Trifluoromethylated 4,5-Dihydro-1,2,4-triazin-6(1H)-ones via (3+3)-Annulation of Nitrile Imines with α-Amino Esters

The synthesis of two series of monocyclic and bicyclic trifluoromethylated 4,5-dihydro-1,2,4-triazin-6(1H)-one derivatives based on (3+3)-annulation of methyl esters derived from natural α-amino acids with in situ generated trifluoroacetonitrile imines has been described. The devised protocol is characterized by a wide scope, easily accessible substrates, remarkable functional group tolerance, and high chemical yield. In reactions with chiral starting materials, no racemization at the stereogenic centers was observed and the respective enantiomerically pure products were obtained. Selected functional group interconversions carried out under catalytic hydrogenation and mild PTC oxidation conditions were also demonstrated.


Introduction
The functionalization of organic molecules with fluorine atom(s) and/or with fluoroalkyl groups has been recognized as an efficient method for the tuning of their physicochemical behavior and biological activity [1][2][3][4]. The introduction of fluorine atoms into the parent non-fluorinated compound enables control on properties such as the metabolic stability, reactivity, acidity, oleophilicity, and conformational effects, among others, which are of general significance for the search of new advanced materials [5][6][7], and compounds of potential medicinal [8][9][10] and agrochemical applications [11]. For this reason, the development of new, efficient synthetic methods leading to fluorinated products, in particular, fluoromethylated N-heterocycles [12][13][14], is highly desirable.
On the other hand, a number of more recent publications reported on nitrile imines functionalized at the C-termini with either CF 3 [25][26][27][28][29][30] or CF 2 H [31,32] groups. They were successfully applied for the synthesis of various 5-membered N-heterocyclic systems formed via (3+2)-cycloadditions, notably, with most of the cases proceeding in a fully On the other hand, a number of more recent publications reported on nitrile imines functionalized at the C-termini with either CF3 [25][26][27][28][29][30] or CF2H [31,32] groups. They were successfully applied for the synthesis of various 5-membered N-heterocyclic systems formed via (3+2)-cycloadditions, notably, with most of the cases proceeding in a fully regioselective manner. The presented protocols also revealed fluorinated nitrile imines as readily available building blocks, which can be generated in situ under mild conditions, i.e., via the base-mediated dehydrohalogenation of bench-stable hydrazonoyl halides (or pseudohalides).
Despite the remarkable progress in (3+2)-cycloaddition reactions in recent years, the chemistry of higher formal (3+n)-cycloadditions of fluorinated nitrile imines, such as 1 with bifunctional reagents, are explored to a limited extent. Some time ago, we demonstrated that nitrile imines 1 can be smoothly trapped with α-mercaptoacetaldehyde, and also with other α-mercaptocarbonyl compounds, to give 1,3,4-thiadiazolines 5 as the exclusive products (Scheme 1d) [42]. Nevertheless, to the best of our knowledge, no reports on cyclocondesations of 1 with other bifunctional compounds, such as α-aminocarbonyls, have been published thus far. Hence, we turned the attention to methyl esters derived from natural amino acids as easily available substrates for the preparation of hitherto unknown trifluoromethylated 1,3,4-triazin-6(1H)-one derivatives 6 and 7 (Scheme 1e).
In recent years, monocyclic and bicyclic 1,2,4-triazine derivatives, including their oxo analogues, attracted considerable attention, as the compounds of that type exhibit a wide range of pharmacological properties, in particular antimicrobial and anticancer activity [43,44]. For example, in 2010 Krauth et al. reported on 1,2,4-triazin-5-ones of type 8, which showed distinct antiproliferative effects against the chronic myeloid leukemia cell line (K-562) combined with remarkably low cytotoxicity ( Figure 1) [45]. Later on, Khan et al. evidenced that the introduction of fluorine atom into aryl substituent improves the biological activity of the resulting 1,2,4-triazinones (such as 9), which were recognized as potent CDK2 and anti-HIV-1 inhibitors [46]. Furthermore, a promising thioredoxin reductase (TrxR) inhibition at submicromolar concentration by trifluoromethylated bicyclic triazinone 10 has also been discovered [47]. Thus, despite the reported progress in the synthesis and biological evaluation of fluorinated 1,2,4-triazinones, further development of synthetic protocols and the evaluation of biological properties of this group of N-heterocycles is of general importance.
with bifunctional reagents, are explored to a limited extent. Some time ago, we dem strated that nitrile imines 1 can be smoothly trapped with α-mercaptoacetaldehyde, a also with other α-mercaptocarbonyl compounds, to give 1,3,4-thiadiazolines 5 as the clusive products (Scheme 1d) [42]. Nevertheless, to the best of our knowledge, no repo on cyclocondesations of 1 with other bifunctional compounds, such as α-aminocarbon have been published thus far. Hence, we turned the attention to methyl esters deriv from natural amino acids as easily available substrates for the preparation of hitherto known trifluoromethylated 1,3,4-triazin-6(1H)-one derivatives 6 and 7 (Scheme 1e).
In recent years, monocyclic and bicyclic 1,2,4-triazine derivatives, including their o analogues, attracted considerable attention, as the compounds of that type exhibit a w range of pharmacological properties, in particular antimicrobial and anticancer activ [43,44]. For example, in 2010 Krauth et al. reported on 1,2,4-triazin-5-ones of type 8, wh showed distinct antiproliferative effects against the chronic myeloid leukemia cell line 562) combined with remarkably low cytotoxicity ( Figure 1) [45]. Later on, Khan et al. e denced that the introduction of fluorine atom into aryl substituent improves the biolog activity of the resulting 1,2,4-triazinones (such as 9), which were recognized as pot CDK2 and anti-HIV-1 inhibitors [46]. Furthermore, a promising thioredoxin reduct (TrxR) inhibition at submicromolar concentration by trifluoromethylated bicyclic t zinone 10 has also been discovered [47]. Thus, despite the reported progress in the s thesis and biological evaluation of fluorinated 1,2,4-triazinones, further developmen synthetic protocols and the evaluation of biological properties of this group of N-hete cycles is of general importance. Here we report on our results aimed at the application of nitrile imines 1 in the s thesis of a series of monocyclic and bicyclic 1,2,4-triazin-6(1H)-ones 6 and 7, respectiv by using selected natural amino acid esters as suitable reaction partners. Particularly, ac ral (glycine) and the selected chiral substrates bearing either primary or secondary am group (proline) were examined. Furthermore, the subsequent transformations of the get products, particularly the oxidations of the core heterocyclic ring and interconversi of selected functional group under catalytic hydrogenation conditions, were studied. nally, taking into account the well-documented biological activity of some fluorinated a non-fluorinated 1,2,4-triazinones and related 1,2,4-triazine-based analogues [43][44][45][46][47][48][49][50][51], cytotoxic properties of representative final compounds were examined against MCF-7 a HL-60 cancer cell lines.

General Information
All commercially available chemicals (solvents, reagents) were used as received not stated otherwise, reactions were performed in flame dried flasks under the atm phere of inert gas with the addition of the reactants using a syringe; subsequent mani lations were conducted in the air. NMR spectra were measured with Bruker AVIII inst ment ( 1 H NMR (600 MHz); 13   Here we report on our results aimed at the application of nitrile imines 1 in the synthesis of a series of monocyclic and bicyclic 1,2,4-triazin-6(1H)-ones 6 and 7, respectively, by using selected natural amino acid esters as suitable reaction partners. Particularly, achiral (glycine) and the selected chiral substrates bearing either primary or secondary amino group (proline) were examined. Furthermore, the subsequent transformations of the target products, particularly the oxidations of the core heterocyclic ring and interconversions of selected functional group under catalytic hydrogenation conditions, were studied. Finally, taking into account the well-documented biological activity of some fluorinated and non-fluorinated 1,2,4-triazinones and related 1,2,4-triazine-based analogues [43][44][45][46][47][48][49][50][51], the cytotoxic properties of representative final compounds were examined against MCF-7 and HL-60 cancer cell lines.

General Information
All commercially available chemicals (solvents, reagents) were used as received. If not stated otherwise, reactions were performed in flame dried flasks under the atmosphere of inert gas with the addition of the reactants using a syringe; subsequent manipulations were conducted in the air. NMR spectra were measured with Bruker AVIII instrument ( 1 H NMR (600 MHz); 13 C NMR (151 MHz); 19 F NMR (565 MHz) Bruker BioSpin AG, Fällanden, Switzerland); chemical shifts are given relative to the residual undeuterated solvent peaks (for CDCl 3 : 1 H NMR δ = 7.16, 13 C NMR δ = 77.16; for CD 3 OD: 1 H NMR δ = 3.31, 13 C NMR δ = 49.00; for DMSO-d 6 : 1 H NMR δ = 2.50, 13 C NMR δ = 39.52) or to CFCl 3 ( 19 F NMR δ = 0.00) used as the external standard. Integrals in accordance with the assignments and coupling constants J are given in Hz. For detailed peak assignments, 2D spectra were measured (i.e., COSY, HMQC). Mass spectra (ESI) were performed with a Varian 500-MS LC Ion Trap (Varian Inc., Palo Alto, CA, USA); high resolution measurements were performed with a Waters Synapt G2-Si mass spectrometer (Waters Corporation, Milford, MA, USA). IR spectra were obtained with a Cary 630 FTIR (Agilent Technologies, Santa Clara, CA, USA) spectrometer, in neat. Elemental analyses were performed with a Vario EL III (Elementar Analysensysteme GmbH, Langenselbold, Germany) instrument. The melting points were determined in the capillaries with a Melt-Temp II (Laboratory Devices, Holliston, MA, USA) apparatus or with a polarizing optical microscope (Opta-Tech, Warsaw, Poland), and they are uncorrected. The ball-milling apparatus was a MM 400 mixer mill (Retsch GmbH, Haan, Germany). The mechanochemical reactions were performed in 5 mL stainless steel jars, at 25 Hz, with three stainless steel balls (ø 7 mm). The optical rotations were determined with a MCP 500 (Anton Paar, Graz, Austria) polarimeter at the temperatures indicated. The enantiopurity was analyzed with 1260 Infinity HPLC (Agilent Technology, Germany) using a column with chiral support (CHIRALPAK AD-H). The required known nitrile imine precursors, i.e., hydrazonoyl bromides 11, were prepared starting with readily available trifluoroacetaldehyde arylhydrazones 14 [52], by NBS-mediated bromination of the latter, as described [25].

Synthetic Protocols
Synthesis of 1,2,4-triazin-6(1H)-ones 6 and 7: An excess Et 3 N (8.0 mmol, 1.12 mL) was added under inert atmosphere to a suspension of amino ester hydrochloride 12 (1.0 mmol) in dry THF (3.0 mL). Then, a solution of hydrazonoyl bromide 11 (1.1 mmol) in dry THF (3.0 mL) was added, and the stirring was continued overnight (the consumption of 11 was confirmed by TLC). The resulting solution was filtered and the precipitate was washed with Et 2 O (2 × 4.0 mL). After the filtrates were combined and the solvents were removed under reduced pressure, the crude product 6 or 7 was purified by standard column chromatography (CC). In certain cases of glycine derivatives, the resulting material was additionally recrystallized from hexane-dichloromethane mixtures by the slow evaporation of the solvents.
Synthesis of 1-(4-tolyl)-3-trifluoromethyl-1,2,4-triazin-6(1H)-one (6s): A mixture of 4,5-dihydro-1,2,4-triazinone 6g (0.5 mmol, 128.5 mg), K 3 Fe(CN) 6 (3.0 mmol, 987 mg), aqueous solution of Na 2 CO 3 (0.5M, 10 mL), and Et 4 NBr (15 mol%) in CH 2 Cl 2 (10 mL) was vigorously stirred at room temperature for 4 h (monitored on TLC). The resulting mixture was extracted with CH 2 Cl 2 (3 × 10 mL), the combined organic layers were dried over anh. Na 2 SO 4 , filtered and the solvents were removed under reduced pressure. The crude product was purified by standard CC (SiO 2 , CH 2 Cl 2 ) to give 6s (99 mg, 78%). Colorless solid, m.p. 80-82 • C. 1  General procedure for catalytic hydrogenation reactions: A solution of the corresponding triazinone (7a or 7h, 0.5 mmol) in EtOH (10 mL) was added Pd/C (5.0 mmol), and the resulting mixture was vigorously shaken in the atmosphere of H 2 (3 atm) for the required time. The mixture was filtered through Celite, washed with EtOH (5 mL), and the solvents were removed under reduced pressure. The resulting mixture was filtered through a short plug of silica (CC) to give the spectroscopically pure product. General procedure for synthesis of hydrazonoyl bromides 11: Following the general literature protocol [25], arylhydrazone 14 (1.0 mmol) was dissolved in dry DMF (3 mL), the solution was cooled to 0 • C, then solid NBS (1.05 mmol, 187 mg) was added and stirring was continued at this temperature. After the starting hydrazone was fully consumed (TLC monitoring, typically ca. 2 h), the resulting mixture was extracted with H 2 O/Et 2 O 1:1 mixture (20 mL), the organic layer was washed with H 2 O (3 × 10 mL), dried over anh. Na 2 SO 4 , filtered and the solvents were removed in vacuo. Crude products were purified by column chromatography. of a base (3.0 equiv.), the slow consumption of the nitrile imine precursor was observed (according to TLC monitoring) to give after 16 h the expected (3+3)-cycloadduct 6a, which was isolated by flash chromatography in a fair 59% yield (Scheme 2). Brief optimization of the reaction conditions revealed that increasing the amount of Et 3 N (8.0 equiv.) enhanced the yield of isolated product 6a (75%) formed as the only product. On the other hand, neither the change of the base (Cs 2 CO 3 , pyridine, DBU), nor the type of solvent used (CH 2 Cl 2 , toluene) resulted in remarkable change of the chemical yield of the studied reaction. Prompted by the often-observed positive effects of mechanochemical activation on reaction outcomes, (e.g., remarkable shortening of reaction times, higher yields, etc.) [53][54][55], the (3+3)-annulation reaction of 11a and 12a was also tested under ball-mill conditions (steel balls, ø 7 mm, 25 Hz). However, in this case, the formation of viscous mixture was observed upon the reaction progress, which enabled effective ball-milling and the reaction could not be completed. Moreover, the use of three-fold excess of glycinate 12a (with respect to bromide 11a) was necessary to obtain the desired product 6a in a comparable yield (77%) and the remarkably shorter reaction time of 2 h, and for these reasons we waved on the mechanochemical approach.
(CH2Cl2, toluene) resulted in remarkable change of the chemical yield of the studied reaction. Prompted by the often-observed positive effects of mechanochemical activation on reaction outcomes, (e.g., remarkable shortening of reaction times, higher yields, etc.) [53][54][55], the (3+3)-annulation reaction of 11a and 12a was also tested under ball-mill conditions (steel balls, ø 7 mm, 25 Hz). However, in this case, the formation of viscous mixture was observed upon the reaction progress, which enabled effective ball-milling and the reaction could not be completed. Moreover, the use of three-fold excess of glycinate 12a (with respect to bromide 11a) was necessary to obtain the desired product 6a in a comparable yield (77%) and the remarkably shorter reaction time of 2 h, and for these reasons we waved on the mechanochemical approach.
With the optimized reaction conditions in hand, a series of variously substituted nitrile imines 1b−1h were checked in the reaction with glycinate 12a (Scheme 2). The required nitrile imine precursors of type 11 were prepared according to the general literature protocols by condensation of arylhydrazines with fluoral hydrate [52], followed by NBS-mediated bromination of the first formed hydrazones 14 (Scheme 3) [25]. As shown in Scheme 2, the expected products were formed in high yields (74-93%), irrespective of the electronic nature of the substituent X. These observations nicely correspond to the previous results reported by Dalloul for reactions of some non-fluorinated nitrile imines with methyl glycinate and other amino acid esters [56,57]. Notably, in the case of highly electron-deficient nitrile imine 1e functionalized at the N-termini with 2,4-dichlorophenyl group, the formation of a mixture of the target material 6e and the first formed acyclic adduct 13e in ca. 3:2 ratio was observed, which nicely evidenced the stepwise character of Scheme 2. Synthesis of 1,2,4-triazin-6(1H)-ones 6a-6h derived from methyl glycinate (scope of nitrile imines 1) and the structure of intermediate 13e; a crude reaction mixture was refluxed for 2 h.
The structure of the isolated product 6a was confirmed based on NMR methods; for example, in 1 H NMR spectrum, a broadened doublet (J ≈ 1.5 Hz) attributed to 5-H 2 was found at δ 4.30, along with absorption (s br ) of the NH (δ 5.31), and the characteristic set of signals (two d br located at δ 7.92 and 8.27, J ≈ 9.2 Hz each) of the C 6 H 4 NO 2 group. Furthermore, two diagnostic quartets attributed to the CF 3 group, and the C-3 atom were found at δ 118.2 ( 1 J C-F = 275.2 Hz) and δ 137.3 ( 2 J C-F = 37.8 Hz), respectively, whereas the absorption (s) of the amide-type C=O group was found at δ 158.1. Finally, the 19 F NMR indicated the presence of a single CF 3 group (singlet at δ −70.6), while the ESI-MS, supplemented by combustion analysis, confirmed the molecular formula of C 10 H 7 F 3 N 4 O 3 . Based on the collected data, the structure of isolated product was established as the hitherto unknown 1-(4-nitrophenyl)-3-trifluoromethyl-4,5-dihydro-1,2,4-triazin-6(1H)-one (6a).
With the optimized reaction conditions in hand, a series of variously substituted nitrile imines 1b-1h were checked in the reaction with glycinate 12a (Scheme 2). The required nitrile imine precursors of type 11 were prepared according to the general literature protocols by condensation of arylhydrazines with fluoral hydrate [52], followed by NBSmediated bromination of the first formed hydrazones 14 (Scheme 3) [25]. As shown in Scheme 2, the expected products were formed in high yields (74-93%), irrespective of the electronic nature of the substituent X. These observations nicely correspond to the previous results reported by Dalloul for reactions of some non-fluorinated nitrile imines with methyl glycinate and other amino acid esters [56,57]. Notably, in the case of highly electron-deficient nitrile imine 1e functionalized at the N-termini with 2,4-dichlorophenyl group, the formation of a mixture of the target material 6e and the first formed acyclic adduct 13e in ca. 3:2 ratio was observed, which nicely evidenced the stepwise character of the studied (3+3)-annulation reaction. Therefore, in the next attempt, the resulting crude mixture was additionally refluxed for 2 h in order to accelerate the second ring-closure step, and after standard work-up, the product 6e was isolated in excellent yield (93%).
Materials 2023, 16, x FOR PEER REVIEW the studied (3+3)-annulation reaction. Therefore, in the next attempt, the resultin mixture was additionally refluxed for 2 h in order to accelerate the second ring step, and after standard work-up, the product 6e was isolated in excellent yield (9 Scheme 3. Synthesis of hydrazonoyl bromides 11a−11h. Next, a series of enantiopure α-substituted (S)-amino esters 12b−12i deriv alanine, valine, leucine, phenylglycine, serine, methionine, aspartic acid, and tryp respectively, were examined in reaction with selected hydrazonoyl bromides 11a to afford the expected optically active products 6i−6q (Schemes 4 and 5). Thus, alo simple alkyl and aryl substituents in 6i−6l, functional groups such as hydroxy (6 oether (6n) and secondary amine (6p) could also be efficiently introduced. Notabl case of methyl aspartate (12h), the presence of the additional CO2Me group did n fere with the subsequent cyclisation step, and the 6-membered product 6o was exclusively in excellent yield (93%). In order to check the optical purity of the resulting products, phenylglycineproduct 6q was selected for a more detailed examination, and a sample of racem triazinone rac-6q was prepared as a reference compound (Scheme 5). Unfortunat ther chiral-HPLC analysis of pure samples of 6q nor 1 H NMR measurements of th stereomeric 1:1 mixtures with (+)-(S)-mandelic acid and with (+)-(R)-(tert-but nyl)phosphonothioic acid [58] selected as chiral solvating agents was successful. Next, a series of enantiopure α-substituted (S)-amino esters 12b-12i derived from alanine, valine, leucine, phenylglycine, serine, methionine, aspartic acid, and tryptophan, respectively, were examined in reaction with selected hydrazonoyl bromides 11a and 11g to afford the expected optically active products 6i-6q (Schemes 4 and 5). Thus, along with simple alkyl and aryl substituents in 6i-6l, functional groups such as hydroxy (6m), thioether (6n) and secondary amine (6p) could also be efficiently introduced. Notably, in the case of methyl aspartate (12h), the presence of the additional CO 2 Me group did not interfere with the subsequent cyclisation step, and the 6-membered product 6o was formed exclusively in excellent yield (93%). the studied (3+3)-annulation reaction. Therefore, in the next attem mixture was additionally refluxed for 2 h in order to accelerate step, and after standard work-up, the product 6e was isolated in Scheme 3. Synthesis of hydrazonoyl bromides 11a−11h.

Conclusions
In the presented study, the synthesis of a series of 4,5-dihydro-1,2,4-triazin-6(1H)-ones functionalized with the CF 3 group is reported. The devised protocol is based on the (3+3)annulation of methyl esters derived from natural α-amino acids with in situ generated trifluoroacetonitrile imines applied as reactive 1,3-dipolar reaction partners. Notably, starting with chiral α-amino esters, no racemization occurred under the optimized reaction conditions, and the expected enantiopure materials were isolated as the only products. Furthermore, with the application of methyl L-prolinate as a model secondary amino ester, the respective fused 1,2,4-triazinones were obtained. The selected functional group interconversions performed under catalytic hydrogenation or mild PTC-oxidation conditions demonstrated remarkable stability of the core heterocycle. Thus, the presented method offers straightforward access to the desired heterocyclic system functionalized not only with simple alkyl and aryl substituents, but also with such functional groups as nitro, cyano, hydroxy, amino, methoxycarbonyl, and sulfide, as well as 1H-indol-3-yl and halogen(s). Taking into account the easy accessibility of the starting materials and the exceptionally mild reaction conditions, the presented approach can be recommended for the synthesis of title 3-trifluoromethylated heterocycles, and nicely supplements previous reports on the synthesis of 1,2,4-triazin-6(1H)-ones exploiting amino acids and their derivatives as key building blocks [56,57,[59][60][61][62][63][64].