H), 7.31–7.26 (m, 3H), 7.15 (d, J = 8.5 Hz, 1H), 7.07–7.04 (m, 2H), 5.35 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3): δ = 162.29 (d, 1J(CF) = 246.8 Hz, C4), 161.71 (C(O)), 150.68 (C(O)), 140.96, 135.53, 131.18 (d, 4J(CCCCF) = 3.2 Hz, C1), 128.94, 128.36 (d, 3J(CCCF) = 7.9 Hz, C2, C6), 123.41, 116.21, 115.98 (d, 2J(CCF) = 21.9 Hz, C3, C5), 114.68, 45.93. Anal. calcd. for C15H11FN2O2: C, 66.66; H, 4.10; N, 10.37. Found: C, 66.83; H, 4.34; N, 10.57.

3.4. General Procedure for Allylation of 1-Benzylquinazolin-2,4-Dione 27a–d

Allyl bromide (2.20 mmol) was added to a suspension of the respective 1-benzylquinazoline-2,4-dione 27a–d (1.00 mmol) and potassium hydroxide (3.00 mmol) in anhydrous acetonitrile (15 mL). The reaction mixture was stirred at 105 °C for 4 h. The solvent was removed in vacuo and the residue was dissolved in methylene chloride (10 mL) and extracted with water (3 × 10 mL). The organic layer was dried (MgSO4), concentrated, and purified by column chromatography with a chloroform–hexane mixture (7:3, v/v), then crystallized from a chloroform–petroleum ether mixture to produce compounds 24a–d.
3-Allyl-1-benzylquinazoline-2,4-dione (24a). According to the general procedure from 1-benzylquinazoline-2,4-dione 27a (0.150 g, 0.594 mmol), potassium hydroxide (0.100 g, 1.78 mmol), and allyl bromide (0.113 mL, 1.31 mmol), 3-allyl-1-benzylquinazoline-2,4-dione 24a (0.174g, 89%) was obtained as a white amorphous solid, m.p. = 101–103 °C. IR (KBr, cm–1) νmax: 3063, 2963, 1701, 1606, 1454, 1341, 1272, 1025, 941, 762. 1H NMR (600 MHz, CDCl3): δ = 8.27 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.58–7.55 (m, 1H), 7.37–7.35 (m, 2H), 7.31–7.28 (m, 3H), 7.25–7.23 (m, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.03 (ddt, 3J = 17.1 Hz, 3J = 10.2 Hz, 3J = 5.8 Hz, 1H, CH2–CH=CH2), 5.42 (s, 2H, CH2Ph), 5.37 (dd, 3J = 17.1 Hz, 2J = 1.3 Hz, 1H, CH2–CH=CHH), 5.26 (dd, 3J = 10.2 Hz, 2J = 1.3 Hz, 1H, CH2–CH=CHH), 4.80 (d, 3J = 5.8 Hz, 2H, CH2–CH=CH2). 13C NMR (150 MHz, CDCl3): δ = 161.46 (C(O)), 151.17 (C(O)), 140.02, 135.74, 135.04, 131.93, 129.10, 128.98, 127.62, 126,48, 123.07, 117.64, 115.93, 115.80, 47.34, 44.01. Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 73.87; H, 5.23; N; 9.77.
3-Allyl-1-(2-fluorobenzyl)quinazoline-2,4-dione (24b). According to the general procedure from 1-(2-fluorobenzyl)quinazoline-2,4-dione 27b (0.250 g, 0.925 mmol), potassium hydroxide (0.156 g, 2.78 mmol), and allyl bromide (0.176 mL, 2.04 mmol), 3-allyl-1-(2-fluorobenzyl)quinazoline-2,4-dione 24b (0.269 g, 94%) was obtained as a white amorphous solid, m.p. = 93–94 °C. IR (KBr, cm–1) νmax: 3086, 2988, 1703, 1660, 1606, 1454, 1339, 1225, 1025, 935, 756. 1H NMR (600 MHz, CDCl3): δ = 8.28 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.61–7.58 (m, 1H), 7.31–7.25 (m, 2H), 7.15–7.11 (m, 3H), 7.10–7.07 (m, 1H), 5.95 (ddt, 3J = 17.1 Hz, 3J = 10.2 Hz, 3J = 5.8 Hz, 1H, CH2–CH=CH2), 5.47 (s, 2H, CH2Ph), 5.36 (dd, 3J = 17.1 Hz, 2J = 1.3 Hz 1H, CH2–CH=CHH), 5.27 (d, 3J = 10.2 Hz, 2J = 1.3 Hz, 1H, CH2–CH=CHH), 4.80 (d, 3J = 5.8 Hz, 2H, CH2–CH=CH2). 13C NMR (150 MHz, CDCl3): δ = 161.39 (C(O)), 160.33 (d, 1J(CF) = 246.2 Hz, C2), 151.23 (C(O)), 139.69, 135.22, 131.85, 129.39 (d, 3J(CCCF) = 8.0 Hz, C6), 129.16, 128.10 (d, 3J(CCCF) = 4.0 Hz, C4), 124.70 (d, 4J(CCCCF) = 3.6 Hz, C5), 123.23, 122.78 (d, 2J(CCF) = 13.8 Hz, C3), 117.96, 115.78, 115.61 (d, 2J(CCF) = 21.7 Hz, C1), 113.97 (d, 5J = 1.7 Hz), 44.03, 40.97 (d, 3J(CCCF) = 5.4 Hz). Anal. calcd. for C18H15FN2O2: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.77; H, 4.60; N, 9.26.
3-Allyl-1-(3-fluorobenzyl)quinazoline-2,4-dione (24c). According to the general procedure from 1-(3-fluorobenzyl)quinazoline-2,4-dione 27c (0.260 g, 0.962 mmol), potassium hydroxide (0.162 g, 2.89 mmol), and allyl bromide (0.183 mL, 2.12 mmol), 3-allyl-1-(3-fluorobenzyl)quinazoline-2,4-dione 24c (0.249 g, 83%) was obtained as a white amorphous solid, m.p. = 115–116 °C. IR (KBr, cm–1) νmax: 3091, 2963, 1700, 1655, 1604, 1485, 1342, 1252, 1001, 940, 929, 765. 1H NMR (600 MHz, CDCl3): δ = 8.28 (dd, J = 7.9 Hz, J = 1.4 Hz 1H), 7.60–7.57 (m, 1H), 7.35–7.32 (m, 1H), 7.29–7.26 (m, 1H), 7.10 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 7.01–6.97 (m, 2H), 5.95 (ddt, 3J = 17.1 Hz, 3J = 10.2 Hz, 3J = 5.8 Hz, 1H, CH2–CH=CH2), 5.40 (s, 2H, CH2Ph), 5.36 (dd, 3J = 17.1 Hz, 2J = 1.3 Hz, 1H, CH2–CH=CHH), 5.24 (d, 3J = 10.2 Hz, 2J = 1.3 Hz, 1H, CH2–CH=CHH), 4.80 (d, 3J = 5.8 Hz, 2H, CH2–CH=CH2). 13C NMR (150 MHz, CDCl3): δ = 163.23 (d, 1J(CF) = 247.6 Hz, C3), 161.33 (C(O)), 151.11 (C(O)), 139.81, 138.38 (d, 3J(CCCF) = 7.0 Hz, C5), 135.12, 131.83, 130.62 (d, 3J(CCCF) = 8.5 Hz, C1), 129.25, 123.26, 122.05 (d, 4J(CCCCF) = 3.1 Hz, C6), 118.05, 115.83, 114.72 (d, 2J(CCF) = 21.3 Hz, C2), 114.14, 113.57 (d, 2J(CCF) = 22.1 Hz, C4), 46.91, 44.05. Anal. calcd. for C18H15FN2O2: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.59; H, 4.58; N, 9.31.
3-Allyl-1-(4-fluorobenzyl)quinazoline-2,4-dione (24d). According to the general procedure from 1-(4-fluorobenzyl)quinazoline-2,4-dione 27d (0.215 g, 0.796 mmol), potassium hydroxide (0.134 g, 2.39 mmol), and allyl bromide (0.150 mL, 1.75 mmol), 3-allyl-1-(4-fluorobenzyl)quinazoline-2,4-dione 24d (0.159 g, 64%) was obtained as a white amorphous solid, m.p. = 120–122 °C. IR (KBr, cm–1) νmax: 3155, 2924, 1702, 1660, 1607, 1485, 1324, 1221, 1051, 967, 743. 1H NMR (600 MHz, CDCl3): δ = 8.28 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.60–7.57 (m, 1H), 7.29–7.25 (m, 3H), 7.13 (d, J = 8.5 Hz, 1H), 7.07–7.04 (m, 2H), 6.02 (ddt, 3J = 16.1 Hz, 3J = 10.2 Hz, 3J = 5.8 Hz, 1H, CH2–CH=CH2), 5.37 (s, 2H, CH2Ph), 5.36 (dd, 3J = 16.1 Hz, 2J = 1.4 Hz, 1H, CH2–CH=CHH), 5.26 (d, 3J = 10.2 Hz, 2J = 1.4 Hz, 1H, CH2–CH=CHH), 4.79 (d, 3J = 5.8 Hz, 2H, CH2–CH=CH2). 13C NMR (150 MHz, CDCl3): δ = 162.24 (d, 1J(CF) = 246.3 Hz, C4), 161.35 (C(O)), 151.13 (C(O)), 139.85, 135.07, 131.87, 131.49 (d, 4J(CCCCF) = 3.1 Hz, C1), 129.21, 128.31 (d, 3J(CCCF) = 7.86 Hz, C2, C6), 123.18, 117.99, 115.93 (d, 2J(CCF) = 21.8 Hz, C3, C5), 115.83, 114.17, 46.69, 44.01. Anal. calcd. for C18H15FN2O2: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.76; H, 4.60; N, 9.27.

3.5. General Procedure for the Synthesis of Isoxazolidines cis-20 and trans-20 As Well As cis-21 and trans-21

Solutions of nitrones 14 or 15 (1.00 mmol) and the respective N3-allylated quinazoline-2,4-dione 24a–d (1.00 mmol) in toluene were stirred at 60 °C until the starting nitrone disappeared. Solvents were evaporated in vacuo and the crude products were obtained as the mixtures of the respective diastereoisomeric isoxazolidines cis-20/trans-20 or cis-21/trans-21, which were then purified on a silica gel purified on silica gel column chromatography with chloroform–hexane mixtures as eluents.
Diethyl trans-5-((1-benzyl-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)-2-methylisoxazolidin-3-yl)phosphonate (trans-20a). Colorless oil. IR (film, cm–1) νmax: 2981, 1706, 1660, 1607, 1484, 1350, 1238, 1052, 1025, 968, 759. NMR signals of trans-20a were extracted from the spectra of 10:90 mixtures of cis-20a and trans-20a, 1H NMR (600 MHz, CDCl3): δ = 8.24 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.56–7.54 (m, 1H), 7.35–7.32 (m, 2H), 7.29–7.26 (m, 3H), 7.23–7.21 (m, 1H), 7.13 (d, J = 8.5 Hz, 1H), 5.41 (AB, JAB = 16.4 Hz, 1H, HCHN), 5.36 (AB, JAB = 16.4 Hz, 1H, HCHN), 4.53–4.48 (m, 2H, HC5, HCHN), 4.26 (dd, 2J = 16.7 Hz, 3J(HC–H5) = 8.4 Hz, 1H, HCHN), 4.23–4.16 (m, 4H, 2 × CH2OP), 3.10 (ddd, 3J(H3–H4β) = 9.7 Hz, 3J(H3–H4α) = 7.0 Hz, 2J(H3–P) = 2.3 Hz, 1H, HC3), 2.91 (s, 3H, CH3N), 2.67 (dddd, 3J(H4α–P) = 19.4 Hz, 2J(H4α–H4β) = 12.4 Hz, 3J(H4α–H3) = 7.0 Hz, 3J(H4β–H5) = 7.0 Hz, 1H, HαC4), 2.42 (dddd, 2J(H4β–H4α) = 12.4 Hz, 3J(H4β–P) = 12.4 Hz, 3J(H4β–H3) = 9.7 Hz, 3J(H4β–H5) = 6.4 Hz, 1H, HβC4), 1.35 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.34 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 161.683 (C(O)), 151.38 (C(O)), 139.97, 135.66, 135.12, 129.15, 128.95, 127.66, 126.49, 123.09, 115.68, 114.38, 74.49 (d, 3J(CCCP) = 7.7 Hz, C5), 63.89 (d, 1J(CP) = 168.7 Hz, C3), 63.11 (d, 2J(COP) = 6.4 Hz, CH2OP), 62.36 (d, 2J(COP) = 6.3 Hz, CH2OP), 47.41 (CH2N), 46.51 (d, 3J(CNCP) = 4.3 Hz, CH3N), 44.32 (CH2Ph), 36.27 (d, 2J(CCP) = 1.7 Hz, C4), 16.50 (d, 3J(CCOP) = 6.2 Hz, CH3CH2OP), 16.46 (d, 3J(CCOP) = 6.3 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.14. Anal.calcd. for. C24H30N3O6P: C, 59.13; H, 6.20; N, 8.62. Found: C, 59.38; H, 5.97; N, 8.91 (obtained on 10:90 mixtures of cis-20a and trans-20a).
Diethyl trans-(5-((1-(2-fluorobenzyl)-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)-2-methylisoxazolidin-3-yl)phosphonate (trans-20b). Colorless oil. IR (film, cm–1) νmax: 2981, 1707, 1661, 1608, 1483, 1351, 1231, 1052, 1023, 967, 756. NMR signals of trans-20b were extracted from the spectra of 8:92 mixtures of cis-20b and trans-20b, 1H NMR (600 MHz, CDCl3): δ = 8.24 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.60–7.57 (m, 1H), 7.28–7.23 (m, 2H), 7.14–7.10 (m, 3H), 7.07–7.05 (m, 1H), 5.46 (AB, JAB = 16.8 Hz, 1H, HCHN), 5.42 (AB, JAB = 16.8 Hz, 1H, HCHN), 4.54–4.48 (m, 2H, HC5, HCHN), 4.29–4.25 (m, 1H, HCHN), 4.24–4.1 (m, 4H, 2 × CH2OP), 3.12–3.09 (m, 1H, HC3), 2.91 (s, 3H, CH3N), 2.68 (dddd, 3J(H4α–P) = 19.8 Hz, 2J(H4α–H4β) = 12.7 Hz, 3J(H4α–H3) = 7.3 Hz, 3J(H4β–H5) = 7.3 Hz, 1H, HαC4), 2.43 (dddd, 2J(H4β–H4α) = 12.7 Hz, 3J(H4β–P) = 12.7 Hz, 3J(H4β–H3) = 11.2 Hz, 3J(H4β–H5) = 6.7 Hz, 1H, HβC4), 1.35 (t, 3J = 7.1 Hz, 3H, CH3CH2OP), 1.34 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 161.62 (C(O)), 160.30 (d, 1J(CF) = 245.6 Hz, C2’), 151.44 (C(O)), 139.65, 135.23, 129.40 (d, 3J(CCCF) = 8.0 Hz, C6’), 129.21, 128.13 (d, 3J(CCCF) = 3.7 Hz, C4’), 124.69 (d, 4J(CCCCF) = 3.3 Hz, C5’), 123.26, 122.69 (d, 2J(CCF) = 14.0 Hz, C3’), 115.66, 115.57 (d, 2J(CCF) = 21.9 Hz, C1’), 113.97 (d, 5J = 1.4 Hz), 74.46 (d, 3J(CCCP) = 7.7 Hz, C5), 63.86 (d, 1J(CP) = 168.6 Hz, C3), 63.15 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.38 (d, 2J(COP) = 7.1 Hz, CH2OP), 46.44 (d, 3J(CNCP) = 3.6 Hz, CH3N), 44.33 (CH2N), 41.01 (d, 3J(CCCF) = 5.2 Hz, CH2Ph), 35.22 (d, 2J(CCP) = 1.6 Hz, C4), 16.50 (d, 3J(CCOP) = 6.3 Hz, CH3CH2OP), 16.46 (d, 3J(CCOP) = 5.9 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 21.95. Anal.calcd. for C24H29FN3O6P × 1.5 H2O: C, 54.13; H, 6.06; N, 7.89. Found: C, 54.16; H, 5.77; N, 7.61 (obtained on 8:92 mixtures of cis-20b and trans-20b).
Diethyl trans-(5-((1-(3-fluorobenzyl)-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)-2-methylisoxazolidin-3-yl)phosphonate (trans-20c). Colorless oil. IR (film, cm–1) νmax: 2983, 1706, 1653, 1609, 1484, 1401, 1346, 1251, 1097, 1024, 967, 762. NMR signals of trans-20c were extracted from the spectra of 8:92 mixtures of cis-20c and trans-20c, 1H NMR (600 MHz, CDCl3): δ = 8.23 (dd, J = 7.9 Hz, J = 1.2 Hz, 1H), 7.57–7.55 (m, 1H), 7.32–7.28 (m, 1H), 7.24–7.22 (m, 1H), 7.07 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.97–6.94 (m, 2H), 5.40 (AB, JAB = 16.6 Hz, 1H, HCHN), 5.31 (AB, JAB = 16.6 Hz, 1H, HCHN), 4.51–4.46 (m, 2H, HC5, HCHN), 4.25 (dd, 2J = 16.4 Hz, 3J(HC–H5) = 8.1 Hz, 1H, HCHN), 4.21–4.14 (m, 4H, 2 × CH2OP), 3.10–3.07 (m, 1H, HC3), 2.89 (s, 3H, CH3N), 2.66 (dddd, 3J(H4α–P) = 19.3 Hz, 2J(H4α–H4β) = 12.8 Hz, 3J(H4α–H3) = 7.0 Hz, 3J(H4β–H5) = 7.0 Hz, 1H, HαC4), 2.40 (dddd, 2J(H4β–H4α) = 12.8 Hz, 3J(H4β–P) = 12.8 Hz, 3J(H4β–H3) = 10.2 Hz, 3J(H4β–H5) = 6.8 Hz, 1H, HβC4), 1.34 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.34 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 163.19 (d, 1J(CF) = 246.9 Hz, C3’), 161.55 (C(O)), 151.32 (C(O)), 139.75, 138.32 (d, 3J(CCCF) = 7.2 Hz, C5’), 135.22, 130.58 (d, 3J(CCCF) = 8.0 Hz, C1’), 129.26, 123.27, 122.10 (d, 4J(CCCCF) = 2.4 Hz, C6’), 115.69, 114.71 (d, 2J(CCF) = 21.1 Hz, C2’), 114.11, 113.60 (d, 2J(CCF) = 22.1 Hz, C4’), 75.43 (d, 3J(CCCP) = 7.7 Hz, C5), 63.88 (d, 1J(CP) = 168.8 Hz, C3), 63.11 (d, 2J(COP) = 6.3 Hz, CH2OP), 62.36 (d, 2J(COP) = 7.2 Hz, CH2OP), 46.95 (CH2N), 46.46 (d, 3J(CNCP) = 4.1 Hz, CH3N), 44.32 (CH2Ph), 35.06 (d, 2J(CCP) = 1.4 Hz, C4), 16.49 (d, 3J(CCOP) = 6.3 Hz, CH3CH2OP), 16.44 (d, 3J(CCOP) = 6.6 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.09. Anal.calcd. for C24H29FN3O6P × 2.5 H2O: C, 52.36; H, 6.23; N, 7.63. Found: C, 52.35; H, 5.95; N, 7.42 (obtained on 8:92 mixtures of cis-20c and trans-20c).
Diethyl trans-(5-((1-(4-fluorobenzyl)-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)-2-methylisoxazolidin-3-yl)phosphonate (trans-20d). Colorless oil. IR (film, cm–1) νmax: 2983, 1706, 1661, 1608, 1483, 1329, 1232, 1052, 967, 1024, 756. NMR signals of trans-20d were extracted from the spectra of 8:92 mixtures of cis-20d and trans-20d, 1H NMR (600 MHz, CDCl3): δ = 8.13 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.60–7.57 (m, 1H), 7.28–7.24 (m, 3H), 7.12–7.28 (d, J = 8.4 Hz, 1H), 7.06–7.03 (m, 2H), 5.31 (AB, JAB = 16.3 Hz, 1H, HCHN), 5.32 (AB, JAB = 16.3 Hz, 1H, HCHN), 4.61–4.52 (m, 1H, HC5), 4.50 (dd, 2J = 12.4 Hz, 3J(HC–H5) = 7.6 Hz, 1H, HCHN), 4.26 (dd, 2J = 12.4 Hz, 3J(HC–H5) = 3.8 Hz, 1H, HCHN), 4.24–4.17 (m, 4H, 2 × CH2OP), 3.14–3.12 (m, 1H, HC3), 2.70 (dddd, 3J(H4α–P) = 19.8 Hz, 2J(H4α–H4β) = 12.6 Hz, 3J(H4α–H3) = 7.3 Hz, 3J(H4β–H5) = 7.3 Hz, 1H, HαC4), 2.43 (dddd, 2J(H4β–H4α) = 12.6 Hz, 3J(H4β–P) = 12.6 Hz, 3J(H4β–H3) = 9.9 Hz, 3J(H4β–H5) = 6.7 Hz, 1H, HβC4), 1.37 (t, 3J = 7.1 Hz, 3H, CH3CH2OP), 1.36 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 162.25 (d, 1J(CF) = 246.3 Hz, C4’), 161.44 (C(O)), 151.35 (C(O)), 139.82, 135.19, 130.89 (d, 4J(CCCCF) = 3.0 Hz, C1’), 129.30, 128.32 (d, 3J(CCCF) = 8.4 Hz, C2’, C6’), 123.24, 115.17 (d, 2J(CCF) = 21.8 Hz, C3’, C6’), 115.71, 114.16, 74.67 (d, 3J(CCCP) = 7.8 Hz, C5), 63.81 (d, 1J(CP) = 163.9 Hz, C3), 63.25 (d, 2J(COP) = 6.4 Hz, CH2OP), 62.49 (d, 2J(COP) = 6.7 Hz, CH2OP), 46.80 (CH2N), 46.30 (d, 3J(CNCP) = 2.0 Hz, CH3N), 44.32 (CH2Ph), 36.16 (C4), 16.51 (d, 3J(CCOP) = 5.5 Hz, CH3CH2OP), 16.4 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 21.70. Anal.calcd. for C24H29FN3O6P × 1.5 H2O: C, 54.13; H, 6.06; N, 7.89. Found: C, 54.32; H, 5.90; N, 7.73 (obtained on 10:90 mixtures of cis-20d and trans-20d).
Diethyl trans-(2-benzyl-5-((1-benzyl-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)isoxazolidin-3-yl)phosphonate (trans-21a). Colorless oil. IR (film, cm–1) νmax: 2979, 1710, 1657, 1607, 1483, 1404, 1231, 1054, 1020, 967, 764. NMR signals of trans-21a were extracted from the spectra of 10:90 mixtures of cis-21a and trans-21a. 1H NMR (600 MHz, CDCl3): δ = 8.25 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 7.56–7.53 (m, 1H), 7.43–7.39 (m, 2H), 7.33–7.31 (m, 2H), 7.29–7.21 (m, 7H), 7.11 (d, J = 8.5 Hz, 1H), 5.40 (AB, JAB = 16.3 Hz, 1H, HCHN), 5.34 (AB, JAB = 16.3 Hz, 1H, HCHN), 4.57–4.53 (m, 1H, HC5), 4.49 (dd, 2J = 12.9 Hz, 3J(HC–H5) = 5.5 Hz, 1H, HCHN), 4.46 (d, 2J = 13.9 Hz, 1H, HCHPh), 4.26–4.18 (m, 5H, 2 × CH2OP, HCHN), 4.09 (d, 2J = 13.9 Hz, 1H, HCHPh), 3.42 (ddd, 3J(H3–H4β) = 9.2 Hz, 3J(H3–H4α) = 7.3 Hz, 2J(H3–P) = 3.0 Hz, 1H, HC3), 2.69 (dddd, 3J(H4α–P) = 17.3 Hz, 2J(H4α–H4β) = 12.6 Hz, 3J(H4α–H3) = 7.3 Hz, 3J(H4β–H5) = 7.3 Hz, 1H, HαC4), 2.42 (dddd, 2J(H4β–H4α) = 12.6 Hz, 3J(H4β–P) = 12.6 Hz, 3J(H4β–H3) = 9.2 Hz, 3J(H4α–H5) = 7.3 Hz, 1H, HβC4), 1.35 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.34 (t, 3J = 7.0 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 161.73 (C(O)), 151.35 (C(O)), 139.98, 137.17, 135.65, 135.11, 129.56, 128.98, 128.07, 127.63, 127.63, 127.18, 126.43, 123.10, 115.71, 114.42, 74.47 (d, 3J(CCCP) = 7.4 Hz, C5), 63.25 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.96 (d, 3J(CNCP) = 5.5 Hz, CH2Ph), 62.44 (d, 2J(COP) = 6.8 Hz, CH2OP), 60.90 (d, 1J(CP) = 170.3 Hz, C3), 47.43 (CH2N), 44.69 (CH2Ph), 35.49 (C4), 16.53 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP), 16.50 (d, 3J(CCOP) = 5.7 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.13. Anal.calcd. for C30H34N3O6P: C, 63.93; H, 6.08; N, 7.46. Found: C, 63.66; H, 6.38; N, 7.20 (obtained on 10:90 mixtures of cis-21a and trans-21a).
Diethyl trans-(2-benzyl-5-((1-(2-fluorobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(2H)-yl)methyl)isoxazolidin-3-yl)phosphonate (trans-21b). Colorless oil. IR (film, cm–1) νmax: 2981, 1710, 1657, 1607, 1482, 1354, 1246, 1054, 1020, 967, 764. NMR signals of trans-21b were extracted from the spectra of 15:85 mixtures of cis-21b and trans-21b. 1H NMR (600 MHz, CDCl3): δ = 8.26 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.60–7.57 (m, 1H), 7.42–7.38 (m, 2H), 7.29–7.21 (m, 5H), 7.14–7.10 (m, 2H), 7.07–7.05 (m, 1H), 7.03–7.00 (m, 1H), 5.45 (AB, JAB = 16.9 Hz, 1H, HCHN), 5.41 (AB, JAB = 16.9 Hz, 1H, HCHN), 4.56 (d, 2J = 13.9 Hz, 1H, HCHPh), 4.50 (dd, 2J = 12.8 Hz, 3J(HC–H5) = 7.5 Hz, 1H, HCHN), 4.40–4.39 (m, 1H, HC5), 4.26–4.18 (m, 5H, 2 × CH2OP, HCHN), 4.08 (d, 2J = 13.9 Hz, 1H, HCHPh), 3.42 (ddd, 3J(H3–H4β) = 9.1 Hz, 3J(H3–H4α) = 7.2 Hz, 2J(H3–P) = 3.0 Hz, 1H, HC3), 2.69 (dddd, 3J(H4α–P) = 19.7 Hz, 2J(H4α–H4β) = 12.8 Hz, 3J(H4α–H3) = 7.2 Hz, 3J(H4β–H5) = 7.2 Hz, 1H, HαC4), 2.42 (dddd, 3J(H4β–P) = 12.8 Hz, 2J(H4β–H4α) = 12.8 Hz, 3J(H4β–H3) = 9.1 Hz, 3J(H4β–H5) = 6.5 Hz, 1H, HβC4), 1.35 (t, 3J = 7.1 Hz, 3H, CH3CH2OP), 1.34 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 161.66 (C(O)), 160.29 (d, 1J(CF) = 245.6 Hz, C2’), 151.40 (C(O)), 139.66, 136.87, 135.30, 129.63, 129.35 (d, 3J(CCCF) = 8.3 Hz, C6’), 129.18, 128.08, 128.05 (d, 3J(CCCF) = 3.5 Hz, C4’), 127.26, 124.75 (d, 4J(CCCCF) = 3.3 Hz, C5’), 123.27 122.67 (d, 2J(CCF) = 14.0 Hz, C3’), 115.70, 115.57 (d, 2J(CCF) = 21.5 Hz, C1’), 113.98, 74.48 (d, 3J(CCCP) = 7.3 Hz, C5), 63.32 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.80 (d, 3J(CNCP) = 5.4 Hz, CH2Ph), 62.47 (d, 2J(COP) = 6.9 Hz, CH2OP), 60.79 (d, 1J(CP) = 169.6 Hz, C3), 44.68 (CH2N), 41.08 (d, 3J(CCCF) = 5.3 Hz, CH2Ph), 35.43 (d, 2J(CCP) = 1.6 Hz, C4), 16.53 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP), 16.49 (d, 3J(CCOP) = 5.8 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.12. Anal.calcd. for C30H33FN3O6P: C, 61.96; H, 5.72; N, 7.23. Found: C, 61.86; H, 5.57; N, 7.35 (obtained on 15:85 mixtures of cis-21b and trans-21b).
Diethyl trans-(2-benzyl-5-((1-(3-fluorobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(2H)-yl)methyl)isoxazolidin-3-yl)phosphonate (trans-21c). Colorless oil. IR (film, cm–1) νmax: 2978, 1704, 1656, 1607, 1483, 1351 1246, 1053, 1022, 968, 765. NMR signals of trans-21c were extracted from the spectra of 15:85 mixtures of cis-21c and trans-21c. 1H NMR (600 MHz, CDCl3): δ = 8.13 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.58 (ddd, J = 8.6 Hz, J = 7.3 Hz, J = 1.6 Hz, 1H), 7.42–7.38 (m, 2H), 7.31–7.21 (m, 5H), 7.07 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 7.00–6.94 (m, 2H), 5.39 (AB, JAB = 16.7 Hz, 1H, HCHN), 5.33 (AB, JAB = 16.7 Hz, 1H, HCHN), 4.56–4.47 (m, 1H, HC5), 4.50 (dd, 2J = 13.8 Hz, 3J(HC–H5) = 5.3 Hz, 1H, HCHN), 4.45 (d, 2J = 13.8 Hz, 1H, HCHPh), 4.26–4.17 (m, 5H, 2 × CH2OP, HCHN), 4.08 (d, 2J = 13.8 Hz, 1H, HCHPh), 3.41 (ddd, 3J(H3–H4β) = 9.2 Hz, 3J(H3–H4α) = 7.1 Hz, 2J(H3–P) = 3.1 Hz, 1H, HC3), 2.69 (dddd, 3J(H4α–P) = 17.3 Hz, 2J(H4α–H4β) = 12.9 Hz, 3J(H4α–H3) = 7.1 Hz, 3J(H4β–H5) = 7.1 Hz, 1H, HαC4), 2.42 (dddd, 2J(H4β–H4α) = 12.9 Hz, 3J(H4β–P) = 12.9 Hz, 3J(H4β–H3) = 9.2 Hz, 3J(H4β–H5) = 6.6 Hz, 1H, HβC4), 1.36 (t, 3J = 7.1 Hz, 3H, CH3CH2OP), 1.35 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 163.20 (d, 1J(CF) = 247.6 Hz, C3’), 161.61 (C(O)), 151.30 (C(O)), 139.78, 138.30 (d, 3J(CCCF) = 6.8 Hz, C5’), 137.11, 135.20, 130.65 (d, 3J(CCCF) = 8.0 Hz, C1’), 129.56, 129.25, 128.06, 127.19, 123.29, 122.01 (d, 4J(CCCCF) = 2.8 Hz, C6’), 115.75, 115.70 (d, 2J(CCF) = 21.0 Hz, C2’), 114.14, 113.57 (d, 2J(CCF) = 22.4 Hz, C4’), 75.45 (d, 3J(CCCP) = 7.1 Hz, C5), 63.25 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.94 (d, 3J(CNCP) = 5.7 Hz, CH2Ph), 62.45 (d, 2J(COP) = 6.8 Hz, CH2OP), 60.87 (d, 1J(CP) = 169.8 Hz, C3), 47.01 (CH2N), 44.73 (CH2Ph), 35.17 (d, 2J(CCP) = 1.7 Hz, C4), 16.51 (d, 3J(CCOP) = 5.7 Hz, CH3CH2OP), 16.48 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.09. Anal.calcd. for C30H33FN3O6P: C, 61.69; H, 5.72; N, 7.23. Found: C, 61.97; H, 5.89; N, 7.11 (obtained on 10:90 mixtures of cis-21c and trans-21c).
Diethyl trans-(2-benzyl-5-((1-(4-fluorobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(2H)-yl)methyl)isoxazolidin-3-yl)phosphonate (trans-21d). Colorless oil. IR (film, cm–1) νmax: 2978, 1710, 1657, 1607, 1482, 1353, 1243, 1054, 1020, 967, 764. NMR signals of trans-21d were extracted from the spectra of 15:85 mixtures of cis-21d and trans-21d. 1H NMR (600 MHz, CDCl3): δ = 8.24 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H), 7.59–7.57 (m, 1H), 7.42–7.38 (m, 2H), 7.27–7.22 (m, 6H), 7.10 (d, J = 8.5 Hz, 1H), 7.03–6.99 (m, 2H), 5.38 (AB, JAB = 15.1 Hz, 1H, HCHN), 5.30 (AB, JAB = 15.1 Hz, 1H, HCHN), 4.56–4.48 (m, 2H, HC5, HCHN), 4.46 (d, 2J = 13.8 Hz, 1H, HCHPh), 4.26–4.18 (m, 5H, 2 × CH2OP, HCHN), 4.08 (d, 2J = 13.8 Hz, 1H, HCHPh), 3.41 (ddd, 3J(H3–H4β) = 9.2 Hz, 3J(H3–H4α) = 7.1 Hz, 2J(H3–P) = 3.1 Hz, 1H, HC3), 2.70 (dddd, 3J(H4α–P) = 19.6 Hz, 2J(H4α–H4β) = 12.07 Hz, 3J(H4α–H3) = 7.1 Hz, 3J(H4β–H5) = 7.1 Hz, 1H, HαC4), 2.42 (dddd, 2J(H4β–H4α) = 12.7 Hz, 3J(H4β–P) = 12.7 Hz, 3J(H4β–H3) = 9.2 Hz, 3J(H4β–H5) = 6.3 Hz, 1H, HβC4), 1.37 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.36 (t, 3J = 7.0 Hz, 3H, CH3CH2OP). 13C NMR (150 MHz, CDCl3): δ = 162.21 (d, 1J(CF) = 246.5 Hz, C4’), 161.63 (C(O)), 151.31 (C(O)), 139.82, 137.18, 135.13, 131.37 (d, 4J(CCCCF) = 3.0 Hz, C1’), 129.52, 129.23, 128.85, 128.25 (d, 3J(CCCF) = 7.9 Hz, C2’, C6’), 128.06, 127.99, 127.18, 123.21, 115.93 (d, 2J(CCF) = 21.3 Hz, C3’, C5’), 115.77, 114.17, 74.42 (d, 3J(CCCP) = 7.0 Hz, C5), 63.23 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.95 (d, 3J(CNCP) = 5.5 Hz, CH2Ph), 62.45 (d, 2J(COP) = 6.8 Hz, CH2OP), 60.92 (d, 1J(CP) = 169.8 Hz, C3), 46.79 (CH2N), 44.75 (CH2Ph), 35.50 (d, 2J(CCP) = 1.8 Hz, C4), 16.52 (d, 3J(CCOP) = 5.4 Hz, CH3CH2OP), 16.48 (d, 3J(CCOP) = 5.4 Hz, CH3CH2OP). 31P NMR (243 MHz, CDCl3): δ = 22.09. Anal.calcd. for C30H33FN3O6P: C, 61.96; H, 5.72; N, 7.23. Found: C, 62.26; H, 5.80; N, 7.00 (obtained on 10:90 mixtures of cis-21d and trans-21d).

3.6. Antiviral Activity Assays

The compounds were evaluated against different herpesviruses, including herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK VZV strain 07-1, and human cytomegalovirus (HCMV) strains AD-169 and Davis as well as vaccinia virus, adenovirus-2, human coronavirus, parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, respiratory syncytial virus (RSV), and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87), and influenza B virus (B/HK/5/72), 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), 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 virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 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.

3.7. 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 108 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.

3.8. Microbial Mutagenicity Assay—The Ames Test

Mutagenicity was determined using a standard microplate AMES MPFTM 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.

4. Conclusions

Good yields (61–96%) and trans/cis diastereoselectivities (d.e = 70–84%) for the functionalized diastereoisomeric 3-(diethoxyphosphonyl)isoxazolidines trans-20/cis-20 and trans-21/cis-21 were observed in 1,3-dipolar cycloadditions of N-methyl- and N-benzyl-C-(diethoxyphosphonyl)nitrones 22 and 23 with selected N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d. The inhibitory activities of alkenes 24a–d and isoxazolidines 20 and 21 were assayed toward a broad panel of DNA and RNA viruses and several isoxazolidines appeared active toward VZV (EC50 = 12.63–58.48 µM). The inseparable mixtures of isoxazolidienes cis-21b/trans-21b (15:85) (EC50 = 12.63 µM) and cis-21a/trans-21a (10:90) (EC50 = 14.5 µM) showed the highest activity against TK+ VZV OKA strain, but much lower than that of the reference compounds acyclovir (EC50 = 0.49 µM) and brivudine (EC50 = 0.026 µM). On the other hand, the mixture of isoxazolidines cis-20c/trans-20c (6:94) inhibited the growth of B. cereus PCM 1948, the bacteria responsible for foodborne illnesses, showing the MIC value of 0.625 mg/mL while not being mutagenic at this concentration. Unfortunately, the observed MIC value was lower than that of the reference drug amikacin (MIC = 0.02 mg/mL).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27196526/s1, Figure S1: 1H NMR Spectrum for trans-20a in CDCl3; Figure S2: 31P NMR Spectrum for trans-20a in CDCl3; Figure S3: 13C NMR Spectrum for trans-20a in CDCl3; Figure S4: 1H NMR Spectrum for trans-20b in CDCl3; Figure S5: 31P NMR Spectrum for trans-20b in CDCl3; Figure S6: 13C NMR Spectrum for trans-20b in CDCl3; Figure S7: 1H NMR Spectrum for trans-20c in CDCl3; Figure S8: 31P NMR Spectrum for trans-20c in CDCl3; Figure S9: 13C NMR Spectrum for trans-20c in CDCl3; Figure S10: 1H NMR Spectrum for trans-20d in CDCl3; Figure S11: 31P NMR Spectrum for trans-20d in CDCl3; Figure S12: 13C NMR Spectrum for trans-20d in CDCl3; Figure S13: 1H NMR Spectrum for trans-21a in CDCl3; Figure S14: 31P NMR Spectrum for trans-21a in CDCl3; Figure S15: 13C NMR Spectrum for trans-21a in CDCl3; Figure S16: 1H NMR Spectrum for trans-21b in CDCl3; Figure S17: 31P NMR Spectrum for trans-21b in CDCl3; Figure S18: 13C NMR Spectrum for trans-21b in CDCl3; Figure S19: 1H NMR Spectrum for trans-21c in CDCl3; Figure S20: 31P NMR Spectrum for trans-21c in CDCl3; Figure S21: 13C NMR Spectrum for trans-21c in CDCl3; Figure S22: 1H NMR Spectrum for trans-21d in CDCl3; Figure S23: 31P NMR Spectrum for trans-21d in CDCl3; Figure S24: 13C NMR Spectrum for trans-21d in CDCl3; Figure S25: 1H NMR Spectrum for 24a in CDCl3; Figure S26: 13C NMR Spectrum for 24a in CDCl3; Figure S27: 1H NMR Spectrum for 24b in CDCl3; Figure S28: 13C NMR Spectrum for 24b in CDCl3; Figure S29: 1H NMR Spectrum for 24c in CDCl3; Figure S30: 13C NMR Spectrum for 24c in CDCl3; Figure S31: 1H NMR Spectrum for 24d in CDCl3; Figure S32: 13C NMR Spectrum for 24d in CDCl3; Figure S33: 1H NMR Spectrum for 26a in CDCl3; Figure S34: 13C NMR Spectrum for 26a in CDCl3; Figure S35: 1H NMR Spectrum for 26b in CDCl3; Figure S36: 13C NMR Spectrum for 26b in CDCl3; Figure S37: 1H NMR Spectrum for 26c in CDCl3; Figure S38: 13C NMR Spectrum for 26c in CDCl3; Figure S39: 1H NMR Spectrum for 26d in CDCl3; Figure S40: 13C NMR Spectrum for 26d in CDCl3; Figure S41: 1H NMR Spectrum for 27a in CDCl3; Figure S42: 13C NMR Spectrum for 27a in CDCl3; Figure S43: 1H NMR Spectrum for 27b in CDCl3; Figure S44: 13C NMR Spectrum for 27b in CDCl3; Figure S45: 1H NMR Spectrum for 27c in CDCl3; Figure S46: 13C NMR Spectrum for 27c in CDCl3; Figure S47: 1H NMR Spectrum for 27d in CDCl3; Figure S48: 13C NMR Spectrum for 27d in CDCl3.

Author Contributions

Conceptualization, M.Ł., I.E.G. and D.G.P.; methodology and investigation, M.Ł., I.E.G., G.A., D.S., R.S., P.L., M.S. and D.G.P.; (M.Ł., I.E.G. and D.G.P. carried out the synthesis of the compounds, interpreted the results, and characterized all the obtained compounds; G.A., D.S. and R.S. conducted the antiviral and cytostatic assays and provided the experimental procedures and results; P.L. and M.S. conducted the antimicrobial assays and provided the experimental procedures and results); resources and project administration, M.Ł., I.E.G. and D.G.P.; writing—original draft preparation, M.Ł., I.E.G. and D.G.P.; writing—review and editing, M.Ł., I.E.G., G.A., P.L., M.S. and D.G.P.; supervision, I.E.G. and D.G.P.; funding acquisition, D.G.P. All authors have read and agreed to the published version of the manuscript.

Funding

The synthetic part of this work and the antimicrobial assays were supported by the Medical University of Lodz internal funds (503/3-014-01/503-31-001 and 503/3-012-03/503-31-001). The virological part of this work was supported by the Rega Foundation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

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

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples are not available.

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Figure 1. Examples of quinazoline-2,4-dione derivatives showing antibacterial and antiviral activity.
Figure 1. Examples of quinazoline-2,4-dione derivatives showing antibacterial and antiviral activity.
Molecules 27 06526 g001
Figure 2. Examples of quinazoline-2,4-dione-containing nucleosides and nucleotides analogues.
Figure 2. Examples of quinazoline-2,4-dione-containing nucleosides and nucleotides analogues.
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Figure 3. Examples of natural nucleosides (uridine, 2′-deoxythymidine) and N3-substituted pyrimidine nucleotide analogues 10–11.
Figure 3. Examples of natural nucleosides (uridine, 2′-deoxythymidine) and N3-substituted pyrimidine nucleotide analogues 10–11.
Molecules 27 06526 g003
Scheme 1. The general structure of compound 12 and retrosynthesis of newly designed quinazoline-2,4-dione-conjugates of isoxazolidine 13.
Scheme 1. The general structure of compound 12 and retrosynthesis of newly designed quinazoline-2,4-dione-conjugates of isoxazolidine 13.
Molecules 27 06526 sch001
Scheme 2. Synthesis of N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d. Reaction and conditions: (a) selected benzyl bromide, NaH, DMF, r.t., 18 h; (b) urea, DMF, reflux, 5 h; (c) allyl bromide, MeCN, 105 °C, 4 h.
Scheme 2. Synthesis of N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d. Reaction and conditions: (a) selected benzyl bromide, NaH, DMF, r.t., 18 h; (b) urea, DMF, reflux, 5 h; (c) allyl bromide, MeCN, 105 °C, 4 h.
Molecules 27 06526 sch002
Scheme 3. Synthesis of isoxazolidines 20 and 21. Reaction and conditions: (a) toluene, 60 °C, 72 h.
Scheme 3. Synthesis of isoxazolidines 20 and 21. Reaction and conditions: (a) toluene, 60 °C, 72 h.
Molecules 27 06526 sch003
Table 1. Cycloadditions of the nitrone 22/23 and N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d.
Table 1. Cycloadditions of the nitrone 22/23 and N3-allyl-N1-benzylquinazoline-2,4-diones 24a–d.
Nitrone 22/23 (R)Alkene 24 (R′) 13cis:trans RatioYield (%)
22 (Me)Ph10:90cis-20a + trans-20a (87) a
22 (Me)2-F-C6H48:92cis-20b + trans-20b (80) a
22 (Me)3-F-C6H46:94cis-20c + trans-20c (96) a
22 (Me)4-F-C6H410:90cis-20d + trans-20d (92) a
23 (Bn)Ph10:90cis-21a + trans-21a (95) a
23 (Bn)2-F-C6H415:85cis-21b + trans-21b (71) a
23 (Bn)3-F-C6H410:90cis-21c + trans-21c (91) a
23 (Bn)3-F-C6H415:85cis-21d + trans-21d (84) a
a Yield of pure mixture of cis- and trans-isomers.
Table 2. Antiviral activity and cytotoxicity against varicella-zoster virus (VZV) in HEL cell cultures.
Table 2. Antiviral activity and cytotoxicity against varicella-zoster virus (VZV) in HEL cell cultures.
CompoundRR′Antiviral Activity EC50 (μM) aCytotoxicity (μM)
TK+ VZV Strain
(OKA)
TK VZV Strain
(07-1)
Cell Morphology
MCC b
cis-20a/trans-20a (10:90)MePh31.68100>100
cis-20b/trans-20b (8:92)Me2-F-C6H431.6858.48>100
cis-20c/trans-20c (6:94)Me3-F-C6H410058.48>100
cis-20d/trans-20d (10:90)Me4-F-C6H458.4838.07>100
cis-21a/trans-21a (10:90)BnPh14.534.2100
cis-21b/trans-21b (15:85)Bn2-F-C6H412.6327.59100
cis-21c/trans-21c (10:90)Bn3-F-C6H42014.4920
cis-21d/trans-21d (15:85)Bn4-F-C6H42020100
24a-Ph100100>100
24b-2-F-C6H4>10064.47>100
24c-3-F-C6H4>100>100>100
24d-4-F-C6H454.69>20100
Acyclovir 0.4923.22>440
Brivudin 0.02612.01>300
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). b Minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology.
Table 3. Antimicrobial activity of isoxazolidines cis-20/trans-20 and cis-2a/trans-2a, and alkene 24.
Table 3. Antimicrobial activity of isoxazolidines cis-20/trans-20 and cis-2a/trans-2a, and alkene 24.
CompoundE. faecalis
ATCC 29212
S. aureus
ATCC 2593
B. cereus
PCM 1948
E. coli
ATCC 25922
P. aeruginosa
ATCC 27853
C. albicans
ATCC 10241
A. brasiliensis
ATCC 16404
MIC a/MBC b
(mg/mL)
MIC a = MBC b
(mg/mL)
cis-20a/trans-20a (10:90)>5/>52.51.251.252.52.55
cis-20b/trans-20b (8:92)>5/>551.252.52.52.55
cis-20c/trans-20c (6:94)>5/>550.6252.52.52.55
cis-20d/trans-20d (10:90)>5/>551.252.52.52.55
cis-21a/trans-21a (10:90)>5/>551.252.52.555
cis-21b/trans-21b (15:85)>5/>551.252.52.52.55
cis-21c/trans-21c (10:90)>5/>552.51.252.52.55
cis-21d/trans-21d (15:85)>5/>551.251.252.52.55
24a>5/>5552.52.555
24b>5/>552.52.52.555
24c>5/>5551.252.52.55
24d>5/>5552.52.52.55
Amikacin0.0006250.0010.020.0006250.02--
Fluconazole-----0.005/>0.0050.005/>0.005
a Minimal inhibitory concentrations. b Minimal bactericidal concentrations.
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Łysakowska, M.; Głowacka, I.E.; Andrei, G.; Schols, D.; Snoeck, R.; Lisiecki, P.; Szemraj, M.; Piotrowska, D.G. Design, Synthesis, Anti-Varicella-Zoster and Antimicrobial Activity of (Isoxazolidin-3-yl)Phosphonate Conjugates of N1-Functionalised Quinazoline-2,4-Diones. Molecules 2022, 27, 6526. https://doi.org/10.3390/molecules27196526

AMA Style

Łysakowska M, Głowacka IE, Andrei G, Schols D, Snoeck R, Lisiecki P, Szemraj M, Piotrowska DG. Design, Synthesis, Anti-Varicella-Zoster and Antimicrobial Activity of (Isoxazolidin-3-yl)Phosphonate Conjugates of N1-Functionalised Quinazoline-2,4-Diones. Molecules. 2022; 27(19):6526. https://doi.org/10.3390/molecules27196526

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Łysakowska, Magdalena, Iwona E. Głowacka, Graciela Andrei, Dominique Schols, Robert Snoeck, Paweł Lisiecki, Magdalena Szemraj, and Dorota G. Piotrowska. 2022. "Design, Synthesis, Anti-Varicella-Zoster and Antimicrobial Activity of (Isoxazolidin-3-yl)Phosphonate Conjugates of N1-Functionalised Quinazoline-2,4-Diones" Molecules 27, no. 19: 6526. https://doi.org/10.3390/molecules27196526

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