Ugi Reaction on α-Phosphorated Ketimines for the Synthesis of Tetrasubstituted α-Aminophosphonates and Their Applications as Antiproliferative Agents

An Ugi three-component reaction using preformed α-phosphorated N-tosyl ketimines with different isocyanides in the presence of a carboxylic acid affords tetrasubstituted α-aminophosphonates. Due to the high steric hindrance, the expected acylated amines undergo a spontaneous elimination of the acyl group. The reaction is applicable to α-aryl ketimines bearing a number of substituents and several isocyanides. In addition, the densely substituted α-aminophosphonate substrates showed in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell line A549 (carcinomic human alveolar basal epithelial cell).


Introduction
In the interdisciplinary research field of chemical biology and drug discovery, diversityoriented synthesis is an interesting model for the production of large chemical libraries of small molecules, bearing multiple functional groups, in order to explore their influence into the biological properties of those substrates [1][2][3]. At the heart of this concept, multicomponent reactions (MCRs) have become a mainstay of medicinal and organic chemistry that allow the preparation of a broad spectrum of compounds with a reduced number of synthetic steps [4,5]. In such synthetic procedures, three reactants or more are combined in the same pot to generate a new substrate, whose structure shows portions of all the starting materials. The atom economy, efficiency, mild conditions and high convergence of MCRs justify a central place in the toolbox of diversity-oriented synthesis [6,7]. Among the numerous MCRs described along the last decades, the Ugi reaction [8] has been verified as one of the most important multicomponent processes for the synthesis of peptide-like structures [9,10]. The Ugi reaction consists on a nucleophilic attack of an isonitrile 6 to an iminium ion 5, a salt composed of a carboxylic acid 4 and an imine 3, which is often generated in situ from a carbonyl derivative 1 and an amine 2. Then, a second nucleophilic attack of the carboxylate anion in the intermediate nitrilium species 7 results in the formation of acyl imidate 8. The reaction ends with an irreversible Mumm rearrangement of species 8, leading to α-amido amide substrates 9 in a very efficient manner (Scheme 1). Remarkably, the whole reaction is driven by the Mumm rearrangement since all other species involved in the mechanism are in equilibrium. Due to its versatility, the Ugi reaction has become increasingly practical in the synthesis of many active complex drugs and natural products [11][12][13]. On the other hand, the α-aminophosphonic acid framework enjoys significant attention in medicinal sciences, due to its unique ability to mimic the transition state of peptide cleavage in an irreversible fashion, thus blocking very efficiently enzymes implicated in proteolysis processes ( Figure 1). For this reason, α-aminophosphonic acid derivatives and their phosphapeptides display an assorted biological activity, including anticancer properties [14][15][16][17][18]. α-Aminophosphonic acids can be considered as structural isosters of αaminoacids, where the flat carboxylic acid group has been replaced by a phosphonic acid group, and one of the most straightforward methods for the preparation of both compounds, α-aminoacids and α-aminophosphonic acids, consists on the addition of carbon nucleophiles to α-iminoesters or α-iminophosphonates, respectively [19,20]. While an Ugi reaction using α-iminoesters to afford α,α-diamino acid derivatives is documented [21,22] no examples are described using α-iminophosphonates as starting materials. In addition, the use of ketones or ketimines as substrates in such reactions, in order to generate structures bearing tetrasubstituted carbons, entails additional obstacles, since the inherent steric factors observed in these systems enhance the difficulty level in these synthetic methodologies [23]. In addition, the use of acyclic ketones typically requires preformation of the imine intermediate in a separate step, and the yields of the Ugi are often modest [24][25][26]. In this context, during the course of our research on the addition of nucleophiles to α-ketiminophosphonates, in the past, we achieved the synthesis of tetrasubtituted α-aminophosphonates [27] using cyanide [28], organometallics [29] and nitromethane [30] as nucleophiles and, more recently, we have reported the first enantioselective Reformatsky reaction using acyclic ketimines as substrates [31]. Continuing with our interest in the chemistry of organophosphorus compounds, we thought that α-ketiminophosphonates would be excellent substrates in Ugi reactions for the generation of phosphorated peptidelike structures bearing tetrasubstituted carbons. Due to the great occurrence of tetrasubstituted carbons in natural products and drugs [32], the high affinity of α-aminophosphonates to proteolytic enzymes and the synthetic versatility of multicomponent reactions, a synthetic protocol of an Ugi reaction using α-phosphorated ketimines would be of great value in organic and medicinal chemistry. On the other hand, the α-aminophosphonic acid framework enjoys significant attention in medicinal sciences, due to its unique ability to mimic the transition state of peptide cleavage in an irreversible fashion, thus blocking very efficiently enzymes implicated in proteolysis processes ( Figure 1). For this reason, α-aminophosphonic acid derivatives and their phosphapeptides display an assorted biological activity, including anticancer properties [14][15][16][17][18]. α-Aminophosphonic acids can be considered as structural isosters of α-aminoacids, where the flat carboxylic acid group has been replaced by a phosphonic acid group, and one of the most straightforward methods for the preparation of both compounds, α-aminoacids and α-aminophosphonic acids, consists on the addition of carbon nucleophiles to α-iminoesters or α-iminophosphonates, respectively [19,20]. While an Ugi reaction using α-iminoesters to afford α,α-diamino acid derivatives is documented [21,22] no examples are described using α-iminophosphonates as starting materials. In addition, the use of ketones or ketimines as substrates in such reactions, in order to generate structures bearing tetrasubstituted carbons, entails additional obstacles, since the inherent steric factors observed in these systems enhance the difficulty level in these synthetic methodologies [23]. In addition, the use of acyclic ketones typically requires preformation of the imine intermediate in a separate step, and the yields of the Ugi are often modest [24][25][26]. On the other hand, the α-aminophosphonic acid framework enjoys significant attention in medicinal sciences, due to its unique ability to mimic the transition state of peptide cleavage in an irreversible fashion, thus blocking very efficiently enzymes implicated in proteolysis processes ( Figure 1). For this reason, α-aminophosphonic acid derivatives and their phosphapeptides display an assorted biological activity, including anticancer properties [14][15][16][17][18]. α-Aminophosphonic acids can be considered as structural isosters of αaminoacids, where the flat carboxylic acid group has been replaced by a phosphonic acid group, and one of the most straightforward methods for the preparation of both compounds, α-aminoacids and α-aminophosphonic acids, consists on the addition of carbon nucleophiles to α-iminoesters or α-iminophosphonates, respectively [19,20]. While an Ugi reaction using α-iminoesters to afford α,α-diamino acid derivatives is documented [21,22] no examples are described using α-iminophosphonates as starting materials. In addition, the use of ketones or ketimines as substrates in such reactions, in order to generate structures bearing tetrasubstituted carbons, entails additional obstacles, since the inherent steric factors observed in these systems enhance the difficulty level in these synthetic methodologies [23]. In addition, the use of acyclic ketones typically requires preformation of the imine intermediate in a separate step, and the yields of the Ugi are often modest [24][25][26]. In this context, during the course of our research on the addition of nucleophiles to α-ketiminophosphonates, in the past, we achieved the synthesis of tetrasubtituted α-aminophosphonates [27] using cyanide [28], organometallics [29] and nitromethane [30] as nucleophiles and, more recently, we have reported the first enantioselective Reformatsky reaction using acyclic ketimines as substrates [31]. Continuing with our interest in the chemistry of organophosphorus compounds, we thought that α-ketiminophosphonates would be excellent substrates in Ugi reactions for the generation of phosphorated peptidelike structures bearing tetrasubstituted carbons. Due to the great occurrence of tetrasubstituted carbons in natural products and drugs [32], the high affinity of α-aminophosphonates to proteolytic enzymes and the synthetic versatility of multicomponent reactions, a synthetic protocol of an Ugi reaction using α-phosphorated ketimines would be of great value in organic and medicinal chemistry. In this context, during the course of our research on the addition of nucleophiles to α-ketiminophosphonates, in the past, we achieved the synthesis of tetrasubtituted α-aminophosphonates [27] using cyanide [28], organometallics [29] and nitromethane [30] as nucleophiles and, more recently, we have reported the first enantioselective Reformatsky reaction using acyclic ketimines as substrates [31]. Continuing with our interest in the chemistry of organophosphorus compounds, we thought that α-ketiminophosphonates would be excellent substrates in Ugi reactions for the generation of phosphorated peptide-like structures bearing tetrasubstituted carbons. Due to the great occurrence of tetrasubstituted carbons in natural products and drugs [32], the high affinity of α-aminophosphonates to proteolytic enzymes and the synthetic versatility of multicomponent reactions, a synthetic protocol of an Ugi reaction using α-phosphorated ketimines would be of great value in organic and medicinal chemistry.

Chemistry
N-tosyl α-ketiminophosphonates 10 can be synthesized by a formal oxidation of trisubstituted aminophosphonates as reported in literature [28,31]. In our first experiment we studied the Ugi reaction of N-tosyl ketimine 10a (R 1 = Me, R 2 = Ph) with phenyl acetic acid 11 and cyclohexyl isocyanide 12a (R 3 = Cy) under the typical reaction conditions (Scheme 2). After stirring a mixture of the three compounds in CH 2 Cl 2 at room temperature for 1 h, NMR showed the complete disappearance of the starting materials and formation of tetrasubstituted α-aminophosphonate 13a. Due to the insolubility of the starting materials, the use of other environmentally friendly solvents led to the formation of substrate 13a in lower yields and longer reaction times.

Chemistry
N-tosyl α-ketiminophosphonates 10 can be synthesized by a formal oxidation of trisubstituted aminophosphonates as reported in literature [28,31]. In our first experiment we studied the Ugi reaction of N-tosyl ketimine 10a (R 1 = Me, R 2 = Ph) with phenyl acetic acid 11 and cyclohexyl isocyanide 12a (R 3 = Cy) under the typical reaction conditions (Scheme 2). After stirring a mixture of the three compounds in CH2Cl2 at room temperature for 1 h, NMR showed the complete disappearance of the starting materials and formation of tetrasubstituted α-aminophosphonate 13a. Due to the insolubility of the starting materials, the use of other environmentally friendly solvents led to the formation of substrate 13a in lower yields and longer reaction times. With this result in hand, next we extended the Ugi protocol to different α-iminophosphonates 10 and isocyanides 12 using phenylacetic acid 11 in CH2Cl2 (Scheme 2). First, different isocyanides 12 were tested in the reaction using ketimine 10a (R 1 = Me, R 2 = Ph) derived from dimethylphosphonate. The reactions proceed fast (1 h) and with good yields, not only using cyclohexyl isocyanide 12a (R 3 = Cy), but also with methyl isocyanoacetate 12b (R 1 = CH2CO2Me) or benzyl isocyanide 12c (R 3 = Bn) to afford α-aminophosphonates 13b-c (Scheme 2).
Next, diethyl, dibenzyl and di-iso-propyl phosphonate substituted ketimines 10b-d (R 1 = Et, Bn, i Pr,) were tested as electrophilic substrates with very good results but different reactivity. In the case of diethylphosphonates 13d-f (R 1 = Et, R 2 = Ph), and dibenzylphosphonates 13g (R 1 = Bn, R 2 = Ph) the reactions proceed to full conversion after 6 h and even longer reaction times of 14 h are needed for di-iso-propylphosphonates 13h-j (R 1 = i Pr, R 2 = Ph) (Scheme 2). These differences in the reactivity related to the size of the phosphonate substituents are in agreement with what has been observed in similar reactions [28,30].
Then, the scope of the reaction was extended to the use of phosphorated ketimines bearing substituted aromatic rings. Aromatic ketimines holding strong electron withdrawing substituents such as a para-nitro group showed very good reactivity and aminophosphonate 13k was obtained in very good yield after 1 h at room temperature (Scheme 2). The reaction is also fast using ketimines with halogenated aromatic groups. Several halogen substituted aromatic ketimines were successfully used in the reaction, including para-substituted aromatic rings containing bromine or chlorine to yield halogenated αaminophosphonates in full conversion after 1 h (Scheme 2, 13l-m). The reaction tolerates also the presence of an ortho-fluor substituted aromatic ring in (Scheme 2, 13n) and even the existence of a perfluorinated phenyl group (Scheme 2, 13o). Besides, when aromatic ketimines substituted by electron donating groups were used as substrates, an increase in the reaction times was observed. However, α-aminophosphonates 13p-q were obtained in full conversion after 14 h (Scheme 2).
Tetrasubstituted α-aminophosphonates 13 were characterized on the basis of their 1 H, 31 P, 19 F and 13 C NMR, IR spectra and high-resolution mass spectra (see Supplementary Materials for the detail). For example, 1 H NMR spectrum α-aminophosphonate 13a presents the signals corresponding to the aliphatic cyclohexyl moiety with several chemical shifts in the interval δ H = 0.92-1.91 ppm for the five methylene groups and an additional multiplet at δ H = 3.77 ppm for the CH bonded to the nitrogen. The phosphonate moiety is seen as two representative doublets at δ H = 3.80 ppm ( 3 J PH = 10.5 Hz) and δ H = 3.99 ppm ( 3 J PH = 10.7 Hz), typical for the diastereotopic methoxy groups at the phosphonate. The presence of the tosyl group is evident from the chemical shift for its para-methyl substituent at δ H = 2.33 ppm, that appears as a singlet, and the two doublets at δ H = 7.00 and 7.16 ppm ( 3 J HH = 8.3 Hz), corresponding to the four aromatic protons, that appear partially overlapped with the five protons of the phenyl substituent in the interval at δ H = 6.99-7.25 ppm. The sulfamide and amide NH protons appear as two doublets that interchange with D 2 O at δ H = 6.47 ppm ( 3 J PH = 8.2 Hz) and δ H = 6.76 ppm ( 3 J HH = 6.4 Hz), respectively. Due to the low interchange rate in such acidic protons, the signal corresponding to the NH of the sulfamide moiety is coupled with the magnetically active phosphorus atom, while the amide NH is coupled with the neighboring CH of the cyclohexyl group.
In addition, in the 13 C NMR spectrum of α-aminophosphonate 13a, the cyclohexyl group can be detected by the chemical shift at δ C = 49.8 ppm, corresponding to its methyne group, bonded to the nitrogen atom and, due to the stereogenic center present in the structure, the other five methylene carbons show five different signals at δ C = 24.5, 24.6, 25.4, 32.1 and 32.3 ppm. Here, again, the two diastereotopic methoxy groups at the phosphonate moiety are seen as two doublets at δ H = 55.8 ppm ( 2 J PC = 8.2 Hz) and δ C = 55.2 ppm ( 2 J PC = 7.5 Hz). The most characteristic chemical shift of α-aminophosphonate 13a in 13 C NMR is certainly the doublet corresponding to the quaternary carbon directly bonded to the phosphonate that appears at δ C = 68.5 ppm and presents a strong coupling with the phosphorus atom ( 1 J PC = 157. 2 Hz). The presence of the tosyl group is here deduced from the chemical shift corresponding to its para-methyl substituent at δ C = 21.6 ppm and the aromatic carbons with two signals at δ C = 126.5 and 129.1 ppm for each of the two couples of the equivalent CH carbons of the aromatic ring, as well as another two signals for the two quaternary carbons at δ C = 142. 4 and 139.2 ppm, the latter seen as a doublet due to the coupling with the phosphorus atom ( 4 J PC = 1.6 Hz). In the aromatic region it also appears the chemical shifts of the carbons corresponding to the phenyl ring, with the signals corresponding to the two pairs of equivalent CH carbons at δ C = 127.9 and 130.2 ppm, the second as a doublet coupled with the phosphorus atom ( 3 J PC = 8. 3 Hz). The fifth aromatic CH appears at δ C = 128.7 ppm and the quaternary carbon as a doublet at δ C = 131.9 ( 2 J PC = 1. 8 Hz). Surprisingly, the amide carbonyl group does not show coupling with the phosphorus atom and the signal appears as a singlet at δ C = 166.1 ppm.
The most relevant absorptions observed in IR spectrum correspond to the amide, sulfamide and phosphonate moieties. The stretching vibration of amide and sulfamide NH groups can be observed at ν = 3426 and 3333 cm −1 , respectively. In addition, two strong bands are observed at ν =1678 and 1256 cm −1 , correspond to the vibration of amide C=O and phosphonate P=O bonds. Finally, the spectrum shows two characteristic absorptions ν = 1333 and 1164 cm −1 that correspond to the asymmetric and symmetric stretching vibration of the sulfonyl group.
Regarding the mechanism of the reaction, we theorized that compounds 13 might be formed by a typical three-component Ugi reaction that leads to the formation of the predicted phosphorated α-amido amide 15, followed by a spontaneous cleavage of the acyl group, due to the high steric hindrance present in the intermediate 15 (Scheme 3). In fact, the same behavior has been observed in the acylcyanation reaction of N-tosyl ketimines 10 (R 2 = Ar, PG = Ts) with pyruvonitrile [28]. In our attempts to detect the acylated intermediate 15, different carboxylic acids were used in the reaction, but αaminophosphonate 13a was obtained in all cases, even when acetic, trifluoroacetic or benzoic acid were used as reagents. Nevertheless, the reaction does not proceed in the absence of a carboxylic acid, which at least indicates that the formation of iminium species is crucial prior to the nucleophilic attack of isocyanide. The most relevant absorptions observed in IR spectrum correspond to the amide, sulfamide and phosphonate moieties. The stretching vibration of amide and sulfamide NH groups can be observed at ν = 3426 and 3333 cm −1 , respectively. In addition, two strong bands are observed at ν =1678 and 1256 cm −1 , correspond to the vibration of amide C=O and phosphonate P=O bonds. Finally, the spectrum shows two characteristic absorptions ν = 1333 and 1164 cm −1 that correspond to the asymmetric and symmetric stretching vibration of the sulfonyl group.
Regarding the mechanism of the reaction, we theorized that compounds 13 might be formed by a typical three-component Ugi reaction that leads to the formation of the predicted phosphorated α-amido amide 15, followed by a spontaneous cleavage of the acyl group, due to the high steric hindrance present in the intermediate 15 (Scheme 3). In fact, the same behavior has been observed in the acylcyanation reaction of N-tosyl ketimines 10 (R 2 = Ar, PG = Ts) with pyruvonitrile [28]. In our attempts to detect the acylated intermediate 15, different carboxylic acids were used in the reaction, but α-aminophosphonate 13a was obtained in all cases, even when acetic, trifluoroacetic or benzoic acid were used as reagents. Nevertheless, the reaction does not proceed in the absence of a carboxylic acid, which at least indicates that the formation of iminium species is crucial prior to the nucleophilic attack of isocyanide. In order to check if the Mumm rearrangement was indeed taking place, next we used N-trityl aldimine 14 (R 2 = H, PG = CPh3) [33] as the electrophile substrate, in the presence of phenylacetic acid 11 and cyclohexyl isocyanide 12a (R 3 = Cy) (Scheme 3). Due to the utilization of an aldimine derived electrophile in the reaction, a less hindered structure is expected in the Ugi adduct, which may result in the isolation of species 15. However, in this case, trisubstituted α-aminophosphonate 16 was obtained in full conversion, where, the formation of α-amido amide 15 is followed by a spontaneous cleavage of the bulky trityl protecting group (Scheme 3).
Although this last experiment supports an Ugi three-component mechanism of the process, still we were skeptical about the real role of the carboxylic acid in the system. It is true that, considering the accepted mechanism for the Ugi reaction, only through the irreversible Mumm rearrangement all the equilibrium in the process can be displaced to the final products. But yet, it might be vaguely possible that, in the case of our ketimines 10, a simple addition of isocyanide to iminium species could afford tetrasubstituted αaminophosphonate 13a after an irreversible hydrolysis of the nitrilium intermediate, due to the presence of traces of water in the solvent. Then the key question to be addressed is: is the third reactant of the multicomponent reaction a carboxylic acid or is it just water?
This matter could be resolved in view of the fact that the isolation of intermediate 15 was achieved when para-fluorophenyl or para-trifluoromethylphenyl substituted α-phos- In order to check if the Mumm rearrangement was indeed taking place, next we used N-trityl aldimine 14 (R 2 = H, PG = CPh 3 ) [33] as the electrophile substrate, in the presence of phenylacetic acid 11 and cyclohexyl isocyanide 12a (R 3 = Cy) (Scheme 3). Due to the utilization of an aldimine derived electrophile in the reaction, a less hindered structure is expected in the Ugi adduct, which may result in the isolation of species 15. However, in this case, trisubstituted α-aminophosphonate 16 was obtained in full conversion, where, the formation of α-amido amide 15 is followed by a spontaneous cleavage of the bulky trityl protecting group (Scheme 3).
Although this last experiment supports an Ugi three-component mechanism of the process, still we were skeptical about the real role of the carboxylic acid in the system. It is true that, considering the accepted mechanism for the Ugi reaction, only through the irreversible Mumm rearrangement all the equilibrium in the process can be displaced to the final products. But yet, it might be vaguely possible that, in the case of our ketimines 10, a simple addition of isocyanide to iminium species could afford tetrasubstituted α-aminophosphonate 13a after an irreversible hydrolysis of the nitrilium intermediate, due to the presence of traces of water in the solvent. Then the key question to be addressed is: is the third reactant of the multicomponent reaction a carboxylic acid or is it just water?
This matter could be resolved in view of the fact that the isolation of intermediate 15 was achieved when para-fluorophenyl or para-trifluoromethylphenyl substituted αphosphorated ketimines 10l,m (R = CF 3 , F) were used as the electrophile unit in the Ugi reaction. Using phenylacetic acid 11 and cyclohexyl isocyanide 12a, phosphorated αamido amides 15a,b were obtained, without the elimination of the amide group (Scheme 4). Although substrate 15b proved to be very stable, trifluoromethyl substituted α-amido amide 15a underwent spontaneous hydrolysis of the amide under the air moisture to yield tetrasubstituted α-aminophosphonate 13r.
phorated ketimines 10l,m (R = CF3, F) were used as the electrophile unit in the Ugi reaction. Using phenylacetic acid 11 and cyclohexyl isocyanide 12a, phosphorated α-amido amides 15a,b were obtained, without the elimination of the amide group (Scheme 4). Although substrate 15b proved to be very stable, trifluoromethyl substituted α-amido amide 15a underwent spontaneous hydrolysis of the amide under the air moisture to yield tetrasubstituted α-aminophosphonate 13r. NMR properties of phosphorated α-amido amides 15 were very similar to the parent substrates 13 except for some significant differences. In the case of substrate 15b, the presence of benzylamide group was evident in 13 C NMR by the existence of the chemical shifts for two carbonyl groups at δC = 176. 4 and 165.3 ppm and a methylene carbon at δC = 45.7 ppm (DEPT). Key features for this compound in 1 H NMR spectrum are mainly the two diastereotopic protons of the benzyl group that appear as doublets at δC = 3.92 and 4.16 ppm with a strong geminal coupling constant 2 JHH = 17.1 Hz. It is also noteworthy the presence of an atypical doublet for two equivalent aromatic protons at δC = 8.26 ppm ( 3 JHH = 7.9 Hz) that corresponds either to the benzyl or the tosyl moiety that appears especially deshielded, which is probably originated by the proximity of both aromatic rings due to the steric crowding present in the structure.
In order to shed more light on this issue, we set up an additional experiment where the three-component reaction was performed using of N-tosyl ketimine 10a, thioacetic acid 17 and cyclohexyl isocyanide 12a in CDCl3. However, after 1h at room temperature a complex mixture was observed in the reaction vessel. We hypothesized that the high steric hindrance due to the presence of the tetrasubstituted carbon together with the higher Van der Waals radius of the sulfur atom versus the oxygen (180 pm vs. 152 pm) could be the reason of such different behavior.
For this reason, next we tried the Ugi reaction using a less sterically demanding isocyanide such as methyl isocyanoacetate 12b (Scheme 5). In this case, formation of thioamide 18 was observed in full conversion. The presence of a sulfur atom in the structure confirms unambiguously the Ugi mechanism of our reaction through the formation of iminium species 19 from α-ketiminophosphonate 10a and thioacid 17, followed by a nucleophilic attack of isocyanide 12b. Then, a second nucleophilic attack of thiocarboxylate anion in the intermediate nitrilium species 20 results in the formation of acyl thioimidate 21. To complete the Ugi sequence, the acyl transfer from thioimidate 21 to the adjacent nitrogen atom yields irreversibly phosphorated α-amido amide 22 that, due to the high steric hindrance owing to the presence of the tetrasubstituted carbon, undergoes a spontaneous cleavage of the acyl group that affords finally tetrasubstituted α-aminophosphonate 18. NMR properties of phosphorated α-amido amides 15 were very similar to the parent substrates 13 except for some significant differences. In the case of substrate 15b, the presence of benzylamide group was evident in 13 C NMR by the existence of the chemical shifts for two carbonyl groups at δ C = 176. 4 and 165.3 ppm and a methylene carbon at δ C = 45.7 ppm (DEPT). Key features for this compound in 1 H NMR spectrum are mainly the two diastereotopic protons of the benzyl group that appear as doublets at δ C = 3.92 and 4.16 ppm with a strong geminal coupling constant 2 J HH = 17.1 Hz. It is also noteworthy the presence of an atypical doublet for two equivalent aromatic protons at δ C = 8.26 ppm ( 3 J HH = 7.9 Hz) that corresponds either to the benzyl or the tosyl moiety that appears especially deshielded, which is probably originated by the proximity of both aromatic rings due to the steric crowding present in the structure.
In order to shed more light on this issue, we set up an additional experiment where the three-component reaction was performed using of N-tosyl ketimine 10a, thioacetic acid 17 and cyclohexyl isocyanide 12a in CDCl 3 . However, after 1h at room temperature a complex mixture was observed in the reaction vessel. We hypothesized that the high steric hindrance due to the presence of the tetrasubstituted carbon together with the higher Van der Waals radius of the sulfur atom versus the oxygen (180 pm vs. 152 pm) could be the reason of such different behavior.
For this reason, next we tried the Ugi reaction using a less sterically demanding isocyanide such as methyl isocyanoacetate 12b (Scheme 5). In this case, formation of thioamide 18 was observed in full conversion. The presence of a sulfur atom in the structure confirms unambiguously the Ugi mechanism of our reaction through the formation of iminium species 19 from α-ketiminophosphonate 10a and thioacid 17, followed by a nucleophilic attack of isocyanide 12b. Then, a second nucleophilic attack of thiocarboxylate anion in the intermediate nitrilium species 20 results in the formation of acyl thioimidate 21.
To complete the Ugi sequence, the acyl transfer from thioimidate 21 to the adjacent nitrogen atom yields irreversibly phosphorated α-amido amide 22 that, due to the high steric hindrance owing to the presence of the tetrasubstituted carbon, undergoes a spontaneous cleavage of the acyl group that affords finally tetrasubstituted α-aminophosphonate 18. Nevertheless, attempts to isolate compound 18 failed due to its decomposition during the workup, but the identity of thioamide 22 was confirmed by NMR of the crude reaction. 31 P NMR showed the disappearance of the starting imine (δP = 6.6 ppm) and the formation of a major compound with a chemical shift at δP = 18.7 ppm. On the other hand, 1 H NMR showed two clear doublets at δH = 3.90 ppm ( 3 JPH = 10.7 Hz) and δH = 3.79 ppm ( 3 JPH = 10.8 Hz), typical for the diastereotopic methoxy groups at the phosphonate, that suggest the formation of a stereogenic carbon close to the phosphorus atom and a broad triplet that interchanges with D2O, at δH = 8.63 ppm ( 1 JNH = 4.0 Hz), that may correspond to the NH of thioamide group, where the proton is coupled with the quadrupolar nucleus of 14 N. More importantly, 13 C NMR shows a doublet for the quaternary C-P (DEPT) at δC = 58.6 ppm ( 1 JPH = 167.2 Hz), and the characteristic chemical shift for the C=S group of thioamides at δC = 199.2 ppm. A similar result was obtained using thiobenzoic acid instead of thioacetic acid.
Additionally, the hydrolysis the phosphonate group to its phosphonic acid derivative 23 can be performed under mild conditions in chloroform by the treatment of 13b with trimethylsilyl bromide at room temperature. The subsequent aqueous workup yields α-aminophosphonic acid 23 in almost quantitative yield (Scheme 6). Scheme 6. Hydrolysis of phosphonate ester 13b.

Biological Results
In vitro cytotoxicity of tetrasubstituted α-aminophosphonate derivatives 13, 15 and 23 was evaluated by testing their antiproliferative activities against A549 cell line (carcinomic human alveolar basal epithelial cell). Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, nonmalignant MRC5 lung fibroblasts were tested for studying selective toxicity [34] and chemotherapeutic doxorubicin is used as reference value. The cell proliferation inhibitory activity is shown as IC50 values (Table 1). Nevertheless, attempts to isolate compound 18 failed due to its decomposition during the workup, but the identity of thioamide 22 was confirmed by NMR of the crude reaction. 31 P NMR showed the disappearance of the starting imine (δ P = 6.6 ppm) and the formation of a major compound with a chemical shift at δ P = 18.7 ppm. On the other hand, 1  Additionally, the hydrolysis the phosphonate group to its phosphonic acid derivative 23 can be performed under mild conditions in chloroform by the treatment of 13b with trimethylsilyl bromide at room temperature. The subsequent aqueous workup yields α-aminophosphonic acid 23 in almost quantitative yield (Scheme 6). Nevertheless, attempts to isolate compound 18 failed due to its decomposition during the workup, but the identity of thioamide 22 was confirmed by NMR of the crude reaction. 31  Additionally, the hydrolysis the phosphonate group to its phosphonic acid derivative 23 can be performed under mild conditions in chloroform by the treatment of 13b with trimethylsilyl bromide at room temperature. The subsequent aqueous workup yields α-aminophosphonic acid 23 in almost quantitative yield (Scheme 6). Scheme 6. Hydrolysis of phosphonate ester 13b.

Biological Results
In vitro cytotoxicity of tetrasubstituted α-aminophosphonate derivatives 13, 15 and 23 was evaluated by testing their antiproliferative activities against A549 cell line (carcinomic human alveolar basal epithelial cell). Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, nonmalignant MRC5 lung fibroblasts were tested for studying selective toxicity [34] and chemotherapeutic doxorubicin is used as reference value. The cell proliferation inhibitory activity is shown as IC50 values (Table 1). Scheme 6. Hydrolysis of phosphonate ester 13b.

Biological Results
In vitro cytotoxicity of tetrasubstituted α-aminophosphonate derivatives 13, 15 and 23 was evaluated by testing their antiproliferative activities against A549 cell line (carcinomic human alveolar basal epithelial cell). Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, nonmalignant MRC5 lung fibroblasts were tested for studying selective toxicity [34] and chemotherapeutic doxorubicin is used as reference value. The cell proliferation inhibitory activity is shown as IC 50 values (Table 1). In a preliminary study, we tested the cytotoxicity of simple phenyl substituted αaminophosphonates 13a-j as lead compounds. Although no grown inhibition activity was observed for dimethyl and diethylphosphonates 13a,f ( Table 1, Entries 1-2), dibenzylphosphonate 13g showed some cytotoxicity against A549 cell line with an IC 50 value of 16.46 ± 1.49 µM and, interestingly, very good selectivity was also obtained towards MRC5 nonmalignant cell line ( Then we studied the introduction of substituents at the aromatic ring of tetrasubstituted aminophosphonates 13. Scarce cytotoxic effect was found for para-nitrophenyl substituted substrate 13k, bearing an electron poor aromatic group (Table 1, Entry 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 [35][36][37]. For this reason, next we tested the in vitro cytotoxicity of fluorine containing α-aminophosphonates 13n. However, ortho-fluorophenyl and para-trifluoromethylphenyl substituted substrates 13n,r presented IC 50 values higher than 50 µM (Table 1, Entries 6, 8). Interestingly, thioether derived α-aminophosphonate 13p, showed a considerable antiproliferative activity with an IC 50 value of 14.56 ± 2.53 µM and a very good selectivity towards MRC5 cell line (Table 1, Entry 7). Phosphorated α-amido amide 15a bearing a para-trifluomethylphenyl substituent showed better toxicity than its parent compound 13r with an IC 50 value of 28.76 ± 3.20 µM and a good selectivity towards nonmalignant cells (Table 1, Entry 8 vs. Entry 9). Finally phosphonic acid derivative 23 did not provide any toxicity against A549 cell line (Table 1, Entry 10).

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, USA) (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%) as external reference (δ = 0.0 ppm) for 31 P 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, USA) 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, USA) using a CHIRALPAK®IA column (5µm, 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 tested α-aminophosphonate derivatives 13, 15 and α-aminophosphonaic acid 23 is >95%, which meets the purity requirement by the Journal.

Experimental Procedures and Characterization Data for Compounds 13, 15, 16 and 23
General Procedure for the Synthesis N-Tosyl α-Iminophosphonates 10 Following a literature procedure, [28,31] to a solution of the corresponding tetrasubstituted N-tosyl α-aminophosphonate (10 mmol) in CH 2 Cl 2 (30 mL) was added trichloroisocyanuric acid (6.97 g, 30 mmol). The resulting suspension was stirred at 0 • C until disappearance of the starting N-tosyl α-aminophosphonate, as monitored by 31 P NMR (14 to 48 h). The solid residue was eliminated by filtration to afford a clear solution of intermediate N-chloro α-aminophosphonate and then, poly(4-vinylpyridine) (3.0 g), previously dried at 100 • C overnight, was added. The resulting suspension was stirred under reflux overnight and the reaction was then filtered and concentrated under reduced pressure. The resulting yellow oily crude was purified by crystallization from diethyl ether.
General Procedure for the Synthesis N-Trityl α-Iminophosphonate 14 Following a literature procedure, [33] N-bromosuccinimide (178 mg, 1 mmol) was added on a solution of dimethyl ((tritylamino)methyl)phosphonate (457 mg, 1 mmol) in CCl 4 (3 mL). The mixture was stirred in quartz flask under UV light until the disappearance of starting α-aminophosphonate as monitored by 31 P-NMR (δ H 30.9 to 10.1 ppm). The resulting suspension was filtered under inert atmosphere to afford a clear solution of dimethyl (E)-((tritylimino)methyl)phosphonate that can be used without any further workup.
General Procedure for the Ugi Reaction of α-Phosphorated Ketimines 10 and 14 A mixture of α-iminophosphonate 10 or 14 (1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and isocyanide 12 (1.1 mmol) in ichloromethane (3 mL) was stirred at room temperature until disappearance of the starting iminophosphonate 10 as monitored by 31 P-NMR. The reaction was concentrated under vacuum and the resulting crude residue was purified by crystallization (Dichomethane/Hexanes 1:3), yielding α-aminophosphonates 13, 15 or 16. In some cases, a purification by column chromatography was necessary as detailed for each compound.

Conclusions
In conclusion, we report an efficient Ugi methodology using ketimines for the preparation of tetrasubstituted α-aminophosphonates holding a variety of substituents. Despite the difficulty often observed for the utilization of ketones or ketimines in Ugi reactions, α-phosphorated ketimines react under mild conditions to give the Ugi adducts after the spontaneous cleavage of the amide moiety. Clear evidences of the Ugi mechanism are provided, using thioacids. Moreover, obtained α-aminophosphonate derivatives 13g, 13h, 13p and 15a showed in vitro cytotoxicity inhibiting the growth of human tumor cell line A549 (carcinomic human alveolar basal epithelial cell), and a high selectivity toward MRC5 nonmalignant lung fibroblasts. As far as we know this is the first example of much hindered tetrasubstituted α-aminophosphonates showing antiproliferative activity.

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).