First synthesis of merged hybrids phosphorylated azirino[2,1-b]benzo [e][1,3]oxazine derivatives as anticancer agents

1 First synthesis of merged hybrids phosphorylated azirino[2,1-b] benzo[e][1,3]oxazine derivatives as anticancer agents. Victor Carramiñana, Ana M. Ochoa de Retana, Jesús M. de los Santos*, Francisco Palacios* Department of Organic Chemistry I, Faculty of Pharmacy and Lascaray Research Center, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria, Spain. E-mail addresses: jesus.delossan[email protected]us / francisco.pa[email protected]s Keywords: antiproliferative effect, aziridines, 2H-azirines, merged hybrid compounds, phosphorus substituted azirino[2,1-b]benzo[e][1,3]oxazines. Abstract: This work describes a straightforward diastereoselective synthetic access to azirino[2,1b]benzo[e][1,3]oxazines containing phosphorus substituents such as phosphonate or phosphine oxide, by means of nucleophilic addition of functionalized phenols to the C–N double bond of 2H-azirine derivatives. In addition, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and human embryonic kidney (HEK293) was also screened. Some azirino[2,1-b]benzo[e][1,3]oxazines 4 and 6 exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over (HEK293), and nonmalignant cells (MCR-5) has been detected. 1. Introduction In the increasing field of irreversible inhibitor drug discovery, there is a new revival of the interest in covalent binding drugs owing to the FDA approval of safe and efficient covalent therapeutics and a better understanding of the profits of the covalent binding mechanism [1,2]. These facts are making irreversible inhibitors a robust comeback in cancer therapeutics [3,4]. Covalent inhibitors own many advantages over reversible inhibitors, including less frequent drug dosing due to the prolonged interval of action, improved biochemical efficiency, the potential to prevent determined drug resistance mechanisms, and a lower effective dose may result in high potential for greater therapeutic index [5]. In recent times, with the growth of targeted covalent inhibitors, many new drugs contain electrophilic moieties acting as “warheads”. Several molecules with a diversity of electrophilic warheads, including ketone, This is the accepted manuscript of the article that appeared in final form in European Journal of Medicinal Chemistry 185 : (2020) // Aritcle ID 111771, which has been published in final form at https://doi.org/10.1016/j.ejmech.2019.111771. © 2019 Elsevier Masson SAS under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 ester, nitrile, α,β-unsaturated carbonyl, epoxide, or aziridine functionalities have been recognized as covalent inhibitors [6]. Therefore, obtaining a scaffold with an appropriate platform for insertion of an electrophilic moiety at the right trajectory relative to the nucleophilic partner is being used as a further challenge. The aziridine three-membered ring is a structural component found in a wide range of biologically active agents, natural products, and related compounds. Aziridine-based inhibitors displayed high selectivity for parasite protozoon cysteine protease [7], while aziridine-2,3dicarboxylate was used as trypanocidal agents targeting the major cysteine protease of Trypanosoma brucei [8]. In addition, a biotinylated dibenzylaziridine-2,3-dicarboxylate was designed by Leippe et al. [9] as an irreversible cysteine protease inhibitor to target the malaria parasite Plasmodium falciparum. The antitumor and antibiotic properties of some of these compounds, including azinomycin B [10,11], mitomycin C [12,13], maduropeptin [14], and FR-900482 [15], a close cousin of mitomycin C since they share a similar structural analogy, are widely known. The therapeutic applications of some others are more varied [16]. For instance, ficellomycin [17] displays high in vivo activity against Gram-positive bacterias, as well as, multidrug resistant strains of Staphylococcus aureus; however, azicemicin [18,19] shows inhibitory activities toward Gram-negative bacterias and mycobacterias (Figure 1). O O O OH N O N H OOH O N OH AcO Azinomycin B NH O Mitomycin C H2N O O N OCONH2 H2N H N ON NH H2N HN OH O Ficellomycin NO OH OHC OH NH OCONH2 FR-900482 HO O OH O OH O O OH H OH Azicemicin A N Figure 1. Representative examples of covalent drugs with aziridine warheads. Electrophiles are highlighted. 3 Mitomycins are a class of pharmaceuticals with very potent antibacterial and anti-cancer activity. For instance, mitomycin C (Figure 1) has been in extensive clinical use for more than 20 years for its activity against bladder tumors, stomach, breast, esophagus, colorectal, and nonsmall-cell lung cancers [20]. This class of chemotherapy agents are the classical bioreductive drugs integrating a quinone moiety for reductive activation in vivo and subsequent electrophilic sites for DNA alkylation and covalent cross-linking. The aziridine ring present in these chemotherapeutics seems to cause DNA monoalkylation via acid-activation to produce a protonated aziridine that ring opens relieving the strain related to the three-membered ring [21,22]. Therefore, aziridines are potent alkylating agents which may act as covalent drugs through their capability to act as DNA cross-linking agents via nucleophilic ring opening of the three-membered heterocycle [23]. To overcome the drastic side effects related to a single drug, the development of hybrid molecules introducing two or more potentially pharmacophores to concurrently modulate several targets of multifactorial diseases, have been recognized as a popular approach for multidrug therapy [24]. Whereas aziridine compounds are valuable as reactive building blocks on their own [25,26,27], several aziridine-containing products have proved to possess biological activity, essentially owing to the presence of the aziridine moiety. Moreover, from a biological viewpoint, organophosphorus compounds are fascinating compounds as it is known that phosphorus substituents may modify the reactivity of heterocycles and control important biological functions [28], including new antileishmanial [29] and antiproliferative agents [30,31]. Strategies for incorporation of organophosphorus functionalities in adequate synthons have been extensively applied in the synthesis of phosphorylated azaheterocycles [32,33], aminophosphonates [34], or phosphinates [35]. Likewise, the benzoxazine framework is present in many active compounds of biological relevance, such as anti-malarials [36], antimycobacterials [37], anti-inflammatory [38], as well as potent inhibitors of methionyl-tRNA synthetase (MRS) [39] or DNA-PK enzyme with antiproliferative activity [40]. Considering these facts, here we wish to account the synthesis of new [1,3]benzoxazineaziridine merged hybrids with phosphorus containing groups such as phosphine oxide or phosphonate, through nucleophilic addition of functionalized salicylaldehydes to 2H-azirines. These new hybrid molecules, which may retain the functional properties of the parent molecules, are obtained by overlapping of [1,3]benzoxazine and aziridine frameworks, directly connected to the phosphorus substituent (Figure 2). Besides, these new hybrid molecules were tested for antiproliferative activity against different human cancer cell lines. Owing to the 4 inherent interest of these new hybrids, both in synthetic and medicinal chemistry, this entail an interesting challenge. As far as we know, this is the first example of the preparation of azirino[2,1-b]benzo[e][1,3]oxazines and their study as antiproliferative agents. O NPR 2 O O NHN PR 2 O 2H-benzo[ e][1,3]oxazine framework Aziridine moiety Introducing organophosphorus functionalities Figure 2. Phosphorylated azirino[2,1-b]benzo[e][1,3]oxazines as new merged hybrid molecules. 2. Results and discussion 2.1. Chemistry At first, we anticipated that nucleophilic addition of functionalized salicylaldehydes 1 to phosphorus substituted 2H-azirines 2, would provide a useful approach to the synthesis of functionalized azirino-benzoxazine derivatives 4 with phosphorus substituents at C–1 (Scheme 1). Therefore, the reaction of 2H-azirines 2 with salicylaldehyde derivatives 1 was assessed. Thus, as outlined in Scheme 1, in an initial experiment the nucleophilic addition of salicylaldehyde 1a (R1 = H) to 2H-azirine-phosphate 2a (R2 = Me, R = OiPr) was readily achieved using Et3N in dichloromethane at room temperature. Two equivalents of the salicyladehyde component were used in order to ensure full conversion. Under these reactions conditions, the azirino-benzoxazine derivative 4a was apparently obtained as a mixture of two isomers in a ratio of 71:29 (Table 1, entry 1), as evidenced by the presence of two sets of signals in 1H and 31P NMR spectra. We have recently reported a diastereoselective synthetic methodology for the preparation of phosphorus substituted cyanoaziridines [41]. These compounds have been obtained as a mixture of two rotamers attributed to the different geometries around the phosphorus atom. Assuming we could obtain a mixture of rotamers around the P–C bond of azirino-benzoxazine derivative 4a, as before, we achieve variable-temperature 1H NMR and 31P NMR studies for 4a 5 (R = OiPr, R1 = H, R2 = Me). These studies supported the hypothesis that the doubling of signals N R 2 PR 2 OCH 2 Cl 2 , 4 Å M.S. 0 ºC to rt 2a: R = O i Pr, R 2 = Me 2b: R = OEt, R 2 = Me 2c: R = Ph, R 2 = Me 2d: R = Ph, R 2 = Et 4 OH O+Et 3 N O N OH R 2 PR 2 O R 1 R 1 R 1 O N R 2 PR 2 OH O CH 2 Cl 2 , 4 Å M.S. 0 ºC to rt Et 3 N 3 7 1a: R 1 = H 1b: R 1 = 3-OMe 1c: R 1 = 5-Me 1d: R 1 = 5-F Scheme 1. Synthesis of azirino[2,1-b]benzo[e][1,3]oxazine phosphonates and phosphine oxides. in 1H, 31P, and 13C NMR spectra was due to the presence of two rotamers of compound 4a, since both rotamers interchange by rotation around the P–C bond (see supplementary data). Variable-temperature 31P NMR study for 4a in DMSO-d6 showed a mixture of two isomers in a ratio of 81:19 at 18 ºC. However, this mixture tend to equilibrate at higher temperatures since at 80 ºC the ratio of rotamers changed to 62:38. Similar results has been observed for variabletemperature 1H NMR study (see supplementary data). A slight difference in rotamers ratio can be detected by changing the solvent from CDCl3 to DMSO-d6 (see Table 1, entry 1). The fact that two rotamers were observed may be connected to the different geometries around the phosphorus atom, as previously observed in a benzofulvene-phosphine oxide [42], or more recently in a DFT study of the most stable rotamer of an aziridine-2-carboxylate [43]. The absence of coupling constant observed in 13C NMR spectrum between the di(iso-propyl) phosphonate substituent at C–1 and the methyl group at C–1a of azirino-benzoxazine 4a, appears to indicate a syn-relationship between both groups [44]. Conclusively, the X-ray diffraction analysis not only established the syn-relationship between the methyl group and the phosphorus atom, but also the anti-relationship between those groups and the hydroxyl group at C–7 (see supplementary data). 6 Table 1 Azirino[2,1-b]benzo[e][1,3]oxazine phosphonates and phosphine oxides 4 obtained. O N OH R 2 PR 2 O R 1 Entry Compound R R1 R2 Yield(%)[a] Rotamers ratio[b] 1 4a O i Pr H Me 81 71:29 (81:19) [ c] 2 4b OEt H Me 76 70:30 3 4c Ph H Me 55 76:24 4 4d Ph H Et 54 81:19 5 4e Ph 3-OMe Me 80 76:24 6 4f Ph 3-OMe Et 51 74:26 7 4g Ph 5-Me Me 84 75:25 8 4h Ph 5-Me Et 75 74:26 9 4i Ph 5-F Me 96 92:8 [a] Yield of isolated purified compounds 4. [b] Rotamers ratio calculated by 1 H NMR in CDCl3 solutions. [c] Rotamers ratio calculated by 1H NMR in DMSO-d6 solutions. Starting from 2H-azirine-phosphonate 2a and salicylaldehyde 1a, azirino-benzoxazine derivative 4a (R = OiPr, R1 = H, R2 = Me) was isolated in 81% yield (Scheme 1, Table 1, entry 1), while the addition of salicylaldehyde 1a to functionalized 2H-azirine-phosphonate 2b provide 76% of azirino-benzoxazine derivative 4b (R = OEt, R1 = H, R2 = Me) (Scheme 1, Table 1, entry 2). A reasonable mechanism for the construction of heterocycles 4 can be explained through initial nucleophilic addition of functionalized phenol 1 at the carbon-nitrogen double bond of 2H-azirine 2 to give intermediate 3. This nucleophilic addition is expected to arise in a diastereoselective way through the less hindered face. Hence, the nearness of phenol group of functionalized salicylaldehyde 1 to the C–N double bond of 2H-azirine 2 on the other side of the phosphonate group appears to be more favorable, due to the high exocyclic dihedral angle of the sp3 hybridized saturated carbon and the presence of the large phosphorus group. We have previously observed this remark in the nucleophilic addition of Grignard reagents [44,45], hydrides [46], alcohols [45], or cyanide anion [41] to 2H-azirines. After the nucleophilic addition, the construction of tricyclic azirino-benzoxazine framework in derivative 4 takes 7 place through a diastereoselective intramolecular nucleophilic attack of the aziridine moiety to the carbonyl group of intermediate 3, in a similar way to the previously reported for the reaction of 2H-azirine with enolates derived from β-keto esters[47]. This process was extended to the nucleophilic addition of salicylaldehyde 1a to 2H-azirinephosphine oxides 2c,d (R = Ph) under the same reaction conditions (Scheme 1). Azirinobenzoxazines 4c (R = Ph, R1 = H, R2 = Me, Table 1, entry 3) and 4d (R = Ph, R1 = H, R2 = Et, Table 1, entry 4) were obtained in moderate chemical yields and as a mixture of two rotamers. Further investigation of the process revealed that this strategy is also suitable to other salicylaldehyde derivatives 1. As illustrated in Table 1, the method is tolerant of a variety of functionalized salicylaldehydes 1 with varying substitutions. For instance, 2H-azirinephosphine oxides 2c,d (R = Ph) reacted with o-vanillin 1b (R1 = OMe), 5methylsalicylaldehyde 1c (R1 = Me), or 5-fluorosalizylaldehyde 1d (R1 = F) in the presence of Et3N, producing the corresponding azidinyl-benzoxazines 4e–i (see Table 1, entries 5–9). In order to define the scope and limitations of the nucleophilic addition of functionalized phenols to 2H-azirines 2 and increase the diversity of substituents in our substrates, this diastereoselective approach was extended to include the reactivity of 2-hydroxyacetophenone 5 toward phosphorus substituted 2H-azirines 2. For this purpose, the nucleophilic addition of 5 to 2H-azirine phosphonate 2a and phosphine oxides 2c,d were performed using Et3N as the base in CH2Cl2 to yield derivatives 6a–c in moderate yields (Table 2). These new azirinobenzoxazines 6 were obtained, as before, as a mixture of two rotamers in a ratio around 85:15. A similar configuration to that found in the case of salicylaldehydes was observed for azirinobenzoxazine 6a and was unambiguously proved by X-ray diffraction analysis (see supplementary data). This methodology describes a straightforward diastereoselective route to functionalized azirino[2,1-b]benzo[e][1,3]oxazine phosphonate and phosphine oxides 4 and 6 by means nucleophilic addition of functionalized phenol, such as salicylaldehyde derivatives 1 and 2hydroxyacetophenone 5, to phosphorus substituted 2H-azirines 2. To our knowledge, this approach represents the first example of an azirino[2,1-b]benzo[e][1,3]oxazine. 8 Table 2 Azirino[2,1-b]benzo[e][1,3]oxazine phosphonate and phosphine oxides 6 obtained. N R 2 PR 2 OCH 2 Cl 2 , 4 Å M.S. 0 ºC to rt 2a: R = O i Pr, R 2 = Me 2c: R = Ph, R 2 = Me 2d: R = Ph, R 2 = Et 6a-c OH O+Et 3 N O N R 2 PR 2 O 5 Me MeHO Entry Compound R R2 Yield(%)[a] Rotamers ratio[b] 1 6a O i Pr Me 35 85:15 (  100:0)[c] 2 6b Ph Me 57 85:15 3 6c Ph Et 62 87:13 [a] Yield of isolated purified compounds 6. [b] Rotamers ratio calculated by 1 H NMR in CDCl 3 solutions. [c] Rotamers ratio calculated by 1H NMR in DMSO-d 6 solutions. 2.2. Biological results In vitro cytotoxicity of our newly prepared phosphorylated azirino[2,1b]benzo[e][1,3]oxazines 4 and 6 was assessed by testing their antiproliferative activities against two different human cancer cell lines: A549 (carcinomic human alveolar basal epithelial cells) and HEK-293 (human embryonic kidney cells). Cell counting kit (CCK-8) assay was applied to evaluate growth inhibition. Table 3 displays cell proliferation inhibitory activities as IC50 values of all synthesized compounds and chemotherapeutic doxorubicin (DOX). As reported in table 3, tested compounds exhibited a wide spectrum of antiproliferative activity against the cancer cell lines tested in culture. In general, compounds 4 and 6 presented a greater selective cytotoxicity in the human lung adenocarcinoma cell line (A549) than in the human embryonic kidney cell line (HEK-293). Likewise, healthy lung cells, such as MRC-5 non-malignant lung fibroblasts were tested for studying selective cytotoxicity [48], and as outlined in table 3, none of the synthesized compounds 4 and 6 exhibited any toxicity toward MRC-5 cells. 9 Table 3 Antiproliferative activity of azirino[2,1-b]benzo[e][1,3]oxazines 4 and 6. O N R 2 PR 2 O R 3 HO R 1 Entry Comp. R R1 R2 R3 Cytotoxicity IC50 (M)[a] lung A549 kidney HEK-293 MRC-5 1 DOX – – – 0.48 ± 0.017[49] 5.68 ± 1.56 [50] >50 [51] 2 4a O i Pr H Me H 3.0  0.29 >50 >50 3 4b OEt H Me H 15.8  2.44 n.r. [b] >50 4 4c Ph H Me H 2.7  0.37 >50 >50 5 4d Ph H Et H 10.4  0.68 >50 >50 6 4e Ph 3-OMe Me H 1.3  0.22 >50 >50 7 4f Ph 3-OMe Et H 20.7  3.75 >50 >50 8 4g Ph 5-Me Me H 3.2  0.80 >50 >50 9 4h Ph 5-Me Et H 12.9  0.90 >50 >50 10 4i Ph 5-F Me H 19.6  0.24 >50 >50 11 6a O i Pr H Me Me 10.3  1.67 >50 >50 12 6b Ph H Me Me 21.1  2.44 >50 >50 13 6c Ph H Et Me 3.2  0.27 >50 >50 [a] The cytotoxicity IC50 values listed are the concentrations corresponding to 50% growth inhibition. [b] Not reported. Regarding the new azirino-benzoxazines 4 against A549 cell line in vitro, diisopropyl 4a and diethyl azirino-benzoxazine phosphonate 4b showed dissimilar IC50 values between 3.0  0.29 and 15.8 2.44 M with the most effective compound being diisopropyl azirinobenzoxazine 4a (R = OiPr, R1 = R3 =H, R2 = Me) (Table 3, compare entries 1 and 2). As previously reported [52], the presence of a bulky group at the phosphonate ester moiety give rise to an increased activity. Azirino-benzoxazine phosphine oxides 4c–i (Table 3, entries 3– 10) showed IC50 values between 1.3  0.22 and 20.7  3.75 M with the most effective derivative being azirino-benzoxazine phosphine oxide 4e (R = Ph, R1 = 3-OMe, R2 = Me, R3 =H) with an IC50 value of 1.3  0.22 M. Concerning the effect of the substitution at C–1a, in 16 107.4 Hz, CH)major, 26.9 (CH2)major, 26.8 (CH2)minor, 9.5 (CH3)major, 9.2 (CH3)minor, ppm; 31P NMR (120 MHz, CDCl3) δ 24.9major, 23.7minor ppm; ESI-HRMS (CI) m/z calcd. for C24H25NO4P ([M+H]+) 422.1521 , found 422.1508. ((1S*,1aS*,7S*)-7-Hydroxy-1a,5-dimethyl-1,1a-dihydro-7Hazirino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (4g). (1.65 g, 84%) as a yellow solid from 2H-azirine 2c (1.27 g, 5 mmol, 1 eq) and 2-hydroxy-5-methylbenzaldehyde 1c (1.36 g, 10 mmol, 2 eq) as described in the general procedure. The crude product was purified by crystallization from hexane/CH2Cl2 (50:50) to afford the title compound 4g. mp 168–170 ºC; IR (neat) vmax 3242, 3056, 2867, 1496, 1438, 1435, 1177, 1069, 724 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.83–6.69 (m, 13H, ArH), 5.75 (s, 1H, CHOH)major, 5.48 (s, 1H, CHOH)minor, 5.23 (bs, 1H, OH), 2.98 (d, 2JPH = 21.8 Hz, 1H, CH-P)major, 2.64 (d, 2JPH = 22.3 Hz, 1H, CHP)minor, 2.25 (s, 3H, CH3)major, 2.23 (s, 3H, CH3)minor, 1.88 (s, 3H, CH3)minor, 1.83 (s, 3H, CH3)major ppm; 13C {1H} NMR (100 MHz, CDCl3)  146.4minor, 146.3major, 132.9, 132.5, 132.2, 132.2, 132.1, 132.0, 131.9, 131.9, 131.8, 131.4, 131.4, 131.3, 131.2, 130.4, 129.3, 129.0, 128.9, 128.8, 128.6, 128.5, 127.7, 120.2major, 118.8minor, 117.1minor, 116.1major (CAr), 80.8 (d, 3JPC = 4.6 Hz, CH)minor, 76.7 (d, 3JPC = 3.6 Hz, CH)major, 74.2 (d, 2JPC = 4.4 Hz, Cquat)major, 74.0 (d, 2JPC = 5.4 Hz, Cquat)minor, 35.8 (d, 1JPC = 106.5 Hz, CH)minor, 32.8 (d, 1JPC = 107.6 Hz, CH)major, 20.9 (CH3)major, 20.8 (CH3)minor, 20.6 (CH3)minor, 20.1 (CH3)major ppm; 31P NMR (120 MHz, CDCl3) δ 25.7major, 24.3minor ppm; ESI-HRMS (CI) m/z calcd. for C23H23NO3P ([M+H]+) 392.1416, found 392.1403. ((1S*,1aS*,7S*)-1a-Ethyl-7-hydroxy-5-dimethyl-1,1a-dihydro-7Hazirino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (4h). (1.57 g, 75%) as a pale yellow solid from 2H-azirine 2d (1.34 g, 5 mmol, 1 eq) and 2-hydroxy-5-methylbenzaldehyde 1c (1.36 g, 10 mmol, 2 eq) as described in the general procedure. The crude product was purified by crystallization from hexane/Et2O (50:50) to afford the title compound 4h. mp 122– 124 ºC; IR (neat) vmax 3243, 3060, 2977, 1619, 1590, 1495, 1438, 1175, 1119, 730, 701 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.83–6.71 (m, 13H, ArH), 5.73 (d, 3JHH = 3.9 Hz, 1H, CHOH)major, 5.48 (bs, 1H, CHOH)minor, 5.21 (d, 3JHH = 3.9 Hz, 1H, OH)major, 3.89 (d, 3JHH = 2.4 Hz, 1H, OH)minor, 3.03 (d, 2JPH = 21.8 Hz, 1H, CH-P)major, 2.69 (d, 2JPH = 22.7 Hz, 1H, CHP)minor, 2.25 (s, 3H, CH3)major, 2.23 (s, 3H, CH3)minor, 2.25–2.06 (m, 2H, CH2), 1.11 (t, 3JHH = 7.4 Hz, 3H, CH3)minor, 1.03 (t, 3JHH = 7.4 Hz, 3H, CH3)major ppm; 13C {1H} NMR (75 MHz, DMSO)  146.4minor, 146.1major, 134.4, 133.3, 133.0, 131.9, 131.8, 131.6, 131.5, 131.4, 131.3, 130.9, 130.8, 130.6, 130.5, 130.1, 129.1, 128.9, 128.8, 128.7, 128.6, 128.6, 128.3, 128.1, 127.1, 17 120.7, 120.5, 119.9, 116.3, 115.9, (CAr), 79.2 (d, 3JPC = 5.0 Hz, CH)minor, 77.3 (d, 2JPC = 5.2 Hz, Cquat)minor, 76.8 (d, 2JPC = 4.5 Hz, Cquat)major, 76.2 (d, 3JPC = 3.5 Hz, CH)major, 35.6 (d, 1JPC = 106.0 Hz, CH)minor, 33.0 (d, 1JPC = 108.4 Hz, CH)major, 26.3 (CH2), 20.4 (CH3)major, 20.2 (CH3)minor, 9.3 (CH3)minor, 9.2 (CH3)major ppm; 31P NMR (120 MHz, CDCl3) δ 25.1major, 23.9minor ppm; ESI-HRMS (CI) m/z calcd. for C24H25NO3P ([M+H]+) 406.1572, found 406.1561. ((1S*,1aS*,7S*)-5-Fluoro-7-hydroxy-1a-methy-1,1a-dihydro-7Hazirino[2,1-b]benzo[e][1,3]oxazin-1-yl) diphenylphosphine oxide (4i). (1.90 g, 96%) as a white solid from 2H-azirine 2c (1.27 g, 5 mmol, 1 eq) and 5-fluorosalicylaldehyde 1d (0.84 g, 10 mmol, 1.2 eq) as described in the general procedure. The crude product was purified by crystallization from hexane/CH2Cl2 (50:50) to afford the title compound 4i. mp 169–171 ºC; IR (neat) vmax 3417, 2930, 2857, 1483, 1432, 1182, 1141, 732, 691 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.87-6.74 (m, 13H, ArH), 5.92 (d, 3JHH = 4.3 Hz, 1H, CHOH)major, 5.69 (d, 3JHH = 4.0 Hz, 1H, OH)major, 5.47 (bs, 1H, CHOH)minor, 4.77 (bs, 1H, OH)minor, 2.91 (d, 2JPH = 21.7 Hz, 1H, CH-P)major, 2.60 (d, 2JPH = 22.0 Hz, 1H, CH-P)minor, 1.85 (s, 3H, CH3)minor, 1.83 (s, 3H, CH3)major ppm; 13C {1H} NMR (75 MHz, DMSO)  159.1, 155.1, 144.6minor, 144.4major, 134.3, 133.0, 133.0, 131.9, 131.7, 131.5, 131.4, 131.0, 130.9, 130.6, 130.5, 128.8, 128.8, 128.7, 128.3, 128.2, 122.0, 119.9, 117.9, 116.6, 116.3, 113.3, 113.0, (CAr), 79.3 (d, 3JPC = 4.7 Hz, CH)minor, 75.7 (d, 3JPC = 3.0 Hz, CH)major, 74.0 (d, 2JPC = 5.3 Hz, Cquat)minor, 73.6 (d, 2JPC = 4.3 Hz, Cquat)major, 35.2 (d, 1JPC = 105.5 Hz, CH)minor, 32.2 (d, 1JPC = 108.0 Hz, CH)major, 20.1 (CH3)minor, 19.7 (CH3)major ppm; 31P NMR (120 MHz, CDCl3) δ 25.9major, 24.5minor ppm; 19F {1H} NMR (282 MHz, CDCl3) δ –119.8 ppm; ESI-HRMS (CI) m/z calcd. for C22H20FNO3P ([M+H]+) 396.1165, found 396.1153. 4.1.3.2. General procedure and spectral data for the reaction of functionalized 2H-azirines 2 with 2-hydroxyacetophenone 5. To a 0 ºC solution of 2H-azirine (5 mmol, 1 eq) 2 in CH2Cl2 (25 mL) and 4 Å M.S., was added dropwise 2-hydroxyacetophenone 5 (1.20 mL, 10 mmol, 2 eq) and Et3N (2.09 mL, 15 mmol, 3 eq) under a nitrogen atmosphere. The reaction mixture was allowed to reach room temperature and stirred at the same temperature until TLC showed the disappearance of 2H-azirine 2 (72–120 h). 4 Å M.S. was filtered through a sintered glass vacuum filtration funnel with celite and washed with CH2Cl2. The reaction mixture was washed with water (315 mL) and extracted with CH2Cl2 (15 mL). The organic layers were dried over anhydrous MgSO4, filtered and concentrated to dryness in vacuum. The crude product was purified by crystallization or flash-column chromatography to afford the corresponding azirinobenzoxazine derivatives 6. 18 Diisopropyl ((1S*,1aS*,7S*)-7-hydroxy-1a,7-dimethyl-1,1a-dihydro-7H-azirino[2,1-b]benzo[e][1,3]oxazin-1-yl)phosphonate (6a). (0.63 g, 35%) as a white solid from 2H-azirine 2a (1.27 g, 5 mmol, 1 eq) as described in the general procedure after stirring at room temperature for 120 h. The crude product was purified by flash-column chromatography (SiO2, AcOEt/hexane 50:50) to afford the title compound 6a. mp 122–124 ºC; IR (neat) vmax 3331, 2981, 2934, 1612, 1587, 1374, 1280, 1228, 1011, 986 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.48–7.46 (m, 1H, ArH), 7.23–7.21 (m, 1H, ArH), 7.06–6.99 (m, 1H, ArH), 6.83–6.81 (m, 1H, ArH), 4.74–4.68 (m, 2H, CH), 3.27 (d, 3JPH = 5.7 Hz, 1H, OH)major, 2.48 (bs, 1H, OH)minor, 2.38 (d, 2JPH = 12.3 Hz, 1H, CH-P)major, 2.14 (d, 2JPH = 12.5 Hz, 1H, CH-P)minor, 1.90 (s, 3H, CH3)minor, 1.84 (s, 3H, CH3)major, 1.83 (s, 3H, CH3)minor, 1.63 (s, 3H, CH3)major, 1.34–1.28 (m, 12H, CH3) ppm; 13C {1H} NMR (100 MHz, CDCl3)  148.1minor, 147.5major, 130.4, 129.5, 126.6, 126.3, 126.2, 125.8, 123.2, 123.0, 117.4, 116.5 (CAr), 83.5 (d, 3JPC = 7.0 Hz, Cquat)minor, 83.5 (d, 3JPC = 4.9 Hz, Cquat)major, 73.7 (d, 2JPC = 4.4 Hz, Cquat), 71.4 (d, 2JPC = 6.4 Hz, OCH), 71.1 (d, 2JPC = 6.7 Hz, OCH), 32.0 (CH3), 31.4 (d, 1JPC = 211.5 Hz, CH), 24.2 (d, 3JPC = 3.4 Hz, CH3), 24.1 (d, 3JPC = 4.4 Hz, CH3), 24.1 (d, 3JPC = 3.0 Hz, CH3), 23.9 (d, 3JPC = 5.5 Hz, CH3), 20.3 (CH3)minor, 20.0 (CH3)major ppm; 31P NMR (120 MHz, CDCl3) δ 19.6major, 19.1minor ppm; ESIHRMS (CI) m/z calcd. for C17H27NO5P ([M+H]+) 356.1627, found 356.1628. ((1S*,1aS*,7S*)-7-Hydroxy-1a,7-dimethyl-1,1a-dihydro-7Hazirino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (6b). (1.11 g, 57%) as a white solid from 2H-azirine 2c (1.27 g, 5 mmol, 1 eq) as described in the general procedure after stirring at room temperature for 96 h. The crude product was purified by crystallization from Et2O and washed several times with pentane to afford the title compound 6b. mp 163–165 ºC; IR (neat) vmax 3250, 2977, 2930, 1613, 1594, 1435, 1185, 1157, 729 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.76–6.82 (m, 14H, ArH), 2.79 (d, 2JPH = 21.5 Hz, 1H, CH-P)major, 2.56 (bs, 1H, OH), 2.46 (d, 2JPH = 23.0 Hz, 1H, CH-P)minor, 2.00 (s, 3H, CH3)minor, 1.90 (s, 3H, CH3)major, 1.61 (s, 3H, CH3)major, 1.20 (s, 3H, CH3)minor ppm; 13C {1H} NMR (100 MHz, CDCl3 + CD3OD)  148.1minor, 147.4major, 132.8, 132.4, 132.4, 132.3, 132.1, 131.5, 131.4, 131.2, 131.1, 131.0, 130.7, 130.3, 129.4, 129.2, 129.0, 128.9, 128.8, 128.7, 128.7, 128.6, 126.6, 126.3, 125.5, 123.3, 123.0, 122.4, 117.3, 116.4 (CAr), 82.7 (d, 3JPC = 3.5 Hz, Cquat), 74.0 (d, 2JPC = 4.7 Hz, Cquat)major, 73.6 (d, 2JPC = 5.7 Hz, Cquat)minor, 35.7 (d, 1JPC = 107.1 Hz, CH)minor, 33.3 (d, 1JPC = 108.8 Hz, CH)major, 32.2 (CH3), 20.5 (CH3)minor, 20.1 (CH3)major ppm; 31P NMR (160 MHz, CDCl3) δ 25.2major, 24.2minor ppm; ESIHRMS (CI) m/z calcd. for C23H23NO3P ([M+H]+) 392.1416, found 392.1403. 19 ((1S*,1aS*,7S*)-1a-Ethyl-7-hydroxy-7-methyl-1,1a-dihydro-7Hazirino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (6c). (1.25 g, 62%) as a yellow solid from 2H-azirine 2d (1.34 g, 5 mmol, 1 eq) as described in the general procedure after stirring at room temperature for 72 h. The crude product was purified by crystallization from Et2O and washed several times with pentane to afford the title compound 6c. mp 147–179 ºC; IR (neat) vmax 3253, 2974, 2924, 1619, 1587, 1442, 1163, 910, 729, 698 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.79–6.85 (m, 14H, ArH), 2.87 (d, 2JPH = 21.7 Hz, 1H, CH-P)major, 2.74 (bs, 1H, OH), 2.53 (d, 2JPH = 23.1 Hz, 1H, CH-P)minor, 2.32–2.12 (m, 2H, CH2), 1.64 (s, 3H, CH3), 1.22–1.12 (m, 2H, CH3) ppm; 13C {1H} NMR (100 MHz, CDCl3)  148.4minor, 147.7major, 133.2, 132.5, 132.2, 132.2, 132.1, 132.1, 132.1, 131.8, 131.7, 131.5, 131.3, 131.2, 129.6, 128.9, 128.7, 128.6, 128.5, 126.4, 124.9, 123.2, 116.8 (CAr), 82.9 (d, 3JPC = 3.4 Hz, Cquat), 78.2 (d, 2JPC = 4.7 Hz, Cquat), 34.7 (d, 1JPC = 108.2 Hz, CH), 32.9 (CH3), 26.9 (CH2), 9.2 (CH3) ppm; 31P NMR (120 MHz, CDCl3) δ 25.0major, 23.9minor ppm; ESI-HRMS (CI) m/z calcd. for C24H25NO3P ([M+H]+) 406.1572, found 406.1558. 4.2. Biology 4.2.1. Materials Reagents and solvents were used as purchased without further purification. All stock solutions of the investigated compounds were prepared by dissolving the powered materials in appropriate amounts of DMSO. The final concentration of DMSO never exceeded 10% (v/v) in reactions. The stock solution was stored at 5 ºC until it was used. 4.2.2. Cytotoxicity assays. Cells were cultured according to the supplier´s instructions. Cells were seeded in 96-well plates at a density of 2–4 x 103 cells per well and incubated overnight in 0.1 mL of media supplied with 10% Fetal Bovine Serum (Lonza) in 5% CO2 incubator at 37 ºC. On day 2, drugs were added and samples were incubated for 48 hours. After treatment, 10 μL of cell counting kit-8 was added into each well for additional 2 hours incubation at 37 ºC. 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