A Multicomponent Protocol for the Synthesis of Highly Functionalized γ-Lactam Derivatives and Their Applications as Antiproliferative Agents

An efficient synthetic methodology for the preparation of 3-amino 1,5-dihydro-2H-pyrrol-2-ones through a multicomponent reaction of amines, aldehydes, and pyruvate derivatives is reported. In addition, the densely substituted lactam substrates show in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma), and A549 (carcinomic human alveolar basal epithelial cell). Given the possibilities in the diversity of the substituents that offer the multicomponent synthetic methodology, an extensive structure-activity profile is presented. In addition, both enantiomers of phosphonate-derived γ-lactam have been synthesized and isolated and a study of the cytotoxic activity of the racemic substrate vs. its two enantiomers is also presented. Cell morphology analysis and flow cytometry assays indicate that the main pathway by which our compounds induce cytotoxicity is based on the activation of the intracellular apoptotic mechanism.


Introduction
Population aging is one of humanity's greatest achievements, but also one of its biggest challenges. From a health point of view, one of the main side effects of the growth in life expectancy is the increasing occurrence of disorders associated with aging. In this regard, cancer diseases are currently one of the biggest health problems worldwide [1] and, accordingly, malignant neoplasms have become nowadays one of the primary targets in medicinal sciences. One of our strongest weapons to combat cancer implies the combination of surgical techniques and chemotherapeutical agents, where antineoplastic drugs are used to kill fast-growing cells in the body [2]. For many years, one of the major goals of cancer therapy has been the specific and effective elimination of tumor cells by apoptosis, a programmed and highly regulated cellular death mechanism triggered by multiple factors that induce cellular stress. In this regard, there are already available some FDA-approved anticancer agents specifically designed to target intracellular apoptotic pathways, and also Pharmaceuticals 2021, 14, 782 2 of 21 novel more selective agents are under development [3]. During the last decades, a plethora of cytotoxic drugs that target actively proliferating cells have been developed, but more efforts are still required in this area, to increase our ammunition to fight against cancer. The identification of new active compounds is an imperative task in medicine and, there, organic and medicinal chemistry plays a crucial role, as the front line of battle, through the production of libraries of organic compounds and the preliminary in vitro evaluation of their cytotoxicity against cancer cell lines.
In this regard, the γ-lactam ring ( Figure 1) is a key structural scaffold in medicinal chemistry that can be found in the structure of many natural and synthetic bioactive compounds [4]. Within this family of heterocycles, 1,5-dihydro-2H-pyrrol-2-ones (Figure 1), present a conjugated ring system, that possesses latent reactivity for further modifications and, for this reason, they are valuable building blocks in synthetic chemistry [5][6][7][8]. Moreover, the structure of these unsaturated γ-lactam derivatives is the essential part of the skeleton of numerous relevant molecules that show a large variety of biological activities [9][10][11]. For example, ascosali pyrrolidone A has been isolated from the marine fungus Ascochyta salicornae and shows antimicrobial activity [12], cryptocin is inactive against human fungi but shows selective activity against plant pathogenic ones [13] and erisothramidine 2 shows anticonvulsive, sedative, and hypnotic properties [14] and has been reported as a neuromuscular blocking agent [15] (Figure 1). approved anticancer agents specifically designed to target intracellular apoptotic pathways, and also novel more selective agents are under development [3]. During the last decades, a plethora of cytotoxic drugs that target actively proliferating cells have been developed, but more efforts are still required in this area, to increase our ammunition to fight against cancer. The identification of new active compounds is an imperative task in medicine and, there, organic and medicinal chemistry plays a crucial role, as the front line of battle, through the production of libraries of organic compounds and the preliminary in vitro evaluation of their cytotoxicity against cancer cell lines.
In this regard, the γ-lactam ring ( Figure 1) is a key structural scaffold in medicinal chemistry that can be found in the structure of many natural and synthetic bioactive compounds [4]. Within this family of heterocycles, 1,5-dihydro-2H-pyrrol-2-ones (Figure 1), present a conjugated ring system, that possesses latent reactivity for further modifications and, for this reason, they are valuable building blocks in synthetic chemistry [5][6][7][8]. Moreover, the structure of these unsaturated γ-lactam derivatives is the essential part of the skeleton of numerous relevant molecules that show a large variety of biological activities [9][10][11]. For example, ascosali pyrrolidone A has been isolated from the marine fungus Ascochyta salicornae and shows antimicrobial activity [12], cryptocin is inactive against human fungi but shows selective activity against plant pathogenic ones [13] and erisothramidine 2 shows anticonvulsive, sedative, and hypnotic properties [14] and has been reported as a neuromuscular blocking agent [15] (Figure 1). Interestingly, some 2-pyrrolone substrates have been recently identified as p53−MDM2 [16] and STAT3 [17] inhibitors, which results in a strong antiproliferative activity and, besides, many other 2-pyrrolone derivatives have been described as antitumoral agents [18][19][20][21][22][23].
Besides, multicomponent protocols are considered as an essential tool in diversityoriented synthesis [36,37], due to the high degree of molecular diversity achieved, and, accordingly, they have become a preferential methodology in the field of medicinal chemistry [38,39]. In this context, a few years ago, we described a sulfuric acid-promoted multicomponent reaction of amines, aldehydes, and ethyl pyruvate to efficiently afford 3-amino 1,5-dihydro-2H-pyrrol-2-ones [24]. More recently, we have reported the asymmetric version of this reaction, using chiral phosphoric acids as catalysts [27], and we have extended this methodology to the synthesis of phosphorated and fluorinated γ-lactam derivatives [40]. We have also described a similar Brönsted acid-catalyzed multicomponent reaction using acetylene carboxylates instead of pyruvate derivatives that affords in this case 4-carboxyl substituted γ-lactam derivatives [29].

Chemistry
The multicomponent protocol for the synthesis of 1,5-dihydro-2H-pyrrol-2-ones 4-12 implies the reaction of aromatic amines 1 with aldehydes 2 and pyruvate derivatives 3 in the presence of a catalytic amount of BINOL derived phosphoric for several hours, at room temperature [27,40]. Although the stoichiometry of the reaction is indeed 2:1:1 (amine/aldehyde/pyruvate), three equivalents of pyruvate derivative are required to achieve good yields in reasonable reaction times. Due to the good solubility of the reagents and phosphoric acid catalyst in chlorinated solvents, dichloromethane was chosen as the optimal reaction media, since the reaction in ethereal solvents, toluene, or AcN afforded γ-lactam derivatives in lower yields and higher reaction times. In addition, the presence of MgSO 4 is necessary to remove the water generated in the process (Scheme 1). Although the reaction proceeds with similar efficiency using other acidic sources (H 2 SO 4 , H 3 PO 4 , Mg(HSO 4 ) 2 ) the use of BINOL-derived phosphoric acid as a catalyst is very convenient since it facilitates the purification process and has no interaction with the drying agent. The mechanism of the reaction comprises an initial formation of imine and enamine species 13 and 14 through a double condensation of the amine 1 with aldehyde 2 and pyruvate derivative 3, followed by an acid promoted Mannich reaction that leads to the formation of intermediate 15. Finally, an intramolecular cyclization between the amine and the ester groups yields γ-lactam substrates 4-12.

Chemistry
The multicomponent protocol for the synthesis of 1,5-dihydro-2H-pyrrol-2-ones 4-12 implies the reaction of aromatic amines 1 with aldehydes 2 and pyruvate derivatives 3 in the presence of a catalytic amount of BINOL derived phosphoric for several hours, at room temperature [27,40]. Although the stoichiometry of the reaction is indeed 2:1:1 (amine/aldehyde/pyruvate), three equivalents of pyruvate derivative are required to achieve good yields in reasonable reaction times. Due to the good solubility of the reagents and phosphoric acid catalyst in chlorinated solvents, dichloromethane was chosen as the optimal reaction media, since the reaction in ethereal solvents, toluene, or AcN afforded γ-lactam derivatives in lower yields and higher reaction times. In addition, the presence of MgSO4 is necessary to remove the water generated in the process (Scheme 1). Although the reaction proceeds with similar efficiency using other acidic sources (H2SO4, H3PO4, Mg(HSO4)2) the use of BINOL-derived phosphoric acid as a catalyst is very convenient since it facilitates the purification process and has no interaction with the drying agent. The mechanism of the reaction comprises an initial formation of imine and enamine species 13 and 14 through a double condensation of the amine 1 with aldehyde 2 and pyruvate derivative 3, followed by an acid promoted Mannich reaction that leads to the formation of intermediate 15. Finally, an intramolecular cyclization between the amine and the ester groups yields γ-lactam substrates 4-12. Keeping into account the mechanism proposed for this transformation, the electronic character of the amine substrate might be the crucial factor in the reactivity of the reaction. Keeping into account the mechanism proposed for this transformation, the electronic character of the amine substrate might be the crucial factor in the reactivity of the reaction. While the use of activated amines may benefit the nucleophilic character of enamine nucleophiles 14 this would result in a decreased electrophilic character of imine substrates 13. On the contrary, the use of deactivated amines would result in activation of imine electrophile 13 and deactivation of enamine nucleophile 14. Following this approach, 38 densely functionalized substrates were synthesized, to illustrate the synthetic potential of the reaction. First γ-lactams 4-5 were prepared using weakly activated p-toluidine (1a, R 1 = p-CH 3 C 6 H 4 ) as amine substrate, as well as diverse aldehydes and pyruvate derivatives (Chart 1). Using ethyl pyruvate (3a, R 3 = H), simple formaldehyde (2a, R 2 = H) or benzaldehyde (2b, R 2 = Ph), aromatic aldehydes bearing electron-donating and electron-withdrawing groups 2c-i, heteroaryl and naphthyl carboxaldehydes 2j-m, and simple aliphatic aldehydes 2n-q were used successfully in the multicomponent reaction (Chart 1, 4a-q). Moreover, other aldehydes such as cinnamaldehyde (2r, R 2 = CH=CHPh) or ethyl glyoxalate (2s, R 2 = CO 2 Et) were also effectively used as substrates (Chart 1, 4r-s). Remarkably, fluorine-containing γ-lactam substrates were also synthesized using perfluorobenzaldehyde (2t, R 2 = C 6 F 5 ) or trifluoroacetaldehyde (2u, R 2 = CF 3 ) (Chart 1, 4t,u), the latter generated in situ from its hydrate, and even phosphorus-containing substrates were efficiently obtained when β-phosphorated aldehydes 2v,w (R 2 = CH 2 P(O)(OEt) 2 , CH 2 P(O)Ph 2 ) were used as starting materials in the multicomponent process (Chart 1, 4v,w). While the use of activated amines may benefit the nucleophilic character of enamine nucleophiles 14 this would result in a decreased electrophilic character of imine substrates 13. On the contrary, the use of deactivated amines would result in activation of imine electrophile 13 and deactivation of enamine nucleophile 14.
Next, the reaction was extended to the use of other amine substrates. The reaction of ethyl pyruvate (3a, R 3 = H) with electron-rich p-anisidine (1b, R 1 = p-CH 3 OC 6 H 4 ) and several aldehydes 2 affords the corresponding γ-lactam derivatives in very good yields (Chart 2, 6a-c) while, if halogen-substituted anilines 1c-g are used as amine substrates in the same reaction, γ-lactam derivatives are again efficiently obtained (Chart 2, 7-10). The reaction even tolerates the use of deactivated aromatic amines such as mtrifluoromethylaniline (1h, R 1 = m-CF 3 C 6 H 4 ) for the formation of substrates containing trifluoromethyl groups (Chart 2, 11a,b) and one example of the utilization of a heteroaromatic amine 1h (R 1 = 2-quinolinyl), that leads to the formation of a quinoline derived substrate, is also shown (Chart 2, 12. In the latter case, a low yield of lactam substrate 12a is obtained, possibly due to the high steric crowding present in the final structure. weakly activated p-toluidine (1a, R 1 = p-CH3C6H4) react with p-nitrobenzaldehyde (2g, R 2 = p-NO2C6H4) or benzaldehyde (2b, R 2 = Ph) to afford 4-substituted γ-lactam derivatives (Chart 1, 5a-c). In this case, the reaction should be heated and performed in methyl tertbutyl ether as a solvent, which may be due to the high steric hindrance present in the final product. Under the same conditions β-phosphorated pyruvate derivatives 3e,f (R 3 = P(O)(OEt)2, P(O)OPh2) react with p-toluidine (1a, R 1 = p-CH3C6H4) and formaldehyde (2a, R 2 = H) yielding phosphorus substituted γ-lactams (Chart 1, 5d,e).
Next, the reaction was extended to the use of other amine substrates. The reaction of ethyl pyruvate (3a, R 3 = H) with electron-rich p-anisidine (1b, R 1 = p-CH3OC6H4) and several aldehydes 2 affords the corresponding γ-lactam derivatives in very good yields (Chart 2, 6a-c) while, if halogen-substituted anilines 1c-g are used as amine substrates in the same reaction, γ-lactam derivatives are again efficiently obtained (Chart 2, 7-10). The reaction even tolerates the use of deactivated aromatic amines such as m-trifluoromethylaniline (1h, R 1 = m-CF3C6H4) for the formation of substrates containing trifluoromethyl groups (Chart 2, 11a,b) and one example of the utilization of a heteroaromatic amine 1h (R 1 = 2-quinolinyl), that leads to the formation of a quinoline derived substrate, is also shown (Chart 2, 12. In the latter case, a low yield of lactam substrate 12a is obtained, possibly due to the high steric crowding present in the final structure. Besides, when we tried to extend the multicomponent protocol to the use of ketone electrophiles instead of aldehydes, our reaction failed. In the case of the reaction using ethyl pyruvate (3a), and p-toluidine (1a), the imine species generated from pyruvate and the amine was found to be more reactive than the alternative ketimine species and a selfcondensation reaction was observed. Accordingly, substrate 16 is obtained when the reaction is performed using one equivalent of p-toluidine (1a) and ethyl pyruvate (3a), where two units of each substrate conform to the structure of the γ-lactam derivative. Additionally, the hydrogenation of 1,5-dihydro-2H-pyrrol-2-one ring in 16 allowed the obtaining of saturated γ-lactam 17 in very good yield and as a single diastereoisomer (Scheme 2).
The relative configuration of the stereocenters in saturated γ-lactam 17 was determined by NOESY. NMR experiments with compound 17 showed NOE effect between both, the proton and the methyl group at the two stereogenic centers, at δ = 4.26 and 1.63 Chart 2. 1,5-Dihydro-2H-pyrrol-2-ones 6-12 obtained (Ring numbering is shown for 6a).
Besides, when we tried to extend the multicomponent protocol to the use of ketone electrophiles instead of aldehydes, our reaction failed. In the case of the reaction using ethyl pyruvate (3a), and p-toluidine (1a), the imine species generated from pyruvate and the amine was found to be more reactive than the alternative ketimine species and a self-condensation reaction was observed. Accordingly, substrate 16 is obtained when the reaction is performed using one equivalent of p-toluidine (1a) and ethyl pyruvate (3a), where two units of each substrate conform to the structure of the γ-lactam derivative. Additionally, the hydrogenation of 1,5-dihydro-2H-pyrrol-2-one ring in 16 allowed the obtaining of saturated γ-lactam 17 in very good yield and as a single diastereoisomer (Scheme 2).
The relative configuration of the stereocenters in saturated γ-lactam 17 was determined by NOESY. NMR experiments with compound 17 showed NOE effect between both, the proton and the methyl group at the two stereogenic centers, at δ = 4.26 and 1.63 ppm, respectively, with the same NMR signal corresponding to one of the protons of the diastereotopic CH 2 group at δ = 2.61 ppm, indicating that these three atoms are oriented in the same direction. As expected, a strong NOE is observed between both diastereotopic protons. The fact that almost no NOE is observed between the signal corresponding to the second of the diastereotopic protons, at δ = 2.46 ppm, and the proton and the methyl group at the stereogenic centers confirms a relative cis configuration between the carboxylate and the amino groups. ppm, respectively, with the same NMR signal corresponding to one of the protons of the diastereotopic CH2 group at δ = 2.61 ppm, indicating that these three atoms are oriented in the same direction. As expected, a strong NOE is observed between both diastereotopic protons. The fact that almost no NOE is observed between the signal corresponding to the second of the diastereotopic protons, at δ = 2.46 ppm, and the proton and the methyl group at the stereogenic centers confirms a relative cis configuration between the carboxylate and the amino groups. Then, to extend the diversity of our substrates, some synthetic transformations of 1,5dihydro-2H-pyrrol-2-one substrates were performed. The hydrogenation of phosphorus and fluorine substituted γ-lactams 4u,v and 6a, in the presence of a palladium catalyst, affords saturated 5-membered ring substrates in excellent yields (Chart 3, 18a-c). Likewise, treatment of γ-lactam 4v with methyllithium followed by the addition of methyl iodide is known to give the dialkylated substrate (Chart 3, 19), while the metalation of 4v with lithium di-iso-propylamide (LDA) followed by the addition of an aldehyde affords conjugated azatrienes 20 through a Horner-Wadsworth-Emmons olefination reaction and a subsequent ring-opening of the γ-lactam core (Chart 3, 20a,b). All our other additional attempts to functionalize the enamine moiety of lactam substrate 4b were unsuccessful but, surprisingly, when a bulky base like LDA was used, to generate the aza-enolate species, oxidation of the substrate was observed, leading to the formation of γ-lactam 21 that could be only isolated in very low yields (Scheme 3). Then, to extend the diversity of our substrates, some synthetic transformations of 1,5dihydro-2H-pyrrol-2-one substrates were performed. The hydrogenation of phosphorus and fluorine substituted γ-lactams 4u,v and 6a, in the presence of a palladium catalyst, affords saturated 5-membered ring substrates in excellent yields (Chart 3, 18a-c). Likewise, treatment of γ-lactam 4v with methyllithium followed by the addition of methyl iodide is known to give the dialkylated substrate (Chart 3, 19), while the metalation of 4v with lithium di-iso-propylamide (LDA) followed by the addition of an aldehyde affords conjugated azatrienes 20 through a Horner-Wadsworth-Emmons olefination reaction and a subsequent ring-opening of the γ-lactam core (Chart 3, 20a,b).
diastereotopic CH2 group at δ = 2.61 ppm, indicating that these three atoms are oriented in the same direction. As expected, a strong NOE is observed between both diastereotopic protons. The fact that almost no NOE is observed between the signal corresponding to the second of the diastereotopic protons, at δ = 2.46 ppm, and the proton and the methyl group at the stereogenic centers confirms a relative cis configuration between the carboxylate and the amino groups. Then, to extend the diversity of our substrates, some synthetic transformations of 1,5dihydro-2H-pyrrol-2-one substrates were performed. The hydrogenation of phosphorus and fluorine substituted γ-lactams 4u,v and 6a, in the presence of a palladium catalyst, affords saturated 5-membered ring substrates in excellent yields (Chart 3, 18a-c). Likewise, treatment of γ-lactam 4v with methyllithium followed by the addition of methyl iodide is known to give the dialkylated substrate (Chart 3, 19), while the metalation of 4v with lithium di-iso-propylamide (LDA) followed by the addition of an aldehyde affords conjugated azatrienes 20 through a Horner-Wadsworth-Emmons olefination reaction and a subsequent ring-opening of the γ-lactam core (Chart 3, 20a,b). 19 and derivatives 20 (Ring numbering is shown for 18a).
All our other additional attempts to functionalize the enamine moiety of lactam substrate 4b were unsuccessful but, surprisingly, when a bulky base like LDA was used, to generate the aza-enolate species, oxidation of the substrate was observed, leading to the formation of γ-lactam 21 that could be only isolated in very low yields (Scheme 3). All our other additional attempts to functionalize the enamine moiety of lactam substrate 4b were unsuccessful but, surprisingly, when a bulky base like LDA was used, to generate the aza-enolate species, oxidation of the substrate was observed, leading to the formation of γ-lactam 21 that could be only isolated in very low yields (Scheme 3). With this batch of structures in our hands, we faced the study of their biological activity. Accordingly, the antiproliferative activity of our γ-lactam derivatives against several cancer cell lines was next investigated.

Biological Results
In vitro cytotoxicity of the γ-lactam derivatives was evaluated by testing their antiproliferative activities against several human cancer cell lines. Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, non-malignant MRC5 lung fibroblasts were tested for studying selective toxicity [57] and chemotherapeutic doxorubicin is used as the reference value.
In a preliminary study, we tested the cytotoxicity of γ-lactam 4b as lead compound against seven human cancer cell lines: HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell). The cell proliferation inhibitory activity of γ-lactam 4b is shown as IC50 values ( Figure 2). Although no grown inhibition activity was observed against HEK293 and MCF7 cell lines, moderate cytotoxicity was observed in the case of HTB81, HeLa, and RKO cell lines. Remarkably, promising IC50 values of γ-lactam 4b against SKOV3 and A549 cell lines and, interestingly very good selectivity was also obtained towards MRC5 non-malignant cell line ( Figure 2). With these results in hand, we extended our study of the cell proliferation inhibitory activities of our family of γ-lactams using RKO, SKOV3, and A549 cell lines. The IC50 values of γ-lactam derivatives 4-12 (Charts 1 and 2) are presented in Table 1. First, γ-lactam derivatives 4 derived from p-toluidine were evaluated. The simplest member of this family is substrate 4a, which is obtained from formaldehyde and holds no substituent at position 5 of the ring. Compound 4a did not show activity against the SKOV3 cell line in vitro, but a slight activity was observed for the A549 cell line, with modest IC50 values of 38.25 ± 3.35 µM (Table 1, Entry 1). The cytotoxicity of γ-lactams 4 was found to be strongly dependent on the substituent at the 5 position of the ring. Phenyl substituted lactam 4b showed improved IC50 values of = 9.62 ± 1.18 and 2.34 ± 0.28 µM against SKOV3 and A549, With this batch of structures in our hands, we faced the study of their biological activity. Accordingly, the antiproliferative activity of our γ-lactam derivatives against several cancer cell lines was next investigated.

Biological Results
In vitro cytotoxicity of the γ-lactam derivatives was evaluated by testing their antiproliferative activities against several human cancer cell lines. Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, non-malignant MRC5 lung fibroblasts were tested for studying selective toxicity [57] and chemotherapeutic doxorubicin is used as the reference value.
In a preliminary study, we tested the cytotoxicity of γ-lactam 4b as lead compound against seven human cancer cell lines: HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell). The cell proliferation inhibitory activity of γ-lactam 4b is shown as IC 50 values ( Figure 2). Although no grown inhibition activity was observed against HEK293 and MCF7 cell lines, moderate cytotoxicity was observed in the case of HTB81, HeLa, and RKO cell lines. Remarkably, promising IC 50 values of γ-lactam 4b against SKOV3 and A549 cell lines and, interestingly very good selectivity was also obtained towards MRC5 non-malignant cell line ( Figure 2). With this batch of structures in our hands, we faced the study of their biological activity. Accordingly, the antiproliferative activity of our γ-lactam derivatives against several cancer cell lines was next investigated.

Biological Results
In vitro cytotoxicity of the γ-lactam derivatives was evaluated by testing their antiproliferative activities against several human cancer cell lines. Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, non-malignant MRC5 lung fibroblasts were tested for studying selective toxicity [57] and chemotherapeutic doxorubicin is used as the reference value.
In a preliminary study, we tested the cytotoxicity of γ-lactam 4b as lead compound against seven human cancer cell lines: HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell). The cell proliferation inhibitory activity of γ-lactam 4b is shown as IC50 values (Figure 2). Although no grown inhibition activity was observed against HEK293 and MCF7 cell lines, moderate cytotoxicity was observed in the case of HTB81, HeLa, and RKO cell lines. Remarkably, promising IC50 values of γ-lactam 4b against SKOV3 and A549 cell lines and, interestingly very good selectivity was also obtained towards MRC5 non-malignant cell line (Figure 2). With these results in hand, we extended our study of the cell proliferation inhibitory activities of our family of γ-lactams using RKO, SKOV3, and A549 cell lines. The IC50 values of γ-lactam derivatives 4-12 (Charts 1 and 2) are presented in Table 1. First, γ-lactam derivatives 4 derived from p-toluidine were evaluated. The simplest member of this family is substrate 4a, which is obtained from formaldehyde and holds no substituent at position 5 of the ring. Compound 4a did not show activity against the SKOV3 cell line in vitro, but a slight activity was observed for the A549 cell line, with modest IC50 values of 38.25 ± 3.35 µM (Table 1, Entry 1). The cytotoxicity of γ-lactams 4 was found to be strongly dependent on the substituent at the 5 position of the ring. Phenyl substituted lactam 4b showed improved IC50 values of = 9.62 ± 1.18 and 2.34 ± 0.28 µM against SKOV3 and A549, With these results in hand, we extended our study of the cell proliferation inhibitory activities of our family of γ-lactams using RKO, SKOV3, and A549 cell lines. The IC 50 values of γ-lactam derivatives 4-12 (Charts 1 and 2) are presented in Table 1. First, γlactam derivatives 4 derived from p-toluidine were evaluated. The simplest member of this family is substrate 4a, which is obtained from formaldehyde and holds no substituent at position 5 of the ring. Compound 4a did not show activity against the SKOV3 cell line in vitro, but a slight activity was observed for the A549 cell line, with modest IC 50 values of 38.25 ± 3.35 µM (Table 1, Entry 1). The cytotoxicity of γ-lactams 4 was found to be strongly dependent on the substituent at the 5 position of the ring. Phenyl substituted lactam 4b showed improved IC 50 values of = 9.62 ± 1.18 and 2.34 ± 0.28 µM against SKOV3 and A549, respectively, and good selectivity with respect to non-malignant lung fibroblasts (Table 1, Entry 2). The introduction of methyl groups into a bioactive structure makes it more lipophilic, often resulting in an improved ability of molecules to cross cell membranes [58,59]. However, in this case, ortho, meta or para methyl-substituted benzene substituents in the position 5 of the ring resulted in a drop in the cytotoxic activity against the A549 cell line, with IC 50 values around 20-40 µM, and a complete loss of the activity in SKOV3 cell line with IC 50 values over 50 µM (Table 1, Entries-3-5).
Although, the effect of the introduction of fluorine atoms in the structure of organic compounds is rather difficult to predict, very often it leads to increased activities [60][61][62]. For this reason, next, we tested the in vitro cytotoxicity of fluorine-containing γ-lactams 4f,i against SKOV3 and A549 cell lines. The introduction of a para-fluorophenyl substituent into the 5-membered ring did not result in improved activity and IC 50  Next, we studied the introduction of aliphatic substituents at position 5 of the γlactam ring. The cytotoxic effect found for methyl-substituted lactam 4n was found to be slightly lower if compared with phenyl substituted lactam 4b, with IC 50 values of 29.16 ± 1.00 and 15.68 ± 0.92 µM in SKOV3 and A549 cell lines, respectively (Table 1, Entry 14). Substitution with larger aliphatic substituents, such as iso-propyl, iso-butyl, or cyclohexyl groups, resulted in a complete loss of the cytotoxic activity of substrates 4o-q against SKOV3 cell line and nearly the same IC 50 values or slightly better against A549 cell line (  50 values of about 10 µM for SKOV3 and A549 cell lines, but not much selectivity with respect to non-malignant cells, and ethoxycarbonyl substituted lactam 4s did not show any cytotoxic activity (Table 1, Entries 18,19).
Continuing with our interest in fluorine-containing molecules, next we faced the study of the effect of perfluoroalkylalted substituents on the antiproliferative activity of our γ-lactam substrates. Nevertheless, perfluorophenyl and trifluoromethyl substituted γ-lactams 4t,u showed lower cytotoxic activity than their parent non-perfluorinated derivatives 4b and 4n (Table 1, Entries 2, 14 vs. 20, 21). In addition, it is well known that structural modifications of active molecules involving the introduction of phosphorus-containing functional groups, very often result in increased or new activities [63][64][65]. For this reason, next, we studied the cell proliferation inhibitory activities of phosphonate and phosphine oxide-derived substrates 4v,w. Phosphonate derivative 4v showed a modest cytotoxic activity, but only against A549 cell line, with an IC 50 value of 11.29 ± 1.80 µM (Table 1,  Entry 22) and, although phosphine oxide derivative 4w presented cytotoxic properties against the three studied cancer cell lines, the selectivity with respect to non-malignant lung fibroblasts was not very significant (Table 1, Entry 23).
After studying the effect of the substitution at position 5 of the ring into the antiproliferative activity of 1,5-dihydro-2H-pyrrol-2-ones 4, we set up our following objective into the study of the effect of the substitution at the 4 position of 1,5-dihydro-2H-pyrrol-2-one ring. Accordingly, γ-lactams 5, synthesized using substituted pyruvate derivatives, were next studied. Although none of those substrates showed any activity against the SKOV3 cell line, some interesting results were obtained with the A549 cell line. Methyl and benzyl substituted γ-lactams 5a,b showed improved IC 50 values of 2.05 ± 0.23 and 9.92 ± 1.15 µM (Table 1, Entries 24,25) if compared with the parent unsubstituted derivative 4g (Table 1,  Entry 7). Remarkably, lactam 5c holding a methyl carboxylate substituent at position 5 of the ring showed a very good IC 50 value of 1.67 ± 0.49 µM against A549 cell line and a very good selectivity if compared with SKOV3 or healthy cells. Moreover, the introduction of a phosphonate substituent at the 4 position results in a dramatic decrease in the cytotoxic power in γ-lactam 5d with an IC 50 value higher than 50 µM (Table 1, Entry 27), which is reflected by the comparison with the IC 50 value observed in its unsubstituted derivative 4a (Table 1, Entry 1). Unfortunately, the activity of diphenylphosphine oxide-derived compound 5e could not be tested due to the low solubility of the substrate in the solvents that are compatible with the tests for in vitro assays (Table 1, Entry 28).
Once the influence of the substituents at the positions 1, 3, and 4 of the γ-lactam ring into the cell proliferation inhibitory activities of our substrates have been described, the next point to be addressed is the impact of the amine substituents in the growth inhibition activity of lactams 6-12 (Chart 2). First, p-anisidine derived substrates 6 were examined. Phenyl substituted lactam 6a showed improved cytotoxicity against the SKOV3 cell line but slightly worse against the A549 cell line than its parent p-toluidine derivative 4b with IC 50 values of 6.84 ± 0.59 and 12.02 ± 1.96 µM. Their selectivity with respect to nonmalignant cells was very satisfactory (  Next, we studied the effect of the introduction of halogen atoms in the structure of the amine moiety of substrates 7-10. No significant changes were observed in the cytotoxic effect of p-chloroaniline derivative 7, holding a p-nitrophenyl at the 5 position of the ring, if compared to its p-toluidine or p-anisidine derivative 4g or 6b (Table 2, Entry 4 vs. Table 1, Entry 7 and Table 2, Entry 2). Likewise, p-bromoaniline derivatives 8a,b did not show any grown inhibition in any of the cell lines (Table 2, Entries 5, 6). Switching the chlorine atom from para to the meta position in the aromatic ring of the amine improved the cytotoxic activity in both cell lines for γ-lactam 9, although in this case, the selectivity against non-malignant cells gets worse (  1, Entry 7). The use of m-trifluoromethyl substituted aniline substrates resulted also in an improvement of the grown inhibition of compounds 11a,b. Unsubstituted formaldehyde derivative 11a showed slightly better IC 50 values than the model non-fluorinated derivative 4a (  1, Entry 7).
The effect of a heteroaromatic amine moiety on the γ-lactam structure was also studied. In this context, quinoline scaffold plays an important role in drug development [66,67] and many of their derivatives have shown their potential as anticancer agents [68] such as wellknown camptothecin and their derivatives [69]. However, although an improvement in the cytotoxic activity and selectivity in the A549 cell line was observed in 2-quinolinylamine substituted lactam 12a with respect to other similar derivatives, like p-toluidine, p-anisidine, p-chloroaniline, and p-bromoaniline substituted lactams 4g, 6b, 7, and 8a (  Table 2, Entries 2, 4 and 5), m-chloroaniline, o-fluoroaniline, and m-trifluoromethylaniline substituted substrates 9, 10 and 11b proved to be superior to 2-quinolinylamine derivative 12a in this respect ( To complete our research, we finally tested the cell proliferation inhibitory activities of other γ-lactam derivatives such as dimeric substrate 16 (see Scheme 2, vide supra) as well as derivatives 17-21, obtained by the modification of some of the γ-lactam derivatives (Chart 3 and Scheme 3, vide supra).
Dimeric lactam derivative 16 showed a modest IC 50 value 13.97 ± 1.05 µM in the A549 cell line, and it presented a high selectivity if compared with SKOV3, RKO, or MRC5 cell lines (Table 3, Entry 1). Unfortunately, the reduction of compound 16 showed to have a negative effect on the cytotoxicity of lactam derivatives, and no activity was observed for the saturated γ-lactam derivative 17 (Table 3, Entry 2). With respect to the activity against A549 cell line, the reduction of dihydropyrrolone ring in compounds 4u,v resulted in an improvement in the cytotoxic activity of p-toluidine derived γ-lactam compounds 18a,b with better IC 50 values of 3.27 ± 0.65 and 4.25 ± 0.65 µM, respectively (Table 3, Entries 3, 4 vs. Table 1, Entries 21,22). However, in the case of p-anisidine derivative 18c, a slightly worse IC 50 value of 19.45 ± 0.34 µM was found if compared with its parent derivative 6a ( Table 3, Entry 5 vs. Table 2, Entry 1). With respect to the SKOV3 cell line, the reduction of dihydropyrrolone ring in compounds 4u,v did not result in any improvement into the grown inhibition activity, and γ-lactam derivatives 18a,b presented IC 50 values higher than 50 µM, as their parent unsaturated compounds 4u,v (Table 3, Entries 3, 4 vs. Table 1, Entries 21,22). Although saturated γ-lactam derived from p-anisidine 18c still showed an IC 50 value of 19.45 ± 0.34 µM, this value was found to be slightly higher than its unsaturated parent compound 6a (Table 3, Entry 5 vs. Table 2, Entry 1). As it has been addressed above, due to the lipophilic character of alkyl groups, the functionalization of bioactive structures with methyl groups often results in stronger activities than the parent structures [59]. For that reason, we had high expectations with respect to the activity of lactam derivative 19 that holds four methyl groups unto its structure. Indeed, a slightly higher IC 50 value of 3.65 ± 0.25 µM against A549 cell line was obtained for functionalized compound 19 with respect to the parent lactam 4v ( Finally, due to the presence of a stereogenic carbon in the structure of our lactam derivatives, we were intrigued about the real activity of the individual enantiomers. For that reason, we performed the enantioselective synthesis of lactam 4v, using both enantiopure isomers of a substituted BINOL phosphoric acid catalyst in the multicomponent reaction (Scheme 4).
showed a very high toxicity against the A549 cell line and moderate activity against the SKOV3 cell line with IC50 values of 0.60 ± 0.08 and 16.72 ± 1.1 µM, respectively. However, compound 21 exhibited also a strong toxicity against healthy cells (Table 3, Entry 9).
Finally, due to the presence of a stereogenic carbon in the structure of our lactam derivatives, we were intrigued about the real activity of the individual enantiomers. For that reason, we performed the enantioselective synthesis of lactam 4v, using both enantiopure isomers of a substituted BINOL phosphoric acid catalyst in the multicomponent reaction (Scheme 4). Both enantiomers of 4v were prepared in 90% ee and, then, two almost enantiopure samples were obtained in 97% ee by a subsequent crystallization. However, no significant differences were observed for R or S enantiomers if compared with the racemic sample with IC50 values of 9.23 ± 1.68 and 10.53 ± 0.80 µM, respectively.
Next, cell morphology analysis and flow cytometry assays were performed, to elucidate the mechanism of the antiproliferative activity of γ-lactam substrates. Cell morphology was analyzed at different time points after the addition of compound 4b to visualize the cellular modifications as a result of the treatment. The first concentration studied was 1 µM, which is below the IC50 of 4b and, accordingly, cells showed a healthy, uniform morphology and cellular growth was recognizable over time, meaning that this concentration was well tolerated by the cells (Figure 3A). On the contrary, after the addition of 5 µM of 4b, alterations of the cellular morphology were observed, as well as a mild disruption of the cellular growth, especially after 6 h of exposure ( Figure 3B). At higher concentrations, the alterations observed in cell morphology clearly evidenced high cytotoxicity ( Figure 3C,D). These results support previously obtained data in relation to the cytotoxicity and the IC50 of 4b.
In addition, flow cytometry assays were performed using compound 4b in A549 cells at 12, 24, and 48 h post-exposure. Early apoptotic cells were FL-1 positive and FL-3 negative, meaning that they had initialized the apoptotic process but were still alive at the time of the measurement. Percentages of these early-apoptotic cells after 12, 24, and 48 h. were, respectively, 31.17 ± 2.04%, 34.97 ± 3.62% and 40.13 ± 0.81%. On the other hand, late apoptotic and necrotic cells were both FL-1 and FL-3 positive, meaning that they activated the apoptotic machinery and were dead at the time of the measurement. Percentages of lateapoptotic and necrotic cells after 12, 24 and 48 h were, respectively, 33.03 ± 1.43%, 37.60 ± 3.37% and 36.90 ± 0.70% (Figure 4). Control live cells were all FL-1 and FL-3 negative, while around 95% of control necrotic cells were FL-3 positive, FL-1 negative (See ESI). Taken together, these results suggest that one of the main mechanisms by which 4b induces cytotoxicity is based on the activation of intracellular apoptotic mechanisms. In fact, among all the dead cells detected, which were FL-1 positive, almost all were simultaneously positive for the FL-3 channel too, while only a small percentage (2% or lower) of dead cells were exclusively FL-1 positive. Therefore, the majority of dead cells detected in Both enantiomers of 4v were prepared in 90% ee and, then, two almost enantiopure samples were obtained in 97% ee by a subsequent crystallization. However, no significant differences were observed for R or S enantiomers if compared with the racemic sample with IC 50 values of 9.23 ± 1.68 and 10.53 ± 0.80 µM, respectively.
Next, cell morphology analysis and flow cytometry assays were performed, to elucidate the mechanism of the antiproliferative activity of γ-lactam substrates. Cell morphology was analyzed at different time points after the addition of compound 4b to visualize the cellular modifications as a result of the treatment. The first concentration studied was 1 µM, which is below the IC 50 of 4b and, accordingly, cells showed a healthy, uniform morphology and cellular growth was recognizable over time, meaning that this concentration was well tolerated by the cells (Figure 3A). On the contrary, after the addition of 5 µM of 4b, alterations of the cellular morphology were observed, as well as a mild disruption of the cellular growth, especially after 6 h of exposure ( Figure 3B). At higher concentrations, the alterations observed in cell morphology clearly evidenced high cytotoxicity ( Figure 3C,D). These results support previously obtained data in relation to the cytotoxicity and the IC 50 of 4b.
In addition, flow cytometry assays were performed using compound 4b in A549 cells at 12, 24, and 48 h post-exposure. Early apoptotic cells were FL-1 positive and FL-3 negative, meaning that they had initialized the apoptotic process but were still alive at the time of the measurement. Percentages of these early-apoptotic cells after 12, 24, and 48 h. were, respectively, 31.17 ± 2.04%, 34.97 ± 3.62% and 40.13 ± 0.81%. On the other hand, late apoptotic and necrotic cells were both FL-1 and FL-3 positive, meaning that they activated the apoptotic machinery and were dead at the time of the measurement. Percentages of late-apoptotic and necrotic cells after 12, 24 and 48 h were, respectively, 33.03 ± 1.43%, 37.60 ± 3.37% and 36.90 ± 0.70% (Figure 4). Control live cells were all FL-1 and FL-3 negative, while around 95% of control necrotic cells were FL-3 positive, FL-1 negative (See ESI). Taken together, these results suggest that one of the main mechanisms by which 4b induces cytotoxicity is based on the activation of intracellular apoptotic mechanisms. In fact, among all the dead cells detected, which were FL-1 positive, almost all were simultaneously positive for the FL-3 channel too, while only a small percentage (2% or lower) of dead cells were exclusively FL-1 positive. Therefore, the majority of dead cells detected in this study were apoptotic cells. In addition, the total percentages of FL-3 positive cells, which comprised early and late apoptotic cells, were over 60%, and the percentage increased over time. this study were apoptotic cells. In addition, the total percentages of FL-3 positive cells, which comprised early and late apoptotic cells, were over 60%, and the percentage increased over time.  It is also worth mentioning that, except for substrates 8a, 8b, and 11b, all other γlactam derivatives fulfill the requirements for orally active drugs in humans, in accordance with Lipinski's rule of five. According to the predictions, most of the described substrates have a high gastrointestinal absorption and the ability to cross the blood-brain barrier (See Table in Supplementary Materials).   It is also worth mentioning that, except for substrates 8a, 8b, and 11b, all other γ lactam derivatives fulfill the requirements for orally active drugs in humans, in accord ance with Lipinski's rule of five. According to the predictions, most of the described sub strates have a high gastrointestinal absorption and the ability to cross the blood-brain barrier (See Table in   It is also worth mentioning that, except for substrates 8a, 8b, and 11b, all other γlactam derivatives fulfill the requirements for orally active drugs in humans, in accordance with Lipinski's rule of five. According to the predictions, most of the described substrates have a high gastrointestinal absorption and the ability to cross the blood-brain barrier (See Table in Supplementary Materials).

Compounds Purity Analysis
All synthesized compounds were analyzed by HPLC to determine their purity. The analyses were performed on Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, United States) (C-18 column, Hypersil, BDS, 5 µm, 0.4 mm × 25 mm) at room temperature. All the tested compounds were dissolved in dichloromethane, and 5 µL of the sample was loaded onto the column. Ethanol and heptane were used as the mobile phase, and the flow rate was set at 1.0 mL/min. The maximal absorbance at the range of 190−400 nm was used as the detection wavelength. The purity of all the tested lactam derivatives 4-12 and 16-21 is >95%, which meets the purity requirement by the Journal.

Representative Experimental Procedures and Characterization Data for Compounds 4-12 and 16-21
Representative Procedure for the Multicomponent Reaction of Amines 1, Aldehydes 2, and Pyruvate Derivatives 3 A solution of amine 1 (4 mmol), aldehyde 2 (2 mmol), pyruvate derivative 3 (6 mmol), and BINOL derived phosphoric acid (70 mg, 0.2 mmol) was stirred in diethyl ether or MTBE (10 mL) in the presence of anhydrous MgSO 4 at room temperature or 55 • C for 48 h (see ESI). The volatiles were distilled off at reduced pressure and the crude residue was purified by crystallization in diethyl ether or by column chromatography (Hexanes/AcOEt) to afford pure lactams 4-12.
Representative Procedure for the Horner-Wadsworth-Emmons Reactions of γ-Lactam 4v A solution of lactam 4v (214 mg, 0.5 mmol) in THF (2 mL) was dropwise added to a cooled solution (−78 • C) of LDA (1.1 mmol, prepared from 690 µL of 1.6 M n-butyl lithium and 150 µL of diisopropylamine in 5 mL of THF) and the mixture was stirred for 1 h at −78 • C. Then, the corresponding aldehyde (0.75 mmol) was added and the reaction was warmed to room temperature overnight. The solution was quenched with 10 mL of water and extracted with dichloromethane (3 × 10 mL), dried with anhydrous MgSO 4 , and concentrated at reduced pressure. The crude residue was purified by column chromatography (Hexanes/AcOEt 9:1) to afford products 20a-b.

Evaluation of Cytotoxicity Mechanisms
Flow cytometry assays were conducted using a FACSCalibur system flow cytometer (Becton Dickinson Bioscience, Franklin Lakes, NJ, USA), to identify apoptotic cells and to differentiate them from necrotic cells. A549 cells were exposed to 5 µM of the cytotoxic compound 4b and cell apoptosis and necrosis were evaluated 12, 24, and 48 h after exposure. For that purpose, treated cells were washed with phosphate-buffered saline (PBS) (Sigma-Aldrich, Spain) and detached with trypsin/EDTA (0.25%) (Gibco, New York, NY, USA). Cells were centrifuged at 1100 rpm for 5 min, and then the resulting pellet was resuspended in cell growth media and transferred to specific flow cytometer tubes. Propidium iodide (Sigma-Aldrich, Spain) at 1:300 dilution was used in each sample to detect necrotic cells and eBioscience™ Annexin V Apoptosis Detection Kit FITC (Fisher Scientific, Spain) was used to detect apoptotic cells following the manufacturer's instructions. The fluorescent signals corresponding to necrotic cells and apoptotic cells were measured at 650 nm (FL3) and 525 nm (FL1), respectively. Non-treated cells, used as negative control samples, were displayed on a forward scatter (FSC) versus side scatter (SSC) dot plot to establish a collection gate and exclude cells debris. Cells treated with 1 µM of camptothecin (Sigma-Aldrich, Spain) served as a positive control for apoptosis and were used to establish cytometer settings and channel compensations. The experiments were carried out in triplicate for each condition. For each sample, 10,000 events were collected.

Visualization of Cell Growth and Morphology
Qualitative analysis of A549 cell growth and morphology after exposure to 1 µM and 5 µM of the cytotoxic compound 4b was conducted using Cytation TM 1 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Cell images were acquired immediately after the addition of the compound and at the following time-points after exposure: 0.5, 1, 1.5, 2, 3, 4, 8, 12, 16, 24, and 48 h.

Conclusions
In conclusion, we report an efficient multicomponent methodology for the preparation of 1,5-dihydro-2H-pyrrol-2-ones holding a variety of substituents at the five-membered ring. This strategy allows the possibility of assorted structural diversity in the resultant scaffold depending on the starting amine, aldehyde, and pyruvate derivative in a single step. Moreover, obtained lactam derivatives showed in vitro cytotoxicity inhibiting the growth of human tumor cell lines HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma), and A549 (carcinomic human alveolar basal epithelial cell), and high selectivity toward MRC5 non-malignant lung fibroblasts. QSAR studies indicate that the cytotoxicity is enhanced, in general, by the presence of aromatic groups bearing lipophilic methyl substituents or fluorine atoms. Moreover, better cytotoxic activity is observed when the γ-lactam core is substituted by a polar ester group at position 4. Compound 5c showed a very good IC 50 value of 1.67 µM against A549 a cell line, and a very high selectivity if compared with the MRC5 cell line. Moreover, γ-lactam 10, derived from m-fluoroalinine, presented IC 50 values of 4.36 and 5.55 µM against A549 and SKOV3 cell lines and very good selectivity, with respect to healthy cells, and substrates 18a and 19, obtained by modification of the lactam structure, display very good IC 50 values of 3.27 and 3.65 µM against A549. Although HTB81, HeLa, and RKO cell lines were proved to be more resistant to γ-lactam derivatives, compound 4b presented IC 50 values of 27.93, 30.23, and 22.07 µM for each cell line respectively and good selectivity towards MRC5 cell line. The main mechanism by which γ-lactams derivatives induce cytotoxicity is based on the activation of intracellular apoptotic mechanisms.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ph14080782/s1, full experimental details, characterization and 1 H, 13 C, 19 F, and 31 P-NMR copies and HPLC chromatograms of compounds 4-12 and 16-21, flow cytometric assays on A-549 cells S156, and Visualization of cell growth and morphology of A-549 cells.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the Supplementary Materials file or on request from the corresponding author ( 1 H, 13 C, 19 F, and 31 P-NMR and HRMS spectra and cytotoxicity essays).