Synthesis of Tetrasubstituted Phosphorus Analogs of Aspartic Acid as Antiproliferative Agents
Abstract
Financial support by Ministerio de Economía, Industria y Competividad (PID2021-122558OB-I00) and Gobierno Vasco (GV-IT1701-22) is gratefully acknowledged. X.d.C. and A.L.-F. thank the Basque Country Government for a predoctoral grant.
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Citation: del Corte, X.; Maestro, A.; López-Francés, A.; Palacios, F.; Vicario, J. Synthesis of Tetrasubstituted Phosphorus Analogs of Aspartic Acid as Antiproliferative Agents. Molecules 2022,27, 8024. https://doi.org/10.3390/ molecules27228024 Academic Editor: Jakub Adamek Received: 16 September 2022 Accepted: 15 November 2022 Published: 18 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Synthesis of Tetrasubstituted Phosphorus Analogs of Aspartic Acid as Antiproliferative Agents Xabier del Corte †, Aitor Maestro †, Adrián López-Francés , Francisco Palacios and Javier Vicario * Departamento de Química Orgánica I, Centro de Investigación y Estudios Avanzados “Lucio Lascaray”, Facultad de Farmacia, University of the Basque Country, UPV/EHU Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain *Correspondence: javier[email protected]; Tel.: +34-945013891 † These authors contributed equally to this work. Abstract: An efficient general method for the synthesis of a wide family of α -aminophosphonate analogs of aspartic acid bearing tetrasubstituted carbons is reported through an aza-Reformatsky reaction of α -iminophosphonates, generated from α -aminophosphonates, in an umpolung process. In addition, the α -aminophosphonate substrates showed in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines A549 (carcinomic human alveolar basal epithelial cell) and SKOV3 (human ovarian carcinoma). In view of the possibilities in the diversity of the substituents that offer the synthetic methodology, an extensive profile structure–activity is presented, measuring IC50 values up to 0.34 µM in the A549 and 9.8 µM in SKOV3 cell lines. Keywords: Reformatsky reaction; tetrasubstituted α -aminophosphonates; aspartic acid; antiproliferative effect 1. Introduction The increase in life expectancy is one of the greatest achievements of humankind, which is also linked to far-reaching consequences with implications for nearly all socioeconomic sectors [ 1 ]. According to a study published in The Lancet in 2018 [ 2 ], by 2040, it is expected that 59 countries will have an average life expectancy of more than 80 years. In particular, the average life expectancy in Spain is predicted to be the highest in the world and will reach 85.8 years. In the past, the main roots of mortality were associated to infectious and parasitic diseases but, due to the phenomenon of population ageing, chronic and degenerative diseases have become the main concern of all healthcare systems worldwide. Accordingly, cancers figure among the leading causes of morbidity and mortality worldwide and have become one of the world’s largest health problems [3]. The systemic treatment of cancer implies a combination of surgery, chemotherapy and radiation. Other options include immunotherapy, targeted therapy, laser or hormonal therapy [ 4 ]. Chemotherapeutic agents possess the ability to travel throughout the body, and selectively destroy fast-growing malignant cells [ 5 ]. Here, in the first front of battle, drug discovery plays a crucial role in this area through the synthesis and characterization of drug candidates and the evaluation of their anticancer properties, prior to the subsequent clinical trials. Despite the strong efforts made in the last decades for the development of efficient chemotherapeutic agents, the continuous search for newer, safer and more potent cytotoxic drugs is an essential task in science, especially due to the known ability of cancer cells to develop resistance to the known therapies [6,7]. Among the innumerable amount of potentially chemotherapeutic molecules, we focused, in this case, on organophosphorus compounds. In particular, phosphonic acids and their esters are a family of compounds, characterized by the presence of a stable C-P bond in their structure, that show interesting and useful biological properties [ 8 ], including anticancer activity, such as the case of cyclophosphamide [ 9 ] or zolendronate [ 10 , 11 ]. Molecules 2022,27, 8024. https://doi.org/10.3390/molecules27228024 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 8024 2 of 22 Specifically, α -aminophosphonic acids are bioisosters of α -amino acids, where the flat carboxylic acid group has been replaced by a tetrahedral phosphonic acid group that shows the fully oxidized phosphorus atom at the core [ 12 ]. Due to this isosteric substitution, α -aminophosphonic acid scaffold is able to mimic the tetrahedral geometry and negative charge development found in the transition state of peptide cleavage, thus inhibiting enzymes implied in proteolysis processes (Figure 1) [ 13 – 15 ]. Considering this, it is easy to anticipate that a number of α -aminophosphonic acid derivatives show interesting biological activities, such as herbicidal [ 16 , 17 ], antimicrobial [ 18 – 21 ] or antioxidant [ 22 , 23 ], and some of them have been reported as potential drugs, in particular, for the treatment of infectious diseases [ 24 , 25 ]. Remarkably, some α -aminophosphonate derivatives have been described as anticancer [26–29] agents. Molecules2022,27,xFORPEERREVIEW2of23 bondintheirstructure,thatshowinterestingandusefulbiologicalproperties[8], includinganticanceractivity,suchasthecaseofcyclophosphamide[9]orzolendronate [10,11].Specifically,α‐aminophosphonicacidsarebioisostersofα‐aminoacids,wherethe flatcarboxylicacidgrouphasbeenreplacedbyatetrahedralphosphonicacidgroupthat showsthefullyoxidizedphosphorusatomatthecore[12].Duetothisisosteric substitution,α‐aminophosphonicacidscaffoldisabletomimicthetetrahedralgeometry andnegativechargedevelopmentfoundinthetransitionstateofpeptidecleavage,thus inhibitingenzymesimpliedinproteolysisprocesses(Figure1)[13–15].Consideringthis, itiseasytoanticipatethatanumberofα‐aminophosphonicacidderivativesshow interestingbiologicalactivities,suchasherbicidal[16,17],antimicrobial[18–21]or antioxidant[22,23],andsomeofthemhavebeenreportedaspotentialdrugs,inparticular, forthetreatmentofinfectiousdiseases[24,25].Remarkably,someα‐aminophosphonate derivativeshavebeendescribedasanticancer[26–29]agents. Figure1.α‐Aminophosphonicacidgroupmimicsthetransitionstateofpeptidecleavage. Asparticacidisoneofthe20buildingblockα‐aminoacidsofproteinsthatisknown tohavepharmacologicalactivityatsomeglutamatereceptors[30].Themostinteresting featureofthestructureofasparticacidisthepresenceofasecondacidicsidechainthat mayinteractwithotheraminoacids,enzymesorproteinsinthebody.Inthecontextof thisresearch,followingthebiosisosterismapproach,twopossiblephosphoratedanalogs maybedesignedfromasparticacidscaffoldI:β‐phosphorylatedα‐aminoacidsIIorβ‐ carboxylicα‐aminophosphonatesIII(Scheme1). Scheme1.Twopossibleisostericsubstitutionsofthecarboxylicmoietybyaphosphonicacidgroup inasparticacidscaffold. Whilethesynthesisofphosphonateanalogsofasparticacidbytheisosteric substitutionoftheβ‐carboxylicgroupiswelldocumented[31–33],thepreparationofthe parentα‐aminophosphonateanalogsIIIbythesubstitutionoftheα‐carboxylicmoietyhas receivedlessattention,andmostofthesubstratesarereportedassingleexamplesof generalmethodsleadingtoα‐aminophosphonatesorasparticacidderivatives[32,34,35]. Inparticular,thereareafewexamplesreportedforthesynthesisoftetrasubstitutedα‐ aminophosphonatesderivedfromasparticacidasconcreteexamplesofthescopeof reactionsthatimplyC‐CorC‐Pbondformation[36–39].Itiswellknownthatthe developmentofreactionsleadingtotheformationoftetrasubstitutedcarbonsisa challengingtask,duetothelackofreactivityofthesubstrates,derivedfromthe generationofahighlycrowdedstructure. Figure 1. α-Aminophosphonic acid group mimics the transition state of peptide cleavage. Aspartic acid is one of the 20 building block α -amino acids of proteins that is known to have pharmacological activity at some glutamate receptors [ 30 ]. The most interesting feature of the structure of aspartic acid is the presence of a second acidic side chain that may interact with other amino acids, enzymes or proteins in the body. In the context of this research, following the biosisosterism approach, two possible phosphorated analogs may be designed from aspartic acid scaffold I:β-phosphorylated α-aminoacids II or β-carboxylic α-aminophosphonates III (Scheme 1). Molecules2022,27,xFORPEERREVIEW2of23 bondintheirstructure,thatshowinterestingandusefulbiologicalproperties[8], includinganticanceractivity,suchasthecaseofcyclophosphamide[9]orzolendronate [10,11].Specifically,α‐aminophosphonicacidsarebioisostersofα‐aminoacids,wherethe flatcarboxylicacidgrouphasbeenreplacedbyatetrahedralphosphonicacidgroupthat showsthefullyoxidizedphosphorusatomatthecore[12].Duetothisisosteric substitution,α‐aminophosphonicacidscaffoldisabletomimicthetetrahedralgeometry andnegativechargedevelopmentfoundinthetransitionstateofpeptidecleavage,thus inhibitingenzymesimpliedinproteolysisprocesses(Figure1)[13–15].Consideringthis, itiseasytoanticipatethatanumberofα‐aminophosphonicacidderivativesshow interestingbiologicalactivities,suchasherbicidal[16,17],antimicrobial[18–21]or antioxidant[22,23],andsomeofthemhavebeenreportedaspotentialdrugs,inparticular, forthetreatmentofinfectiousdiseases[24,25].Remarkably,someα‐aminophosphonate derivativeshavebeendescribedasanticancer[26–29]agents. Figure1.α‐Aminophosphonicacidgroupmimicsthetransitionstateofpeptidecleavage. Asparticacidisoneofthe20buildingblockα‐aminoacidsofproteinsthatisknown tohavepharmacologicalactivityatsomeglutamatereceptors[30].Themostinteresting featureofthestructureofasparticacidisthepresenceofasecondacidicsidechainthat mayinteractwithotheraminoacids,enzymesorproteinsinthebody.Inthecontextof thisresearch,followingthebiosisosterismapproach,twopossiblephosphoratedanalogs maybedesignedfromasparticacidscaffoldI:β‐phosphorylatedα‐aminoacidsIIorβ‐ carboxylicα‐aminophosphonatesIII(Scheme1). Scheme1.Twopossibleisostericsubstitutionsofthecarboxylicmoietybyaphosphonicacidgroup inasparticacidscaffold. Whilethesynthesisofphosphonateanalogsofasparticacidbytheisosteric substitutionoftheβ‐carboxylicgroupiswelldocumented[31–33],thepreparationofthe parentα‐aminophosphonateanalogsIIIbythesubstitutionoftheα‐carboxylicmoietyhas receivedlessattention,andmostofthesubstratesarereportedassingleexamplesof generalmethodsleadingtoα‐aminophosphonatesorasparticacidderivatives[32,34,35]. Inparticular,thereareafewexamplesreportedforthesynthesisoftetrasubstitutedα‐ aminophosphonatesderivedfromasparticacidasconcreteexamplesofthescopeof reactionsthatimplyC‐CorC‐Pbondformation[36–39].Itiswellknownthatthe developmentofreactionsleadingtotheformationoftetrasubstitutedcarbonsisa challengingtask,duetothelackofreactivityofthesubstrates,derivedfromthe generationofahighlycrowdedstructure. Scheme 1. Two possible isosteric substitutions of the carboxylic moiety by a phosphonic acid group in aspartic acid scaffold. While the synthesis of phosphonate analogs of aspartic acid by the isosteric substitution of the β -carboxylic group is well documented [ 31 – 33 ], the preparation of the parent α -aminophosphonate analogs III by the substitution of the α -carboxylic moiety has received less attention, and most of the substrates are reported as single examples of general methods leading to α -aminophosphonates or aspartic acid derivatives [ 32 , 34 , 35 ]. In particular, there are a few examples reported for the synthesis of tetrasubstituted α -aminophosphonates derived from aspartic acid as concrete examples of the scope of reactions that imply C-C or C-P bond formation [ 36 – 39 ]. It is well known that the development of reactions leading to the formation of tetrasubstituted carbons is a challenging task, due to the lack of reactivity of the substrates, derived from the generation of a highly crowded structure. In this context, the aza-Reformatsky reaction is a widely used method for the synthesis of β -amino acids [ 40 – 43 ], as well as for the synthesis of biologically active molecules [44–46] . During the last years, the use of dialkylzinc reagents has emerged as an alternative to Zn dust [ 47 – 50 ]. In this regard, very recently, we have reported an enantioselective aza-
Molecules 2022,27, 8024 3 of 22 Reformatsky reaction of α -phosphorylated ketimines that leads to the formation of tetrasubstituted α -aminophosphonate derivatives [ 51 ]. In view of the interesting properties of aspartic acid derivatives and the potential of the isosteric substitution of a carboxylate by a phosphonate group, the corresponding phosphorus analogs of aspartic acid may be very interesting substrates from a biological point of view. According to this, as part of our ongoing research into the identification of new chemotherapeutic agents [ 52 – 54 ], with a special focus on organophosphorus derivatives [ 55 – 57 ], we thought that the preparation of a wide family of phosphorated analogs of aspartic acid and the study of their anticancer properties would be an interesting contribution to the field of organic and medicinal chemistry. For all the reasons mentioned above, herein, we report a general method for the synthesis of tetrasubstituted phosphorated analogs of aspartic acid through an aza-Reformatsky reaction of α -ketiminophosphonates and the evaluation of their in vitro cytotoxic activity against several cancer cell lines. 2. Results and Discussion 2.1. Chemistry During the last years, we have been involved in the synthesis of α -aminophosphonate derivatives through the addition of carbon nucleophiles to imines. The most remarkable feature of our approach is that α -iminophosphonate electrophiles are generated by the oxidation of the parent α -aminophosphonates. Thus, the global reaction can be considered as an umpolung process, where the nucleophilic character of α -aminophosphonate species has been inverted. In particular, following this approach, we have reported the enantioselective synthesis of indolyl phosphoglycines [ 58 ] by the addition of indole derivatives to α - phosphorated aldimines and the nucleophilic addition of cyanide [ 59 ], organometallics [ 60 ] or nitromethane species [ 61 ] to α -phosphorated ketimines, for the preparation of diverse tetrasubstituted α -aminophosphonate derivatives. More recently, we have extended this strategy to the enantioselective Reformatsky reaction, using α -ketiminophosphonates as the electrophile source [51]. Following this last approach, we tested the aza-Reformatsky reaction of different imines 1–4 with ethyl iodoacetate, under dry air atmosphere, in the presence of dimethylzinc, affording the corresponding β -aminoesters 4–6 very good yields when aldimines or activated ketimines were used (Scheme 2). Molecules2022,27,xFORPEERREVIEW3of23 Inthiscontext,theaza‐Reformatskyreactionisawidelyusedmethodforthe synthesisofβ‐aminoacids[40–43],aswellasforthesynthesisofbiologicallyactive molecules[44–46].Duringthelastyears,theuseofdialkylzincreagentshasemergedas analternativetoZndust[47–50].Inthisregard,veryrecently,wehavereportedan enantioselectiveaza‐Reformatskyreactionofα‐phosphorylatedketiminesthatleadsto theformationoftetrasubstitutedα‐aminophosphonatederivatives[51].Inviewofthe interestingpropertiesofasparticacidderivativesandthepotentialoftheisosteric substitutionofacarboxylatebyaphosphonategroup,thecorrespondingphosphorus analogsofasparticacidmaybeveryinterestingsubstratesfromabiologicalpointofview. Accordingtothis,aspartofourongoingresearchintotheidentificationofnew chemotherapeuticagents[52–54],withaspecialfocusonorganophosphorusderivatives [55–57],wethoughtthatthepreparationofawidefamilyofphosphoratedanalogsof asparticacidandthestudyoftheiranticancerpropertieswouldbeaninteresting contributiontothefieldoforganicandmedicinalchemistry.Forallthereasonsmentioned above,herein,wereportageneralmethodforthesynthesisoftetrasubstituted phosphoratedanalogsofasparticacidthroughanaza‐Reformatskyreactionofα‐ ketiminophosphonatesandtheevaluationoftheirinvitrocytotoxicactivityagainst severalcancercelllines. 2.ResultsandDiscussion 2.1.Chemistry Duringthelastyears,wehavebeeninvolvedinthesynthesisofα‐ aminophosphonatederivativesthroughtheadditionofcarbonnucleophilestoimines. Themostremarkablefeatureofourapproachisthatα‐iminophosphonateelectrophiles aregeneratedbytheoxidationoftheparentα‐aminophosphonates.Thus,theglobal reactioncanbeconsideredasanumpolungprocess,wherethenucleophiliccharacterof α‐aminophosphonatespecieshasbeeninverted.Inparticular,followingthisapproach, wehavereportedtheenantioselectivesynthesisofindolylphosphoglycines[58]bythe additionofindolederivativestoα‐phosphoratedaldiminesandthenucleophilicaddition ofcyanide[59],organometallics[60]ornitromethanespecies[61]toα‐phosphorated ketimines,forthepreparationofdiversetetrasubstitutedα‐aminophosphonate derivatives.Morerecently,wehaveextendedthisstrategytotheenantioselective Reformatskyreaction,usingα‐ketiminophosphonatesastheelectrophilesource[51]. Followingthislastapproach,wetestedtheaza‐Reformatskyreactionofdifferent imines1–4withethyliodoacetate,underdryairatmosphere,inthepresenceof dimethylzinc,affordingthecorrespondingβ‐aminoesters4–6verygoodyieldswhen aldiminesoractivatedketimineswereused(Scheme2). Scheme2.Aza‐Reformatskyreactionofimines1–4. Scheme 2. Aza-Reformatsky reaction of imines 1–4. Initially, the simple N-tosylimine 1 derived from benzaldehyde (R = H) was proved to be an excellent substrate for the reaction, affording β -phenylalanine derivative 5 a very good yield (Scheme 2). Next, we were intrigued whether the reaction could also be applicable to α -iminoesters and, for this reason, we tried the same reaction conditions using N-tosyl-protected α -iminoester 2 (R = CO 2 Et) as a substrate. In this case, the reaction
Molecules 2022,27, 8024 4 of 22 also proceeded efficiently to afford quaternary aspartic acid derivative 6 (Scheme 2). In a similar way, using benzaldehyde-derived α -iminophosphonate 3a (R = P(O)(OMe) 2 ), the tetrasubstituted aspartic acid derivative 7a was obtained in an excellent yield (Scheme 2). However, the reaction conditions, including the required dimethylzinc and the reaction time, presented high variations depending on the structure of the imine. On the other hand, we also tested a non-activated ketimine 4 , derived from acetophenone. Unfortunately, in this case, the Reformatsky product 8 was not observed, and the starting materials were recovered unaltered. The substrates obtained from the aza-Reformatsky reaction were fully characterized on the basis of their 1 H, 31 P, and 13 C NMR, IR spectra and HRMS (see Supplementary Materials). The most characteristic pattern for these compounds in the 1 H NMR spectrum is the signals corresponding to the two protons of the methylene group next to the tetrasubstituted carbon at δ ~3.5 ppm, which, because of the presence of a chiral center in the structure, have a diastereotopic character and appear as two independent signals. In the particular case of phosphorated aspartic acid derivative 7a , those signals appear as two double doublets at δ = 3.59 and 3.46 ppm, showing a reciprocal geminal coupling of 2 J HH = 16.4 Hz and additional vicinal couplings with the phosphorus atom of 3 J PH = 22.7 and 10.7 Hz, respectively. Accordingly, the dimethyl phosphonate moiety in 7a is seen as two representative intense doublets at δH = 3.46 ppm ( 3 J PH = 10.7 Hz) and δH = 3.48 ppm ( 3 J PH = 10.5 Hz), typical for the diastereotopic methoxy groups at the phosphonate moiety. Remarkably, the signal corresponding to the NH group of 6a appears as a thin doublet at δH = 6.17 ppm, that slowly interchanges with D 2 O, showing a strong coupling with the phosphorus atom of 3 J PH = 11.2 Hz, which may be attributable to a weak acidic character of the sulfonamide moiety. Likewise, in the 13 C NMR spectrum of phosphorylated derivative 7a , undoubtedly, the most characteristic signal is the doublet corresponding to the chiral quaternary carbon (DEPT) at δC = 62.1 ppm, which shows a very strong ipso coupling with the phosphorus atom of 1 J PC = 153.8 Hz. The methylene group next to the chiral carbon appears as a doublet at δC = 54.0 ppm with a geminal coupling with the phosphorus atom of 2JCP = 7.0 Hz , weaker than expected, possibly due to an unfavorable angle in terms of the coupling, which can be related to a distortion at the topology at the sp 3 chiral carbon, attributed to the high steric hindrance present at the quaternary center. The presence of the ester group is evident from the chemical shift at δC = 170.2 ppm, typical for carboxylic groups, which appears as a doublet, coupled with the phosphorus atom with a vicinal coupling constant of 3 J PC = 8.0 Hz. The fact that the vicinal C-P coupling is stronger than the geminal supports the proposed distortion of the bonding angles at the quaternary carbon, as expected from the high steric crowding. In congruity with the proposed structure, the Heteronuclear Multiple Bond Correlation Spectroscopy (HMBC) spectrum of 7a presents clear correlations of both diastereotopic methylene protons with the carbonyl group, the chiral tetrasubstituted carbon and the quaternary aromatic carbon of the phenyl substituent. Next, in view of the efficient protocol achieved for the aza-Reformatsky reaction, we focused our efforts on the extension of the reaction to the use of several α -ketiminophosphonate substrates 3 . In this regard, we considered the synthesis of α -iminophosphonate substrates 3 from a formal oxidation of tertiary aminophosphonates 9 . Then, the subsequent addition of a nucleophile species would afford tetrasubstituted aminophosphonates 11 . Therefore, this synthetic approach can be considered globally as a route for the generation of tetrasubstituted α -aminophosphonates by the substitution of hydrogen in a trisubstituted α - aminophosphonate by a nucleophilic reagent and the complementary process (‘umpolung reaction’) to the typical electrophilic substitution of trisubstituted α -amino-phosphonates 9 leading to functionalized α-aminophosphonates 10 (Scheme 3). Following this approach, α -ketiminophosphonates 3 were first generated by a formal oxidation of α -aminophosphonates 9 , following the procedure developed by our research group. Then, using dimethylphosphonate-substituted imines and ethyl iodoacetate, 19 tetra-
Molecules 2022,27, 8024 5 of 22 substituted aspartic acid analogs 7 , bearing different alpha-aromatic substituents, were efficiently synthesized (Scheme 4). Molecules2022,27,xFORPEERREVIEW5of23 complementaryprocess(‘umpolungreaction’)tothetypicalelectrophilicsubstitutionof trisubstitutedα‐amino‐phosphonates9leadingtofunctionalizedα‐aminophosphonates 10(Scheme3). Scheme3.Generalstrategiesforthesynthesisofquaternaryα‐aminophosphonates. Followingthisapproach,α‐ketiminophosphonates3werefirstgeneratedbyaformal oxidationofα‐aminophosphonates9,followingtheproceduredevelopedbyourresearch group.Then,usingdimethylphosphonate‐substitutediminesandethyliodoacetate,19 tetrasubstitutedasparticacidanalogs7,bearingdifferentalpha‐aromaticsubstituents, wereefficientlysynthesized(Scheme4). Scheme4.Phosphorusanalogsofasparticacid7,synthesizedthroughanaza‐Reformatskyreaction. Inadditiontothemodelreaction,usinganiminewithasimplephenylsubstituent (Scheme4,7a),thereactiontoleratesthepresenceofparaandmetaalkylsubstituentsatthe aromaticring(Scheme4,7b–c),aswellasstrongelectrondonatinggroupsatthepara positionofthearomaticimine(Scheme4,7d).Severalhalogen‐substitutedaromatic ketimineswerealsosuccessfullyusedinthereaction,includingaromaticringscontaining bromine(Scheme4,7e),chlorine(Scheme4,7f–i)orfluorine(Scheme4,7j–n)atomsat diversepositionsandincludingaperfluorophenylsubstituent(Scheme4,7o). Furthermore,anexcellentresultwasobservedusingaromaticiminessubstitutedwith electron‐withdrawinggroupssuchasp‐trifluoromethylorp‐nitrosubstituents(Scheme4, Scheme 3. General strategies for the synthesis of quaternary α-aminophosphonates. Molecules2022,27,xFORPEERREVIEW5of23 complementaryprocess(‘umpolungreaction’)tothetypicalelectrophilicsubstitutionof trisubstitutedα‐amino‐phosphonates9leadingtofunctionalizedα‐aminophosphonates 10(Scheme3). Scheme3.Generalstrategiesforthesynthesisofquaternaryα‐aminophosphonates. Followingthisapproach,α‐ketiminophosphonates3werefirstgeneratedbyaformal oxidationofα‐aminophosphonates9,followingtheproceduredevelopedbyourresearch group.Then,usingdimethylphosphonate‐substitutediminesandethyliodoacetate,19 tetrasubstitutedasparticacidanalogs7,bearingdifferentalpha‐aromaticsubstituents, wereefficientlysynthesized(Scheme4). Scheme4.Phosphorusanalogsofasparticacid7,synthesizedthroughanaza‐Reformatskyreaction. Inadditiontothemodelreaction,usinganiminewithasimplephenylsubstituent (Scheme4,7a),thereactiontoleratesthepresenceofparaandmetaalkylsubstituentsatthe aromaticring(Scheme4,7b–c),aswellasstrongelectrondonatinggroupsatthepara positionofthearomaticimine(Scheme4,7d).Severalhalogen‐substitutedaromatic ketimineswerealsosuccessfullyusedinthereaction,includingaromaticringscontaining bromine(Scheme4,7e),chlorine(Scheme4,7f–i)orfluorine(Scheme4,7j–n)atomsat diversepositionsandincludingaperfluorophenylsubstituent(Scheme4,7o). Furthermore,anexcellentresultwasobservedusingaromaticiminessubstitutedwith electron‐withdrawinggroupssuchasp‐trifluoromethylorp‐nitrosubstituents(Scheme4, Scheme 4. Phosphorus analogs of aspartic acid 7 , synthesized through an aza-Reformatsky reaction. In addition to the model reaction, using an imine with a simple phenyl substituent (Scheme 4, 7a ), the reaction tolerates the presence of para and meta alkyl substituents at the aromatic ring (Scheme 4, 7b–c ), as well as strong electron donating groups at the para position of the aromatic imine (Scheme 4, 7d ). Several halogen-substituted aromatic ketimines were also successfully used in the reaction, including aromatic rings containing bromine (Scheme 4, 7e ), chlorine (Scheme 4, 7f–i ) or fluorine (Scheme 4, 7j–n ) atoms at diverse positions and including a perfluorophenyl substituent (Scheme 4, 7o ). Furthermore, an excellent result was observed using aromatic imines substituted with electron-withdrawing groups such as p-trifluoromethyl or p-nitro substituents (Scheme 4, 7p–q ). The reaction can even be extended to the use of ketimines holding heteroaromatic or biphenyl substituents (Scheme 4,7r–s). Next, the synthetic procedure was extended to the use of other different alkyl iodoacetates. The reaction using methyl iodoacetate and a p-fluorophenyl substituted α-ketiminophosphonate affords the corresponding tetrasubstituted α-aminophosphonate 12 an excellent yield (Scheme 5). Under the same conditions, the reaction using benzyl iodoacetate efficiently yields the benzyl-protected analogs of aspartic acid 13a–b (Scheme 5).
Molecules 2022,27, 8024 6 of 22 Molecules2022,27,xFORPEERREVIEW6of23 7p–q).Thereactioncanevenbeextendedtotheuseofketiminesholdingheteroaromatic orbiphenylsubstituents(Scheme4,7r–s). Next,thesyntheticprocedurewasextendedtotheuseofotherdifferentalkyl iodoacetates.Thereactionusingmethyliodoacetateandap‐fluorophenylsubstitutedα‐ ketiminophosphonateaffordsthecorrespondingtetrasubstitutedα‐ aminophosphonate 12anexcellentyield(Scheme5).Underthesameconditions,thereactionusingbenzyl iodoacetateefficientlyyieldsthebenzyl‐protectedanalogsofasparticacid13a–b(Scheme 5). Scheme5.Extensionoftheaza‐Reformatskyreactiontootheralkylhaloacetatederivatives. Inordertoobtainthecarboxylicacidand/orphosphonicacidderivativesofthe aspartateanalogs,thehydrolysisoftheobtainedsubstrateswasattempted.However, underacidicorbasictreatment,compounds7a,12or13bledtotheformationofcomplex mixtures.Onthecontrary,thetreatmentofbenzylester13bunderhydrogenpressurein thepresenceofapalladiumcatalystaffordedthecorrespondingcarboxylicacid14an almostquantitativeyield(Scheme6). Scheme6.Hydrogenolysisofbenzylester13b. Whiletryingtofurtherunderstandthenatureofthereaction,somecontrol experimentswereperformedusingnon‐conventionalhaloacetatederivatives.For instance,theuseofα‐branchediodoacetate(Scheme7a)resultedinacompletelossofthe reactivitythatmaybeexplainedduetotheelectroniceffectofthefluorine,whichreduces thenucleophilicityoftheintermediatespecies.Incontrast,theuseofabulkier phosphonateinsteadoftheestergroupincreasesthestericdemandofthenucleophile (Scheme7b).Finally,whenusingethyl3‐iodopropionate,theformationoftheenolate intermediatedoesnotoccur,andthecorrespondingorganozinchalidespeciesmaybe formed,whicharenotusualinnucleophilicadditionsduetotheirlowreactivity(Scheme 7c). Scheme 5. Extension of the aza-Reformatsky reaction to other alkyl haloacetate derivatives. In order to obtain the carboxylic acid and/or phosphonic acid derivatives of the aspartate analogs, the hydrolysis of the obtained substrates was attempted. However, under acidic or basic treatment, compounds 7a , 12 or 13b led to the formation of complex mixtures. On the contrary, the treatment of benzylester 13b under hydrogen pressure in the presence of a palladium catalyst afforded the corresponding carboxylic acid 14 an almost quantitative yield (Scheme 6). Molecules2022,27,xFORPEERREVIEW6of23 7p–q).Thereactioncanevenbeextendedtotheuseofketiminesholdingheteroaromatic orbiphenylsubstituents(Scheme4,7r–s). Next,thesyntheticprocedurewasextendedtotheuseofotherdifferentalkyl iodoacetates.Thereactionusingmethyliodoacetateandap‐fluorophenylsubstitutedα‐ ketiminophosphonateaffordsthecorrespondingtetrasubstitutedα‐ aminophosphonate 12anexcellentyield(Scheme5).Underthesameconditions,thereactionusingbenzyl iodoacetateefficientlyyieldsthebenzyl‐protectedanalogsofasparticacid13a–b(Scheme 5). Scheme5.Extensionoftheaza‐Reformatskyreactiontootheralkylhaloacetatederivatives. Inordertoobtainthecarboxylicacidand/orphosphonicacidderivativesofthe aspartateanalogs,thehydrolysisoftheobtainedsubstrateswasattempted.However, underacidicorbasictreatment,compounds7a,12or13bledtotheformationofcomplex mixtures.Onthecontrary,thetreatmentofbenzylester13bunderhydrogenpressurein thepresenceofapalladiumcatalystaffordedthecorrespondingcarboxylicacid14an almostquantitativeyield(Scheme6). Scheme6.Hydrogenolysisofbenzylester13b. Whiletryingtofurtherunderstandthenatureofthereaction,somecontrol experimentswereperformedusingnon‐conventionalhaloacetatederivatives.For instance,theuseofα‐branchediodoacetate(Scheme7a)resultedinacompletelossofthe reactivitythatmaybeexplainedduetotheelectroniceffectofthefluorine,whichreduces thenucleophilicityoftheintermediatespecies.Incontrast,theuseofabulkier phosphonateinsteadoftheestergroupincreasesthestericdemandofthenucleophile (Scheme7b).Finally,whenusingethyl3‐iodopropionate,theformationoftheenolate intermediatedoesnotoccur,andthecorrespondingorganozinchalidespeciesmaybe formed,whicharenotusualinnucleophilicadditionsduetotheirlowreactivity(Scheme 7c). Scheme 6. Hydrogenolysis of benzylester 13b. While trying to further understand the nature of the reaction, some control experiments were performed using non-conventional haloacetate derivatives. For instance, the use of α -branched iodoacetate (Scheme 7a) resulted in a complete loss of the reactivity that may be explained due to the electronic effect of the fluorine, which reduces the nucleophilicity of the intermediate species. In contrast, the use of a bulkier phosphonate instead of the ester group increases the steric demand of the nucleophile (Scheme 7b). Finally, when using ethyl 3-iodopropionate, the formation of the enolate intermediate does not occur, and the corresponding organozinc halide species may be formed, which are not usual in nucleophilic additions due to their low reactivity (Scheme 7c). Molecules2022,27,xFORPEERREVIEW7of23 Scheme7.ReactionconditionsasshownintheScheme5.(a)Reactionwithα‐fluoroiodoacetate.(b) Reactiontestwithα‐iodophosphonate.(c)Remotereformatskyreaxction. Basedonthesecontrolreactions,aswellasonthereactionpathwaysproposedfor similarprocesses[62,63],wetheorizeatentativecatalyticcycleinwhichacombinationof electronicandstericeffectsandthestabilityofthezinc‐enolateintermediateare considered(Scheme8). Scheme8.Proposedreactionmechanism. Ashasbeenaddressedabove,asparticacidderivativeshaveprovedtohaveassorted pharmacologicalactivities[30].Thus,inviewofthedescribedbenefitsoftheisosteric substitutionofacarboxylatebyaphosphonategroup[12],next,weexploredthe applicationsofthesynthesizedphosphoratedanalogsofasparticacidasanticancer agents. 2.2.BiologicalResults Invitrocytotoxicityofthephosphoratedasparticacidderivativeswasevaluatedby testingtheirantiproliferativeactivitiesagainstseveralhumancancercelllines.Cell countingkit(CCK‐8)assaywasusedfortheevaluationofgrowthinhibition.Moreover, non‐malignantMRC5lungfibroblastsweretestedforstudyingselectivetoxicity[64]and chemotherapeuticdoxorubicinisusedasareferencevalue.Inaddition,trisubstituted aminophosphonate9a(Imineprecursor,R1=H,R2=P(O)(OMe)2)[59]andtetrasubstituted aminophosphonate18(R1=Me,R2=P(O)(OMe)2)[60]areusedastemplatesinorderto evaluatetheeffectofthesubstituents(Table1). Scheme 7. Reaction conditions as shown in the Scheme 5. ( a ) Reaction with α -fluoroiodoacetate. (b) Reaction test with α-iodophosphonate. (c) Remote reformatsky reaxction.
Molecules 2022,27, 8024 7 of 22 Based on these control reactions, as well as on the reaction pathways proposed for similar processes [ 62 , 63 ], we theorize a tentative catalytic cycle in which a combination of electronic and steric effects and the stability of the zinc-enolate intermediate are considered (Scheme 8). Molecules2022,27,xFORPEERREVIEW7of23 Scheme7.ReactionconditionsasshownintheScheme5.(a)Reactionwithα‐fluoroiodoacetate.(b) Reactiontestwithα‐iodophosphonate.(c)Remotereformatskyreaxction. Basedonthesecontrolreactions,aswellasonthereactionpathwaysproposedfor similarprocesses[62,63],wetheorizeatentativecatalyticcycleinwhichacombinationof electronicandstericeffectsandthestabilityofthezinc‐enolateintermediateare considered(Scheme8). Scheme8.Proposedreactionmechanism. Ashasbeenaddressedabove,asparticacidderivativeshaveprovedtohaveassorted pharmacologicalactivities[30].Thus,inviewofthedescribedbenefitsoftheisosteric substitutionofacarboxylatebyaphosphonategroup[12],next,weexploredthe applicationsofthesynthesizedphosphoratedanalogsofasparticacidasanticancer agents. 2.2.BiologicalResults Invitrocytotoxicityofthephosphoratedasparticacidderivativeswasevaluatedby testingtheirantiproliferativeactivitiesagainstseveralhumancancercelllines.Cell countingkit(CCK‐8)assaywasusedfortheevaluationofgrowthinhibition.Moreover, non‐malignantMRC5lungfibroblastsweretestedforstudyingselectivetoxicity[64]and chemotherapeuticdoxorubicinisusedasareferencevalue.Inaddition,trisubstituted aminophosphonate9a(Imineprecursor,R1=H,R2=P(O)(OMe)2)[59]andtetrasubstituted aminophosphonate18(R1=Me,R2=P(O)(OMe)2)[60]areusedastemplatesinorderto evaluatetheeffectofthesubstituents(Table1). Scheme 8. Proposed reaction mechanism. As has been addressed above, aspartic acid derivatives have proved to have assorted pharmacological activities [ 30 ]. Thus, in view of the described benefits of the isosteric substitution of a carboxylate by a phosphonate group [ 12 ], next, we explored the applications of the synthesized phosphorated analogs of aspartic acid as anticancer agents. 2.2. Biological Results In vitro cytotoxicity of the phosphorated aspartic acid derivatives was evaluated by testing their antiproliferative activities against several human cancer cell lines. Cell counting kit (CCK-8) assay was used for the evaluation of growth inhibition. Moreover, non-malignant MRC5 lung fibroblasts were tested for studying selective toxicity [ 64 ] and chemotherapeutic doxorubicin is used as a reference value. In addition, trisubstituted aminophosphonate 9a (Imine precursor, R 1 = H, R 2 = P(O)(OMe) 2 ) [ 59 ] and tetrasubstituted aminophosphonate 18 (R 1 = Me, R 2 = P(O)(OMe) 2 ) [ 60 ] are used as templates in order to evaluate the effect of the substituents (Table 1). Table 1. Antiproliferative activity of substrates 5–7, 9 and 18 against lung and ovarian cancer cell lines. Molecules2022,27,xFORPEERREVIEW8of23 Table1.Antiproliferativeactivityofsubstrates5–7,9and18againstlungandovariancancercell lines. EntryComp.R1R2IC50(μM) SKOV3A549MRC5 15CH2CO2EtH>5018.68±2.16>50 26CH2CO2EtCO2Et>5014.17±0.41>50 37aCH2CO2EtP(O)(OMe)2>502.66±0.26>50 49aHP(O)(OMe)2>5017.56±1.3>50 518CH3P(O)(OMe)2>50>50n.d. 6Doxorubicin0.13±0.098<0.1>50 Inapreliminarystudy,themostsimpleβ‐alaninesubstrate5showedsome cytotoxicityagainsttheA549cellline,withmodestIC50valuesof18.68±2.16μMbuthigh selectivityifcomparedwiththeMCR5cellline(Table1,Entry1).AslightlyimprovedIC50 valueof14.17±0.41μMwasobservedforasparticacidester6inthesamecelllineand, remarkably,theisostericsubstitutionoftheestergroupbyaphosphonategroupin7a resultedinasignificantlybetterIC50valueof2.66±0.26μM,stillwithahighselectivity towardnon‐malignantcells(Table1,Entries2–3). Thepresenceoftheestergroup,however,provedtobeveryrelevanttothe antiproliferativeactivityofcompound7asincetetrasubstitutedα‐aminophosphonate18, lackingsuchasubstituent,showednocytotoxicityagainsttheA549celllineand trisubstitutedα‐aminophosphonate9ashowedahigherIC50valueof17.56±1.3μM(Table 1,Entries4–5).NoneofthetestedcompoundsshowedinvitrotoxicityagainsttheSKOV3 cellline. Thisfirsttestofoursubstratesdemonstratedthatthebestresultsinactivityare obtainedwhenbothα‐phosphonateandβ‐carboxylategroupsarepresentinthestructure, formingaphosphorusanalogofasparticacid.Forthisreason,next,weperformedastudy oftheeffectofthesubstitutionattheα‐aromaticringoftheasparticacidanalogsintothe cytotoxicityagainsttheSKOV3andA549celllines(Table2). Table2.Antiproliferativeactivityofphosphoratedanalogsofasparticacid7,12–14againstlung andovariancancercelllines. EntryComp.R1R2IC50(μM) SKOV3A549MRC5 17aPhEt>502.66±0.26>50 27b4‐Me‐C6H4Et>500.34±0.0429.62±2.98 37c3‐Me‐C6H4Et>502.00±0.52>50 47d4‐Cl3CS‐C6H4Et6.43±0.644.41±0.292.47±0.30 57e4‐Br‐C6H4Et>501.08±0.09>50 67f4‐Cl‐C6H4Et24.12±1.453.09±0.14>50 77g3‐Cl‐C6H4Et>502.96±0.3438.05±1.61 87h3,4‐Cl2‐C6H3Et6.94±0.631.05±0.4211.29±1.16 97i3‐Cl‐4‐MeO‐C6H3Et>501.44±0.15>50 Entry Comp. R1R2IC50 (µM) SKOV3 A549 MRC5 15CH2CO2Et H >50 18.68 ±2.16 >50 26CH2CO2Et CO2Et >50 14.17 ±0.41 >50 37a CH2CO2Et P(O)(OMe) 2>50 2.66 ±0.26 >50 49a H P(O)(OMe) 2>50 17.56 ±1.3 >50 518 CH3 P(O)(OMe) 2>50 >50 n.d. 6Doxorubicin 0.13 ±0.098 <0.1 >50 In a preliminary study, the most simple β -alanine substrate 5 showed some cytotoxicity against the A549 cell line, with modest IC 50 values of 18.68 ± 2.16 µ M but high selectivity if compared with the MCR5 cell line (Table 1, Entry 1). A slightly improved IC 50 value of
Molecules 2022,27, 8024 8 of 22 14.17 ± 0.41 µ M was observed for aspartic acid ester 6 in the same cell line and, remarkably, the isosteric substitution of the ester group by a phosphonate group in 7a resulted in a significantly better IC 50 value of 2.66 ± 0.26 µ M, still with a high selectivity toward non-malignant cells (Table 1, Entries 2–3). The presence of the ester group, however, proved to be very relevant to the antiproliferative activity of compound 7a since tetrasubstituted α-aminophosphonate 18, lacking such a substituent, showed no cytotoxicity against the A549 cell line and trisubstituted α - aminophosphonate 9a showed a higher IC 50 value of 17.56 ± 1.3 µ M (Table 1, Entries 4–5 ). None of the tested compounds showed in vitro toxicity against the SKOV3 cell line. This first test of our substrates demonstrated that the best results in activity are obtained when both α -phosphonate and β -carboxylate groups are present in the structure, forming a phosphorus analog of aspartic acid. For this reason, next, we performed a study of the effect of the substitution at the α -aromatic ring of the aspartic acid analogs into the cytotoxicity against the SKOV3 and A549 cell lines (Table 2). Table 2. Antiproliferative activity of phosphorated analogs of aspartic acid 7, 12–14 against lung and ovarian cancer cell lines. Molecules2022,27,xFORPEERREVIEW8of23 Table1.Antiproliferativeactivityofsubstrates5–7,9and18againstlungandovariancancercell lines. EntryComp.R1R2IC50(μM) SKOV3A549MRC5 15CH2CO2EtH>5018.68±2.16>50 26CH2CO2EtCO2Et>5014.17±0.41>50 37aCH2CO2EtP(O)(OMe)2>502.66±0.26>50 49aHP(O)(OMe)2>5017.56±1.3>50 518CH3P(O)(OMe)2>50>50n.d. 6Doxorubicin0.13±0.098<0.1>50 Inapreliminarystudy,themostsimpleβ‐alaninesubstrate5showedsome cytotoxicityagainsttheA549cellline,withmodestIC50valuesof18.68±2.16μMbuthigh selectivityifcomparedwiththeMCR5cellline(Table1,Entry1).AslightlyimprovedIC50 valueof14.17±0.41μMwasobservedforasparticacidester6inthesamecelllineand, remarkably,theisostericsubstitutionoftheestergroupbyaphosphonategroupin7a resultedinasignificantlybetterIC50valueof2.66±0.26μM,stillwithahighselectivity towardnon‐malignantcells(Table1,Entries2–3). Thepresenceoftheestergroup,however,provedtobeveryrelevanttothe antiproliferativeactivityofcompound7asincetetrasubstitutedα‐aminophosphonate18, lackingsuchasubstituent,showednocytotoxicityagainsttheA549celllineand trisubstitutedα‐aminophosphonate9ashowedahigherIC50valueof17.56±1.3μM(Table 1,Entries4–5).NoneofthetestedcompoundsshowedinvitrotoxicityagainsttheSKOV3 cellline. Thisfirsttestofoursubstratesdemonstratedthatthebestresultsinactivityare obtainedwhenbothα‐phosphonateandβ‐carboxylategroupsarepresentinthestructure, formingaphosphorusanalogofasparticacid.Forthisreason,next,weperformedastudy oftheeffectofthesubstitutionattheα‐aromaticringoftheasparticacidanalogsintothe cytotoxicityagainsttheSKOV3andA549celllines(Table2). Table2.Antiproliferativeactivityofphosphoratedanalogsofasparticacid7,12–14againstlung andovariancancercelllines. EntryComp.R1R2IC50(μM) SKOV3A549MRC5 17aPhEt>502.66±0.26>50 27b4‐Me‐C6H4Et>500.34±0.0429.62±2.98 37c3‐Me‐C6H4Et>502.00±0.52>50 47d4‐Cl3CS‐C6H4Et6.43±0.644.41±0.292.47±0.30 57e4‐Br‐C6H4Et>501.08±0.09>50 67f4‐Cl‐C6H4Et24.12±1.453.09±0.14>50 77g3‐Cl‐C6H4Et>502.96±0.3438.05±1.61 87h3,4‐Cl2‐C6H3Et6.94±0.631.05±0.4211.29±1.16 97i3‐Cl‐4‐MeO‐C6H3Et>501.44±0.15>50 Entry Comp. R1R2IC50 (µM) SKOV3 A549 MRC5 17a Ph Et >50 2.66 ±0.26 >50 27b 4-Me-C6H4Et >50 0.34 ±0.04 29.62 ±2.98 37c 3-Me-C6H4Et >50 2.00 ±0.52 >50 47d 4-Cl3CS-C6H4Et 6.43 ±0.64 4.41 ±0.29 2.47 ±0.30 57e 4-Br-C6H4Et >50 1.08 ±0.09 >50 67f 4-Cl-C6H4Et 24.12 ±1.45 3.09 ±0.14 >50 77g 3-Cl-C6H4Et >50 2.96 ±0.34 38.05 ±1.61 87h 3,4-Cl2-C6H3Et 6.94 ±0.63 1.05 ±0.42 11.29 ±1.16 97i 3-Cl-4-MeO-C6H3Et >50 1.44 ±0.15 >50 10 7j 4-F-C6H4Et >50 7.15 ±0.24 >50 11 7k 3-F-C6H4Et >50 0.59 ±0.09 >50 12 7l 2-F-C6H4Et >50 0.90 ±0.12 >50 13 7m 2,4-F2-C6H3Et >50 2.24 ±0.31 >50 14 7n 3,4-F2-C6H3Et >50 5.70 ±0.70 >50 15 7o C6F5Et 20.46 ±2.75 3.65 ±0.21 >50 16 7p 4-CF3-C6H4Et 9.80 ±0.60 20. 30 ±1.14 >50 17 7q 4-NO2-C6H4Et >50 0.67 ±0.06 >50 18 7r 5-Cl-2-thienyl Et 11.77 ±0.60 1.04 ±0.28 >50 19 7s 4-Ph-C6H4Et 17.01 ±1.22 1.48 ±0.40 >50 20 12 3-F-C6H4Me >50 24.11 ±4.01 n.d. 21 13a 3-F-C6H4Bn >50 12.75 ±2.38 n.d. 22 13b Ph Bn >50 5.20 ±0.16 >50 23 14 Ph H >50 20.61 ±1.05 >50 24 Doxorubicin 0.13 ±0.098 <0.1 >50
Molecules 2022,27, 8024 9 of 22 The introduction of methyl groups into a bioactive structure results in a more lipophilic character, often resulting in an improved ability of molecules to cross cell membranes [ 65 , 66 ]. Indeed, para and meta tolyl-substituted aminophosphonates 7b and 7c presented improved IC 50 values of 0.34 ± 0.04 and 2.00 ± 0.52 µ M in the A549 cell line if compared with the model structure 7a (Table 2, Entries 2–3 vs. Entry 1). Although compound 7c showed a high selectivity if compared with the non-malignant cells, derivative 7b showed some toxicity toward the MCR5 cell line. Unfortunately, no activity was observed against the SKOV3 cell line for both compounds (Table 2, Entries 2–3). In addition, p-trichloromethylthiophenyl derivative 7d showed good IC 50 values of 6.43 ± 0.64 and 4.41 ± 0.29 µ M in the SKOV3 and A549 cell lines, respectively, but it presented a very low selectivity toward the MRC5 cell line (Table 2, Entry 4). Next, the effect of the substitution of the α -aromatic ring with different halogen atoms was explored. First, p-bromo-substituted derivative 7e proved to be a very good growth inhibitor of the A549 cell line with an IC 50 value of 1.08 ± 0.09 µ M, and a good selectivity if compared to the SKOV3 or MCR5 cell lines, which showed values over 50 µ M (Table 2, Entry 5). p-Chlorophenyl derivative 7f showed very good toxicity against the A549 cell line and some activity in the SKOV3 cell line with IC 50 values of 3.09 ± 0.14 and 24.12 ±1.45 µM , respectively, and a good selectivity against the healthy cells (Table 2, Entry 6). However, the m-chlorophenyl isomer 7g was found to be less effective than the para isomer and toxic against the healthy cells (Table 2, Entry 7). Remarkably, the combination of both substituents in substrate 7h resulted in a strong cytotoxic effect against all the cell lines and a total lack of selectivity (Table 2, Entry 8). Likewise, the combination of a meta-chloro and apara-methoxy group in the aromatic ring of 7i resulted in a very selective activity against the A549 cell line, with an IC50 value of 1.44 ±0.15 µM (Table 2, Entry 9). Although, generally, the effect of fluorine substituents on the activity of organic compounds is rather difficult to predict, it is well known that the introduction of fluorine atoms into bioactive molecules very often leads to increased activities [ 67 – 69 ]. With this in mind, we explored the effect of the introduction of different fluorine-containing phosphorated analogs of aspartic acid 7j–p . First, the introduction of a para-fluorine substituent in 7j had a negative effect on the cytotoxicity if compared with the model phenyl-substituted compound 7a (Table 2, Entry 10 vs. Entry 1). On the contrary, the metaand ortho-substituted isomers 7k and 7l , compared with the model substrate 7a , showed a very good cytotoxic effect and a high selectivity against the A549 cell line and improved IC 50 values of 0.59 ±0.09 and 0.90 ±0.12 µM were observed, respectively (Table 2, Entries 11 and 12 vs. Entry 1). Those values could not be improved by the combination of two fluorine substituents at the aromatic ring and, although good IC 50 values of 2.24 ± 0.31 and 5.70 ±0.70 µM were obtained for difluoro-substituted substrates 7m–n in the A549 cell line, those were significantly higher than the values obtained for mono-substituted substrates 7k-l (Table 2, Entries 13 and 14 vs. Entries 11 and 12). Moreover, perfluorophenyl derivative 7o was found to be active against the A549 and SKOV3 cell lines with IC 50 values of 3.65 ±0.21 and 20.46 ± 2.75 µ M, respectively, with a good selectivity toward the healthy cells (Table 2, Entry 15). Surprisingly, the introduction of a para-trifluoromethyl electron-withdrawing group at the aromatic ring had a positive effect on the cytotoxicity of substrate 7p in the SKOV3 cell line, while it had a negative effect for the A549 cell line. In this case, IC 50 values of 9.80 ±0.60 and 20.3 ± 1.14 µ M were obtained for each cell line (Table 2, Entry 16 vs. Entry 1) and no toxicity was observed in the MCR5 cells. Aspartic acid analog 7q , bearing other electron poor aromatic substituents, such as a p-nitrophenyl group, showed very good toxicity in the A549 cell line and a high selectivity with an IC 50 value of 0.67 ±0.06 µM (Table 2, Entry 17). Finally, heteroaromatic or biphenyl α -substituted substrates 7r and 7s presented very good IC 50 values of 1.04 ± 0.28 and 1.48 ± 0.40 µ M in the A549 cell line and moderate cytotoxicity against the SKOV3 cell line, with IC 50 values of 11.77 ± 0.60 and 17.01 ± 1.22 µ M, respectively. Both compounds were found to be selective if compared with the non-malignant cells (Table 2, Entries 18 and 19).
Molecules 2022,27, 8024 16 of 22 = 7.3 Hz, CH 3 ), 54.4 (d, 2 J PC = 7.6 Hz, CH 3 ), 38.2 (CH 2 ), 21.6 (CH 3 ), 14.2 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.7. 19 F NMR (282 MHz, CDCl 3 ) δ− 137.6, − 138.0. FTIR (neat) νmax 3262 (N-H), 1738 (C=O), 1338 (O=S=O), 1249 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C20H25F2NO7PS [M + H]+492.1052, found 492.1060. Ethyl 3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido)-3-(perfluorophenyl) propanoate (7o) . The general procedure was followed, starting form imine 3o (457 mg, 1 mmol) to afford 501 mg (92%) of 7o as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 7.52 (d, 3 J HH = 8.3 Hz, 2H), 7.16 (d, 3 J HH = 8.3 Hz, 2H), 6.23 (d, 3 J PH = 10.5 Hz, 1H), 4.18–4.07 (m, 2H), 3.98 (m, 1H), 3.80 (d, 3 J PH = 10.9 Hz, 3H), 3.75 (d, 3 J PH = 10.9 Hz, 3H), 3.45 (m, 1H), 2.36 (s, 3H), 1.24 (t, 3 J HH = 7.1 Hz, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 170.1 (d, 3 J PC = 8.4 Hz, C quat ), 145.9 (m, C quat ), 144.1 (C quat ), 140.8 (m, C quat ), 137.7 (d, 4 J PC = 1.9 Hz, Cquat), 137.5 (m, Cquat), 129.2 (CH), 127.1 (CH), 111.2 (m, Cquat), 61.0 (CH2), 60.0 (d, 1JPC = 153.3 Hz, C quat ), 55.7 (d, 2 J PC = 7.2 Hz, CH 3 ), 54.9 (d, 2 J PC = 7.8 Hz, CH 3 ), 40.3 (d, 4 J FC = 5.3 Hz, CH 2 ), 21.4 (CH 3 ), 14.1 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.7. 19 F NMR (282 MHz, CDCl 3 ) δ− 135.0, − 154.3, − 162.8. FTIR (neat) νmax 3284 (N-H), 1741 (C=O), 1333 (O=S=O), 1254 (P=O), 1166 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C 20 H 22 F 5 NO 7 PS [M + H]+546.0769, found 546.0781. Ethyl 3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido)-3-(4-(trifluoromethyl) phenyl)propanoate (7p) . The general procedure was followed, starting form imine 3p (435 mg, 1 mmol) to afford 455 mg (87%) of 7p as a white solid. M.p. (CH 2 Cl 2 -hexanes). 114–115 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 ( 3 J HH = 8.8 Hz, 3 J FH = 2.3 Hz, 2H), 7.42 (d, 3 J HH = 8.4 Hz, 2H), 7.34 (d, 3 J HH = 8.4 Hz, 2H), 7.12 (d, 3 J HH = 8.2 Hz, 2H), 6.24 (d, 3 J PH = 10.1 Hz, 1H), 4.18 (qd, 3 J HH = 7.2 Hz, 3 J FH = 1.7 Hz, 2H), 3.68 (dd, 3 J PH = 23.8 Hz, 2 J HH = 16.2 Hz, 1H), 3.58 (d, 3 J PH = 10.8 Hz, 3H), 3.49 (d, 3 J PH = 10.8 Hz, 3H), 3.44 (dd, 3 J PH = 8.2 Hz, 2 J HH = 16.2 Hz, 1H), 2.39 (s, 3H), 1.28 (t, 3 J HH = 7.1 Hz, 3H). 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ 170.0 (d, 3 J PC = 6.8 Hz, C quat ), 143.7 (C quat ), 138.9 (C quat ), 138.6 (d, 2 J PC = 6.6 Hz, C quat ), 130.4 (dq, 2 J FC = 32.8 Hz, 5 J PC = 3.0 Hz, C quat ), 129.3 (CH), 128.8 (d, 3 J PC = 5.0 Hz, CH), 127.5 (CH), 124.7 (m, CH), 123.9 (q, 1 J FC = 272.4 Hz, C quat ), 62.0 (d, 1 J PC = 152.8 Hz, C quat ), 61.3 (CH 2 ), 54.8 (d, 2 J PC = 7.4 Hz, CH 3 ), 54.4 (d, 2 J PC = 7.6 Hz, CH 3 ), 38.1 (CH 2 ), 21.6 (CH 3 ), 14.3 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.9. 19 F NMR (282 MHz, CDCl 3 ) δ − 63.4. FTIR (neat) νmax 3261 (N-H), 1735 (C=O), 1327 (O=S=O), 1263 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C21H26F3NO7PS [M + H]+524.1114, found 524.1121. Ethyl 3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido)-3-(4-nitrophenyl)propanoate (7q) . The general procedure was followed, starting form imine 3q (412 mg, 1 mmol) to afford 425 mg (85%) of 7q as a white solid. M.p. (CH 2 Cl 2 -hexanes). 129–130 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.96 (d, 3 J HH = 9.0 Hz, 2H), 7.56 (dd, 3 J HH = 9.0 Hz, 3 J PH = 2.3 Hz, 2H), 7.50 (d, 3 J HH = 8.2 Hz, 2H), 7.17 (d, 3 J HH = 8.2 Hz, 2H), 6.30 (d, 3 J PH = 10.3 Hz, 1H), 4.16 (m, 2H), 3.62 (dd, 2 J HH = 16.5 Hz, 3 J PH = 6.2 Hz, 1H), 3.58 (d, 3 J PH = 10.8 Hz, 3H), 3.55 (d, 3 J PH = 10.7 Hz, 3H), 3.48 (dd, 2 J HH = 16.5 Hz, 3 J PH = 8.2 Hz, 1H), 2.41 (s, 3H), 1.27 (t, 3 J HH = 7.2 Hz, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 169.7 (d, 3 J PC = 7.6 Hz, C quat ), 147.4 (d, 5 J PC = 3.4 Hz, C quat ), 144.1 (C quat ), 142.5 (d, 2 J PC = 6.6 Hz, C quat ), 138.8 (d, 4 J PC = 1.4 Hz, C quat ), 129.4 (CH), 129.3 (d, 3 J PC = 5.0 Hz, CH), 127.5 (CH), 122.8 (d, 4 J PC = 2.6 Hz, CH), 62.3 (d, 1 J PC = 152.2 Hz, C quat ), 61.4 (CH 2 ), 54.9 (d, 2 J PC = 7.3 Hz, CH 3 ), 54.6 (d, 2 J PC = 7.5 Hz, CH 3 ), 38.3 (CH 2 ), 21.7 (CH 3 ), 14.2 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.2. FTIR (neat) νmax 3272 (N-H), 1743 (C=O), 1349 (O=S=O), 1244 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C20H26N2O9PS [M + H]+501.1091, found 501.1098. Ethyl 3-(5-chlorothiophen-2-yl)-3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido) propanoate (7r) . The general procedure was followed, starting form imine 3r (407 mg, 1 mmol) to afford 378 mg (76%) of 7r as a pale brown solid. M.p. (CH 2 Cl 2 -hexanes). 93–94 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.48 (d, 3 J HH = 8.3 Hz, 2H), 7.17 (d, 3 J HH = 8.3 Hz, 2H), 6.79 (dd, 3 J HH = 4.0 Hz, 4 J PH = 3.4 Hz, 1H), 6.61 (d, 3 J HH = 4.0 Hz, 1H), 6.23 (d, 3 J PH = 7.2 Hz, 1H), 4.20 (q, 3 J HH = 7.2 Hz, 2H), 3.68 (d, 3 J PH = 10.7 Hz, 3H), 3.66 (d, 3 J PH = 10.5 Hz, 3H), 3.61 (m, 1H), 3.21 (dd, 2 J HH = 15.5 Hz, 3 J PH = 7.1 Hz, 1H), 2.40 (s, 3H), 1.30 (t, 3 J HH = 7.2 Hz, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 169.5 (d, 3 J PC = 5.6 Hz, C quat ), 143.6 (C quat ),
Molecules 2022,27, 8024 17 of 22 138.7 (d, 4 J PC = 1.4 Hz, C quat ), 135.9 (d, 2 J PC = 7.7 Hz, C quat ), 132.3 (d, 5 J PC = 3.6 Hz, C quat ), 129.3 (CH), 128.6 (d, 3 J PC = 7.3 Hz, CH), 127.5 (CH), 125.3 (d, 4 J PC = 2.8 Hz, CH), 61.4 (CH 2 ), 60.0 (d, 1 J PC = 161.5 Hz, C quat ), 55.2 (d, 3 J PC = 7.3 Hz, CH 3 ), 54.5 (d, 3 J PC = 7.6 Hz, CH 3 ), 39.0 (CH 2 ), 21.7 (CH 3 ), 14.2 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 20.3. FTIR (neat) νmax 3270 (N-H), 1735 (C=O), 1341 (O=S=O), 1243 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C18H24ClNO7PS2[M + H]+496.0415, found 496.0444.. Ethyl 3-([1,1’-biphenyl]-4-yl)-3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido) propanoate (7s) . The general procedure was followed, starting form imine 3s (443 mg, 1 mmol) to afford 478 mg (90%) of 7s as a white solid. M.p. (CH 2 Cl 2 -hexanes). 112–113 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.56–7.46 (m, 4H), 7.45–7.40 (m, 5H), 7.39–7.33 (m, 2H), 7.13 (d, 3 J HH = 8.3 Hz, 2H), 6.21 (d, 3 J PH = 10.5 Hz, 1H), 4.20 (q, 3 J HH = 7.2 Hz, 2H), 3.69 (dd, 3 J PH = 23.4 Hz, 2 J HH = 16.4 Hz, 1H), 3.56 (d, 3 J PH = 10.7 Hz, 3H), 3.49 (m, 1H), 3.46 (d, 3 J PH = 10.6 Hz, 3H), 2.38 (s, 3H), 1.29 (t, 3 J HH = 7.2 Hz, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 170.3 (d, 3 J PC = 7.3 Hz, C quat ), 143.3 (C quat ), 141.0 (d, 5 J PC = 3.1 Hz, C quat ), 140.1 (d, 4 J PC = 1.4 Hz, C quat ), 139.2 (C quat ), 129.2 (CH), 129.0 (CH), 128.8 (d, 3 J PC = 5.1 Hz, CH), 128.3 (C quat ), 127.8 (CH), 127.7 (CH), 127.1 (CH), 126.5 (d, 4 J PC = 2.8 Hz, CH), 62.1 (d, 1 J PC = 153.9 Hz, C quat ), 61.1 (CH 2 ), 54.7 (d, 2 J PC = 7.4 Hz, CH 3 ), 54.2 (d, 2 J PC = 7.6 Hz, CH 3 ), 38.2 (CH 2 ), 21.7 (CH 3 ), 14.3 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 22.2. FTIR (neat) νmax 3308 (N-H), 1729 (C=O), 1332 (O=S=O), 1241 (P=O), 1161 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C26H31NO7PS [M + H]+532.1553, found 532.1555. Methyl 3-(dimethoxyphosphoryl)-3-(3-fluorophenyl)-3-((4-methylphenyl)sulfonamido) propanoate (12) . The general procedure was followed, starting form imine 3k (417 mg, 1 mmol) to afford 372 mg (81%) of 12 as a white solid. M.p. (CH 2 Cl 2 -hexanes). 115–116 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.48 (d, 3 J HH = 8.3 Hz, 2H), 7.20–7.09 (m, 4H), 7.00 (m, 1H), 6.91 (m, 1H), 6.21 (br d, 3 J HH = 7.8 Hz, 1H), 3.73 (s, 3H), 3.64 (dd, 3 J PH = 24.4 Hz, 2 J HH = 16.2 Hz, 1H), 3.56–3.50 (m, 6H), 3.46 (dd, 2 J HH = 16.2 Hz, 3 J PH = 7.6 Hz, 1H), 2.39 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 170.6 (d, 3 J PC = 6.8 Hz, C quat ), 162.3 (dd, 1 J FC = 246.0 Hz, 4 J PC = 3.0 Hz, C quat ), 143.7 (C quat ), 139.0 (C quat ), 137.1 (m, C quat ), 129.5 (dd, 3 J FC = 8.1 Hz, 4 J PC 2.8 Hz, CH), 129.3 (CH), 127.5 (CH), 123.7 (dd, 3 J PC = 5.1 Hz, 4 J FC = 3.0 Hz, CH), 116.1 (dd, 2 J FC = 24.1 Hz, 3 J PC = 4.8 Hz, CH), 115.5 (dd, 2 J FC = 21.1 Hz, 5 J PC = 2.9 Hz, CH), 62.0 (dd, 1 J PC = 153.7 Hz, 4 J FC = 1.4 Hz, C quat ), 54.9 (d, 2 J PC = 7.7 Hz, CH 3 ), 54.4 (d, 2 J PC = 7.6 Hz, CH 3 ), 52.3 (CH 3 ), 38.2 (CH 2 ), 21.6 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.7. 19 F NMR (282 MHz, CDCl 3 ) δ− 112.9. FTIR (neat) νmax 3281 (N-H), 1729 (C=O), 1330 (O=S=O), 1243 (P=O), 1159 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C 19 H 24 FNO 7 PS [M + H] + 460.0990, found 460.1004. Benzyl 3-(dimethoxyphosphoryl)-3-(3-fluorophenyl)-3-((4-methylphenyl)sulfonamido) propanoate (13a) . The general procedure was followed, starting form imine 3k (417 mg, 1 mmol) to afford 449 mg (84%) of 13a as a white solid. M.p. (CH 2 Cl 2 -hexanes). 78–79 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 (d, 3 J HH = 8.2 Hz, 2H), 7.41–7.31 (m, 5H), 7.17–7.08 (m, 4H), 7.03–6.98 (m, 1H), 6.94–6.87 (m, 1H), 6.24 (d, 3 J PH = 10.2 Hz, 1H), 5.19 (d, 2 J HH = 12.3 Hz. 1H), 5.13 (d, 2 J HH = 12.3 Hz, 1H), 3.73–3.63 (m, 1H), 3.57–3.34 (m, 7H), 2.38 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 169.9 (d, 3 J PC = 6.9 Hz, C quat ), 162.3 (dd, 1 J FC = 246.0 Hz, 4 J PC = 3.0 Hz, C quat ), 143.6 (C quat ), 139.0 (d, 4 J PC = 1.3 Hz, C quat ), 137.1 (C quat ), 135.6 (C quat ), 129.4 (dd, 3 J FC = 8.2 Hz, 4 J PC = 2.5 Hz, CH), 129.3 (CH), 128.7 (CH), 128.7 (CH), 128.5 (CH), 127.5 (CH), 123.7 (dd, 3 J PC = 5.0, 4 J FC = 2.9 Hz, CH), 116.0 (dd, 2 J FC = 24.0 Hz, 3 J PC = 4.7 Hz, CH), 115.4 (dd, 2 J FC = 21.1 Hz, 5 J PC = 3.1 Hz, CH), 67.0 (CH 2 ), 61.9 (dd, 1 J PC = 153.6 Hz, 4 J FC = 1.6 Hz, C quat ), 54.8 (d, 2 J PC = 7.5 Hz, CH 3 ), 54.3 (d, 2 J PC = 7.7 Hz, CH 3 ), 38.2 (CH 2 ), 21.6 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.6. 19 F NMR (282 MHz, CDCl 3 ) δ− 112.9. FTIR (neat) νmax 3281 (N-H), 1735 (C=O), 1331 (O=S=O), 1247 (P=O), 1154 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C25H28FNO7PS [M + H]+536.1303, found 536.1322. Benzyl 3-(dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido)-3-phenylpropanoate (13b) . The general procedure was followed, starting form imine 3a (367 mg, 1 mmol) to afford 476 mg (92%) of 13b as a white solid. M.p. (CH 2 Cl 2 -hexanes). 84–85 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 7.46 (d, J= 8.3 Hz, 2H), 7.37–7.29 (m, 7H), 7.20–7.15 (m, 1H), 7.12–7.06 (m, 4H),
Molecules 2022,27, 8024 18 of 22 6.22 (d, 3 J PH = 11.1 Hz, 1H), 5.14 (d, 2 J HH = 12.2 Hz, 1H), 5.09 (d, 2 J HH = 12.2 Hz, 1H), 3.65 (dd, 3JPH = 22.3 Hz, 2JHH = 16.6 Hz, 1H), 3.49 (dd, 2JHH = 16.6 Hz, 3JPH = 7.5 Hz, 1H), 3.37 (d, 3 J PH = 10.7 Hz, 3H), 3.31 (d, 3 J PH = 10.7 Hz, 3H), 2.34 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 169.8 (d, 3 J PC = 8.1 Hz, C quat ), 143.1 (C quat ), 139.0 (d, 4 J PC = 1.4 Hz, C quat ), 135.5 (C quat ), 134.1 (d, 2 J PC = 7.3 Hz, C quat ), 129.0 (CH), 128.4 (CH), 128.4 (CH), 128.2 (CH), 128.2 (CH), 128.0 (d, 3 J PC = 5.0 Hz, CH), 127.8 (d, 4 J PC = 2.6 Hz, CH), 127.4 (CH), 66.5 (CH 2 ), 61.9 (d, 1 J PC = 154.1 Hz, C quat ), 54.4 (d, 2 J PC = 7.4 Hz, CH 3 ), 53.8 (d, 2 J PC = 7.7 Hz, CH 3 ), 37.7 (CH 2 ), 21.4 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 22.0. FTIR (neat) νmax 3322 (N-H), 1739 (C=O), 1337 (O=S=O), 1248 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd for C25H29NO7PS [M + H]+518.1397, found 518.1372. Procedure for the Obtention of 3-(Dimethoxyphosphoryl)-3-((4-methylphenyl)sulfonamido)- 3-phenylpropanoic acid 14 A mixture of aminophosphonate 13b (518 mg, 1 mmol) and Pd-C 10% (106 mg, 0.1 mmol) in MeOH (50 mL) were stirred for 12 h under H 2 atmosphere (75 psi). The mixture was then filtered on celite and concentrated under reduced pressure to yield product 14 as a white solid (402 mg, 94%), after crystallization in MeOH. M.p. (MeOH). 145–146 ◦ C. 1 H NMR (400 MHz, CDCl 3 ) δ 9.91 (br s, 1H), 7.48 (d, 3 J HH = 8.2 Hz, 2H), 7.33 (d, 3 J HH = 7.6 Hz, 2H), 7.20 (m, 1H), 7.17–7.09 (m, 4H), 6.60 (d, 3 J PH = 10.6 Hz, 1H), 3.67 (dd, 3 J PH = 23.9 Hz, 2 J HH = 16.0 Hz, 1H), 3.52 (d, 3 J PH = 10.8 Hz, 3H), 3.49 (m, 1H), 3.47 (d, 3 J PH = 10.6 Hz, 3H), 2.38 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl 3 ) δ 173.5 (d, 3 J PC = 7.7 Hz, C quat ), 143.3 (C quat ), 139.2 (d, 4 J PC = 1.4 Hz, C quat ), 134.1 (d, 2 J PC = 7.6 Hz, C quat ), 129.2 (CH), 128.4 (d, 5 J PC = 3.0 Hz, CH), 128.3 (d, 3 J PC = 5.1 Hz, CH), 128.0 (d, 4 J PC = 2.6 Hz, CH), 127.6 (CH), 62.1 (d, 1 J PC = 155.6 Hz, C quat ), 55.1 (d, 2 J PC = 7.3 Hz, CH 3 ), 54.5 (d, 2 J PC = 7.9 Hz, CH 3 ), 38.0 (CH 2 ), 21.6 (CH 3 ). 31 P NMR (120 MHz, CDCl 3 ): δ 21.8. FTIR (neat) νmax 3500–2500 (O-H st), 3271 (N-H st), 1714 (C=O st), 1337 (O=S=O st as), 1235 (P=O st), 1163 (O=S=O st sim) cm –1 . HRMS (ESI-TOF) m/z: calcd for C 18 H 23 NO 7 PS [M + H] + 428.0927, found 428.0901. Procedure for the Obtention of Dimethyl (1-((4-methylphenyl)sulfonamido)-1-phenylethyl) phosphonate 18 A solution of 3a (367 mg, 1 mmol) in dry CH 3 CN (3 mL) was stirred at room temperature under N 2 atmosphere. To this mixture, Me 2 Zn (1.7 mL, 1.2 M in toluene, 2 mmol) was added and the mixture was stirred for 2h at room temperature. The reaction was quenched by a slow addition of a saturated aqueous solution of NH 4 Cl (1 mL) and dried over anhydrous MgSO 4 . The solid was removed by filtration and washed with AcOEt, and the filtrate was concentrated at reduced pressure to yield the crude product, which was purified by column chromatography in silica gel (AcOEt/Hexanes) to give 326 mg (85%) of 18 as a white solid [ 60 ]. M.p. (Et 2 O/pentane). 163–164 ◦ C. Lit. 161–162 (Et 2 O). 1 H NMR (400 MHz, CDCl 3 ) δ 7.53 (d, 3 J HH = 8.3 Hz, 2H), 7.40 (m, 2H), 7.22–7.18 (m, 3H), 7.11 (d, 3 J HH = 8.2 Hz, 2H), 5.78 (d, 3 J PH = 8.1 Hz, 1H), 3.70 (d, 3 J PH = 10.4 Hz, 3H), 3.35 (d, 3 J PH = 10.4 Hz, 3H), 2.36 (s, 3H), 1.97 (d, 3 J PH = 16.8 Hz, 3H). 13 C { 1 H} NMR (75 MHz, CDCl 3 ) δ 142.3 (C quat ), 141.8 (d, 4 J PC = 1.7 Hz, C quat ), 133.4 (C quat ), 128.6 (CH), 128.4 (d, 3 J PC = 6.0 Hz, CH), 128.1 (d, 4 J PC = 2.2 Hz, CH), 128.0 (d, 5 J PC = 2.9 Hz, CH), 126.7 (CH), 61.1 (d, 1 J PC = 152.2 Hz, C quat ), 54.5 (d, 2 J PC = 7.1 Hz, CH 3 ), 53.9 (d, 2 J PC = 7.0 Hz, CH 3 ), 21.3 (CH 3 ), 20.4 (d, 2 J PC = 5.2 Hz CH 3 ). 31 P NMR (120 MHz, CDCl 3 ) δ 26.1. FTIR (neat) νmax 3315 (N-H), 1327 (O=S=O), 1242 (P=O), 1166 (O=S=O). HRMS (ESI-TOF) m/z: calcd. for C 17 H 22 NO 5 PS [M + Na]+406.0848, found 406.0856. 3.2. Biology 3.2.1. Materials Reagents and solvents were used as purchased without further purification. All stock solutions of the investigated compounds were prepared by dissolving the powered materials in appropriate amounts of dimethylsulfoxide (DMSO). The final concentration
Molecules 2022,27, 8024 19 of 22 of DMSO never exceeded 5% (v/v) in the reactions. The stock solution was stored at 5 ◦ C until it was used. 3.2.2. Cell Culture Human epithelial lung carcinoma cells (A549) (ATCC ® CCL-185 ™ , ATCC, Manassas, VA, USA) were grown in Kaighn’s Modification of Ham’s F-12 Medium (ATCC ® 30-2004 ™ , ATCC, Manassas, VA, USA) and lung fibroblast cells (MRC5) (ATCC ® CCL-171 ™ , ATCC, Manassas, VA, USA) were grown in Eagle’s Minimum Essential Medium (EMEM, ATCC ® 30-2003 ™ , ATCC, Manassas, VA, USA). Epithelial ovary adenocarcinoma cells (SKOV3) (ATCC ® HTB-77 ™ , ATCC, Manassas, VA, USA) were grown in McCoy’s 5A medium (ATCC ® 30-2007 ™ , ATCC, Manassas, VA, USA). All of them were supplemented with 10% of fetal bovine serum (FBS) (Sigma-Aldrich, Madrid, Spain) and with 1% of NORMOCIN solution (Thermo Fisher, Waltham, MA, USA). Cells were incubated at 37 ◦ C and 5% CO 2 atmosphere, and were split every 3–4 days to maintain monolayer coverage. For the cytotoxicity experiments, the A549 and SKOV3 cells were seeded in 96-well plates at a density of 2.5–3 × 10 3 cells per well and incubated overnight to achieve 70% of confluence at the time of exposition to the cytotoxic compound. 3.2.3. Cytotoxicity Assays Cells were exposed to different concentrations of the cytotoxic compounds and were incubated for 48 h. Then, 10 µ L of cell counting kit-8 was added into each well for an additional two hours’ incubation at 37 ◦ C. The absorbance of each well was determined by an Automatic Elisa Reader System (Thermo Scientific Multiskan FC Automatic Elisa Reader System, Thermo Scientific, Shanghai, China) at 450 nm wavelength. 4. Conclusions In conclusion, we report an efficient methodology for the preparation of phosphonate analogs of aspartic acid, holding a variety of substituents at their α -aromatic ring. α -Ketiminophosphonates are generated by the oxidation of their parent tertiary α -aminophosphonates and a subsequent aza-Reformatsky reaction with alkyl iodoacetate derivatives. Moreover, this methodology has been successfully extended to aldimines and activated ketimines, affording the Reformatsky products in high yields. This strategy allows the possibility of assorted structural diversity in the resultant scaffold depending on the starting imine or alkyl iodoacetate. Moreover, the phosphorated analogues of aspartic acid 6 showed in vitro cytotoxicity inhibiting the growth of human tumor cell lines SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell), and a high selectivity toward the MRC5 non-malignant lung fibroblasts. The most active substrates were proved to be ethyl ester derivatives. Although p-tolyl derivatives showed the best result against A549, the introduction of a trifluoromethylphenyl moiety in the para position exhibited the most remarkable IC 50 value against the SKOV3 cell line. Moreover, the majority of the compounds were selective toward the non-malignant cells. The best IC 50 values obtained are 9.80 µ M in the SKOV3 cell line for α -aminophosphonate 7p , with a p-trifluorophenyl substituent, and 0.34 µ M in the A549 cell line for substrate 7k , holding a p-methylphenyl moiety. Most of the compounds presented in this study show low micromolar activity and a high selectivity toward the non-malignant cells. It has also been proved that the absolute configuration of the tetrasubstituted stereocenter does not have any influence on the biological activity of the phosphorated aspartic acid derivatives, since both enantiomers of substrate 7k showed similar IC50 values. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27228024/s1, 1 H, 13 C, 31 P and 19 F copies of compounds 5,6,7,12,13,14 and 18. 2D-NMR of compound 7a. Author Contributions: Conceptualization, X.d.C., A.M., F.P. and J.V.; methodology: evaluation of the in vitro antiproliferative activity, X.d.C.; methodology: organic synthesis, A.M. and A.L.-F.;
Molecules 2022,27, 8024 20 of 22 software, X.d.C. and A.M.; validation, J.V.; formal analysis, X.d.C. and A.M.; investigation, X.d.C., A.M. and A.L.-F.; resources, F.P. and J.V.; data curation, X.d.C., A.M. and A.L.-F.; writing—original draft preparation, J.V.; writing—review and editing, X.d.C., A.M., A.L.-F., F.P. and J.V.; visualization, F.P. and J.V.; supervision, J.V.; project administration, F.P. and J.V.; funding acquisition, F.P. and J.V. All authors have read and agreed to the published version of the manuscript. Funding: Financial support by Ministerio de Economía, Industria y Competividad (PID2021-122558OBI00) and Gobierno Vasco (GV-IT1701-22) is gratefully acknowledged. X.d.C. and A.L.-F. thank the Basque Country Government for a predoctoral grant. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the supplementary materials file or on request from the corresponding author ( 1 H, 13 C, 19 F and 31 P-NMR and HRMS spectra and cytotoxicity essays). Acknowledgments: The authors thank for the technical and human support provided by SGIker (UPV/EHU/ERDF, EU). Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are not available. References 1. Légaré, J. Population Aging: Economic and Social Consequences. In International Encyclopedia of the Social & Behavioral Sciences, 2nd ed.; Elsevier: Oxford, UK, 2015; p. 540. 2. Foreman, K.J.; Marquez, N.; Dolgert, A.; Fukutaki, K.; Fullman, N.; McGaughey, M.; Pletcher, M.A.; Smith, A.E.; Tang, K.; Yuan, C.-W.; et al. Forecasting life expectancy, years of life lost, and all-cause and cause-specific mortality for 250 causes of death: Reference and alternative scenarios for 2016–40 for 195 countries and territories. Lancet 2018,392, 2052. [CrossRef] 3. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015 ,136, E359. [CrossRef] [PubMed] 4. Bidram, E.; Esmaeili, Y.; Ranji-Burachaloo, H.; Al-Zaubai, N.; Zarrabi, A.; Stewart, A. A concise review on cancer treatment methods and delivery systems. J. Drug Deliv. Sci. Technol. 2019,54, 101350. [CrossRef] 5. Dickens, E.; Ahmed, S. Principles of cancer treatment by chemotherapy. Surgery 2018,36, 134–138. 6. Cree, I.A.; Charlton, P. Molecular chess? Hallmarks of anti-cancer drug resistance. BMC Cancer 2017 ,17, 10. [CrossRef] [PubMed] 7. Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Snyder, N.; Sarkar, S. Drug resistance in cancer: An overview. Cancers 2014,6, 1769. [CrossRef] 8. Horsman, G.P.; Zechel, D.L. Phosponate biochemistry. Chem. Rev. 2017,117, 5704–5783. [CrossRef] 9. Emadi, A.; Jones, R.J.; Brodsky, R.A. Cyclophosphamide and cancer: Golden anniversary. Nat. Rev. Clin. Oncol. 2009 ,6, 638. [CrossRef] 10. Msaouel, P.; Galanis, E.; Koutsilieris, M. Somatostatin and somatostatin receptors: Implications for neoplastic growth and cancer biology. Expert Op. Investig. Drugs 2009,18, 1297. [CrossRef] 11. Caraglia, M.; D’Alessandro, A.M.; Marra, M.; Giuberti, G.; Vitale, G.; Viscomi, C.; Colao, A.M.; del Prete, S.; Tagliaferri, P.; Tassone, P.; et al. The farnesyl transferase inhibitor R115777 (Zarnestra ® ) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa®) and Pamidronate. Oncogene 2004,23, 6900. [CrossRef] 12. Kukhar, V.P.; Hudson, H.R. (Eds.) Aminophosphonic and Aminophosphinic Acids. In Chemistry and Biological Activity; Wiley: Chichester, UK, 2000. 13. Moonen, K.; Laureyn, I.; Stevens, C.V. Synthetic Methods for Azaheterocyclic Phosphonates and Their Biological Activity. Chem. Rev. 2004,104, 2177. [CrossRef] [PubMed] 14. Berlicki, L.; Kafarski, P. Computer-Aided Analysis and Design of Phosphonic and Phosphinic Enzyme Inhibitors as Potential Drugs and Agrochemicals. Curr. Org. Chem. 2005,9, 1829. [CrossRef] 15. Kafarski, P.; Lejczak, B. Aminophosphonic Acids of Potential Medical Importance. Curr. Med. Chem. Anti Cancer Ag. 2001 ,1, 301. [CrossRef] 16. Reddy, M.V.N.; Balakrishna, A.; Kumar, M.A.; Reddy, G.C.S.; Sankar, A.U.R.; Reddy, C.S.; Krishna, T.M. One-Step Synthesis and Bioassay of N-Phosphoramidophosphonates. Chem. Pharm. Bull. 2009,57, 1391. [CrossRef] 17. Che, J.-Y.; Xu, X.-Y.; Tang, Z.-L.; Gu, Y.-C.; Shi, D.-G. Synthesis and herbicidal activity evaluation of novel α -amino phosphonate derivatives containing a uracil moiety. Bioorg. Med. Chem. Lett. 2016,26, 1310. [CrossRef] [PubMed] 18. Reddy, M.V.N.; Kumar, B.S.; Balakrishna, A.; Reddy, C.S.; Nayak, S.K.; Reddy, C.D. One-pot synthesis of novel α -amino phosphonates using tetramethylguanidine as a catalyst. Arkivoc 2007,15, 246. [CrossRef]
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