Formation of 5-Aminomethyl-2,3-dihydropyridine-4(1H)-ones from 4-Amino-tetrahydropyridinylidene Salts

Various 4-aminotetrahydropyridinylidene salts were treated with aldehydes in an alkaline medium. Their conversion to 5-substituted β-hydroxyketones in a one-step reaction succeeded only with an aliphatic aldehyde. Instead, aromatic aldehydes gave 5-substituted β-aminoketones or a single δ-diketone. The new compounds were characterized using spectroscopic methods and a single crystal structure analysis. Some of them showed anticancer and antibacterial properties.

2,3-Dihydropyridin-4(1H)-one is a partial structure of cenocladamide, an alkaloid from Piper cenocladum [24].This alkaloid and derivatives thereof have previously been investigated for their anticancer potency [25].Furthermore, the dihydropyridin-4(1H)one moiety is part of derivatives of piperlongumine, which have been investigated for their anticancer activity [26] as well as of the potent antibacterial MRX-I [27] (Figure 1).Therefore, we investigated the new compounds for their anticancer activities as well as their antibacterial potency in vitro.
2,3-Dihydropyridin-4(1H)-one is a partial structure of cenocladamide, an alkaloid from Piper cenocladum [24].This alkaloid and derivatives thereof have previously been investigated for their anticancer potency [25].Furthermore, the dihydropyridin-4(1H)-one moiety is part of derivatives of piperlongumine, which have been investigated for their anticancer activity [26] as well as of the potent antibacterial MRX-I [27] (Figure 1).Therefore, we investigated the new compounds for their anticancer activities as well as their antibacterial potency in vitro.
or an alkenyl substituent in ring position 5 of a dihydropyridine-4-one has been reported [22,23].
2,3-Dihydropyridin-4(1H)-one is a partial structure of cenocladamide, an alkaloid from Piper cenocladum [24].This alkaloid and derivatives thereof have previously been investigated for their anticancer potency [25].Furthermore, the dihydropyridin-4(1H)-one moiety is part of derivatives of piperlongumine, which have been investigated for their anticancer activity [26] as well as of the potent antibacterial MRX-I [27] (Figure 1).Therefore, we investigated the new compounds for their anticancer activities as well as their antibacterial potency in vitro.

Chemistry
The different 4-dialkylaminotetrahydropyridinylidene salts 1a-1d used as starting products were prepared as described earlier [1].They were exposed for several days to aromatic aldehydes or aliphatic aldehydes in an alkaline medium at room temperature.Hydrolysis of 1a-1d in ring position 4 was taken into account, but the expected β-hydroxyketones 11 and 12 were only formed as the main product from the aliphatic cyclohexane carbaldehyde and pivalaldehyde, whereas the β-aminoketones 6a and 6b were isolated as by-products.The reaction of salts 1a-1d with aromatic aldehydes afforded mainly β-aminoketones (compounds 2-5 and 7-10) (Scheme 2).
The different 4-dialkylaminotetrahydropyridinylidene salts 1a-1d used as starting products were prepared as described earlier [1].They were exposed for several days to aromatic aldehydes or aliphatic aldehydes in an alkaline medium at room temperature.Hydrolysis of 1a-1d in ring position 4 was taken into account, but the expected βhydroxyketones 11 and 12 were only formed as the main product from the aliphatic cyclohexane carbaldehyde and pivalaldehyde, whereas the β-aminoketones 6a and 6b were isolated as by-products.The reaction of salts 1a-1d with aromatic aldehydes afforded mainly β-aminoketones (compounds 2-5 and 7-10) (Scheme 2).

Entry
R Due to the low solubility in water of solid ferrocenyl carbaldehyde and 4-ethoxy-3methoxybenzaldehyde, ethanol was added to the reaction mixture.Other than the so far reported reactions, these electron-sufficient aldehydes resulted in dimers 13 and 14 (Scheme 3).
In order to confirm the initial steps of the proposed mechanism, we treated the 4dialkylaminotetrahydropyridinylidene salt 1a with potassium hydroxide for 4 days to yield dihydropyridone 15, quantitatively.Then, a mixture of pyrrolidine and benzaldehyde in alkaline solution was added and considerable amounts of 2a were formed overnight.The reaction of 15 without secondary amine resulted in a high yield of the hydroxy analog 11 (Scheme 4).Due to the low solubility in water of solid ferrocenyl carbaldehyde and 4-ethoxy-3-methoxybenzaldehyde, ethanol was added to the reaction mixture.Other than the so far reported reactions, these electron-sufficient aldehydes resulted in dimers 13 and 14 (Scheme 3).
In order to confirm the initial steps of the proposed mechanism, we treated the 4-dialkylaminotetrahydropyridinylidene salt 1a with potassium hydroxide for 4 days to yield dihydropyridone 15, quantitatively.Then, a mixture of pyrrolidine and benzaldehyde in alkaline solution was added and considerable amounts of 2a were formed overnight.The reaction of 15 without secondary amine resulted in a high yield of the hydroxy analog 11 (Scheme 4).

Structure Elucidation
The structures of the obtained compounds were elucidated using NMR spectroscopy: In the 13 C-spectra, a signal shift from 162 to 189 ppm was observed for C-4 due to ketone formation.Furthermore, the resonance of the proton in position 5 disappeared in the 1 H spectra.The remaining olefinic proton showed a coupling to the proton in ring position 1.Connectivity was proven by cross peaks of the newly formed methine proton to C-4 and C-5 as well as to NCH2 and aromatic carbons in the HMBC spectra.Through-space couplings were detected in NOE experiments (Nuclear Overhauser experiments) (Figure 2).Both can be seen in the Supplementary Materials.

Structure Elucidation
The structures of the obtained compounds were elucidated using NMR spectroscopy: In the 13 C-spectra, a signal shift from 162 to 189 ppm was observed for C-4 due to ketone formation.Furthermore, the resonance of the proton in position 5 disappeared in the 1 H spectra.The remaining olefinic proton showed a coupling to the proton in ring position 1.Connectivity was proven by cross peaks of the newly formed methine proton to C-4 and C-5 as well as to NCH 2 and aromatic carbons in the HMBC spectra.Through-space couplings were detected in NOE experiments (Nuclear Overhauser experiments) (Figure 2).Both can be seen in the Supplementary Materials.Finally, evidence of the structure of compound 6a was achieved using a single X-ray crystal analysis which confirmed the compound as 5-[cyclohexyl(pyrrolidin-1-yl)methyl]-2,2-dimethyl-2,3-dihydropyridin-4(1H)-one.This is the first determination of a structure containing a (pyrrolidin-1-yl) substituent in a 2,3-dihydropyridin-4(1H)-one (Figure 3).All atoms lie on general positions.The compound is a racemate.

Biological Activities
Most of the new compounds were tested for their anti-proliferative activity against human leukemia cells (CCRF-CEM) as well as against non-tumorigenic human lung fibroblasts (MRC-5) using an XTT assay.Cells were exposed to compounds at concentrations of 5 and 50 µM for a time period of 72 h.The results are presented in Figure 4A,B.Finally, evidence of the structure of compound 6a was achieved using a single X-ray crystal analysis which confirmed the compound as 5-[cyclohexyl(pyrrolidin-1-yl)methyl]-2,2-dimethyl-2,3-dihydropyridin-4(1H)-one.This is the first determination of a structure containing a (pyrrolidin-1-yl) substituent in a 2,3-dihydropyridin-4(1H)-one (Figure 3).All atoms lie on general positions.The compound is a racemate.Finally, evidence of the structure of compound 6a was achieved using a single X-ray crystal analysis which confirmed the compound as 5-[cyclohexyl(pyrrolidin-1-yl)methyl]-2,2-dimethyl-2,3-dihydropyridin-4(1H)-one.This is the first determination of a structure containing a (pyrrolidin-1-yl) substituent in a 2,3-dihydropyridin-4(1H)-one (Figure 3).All atoms lie on general positions.The compound is a racemate.

Biological Activities
Most of the new compounds were tested for their anti-proliferative activity against human leukemia cells (CCRF-CEM) as well as against non-tumorigenic human lung fibroblasts (MRC-5) using an XTT assay.Cells were exposed to compounds at concentrations of 5 and 50 µM for a time period of 72 h.The results are presented in Figure 4A,B.

Biological Activities
Most of the new compounds were tested for their anti-proliferative activity against human leukemia cells (CCRF-CEM) as well as against non-tumorigenic human lung fibroblasts (MRC-5) using an XTT assay.Cells were exposed to compounds at concentrations of 5 and 50 µM for a time period of 72 h.The results are presented in Figure 4A,B.
At concentrations of 5 µM, only the 2-(trifluoromethyl)phenyl aminoketone 4a showed moderate activity against a leukemia cell line.All other test compounds were inactive at this concentration.At 50 µM, the (tert-butylamino)ketone 2d as well as the dimer 11 were still non-effective and the 4-(trifluoromethyl)phenyl aminoketones 5a and 5b had moderate activity.All other compounds showed a selective anti-leukemic effect.However, the aminoketones 6b and 8a, dimer 12, and the β-hydroxyketone 13 were comparably toxic against lung fibroblasts.The most promising selectivity was observed for the 2-(trifluoromethyl)phenyl aminoketone 4a, which showed good anti-leukemic effect paired with low cytotoxicity in fibroblasts at 5 µM.At concentrations of 5 µM, only the 2-(trifluoromethyl)phenyl aminoketone 4a showed moderate activity against a leukemia cell line.All other test compounds were inactive at this concentration.At 50 µM, the (tert-butylamino)ketone 2d as well as the dimer 11 were still non-effective and the 4-(trifluoromethyl)phenyl aminoketones 5a and 5b had moderate activity.All other compounds showed a selective anti-leukemic effect.However, the aminoketones 6b and 8a, dimer 12, and the β-hydroxyketone 13 were comparably toxic against lung fibroblasts.The most promising selectivity was observed for the 2-(trifluoromethyl)phenyl aminoketone 4a, which showed good anti-leukemic effect paired with low cytotoxicity in fibroblasts at 5 µM.
The results of the antibacterial assay against Gram-negative (E.coli) and Grampositive bacteria (B.sub.) are listed in Table 1.The results of the antibacterial assay against Gram-negative (E.coli) and Gram-positive bacteria (B.sub.) are listed in Table 1.

Substance
Escherichia coli Bacillus subtilis
The most active compounds against Escherichia coli were aminoketones 3a, 4b, 5a, and 5b with fluorophenyl or (trifluoromethyl)phenyl substitution as well as their cyclohexyl analog 6a.The most active compound against Bacillus subtilis was the β-hydroxyketone 11.

Instrumentation and Chemicals
Solvents were used without further purification.Aldehydes were purified via chromatographic separation on a small column filled with aluminum oxide 60 active basic (activity I) (Merck, Darmstadt, Germany) to remove acidic impurities.For thin-layer chromatography (TLC), TLC plates with silica gel 60 F254 (Merck, Darmstadt, Germany) were used.Melting points were obtained on an Electrothermal IA 9200 melting point apparatus.IR spectra: Bruker Alpha Platinum ATR FTIR spectrometer (KBr discs); frequencies are reported in cm −1 .The structures of all newly synthesized compounds were determined using one-and two-dimensional NMR spectroscopy.The NMR spectra were acquired with Varian UnityInova 400 (298 K) or Bruker Avance Neo 400 instruments in 5 mm tubes.Some spectra were acquired in CDCl 3 containing 0.03% TMS.Chemical shifts were recorded in parts per million (ppm).For the 1 H spectra, TMS (0.00) was used as the internal standard, and for the 13 C spectra, the central peak of the CDCl 3 peak was used as the internal reference (77.0).Most of the spectra were acquired in DMSO-d 6 .In this case, the central peaks of the solvent signal at 2.49 ppm in the 1 H spectra and at 39.7 ppm in the 13 C spectra served as the internal references.Shifts in the 1 H NMR (400 MHz) and the 13 C NMR (100 MHz) spectra are reported in ppm; 1 H-and 13 C-resonances were assigned using 1 H, 1 Hand 1 H, 13 C-correlation spectra and are numbered as given in Scheme 1. Abbreviations: aromatic H, ArH; aromatic C, ArC; and quaternary aromatic C, ArC q .Signal multiplicities are abbreviated as follows: s, singlet; d, doublet; dd, double doublet; dsept, double septet; quin, quintet; t, triplet; m, multiplet; and br, broad.Coupling constants (J) are reported in Hertz (Hz).Assignments marked with an asterisk are interchangeable.HRMS: Micromass Tofspec 3E spectrometer (MALDI) and GCT Premier (Waters, Milford, MA, USA) (EI, 70 eV) or Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer, Thermo Fisher Scientific, Austin, TX, USA (HESI, capillary voltage 3.5 kV).Electron ionization (EI+, 70 eV, source at 250 • C) mass spectra were acquired on a JMS-T2000GC (AccuTOF TM GC-Alpha) from JEOL Ltd. (Tokyo, Japan) equipped with a direct insertion probe (DIP).The 1 H NMR and 13 C NMR spectra of new compounds are provided in the Supplementary Materials.

Syntheses
Compounds 1a-1d were prepared earlier and their NMR data are in accordance with the reported literature [1].
Compound 11 from 15: Compound 15 (0.412 g (3.29 mmol)) was dissolved in a solution of KOH (0.752 g (13.4 mmol)) in distilled water (15 mL) with sonication.To this solution, cyclohexyl carbaldehyde (0.375 g (3.24 mmol)) was added and the mixture stirred at r.t.After a few minutes a white precipitate was formed.To complete the reaction, the mixture was stirred for a further 4 days at r.t.The white precipitate was sucked off, washed with water, and dried over phosphorous pentaoxide in a desiccator under reduced pressure.Yield: 0.712 g (91%) of pure alcohol 11.
Cytotoxicity was measured via cell metabolic activity, which was determined using XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide).Cells in the logarithmic growth phase were seeded into 96-well plates (flat bottom; 100 µL/well) at a density of 10,000 cells/well.CCRF-CEM cells were immediately used for experiments, whereas MRC-5 cells were incubated overnight before being used for experiments.Both cell lines were treated with compounds (5 and 50 µM) for 72 h before cell metabolic activity was determined using a commercial kit (Cell proliferation kit II, Roche) according to the manufacturer's instructions.CCRF-CEM and MRC-5 cells were incubated with the XTT solution for 4 h and 2 h, respectively.Then, absorbance was measured at 490 nm using a Hidex Sense microplate reader (Hidex, Turku, Finland).Experiments were performed as two independent experiments carried out in triplicates.Cell viability was calculated relative to mock-treated cells (0.1% DMSO) using blank-corrected values.Vinblastine (100 nM) served as a positive control.

Antibacterial Activity
All compounds were dissolved in DMSO at a concentration of 0.01 mg/µL and the disc plate method [31] was used to detect bioactivity against Gram-positive (B.subtilis) and Gram-negative (E.coli) bacteria.The results were noted as the ability of the compound to inhibit the growth of the corresponding test organism by noting the zone of inhibition (ZOI) around a disc (6 mm) as follows: mild clearance = detectable activity (±) (ZOI = 7-9 mm), clearance = active (+) (ZOI = 10 mm), and clear clearance = higher activity (++) (ZOI > 10 mm).Compound 6a has cyclohexane moiety that could have interfered with its ability to penetrate the bacterial outer membrane (lipopolysaccharide layer), disrupting this layer that is structurally significant in Gram-negative bacteria [32], while the addition of fluorine improved Gram-negative activity in 3a, 4b, 5a, 5b.Fluorine has the ability to change the electron distribution of a molecule, leading to modifications in the molecule's pKa, dipole moment, chemical reactivity, and stability.The introduction of fluorine can reduce the basicity of compounds, resulting in improved bioavailability as a result of better permeation via cellular membranes.

Conclusions
Exposition of 4-amino-tetrahydropyridinylidene salts to a series of aldehydes in an alkaline medium afforded 5-substituted dihydropyridin-4(1H)-ones. The expected β-hydroxyketone was only isolated after reaction with cyclohexane carbaldehyde.Aromatic aldehydes were yielded in most cases β-aminoketones and less frequently δ-diketones.Most of the aminoketones exhibited anti-proliferative activity against human leukemia cells.A few of these compounds showed high inhibitory activity against Escherichia coli, whereas the β-hydroxyketone was the most active against Bacillus subtilis.These compounds could be a base for further investigation in order to produce leading compounds that could be further refined and optimized for possible applications.

Figure 2 .
Figure 2. Through-space couplings indicated as arrows in compound 2a.

Figure 3 .
Figure 3. Stereoscopic ORTEP [28] plot of 6a showing the atomic numbering scheme.The probability ellipsoids are drawn at the 50% probability level.The H atoms are drawn with arbitrary radii.Nitrogen atoms are blue, oxygen atoms are red.

Figure 2 .
Figure 2. Through-space couplings indicated as arrows in compound 2a.

Figure 2 .
Figure 2. Through-space couplings indicated as arrows in compound 2a.

Figure 3 .
Figure 3. Stereoscopic ORTEP [28] plot of 6a showing the atomic numbering scheme.The probability ellipsoids are drawn at the 50% probability level.The H atoms are drawn with arbitrary radii.Nitrogen atoms are blue, oxygen atoms are red.

Figure 3 .
Figure 3. Stereoscopic ORTEP [28] plot of 6a showing the atomic numbering scheme.The probability ellipsoids are drawn at the 50% probability level.The H atoms are drawn with arbitrary radii.Nitrogen atoms are blue, oxygen atoms are red.

Figure 4 .
Figure 4. Anti-proliferative activity of compounds 2b-14 against human leukemia cell line CCRF-CEM (A) and against non-tumorigenic MRC-5 lung fibroblasts (B) expressed as mean ± SEM.Cells were treated with 50 and 5 µM of the compounds for 72 h.Cell viability was measured via XTT assay.Viability rates were expressed as a percentage of vehicle-treated control cells (0.1% DMSO).Vinblastine (VB) at a concentration of 100 nM served as positive control.

Figure 4 .
Figure 4. Anti-proliferative activity of compounds 2b-14 against human leukemia cell line CCRF-CEM (A) and against non-tumorigenic MRC-5 lung fibroblasts (B) expressed as mean ± SEM.Cells were treated with 50 and 5 µM of the compounds for 72 h.Cell viability was measured via XTT assay.Viability rates were expressed as a percentage of vehicle-treated control cells (0.1% DMSO).Vinblastine (VB) at a concentration of 100 nM served as positive control.