Exploring the Synthetic Potential of γ-Lactam Derivatives Obtained from a Multicomponent Reaction—Applications as Antiproliferative Agents

A study on the reactivity of 3-amino α,β-unsaturated γ-lactam derivatives obtained from a multicomponent reaction is presented. Key features of the substrates are the presence of an endocyclic α,β-unsaturated amide moiety and an enamine functionality. Following different synthetic protocols, the functionalization at three different positions of the lactam core is achieved. In the presence of a soft base, under thermodynamic conditions, the functionalization at C-4 takes place where the substrates behave as enamines, while the use of a strong base, under kinetic conditions, leads to the formation of C-5-functionalized γ-lactams, in the presence of ethyl glyoxalate, through a highly diastereoselective vinylogous aldol reaction. Moreover, the nucleophilic addition of organometallic species allows the functionalization at C-3, through the imine tautomer, affording γ-lactams bearing tetrasubstituted stereocenters, where the substrates act as imine electrophiles. Taking into account the advantage of the presence of a chiral stereocenter in C-5 substituted γ-lactams, further diastereoselective transformations are also explored, leading to novel bicyclic substrates holding a fused γ and δ-lactam skeleton. Remarkably, an example of a highly stereoselective formal [3+3] cycloaddition reaction of chiral γ-lactam substrates is reported for the synthesis of 1,4-dihidropyridines, where a non-covalent attractive interaction of a carbonyl group with an electron-deficient arene seems to drive the stereoselectivity of the reaction to the exclusive formation of the cis isomer. In order to unambiguously determine the substitution pattern resulting from the diverse reactions, an extensive characterization of the substrates is detailed through 2D NMR and/or X-ray experiments. Likewise, applications of the substrates as antiproliferative agents against lung and ovarian cancer cells are also described.

A few years ago, we reported a Brönsted-acid-catalyzed enantioselective synthesis of 3-amino 3-pyrrolin-2-ones through a multicomponent reaction, making use of amines, aldehydes, and pyruvate derivatives as starting materials [24]. More recently, we have extended this strategy to the preparation of the corresponding phosphorus and fluorine-containing analogs, using in this case fluorine and/or phosphonate-substituted aldehydes and pyruvates [25]. Remarkably, many of those molecules have shown strong antiproliferative activity, inhibiting the growth of several carcinoma human tumor cell lines through the activation of the apoptotic mechanism [10,11]. Key features of the structure of those substrates are the very reactive cyclic enamine moiety and the presence of a chiral stereocenter at the 5-membered ring. Taking the advantage of those two characteristics, we have used 3-amino 3-pyrrolin-2-ones as substrates in Lewis-acid-catalyzed formal [3+3] annulation reactions, for the preparation of bicyclic 1,4-dihidropyridines with a remarkably high degree of stereoselectivity [12]. Considering the easiness of the In addition, from a synthetic point of view, 3-amino 3-pyrrolin-2-ones are cyclic enamine substrates and, therefore, they are outstanding tools in chemical synthesis [41]. Interestingly, some pyrrolone-based systems have been identified as p53−MDM2 [42] and STAT3 [43] inhibitors, which result in strong antiproliferative activity, and, in addition, many other molecules containing the pyrrolone scaffold have been described as efficient antitumoral agents [44][45][46][47][48].
A few years ago, we reported a Brönsted-acid-catalyzed enantioselective synthesis of 3-amino 3-pyrrolin-2-ones through a multicomponent reaction, making use of amines, aldehydes, and pyruvate derivatives as starting materials [24]. More recently, we have extended this strategy to the preparation of the corresponding phosphorus and fluorinecontaining analogs, using in this case fluorine and/or phosphonate-substituted aldehydes and pyruvates [25]. Remarkably, many of those molecules have shown strong antiproliferative activity, inhibiting the growth of several carcinoma human tumor cell lines through the activation of the apoptotic mechanism [10,11]. Key features of the structure of those substrates are the very reactive cyclic enamine moiety and the presence of a chiral stereocenter at the 5-membered ring. Taking the advantage of those two characteristics, we have used 3-amino 3-pyrrolin-2-ones as substrates in Lewis-acid-catalyzed formal [3+3] annulation reactions, for the preparation of bicyclic 1,4-dihidropyridines with a remarkably high degree of stereoselectivity [12]. Considering the easiness of the multicomponent preparation of 3-amino 3-pyrrolin-2-ones and their synthetic potential in organic synthesis, we thought that a study on the synthetic applications of such lactam substrates would be of great interest in the field. Taking into account all the considerations mentioned above, here we Following this procedure, several 3-amino 3-pyrrolin-2-ones 4,5 were synthesized and their reactivity was explored in diverse reactions. As has been addressed above, 3-amino 3-pyrrolin-2-ones derivatives 4 and 5 contain the enamine moiety in their structure. In view of the potential nucleophilic character of those substrates, first we tested the reactivity of the enamine functionality as a nucleophile using first the most simple γ-lactam substrates derived from ethyl pyruvate and formaldehyde 4a,b (Scheme 2). The conjugate addition of the cyclic enamines 4 to acetylene carboxylates 6 (R 2 = OAlk) can be performed by the generation of the aza-enolate through an initial treatment with LDA at low temperature, followed by the addition of the electrophile. The reaction proceeds with a high degree of regioselectivity at the position C-4 of the five-membered heterocycle. Following this procedure, the functionalization reaction was successfully applied to methyl, ethyl, tert-butyl, and 2-naphthyl carboxylate derivatives 7a-f in excellent yields (Scheme 2).
Under the same reaction conditions, the conjugate addition of γ-lactams 4 to acetyl acetylene (R 2 = CH3) failed to give the nucleophilic addition derivatives. However, the new set of reaction conditions such as stoichiometric amounts of a Lewis acid Yb(OTf)3 led to the formation of functionalized lactams 7g-h in excellent yields (Scheme 2).
γ-Lactams 7 were fully characterized on the basis of their 1 H and 13 C NMR and IR spectrum and HRMS. Functionalized γ-lactam 7a was selected in order to unambiguously establish the structure of the reaction products obtained from the conjugate addition to activated alkynes. The functionalization at C-4 is evident by the presence of a singlet at δH = 4.41 ppm in 1 H NMR spectrum that integrates to two protons, corresponding to the methylene group at the 5-membered ring and the two doublets at δH = 7.56 and 5.63 ppm for the alkene group with a typical coupling constant for a trans configuration of the C=C double bond ( 3 JHH = 15.7 Hz). As expected, both protons at the Scheme 1. MCR for the synthesis of γ-lactams 4-5.
Following this procedure, several 3-amino 3-pyrrolin-2-ones 4,5 were synthesized and their reactivity was explored in diverse reactions. As has been addressed above, 3-amino 3-pyrrolin-2-ones derivatives 4 and 5 contain the enamine moiety in their structure. In view of the potential nucleophilic character of those substrates, first we tested the reactivity of the enamine functionality as a nucleophile using first the most simple γ-lactam substrates derived from ethyl pyruvate and formaldehyde 4a,b (Scheme 2). The conjugate addition of the cyclic enamines 4 to acetylene carboxylates 6 (R 2 = OAlk) can be performed by the generation of the aza-enolate through an initial treatment with LDA at low temperature, followed by the addition of the electrophile. The reaction proceeds with a high degree of regioselectivity at the position C-4 of the five-membered heterocycle. Following this procedure, the functionalization reaction was successfully applied to methyl, ethyl, tertbutyl, and 2-naphthyl carboxylate derivatives 7a-f in excellent yields (Scheme 2).
Under the same reaction conditions, the conjugate addition of γ-lactams 4 to acetyl acetylene (R 2 = CH 3 ) failed to give the nucleophilic addition derivatives. However, the new set of reaction conditions such as stoichiometric amounts of a Lewis acid Yb(OTf) 3 led to the formation of functionalized lactams 7g-h in excellent yields (Scheme 2).
γ-Lactams 7 were fully characterized on the basis of their 1 H and 13 C NMR and IR spectrum and HRMS. Functionalized γ-lactam 7a was selected in order to unambiguously establish the structure of the reaction products obtained from the conjugate addition to activated alkynes. The functionalization at C-4 is evident by the presence of a singlet at δ H = 4.41 ppm in 1 H NMR spectrum that integrates to two protons, corresponding to the methylene group at the 5-membered ring and the two doublets at δ H = 7.56 and 5.63 ppm for the alkene group with a typical coupling constant for a trans configuration of the C=C double bond ( 3 J HH = 15.7 Hz). As expected, both protons at the alkene bond proved to be coupled together in the homonuclear correlation spectroscopy (COSY) spectrum. In addition, the 13 C NMR spectrum shows a signal at δ C = 49.2 ppm which was assigned to a CH 2 carbon by distortionless enhancement by polarization transfer (DEPT) experiments. The proton and carbon signals assigned to the methylene group show a correlation in the heteronuclear single quantum coherence spectroscopy (HSQC) spectrum. Moreover, some other characteristic features of the 13 C NMR spectrum of 6a are the two signals corresponding to the two enamine quaternary carbons (DEPT) at δ C = 136.6 and 107.5 ppm as well as the signals corresponding to the methine carbons (DEPT) of the alkene group at δ C = 135.5 and 113.2 ppm. group show a correlation in the heteronuclear single quantum coherence spectroscopy (HSQC) spectrum. Moreover, some other characteristic features of the 13 C NMR spectrum of 6a are the two signals corresponding to the two enamine quaternary carbons (DEPT) at δC = 136.6 and 107.5 ppm as well as the signals corresponding to the methine carbons (DEPT) of the alkene group at δC = 135.5 and 113.2 ppm. In congruity with the proposed structure, the heteronuclear multiple bond correlation spectroscopy (HMBC) spectrum of 6a presents two clear correlations of the methylene protons at the heterocycle with the two enamine carbons, as well as a third one with the alkene carbon next to the γ-lactam ring at δC = 135.5 ppm. As expected, the less shielded signal of the olefinic protons at δH = 7.56 ppm, corresponding to the methine group next to the heterocycle, shows correlation with the two enamine carbons and the methylene group at the 5-membered ring, as well as with the second carbon of the alkene moiety at δC = 113.2 ppm and the carbonyl of the very conjugated ester group at δC = 165.6 ppm. The most shielded signal of the olefinic protons at δH = 5.63 ppm, assigned to the CH group next to the carboxyl group, shows a relationship with the carboxyl group and the other carbon of the alkene moiety at δC = 135.5 ppm. Moreover, this olefinic proton only shows correlation with one of the quaternary enamine carbons at δC = 107.5 ppm.
In order to extend further the synthetic applications of γ-lactam substrates, next we explored the reactivity of the different functionalized derivatives 7. Our first attempt to promote an intramolecular cyclization from acrylate substituted γ-lactam 7a under basic conditions failed, which was attributed to an unfavorable trans configuration of the alkene bond. For this reason, next, we performed the double reduction of the alkene and enamine moieties under hydrogen pressure in the presence of palladium as the catalyst, affording saturated γ-lactam derivative 8 in excellent yield with a complete degree of In congruity with the proposed structure, the heteronuclear multiple bond correlation spectroscopy (HMBC) spectrum of 6a presents two clear correlations of the methylene protons at the heterocycle with the two enamine carbons, as well as a third one with the alkene carbon next to the γ-lactam ring at δ C = 135.5 ppm. As expected, the less shielded signal of the olefinic protons at δ H = 7.56 ppm, corresponding to the methine group next to the heterocycle, shows correlation with the two enamine carbons and the methylene group at the 5-membered ring, as well as with the second carbon of the alkene moiety at δ C = 113.2 ppm and the carbonyl of the very conjugated ester group at δ C = 165.6 ppm. The most shielded signal of the olefinic protons at δ H = 5.63 ppm, assigned to the CH group next to the carboxyl group, shows a relationship with the carboxyl group and the other carbon of the alkene moiety at δ C = 135.5 ppm. Moreover, this olefinic proton only shows correlation with one of the quaternary enamine carbons at δ C = 107.5 ppm.
In order to extend further the synthetic applications of γ-lactam substrates, next we explored the reactivity of the different functionalized derivatives 7. Our first attempt to promote an intramolecular cyclization from acrylate substituted γ-lactam 7a under basic conditions failed, which was attributed to an unfavorable trans configuration of the alkene bond. For this reason, next, we performed the double reduction of the alkene and enamine moieties under hydrogen pressure in the presence of palladium as the catalyst, affording saturated γ-lactam derivative 8 in excellent yield with a complete degree of syn-diastereoselection (Scheme 3). As a consequence of the less strained structure obtained in the saturated derivative 8, in this case the treatment under basic conditions leads to the formation of bicyclic γ-lactam 9 in almost quantitative yield (Scheme 3). As far as we are concerned, this represents the first example of a synthetic methodology leading to the diastereoselective formation of a bicyclic substrate comprising both γ and δ-lactam units.
Additionally, the selective hydrolysis of the enamine moiety can be performed under acidic conditions to yield isotetramic acid derivative 10 in an almost quantitative yield.
syn-diastereoselection (Scheme 3). As a consequence of the less strained structure obtained in the saturated derivative 8, in this case the treatment under basic conditions leads to the formation of bicyclic γ-lactam 9 in almost quantitative yield (Scheme 3). As far as we are concerned, this represents the first example of a synthetic methodology leading to the diastereoselective formation of a bicyclic substrate comprising both γ and δ-lactam units. Additionally, the selective hydrolysis of the enamine moiety can be performed under acidic conditions to yield isotetramic acid derivative 10 in an almost quantitative yield  The relative configuration of the stereocenters in γ-lactams 8 and 9 was determined by nuclear Overhauser enhancement spectroscopy (NOESY) experiments (see ESI). NMR experiments with compound 8 showed a NOE of 3.98% between both protons at the two stereogenic centers, at δ = 4.13 and 2.91 ppm, respectively, indicating that these two atoms are oriented in the same direction. This relative configuration is in agreement with the accepted mechanism for catalytic hydrogenation of a carbon-carbon double bond, consisting in a syn addition of hydrogen.
Continuing with our interest in the nucleophilic addition reactions of enamine-derived γ-lactams, next, we explored the functionalization of substrates 4c,d, holding a phosphorated substituent at the position 4 of the ring. Accordingly, the treatment of γ-lactams 4c,d with LDA at low temperature, followed by the addition of ethyl glyoxalate, led to the nucleophilic addition products 12a,b, where the 5-membered ring is selectively functionalized at C-5. In this case, the presence of the bulky phosphonate or phosphine oxide substituents may block the β-enamine functionalization and the reaction presumably proceeds through the generation of aromatic enol-type intermediate 11, under kinetic conditions instead of the aza-enolate. Then, the subsequent selective vinylogous nucleophilic addition of anionic species 11 to the aldehyde electrophile proceeds selectively at position 5 of the 5-membered ring, leading to the formation of α-hydroxyester-functionalized γ-lactams 12a,b in good yields (Scheme 4). It should be noted that these are the first examples of C-5-functionalized phosphorus-substituted γ-lactams holding an enamine structure, since such substrates are known to rapidly provide the enol-derived γ-lactams through the spontaneous hydrolysis of the enamine moiety, as a consequence of the high steric hindrance present in the 5-membered heterocycle [21]. The relative configuration of the stereocenters in γ-lactams 8 and 9 was determined by nuclear Overhauser enhancement spectroscopy (NOESY) experiments (see ESI). NMR experiments with compound 8 showed a NOE of 3.98% between both protons at the two stereogenic centers, at δ = 4.13 and 2.91 ppm, respectively, indicating that these two atoms are oriented in the same direction. This relative configuration is in agreement with the accepted mechanism for catalytic hydrogenation of a carbon-carbon double bond, consisting in a syn addition of hydrogen.
Continuing with our interest in the nucleophilic addition reactions of enamine-derived γ-lactams, next, we explored the functionalization of substrates 4c,d, holding a phosphorated substituent at the position 4 of the ring. Accordingly, the treatment of γ-lactams 4c,d with LDA at low temperature, followed by the addition of ethyl glyoxalate, led to the nucleophilic addition products 12a,b, where the 5-membered ring is selectively functionalized at C-5. In this case, the presence of the bulky phosphonate or phosphine oxide substituents may block the β-enamine functionalization and the reaction presumably proceeds through the generation of aromatic enol-type intermediate 11, under kinetic conditions instead of the aza-enolate. Then, the subsequent selective vinylogous nucleophilic addition of anionic species 11 to the aldehyde electrophile proceeds selectively at position 5 of the 5-membered ring, leading to the formation of α-hydroxyester-functionalized γ-lactams 12a,b in good yields (Scheme 4). It should be noted that these are the first examples of C-5-functionalized phosphorus-substituted γ-lactams holding an enamine structure, since such substrates are known to rapidly provide the enol-derived γ-lactams through the spontaneous hydrolysis of the enamine moiety, as a consequence of the high steric hindrance present in the 5-membered heterocycle [21]. Remarkably, the nucleophilic addition reaction proceeds with a high degree of stereoselectivity and a single diastereoisomer is observed in the NMR spectra of the crude reaction. Unfortunately, NOESY experiments on the functionalized γ-lactams did not provide solid evidence that could lead to the unambiguous determination of the relative configuration of the adduct. For this reason, a single crystal of substrate 12b was isolated from a mixture of CH2Cl2/hexanes and, next, the structure obtained by X-ray diffraction experiments revealed unequivocally a relative configuration derived from the anti-addition of the γ-lactam core to the aldehyde electrophile ( Figure 2). A plausible tran- Remarkably, the nucleophilic addition reaction proceeds with a high degree of stereoselectivity and a single diastereoisomer is observed in the NMR spectra of the crude reaction. Unfortunately, NOESY experiments on the functionalized γ-lactams did not provide solid evidence that could lead to the unambiguous determination of the relative configuration of the adduct. For this reason, a single crystal of substrate 12b was isolated from a mixture of CH 2 Cl 2 /hexanes and, next, the structure obtained by X-ray diffraction experiments revealed unequivocally a relative configuration derived from the anti-addition of the γlactam core to the aldehyde electrophile ( Figure 2). A plausible transition state leading to the anti-derivative may consist of an electrostatic interaction between the aldehyde and the lithium cation, directing the carbonyl group towards the pyrrole ring. A possible additional stabilization by hydrogen bonding between the NH substituent and the carbonyl oxygen may provide a very rigid arrangement of the aldehyde, which orients its terminal hydrogen in a gauche conformation relative to the bulky phosphorus substituent, as drawn in the Newman projection shown in Figure 2. Remarkably, the nucleophilic addition reaction proceeds with a high degree of stereoselectivity and a single diastereoisomer is observed in the NMR spectra of the crude reaction. Unfortunately, NOESY experiments on the functionalized γ-lactams did not provide solid evidence that could lead to the unambiguous determination of the relative configuration of the adduct. For this reason, a single crystal of substrate 12b was isolated from a mixture of CH2Cl2/hexanes and, next, the structure obtained by X-ray diffraction experiments revealed unequivocally a relative configuration derived from the anti-addition of the γ-lactam core to the aldehyde electrophile ( Figure 2). A plausible transition state leading to the anti-derivative may consist of an electrostatic interaction between the aldehyde and the lithium cation, directing the carbonyl group towards the pyrrole ring. A possible additional stabilization by hydrogen bonding between the NH substituent and the carbonyl oxygen may provide a very rigid arrangement of the aldehyde, which orients its terminal hydrogen in a gauche conformation relative to the bulky phosphorus substituent, as drawn in the Newman projection shown in Figure 2. Surprisingly, under the same reaction conditions, for the special case of the nucleophilic addition of phosphorated γ-lactam 4d to p-nitrophenyl isocyanate, the introduction of an imine moiety into the five-membered ring was observed to give γ-lactam 13 (Scheme 5). An explanation for this behavior may consist of an initial formation of the amide 14 by the typical nucleophilic addition to an isocyanate substrate. Then, a spontaneous oxidation of the 5-membered heterocycle is proposed to yield intermediate 15.
Next, an intramolecular conjugate addition of the amide nitrogen to the heterocycle would afford the instable three-membered aziridinone species 16, and a subsequent rearrangement of the aziridinone and the enamine moieties at the γ-lactam core would lead to N-formamide 17 that undergoes a spontaneous cleavage of the formyl residue to afford imine-functionalized γ-lactam 13. Surprisingly, under the same reaction conditions, for the special case of the nucleophilic addition of phosphorated γ-lactam 4d to p-nitrophenyl isocyanate, the introduction of an imine moiety into the five-membered ring was observed to give γ-lactam 13 (Scheme 5). An explanation for this behavior may consist of an initial formation of the amide 14 by the typical nucleophilic addition to an isocyanate substrate. Then, a spontaneous oxidation of the 5-membered heterocycle is proposed to yield intermediate 15. Next, an intramolecular conjugate addition of the amide nitrogen to the heterocycle would afford the instable threemembered aziridinone species 16, and a subsequent rearrangement of the aziridinone and the enamine moieties at the γ-lactam core would lead to N-formamide 17 that undergoes a spontaneous cleavage of the formyl residue to afford imine-functionalized γ-lactam 13.
Due to the complexity of the transformation proposed and, in view of the simple pattern obtained in the 1 H NMR spectrum, which only shows aromatic protons and the acidic NH group, we were still skeptical about the real identity of the substrate obtained in the reaction. Although the exact mass was consistent with the proposed structure, in order to wipe away any doubt regarding the identity of substrate 13, a monocrystal of γ-lactam derivative 13 was isolated and its structure was unambiguously established by X-ray diffraction (Figure 3). Key features of the crystal structure of 13 are the parallel conformation of the N-aryl groups at the lactam nitrogen and the imine group, possibly Scheme 5. Unexpected γ-lactam derivative 13 obtained by nucleophilic addition of γ-lactam 4d to isocyanates.
Due to the complexity of the transformation proposed and, in view of the simple pattern obtained in the 1 H NMR spectrum, which only shows aromatic protons and the acidic NH group, we were still skeptical about the real identity of the substrate obtained in the reaction. Although the exact mass was consistent with the proposed structure, in order to wipe away any doubt regarding the identity of substrate 13, a monocrystal of γ-lactam derivative 13 was isolated and its structure was unambiguously established by X-ray diffraction ( Figure 3). Key features of the crystal structure of 13 are the parallel conformation of the N-aryl groups at the lactam nitrogen and the imine group, possibly stabilized by π-stacking, and the intramolecular hydrogen bonding observed between the enamine NH and the phosphoryl oxygen. Scheme 5. Unexpected γ-lactam derivative 13 obtained by nucleophilic addition of γ-lactam 4d to isocyanates.
Due to the complexity of the transformation proposed and, in view of the simple pattern obtained in the 1 H NMR spectrum, which only shows aromatic protons and the acidic NH group, we were still skeptical about the real identity of the substrate obtained in the reaction. Although the exact mass was consistent with the proposed structure, in order to wipe away any doubt regarding the identity of substrate 13, a monocrystal of γ-lactam derivative 13 was isolated and its structure was unambiguously established by X-ray diffraction ( Figure 3). Key features of the crystal structure of 13 are the parallel conformation of the N-aryl groups at the lactam nitrogen and the imine group, possibly stabilized by π-stacking, and the intramolecular hydrogen bonding observed between the enamine NH and the phosphoryl oxygen. Next, the functionalization reaction was extended to the use of chiral γ-lactam derivatives 5 holding a substituent at position 5 of the heterocycle. Under similar conditions, the treatment of substituted γ-lactam 5a with LDA at low temperature, followed by the addition of methyl iodide, afforded the alkylated substrate 18 in moderate yield (51%), together with a small amount of the hydrolyzed enol-derived lactam 19 (26%) (Scheme 6). The same behavior has already been observed in similar highly functionalized 3-amino 3-pyrrolin-2-ones and was attributed to the high steric hindrance at the 5-membered ring, due to the high degree of substitution [20][21][22][23][24][25]. In addition, enol-derived substrate 19 can be prepared in an almost quantitative yield by the hydrolysis of enamine-derived γ-lactam 18 in 0.1 M aqueous hydrochloric acid. Unfortunately, under the same reaction conditions, the nucleophilic addition of γ-lactam 5a to other electrophiles, such as acetylene carboxylates, acrylates, or bulkier alkyl halides, failed, which was attributed to the high steric crowding present in the expected densely functionalized resulting substrates. Next, the functionalization reaction was extended to the use of chiral γ-lactam derivatives 5 holding a substituent at position 5 of the heterocycle. Under similar conditions, the treatment of substituted γ-lactam 5a with LDA at low temperature, followed by the addition of methyl iodide, afforded the alkylated substrate 18 in moderate yield (51%), together with a small amount of the hydrolyzed enol-derived lactam 19 (26%) (Scheme 6). The same behavior has already been observed in similar highly functionalized 3-amino 3-pyrrolin-2-ones and was attributed to the high steric hindrance at the 5-membered ring, due to the high degree of substitution [20][21][22][23][24][25]. In addition, enol-derived substrate 19 can be prepared in an almost quantitative yield by the hydrolysis of enamine-derived γ-lactam 18 in 0.1 M aqueous hydrochloric acid. Unfortunately, under the same reaction conditions, the nucleophilic addition of γ-lactam 5a to other electrophiles, such as acetylene carboxylates, acrylates, or bulkier alkyl halides, failed, which was attributed to the high steric crowding present in the expected densely functionalized resulting substrates. The alkylation at C-5 in γ-lactam derivative 18 was verified by an extensive NMR study. The selective functionalization is deduced in the 1 H NMR spectrum from the disappearance of the two doublets at δH = 6.01 and 5.63 ppm ( 3 JHH = 2.6 Hz), corresponding to the two CH groups of the γ-lactam ring in starting substrate 5a, and the appearance of a new singlet at δH = 5.33 ppm assigned, in principle, to the proton at the chiral carbon of the heterocycle in 18. The presence of the methyl group is evident from the presence of an intense singlet at δH = 1.60 ppm.
The most characteristic signals found in the 13 C NMR spectrum of 18 are the four carbons of the 5-membered heterocycle, comprising the chiral CH carbon at δC = 67.6 ppm, the two quaternary enamine carbons at δC = 125.7 and 131.3 ppm, and the conjugated amide carbonyl group at δC = 168.2 ppm. Likewise, the presence of a dimethylaminomethyl group is evidenced from the appearance of an intense signal at δC = 13.6 ppm. The multiplicity of all carbon signals was verified by DEPT experiments. In addition, a strong C-H interaction was observed in HSQC spectrum between the 1 H NMR and 13 C NMR signals attributed to the CH, and methyl groups.
The alkylation at C-5 in γ-lactam derivative 18 was verified by an extensive NMR study. The selective functionalization is deduced in the 1 H NMR spectrum from the disappearance of the two doublets at δ H = 6.01 and 5.63 ppm ( 3 J HH = 2.6 Hz), corresponding to the two CH groups of the γ-lactam ring in starting substrate 5a, and the appearance of a new singlet at δ H = 5.33 ppm assigned, in principle, to the proton at the chiral carbon of the heterocycle in 18. The presence of the methyl group is evident from the presence of an intense singlet at δ H = 1.60 ppm.
The most characteristic signals found in the 13 C NMR spectrum of 18 are the four carbons of the 5-membered heterocycle, comprising the chiral CH carbon at δ C = 67.6 ppm, the two quaternary enamine carbons at δ C = 125.7 and 131.3 ppm, and the conjugated amide carbonyl group at δ C = 168.2 ppm. Likewise, the presence of a dimethylaminomethyl group is evidenced from the appearance of an intense signal at δ C = 13.6 ppm. The multiplicity of all carbon signals was verified by DEPT experiments. In addition, a strong C-H interaction was observed in HSQC spectrum between the 1 H NMR and 13 C NMR signals attributed to the CH, and methyl groups.
HMBC experiments provide additional evidence for the functionalization at C-4 in substrate 18. Accordingly, a correlation is observed for both, the CH and methyl protons, with the two quaternary carbons of the enamine moiety. Moreover, the 1 H NMR signal assigned to the methyl group shows a correspondence with the stereogenic carbon at the 5-membered heterocycle, while the methine proton presents the reciprocal correlation.
On the other hand, the presence of an ester group at the stereogenic carbon of the γ-lactam ring provides an additional acidic character to the proton at C-5. Accordingly, the vinylogous Mannich reaction of 3-pyrrolin-2-one 5g is observed upon treatment with trimethylamine in the presence of Eschenmoser salt, providing, in good yield, γ-lactam 20, bearing a tetrasubstituted carbon (Scheme 7).
the two CH groups of the γ-lactam ring in starting substrate 5a, and the appearance of a new singlet at δH = 5.33 ppm assigned, in principle, to the proton at the chiral carbon of the heterocycle in 18. The presence of the methyl group is evident from the presence of an intense singlet at δH = 1.60 ppm.
The most characteristic signals found in the 13 C NMR spectrum of 18 are the four carbons of the 5-membered heterocycle, comprising the chiral CH carbon at δC = 67.6 ppm, the two quaternary enamine carbons at δC = 125.7 and 131.3 ppm, and the conjugated amide carbonyl group at δC = 168.2 ppm. Likewise, the presence of a dimethylaminomethyl group is evidenced from the appearance of an intense signal at δC = 13.6 ppm. The multiplicity of all carbon signals was verified by DEPT experiments. In addition, a strong C-H interaction was observed in HSQC spectrum between the 1 H NMR and 13 C NMR signals attributed to the CH, and methyl groups.
HMBC experiments provide additional evidence for the functionalization at C-4 in substrate 18. Accordingly, a correlation is observed for both, the CH and methyl protons, with the two quaternary carbons of the enamine moiety. Moreover, the 1 H NMR signal assigned to the methyl group shows a correspondence with the stereogenic carbon at the 5-membered heterocycle, while the methine proton presents the reciprocal correlation.
On the other hand, the presence of an ester group at the stereogenic carbon of the γ-lactam ring provides an additional acidic character to the proton at C-5. Accordingly, the vinylogous Mannich reaction of 3-pyrrolin-2-one 5g is observed upon treatment with trimethylamine in the presence of Eschenmoser salt, providing, in good yield, γ-lactam 20, bearing a tetrasubstituted carbon (Scheme 7). In order to provide evidence of the selective functionalization at C-5, an extensive NMR study of substrate 20 was implemented. In this case, the 1 H NMR spectrum of substrate 20 shows a singlet at δH = 5.95 ppm for the methine group and two additional doublets at δH = 2.74 and 3.17 ppm, characteristic of the two diastereotopic protons of the methylene group, showing a typical geminal coupling constant ( 2 JHH = 14.0 Hz). As expected, the doublets corresponding to the diastereotopic protons show a strong interaction in the COSY spectrum.
The most characteristic signals found in the 13 C NMR spectrum are the four carbons of the 5-membered heterocycle, comprising the chiral quaternary carbon at δC = 72.7 ppm, the two enamine carbons at δC = 105 and 134 ppm, and the amide carbonyl group at δC = 168.3 ppm. The functionalization at C-5 in 20 is deduced from the appearance of the quaternary carbon at higher field than in C-4-functionalized substrate 18 (72.7 vs. 125.7 Scheme 7. Vinylogous reaction of γ-lactam 5b with Eschenmoser salt. In order to provide evidence of the selective functionalization at C-5, an extensive NMR study of substrate 20 was implemented. In this case, the 1 H NMR spectrum of substrate 20 shows a singlet at δ H = 5.95 ppm for the methine group and two additional doublets at δ H = 2.74 and 3.17 ppm, characteristic of the two diastereotopic protons of the methylene group, showing a typical geminal coupling constant ( 2 J HH = 14.0 Hz). As expected, the doublets corresponding to the diastereotopic protons show a strong interaction in the COSY spectrum.
The most characteristic signals found in the 13 C NMR spectrum are the four carbons of the 5-membered heterocycle, comprising the chiral quaternary carbon at δ C = 72.7 ppm, the two enamine carbons at δ C = 105 and 134 ppm, and the amide carbonyl group at δ C = 168.3 ppm. The functionalization at C-5 in 20 is deduced from the appearance of the quaternary carbon at higher field than in C-4-functionalized substrate 18 (72.7 vs. 125.7 ppm), suggesting a sp 3 hybridization of such carbon. Moreover, the fact that the CH carbon appears at significantly lower field in the 13 C NMR spectrum of 20 than in substrate 17 (104.9 vs. 67.6 ppm) suggests that this signal corresponds to an olefinic methine carbon. As usual, the multiplicity of the carbons was verified by DEPT experiments and could also be deduced from the interactions observed in the HSQC spectrum.
Some additional evidences of the functionalization at C-5 in substrate 20 can be gathered from HMBC experiments. In this case, the principal argument to consider a C-5 functionalization arises from the strong correlation observed between the diastereotopic methylene protons and the carboxylic carbon, corresponding to the ester moiety. Moreover, both diastereotopic protons show an additional interaction with the enamine CH group, while no correspondence with the quaternary enamine carbon is observed.
Considering the presence of a chiral stereocenter in C-5-substituted γ-lactam substrates 5, next, some other diastereoselective transformations were explored. A simple transformation of substrate 5b consists of the hydrogenation of the endocyclic enamine moiety under hydrogen pressure (80 psi) in the presence of palladium as catalyst, leading to the formation of a single diastereoisomer of saturated γ-lactam derivative 21 in excellent yield (Scheme 8).
group, while no correspondence with the quaternary enamine carbon is observed.
Considering the presence of a chiral stereocenter in C-5-substituted γ-lactam substrates 5, next, some other diastereoselective transformations were explored. A simple transformation of substrate 5b consists of the hydrogenation of the endocyclic enamine moiety under hydrogen pressure (80 psi) in the presence of palladium as catalyst, leading to the formation of a single diastereoisomer of saturated γ-lactam derivative 21 in excellent yield (Scheme 8).

Scheme 8. Diastereoselective reactions of γ-lactam 5g.
The relative configuration of the stereocenters in saturated γ-lactam 21 was determined by NOESY (see ESI). The fact that the signals corresponding to both protons at the two stereogenic centers, at δ = 4.78 and 4.16 ppm, show a strong NOE effect with the same proton at the diastereotopic methylene group at δ = 3.10 ppm, suggests that these three atoms are oriented in the same direction. In addition, no significant NOE is observed between the signal corresponding to the second of the diastereotopic protons, at δ = 2.08 ppm, and the two protons at both stereogenic centers, which confirms a relative cis configuration between the carboxylate and the amino groups in structure 21. Thus, the conventional mechanism for a catalytic hydrogenation of a carbon-carbon double bond is deduced from this configuration, where a syn addition of hydrogen takes place from the less hindered face, that is, the opposite to the carboxylate substituent at the stereogenic carbon.
In addition, similar cyclic enamines have proved to be excellent substrates in [3+3] annulation reactions [12]. For this reason, enamine-derived chiral lactam 5b was treated with β,γ-unsaturated α-ketoester 22 in the presence of a catalytic amount of ytterbium triflate, leading to the formation of a single diastereoisomer of bicyclic dihydropyridine 23 in excellent yield (Scheme 8). In this case, NOESY experiments showed a strong correlation between both protons at the chiral centers at δ = 4.93 and 5.05 ppm, revealing a cis relative configuration of both substituents at the stereogenic carbons (see ESI). In this Scheme 8. Diastereoselective reactions of γ-lactam 5g.
The relative configuration of the stereocenters in saturated γ-lactam 21 was determined by NOESY (see ESI). The fact that the signals corresponding to both protons at the two stereogenic centers, at δ = 4.78 and 4.16 ppm, show a strong NOE effect with the same proton at the diastereotopic methylene group at δ = 3.10 ppm, suggests that these three atoms are oriented in the same direction. In addition, no significant NOE is observed between the signal corresponding to the second of the diastereotopic protons, at δ = 2.08 ppm, and the two protons at both stereogenic centers, which confirms a relative cis configuration between the carboxylate and the amino groups in structure 21. Thus, the conventional mechanism for a catalytic hydrogenation of a carbon-carbon double bond is deduced from this configuration, where a syn addition of hydrogen takes place from the less hindered face, that is, the opposite to the carboxylate substituent at the stereogenic carbon.
In addition, similar cyclic enamines have proved to be excellent substrates in [3+3] annulation reactions [12]. For this reason, enamine-derived chiral lactam 5b was treated with β,γ-unsaturated α-ketoester 22 in the presence of a catalytic amount of ytterbium triflate, leading to the formation of a single diastereoisomer of bicyclic dihydropyridine 23 in excellent yield (Scheme 8). In this case, NOESY experiments showed a strong correlation between both protons at the chiral centers at δ = 4.93 and 5.05 ppm, revealing a cis relative configuration of both substituents at the stereogenic carbons (see ESI). In this case, in agreement with the proposed pathway for this reaction [49], the mechanism of the process may consist of a Michael addition of the enamine moiety to the conjugated double bond of β,γ-unsaturated α-ketoester and a second nucleophilic addition of the enamine nitrogen to the carbonyl group. A non-covalent attractive interaction of the carbonyl moiety with the electron-deficient arene bearing the nitro-electron-withdrawing substituent [50] may drive the stereoselectivity of the reaction to the exclusive formation of the cis diastereoisomer as shown in Figure 4. case, in agreement with the proposed pathway for this reaction [49], the mechanism of the process may consist of a Michael addition of the enamine moiety to the conjugated double bond of β,γ-unsaturated α-ketoester and a second nucleophilic addition of the enamine nitrogen to the carbonyl group. A non-covalent attractive interaction of the carbonyl moiety with the electron-deficient arene bearing the nitro-electron-withdrawing substituent [50] may drive the stereoselectivity of the reaction to the exclusive formation of the cis diastereoisomer as shown in Figure 4. Next, taking the advantage of the possible in situ tautomerization of the enamine into an imine moiety, the potential of unsaturated γ-lactam substrates as electrophiles was studied. Accordingly, the treatment of 5-substituted γ-lactam 5a derived from p-toluidine (R 1 = p-MeC6H4) with methyllithium in THF at low temperature led to the ef- Next, taking the advantage of the possible in situ tautomerization of the enamine into an imine moiety, the potential of unsaturated γ-lactam substrates as electrophiles was studied. Accordingly, the treatment of 5-substituted γ-lactam 5a derived from ptoluidine (R 1 = p-MeC 6 H 4 ) with methyllithium in THF at low temperature led to the efficient formation of unsaturated lactam derivative 25a, holding a tetrasubstituted chiral carbon (Scheme 9). Next, the reaction was extended to the use of n-butyllithium, obtaining almost identical results under the same reaction conditions (Scheme 9, 25b). The scope of the reaction using n-butyllithium as nucleophilic reagent was also extended to the use of other γ-lactam substrates 5c-e, derived from different aromatic amines such as p-anisidine, p-bromoaniline and m-chloroaniline (R 1 = p-MeOC 6 H 4 , p-BrC 6 H 4 , m-ClC 6 H 4 ), providing tetrasubstituted γ-lactam derivatives 25c-e also in very good yields (Scheme 9). Next, taking the advantage of the possible in situ tautomerization of the enamine into an imine moiety, the potential of unsaturated γ-lactam substrates as electrophiles was studied. Accordingly, the treatment of 5-substituted γ-lactam 5a derived from p-toluidine (R 1 = p-MeC6H4) with methyllithium in THF at low temperature led to the efficient formation of unsaturated lactam derivative 25a, holding a tetrasubstituted chiral carbon (Scheme 9). Next, the reaction was extended to the use of n-butyllithium, obtaining almost identical results under the same reaction conditions (Scheme 9, 25b). The scope of the reaction using n-butyllithium as nucleophilic reagent was also extended to the use of other γ-lactam substrates 5c-e, derived from different aromatic amines such as p-anisidine, p-bromoaniline and m-chloroaniline (R 1 = p-MeOC6H4, p-BrC6H4, m-ClC6H4), providing tetrasubstituted γ-lactam derivatives 25c-e also in very good yields (Scheme 9). Scheme 9. Nucleophilic addition of organolithium reagents to γ-lactams 5.
The formation of substrates 25 was evident by the presence of a chemical shift in the 1 H NMR spectrum of compound 25a (R 1 = p-Me-C6H4, R 2 = Me) at δ = 5.63 ppm, corresponding to the olefinic proton at the 5-membered ring, together with the two character-Scheme 9. Nucleophilic addition of organolithium reagents to γ-lactams 5.
The formation of substrates 25 was evident by the presence of a chemical shift in the 1 H NMR spectrum of compound 25a (R 1 = p-Me-C 6 H 4 , R 2 = Me) at δ = 5.63 ppm, corresponding to the olefinic proton at the 5-membered ring, together with the two characteristic signals in 13 C NMR spectrum at δ = 131.9 and 94.8 ppm, which correspond to the quaternary carbon and the CH group of the alkene bond, respectively. Moreover the exact mass of compound 25a showed a value of 368.1889 amu, which is in agreement with the proposed formula. In addition, the heteronuclear multiple bond correlation experiment (HMBC) showed a correlation between the singlet in 1 H NMR, corresponding to the methyl group at δ = 1.45 ppm, and the signals of the alkene CH and the amide carbonyl groups in 13 C NMR at δ = 94.8 and 175.5 ppm, respectively, while no correlation was observed between the methyl and the phenyl substituents (see ESI). These observations imply unequivocal evidence of the substitution at C-3 of the γ-lactam ring.
The reaction seems to proceed through an initial nucleophilic addition of the organometallic reagent to the imine tautomer of 5, followed by spontaneous oxidation of the 5-membered ring under exposure to the atmosphere (Scheme 9). In order to prove this hypothesis, the hydrogenation reaction of the alkene bond was performed in the presence of a catalytic amount of Pd on carbon. After a quick filtration of the reaction, the 1 H NMR spectrum of the crude reaction showed the presence of the characteristic signals corresponding to the diastereotopic protons of the CH 2 group of saturated intermediate 24 that rapidly disappeared to show again the spectrum corresponding to 25a (Scheme 9). However, we were unable to isolate a pure sample of intermediate 24.

Biological Results
As has been addressed above, some 3-pyrrolin-2-one derivatives have been demonstrated to induce apoptosis in several cancer cell lines [10,11]. For this reason and in order to underscore the usefulness of our functionalized γ-lactam substrates, next, the in vitro cytotoxicity of some of the heterocycles 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 [51]. Chemotherapeutic doxorubicin was used as reference value and 4,5-unsubstituted 3-amino 3-pyrrolin-2-one 27 [11] (R 1 = p-MeC 6 H 4 , R 2 = R 3 = H) was used in order to evaluate the influence of the introduction of the different substituents.
First, the cytotoxicity of the simplest substrates 7 derived from C-5-unsubstituted γ-lactams was evaluated. Although most of alkene-substituted γ-lactams 7 showed no activity, substrate 7d derived from p-toluidine and obtained from the functionalization with tert-butyl acetylene carboxylate presented some toxicity against SKOV3 cell line with an IC 50 value of 21.21 ± 2.36 µM and high selectivity towards A549 and MRC5 cell lines ( Table 1, Entry 1). the 5-membered ring under exposure to the atmosphere (Scheme 9). In order to prove this hypothesis, the hydrogenation reaction of the alkene bond was performed in the presence of a catalytic amount of Pd on carbon. After a quick filtration of the reaction, the 1 H NMR spectrum of the crude reaction showed the presence of the characteristic signals corresponding to the diastereotopic protons of the CH2 group of saturated intermediate 24 that rapidly disappeared to show again the spectrum corresponding to 25a (Scheme 9). However, we were unable to isolate a pure sample of intermediate 24.

Biological Results
As has been addressed above, some 3-pyrrolin-2-one derivatives have been demonstrated to induce apoptosis in several cancer cell lines [10,11]. For this reason and in order to underscore the usefulness of our functionalized γ-lactam substrates, next, the in vitro cytotoxicity of some of the heterocycles 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 [51]. Chemotherapeutic doxorubicin was used as reference value and 4,5-unsubstituted 3-amino 3-pyrrolin-2-one 27 [11] (R 1 = p-MeC6H4, R 2 = R 3 = H) was used in order to evaluate the influence of the introduction of the different substituents.
First, the cytotoxicity of the simplest substrates 7 derived from C-5-unsubstituted γ-lactams was evaluated. Although most of alkene-substituted γ-lactams 7 showed no activity, substrate 7d derived from p-toluidine and obtained from the functionalization with tert-butyl acetylene carboxylate presented some toxicity against SKOV3 cell line with an IC50 value of 21.21 ± 2.36 μM and high selectivity towards A549 and MRC5 cell lines ( Table 1, Entry 1). Next, the growth inhibition activity of chiral C-5-substituted γ-lactams was explored. In this context, due to their chemical similitude to natural phosphate metabolites, phosphonate derivatives show multiple biological activities and, for this reason, they have found numerous applications in medicine and agrochemistry [52][53][54][55][56]. For this reason, the antiproliferative activity of γ-lactam derivative 12a bearing a phosphonate group at C-4 was studied. However, for this special case, no significant toxicity was observed in A549 or SKOV3 cell lines (Table 1, Entry 2). In addition, although the appearance of phosphine oxides in drug discovery is rare, if compared with their counterparts, phosphates, phosphonates, or phosphoramidates, a few of those derivatives have proven to be excellent drug candidates such as anticancer drugs ridaforolimus [57,58] or brigatinib [59,60]. Accordingly, phosphine-oxide-substituted γ-lactam derivative 12b presented an excellent IC 50 value of 3.6 ± 0.20 µM with also high selectivity towards SKOV3 and MRC5 cell lines (Table 1, Entry 3). The IC 50 values observed for compound 12b suggest that the presence of the phosphine oxide moiety is critical in order to obtain both good values of toxicity and selectivity (Table 1, Entry 3 vs. Entry 9). It should be noted that other attempts to measure the cytotoxicity of C-5-unsubstituted phosphorated γ-lactams, like 4d, failed due to the low solubility of phosphine oxides in the solvents compatible with the biological tests [11]. However, in this case, the C-5 functionalization of 4d with ethyl glyoxalate provides a solvent-friendly structure in 12b, suitable for the evaluation of its antiproliferative activity.
Continuing with the SAR study on the γ-lactam derivatives, the cytotoxicity of unsaturated lactams 25 bearing a tetrasubstituted carbon was evaluated. Substrate 25a derived from p-toluidine and, when holding a methyl substituent at the tetrasubstituted carbon, showed good toxicity against A549 and SKOV3 cell lines, with IC 50 values of 15.16 ± 1.84 and 9.73 ± 1.27 µM, respectively. However, compound 25a did not show any selectivity towards non-malignant cells (Table 1, Entry 4). Substitution of the methyl group by a more lipophilic n-butyl group in γ-lactam derivative 25b resulted in an increase of the cytotoxicity, showing IC 50 values of 3.35 ± 0.42 and 6.93 ± 0.35µM in A549 and SKOV3 cell lines, respectively. Nonetheless, a similar increase in the toxicity towards MRC5 cell line was also observed in this case (Table 1, Entry 5). Almost identical results were obtained for other n-butyl-substituted γ-lactams 25c-e derived from different anilines with strong toxicity against cancer cells but non-selectivity towards non-malignant ones ( Table 1, Entries 6-8). The best candidate of this family was found to be p-chloroaniline-derived lactam 25e, which showed IC 50 values of 4.71 ± 1.40 and 4.64 ± 0.62 µM in A549 and SKOV3 cell lines, respectively, and an IC 50 value five times higher in MRC5 cell line ( It is also worth mentioning that, with the exception of substrate 25d, all other γ-lactam 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 high gastrointestinal absorption and the ability to cross the blood-brain-barrier (see Table  in ESI).

General Experimental Information
Solvents for extraction and chromatography were technical grade. All solvents used in reactions were freshly distilled from appropriate drying agents before use. All other reagents were recrystallized or distilled as necessary. All reactions were performed under an atmosphere of dry nitrogen. Analytical TLC was performed with silica gel 60 F 254 plates. Visualization was accomplished by UV light. 1 H, 13 C, 31 P, and 19 F-NMR spectra were recorded on a Varian Unity Plus (Varian Inc., NMR Systems, Palo Alto (CA), United States) (at 300 MHz, 75 MHz, 120 MHz, and 282 MHz, respectively) and on a Bruker Avance 400 (Bruker BioSpin GmbH, Rheinstetten, Germany) (at 400 MHz for 1 H, and 100 MHz for 13 C). Chemical shifts (δ) are reported in ppm relative to residual CHCl 3 (δ = 7.26 ppm for 1 H and δ = 77.16 ppm for 13 C NMR) and using phosphoric acid (50%) or HF as external reference (δ = 0.0 ppm) for 31 P and 19 F NMR spectra. Coupling constants (J) are reported in Hertz. Data for 1 H NMR spectra are reported as follows: chemical shift, multiplicity, coupling constant, integration. Multiplicity abbreviations are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. 13 C NMR peak assignments were supported by distortionless enhanced polarization transfer (DEPT). High-resolution mass spectra (HRMS) were obtained by positive-ion electrospray ionization (ESI). Data are reported in the form m/z (intensity relative to base = 100). Infrared spectra (IR) were taken in a Nicolet iS10 Thermo Scientific spectrometer (Thermo Scientific Inc., Waltham, Massachusetts (MA), United States) as neat solids. Peaks are reported in cm −1 .

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) using a CHIRALPAK ® IA column (5 µm, 0.54 cm ø × 25 cm, Daicel Chiral Technologies, Illkirch Cedex, France) 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 derivatives tested in biological essays is >95%, which meets the purity requirement by the Journal. Following a known procedure [11], a solution of amine 1 (10 mmol), aldehyde 2 (5 mmol), ethyl pyruvate 3 (1.74 g, 15 mmol), and 1,1 -binaphthyl-2,2 -diyl hydrogen phosphate (174 mg, 10 mol%) was stirred in Et 2 O (25 mL) at room temperature or MTBE (25 mL) at 55 • C (heating plate with Heat-On) in the presence of anhydrous MgSO 4 (2.5 g) for 48 h. The volatiles were distilled off at reduced pressure and the crude residue was purified by chromatography (AcOEt/hexanes 9:1) to afford pure γ-lactams 4 and 5.
General Procedure for the Nucleophilic Conjugate Addition of γ-Lactams 4 to Activated Alkynes Procedure A (Esters). The corresponding γ-lactam 4 (1 mmol) was added to a freshly prepared solution of LDA (1.2 mmol) in THF (3 mL) at −78 • C under N 2 atmosphere. After 1 h, the selected alkyl propiolate (1.2 mmol) was added and the reaction was stirred at room temperature until the disappearance of the starting materials (monitored by TLC and/or 1 H RMN,~4 h). The reaction was acidified with a 0.5 M aqueous solution of HCl (10 mL), and extracted with dichloromethane (2 × 10 mL). The combined organic layers were washed with water (2 × 10 mL), dried over MgSO 4 , and concentrated under vacuum. The crude residue was purified by crystallization, affording pure functionalized γ-lactam derivatives 6. In some cases, a previous purification by chromatography was necessary as detailed for each compound.
Procedure B (Ketones). A solution of the corresponding γ-lactam 4 (1 mmol), 3-butn-2one (0.097 mL, 1.2 mmol) and Yb(OTf) 3 (1 mmol, 620 mg) in dichloromethane (3 mL) was stirred at room temperature until the disappearance of the starting (monitored by TLC, 4 to 20 h). The reaction mixture was filtered through celite and concentrated under vacuum and the crude residue was purified by crystallization, affording pure functionalized γlactam derivatives 7. In some cases, previous purification by chromatography column was necessary as detailed for each compound.

Conclusions
The Brönsted-acid-catalyzed multicomponent reaction of amines, aldehydes, and pyruvate derivatives is a straightforward method for the synthesis of 3-amino α,β-unsaturated γ-lactam derivatives. Selective functionalization at C-4 or C-5 of those substrates is feasible, making use of enamine chemistry or vinylogous nucleophilic additions, while the addition of organometallic species allows functionalization at C-3. Remarkably, the vinylogous aldol reaction with ethyl glyoxalate leads to the exclusive formation of the anti-adduct. In addition, chiral-functionalized γ-lactams are excellent precursors for diastereoselective synthetic transformations. The hydrogenation of the enamine moiety proceeds with high degree of syn diastereoselectivity, leading to the saturated substrates suitable for the further preparation of bicyclic compounds through intramolecular reactions. In addition, an example of a formal [3+3] annulation reaction is described with high cis stereocontrol. Moreover, some of the obtained γ-lactam derivatives showed in vitro cytotoxicity, inhibiting the growth of human tumor cells SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell) with selectivity toward MRC5 non-malignant lung fibroblasts. Although none of the substrates were proved to be superior to chemotherapeutic agent Doxorubicin, substrate 12b showed a promising 3.6 µM IC 50 value in A549 cell line, whereas the best result in SKOV3 cell line, 4.64 µM, was measured for compound 25e.

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).