α-(Imino)pyridyldifluoroethyl Phosphonates: Novel Promising Building Blocks in Synthesis of Biorelevant Aminophosphonic Acids Derivatives

A convenient synthetic approach to previously unknown NH-iminophosphonates bearing 2-, 3-, and 4-pyridyldifluoromethyl groups at the imine carbon atom was developed. The synthetic potential of these novel building blocks was demonstrated by their conversion into highly functionalized acyclic and heterocyclic aminophosphonates and phosphonic acids combining in their structure biorelevant aminophosphonic fragment, difluoromethyl group, and pyridyl, piperidyl, thiazolidin-4-one, or thiazidinan-4-one heterocyclic moieties in a single molecular platform.


Introduction
α-Aminophosphonates are phosphorus analogs of amino acids in which the planar carboxylic group is replaced with a tetrahedral bioisosteric phosphonic unit. They exhibit a wide range of biological activity and play a significant role in the development of antibiotics, antiviral, antihypertensive, antitumor agents and other bioactive substances [1][2][3][4][5]. Of particular interest are fluorinated aminophosphonates. Modification of organic molecule with fluorine has become almost a standard tool in modern drug design. It is generally accepted that introduction of a fluorine containing group may improve the pharmacodynamic and the pharmacokinetic profiles of the compound by concomitant alteration of its electronic, lipohilic, and steric characteristics as well as its metabolic stability [6][7][8][9].
α-Iminophosphonates, i.e., the compounds bearing (RO) 2 P(O)C=N-fragment, are valuable precursors of α-aminophosphonates. Unprotected NH-iminophosphonates are especially promising in this respect. They show enhanced reactivity and allow straightforward preparation of aminophosphonates with a free NH 2 group avoiding tedious N-deprotection step. Recently we have prepared the first representatives of NH-iminophosphonates and demonstrated their potential for the introduction of pharmacophore α-fluoroalkylated aminophosphonic unit into various structures.
NH-Iminophosphonates incorporating heterocyclic residue were unknown so far. In the present work we report the synthesis of NH-iminophosphonates bearing α-, β-, or γ-pyridyldifluoromethyl group at the imine carbon atom and their use for the preparation of aminophosphonic acids derivatives bearing difluoromethyl group and heterocyclic residue. Note, that compounds with 2-pyridyldifluoromethyl fragment were used for the development of effective thrombin and trypsin inhibitors [10] and highly selective antifungal agents [11]. The presence of a pyridine ring offers additional synthetic possibilities connected with the hydrogenation of heterocyclic ring and preparation of compounds Thus, the results of reactions of hydrophosphoryl compounds with nitriles depend on reactants structure and conditions.
We have found that all three isomeric pyridyldifluoroacetonitriles 1a-c react with diethyl phosphite under mild conditions (20 mol.% Et3N, r.t.) to afford respective NHiminophosphonates 2a-c (Scheme 2). It should be noted that 2-pyridyl nitrile 1a reacts much more slowly than the 3-and 4-isomers 1b,c: under the same conditions the reaction is completed within 120 and 12 h, respectively. Similar differences have previously been noted for pKa values of the isomeric pyridineacetic acids [20] which may be associated with the specific effect of the α-nitrogen atom.  Thus, the results of reactions of hydrophosphoryl compounds with nitriles depend on reactants structure and conditions.
We have found that all three isomeric pyridyldifluoroacetonitriles 1a-c react with diethyl phosphite under mild conditions (20 mol.% Et 3 N, r.t.) to afford respective NHiminophosphonates 2a-c (Scheme 2). It should be noted that 2-pyridyl nitrile 1a reacts much more slowly than the 3-and 4-isomers 1b,c: under the same conditions the reaction is completed within 120 and 12 h, respectively. Similar differences have previously been noted for pKa values of the isomeric pyridineacetic acids [20] which may be associated with the specific effect of the α-nitrogen atom. s 2021, 2, FOR PEER REVIEW 2 development of effective thrombin and trypsin inhibitors [10] and highly selective antifungal agents [11]. The presence of a pyridine ring offers additional synthetic possibilities connected with the hydrogenation of heterocyclic ring and preparation of compounds containing aminophosphonic unit, difluoromethyl group and piperidine fragment in the same molecule. It is worth of noting that according to [12] piperidine and pyridine are most frequent nitrogen heterocycles in U.S. FDA approved drugs.

Results and Discussion
Analysis of the literature data shows that hydrophosphoryl compounds can react with nitriles by different schemes. Thus, reaction of dialkylphosphites with alkyl, cycloalkyl, aryl, or pyridyl nitriles in the presence of acids [13,14] or under free radical conditions [15] leads to aminobisphosphonates I, as the main products, formed in low to moderate yields (Scheme 1). At the same time, under basic conditions benzonitrile reacts with dialkyl or diphenyl phosphites to form phosphorylamino phosphonates II [16]. Recently we have shown that highly electrophilic polyfluoroalkylated nitriles in the reactions with dialkyl or diphenyl phosphite afford first representatives of NH-iminophosphonates III [17,18]. At the same time, mixed perfluoro(chloro) acetonitriles react with (RO)2POH (R = Et, Ph) by two competitive routes: addition to the C≡N bond affording the respective N-unprotected iminophosphonates III, or reductive dehalogenation leading to chloro(fluoro) acetonitriles IV and the respective halogenophosphates V [19]. Thus, the results of reactions of hydrophosphoryl compounds with nitriles depend on reactants structure and conditions.
We have found that all three isomeric pyridyldifluoroacetonitriles 1a-c react with diethyl phosphite under mild conditions (20 mol.% Et3N, r.t.) to afford respective NHiminophosphonates 2a-c (Scheme 2). It should be noted that 2-pyridyl nitrile 1a reacts much more slowly than the 3-and 4-isomers 1b,c: under the same conditions the reaction is completed within 120 and 12 h, respectively. Similar differences have previously been noted for pKa values of the isomeric pyridineacetic acids [20] which may be associated with the specific effect of the α-nitrogen atom. Formation of the P-C=N-H fragment is confirmed by 13 C NMR spectra of compounds 2, in which the imine C-atom signal (δ C 171-173 ppm) with a large direct C-P coupling constant ( 1 J CP 153-154 Hz for Z-izomers 2a-c; 210 Hz for E-isomer 2a) was identified, and 1 H, 31 P NMR spectra showing characteristic spin-spin interaction of the N-H proton and phosphorus atom ( 3 J P-C-N-H 40-62 Hz).
Iminophosphonates 2a-c are formed as an equilibrium mixture of Z/E isomers and, similarly to other fluoroalkylated NH-iminophosphonates, a more sterically hindered Z configuration is preferred. We have shown previously that substituent at the C=N bond of iminophosphonates markedly affect E/Z isomeric ratio: C-trifluoromethylated iminophosphonates exist preferentially in Z configuration, whereas for their C-arylated analogs E-configuration prevails [21,22]. Table 1 shows the values of equilibrium Z/E ratios and phosphorus chemical shifts for pyridyldifluoromethylphosphonates 2a-c and some other related iminophosphonates. It is seen that 31 P NMR data and Z/E ratios for iminophosphonates 2b,c are very close and markedly differ from those for isomer 2a. It is very interesting to note from analysis of Table 1 that imines 2b,c bearing more strong electron withdrawing 3-and 4-pyridyl substituent (σ p 0.25 and 0.44, respectively [23] are in this respect more similar to trifluoromethylated analog 3, whereas isomer 2a with less electron withdrawing 2-pyridyl group (σ p 0.17) is more like benzimidoyl phosphonates 4, 5. Table 1. Z/E isomeric ratio and selected 13 C, 31 P NMR data of the compounds 2 and some related iminophosphonates.

Compound
Z Identification of Z,E-isomers 2a-c is based mainly on the significant difference in coupling constants of the N-H proton and phosphorus atom ( 3 J P-C-N-H~4 0 and~61-62 Hz, respectively) and high-field shift of phosphorus signals of Z-isomers in 31 P NMR spectra, previously noted for other α-iminophosphonates [17,18,21,22]. Iminophosphonates 2 contain a polarized C=N bond activated by the presence of electron-withdrawing group at the imine carbon atom. Thus, imines 2a-c readily add methanol across the C=N bond to afford adduct 6a-c (Scheme 3). The reactivity decreases in the order 2a > 2b,c: after 8 h the conversion is 100%, 44%, and 46%, respectively. The complete conversion of 2b,c proceeds within 2 days.
Formation of the P-C=N-H fragment is confirmed by 13 C NMR spectra of compounds 2, in which the imine C-atom signal (δC171-173 ppm) with a large direct C-P coupling constant ( 1 JCP 153-154 Hz for Z-izomers 2a-c; 210 Hz for E-isomer 2a) was identified, and 1 H, 31 P NMR spectra showing characteristic spin-spin interaction of the N-H proton and phosphorus atom ( 3 JP-C-N-H 40-62 Hz).
Iminophosphonates 2a-c are formed as an equilibrium mixture of Z/E isomers and, similarly to other fluoroalkylated NH-iminophosphonates, a more sterically hindered Z configuration is preferred. We have shown previously that substituent at the C=N bond of iminophosphonates markedly affect E/Z isomeric ratio: C-trifluoromethylated iminophosphonates exist preferentially in Z configuration, whereas for their C-arylated analogs E-configuration prevails [21,22]. Table 1 shows the values of equilibrium Z/E ratios and phosphorus chemical shifts for pyridyldifluoromethylphosphonates 2a-c and some other related iminophosphonates. It is seen that 31 P NMR data and Z/E ratios for iminophosphonates 2b,c are very close and markedly differ from those for isomer 2a. It is very interesting to note from analysis of Table 1 that imines 2b,c bearing more strong electron withdrawing 3-and 4-pyridyl substituent (σp 0.25 and 0.44, respectively [23] are in this respect more similar to trifluoromethylated analog 3, whereas isomer 2a with less electron withdrawing 2-pyridyl group (σp 0.17) is more like benzimidoyl phosphonates 4, 5. Table 1. Z/E isomeric ratio and selected 13 C, 31 P NMR data of the compounds 2 and some related iminophosphonates.
Identification of Z,E-isomers 2a-c is based mainly on the significant difference in coupling constants of the N-H proton and phosphorus atom ( 3 JP-C-N-H ~40 and ~61-62 Hz, respectively) and high-field shift of phosphorus signals of Z-isomers in 31 P NMR spectra, previously noted for other α-iminophosphonates [17,18,21,22].
Iminophosphonates 2 contain a polarized C=N bond activated by the presence of electron-withdrawing group at the imine carbon atom. Thus, imines 2a-c readily add methanol across the C=N bond to afford adduct 6a-c (Scheme 3). The reactivity decreases in the order 2a > 2b,c: after 8 h the conversion is 100%, 44%, and 46%, respectively. The complete conversion of 2b,c proceeds within 2 days.
It is very interesting to note, that addition of methanol to iminophosphonates 2 is accompanied by the change in the Z/E ratio of the starting compounds and Z-E equilibrium is established very quickly. Thus, immediately after dissolution of imines 2a-c in methanol, Z/E ratios of imines 2a-c, according to 31 P, 19 F NMR data, are 1:1, 2.5:1, and 2:1, respectively.
Evaporation of methanol and dissolution in deuterochloroform leads to recovery of Z/E isomeric ratios, presented in Table 1, i.e., 2:1, 6:1, and 7:1, respectively. Similar increase of E-isomer content on going from aprotic to protic solvent was shown recently for the sodium salts of N-methyliminophosphonates, bearing aryl or hetaryl substituents at the imine carbon atom [22].
Synthesis of biorelevant pirydyldifluoroemethylated aminophosphonates and aminophosphonic acids. We have found that selective reduction of the C=N bond in iminophosphonates 2a-c with borane-dimethylsulfide (BMS) proceeds under mild conditions to give aminophosphonates 7a-c incorporating pyridyldifluoromethyl residue (Scheme 4). Free aminophosphonates 7a-c are not very stable (the stability decreases in the order 7c > 7b > 7a) and should be stored at the reduced temperature in refrigerator.
Organics 2021, 2, FOR PEER REVIEW 4 It is very interesting to note, that addition of methanol to iminophosphonates 2 is accompanied by the change in the Z/E ratio of the starting compounds and Z-E equilibrium is established very quickly. Thus, immediately after dissolution of imines 2a-c in methanol, Z/E ratios of imines 2a-c, according to 31 P, 19 F NMR data, are 1:1, 2.5:1, and 2:1, respectively. Evaporation of methanol and dissolution in deuterochloroform leads to recovery of Z/E isomeric ratios, presented in Table 1, i.e., 2:1, 6:1, and 7:1, respectively. Similar increase of E-isomer content on going from aprotic to protic solvent was shown recently for the sodium salts of N-methyliminophosphonates, bearing aryl or hetaryl substituents at the imine carbon atom [22].
Synthesis of biorelevant pirydyldifluoroemethylated aminophosphonates and aminophosphonic acids. We have found that selective reduction of the C=N bond in iminophosphonates 2a-c with borane-dimethylsulfide (BMS) proceeds under mild conditions to give aminophosphonates 7a-c incorporating pyridyldifluoromethyl residue (Scheme 4). Free aminophosphonates 7a-c are not very stable (the stability decreases in the order 7c > 7b > 7a) and should be stored at the reduced temperature in refrigerator. Preparation of heterocycle-containing aminophosphonic acids from their esters are often complicated by side processes. It was found that hydrolysis of aminophosphonates 7a-c with conc. HCl proceeds cleanly and affords the first representatives of fluoroalkylated aminophosphonic acids 8a-c, incorporating heterocyclic moiety in the β-position.
The possibility of reducing the pyridine ring was studied using compound 7c as the most stable pyridyl substituted aminophosphonate. It was found that catalytic hydrogenation of 7c leads to complete reduction of pyridine ring to afford 4-piperidyl substituted aminophosphonate 9 (Scheme 5).
The latter should be stored at a reduced temperature, since under ordinary conditions it gradually decomposes, most likely undergoing O-N transfer of the ethyl group. More stable free aminophosphonic acid 10 was obtained by hydrolysis of 9 followed by treatment with propylene oxide. Cyclocondensation with mercaptocarboxylic acids. Highly polarized azomethyne bond in the imines 2 can be readily functionalized by the reactions with the bifunctional compounds. Thus, cyclocondensation of iminophosphonates 2a-c with thioglycolic or 3-mercaptopropionic acid leads to highly functionalized thiazolidin-4-ones 11a-c or thiazidinan-4-ones 12a-c, respectively (Scheme 6). Obviously, the reaction proceeds via primary nucleophilic addition to the C=N bond followed by intramolecular ring closure in the intermediate A. The ease of cyclization of the latter is due to the spatial accessibility of the unsubstituted N-nucleophilic center, and the experimentally found noticeably faster for- Preparation of heterocycle-containing aminophosphonic acids from their esters are often complicated by side processes. It was found that hydrolysis of aminophosphonates 7a-c with conc. HCl proceeds cleanly and affords the first representatives of fluoroalkylated aminophosphonic acids 8a-c, incorporating heterocyclic moiety in the β-position.
The possibility of reducing the pyridine ring was studied using compound 7c as the most stable pyridyl substituted aminophosphonate. It was found that catalytic hydrogenation of 7c leads to complete reduction of pyridine ring to afford 4-piperidyl substituted aminophosphonate 9 (Scheme 5).
It is very interesting to note, that addition of methanol to iminophosphonates 2 is accompanied by the change in the Z/E ratio of the starting compounds and Z-E equilibrium is established very quickly. Thus, immediately after dissolution of imines 2a-c in methanol, Z/E ratios of imines 2a-c, according to 31 P, 19 F NMR data, are 1:1, 2.5:1, and 2:1, respectively. Evaporation of methanol and dissolution in deuterochloroform leads to recovery of Z/E isomeric ratios, presented in Table 1, i.e., 2:1, 6:1, and 7:1, respectively. Similar increase of E-isomer content on going from aprotic to protic solvent was shown recently for the sodium salts of N-methyliminophosphonates, bearing aryl or hetaryl substituents at the imine carbon atom [22].
Synthesis of biorelevant pirydyldifluoroemethylated aminophosphonates and aminophosphonic acids. We have found that selective reduction of the C=N bond in iminophosphonates 2a-c with borane-dimethylsulfide (BMS) proceeds under mild conditions to give aminophosphonates 7a-c incorporating pyridyldifluoromethyl residue (Scheme 4). Free aminophosphonates 7a-c are not very stable (the stability decreases in the order 7c > 7b > 7a) and should be stored at the reduced temperature in refrigerator.

Scheme 4. Selective reduction of the C=N bond in the iminophosphonates 2a-c.
Preparation of heterocycle-containing aminophosphonic acids from their esters are often complicated by side processes. It was found that hydrolysis of aminophosphonates 7a-c with conc. HCl proceeds cleanly and affords the first representatives of fluoroalkylated aminophosphonic acids 8a-c, incorporating heterocyclic moiety in the β-position.
The possibility of reducing the pyridine ring was studied using compound 7c as the most stable pyridyl substituted aminophosphonate. It was found that catalytic hydrogenation of 7c leads to complete reduction of pyridine ring to afford 4-piperidyl substituted aminophosphonate 9 (Scheme 5).
The latter should be stored at a reduced temperature, since under ordinary conditions it gradually decomposes, most likely undergoing O-N transfer of the ethyl group. More stable free aminophosphonic acid 10 was obtained by hydrolysis of 9 followed by treatment with propylene oxide. Cyclocondensation with mercaptocarboxylic acids. Highly polarized azomethyne bond in the imines 2 can be readily functionalized by the reactions with the bifunctional compounds. Thus, cyclocondensation of iminophosphonates 2a-c with thioglycolic or 3-mercaptopropionic acid leads to highly functionalized thiazolidin-4-ones 11a-c or thiazidinan-4-ones 12a-c, respectively (Scheme 6). Obviously, the reaction proceeds via primary nucleophilic addition to the C=N bond followed by intramolecular ring closure in the intermediate A. The ease of cyclization of the latter is due to the spatial accessibility of the unsubstituted N-nucleophilic center, and the experimentally found noticeably faster for-Scheme 5. Catalytic hydrogenation of the aminophosphonate 7c.
The latter should be stored at a reduced temperature, since under ordinary conditions it gradually decomposes, most likely undergoing O-N transfer of the ethyl group. More stable free aminophosphonic acid 10 was obtained by hydrolysis of 9 followed by treatment with propylene oxide.
Cyclocondensation with mercaptocarboxylic acids. Highly polarized azomethyne bond in the imines 2 can be readily functionalized by the reactions with the bifunctional compounds. Thus, cyclocondensation of iminophosphonates 2a-c with thioglycolic or 3-mercaptopropionic acid leads to highly functionalized thiazolidin-4-ones 11a-c or thiazidinan-4-ones 12a-c, respectively (Scheme 6). Obviously, the reaction proceeds via primary nucleophilic addition to the C=N bond followed by intramolecular ring closure in the intermediate A. The ease of cyclization of the latter is due to the spatial accessibility of the unsubstituted N-nucleophilic center, and the experimentally found noticeably faster formation of thiazolidinones 11 as compared to thiazinanones 12 is explained by the proximity of the reaction centers upon creation of the 5-membered cycle. We were able to detect by 31 P NMR adduct A (Scheme 6, n = 2) resulting from addition of 3-mercaptopropionic acid (δ P 16 ppm).
Organics 2021, 2, FOR PEER REVIEW 5 mation of thiazolidinones 11 as compared to thiazinanones 12 is explained by the proximity of the reaction centers upon creation of the 5-membered cycle. We were able to detect by 31 P NMR adduct A (Scheme 6, n = 2) resulting from addition of 3-mercaptopropionic acid (δP 16 ppm).

Conclusions
In summary, the triethylamine catalyzed addition of diethyl phosphite to isomeric pyridyldifluoroacetonitriles leads to α-iminopyridyldifluoroethyl phosphonates, existing as equilibrium mixture of E-Z isomers. The synthetic potential of these novel electrophilic building blocks was demonstrated by their reduction to biorelevant compounds combining in their structure aminophosphonic residue, difluoromethyl group and isomeric pyridine or piperidine moiety. Cyclocondensation with mercaptocarboxylic acids allow preparation of hybrid molecules bearing two bioactive heterocyclic moieties (pyridine and thiazolidine or thiazinane) and aminophosphonic fragment in a single molecular platform. These reactions proceed under mild neutral conditions and lead directly to N-unprotected aminophosphonic derivatives.

Materials and Methods
1 H, 19 F, and 13 C NMR spectra were recorded using Bruker Avance NMR spectrometers operating at 302, 400 and 499.

Conclusions
In summary, the triethylamine catalyzed addition of diethyl phosphite to isomeric pyridyldifluoroacetonitriles leads to α-iminopyridyldifluoroethyl phosphonates, existing as equilibrium mixture of E-Z isomers. The synthetic potential of these novel electrophilic building blocks was demonstrated by their reduction to biorelevant compounds combining in their structure aminophosphonic residue, difluoromethyl group and isomeric pyridine or piperidine moiety. Cyclocondensation with mercaptocarboxylic acids allow preparation of hybrid molecules bearing two bioactive heterocyclic moieties (pyridine and thiazolidine or thiazinane) and aminophosphonic fragment in a single molecular platform. These reactions proceed under mild neutral conditions and lead directly to N-unprotected aminophosphonic derivatives.

Materials and Methods
1 H, 19 F, and 13 C NMR spectra were recorded using Bruker Avance NMR spectrometers operating at 302, 400 and 499.

Reactions of Iminophosphonates 2a-c with Methanol
Iminophosphonates 2a-c (0.02 g, 0.07 mmol) were dissolved in methanol (0.6 mL) at room temperature. According to 31 P, 19 F NMR the reaction was completed after 8 h (2a) or 2 days (2b,c). The spectral data are similar to those of methanol adduct with trifluoromethyl analog [17].