Study of 2-aminoquinolin-4(1H)-one under Mannich and retro-Mannich reaction

2-Aminoquinolin-4(1H)-one was reacted with various primary/secondary amines and paraformaldehyde under Mannich reaction conditions. In the case of secondary amines, the reaction in N,N-dimethylformamide yielded expected Mannich products accompanied with 3,3'-methylenebis(2-aminoquinolin-4(1H)-one). Except these main products, the pyrimido[4,5-b]quinolin-5-one derivative was also identified as co-product. The reaction with primary amines led to the formation of pyrimido[4,5-b]quinolin-5-ones. The Mannich reaction products were thermally unstable and afforded a mixture of bis-(2-aminoquinolin-4(1H)-one) and tris-(2-aminoquinolin-4(1H)-one) derivative, probably via reactive methylene species. This retro-Mannich reaction was tested in reaction with indole and thiophenole as nucleophilles, and appropriate conjugates were formed. The mechanism of above discussed reactions in which 2-aminoquinolinone displays the nucleophilicity on C3 carbon as well as N2 nitrogen is discussed.


Introduction
Due to the outstanding position of the quinolin-4(1H)-one scaffold in the field of medicinal chemistry, 2-aminoquinolin-4(1H)-ones have been widely studied as potential pharmacological agents in different areas. The first paper in this field published in 1974 was devoted to the synthesis and evaluation of antimicrobial activity of selected 2-amino-4-alkoxyquinolines. [1] Recently, 3-acetyl-2-aminoquinolin-4(1H)-ones were reported as potent and selective calpain inhibitors. [2] 2-[2-Substituted-3-(3,4-dichlorobenzylamino)propylamino]qui-nolin-4-ones were found to possess antibacterial activity against various strains, mainly Staphylococcus aureus and Enterococci. [3] Derivatives of 2-aminoquinolin-4-ol have been identified as suitable structural motifs for the preparation of novel oligonucleotide conjugates to enhance binding affinities for complementary RNA targets. [4] Furthermore, the latest results show that compounds based on 2-aminoquinolin-4-ol promote a significant telomere dysfunction leading to long-term anti-tumor activity. [5][6][7][8]  same scaffold was included in the structure of ferrocenes with leishmanicidal activity. [9] Although the number of 2-aminoquinolin-4(1H)-one derivatives were described, they were almost exclusively synthesized by scaffold construction. Modification of 2-amino-4-alkoxyquinoline scaffold was described rarely. The attack of the C 3 carbon with electrophiles was reported only for a coupling reaction with aryl diazonium salts yielding corresponding azocompounds. [10] Formation of benzo[b] [1,8]naphthyridine scaffold was described via a reaction with 3-formylchromone [11] or arylmalonates, [12,13] followed by the condensation of the amino group in the position 2. The reaction of amino group itself was reported only in an acylation reaction. [10,14] The combination of the nucleophilic C 3 carbon and the amino group in position 2 is challenging for the potential use of 2-aminoquinolin-4(1H)-one as the starting material in the Mannich reaction, in which the compound can behave as both C-and N-nucleophile. Although the Mannich reaction belongs to one of the most powerful synthetic strategies for carbon-carbon bond formation and has found numerous applications in the syntheses of natural and biologically active compounds, [15,16] little attention was given to its use for the modification of quinolin-4(1H)-ones. Only several studies were reported, in which the Mannich reaction was used for the modification of 2-methyl-quinolin-4(1H)-ones [17,18] with the aim to prepare novel antibacterial agents.
In this article, we report the results of the study of 2-aminoquinolin-4(1H)-one modification via the Mannich reaction to enlarge the portfolio of synthetic strategies applicable for the preparation of new biologically relevant compounds.

Results and discussion Synthesis
The study of the Mannich reaction employing 2-aminoquinolin-4(1H)-one 1 was performed with use of selected primary amines (β-alanine, 1-phenylethanamine, propylamine) and secondary amines (dimethylamine, piperidine, morpholine) (Scheme 1). Although the Mannich reaction of aminoquinolinone 1 with secondary amines afforded mainly the expected compounds 2a-c, it was also accompanied with numerous by-products. In the case of morpholine and piperidine, the major by-product in a yield ranging from 15 to 20% was isolated and identified as 3,3'-methylenebis(2-aminoquinolin-4(1H)-one) 3. In the case of dimethylamine, the expected product 2a was accompanied with pyrimido [4,5-b]quinolin-5-one 4 formed in a yield of 25%. When reaction was carried out in ethanol instead of N,N-dimethylformamide (DMF), pure compounds 2a-c without the formation of side products were isolated. In contrast to secondary amines, the Mannich reaction with primary amines in ethanol did not provide expected products, but formation of tetrahydropyrimidine derivatives 5a-c was observed. As it was expected, the purity and yield of compounds 5a-c were higher when the quantity of paraformaldehyde was raised to 2 equiv. (Fig 1).
Formation of tetrahydropyrimido [4,5-b]quinolin-5-ones 5 clearly demonstrates the ability of 2-amino-4(1H)-quinolinone to act as both C/N-nucleophile in the cascade reaction. The reaction mechanism probably involved formation of the standard Mannich-type intermediate A, which was converted by paraformaldehyde to the corresponding iminium salt B. The reaction sequence was accomplished by the intramolecular nucleophilic addition to give the tetrahydropyrimidine scaffold of derivative 5 (Fig 2).
A similar reaction was undoubtedly responsible for the formation of compound 4 (Scheme 3) from the starting material 1, in situ formed derivative 2a and paraformaldehyde. A significant role was probably played by different nucleophilicity of amino groups in intermediate E, in which the secondary amine reacts predominantly with formaldehyde to afford iminium salt F, which is subsequently transformed to final product 4. (Fig 3) When the aminoquinolinone 1 was treated only with paraformaldehyde, the quinolinone dimer 3 was formed as the main product at ambient temperature, while at 90˚C a significant  (Retro)Mannich reaction of aminoquinolinone amount of derivative 6 was observed as co-product. More surprisingly, the same mixture of products was observed in LC/MS spectra when Mannich derivatives 2a-c were heated in DMF at the same temperature (Fig 4).
This fact can be explained by the possible formation of the intermediate H originating from the reaction of quinolinone 1 with paraformaldehyde or from decomposition of Mannich products 2a-c ( Fig 5). The intermediate H reacts under Michael addition with 2-aminoquinolin-4(1H)-one 1 to afford intermediate I, followed by the final tautomerization yielding the product 3 ( Fig 5). The dimerization of the similar 2-amino-quinolinone derivatives via reaction of quinolinone with paraformaldehyde was previously observed by Bany et al., [19] but the mechanism has not been discussed to date. Tris-(2-aminoquinolin-4(1H)-one) 6 was finally formed by reaction of derivative 3 with in-situ generated intermediate H (Fig 5).
Our effort to prove the existence of intermediate G or H was not successful, probably due to their instability and rapid transformation to the product 3. When 2-aminoquinolin-4 (1H)-one 1 was subjected to the reaction with paraformaldehyde (1, 3 or 6 equiv.) at ambient temperature without the presence of amines, only the target 3,3'-methylenebis(2-aminoquinolin-4(1H)-one) 3 was obtained, whereas the suggested intermediates G or H were not detected.
The theory of the intermediate H formation was indirectly confirmed by the method of crossed reactions when compound 2a was heated in the presence of indol as a concurrent Cnucleophile. In accordance with our expectation, the corresponding 3-((1H-indol-3-yl) methyl)-2-aminoquinolin-4(1H)-one 7 was isolated. This fact points to the retro-Mannich mechanism of the reaction (Fig 6). When thiophenol was used instead of indol, the sulphidic derivative 8 was obtained.

Structural analysis of prepared compounds
Molecular structures of all compounds were determined by solution NMR spectroscopy. For the univocal structure determination of compound 6 measurements and elaborate analysis of 1D and 2D spectra, including 1H-15N correlation spectra and spectra recorded at variable temperature were performed. The spectra and their detailed discussion are given in Supplementary Information (see S20, S21, S34 and S37-S43 Figs). In addition to NMR spectroscopy, structures of derivatives 2b and 5a were unambiguously confirmed by single-crystal X-ray analysis (Fig 7).

Conclusion
In this article, we reported the study of 2-amino-4(1H)-quinolinone reactivity under Mannich reaction conditions. Apart from expected products, the reaction provided various unexpected compounds exhibiting interesting structures. Further, we observed a thermal instability of Mannich products leading to the plausible formation of reactive methylene intermediate, which can allow synthesis of polycyclic heterocycles via retro-Mannich reaction or enable conjugation with other nucleophiles. The developed procedures can be applied not only for modification of 2-amino-4(1H)-quinolinone, but also for a synthesis of quite new heterocyclic scaffolds with application in any area of chemistry.
High resolution mass spectrometer Exactive based on orbitrap mass analyser was equipped with Heated Electrospray Ionization (HESI). The spectrometer was tuned to obtain maximum response for m/z 70-800. The source parameters were set to the following values: HESI temperature 150˚C, spray voltage +3.5 kV, -3 kV; transfer capillary temperature 320˚C, sheath gas/ aux gas (nitrogen) flow rates 40/20. The HRMS spectra of target peaks allowed to evaluate their elemental composition with less than 1 ppm difference between experimental and theoretically calculated value.  The compounds were purified by reversed phase semipreparative HPLC chromatograph (Agilent Technologies, 1200 Series, USA) consisted of two pumps enabling high-pressure gradient elution, manual valve injector with 1-ml injection loop, UV-VIS detector and fraction collector. The column C18 Pro (particle size 5 μm, length 100mm, I. D. 20 mm, YMC, Japan) was applied for chromatographic separation. The linear gradient elution consisted of 80:20% 0.01 M ammonium acetate buffer:acetonitrile to 10:90% in 13 min and then the composition of mobile phase was kept for 2 min to wash the column. The column was isocraticaly equilibrated for 5 min for next separations. The mobile phase flow rate was set to 15 mL min-1. 200 μL of crude sample was repeatedly injected for separation. The software ChemStation (version B 04.02) was applied for controlling of the instrument and data evaluation.

Synthetic procedures
General procedure for compounds 2a-c. 2-Amino-1H-quinolin-4-one 1 (200 mg, 1.2 mmol) was dissolved in ethanol (5 mL) followed by addition of paraformaldehyde (37.5 mg, 1.2 mmol) and secondary alkylamine Ã (1.2 mmol). The mixture was stirred at 50˚C for 6 hours. The solvent was evaporated in vacuum and the residual solid was suspended in water. The resulting product 2 was filtered off and dried.