Facile synthesis of 6-organyl-4-(trifluoromethyl)pyridin-2(1H)-ones and their polyfluoroalkyl-containing analogs

The three-component cyclization of 3-polyfluoroalkyl-3-oxopropanoates and methyl ketones with ammonium acetate affords 6-organyl-4-(polyfluoroalkyl)pyridin-2(1H)-ones (organyl is alkyl, aryl, or hetaryl). The synthesized pyridones were evaluated for antifungal, antibacterial, and analgesic activity.

T he pyridin-2-one scaff old is an important structural motif in organic chemistry. Compounds of this series are not only used as the starting reagents for the generation of new organic molecules 1-3 but also have found a recent application as ligands in various palladiumcatalyzed reactions. 4-6 These heterocyclic molecules also have attracted attention due to their biological properties. The pyridin-2-one scaff old is present in many biologically active natural products, 2,3,7,8 such as the antibiotic ilicocolin H, 9 the cytostatic agent pyridoxatin, 10 the mycotoxin sambutoxin, 11 thrombostatics calipyridones, 12 and numerous polycyclic alkaloid derivatives, 2, 13 in particular (±)-cytisine, irinotecan, (+)-hosieine, leporin A, huperzine A, 14 camptothecin, 15 and fredericamycin A. 16 The following synthetic drugs were designed based on the pyridin-2-one scaff old: 17-22 amrinone 17 (D1), milrinone 18 (D2), pirfenidone 19 (D3), perampanel 20 (D4), ricinine 21 (D5), and ciclopirox 22 (D6). Besides, pyridin-2-ones were used to produce biosensors. 23 Natural and synthetic pyridin-2-one derivatives proved to be anticancer, antibacterial, antifungal, antiviral, anti-infl ammatory, and psychotherapeutic agents. 3,24-26 Recently, pyridin-2-one natural products were found to act as effi cient SARS-CoV-2 main protease inhibitors. 27 Of particular promise in the synthesis of biologically active compounds are CF 3 -substituted heterocycles 28 because the presence of a trifl uoromethyl group in organic molecules has a signifi cant eff ect on their physical and chemical properties, metabolic stability, solubility, and lipophilicity. 29 Some 4-(trifl uoromethyl)pyridin-2-ones exhibit antiretroviral activity, and their representative, doravirine (D7), was approved as the initial therapy in patients with HIV/AID. 30 3-Cyano-4-(trifl uoromethyl)pyridin-2-ones can inhibit α-synuclein * Dedicated to Academician of the Russian Academy of Sciences V. A. Tartakovsky on the occasion of his 90th birthday. aggregation and prevent degeneration of dopaminergic neurons. One compound of this family, SynuClean-D (D8), is considered as a promising drug candidate for the treatment of Parkinson´s disease. 31 Trifl uoromethylcontaining pyridin-2-ones proved to be effi cient ligands in Pd-catalyzed CH-functionalization reactions. 32 Numerous approaches were developed for the formation of 4-(trifl uoromethyl)pyridin-2-ones 33- 41 and their non-fl uorinated analogs. 42-46 However, despite the relative variability of the synthetic approaches, all these methods suff er from drawbacks, such as the involvement of many steps, the use of diffi cultly accessible reagents, and the application of special cryogenic equipment. Therefore, the development of a facile synthesis of such attractive compounds as 4-(trifl uoromethyl)pyridin-2-ones is a challenging problem.
Recently, we proposed to use the three-component cyclization of 3-polyfl uoroalkyl-3-oxopropanoates with methyl ketones and diamines or amino alcohols as a versatile route to diverse hydrogenated bi-, tri-, and tetracyclic pyridin-2-one derivatives, some of which exhibit antiviral, anticancer, antifungal, and analgesic activity. 47-51 A series of N-functionalized 4-(polyfl uoroalkyl)pyridin-2-ones with tuberculostatic activity were synthesized by the acid cleavage of 7-hydroxy-7poly fluoroalkylhexahydroimidazo[1,2-a]pyridin-5ones. 52 Here, we made an attempt to apply this one-pot method to synthesize hydroxydihydropyridinones 3 and 4-(polyfl uoroalkyl)pyridin-2(1H)-ones 4 by using ammonia in the cyclization with 3-polyfl uoroalkyl-3oxopropanoates 1 and methyl ketones 2. The utilization of ammonia was expected to provide the formation of the pyridine ring unlike the similar transformations of alkylamines studied previously, which resulted in the alternative formation of aminocyclohexenones. 53 The employment of various fl uorine-containing keto esters 1a-d and alkyl, aryl, and hetaryl methyl ketones 2a-k makes it possible to vary the fl uorinated and non-fl uorinated substituents at the C(4) and C(6) positions in the resulting pyridones. First, we studied the reaction of trifl uoroacetoacetate 1a and methyl ketones 2a-c,f-j with 30% aqueous ammonia (Scheme 1, method A). We used 1,4-dioxane as the solvent, which was previously utilized for the synthesis of fused pyridones by three-component cyclizations. 47-51 The progress of the reactions was monitored by GLC and 19 F NMR spectroscopy. At room temperature, the reactions with aqueous ammonia proved to be ineffi cient. Therefore, we performed the synthesis at 60 C. The heating for 10-12 h resulted in the formation of aldol A as the major product (~65-78%). Previously, we obtained this aldol in the reaction of keto ester 1a with acetone 2a. 53 Meanwhile, crystals were obtained within 3-7 days by concentrating the reaction mixture. According to GLC and NMR analysis, these crystals were either hydroxydihydropyridin-5-ones 3 or pyridin-5-ones 4.
The reactions of ester 1a with alkyl methyl ketones 2a-c and aqueous ammonia gave hydroxydihydropyridinones 3a-c as the major products with a small impurity of dehydrated analogs 4a-c (~5-10%). The latter compounds apparently formed during the isolation. To confi rm this assumption, we heated compounds 3a-c in 1,4-dioxane and found that, under these conditions, the dehydration was slow. The effi ciency of the dehydration was higher when the reaction was per-formed under refl ux in toluene in the presence of p-toluenesulfonic acid. In this case, we obtained 2-pyridones 4a-c (see Scheme 1,method B).
As opposed to the transformations using alkyl methyl ketones 2a-c, the reactions with aryl methyl ketones 2f-j accomplished under similar conditions (see Scheme 1, method A) immediately aff orded dehydrated pyridin-2-ones 4f-j. Apparently, the hydroxy derivatives containing the conjugated (het)aryl substituent have a lower dehydration barrier, resulting in the ease of their formation.
Evidently, the presence of water adversely aff ects the reaction. Therefore, were decided to perform the three-component reaction of keto ester 1a and acetone 2a with ammonium acetate in the presence of trimethylamine, which is required for the transformation of the salt into the base (Scheme 2).
Using this reaction, we conducted the optimization of the reaction conditions ( Table 1, runs 1-9). The following optimal conditions were found for the formation of product 4a: the heating in 1,4-dioxane at 60 C (run 8). Under these conditions, product 4a was ob- tained in the highest yield, with the minimum amount of enamine E as the by-product, which was previously obtained in the reaction of ester 1a with ammonia. 54 The use of a twofold excess of keto ester 1a in the reaction with acetone 2a and ammonium acetate allowed the synthesis of pyridone 5 (see Table 1, run 8).
A large series of three-component reactions of 3-polyfl uoroalkyl-3-oxopropanoates 1a-d and ammonium acetate with methyl ketones 2a-k were performed under the optimal conditions, resulting in the preparation of a series of 4-(polyfl uoroalkyl)pyridin-2ones 4a-q in 23-81% yields (Scheme 3, method C). However, the reaction with butan-2-one 2b aff orded, apart from pyridone 4b, a small amount of 5,6-dimethyl-substituted heterocycle 6 (method C) that formed through the addition of the methylene group of the ethyl substituent to the trifl uoroacyl moiety. This reaction in ethanol allowed us to isolate product 6 in 19% yield (method D).
It is worth noting that the reactions with alkyl methyl ketones 2a-e occur more slowly and aff ord the target products in lower yields compared to the reactions with (het)aryl methyl ketones 2f-l. Besides, products 4a-d,m,n,p were purifi ed by column chromatography, which also led to a decrease in their yield. By contrast, the reactions with (het)aryl methyl ketones 2f-l occurred at a high rate and were more effi cient. Besides, products 4e-l,o,q precipitated, which accelerated the conversion of the starting reagents.
We also studied the transformations of 3-oxo esters 1b-d bearing a longer polyfl uorinated substituent with ammonium acetate and acetone 2a or 2-acetylfuran 2i (see Scheme 3, method C). It was found that an increase in the length of the fl uorinated substituent leads to a decrease in the yield of pyridinones 4m-q. Nevertheless, the trends observed in the reactions of trifl uoroacetoacetate 1a are retained. Thus, the yields of pyridones 4n,o,q derived from 2-acetylfuran 2i were somewhat higher compared to analogs 4m,p synthesized from acetone 2a.
The reaction using a twofold excess of keto ester 1a with acetone 2a and ammonium acetate enabled us to prepare compound 5 (see Table 1, run 8). Evidently, this compound is the product of further transformations of pyridone 4a in the presence of keto ester 1a. Actually, the methyl group of pyridone 4a can easily bind to the trifl uoroacetyl moiety of compound 1a in the presence of proline, which effi ciently catalyzes the aldolyzation (Scheme 4).
The structures of pyridinones 3-6 were confi rmed by IR spectroscopy, 1 H, 19 F, and 13 C NMR scopy, high-resolution mass spectrometry, and elemental analysis. Particular attention was given to the lactam-lactim tautomerism typical of pyridin-2-ones. 2, 23 It was noted 55 that the lactam form predominates both in the solid state and in solution although it can be transformed into the lactim form depending on the solvent and the electronic nature of the substituents of the heterocycle. The 13 C NMR spectroscopic data do not allow the discrimination between the tautomeric forms of compounds 4a-q, 5, and 6 because, in all cases, the carbon atom C(2) resonates at δ 161-163, which is characteristic of the sp 2 -hybridized carbon atom of both the amide and imine forms. The tautomeric structures of heterocycles 4a-l, 5, and 6 were determined from the IR spectra recorded for the solid state and solutions. According to the IR spectra of the solid samples, pyr-idin-2-ones 4a-k,m-q, 5, and 6 exist in the lactam form (Scheme 5) because their spectra show absorption bands of C=O stretching vibrations at ν 1645-1672 cm -1 characteristic of the carbonyl group involved in intermolecular hydrogen bonding. 56 The exception is 4-pyridinyl-substituted pyridine 4l, which exists in the lactim form in the solid state, because the IR spectrum of 4l has no absorption of the C=O group but shows broadened absorption bands of the OH group at 2506 cm -1 . However, the IR spectrum of 4l in weakly polar chloroform demonstrated structural changes, as evidenced by an absorption band at 1673 cm -1 assigned to C=O vibrations of the amide function. The IR spectra of 4a-d,k in solution (MeCN or CHCl 3 ) show C=O absorption bands at 1678-1683 cm -1 like the spectra of the solid samples. Therefore, our study demonstrated that the lactam-lactim tautomerism is characteristic only of pyridin-4-yl-substituted pyridone 4l (Scheme 5).
The use aqueous ammonia, which slowed down the formation of target pyridones 3 and 4, allowed us to propose the mechanism for the formation of the reaction products (Scheme 6). Apparently, the fi rst step of the reaction involves the aldol addition of methyl ketones 2 at the polyfl uoroacyl substituent of keto esters 1 to form aldols A.
Ammonia acts as the base that catalyzes the aldol addition. Therefore, these reactions can be considered as autocatalytic. Then ammonia can react with aldols A through two equally probable pathways: at the ethoxycarbonyl group to from amides Х1 or at the keto group to form amines X2 (see Scheme 6). Both these intermediates can undergo intramolecular cyclization to dihydropyridin-2-ones 3. The dehydration of the latter aff ords pyridones 4. Besides, the side condensation reaction of ester 1 with ammonia generates enamine E. It should be noted that if enamines E were intermediates in these transformations, the reactions would give isomeric 2-(polyfl uoroalkyl)pyridin-4-ones rather than 4-(polyfl uoroalkyl)pyridin-2-ones, but it was not the case.
Taking into account the great potential of pyridin-2-ones as biologically active compounds, we evaluated the synthesized trifl uoromethylated representatives for selected biological activities. First, we focused on the antimycotic properties because the known synthetic antifungal agent ciclopirox was designed based on pyridin-2-ones. 22 Pyridones 4a-d,f,g,i-  which the growth of pathogenic fungi was inhibited, were determined using fl uconazole as the positive control. 6-Furanylpyridin-2-one 4i showed weak inhibitory activity (MIC 50 g mL -1 ) against the strain T. interdigitale, and its thiophene and tolyl analogs 4g,j exhibited weak activity against the strain E. fl occosum (MIC 50 g mL -1 ). 6-Phenylpyridin-2-one 4f showed moderate inhibitory activity (MIC 25 g mL -1 ) against the strains T. tonsurans and E. fl occosum.
According to the literature data, the pyridin-2-one moiety is an appropriate structural scaff old for the synthesis of cannabinoid CВ2 receptor ligands promising for the design of analgesics. 58-60 Besides, the antiepileptic agent perampanel exhibited signifi cant analgesic activity in a series of acute and chronic pain tests. 61 We tested the analgesic eff ect and acute toxicity of pyridones 4a,c,f,i,l in in vivo experiments. The acute toxicity was evaluated in CD-1 mice by the intraperitoneal route. The analgesic activity was assessed at a dose of 15 mg kg -1 (intraperitoneal administration) using the hot-plate test in SD rats. 62, 63 The response of animals to the nociceptive stimulus was recorded 1 and 2 h after the administration of the tested compounds ( Table 2). Diclofenac at a dose of 10 mg kg -1 was used as the reference agent.
It was demonstrated that all the tested compounds were non-toxic at a dose of 150-300 mg kg -1 and exhibited the antinociceptive eff ect to a certain degree. The analgesic activity of alkyl-and phenyl-containing pyridones 4a,c,f proved to be lower than that of hetarylsubstituted analogs 4i,l (see Table 2). The highest antinociceptive eff ect was observed for 6-(pyridin-4-yl) pyridin-2-one 4l over the fi rst hour of measurements (75%), which was comparable with that of diclofenac. However, the activity decreased to 53% during the second hour. The structure-activity relationship demonstrates that the introduction of diff erent heteroatomcontaining substituents at the C(6) position is promising for the further structural optimization.
In summary, we developed a new facile one-pot three-component synthesis of 4-trifl uoromethylpyridin-2-ones and their polyfl uoroalkyl-substituted analogs based on the reactions of commercially available 3-oxo esters, methyl ketones, and ammonium acetate. The new method satisfi es the green chemistry principles and has good variability. Thus, the new method allows the variation of the fl uorinated substituent at the C(4) position in the newly formed pyridone ring and the introduction of diff erent alkyl, aryl, or hetaryl substituents at the C(6) position, leaving the C(3) and C(5) positions of the heterocycle accessible for subsequent modifi cations in CH-functionalization reactions. This may be useful for the synthesis of new biologically active compounds. The prospects of the synthesized pyridones in this fi eld are evidenced by the moderate antimycotic, high antibacterial, and signifi cant analgesic activity of the synthesized pyridones.

Synthesis of dihydropyridin-2-ones 3 and 4 (general procedure). Method A.
A mixture of ethyl 4,4,4-trifl uoroacetoacetate 1a (920 mg, 5 mmol) and methyl ketone 2 (5 mmol) in 1,4-dioxane (5 mL) was placed in a 30 mL septum-sealed screw cap vial. Then aqueous ammonia (340 mg, 20 mmol) was added, and the reaction mixture was magnetically stirred with heating to 60 C for 10-12 h. The progress of the reaction was monitored by TLC and 1 9 F NMR spectroscopy. Then the reaction mixture was concentrated and stored in a Petri dish for 3-7 days until the formation of a crystalline precipitate. The precipitate was triturated with Et 2 O, fi ltered off , recrystallized from acetonitrile, and dried in a drying oven.
Dehydration of dihydropyridin-2-ones 3. Method B. A mixture of hydroxydihydropyridone 3a-c (1 mmol) and p-toluenesulfonic acid (247 mg, 1.3 mmol) in toluene (30 mL) was placed in a round-bottom fl ask. The reaction mixture was refl uxed for 6-7 h. The progress of the reaction was monitored by TLC. Then the solvent was removed in a rotary evaporator, and the residue was purifi ed by column chromatography using chloroform as the eluent.
Synthesis of pyridin-2-ones 4. Method C. A mixture of keto ester 1 (5 mmol) and methyl ketone 2 (5 mmol) in 1,4-dioxane (5 mL) was placed in a 30 mL septum-sealed screw cap vial. Then ammonium acetate (385 mg, 5 mmol) and triethylamine (505 mg, 5 mmol) were added. The reaction mixture was magnetically stirred with heating to 60 C for 15-18 h. The progress of the reaction was monitored by TLC and 19 F NMR spectroscopy. After completion of the reaction, in the case of products 4a-d,m,n,p the solvent was removed by evaporation, and the resulting mixture was purifi ed by column chromatography using the appropriate eluent (chloroform, dichloromethane, ethyl acetate). The precipitate that formed in the reaction mixture in the case of products 4e-l,o,q was fi ltered off , recrystallized from an appropriate solvent (acetonitrile, ethanol), and dried in a drying oven.