Isoxazolidine Conjugates of N3-Substituted 6-Bromoquinazolinones—Synthesis, Anti-Varizella-Zoster Virus, and Anti-Cytomegalovirus Activity

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

Despite the wide range of available drugs, effective treatment of viral infections is still limited. This is mainly due to the specificity of viral infections and the ability of viruses to mutate, which is associated with the acquisition of drug resistance. Therefore, new compounds with potential biological activity are being sought all the time. Quinazolinone and its derivatives have been extensively studied because of their wide range of biological activities, including antiviral [6], anticancer [7,8], antifungal [9], antibacterial [10,11], antitubercular [12,13], anti-inflammatory [11,14], anticonvulsant [15], hypolipidemic [16], analgesic [17], or immunotropic properties [18].

Chemistry
The respective 3-substituted 6-bromo-2-vinyl-3H-quinazolin-4-ones 13a-k were synthesized starting from the commercially available isatoic acid anhydride, which was first reacted with bromine and then transformed into 5-bromoanthranilic acid amide 14 according to the procedure in the literature [27,28]. Reaction 5-bromoanthranilic acid amide 14 with 3-chloropropionyl chloride followed by cyclization and dehydrohalogenation led to the formation of 6-bromo-2-vinyl-3H-quinazolin-4-one 13a, which was then used as a key intermediate in the preparation of N3-substituted derivatives 13b-13k via alkylation with the respective benzyl bromides, as well as with methyl or ethyl iodide [29] (Scheme 2).
Almost all synthesized isoxazolidines inhibited the replication of both TK + and TK − VZV strains at EC 50 's in the 5.4-13.6 µM range (Table 2), at the same time they were inactive toward other tested viruses, except for HCMV. In general, the inhibitory activity of the tested compounds toward TK − VZV 07-1 strain was better than that of the reference drugs acyclovir and brivudin (EC 50 = 39.2 µM and EC 50 = 31.9 µM, respectively), which require activation by the viral enzyme. On the other hand, the activity of these compounds against the TK + VZV OKA strain was five to nine-fold and 360 to 587-fold lower than that of the reference drugs acyclovir and brivudin, respectively. While exhibiting significant activity toward both TK + and TK − VZV stains, compounds trans-11b, trans-11d, trans-11g, and trans-11h exhibited the lowest cytotoxicity (CC 50 = 33-42 µM). It is worth mentioning that incorporation of the bromine atom at C6 in the quinazolinone skeleton resulted in a significant increase in potency of isoxazolidine-conjugates 11b-11i against VZV (up to five-fold higher) when compared with previously described analogous conjugates 10b-10i having an unsubstituted quinazoline skeleton [25]. The synthesized isoxazolidine phosphonates also showed marked activity against human cytomegalovirus (HCMV) ( Table 3) and, among them, trans-11d, trans-11g, trans-11h, and trans-11i/cis-11i (92:8) were the most potent ones. Activities of the tested compounds against both AD-169 and Davis strains were comparable (EC 50 = 8.9-12.5 µM) to that of Ganciclovir (EC 50 = 16.9 and 7.7 µM) used as a reference drug, but still lower than that of the other reference drug, Cidofovir (EC 50 = 1.5-1.7 µM).

General Procedure for the Synthesis of Isoxazolidines trans-11 and cis-11
A solution of the nitrone (0.195 g, 1.00 mmol) and the respective 6-bromo-2-vinylquinazolin-4(3H)-one (1.00 mmol) in toluene (2 mL) was stirred at 70 • C until the disappearance (TLC) of the starting nitrone. Solvents were evaporated in vacuo and crude products were subjected to chromatography on silica gel columns with chloroform:methanol (100:1, 50:1, 20:1, v/v) mixtures.  3.05 (s, 3H, CH 3 N), 1.42 (t, 3 J = 7.0 Hz, 3H, CH 3 CH 2 OP), 1.39 (t, 3 J = 6.9 Hz, 3H, CH 3 CH 2 OP). 13  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 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. Alternatively, the cytostatic activity of the test compounds was measured based on inhibition of cell growth. HEL cells were seeded at a rate of 5 × 10 3 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After three days of incubation at 37 • C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC 50 , or the compound concentration required to reduce cell proliferation by 50% relative to the number of cells in the untreated controls.

Cytostatic Activity against Immortalized Cell Lines
Murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa), and immortalized human dermal microvascular endothelial cells (HMEC-1) were suspended at 300,000-500,000 cells/mL of culture medium, and 100 µL of a cell suspension was added to 100 µL of an appropriate dilution of the test compounds in 200 µL-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 IC 50 was defined as the compound concentration required to inhibit cell proliferation by 50%.
It was proved that installation of functionalized benzyl groups at N3 in the quinazolinone moiety is essential for inhibitory properties toward both VZV and HCMV. At the same time, it was noticed that incorporation of the bromine atom at C6 in a quinazolinone skeleton resulted in a significant increase in potency of isoxazolidine-conjugates 11b-11i toward both VZV (up to five-fold higher) and HCMV (up to three-fold higher) when compared with previously described analogous conjugates 10b-10i lacking a bromine substituent at C6 of the quinazolinone moiety. Moreover, inhibitory properties of the newly synthesized compounds 11b-11k toward tested cell lines were also slightly higher than those of previously described analogues 10b-10k.
Author Contributions: The research group from the Medical University of Lodz (D.G.P. and M.G.D.) conceived the research project, participated in all steps of the research, interpreted the results, discussed the experimental data, and prepared the manuscript. The research group from KU Leuven (G.A., D.S., and R.S.) conducted the biological assays and provided the experimental procedures and results. All authors read, commented on, and approved the manuscript.