Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3–H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5–97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at β-lactamase, thus protecting the antibiotic from undesirable biotransformation.


Introduction
The compounds containing azetidinone are of special importance both in chemistry and medicine. Since the discovery of penicillin, the application of azetidinone derivatives has been mainly associated with their antibacterial activity [1]. The family of azetidinone antibiotics (β-lactam antibiotics) includes penems, cephalosporins, monobactams and carbapenems, among others [2][3][4][5][6]. On the other hand, the azetidinone ring is a common structural motif of a vast number of compounds possessing a wide range of other biological properties, including antimalarial [7], antitubercular [8], anti-inflammatory [9], antifungal [10], antidepressant [11] and nootropic activity [11]. Azetidinone derivatives are also known as cholesterol absorption [12,13], human tryptase [14,15] and chymase Compounds with antiviral properties could be found among those containing an azetidinone unit in their structures. For example, non-nucleoside analogues of azetidinone 5 ( Figure 2) exhibited activity towards human cytomegalovirus (HCMV) [26,27]. Peptide linked monocyclic azetidinones 6 showing an inhibitory activity against human cytomegalovirus protease have been also synthesized by Dézeil [28]. Similarly, Sperka and co-workers discovered compounds 7 as inhibitors of HIV-1 protease [29]. Moreover, D'hooghe et al. obtained compounds 8 by introduction of a modified purine nucleobase into an azetidinone ring [19]. Purine β-lactam hybrids showed moderate to good activities against different viruses, i.e., human respiratory syncytial virus (RSV), chikungunya virus (ChikV), HCMV, hepatitis B virus (HBV) and coxsackie B virus (CoxV) [19]. The results of antiviral and cytotoxic activity studies on compounds 9 were so encouraging that identification of several new lead structures among this type of compounds was possible [19,30,31]. Compounds with antiviral properties could be found among those containing an azetidinone unit in their structures. For example, non-nucleoside analogues of azetidinone 5 ( Figure 2) exhibited activity towards human cytomegalovirus (HCMV) [26,27]. Peptide linked monocyclic azetidinones 6 showing an inhibitory activity against human cytomegalovirus protease have been also synthesized by Dézeil [28]. Similarly, Sperka and co-workers discovered compounds 7 as inhibitors of HIV-1 protease [29]. Moreover, D'hooghe et al. obtained compounds 8 by introduction of a modified purine nucleobase into an azetidinone ring [19]. Purine β-lactam hybrids showed moderate to good activities against different viruses, i.e., human respiratory syncytial virus (RSV), chikungunya virus (ChikV), HCMV, hepatitis B virus (HBV) and coxsackie B virus (CoxV) [19]. The results of antiviral and cytotoxic activity studies on compounds 9 were so encouraging that identification of several new lead structures among this type of compounds was possible [19,30,31].
The search for effective antiviral drugs, among newly designed compounds as well as already known ones, became even more challenging in the eyes of the coronavirus pandemic . In fact, symptomatic therapy is appropriate in the treatment of milder illnesses, and antimicrobial drugs are often necessary when bacterial complications occur. Especially, the methicillin resistant Staphylococcus aureus (MRSA) is a Gram-positive member of the most problematic bacteria in clinical treatment, so-called ESKAPE [32]. Various clinical isolates of MRSA are multidrug resistant (MDR), i.e., resistant to antibiotics representing different classes, including β-lactams, macrolides, tetracyclines, etc. Taking into account the structural analogy between β-lactam antibiotics and functionalized derivatives of azetidinones, the latter ones provide some hope in search for effective agents against MRSA, either as new antibacterials less susceptible to MDR mechanisms or as antibiotic "adjuvants" that, being bioisosteres of antibiotics, may be mistakenly recognized as substrates of various bacterial MDR proteins. Thus, further extensive pharmacomodulations among azetidinones are an important challenge for current medicinal chemistry in order to search for innovative therapeutic solutions in the treatment of complex infectious diseases. The search for effective antiviral drugs, among newly designed compounds as well as already known ones, became even more challenging in the eyes of the coronavirus pandemic . In fact, symptomatic therapy is appropriate in the treatment of milder illnesses, and antimicrobial drugs are often necessary when bacterial complications occur. Especially, the methicillin resistant Staphylococcus aureus (MRSA) is a Gram-positive member of the most problematic bacteria in clinical treatment, so-called ESKAPE [32]. Various clinical isolates of MRSA are multidrug resistant (MDR), i.e., resistant to antibiotics representing different classes, including β-lactams, macrolides, tetracyclines, etc. Taking into account the structural analogy between β-lactam antibiotics and functionalized derivatives of azetidinones, the latter ones provide some hope in search for effective agents against MRSA, either as new antibacterials less susceptible to MDR mechanisms or as antibiotic "adjuvants" that, being bioisosteres of antibiotics, may be mistakenly recognized as substrates of various bacterial MDR proteins. Thus, further extensive pharmacomodulations among azetidinones are an important challenge for current medicinal chemistry in order to search for innovative therapeutic solutions in the treatment of complex infectious diseases.
Recently, we communicated a convenient method for the synthesis of 4phosphonylated azetidin-2-ones substitued with various aryl groups at C3 [33]. The proposed methodology relied on the application of Kinugasa reaction of N-methyl Cphosphonylated nitrone with terminal acetylenes. In this paper, a full account of studies on preparation of the series of N-substitued 3-aryl-4-(diethoxyphosphonyl)azetidion-2ones of the general formulae 10 and 11 (Scheme 1) is presented together with the results of their antiviral, cytostatic and antimicrobial/antibiotic adjuvant properties. Recently, we communicated a convenient method for the synthesis of 4-phosphonylated azetidin-2-ones substitued with various aryl groups at C3 [33]. The proposed methodology relied on the application of Kinugasa reaction of N-methyl C-phosphonylated nitrone with terminal acetylenes. In this paper, a full account of studies on preparation of the series of N-substitued 3-aryl-4-(diethoxyphosphonyl)azetidion-2-ones of the general formulae 10 and 11 (Scheme 1) is presented together with the results of their antiviral, cytostatic and antimicrobial/antibiotic adjuvant properties.

Chemistry
As reported earlier, standard conditions were applied for the Kinugasa reaction of nitrone 12 to terminal arylacetylenes 14a-c and 14e [33], namely 3 equivalents of CuI and triethylamine to generate respective copper(I)arylacetylide. After the optimization of the reaction conditions [33], cycloadditions of the nitrone 12 to arylalkynes 14 were carried out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2).

Chemistry
As reported earlier, standard conditions were applied for the Kinugasa reaction of nitrone 12 to terminal arylacetylenes 14a-c and 14e [33], namely 3 equivalents of CuI and triethylamine to generate respective copper(I)arylacetylide. After the optimization of the reaction conditions [33], cycloadditions of the nitrone 12 to arylalkynes 14 were carried out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2). nitrone 12 to terminal arylacetylenes 14a-c and 14e [33], namely 3 equivalents of CuI and triethylamine to generate respective copper(I)arylacetylide. After the optimization of the reaction conditions [33], cycloadditions of the nitrone 12 to arylalkynes 14 were carried out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2).

cis-10 trans-10
a [33] triethylamine to generate respective copper(I)arylacetylide. After the optimization of the reaction conditions [33], cycloadditions of the nitrone 12 to arylalkynes 14 were carried out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2). b [33] reaction conditions [33], cycloadditions of the nitrone 12 to arylalkynes 14 were carried out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2).  c [33] out using 1.5 equivalent of the respective arylalkyne in the presence of catalytic amounts of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2).  cis-10c-7% trans-10c-55% d of CuI, triethylamine and DMAP under microwave irradiation, which significantly shortened the time required for full conversion of the nitrone (4 h vs. 72 h) (Scheme 2, Table 1). For the purpose of this project, the set of alkynes applied in this reaction was expanded by arylacetylene 14d and 14f (Scheme 2, Table 1, entry d and f). Moreover, N-benzyl-C-(diethoxyphosphoryl)nitrone 13 was also used in Kinugasa reaction to fill the library of the azetidinones for biological studies (Scheme 2, Table 2).   1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.  1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification. cis-10f-17% trans-10f-31% 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.
The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in the 1 H NMR spectra of cis-10 and cis-11, vicinal couplings for H3-H4 protons in the 5.5-6.9 Hz range were observed, whereas in the series of trans-10 and trans-11, significantly lower coupling values were noticed for H3-H4 protons ( 3 J H3-H4 = 2.4-2. 9 Hz). Furthermore, the one-bond phosphorus-carbon coupling constant values ( 1 J C-P ) also appeared to be useful since diagnostic differences in coupling constants were found in the series of all 3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones cis-10 and cis-11 in comparison to analo-gous trans-10 and trans-11. The observed values of couplings for all cis-isomers were higher ( 1 J C-P = 170.6-173.0 Hz) when compared to the coupling constants for the other trans-configured diastereoisomers ( 1 J C-P = 164.6-166.6 Hz).  1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification. The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 19 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.  1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.
The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 18 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.  1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.
The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 20 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification. The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 18 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification. The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 18 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.  1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification.
The ratios of diastereoisomers were calculated from 31 P and 1 H NMR spectra of crude reaction mixtures. Diastereoisomeric azetidinones were successfully separated by column chromatography and both pure isomers (except for cis-10c and cis-11c) were isolated in almost all cases, namely cis-10a, cis-10b, cis-10d-f and trans-10a-f as well as cis-11a, cis-11b, cis-11d-f and trans-11a-f (Tables 1 and 2).
The correlation between the cis and trans configuration of 3,4-disubstituted azetidinones and the observed values of vicinal H3-H4 coupling constants has been well recognized [34][35][36]. In the case of compounds cis-10 and cis-11, as well as trans-10 and trans-11 for configurational assignments, the analogous correlation was also applied. Thus, in 19 cis-11f-6% trans-11f-35% 1 The cis/trans ratio was calculated from the 31 P NMR spectra of crude reaction mixtures. 2 Total yield = yield of pure isomers and mixtures of isomers after column chromatography. 3 Yield of pure isomers obtained after chromatographic purification. subtypes) and influenza B virus. Ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight 10,000, DS-10,000), mycophenolic acid, and Urtica dioica agglutinin (UDA) were used as reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses). The cytotoxicity of the tested compounds toward the uninfected HEL, HeLa, Vero and MDCK cells was defined as the minimum cytotoxic concentration (MCC) that causes a microscopically detectable alteration of normal cell morphology. The 50% cytotoxic concentration (CC 50 ), causing a 50% decrease in cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay system. Among all tested compounds, the stereoisomers having 3-methyl-4-fluorophenyl group at C3 in azetidinone ring (11f) showed modest antiviral activity (Figure 3). The isomeric azetidinone trans-11f was able to inhibit the replication of human coronavirus (229E) (EC 50 = 45 µM) and its activity was almost 2.5-fold higher than that of a reference drug ribavirin (EC 50 = 112 µM). Moreover, activity of azetidinone trans-11f toward cytomegalovirus AD-169 strain (EC 50 = 54.69 µM) was also noticed, although it was less active than ganciclovir and cidofovir used as reference drugs. At the same time the compound trans-11f did not affect normal cell morphology. On the other hand, the isomer cis-11f appeared to be active against influenza A virus H1N1 subtype (EC 50 = 12 µM by visual CPE score; EC 50 = 8.3 µM by TMS score; MCC > 100 µM, CC 50 = 39.9 µM) in Madin Darby canine kidney cells (MDCK) and its potency was comparable to ribavirin used as a reference compound (EC 50 = 8.9 µM by visual CPE score; EC 50 = 6.6 µM by TMS score; MCC > 100 µM, CC 50 ≥ 100 µM), but much lower than that of zanamivir, amantadine and rimantadine. None of the compounds described herein were active against the other tested DNA and RNA viruses and none was cytotoxic toward used cell lines at concentrations up to 100 µM.  In regard to the structure-activity relationship, the introduction of benzyl instead of methyl group at nitrogen together with 3-methyl-4-fluorophenyl group at C3 in azetidinone ring seems to be crucial for the observed antiviral activity. Surprisingly, the presence of a monosubstituted phenyl function at C3, regardless of the position of the substituent, is insufficient to maintain the activity. Moreover, the stereochemistry of the azetidinone ring appeared to be important for the activity and selectivity of 11f toward the targeted viruses, i.e., trans-isomer displayed selective action for coronavirus CoV-229 and cytomegalovirus HMCV (AD-169), while cis-for influenza A (H1N1). An additional advantage of the 3-methyl-4-fluorophenyl derivatives of azetidinone is their safety (no cytotoxic effects) for the whole tested panel of uninfected cell lines.

Pharmacology
Although these initial results are not sufficient to recognize and explore likely molecular mechanisms of the antiviral activities of 11f isomers, they mark the 3-methyl-4fluorophenyl scaffold as an important pharmacophore feature worth to be considered in further search for antiviral drugs among the azetidine derivatives.

Antibacterial Action
All synthesized azetidinones cis-10 and trans-10, as well as cis-11 and trans-11, were screened for their antibacterial activity against the Gram-positive S. aureus, including reference strain ATCC 25,923 and the multidrug resistant clinical isolate MRSA HEMSA 5. The tested compounds did not inhibit the growth of either S. aureus strains at concentrations up to 2 mM. Thus, their antibacterial activity can be considered negligible.

Antibiotics Enhancer Properties
Since the lack of direct antibacterial activity of the obtained azetidinones was observed, all isomers trans and cis were investigated on their "adjuvant" properties, i.e., an ability to enhance the effectiveness of antibiotics against S. aureus strains. Thus, the com- In regard to the structure-activity relationship, the introduction of benzyl instead of methyl group at nitrogen together with 3-methyl-4-fluorophenyl group at C3 in azetidinone ring seems to be crucial for the observed antiviral activity. Surprisingly, the presence of a monosubstituted phenyl function at C3, regardless of the position of the substituent, is insufficient to maintain the activity. Moreover, the stereochemistry of the azetidinone ring appeared to be important for the activity and selectivity of 11f toward the targeted viruses, i.e., trans-isomer displayed selective action for coronavirus CoV-229 and cytomegalovirus HMCV (AD-169), while cis-for influenza A (H1N1). An additional advantage of the 3methyl-4-fluorophenyl derivatives of azetidinone is their safety (no cytotoxic effects) for the whole tested panel of uninfected cell lines.
Although these initial results are not sufficient to recognize and explore likely molecular mechanisms of the antiviral activities of 11f isomers, they mark the 3-methyl-4fluorophenyl scaffold as an important pharmacophore feature worth to be considered in further search for antiviral drugs among the azetidine derivatives.

Antibacterial Action
All synthesized azetidinones cis-10 and trans-10, as well as cis-11 and trans-11, were screened for their antibacterial activity against the Gram-positive S. aureus, including reference strain ATCC 25,923 and the multidrug resistant clinical isolate MRSA HEMSA 5. The tested compounds did not inhibit the growth of either S. aureus strains at concentrations up to 2 mM. Thus, their antibacterial activity can be considered negligible.

Antibiotics Enhancer Properties
Since the lack of direct antibacterial activity of the obtained azetidinones was observed, all isomers trans and cis were investigated on their "adjuvant" properties, i.e., an ability to enhance the effectiveness of antibiotics against S. aureus strains. Thus, the compounds were tested in combination with the known β-lactam antibiotic, oxacillin, in the microdilution assays. The ability of the tested azetidinones to reduce minimum inhibitory concentration (MIC) of oxacillin against both, the referenced and the resistant S. aureus strains, was assessed. In the absence of the tested compounds, oxacillin showed MIC value of 0.5 µg/mL against the ATCC 25,923 strain, while 512 µg/mL for the MRSA HEMSA 5, the strain highly resistant to this antibiotic. The azetidinones were tested in the 0.5 mM, i.e., the inactive concentration of each compound (≤ 1 4 MIC) against both strains. Results are shown in Table 3. Among all tested compounds, the strong chemosensitizing effect was demonstrated by compound trans-11f, which reduced the MIC of oxacillin 16-fold against MRSA HEMSA 5 (oxacillin MIC in the presence of the tested compound reduced to 32 µg/mL). Other azetidinones did not improve the susceptibility of MRSA to oxacillin in a significant manner. On the other hand, none of the tested compounds had an impact on the oxacillin activity toward the S. aureus ATCC 25,923 strain, and an even higher concentration of the antibiotic was necessary to inhibit growth of the bacteria when the tested compound was added.

Cytostatic Activity
The 50% cytostatic inhibitory concentration (IC 50 ) causing a 50% decrease in cell proliferation was determined for all obtained compounds toward 9 cancerous cell lines, i.e.,  Table 4. Table 4. Inhibitory effect of azetidinones cis-10/trans-10 and cis-11/trans-11 against the proliferation of cancerous cells. Among all tested compounds, none were active against DND-41, HL-60, K-562, MM.1S and Z-138 cancer cells at the concentrations up to 100 µM, except the compound trans-11f which showed low activity against DND-41 cells (IC 50 = 65.8 µM). Most of the compounds described herein were also not toxic or showed negligible toxicity to non-cancerous retina cells (hERT RPE-1), except trans-11c, trans-11d, trans-11e and trans-11f which exhibited noticeable antiproliferation activities (IC 50 = 45.9, 73.0, 25.6 and 33.5, µM, respectively) ( Table 4). All of the tested azetidinones 10 and 11, except cis-10d (Ar = 4-F-C 6 H 4 ), exhibited moderate activity against pancreatic adenocarcinoma cells (Capan-1) (IC 50 from 19.6 to 95.2 µM), and among them trans-10c (Ar = 3-F-C 6 H 4 ) was the most active with IC 50 value of 19.6 µM, but the inhibitory concentration was much lower than that of the reference drugs (Table 4). On the other hand, the highest inhibitory effect against the proliferation of chronic myeloid leukemia (Hap1) was observed for compounds cis-10b (Ar = 2-F-C 6 H 4 ) (IC 50 = 14.5 µM), however in most cases the activity values of the tested compounds toward chronic myeloid leukemia (Hap1) were slightly lower than these observed for the same series of compounds toward Capan-1 cells. Compound trans-11e having 2,4difuorophenyl moiety at C3 in the azetidinone ring appeared to be the most active toward lung carcinoma (NCI-H460) (IC 50 = 24.4 µM) but unfortunately no selectivity was observed when compared to normal retina (non-cancerous) cells (hTERT RPE-1) (IC 50 = 25.6 µM). Interestingly, N-benzylated azetidinones 11 exhibited moderate activity toward colorectal carcinoma cells (HCT-116) (IC 50 = 35.3 to 87.9 µM), whereas most of analogous N-methyl azetidinones 10 were inactive at the concentration up to 100 µM, except for trans-10b and cis-10d (IC 50 = 44.1 and 86.5 µM, respectively). In the case of the other cancerous cell lines, no significant correlation between structure and the observed activity was noticed.

Computer-Aided Insight into Antibiotic "Adjuvant" Action
The semi-synthetic β-lactam antibiotic oxacillin used in this study is known as antistaphylococcal penicillin, which is resistant to hydrolysis by most staphylococcal β-lactamases [36]. Its bactericidal activity results from the inhibition of bacterial cell wall biosynthesis via interaction with penicillin binding proteins (PBPs) [37]. The resistance to oxacillin primarily stems from the acquisition of the mecA gene encoding PBP2a with lower affinity to β-lactams [38] but various other mechanisms are also possible. Taking into account both, the high structural similarity of investigated 3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones to β-lactam antibiotics and unknown modifications of β-lactamase in the tested XDS strain HEMSA-5, a competitive displacement of oxacillin by trans-11f at β-lactamase seems to be a probable mechanism as well. Therefore, we decided to estimate either β-lactamase or PBP2a as possible targets involved into the oxacillin-enhancing action of trans-11f. In this context, advanced molecular modelling studies for four possible stereoisomers of 11f, namely (3R,4R)-11f, (3S,4S)-11f, (3R,4S)-11f and (3S,4R)-11f (Figure 4), and oxacillin have been performed. trans-10b and cis-10d (IC50 = 44.1 and 86.5 µM, respectively). In the case of the other cancerous cell lines, no significant correlation between structure and the observed activity was noticed.

Computer-Aided Insight into Antibiotic "Adjuvant" Action
The semi-synthetic β-lactam antibiotic oxacillin used in this study is known as antistaphylococcal penicillin, which is resistant to hydrolysis by most staphylococcal β-lactamases [36]. Its bactericidal activity results from the inhibition of bacterial cell wall biosynthesis via interaction with penicillin binding proteins (PBPs) [37]. The resistance to oxacillin primarily stems from the acquisition of the mecA gene encoding PBP2a with lower affinity to β-lactams [38] but various other mechanisms are also possible. Taking into account both, the high structural similarity of investigated 3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones to β-lactam antibiotics and unknown modifications of β-lactamase in the tested XDS strain HEMSA-5, a competitive displacement of oxacillin by trans-11f at β-lactamase seems to be a probable mechanism as well. Therefore, we decided to estimate either β-lactamase or PBP2a as possible targets involved into the oxacillin-enhancing action of trans-11f. In this context, advanced molecular modelling studies for four possible stereoisomers of 11f, namely (3R,4R)-11f, (3S,4S)-11f, (3R,4S)-11f and (3S,4R)-11f ( Figure  4), and oxacillin have been performed.

Influence on β-Lactamase
Due to the structural resemblance of the newly synthesized compounds to oxacillin, the possibility of them being the potential β-lactamase substrate was examined. Four stereoisomers of compound 11f (Figure 4) were tested in docking and molecular dynamics (MD) simulations with β-lactamase from Staphylococcus aureus. For reference, oxacillin was also modelled in the same conditions. The structure of PC1 β-lactamase was used, which is the class A β-lactamase (class D β-lactamases are supposed to hydrolyze oxacillin; however, their crystal structures for Staphylococcus aureus are not available). The docking results of cis-11f (i.e., (3R,4R)-11f and (3S,4S)-11f) and trans-11f (i.e., (3R,4S)-11f and (3S,4R)-11f) are presented in Figure 5. Poses of all analyzed compounds are overlaid with the oxacillin orientation in the binding site.

Influence on β-Lactamase
Due to the structural resemblance of the newly synthesized compounds to oxacillin, the possibility of them being the potential β-lactamase substrate was examined. Four stereoisomers of compound 11f (Figure 4) were tested in docking and molecular dynamics (MD) simulations with β-lactamase from Staphylococcus aureus. For reference, oxacillin was also modelled in the same conditions. The structure of PC1 β-lactamase was used, which is the class A β-lactamase (class D β-lactamases are supposed to hydrolyze oxacillin; however, their crystal structures for Staphylococcus aureus are not available). The docking results of cis-11f (i.e., (3R,4R)-11f and (3S,4S)-11f) and trans-11f (i.e., (3R,4S)-11f and (3S,4R)-11f) are presented in Figure 5. Poses of all analyzed compounds are overlaid with the oxacillin orientation in the binding site. The analysis of the initial compound positions in docking indicates that most similar pose to this of oxacillin is obtained by (3S,4S)-11f. However, MD simulations showed that the docking poses were not very stable during MD and that the compound conformations were varying in the subsequent frames. The MD simulations revealed the possible mechanism of restoring oxacillin activity by trans-11f. One of its enantiomers, (3R,4S)-11f, is the only compound which (similarly to oxacillin) did not leave the β-lactamase active site and remains in the relatively similar position during the whole simulation (Figures 6 and 7). On the other hand, all other compounds diffused away from their positions obtained in docking and they are unlikely to be the substrates for the β-lactamase. The analysis of the initial compound positions in docking indicates that most similar pose to this of oxacillin is obtained by (3S,4S)-11f. However, MD simulations showed that the docking poses were not very stable during MD and that the compound conformations were varying in the subsequent frames. The MD simulations revealed the possible mechanism of restoring oxacillin activity by trans-11f. One of its enantiomers, (3R,4S)-11f, is the only compound which (similarly to oxacillin) did not leave the β-lactamase active site and remains in the relatively similar position during the whole simulation (Figures 6 and 7). On the other hand, all other compounds diffused away from their positions obtained in docking and they are unlikely to be the substrates for the β-lactamase.     The quantitative data were also in line with the qualitative analysis (Figure 7), where only (3R,4S)-11f and oxacillin remained in the same region during the whole MD simulation and did not diffuse away from the binding site. On the other hand, all the other compounds were not strongly fitted to β-lactamase and spent some simulation time away from the protein.
Due to the relatively stable position of (3R,4S)-11f in the β-lactamase active site during MD and its structural resemblance to oxacillin, we suggest that (3R,4S)-11f restores the oxacillin activity in MRSA via being a substrate for β-lactamase, which transforms (3R,4S)-11f instead of oxacillin, and thus oxacillin can play its antibacterial role in the unchanged form.

Interactions with PBP2a
In the next step, the hypothesis of PBP2a being a potential target was examined. We verified the scheme of interactions of different isomers of compound 11f with the PBP2a protein, the alternative penicillin binding protein with the reduced affinity for β-lactam antibiotics. Analogously, docking and MD simulations were applied, as for studies with β-lactamase. The docking results are shown in Figure 8. The quantitative data were also in line with the qualitative analysis (Figure 7), where only (3R,4S)-11f and oxacillin remained in the same region during the whole MD simulation and did not diffuse away from the binding site. On the other hand, all the other compounds were not strongly fitted to β-lactamase and spent some simulation time away from the protein.
Due to the relatively stable position of (3R,4S)-11f in the β-lactamase active site during MD and its structural resemblance to oxacillin, we suggest that (3R,4S)-11f restores the oxacillin activity in MRSA via being a substrate for β-lactamase, which transforms (3R,4S)-11f instead of oxacillin, and thus oxacillin can play its antibacterial role in the unchanged form.

Interactions with PBP2a
In the next step, the hypothesis of PBP2a being a potential target was examined. We verified the scheme of interactions of different isomers of compound 11f with the PBP2a protein, the alternative penicillin binding protein with the reduced affinity for β-lactam antibiotics. Analogously, docking and MD simulations were applied, as for studies with β-lactamase. The docking results are shown in Figure 8. The docking poses to the PBP2a active site indicate a very similar orientation of both cis-11f enantiomers, and (3R,4S)-11f was also docked similarly. On the other hand, (3S,4R)-11f adopted a significantly different pose, which is, however, the furthest away from the active-site serine (S403).
In order to validate the docking poses and examine their stability in the binding pocket, MD simulations were carried out (Figure 9). The analysis of compound poses obtained at different time points of the simulation indicates that their poses were very unstable during MD. If not immediately (as (3S,4R)-11f), all compounds left the active site of PBP2a after less than 200 ns of simulation. These results indicate that the interaction of The docking poses to the PBP2a active site indicate a very similar orientation of both cis-11f enantiomers, and (3R,4S)-11f was also docked similarly. On the other hand, (3S,4R)-11f adopted a significantly different pose, which is, however, the furthest away from the active-site serine (S403).
In order to validate the docking poses and examine their stability in the binding pocket, MD simulations were carried out (Figure 9). The analysis of compound poses obtained at different time points of the simulation indicates that their poses were very unstable during MD. If not immediately (as (3S,4R)-11f), all compounds left the active site of PBP2a after less than 200 ns of simulation. These results indicate that the interaction of PBP2a with examined compounds is very unstable and suggest rather low probability that any stereoisomers of 11f is the PBP2a agent. PBP2a with examined compounds is very unstable and suggest rather low probability that any stereoisomers of 11f is the PBP2a agent. Thus, the most probable mechanism of the "adjuvant" action of trans-11f seems to be mediated by β-lactamase, in which the enantiomer (3R,4S)-11f is probably the responsible component due to predominant ability of β-lactamase substrate.

Chemistry
General information-1 H NMR spectra were taken in CDCl3 on a Bruker Avance III (600 MHz); chemical shifts δ are given in ppm with respect to TMS and coupling constants J in Hz. 13 C NMR and 31 P NMR spectra were recorded in a 1 H-decoupled mode for CDCl3 solutions on the Bruker Avance III (600 MHz) spectrometer at 151 and 243 MHz, respectively. IR spectral data were measured on a Bruker Alpha-T FT-IR spectrometer. Melting points were determined on a Boetius apparatus and are uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of the Faculty of Pharmacy (Medical University of Lodz) on a Perkin Elmer PE 2400 CHNS analyzer and their results were found to be in good agreement (±0.3%) with the theoretical values.
The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh), analytical TLC, Merck TLC plastic sheets silica gel 60 F254. TLC plates were developed in chloroform-methanol solvent systems. Visualization of spots was effected with iodine vapours. All solvents were purified by methods described in the literature. The nitrones 12 and 13 were obtained according to the literature procedure [39,40]. The purity of the samples of all synthesised compounds 10 and 11 used for biological studies was established as ≥99.99%. Thus, the most probable mechanism of the "adjuvant" action of trans-11f seems to be mediated by β-lactamase, in which the enantiomer (3R,4S)-11f is probably the responsible component due to predominant ability of β-lactamase substrate.

Chemistry
General information-1 H NMR spectra were taken in CDCl 3 on a Bruker Avance III (600 MHz); chemical shifts δ are given in ppm with respect to TMS and coupling constants J in Hz. 13 C NMR and 31 P NMR spectra were recorded in a 1 H-decoupled mode for CDCl 3 solutions on the Bruker Avance III (600 MHz) spectrometer at 151 and 243 MHz, respectively. IR spectral data were measured on a Bruker Alpha-T FT-IR spectrometer. Melting points were determined on a Boetius apparatus and are uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of the Faculty of Pharmacy (Medical University of Lodz) on a Perkin Elmer PE 2400 CHNS analyzer and their results were found to be in good agreement (±0.3%) with the theoretical values.
The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh), analytical TLC, Merck TLC plastic sheets silica gel 60 F 254 . TLC plates were developed in chloroform-methanol solvent systems. Visualization of spots was effected with iodine vapours. All solvents were purified by methods described in the literature. The nitrones 12 and 13 were obtained according to the literature procedure [39,40]. The purity of the samples of all synthesised compounds 10 and 11 used for biological studies was established as ≥99.99%. 1 H, 13 C and 31 P NMR spectra of all new synthesized compounds are provided in Supplementary Materials.

General
Procedures for the Synthesis of Azetidine-2-Ones cis-10/trans-10 and cis-11/trans-11 3.2.1. General Procedure A A solution of alkyne 14 (3.0 mmol) in MeCN (1 mL) was cooled to 0 • C under argon atmosphere and CuI (3 mmol) was added, followed by Et 3 N (3 mmol). After 30 min the temperature was allowed to reach 25 • C, the respective nitrone 12 or 13 (1 mmol) in MeCN (1 mL) was added, and the reaction mixture was stirred for 72 h. Subsequently, the reaction mixture was diluted with MeCN and the suspension was filtered through the layer of Celite. The solution was concentrated and the crude product was purified on a silica gel column with chloroform:methanol (100:1, 50:1, v/v) and in some cases also by high-performance liquid chromatography (HPLC) using a X Bridge Prep, C18, 5 µm, OBD (Optimum Bed Density), 19 × 100 mm column and methanol:water mixture (62:38, 60:40, 55:45, v/v) as eluent.

General Procedure B
A solution of alkyne 14 (1.5 mmol) in MeCN (1 mL) was cooled to 0 • C under argon atmosphere and CuI (0.1 mmol) was added, followed by Et 3 N (0.05 mmol) and DMAP (0.05 mmol). After 30 min the temperature was allowed to reach 25 • C, the respective nitrone 12 or 13 (1 mmol) in MeCN (1 mL) was added, and the reaction mixture was irradiated in the Plazmatronika RM800 microwave reactor at 30-40 • C for 4 h. Subsequently, the reaction mixture was diluted with MeCN, and the suspension was filtered through the layer of Celite. The solution was concentrated and the crude product was purified on a silica gel column with chloroform:methanol (100:1, 50:1) and in some cases also by high-performance liquid chromatography (HPLC) using a X Bridge Prep, C18, 5 µm, OBD (Optimum Bed Density), 19 × 100 mm column and methanol:water mixture (62:38, 60:40, 55:45, v/v) as eluent.

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 (ACV r ), herpes simplex virus type 2 (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK − VZV strain 07-1, 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), were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey kidney cells (Vero), human epithelial cervix carcinoma cells (HeLa) or Madin Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (1 CCID 50 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 EC 50 or compound concentration required reducing 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.

Cytostatic Activity against Immortalized Cell Lines
All assays were performed in 96-well microtiter plates. To each well was added (5-7.5) × 10 4 tumor cells and a given amount of the tested compound. The cells were allowed to proliferate at 37 • C in a humidified, CO 2 -controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.

Bacterial Assays
The in-vitro antibacterial property and the capacity of tested compounds to increase the efficacy of antibiotics were evaluated in two Staphylococcus aureus strains, i.e., the reference clonal complex 5 (CC5) methicillin-susceptible (MSSA) strain ATCC 25923, and the methicillin-resistant (MRSA) extensively drug-resistant (XDR) clinical isolate HEMSA-5 [46].
In order to assess the increase of antibiotic efficacy, the assays were conducted by determining if/to what extent the investigated compounds reduce MICs of oxacillin by means of a serial dilution broth microplate method, in accordance with the CLSI requirements [47]. The concentrations of compounds used in the MICs reduction assay were no greater than 1/4 of their respective MICs to ensure that cell viability was not affected by the intrinsic antibacterial activity of the molecules. Serial two-fold dilutions of oxacillin (Sigma-Aldrich; St. Louis, MI, USA, cat. no. 28221), were prepared in 65 mL of the Mueller-Hinton broth (Merck; Darmstadt, Germany, cat. no. 1102930500). Suitable concentrations of the compounds (total volume 10 mL) were then added. Bacterial suspensions were diluted to OD 1 4 0.5. The resulting suspensions were then diluted 1:100 and added in the volume of 75 mL into the oxacillin serial dilutions with the compounds. The results were read after 20-h incubation at 37 • C.
The ability of compounds to improve antibiotic efficacy was expressed as the activity gain [A] parameter calculated according to the formula given in Figure 10.