PMDTA-catalyzed multicomponent synthesis and biological activity of 2-amino-4 H -chromenes containing a phosphonate or phosphine oxide moiety †

A new approach for the preparation of (2-amino-3-cyano-4 H -chromen-4-yl)phosphonate derivatives is described. The multicomponent reaction of salicylaldehydes, malononitrile and dialkyl phosphites catalyzed by pentamethyldiethylenetriamine (PMDTA) provided the bicyclic derivatives in high yields. The method developed did not require chromatographic separation, since the products could be recovered from the reaction mixture by simple ﬁ ltration. Our approach made also possible condensation with secondary phosphine oxides, and this reaction has not been previously reported in the literature. The crystal structures of ﬁ ve derivatives were studied by single-crystal XRD analysis. The in vitro cytotoxicity on di ﬀ erent cell lines and the antibacterial activity of the (2-amino-4 H -chromen-4-yl)phosphonates synthesized were also explored. According to the IC 50 values determined, several derivatives showed moderate or promising activity against mouse ﬁ broblast (NIH/3T3) and human promyelocytic leukemia (HL-60) cells. Furthermore, three (2-amino-3-cyano-4 H -chromen-4-yl)phosphine oxides were active against selected Gram-positive bacteria.


Introduction
Phosphorus-substituted heterocycles represent an important group within organophosphorus compounds.][3] In the last few decades, (2-amino-4H-chromen-4-yl)phosphonates have received significant attention.These compounds are analogues of 2-amino-4H-chromenes, which have valuable applications as pharmaceutical agents, 4 and are widely employed as cosmetics, pigments and potential biodegradable agrochemicals. 5A few (2-amino-4H-chromen-4-yl) phosphonates showed a moderate antioxidant effect 6 and a modest anticancer activity against human lung adenocarcinoma (A549) and human epidermoid cancer (KB) cell lines. 7Therefore, the introduction of a phosphonate moiety on the 2-amino-4H-chromene ring may also have a synergistic effect on the various biological properties of this scaffold.Consequently, the development of green and efficient methods for the synthesis of (2-amino-4H-chromen-4-yl)phosphonates is of great importance.
Multicomponent reactions may represent an efficient method for the one-pot synthesis of complex ring systems. 8ue to their simplicity, high atom efficiency and the ability to provide easy access to large compound libraries, these transformations have attracted significant attention in the field of organic chemistry. 9(2-Amino-4H-chromen-4-yl)phosphonates may be prepared by the three-component reaction of salicylaldehyde derivatives, CH-acidic nitriles (e.g.malononitrile or ethyl cyanoacetate) and dialkyl-or trialkyl phosphites. 10Most procedures utilizing the above synthesis route were carried out in the presence of both a catalyst and a solvent.Diethylamine, 11 dibutylamine, 7 triethylamine, 12 dimethylaminopyridine, 13 imidazole, 14 ethylenediamine diacetate, 15 lithium hydroxide, 16 potassium phosphate, 17 magnesium oxide 18 or indium chloride 19 were tried out in ethanol, and iron oxide, 20 iodine 21 or β-cyclodextrin 22 in water.Water may be considered as a green solvent; however, it requires an additional extraction step during the reaction workup.In a few cases, special solvents, such as PEG, 23 ionic liquids 24,25 or a mixture of urea and choline chloride, 26 were used, which also served as a catalyst.Only four solvent-free variations can be found in the literature; [27][28][29][30] however, in these procedures, special catalysts were used, or a simple catalyst was required in a large excess.The condensation of salicylaldehydes, malononitrile and trialkyl phosphites was carried out in the presence of a silica-bonded 2-HEAA-3 catalyst at room temperature 27 or applying iodine at 50 °C. 28In other cases, dialkyl phosphites were used as phosphorus reagents, and the reactions were carried out applying ZnO nanorods at 100 °C, 29 or in the presence of a large excess (3.5 equiv.) of tetramethylguanidine catalyst at 25 °C. 30lthough many variations have been reported on the multicomponent synthesis of (2-amino-4H-chromen-4-yl)phosphonates, a suitable method using a simple basic catalyst under solvent-free conditions is still required.Furthermore, an extensive literature survey has revealed that there are no reports at all on the preparation of (2-amino-4H-chromen-4-yl)phosphine oxides.
Based on the above considerations, in this paper, we report on a fast, cheap and efficient PMDTA-catalyzed solvent-free process for the synthesis of novel (2-amino-3-cyano-4Hchromen-4-yl)phosphonates by the domino Knoevenagelphospha-Michael reaction of salicylaldehydes, malononitrile and dialkyl phosphites.Our approach made also possible condensation with secondary phosphine oxides, and this reaction has not been previously reported in the literature.The biological activity of the synthesized compounds was investigated in terms of antibacterial activity and in vitro cytotoxicity assays.

Synthesis of (2-amino-3-cyano-4H-chromen-4-yl)phosphonates
To optimize the three-component reaction of salicylaldehyde, malononitrile and diethyl phosphite, at first, the effect of various basic catalysts was studied (Table 1).The reactions were monitored by HPLC, and the reaction mixtures were analyzed by HPLC-MS.The condensations were carried out in a molar ratio of 1 : 1 : 1 in the presence of 5 mol% of an organic base at 60 °C for 30 min in an oil bath.At first, organic bases containing one nitrogen atom were tested as catalysts (Table 1, entries 1-3).Using dipropylamine (DPA) in the absence of any solvent, the conversion was complete, and the reaction mixture contained 65% of 2-(2-hydroxybenzylidene)malononitrile (1) ([M + H] + = 171.1)as an intermediate formed by the Knoevenagel condensation of salicylaldehyde with malononitrile and 35% of diethyl (2-amino-3-cyano-4H-chromen-4-yl) phosphonate (2a) (Table 1, entry 1).Performing the three-component reaction in the presence of triethylamine (TEA) or dipropylethylamine (DIPEA), the ratio of intermediate 1 and product 2a was 62 : 38 or 58 : 42, respectively (Table 1, entries 2 and 3).After that, the condensation was investigated using organic bases containing two nitrogen atoms, such as dimethylaminopridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7ene (DBU), diazobicyclooctane (DABCO) or tetramethylethylenediamine (TMEDA) (Table 1, entries 4-7).In these reactions, the desired diethyl (2-amino-3-cyano-4H-chromen-4-yl) phosphonate (2a) was formed in 27%, 41%, 53% and 58% yield, respectively.Finally, the domino Knoevenagel-phospha-Michael reaction was carried out by the addition of pentamethyldiethylenetriamine (PMDTA) as a catalyst containing three nitrogen atoms, and the proportion of product 2a was increased to 86% (Table 1, entry 8).As expected, the conden- sation was significantly affected by the number of basic nitrogen atoms in the catalyst, which provided more active sites, and a few steric effects were also observed.Among the catalysts tested, PMDTA was found to be the most effective; therefore, further experiments were performed in the presence of this catalyst.To study the effect of solvent, a reaction was also performed in ethanol, which is a common solvent used in the synthesis of similar derivatives, and in acetonitrile (Table 1, entries 9 and 10).In both reactions, the proportion of the expected chromenylphosphonate (2a) decreased dramatically, and only 35% or 30% of product 2a was present in the mixtures.
The presence of the solvent in the reaction carried out at 60 °C for 30 min reduced the reaction rate, thus, the solventfree reaction allowed a higher proportion of product 2a under the same conditions (Table 1, entries 8 vs. 9 and 10).Although in a few literature examples, salicylaldehyde and malononitrile were reacted with triethyl phosphite (TEP) instead of DEP as the P-reagent, according to our experience only 2-(2-hydroxybenzylidene)malononitrile (1) was formed, and no product 2a in the reaction mixture was obtained at 60 °C for 30 min in the absence of any solvent (Table 1, entry 11).Therefore, under the conditions applied, the reactivities of DEP and TEP were significantly different.When the condensation was performed in water, 73% of chromenylphosphonate (2a) was present in the reaction mixture (Table 1, entry 12).This may be explained by the base-catalyzed hydrolysis of TEP to DEP in the presence of water, which then reacts in the condensationa similar observation was previously described by our group in the case of Kabachnik-Fields reactions. 31he model reaction was further optimized with respect to the reaction time and temperature, as well as the catalyst amount; furthermore, the condensation was extended to various dialkyl phosphites and ethyl phenyl-H-phosphinate (Table 2).
First, the reaction time was gradually increased from 30 min to 60 min, and then, a higher temperature of 80 °C was also tried out; however, the ratio of product 2a did not change significantly (Table 2, entries 1-4).By increasing the amount of catalyst to 10 mol% at 60 °C, a product ratio of 91% was reached after 15 min, which increased to 100% after 30 min (Table 2, entries 4 and 5).Diethyl (2-amino-3-cyano-4Hchromen-4-yl)phosphonate (2a) was isolated in a yield of 92% (Table 2, entry 6).When carrying out the domino Knoevenagel-phospha-Michael reaction with dimethyl phosphite, a reaction time of 15 min was enough for a complete conversion, and the corresponding chromenylphosphonate (2b) was obtained in a yield of 90% (Table 2, entry 7).Applying dibutyl-or diisopropyl phosphite as the P-reagent, a reaction temperature of 80 °C and a reaction time of 30 min were necessary to efficiently obtain products 2c and 2d, respectively (Table 2, entries 8-11).The condensation with dibenzyl-and diphenyl phosphite was similar to the reaction of diethyl phosphite, and at 60 °C for 30 min the proportions of the corres- ponding (2-amino-3-cyano-4H-chromen-4-yl)phosphonates (2e and 2f ) were 97% and 98%, respectively.Finally, the threecomponent reaction was performed using ethyl phenyl H-phosphinate at 60 °C for 15 min, and the desired chromenylphosphinate (2g) was obtained in a ratio of 97%, and it was isolated in a yield of 86% (Table 2, entry 14).Due to the second chiral center on the phosphorus atom, compound 2g was obtained as a mixture of two diastereomers in a ratio of 42 : 58 based on the 31 P NMR spectrum.
In the next part of our work, the three-component reaction of substituted salicylaldehydes, malononitrile and dialkyl phosphites was studied under the optimal conditions determined for each phosphite (Scheme 1).Using diethyl phosphite, the condensations were complete at 60 °C for 30 min, similar to the reaction of salicylaldehyde.Starting from 5-fluoro-or 2-chlorosalicylaldehyde, the desired products (3a or 4a) were isolated in yields of 91% and 90%, respectively.3-Bromosalicylaldehyde was found to be slightly less reactive at 60 °C for 30 min than salicylaldehydes bearing 5-F or 2-Cl substituents.Using 3-ethoxysalicylaldehyde, an excellent yield (96%) was obtained.According to our experiences, 3-hydroxysubstituted salicylaldehyde was less reactive as compared to other substituted salicylaldehydes, and product 7a was isolated in a yield of 82%.After that, 5-fluoro-and 2-chloro-salicylaldehyde was reacted with malononitrile and dibutyl phosphite at 80 °C for 30 min, and the corresponding dibutyl chromenylphosphonates (3c and 4c) were obtained in high yields (88% and 90%, respectively).Finally, the condensations were performed with dibenzyl phosphite at 60 °C for 30 min.As in the previous examples, starting from salicylaldehyde or 5-fluoro-, 2-chloro-or 3-ethoxysalicylaldehyde, better results were obtained (81-85% yield), while the derivative containing a bromine substituent at position eight (6e) was obtained in a yield of 70%.The method developed could be effectively applied for various substituted salicylaldehydes.
In contrast to the previous reports dealing with a similar topic, the multicomponent approach developed is a fast, cheap and simple solvent-free methodology for the synthesis of (2-amino-3-cyano-4H-chromen-4-yl)phosphonates.Besides a comprehensive optimization, we have provided exact product compositions, including the amount of the "Knoevenagel product" (1).

Paper
Organic & Biomolecular Chemistry oxides.In these experiments, a small amount of acetonitrile as a solvent was used to overcome the heterogeneity.First, the condensation of salicylaldehyde, malononitrile and diphenylphosphine oxide was carried out in the presence of 5 mol% of PMDTA (Table 3).Stirring the reaction mixture at room temperature for 60 min, the conversion was 80%, and the mixture comprised 17% of the Knoevenagel intermediate ( 1) and 83% of the desired chromenylphosphine oxide (8a) (Table 3, entry 1).On increasing the reaction time to 180 min, the condensation was almost complete; however, the ratio of 1 and 8a was 11 : 89, respectively (Table 3, entry 1).Performing the reaction at 60 °C for 10 min, a complete conversion was achieved, and the proportion of the diphenyl (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxide (8a) was 92%.By increasing the reaction time to 15 min, only product 8a was formed.
Next, the three-component reaction of various salicylaldehydes, malononitrile and secondary phosphine oxides was studied under the optimized conditions (5 mol% PMTDA, 60 °C, 15 min) (Scheme 3).It was found that all the target derivatives (8a-12d) could be efficiently prepared using the developed procedure.It should be noted that the purification did not require chromatographic separation, and the products could be recovered from the reaction mixture by simple fil-tration.Altogether 20 new (2-amino-3-cyano-4H-chromen-4-yl) phosphine oxides (8a-12d) were synthesized in high yields (82-97%).These derivatives can be considered as a new family among O-heterocyclic organophosphorus compounds.
The PMDTA-catalyzed multicomponent method developed is a fast, efficient and convenient methodology for the synthesis of (2-amino-3-cyano-4H-chromen-4-yl)phosphonates and a novel method for the preparation of (2-amino-3-cyano-4Hchromen-4-yl)phosphine oxides, which utilizes a cheap base catalyst, and in most cases, solvent-free conditions and short reaction time.The scope of the reaction was extended to 38 derivatives, of which 33 are new compounds.

X-ray diffraction study
In addition to the spectroscopic analysis, we have determined the crystal structures of compounds 2c, 6e and 8a-c.Singlecrystal XRD analysis confirmed the molecular structures (Fig. 1 and Fig. S1 †) and revealed the formation of intermolecular N-H⋯OvP and N-H⋯N hydrogen bonding leading to hydrogenbonded chains or layers (Table S2 †).Although the studied compounds possessed a similar chromenylphosphonate scaffold, their crystal structures showed a range of H-bonding interactions.The intermolecular N-H⋯OvP hydrogen Scheme 2 Proposed mechanism for the PMDTA-catalyzed synthesis of (2-amino-3-cyano-4H-chromen-4-yl)phosphonates.bonding between the amino group and the phosphonate oxygen atom was found to be a robust structural motif, since it was found in all structures under study.It was also observed that in structures 2c and 14a-c, the amino group as a hydrogen bond donor is involved in the formation of two interactions, while in 6e, the amino group is involved only in one interaction.
In compound 2c, the amino group is involved in the formation of the centrosymmetric N-H⋯N hydrogen bond with the cyano group and in the formation of the N-H⋯OvP interaction with two adjacent molecules, thus enabling the formation of hydrogen-bonded layers (Fig. 2).On the other hand, in compound 6e, the amino group is involved only in the formation of centrosymmetric N-H⋯OvP interactions, and the hydrogen-bonded chain is formed through the centrosymmetric C-H⋯N interaction connecting the chromenyl scaffold with the cyano group of the adjacent molecule (Fig. 2).
In the structures of 8a•CH 3 CN, 8b and 8c, three different packing motifs were observed.In 8a•CH 3 CN and 8c, the amino group is involved in a centrosymmetric N-H⋯N interaction with the cyano group of the adjacent molecule, and the hydrogen-bonded layer is formed through the N-H⋯OvP interaction between the amino group and the phosphonate oxygen atom of the adjacent molecule (Fig. 3).Although in 8a and 8c, similar hydrogen bonds are present, and layer structures are formed, the supramolecular structure of 8a possesses a honeycomb-like structure, which is not the case in 8c.In 8b, a hydrogen-bonded chain is formed through two centrosymmetric hydrogen bonds both formed by the amino group, namely, a   View Article Online centrosymmetric N-H⋯N interaction with the cyano group and a centrosymmetric N-H⋯OvP interaction with the phosphonate oxygen atom of two adjacent molecules (Fig. 3).

Biological activity studies
The in vitro cytotoxicity and the antibacterial activity of the synthesized (2-amino-4H-chromen-4-yl)phosphonates and (2-amino-4H-chromen-4-yl)phosphine oxides were also investigated.The cytotoxicity evaluations were performed on three different cell lines, such as human lung adenocarcinoma (A549), mouse fibroblasts (NIH/3T3) as a healthy cell line and human promyelocytic leukemia (HL-60), using the fluorescent resazurin assay as described previously. 32Positive controls were doxorubicin for A549 and NIH/3T3 (IC 50 = 0.31 ± 0.24 µM and 5.65 ± 0.81 µM, respectively) and bortezomib for HL60 (IC 50 = 7.42 ± 2.60 nM).The antibacterial activity of the compounds was tested on green fluorescent protein (GFP) producing Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) bacterial cells.The GFP producing bacteria are effective tools for screening the antibacterial activity, since the GFP signal measured by fluorimetry is proportional to the number of bacterial cells.Active compounds kill bacterial cells, which results in a decrease in the GFP fluorescence signal; therefore it is suitable for evaluating the antimicrobial effects of different agents.Positive controls were doxycycline and gentamicin for Bacillus subtilis (IC 50 = 0.04 ± 0.01 µM and 0.49 ± 0.14 µM) and Escherichia coli (IC 50 = 0.10 ± 0.02 µM and 4.23 ± 0.99 µM) bacterial cells.The IC 50 values (50% inhibiting concentration) determined are shown in Table 4.

Conclusions
In summary, a facile and efficient PMDTA-catalyzed synthetic method was developed for the preparation of novel (2-amino-3cyano-4H-chromen-4-yl)phosphonates by the domino Knoevenagel-phospha-Michael reaction.Our approach made also possible condensation with secondary phosphine oxides, and this reaction has not been reported previously in the literature.The model reaction of salicylaldehydes, malononitrile and dialkyl phosphites was optimized in detail, and then, the condensation was extended to various substituted salicylaldehydes and secondary phosphine oxides.The approach developed did not require chromatographic separation, since the products could be recovered from the reaction mixture by simple filtration.Altogether 18 (2-amino-3-cyano-4H-chromen-4-yl)phosphonate derivatives and 20 (2-amino-3-cyano-4Hchromen-4-yl)phosphine oxides were prepared in good to high yields, and fully characterized.Except five chromenylphosphonates (2a-d and 6a), all of them are new compounds.The crystal structure of compounds 2c, 6e and 8a-c was studied by single-crystal XRD analysis.The in vitro cytotoxicity and the antibacterial activity of the chromenylphosphonates and chromenylphosphine oxides synthesized were also investigated.Several chromenylphosphonates showed moderate and promising activities against the tested cell lines, especially the dibenzyl (2-amino-3-cyano-4H-chromen-4-yl)phosphonates, which had their IC 50 values in the 8-9 micromolar range against NIH/3T3 cells, and in the 3-7 micromolar range against HL-60 cells.None of the prepared derivatives reduced the growth of Gram-negative bacteria; however, chromenylphosphine oxides containing 3,5-dimethylphenyl groups on the phosphorus atom were active against selected Gram-positive bacteria.Two of the latter derivatives also showed activity in the 10 micromolar range against HL-60 cells.