Introduction

Emergence of antimicrobial resistance, especially multi-drug bacterial resistance has posed worldwide problem in medicine [1, 2]. The Center for Disease Control and Prevention (CDC) estimates that more than 2 million individuals become infected with drug-resistant bacteria annually, and greater than 23,000 individual dies as a result of those infections [3]. Gram-positive bacterium Staphylococcus aureus (Staph) is the causative agent of multitude of diseases including bacteremia or sepsis, pneumonia, endocarditis, and osteomyelitis. Staph became resistant to several antibiotics such as methicillin-resistant Staphylococcus aureus (MRSA) [4,5,6,7,8,9,10], vancomycin-intermediate Staphylococcus aureus (VISA) [11, 12], and vancomycin-resistant Staphylococcus aureus (VRSA) [13,14,15,16]. Micrococci luteus are another Gram-positive bacterium that have been reported to cause pneumonia, meningitis associated with ventricular shunts, septic arthritis, bacteremia, peritonitis, endophthalmitis, and endocarditis. Micrococci have shown resistance to several antibiotics including streptomycin [17]. Pseudomonas aeruginosa is a Gram-negative bacterium that tends to infect people with immunodeficiency and those with indwelling catheters or on respirators. Infection with pseudomonas can lead to urinary tract infections (UTI), sepsis, pneumonia, and pharyngitis. Pseudomonas colonizes the lungs of patients with cystic fibrosis (CF) and contributes to the chronic progressive pulmonary disease and death rate in CF. P. aeruginosa is a Gram-negative bacterium that is intrinsically resistant to β-lactam-based antibiotics such as cephalothin and ampicillin [18,19,20,21,22]. Escherichia coli is the most common Gram-negative pathogen in human, and multiple antibiotic resistance E. coli strains have been reported [23,24,25,26].

2-Oxonicotinonitriles (2-ONN) ring is a core moiety in several biologically important compounds [27, 28]. This motif has been found in HIV-1-non-nucleoside reverse transcriptase inhibitors [27, 29, 30], non-nucleosides HBV inhibitors, and HCV NS5B polymerase inhibitors [31,32,33,34,35], anti-HSV-1 agents [36] (Fig. 1). In addition to their antiviral activity, 2-pyridinone derivatives are known to possess activity against wild type [37] and resistant bacterial strains [38]. Pyridone derivatives have also shown antifungal [39] and antiprotozoal activity [40, 41]. We have identified 2-ONN derivatives with significant anti-SARS and anti-Bacillus subtitis activity (1, 2; Fig. 1) [42]. The structural features of the 2-ONNs derivatives 1, 2 have two aryl substituents and a glycosyl/polar alkyl moieties at the 4 and 6, and N-1 positions, respectively. Though the molecular target for these 2-ONNs is still unknown, it is legitimate, and the structure activity relationship studies of these entities could provide useful antimicrobial agents. It is of interest to probe the effect of substituent at the C-6 and N-1 positions on the antimicrobial activities. Herein, we report on the synthesis of the 4-(p-methoxyphenyl)-6-cyclohexyl-2-ONNs derivatives 3–20 (Table 2; general structures I-II, Fig. 1) and their antimicrobial evaluations.

Fig. 1
figure 1

Structures of biologically active pyridone derivatives (1–2) and the newly synthesized 2-ONN (I, II) derivatives

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Results and discussions

Chemistry

Methods available in the literature for the synthesis of 4,6-disubstituted 2-ONN derivatives involve condensation of 2-substituted acrylamide with enolates [43], one-pot four-component condensation reaction of aromatic aldehyde, ketone, ethyl cyanoacetate in the presence of ammonium acetate [44,45,46,47,48].The later procedure was adapted with minor modification for the synthesis of the 2-ONN derivative 3. Treatment of cyclohexyl methyl ketone with equimolar amounts of p-anisaldehyde, ethyl cyanoacetate, and NH4OAc in reflux EtOH gave the 2-ONN derivative 3 in good yield (Scheme 1).

Scheme 1
scheme 1

Reagents and conditions. a EtOH, 24 h, reflux, 75%

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A schematic representation of the reaction mechanism for the one-pot four-component reaction is illustrated in Fig. 2. The reaction may commence with aldol condensation providing the chalcone intermediate [I], which then reacts with the ethyl cyanoacetate to give the intermediate [II] which cyclizes with ammonia generated from ammonium acetate to give the corresponding dihydropyridine ring [III]. Oxidation of the intermediate [III] would give the pyridone derivative 3. 2-Pyridones are known to exist in two tautomeric forms: amide form and 2-hydroxypyridine form [49, 50], where the distribution ratio of these tautomers, among other factors, could affect the N-/O-alkylation selectivity at the 2-ONN ring. Keto–enol tautomerism in 2-pyridone/2-hydroxypyridine is considered as an intramolecular hydrogen transfer reaction, where the hydrogen atom is transferred between the N and the O sites of the molecule through a transition state (A3) (Fig. 3). Spectroscopic data show the predominance of the keto form over the enol form. IR spectrum of 3 showed νC=O 1647 cm−1, and its13C-NMR spectrum showed the signal for (C2) at 161.8 ppm. Energies of the tautomers of the 2-ONN derivative 3 as well as that of the transition state were calculated via density functional theory (DFT) at ωB97X-D/6-31G* and level of theory [51]. Energies listed in Table 1 coincide with the experimental results supporting the predominance of the keto form (A1) for 3 over the enol form (A3) tautomer (Fig. 4). The calculation results show that the energy of the transition state closer to the enol form compared with the keto form (Table 1). The local ionization potential and electrostatic potential maps are used to identify the nucleophilic or the electrophilic centers in a molecule. The centers for electrophilic attack in 3 are N1 and O2 positions (Fig. 4).

Fig. 2
figure 2

A plausible mechanism for the one-pot four-component synthesis of 3

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Fig. 3
figure 3

Optimized structures of 2-ONN tautomers and their transition state (TS) using DFT-ωB97X-D level of theory

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Table 1 Geometrical optimization of compound 3‘s tautomers and their TS
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Fig. 4
figure 4

A1, A3 Minimized energy for the tautomers of the 2-ONN derivative 3. B1, B2 Electrostatic potential map for the tautomers of the 2-ONN derivative 3. C1, C2 Local ionization map for the tautomers of the 2-ONN derivative 3

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The intrinsic low solubility of the diaryl-2-ONN derivatives in aqueous and most of the organic solvents creates a barrier for those entities to show biological activity. Branching of the diaryl-2-ONN derivatives with either transient or permanent solubilizing groups could overcome their low solubility problem. Glycosylation of small molecules, especially biologically active compounds, with specific mono- and disaccharides has been utilized to modulate their water solubility or their cellular uptake, and hence their pharmacological characteristics [52, 53]. In view of that, the 2-ONN derivative 3 was glycosylated with three glycosyl bromides under basic conditions (Table 2). Treatment of 3 with peracetylated α-D-glucopyranosyl bromide [54] in the presence of K2CO3 in DMF at room temperature gave corresponding N-glucoside derivative 4 in 57% yield along with traces of the corresponding O-glycosyl derivative. In a similar manner, the N-lactoside derivative 6 was prepared by coupling of 3 with peracetylated 1-α-D-lactosyl bromide [55] in 51% yield (Table 2). The N1-glycosidation in 4 and 6 was confirmed by the presence of the amide νC=O signals in the range of 1644–1641 cm−1 in their IR spectra. The diaxial coupling between the anomeric proton and the H-2′ (J1′,2′ = 7.86–8.82 Hz) confirms the β-configuration at the anomeric position of the 2-ONN glycosides (4–6). The observed preference of N-glycosylation over O-glycosylation coincides with the DFT conclusion of the predominance of the keto form of the 2-ONN derivative 3, despite the steric hindrance at the C-6 position. Treatment of the N-glycosides 4 and 6 with triethylamine (TEA) in H2O/MeOH gave the corresponding free N-glycosides 5 and 7, respectively, in good yields. Branching at the amide moiety 2-ONN derivatives with acyclic sugar mimics, alkyl or substituted alkyl derivatives, has resulted in better biological activity compared with the native 2-ONN [42]. Base-catalyzed coupling of 3 with 4-bromobutyl acetate gave a separable mixture of N-butyl acetate derivative 8 and the corresponding O-butyl acetate derivative 10 in good yield (Table 2). Treatment of 3 with 2-bromomethoxyethyl acetate gave a mixture of the corresponding N-acyclic nucleoside mimic 12 and the corresponding O-alkyl derivative 14 in good isolated yields (Table 2). The site of alkylation in the above mentioned reactions is determined by IR and 13C-NMR spectroscopy. For instance, the N-alkylation in 12 is supported by the presence of νC=O signal (1647 cm−1) in its IR spectrum and 170.3 ppm (C2) signal in its 13C-NMR spectrum. Deacetylation of 8, 10, 12, and 14 was performed with Et3N in MeOH/H2O to give 9, 11, 13, and 15, respectively, in good yields. Treatment of 3 with propargyl bromide under (K2CO3/acetone/reflux) conditions gave a separable mixture of the N- and O-propargyl derivatives 16 and 17 in good yields (Table 2). In a similar manner, treatment of 3 with allyl bromide under the same conditions gave a separable mixture of N-allyl and O-allyl derivatives 18 and 19, respectively, in good yields (Table 2). On the other hand, treatment of 3 with epichlorohydrin under the same conditions (K2CO3/acetone/reflux) none of the O-alkyl derivative was isolated, but the N-alkyl derivative 20 was isolated in 42% yield (Table 2).

Table 2 Glycosylation and alkylation of the 2-ONN derivative 3
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Biology

Evaluation of antimicrobial activities

The new synthesized 2-ONN derivatives were screened in vitro for antibacterial activity against the Gram-positive bacteria: Staphylococcus aureus ATCC 6538 and M. luteus ATCC 10240 and Gram-negative bacteria: P. aeruginosa ATCC 9027 and Escherichia coli ATCC 10536. The antifungal activity of the 2-ONN derivatives was assessed against Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404. Agar-diffusion method [42] was used to assess the antibacterial and antifungal activity of the tested compounds. The diameter of inhibition zone (IZ) of the bacterial and fungal growth in mm was used to assess the preliminary antimicrobial activity of the tested compounds [42].

Concentration of the tested samples was 4 mg/mL, ND—not determined

The parent 2-ONN derivative 3 showed significant activity against the Gram-negative bacteria: E. coli ATCC-10536 and P. aeruginosa ATCC-9027, while a moderate activity against the Gram-positive bacteria: S. aureus ATCC-6538 and M. luteus ATCC-10242, was observed (Table 3). Additionally, a significant antifungal activity was observed with the 2-ONN derivative 3, and its N-glucoside derivatives 4 and 5 against Candida albicans and Aspergillus niger (Table 3). It is notable that weak to moderate antibacterial activity is observed with O-alkyl derivatives 11, 17, and 19 and the N-oxiranylmethyl derivative 20. The antifungal activity of selected number of the newly synthesized compounds was evaluated against Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404. The parent compound 3, N-glucosyl acetate derivative 4, its free derivative 5, and N-(2-hydroxyethoxy)methyl derivative 13 showed superior antifungal activity against both strains compared with nystatin. The data observed with compounds 4, 5, 11, 13, 16–20 imply that branching of the parent 2-ONN derivative 3 with solubilizing groups affects the antimicrobial activity.

Table 3 Antibacterial and antifungal activity of selected 2-ONNs derivatives
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Conclusions

A one-pot four-component reaction was utilized to synthesize the 6-cyclohexyl-4-methoxy-2-ONN derivative 3 in good yield. DFT calculations of tautomeric forms of 3 show the predominance of the 2-keto form, (A1) over the enol form (A2). Branching of the amide moiety of 3 by glycosylation gave the corresponding N-glycosides in good yields. Alkylation of 3 with several alkyl halides gave separable mixtures of the corresponding N- and O-alkyl derivatives. Significant antibacterial activity was observed with 13, 16, and 18 against S. aureus ATCC-6538 and M. luteus ATCC-10242, while the parent 2-ONN derivative 3 showed good activity against E. coli. On the other hand, a significant antifungal activity was observed in 2-ONN derivative 3, its N-glucoside derivatives 4 and 5 against Candida albicans and Aspergillus niger. The results of this study provide hits for further optimizations for the discovery of potent antimicrobial agents.

Experimental results and protocols

Material, instruments, and general considerations

Starting materials and reagents were purchased from Acros-Organics, Alpha Aesar, and Sigma-Aldrich. 4-Bromobutyl acetate [56] and 2-bromomethoxyethyl acetate [57, 58] were prepared according to the literature procedures. Reaction’s progress was monitored by TLC analysis using aluminum-backed plates pre-coated with Merck silica gel 60 F254. Column chromatography was carried out on Agela Technologies flash silica 40–60 mesh. Melting points were recorded on Electrothermal IA 9100 apparatus and were uncorrected. IR spectra (KBr disk) were recorded on either a Pye Unicam Sp 3-300 or a Shimadzu FTIR 8101 PC infrared spectrometer. 1H and 13C-NMR spectra were recorded on Bruker 400 MHz spectrometer. Chemical shifts (δ) were reported in ppm and were referenced to TMS as a standard (0.00 ppm). Chemical shifts for 13C-NMR were referenced relative to DMSO (39.50 ppm). Elemental microanalysis was recorded on PerkinElmer 240 analyzer. Analyses indicated by the symbols of the elements were within 0.4% of the theoretical values.

Chemistry

6-Cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (3)

A mixture of 4-methoxybenzaldehyde (6.80 g, 0.05 mol), cyclohexyl methyl ketone (6.31 g, 0.05 mol), and ammonium acetate (30.8 g, 0.4 mol) in ethanol (150 mL) was refluxed for 48 h. The reaction mixture was cooled to room temperature, and precipitate was filtered off, washed with ethanol, and dried under vacuum. The precipitate was recrystallized from a mixture of acetic acid/ethanol (1:5 v:v) to give 3 (11.56 g, 75%) as a pale yellow crystals: m.p. 242–244 °C; IR (KBr): 3286 cm−1 (NH), 2036 cm−1 (CN) and 1647 cm−1 (C=O, amide); 1H-NMR (DMSO-d6) δ 1.23–1.84 (11H, m, cyclohexyl), 3.83 (3H, s, OCH3), 6.26 (1H, s, H-5, pyridone), 7.07 (2H, d, Ar–H, J = 8.7 Hz), 7.60 (2H, d, Ar–H,J = 8.7 Hz), 12.36 (1H, s, NH); 13C-NMR (DMSO-d6)δ25.03, 25.66, 30.80, 41.64, 55.39, 88.9, 104.0, 114.2, 116.9, 128.2, 129.8, 159.6, 159.8, 161.0, 161.8; Anal. Calcd for C19H20N2O2 (308.37): C, 74.00; H, 6.54; N, 9.08. Found: C, 74.09; H, 6.55; N, 9.09.

1-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyranosyl)-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (4)

A mixture of 3 (1 g, 3.24 mmol) and anhydrous K2CO3 (0.45 g, 3.27 mmol) in dry DMF (10 mL) was stirred for 30 min. at 0 °C. 1-Bromo-2,3,4,6-tetra-O-acetyl-D-glucopyranose [54] (1.23 g, 3.0 mmol) was added to reaction mixture in portions, and the mixture was stirred overnight at room temperature. Aqueous 1 N NH4Cl (5 mL) was added, and the solvents were evaporated under reduced pressure. The residue was partitioned between EtOAc and H2O; the organic phase was dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by a silica gel column chromatography (eluate: 10% EtOAc/hexanes) to give 4 (1.18 g,57%yield) as a white powder: m.p. 184–186 °C; IR (KBr) 2228 cm−1 (CN) and broad band at 1754 cm−1 (C=O, acetoxy and amide); 1H-NMR (DMSO-d6) δ 1.30–1.85 (10H, m, cyclohexyl), 1.97, 1.98, 2.00, 2.02 (12H, 4 s, 4CH3CO), 2.74 (1H, t, H1-cyclohexyl, J = 2.80 Hz), 3.83 (3H, s, OCH3), 4.01 (1H, d, H-6′, J6′,5′ = 4.96 Hz, J6′,6″ = 11.71 Hz), 4.18 (1H, dd, H-6″,J6′’,5′ = 5.60 Hz, J6′’,6′ = 5.60 Hz), 4.32 (1H, m, H-5′), 5.03 (1H, t, H-3′,J3′,2′ = 9.60 Hz, J3′,4′ = 10.0 Hz), 5.17 (1H, t, H-2′,J2′,3′ = 4.80 Hz,J2′,1′ = 8.39 Hz), 5.60 (1H, t, H-4′,J4′,5′ = 9.6 Hz, J4′,3′ = 9.6 Hz), 6.48 (1H, d, H-1′, J1′,2′ = 8.39 Hz), 7.12 (1H, d, Ar–H,J = 8.8 Hz,), 7.21 (1H, s, pyridone-H-5), 7.64 (1H, d, Ar–H, J = 8.8 Hz);13C-NMR (DMSO-d6)δ 20.18, 20.24, 20.27, 20.32, 25.36, 25.67, 25.72, 31.25, 55.34, 61.85, 68.13, 70.06, 71.45, 72.02, 91.22, 93.24, 114.2, 114.3, 116.7, 127.5, 130.0, 155.5, 160.7, 161.7, 168.3, 168.7, 169.2, 169.5, 169.8; Anal. Calcd for C33H38N2O11 (638.66): C, 62.06; H, 6.00; N, 4.39. Found: C, 62.07; H, 6.01; N, 4.37.

6-Cyclohexyl-1-(β-D-glucopyranosyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (5)

A mixture of 4 (0.63 g, 1.0 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was crystallized from 95% ethanol gave 5 (0.42 g, 90% yield) as a white powder: m.p. 246–248 °C; IR (KBr) 3427 cm−1 (4OH), 2217 cm−1 (CN) and 1644 cm−1 (C=O, amide); 1H-NMR (DMSO-d6-D2O) δ1.16–1.84 (10H, m, cyclohexyl), 2.60 (1H, t, cyclohexyl-H-1, J = 2.82 Hz), 3.1–3.5 (6H, m, H-6′, H-6″, H-5′, H-4′, H-3′ and H-2′), 3.83 (3H, s, OCH3), 6.27 (1H, d, H-1′, J1′,2′ = 8.21 Hz), 6.98 (1H, s, pyridone-H-5), 7.08 (2H, d, Ar–H, J = 8.84 Hz), 7.61 (2H, d, Ar–H, J = 8.84 Hz); Anal. Calcd for C25H30N2O7 (470.51): C, 63.82; H, 6.43; N, 5.95. Found: C, 63.80; H, 6.42; N, 5.96.

1-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl-(1 → 4)-(2′,3′,6′-tri-O-acetyl-β-D-glucopyranosyl)-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (6)

A mixture of 3 (1 g, 3.24 mmol) and anhydrous K2CO3 (0.45 g, 3.27 mmol) in dry DMF (10 mL) was stirred for 30 min. at 0 °C. 1-Bromo-2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl-(1 → 4)-(2′,3′,6′-tri-O-acetyl-β-D-glucopyranose [55] (1.85 g, 3.0 mmol) was added to the reaction mixture in portions, and the mixture was stirred for 14 h at room temperature. Aqueous 1 N NH4Cl (5 mL) was added, and the solvents were evaporated under reduced pressure. The residue was partitioned between EtOAc and H2O; the organic phase was dried (Na2SO4) and evaporated under reduced pressure. The residue was partitioned between EtOAc and H2O; the organic phase was separated, dried (Na2SO4), and evaporated under reduced pressure. The residue was purified by a silica gel column chromatography to give 6 (1.47 g, yield 49%) as a pale yellow powder: m.p.150–152 °C. IR (KBr) 2227 cm−1 (CN) and broad band at 1752 cm−1 (C=O, acetoxy), 1644 cm−1 (C=O, amide) amidic groups); 1H-NMR (DMSO-d6) δ 1.12–1.85 (10H, m, cyclohexyl), 1.94, 1.96, 1.97, 2.01, 2.03, 2.05, 2.06 (21H, 7 s, 7CH3CO), 2.73 (1H, t, cyclohexyl-H-1,J = 2.86 Hz), 3.83 (3H, s, OCH3), 4.03–4.08 (3H, m, H-2′b, H-6′a and H-6′b), 4.15 (1H, dd, H-6″a, J6″a,6′a = 11.49, J6″a,5′a = 5.61 Hz), 4.26 (1H, m, H-5′b), 5.03 (1H, dd, H-6″b,J6″b,5′b = 6.32 Hz), 5.07 (1H, m, H-5′a), 5.19 (1H, dd, H-1′b, J1′b,2′b = 7.86 Hz), 5.22 (1H, dd, H-4′b,J4′b,3′b = 3.42, J4′b,5′b = 3.82 Hz), 5.36 (1H, dd, H-2′a, J2′a,1′a = 8.97, J2′a,3′a = 8.40 Hz), 5.38 (1H, dd, H-4′a, J4′a,3′a = 9.15, J4′a,5′a = 6.90 Hz), 5.64 (1H, d, H-3′b,J3′b,4′b = 3.45 Hz), 6.44 (1H, d, H-3′a, J3′a,2′a = 6.40 Hz), 6.56 (1H, d, H-1′a,J1′a,2′a = 8.82 Hz), 7.12 (1H, d, Ar–H, J = 7.99 Hz), 7.23 (1H, s,pyridone-H-5), 7.63 (1H, d, Ar–H, J = 8.39 Hz); Anal. Calcd for C45H54N2O19 (926.91): C, 58.31; H, 5.87; N, 3.02. Found: C, 58.30; H, 5.88; N, 3.02.

1-(β-D-galactopyranosyl-(1 → 4)-(β-D-glucopyranosyl)-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (7). A mixture of 6 (0.92 g, 1.0 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was crystallized from 95% ethanol to give 7 (0.55 g, 87% yield) as a pale yellow powder: m.p. 220–222 °C; IR (KBr) 3412 cm−1 (7OH), 2224 cm−1 (CN) and 1641 cm−1 (C=O, amide); 1H-NMR (DMSO-d6/D2O) δ 1.16–1.89 (10H, m, cyclohexyl), 2.83 (1H, t, H-1cyclohexyl, J = 2.83 Hz), 3.85 (3H, s, OCH3), 3.94–4.12 (3H, m, H-2′b, H-6′a and H-6′b), 4.16 (1H, dd, H-6″a, J6″a,6′a = 11.59, J6″a,5′a = 5.64 Hz), 4.22 (1H, m, H-5′b), 4.56 (1H, dd, H-6″b, J6″b,5′b = 6.35 Hz), 4.98 (1H, m, H-5′a), 5.14 (1H, dd, H-1′b, J1′b,2′b = 7.76 Hz), 5.20 (1H, dd, H-4′b, J4′b,3′b = 3.62, J4′b,5′b = 3.86 Hz), 5.31 (1H, dd, H-2′a, J2′a,1′a = 8.87 Hz, J2′a,3′a = 8.44 Hz), 5.34 (1H, dd, H-4′a, J4′a,3′a = 9.05, J4′a,5′a = 6.56 Hz), 5.62 (1H, d, H-3′b, J3′b,4′b = 3.55 Hz), 6.24 (1H, d, H-3′a, J3′a,2′a = 6.43 Hz), 6.76 (1H, d, H-1′a, J1′a,2′a = 8.80 Hz), 7.15 (1H, d, Ar–H, J = 7.99 Hz), 7.25 (1H, s, pyridone-H-5), 7.69 (1H, d, Ar–H, J = 8.39 Hz); Anal. Calcd for C31H40N2O12 (632.66): C, 58.85; H, 6.37; N, 4.43. Found: C, 58.86; H, 6.37; N, 4.43.

4-(3-Cyano-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)butyl acetate (8) and 4-((3-Cyano-6-cyclohexyl-4-(4-methoxyphenyl)pyridin-2-yl)oxy)butyl acetate (10). A mixture of 3 (1 g, 3.24 mmol) with 4-bromobutyl acetate (0.58 g, 3.0 mmol) was added to dry acetone (10 mL) in the presence of anhydrous K2CO3 (0.45 g, 3.27 mmol); the reaction mixture was heated for 15 h at reflux temperature. The reaction mixture was cooled to room temperature, and the insoluble were removed by filtration. The filtrate was evaporated under reduced pressure and then the formed solid product was separated by silica gel chromatography (eluate; CH2Cl2to 5% EtOAc/CH2Cl2) to give 8 (0.44 g, 32% yield) as a pale yellow oil and 10 (0.65 g, 48% yield) as a pale yellow oil. Spectral and analytical data for 8: IR (KBr): 2219 cm−1 (CN), 1734 cm−1 (C=O, acetoxy) and 1644 cm−1 (C = O, amide);1H NMR (DMSO-d6)δ1.13–1.86 (14H, m, cyclohexyl, CH2b, and CH2c), 2.01 (3H, s, COCH3), 2.55 (1H, t, H-1cyclohexyl, J = 2.82 Hz), 3.83 (3H, s, OCH3), 4.07 (t, 1H,CH2a, J = 5.60, 5.95 Hz), 4.46 (t, 1H,CH2d, J = 7.93, 12.81 Hz), 6.41 (s, 1H, H-5 pyridone), 7.09 (2H, d, Ar–H, J = 8.00 Hz), 7.63 (2H, d, Ar–H, J = 9.19 Hz); Anal. Calcd for C25H30N2O4 (422.52): C, 71.07; H, 7.16; N, 6.63. Found: C, 71.08; H, 7.19; N, 6.66. Spectral and analytical data for 10: IR (KBr)2223 cm−1 (CN) and 1734 cm−1 (C=O, acetoxy); 1H-NMR (DMSO-d6)δ1.12–1.87 (14H, m, cyclohexyl, CH2b, and CH2c), 1.97 (3H, s, COCH3), 2.62 (1H, t, cyclohexyl-H-1, J = 2.81 Hz), 3.81 (3H, s, OCH3), 4.05 (1H, t, CH2a, J = 6.01 Hz), 4.45 (1H, t, CH2(d), J = 5.62 Hz), 7.03 (1H, s, pyridone-H-5), 7.07 (2H, d, Ar–H, J = 8.69 Hz), 7.58 (2H, d, Ar, J = 8.69 Hz);Anal. Calcd. for C25H30N2O4 (422.52): C, 71.07; H, 7.16; N, 6.63. Found: C, 71.09; H, 7.15; N, 6.64.

6-Cyclohexyl-1-(4-hydroxybutyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (9). A mixture of 8 (0.5 g, 1.18 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was purified by silica gel column chromatography (eluate 10% MeOH/CH2Cl2) to give 9 (0.36 g, 80% yield) as a pale yellow syrup: IR (KBr) 3428 cm−1 (OH), 2221 cm−1 (CN) and 1642 cm−1 (C=O, amide); 1H-NMR (DMSO-d6/D2O) δ 1.22–1.87 (14H, m, cyclohexyl, and CH2b, and CH2c), 2.68 (1H, t, H1-cyclohexyl, J = 2.80 Hz), 3.44 (2H, t, CH2d, J = 6.42 Hz), 3.81 (3H, s, OCH3), 4.43 (2H, t, CH2a, J = 6.82 Hz), 7.02 (1H, s, H-5 pyridone), 7.08 (2H, d, Ar–H, J = 7.64 Hz), 7.59 (2H, d, Ar–H, J = 7.64 Hz); Anal. Calcd for C23H28N2O3 (380.48): C, 72.60; H, 7.42; N, 7.36. Found: C, 72.63; H, 7.45; N, 7.35.

6-Cyclohexyl-2-(4-hydroxybutoxy)-4-(4-methoxyphenyl)nicotinonitrile (11). A mixture of 10 (0.42 g, 1.0 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was purified by a silica gel column chromatography (eluate: 7% MeOH/CH2Cl2) to give 11 (0.31 g, 83% yield) as a colorless syrup: IR (KBr) 3426 cm−1 (OH) and 2222 cm−1 (CN); 1H-NMR (DMSO-d6) δ 1.33–1.89 (14 H, m, cyclohexyl, CH2b, and CH2c), 2.68 (1H, t, H-1cyclohexyl, J = 2.80 Hz), 3.08 (1H, d, exchangeable with D2O, OH, J = 7.21 Hz), 3.43 (2H, t, CH2d, J = 5.62, J = 6.40 Hz), 3.83 (3H, s, OCH3), 4.43 (2H, t, CH2a), J = 6.42, J = 6.01 Hz), 7.04 (1H, s, H-5pyridone), 7.08 (2H, d, Ar–H, J = 8.46 Hz), 7.60 (2H, d, Ar–H, J = 8.46 Hz); Anal. Calcd for C23H28N2O3 (380.48): C, 72.60; H, 7.42; N, 7.36. Found: C, 72.65; H, 7.44; N, 7.39.

2-((3-Cyano-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)methoxy)ethyl acetate (12) and 2-(((3-Cyano-6-cyclohexyl-4-(4-methoxyphenyl)pyridin-2-yl)oxy)methoxy)ethyl acetate (14). A mixture of 3 (1 g, 3.24 mmol) with (2-acetoxyethoxy)methyl bromide (0.59 g, 3.0 mmol) was added to dry acetone (10 mL) in the presence of anhydrous K2CO3 (0.45 g, 3.27 mmol); the reaction mixture was heated for 15 h under reflux temperature. The reaction mixture was cooled to room temperature, and the inorganic residue was removed by filtration. The solvent was evaporated under reduced pressure and then the residue was purified by silica gel chromatography (eluate: CH2Cl2to 3% EtoAc in CH2Cl2) to give 12 (0.37 g, 27% yield) as a colorless oil and 14 (0.57 g, 42% yield) as a pale yellow syrup. Spectral and analytical data for 12: IR (KBr) 2222 cm−1 (CN), 1735 cm−1 (C=O, acetoxy) and 1647 cm−1 (C=O, amide); 1H-NMR (DMSO-d6) δ 1.58–1.84 (10H, m, cyclohexyl), 2.01 (3H, s, COCH3), 2.26 (1H, t, H1-cyclohexyl-, J = 2.84 Hz), 3.79 (3H, s, OCH3), 4.06 (3H, t, CH2(c), J = 4.81, 6.00 Hz), 4.47 (3H, t, CH2(d), J = 5.63, 4.81 Hz), 4.60 (2H, s, CH2(a)), 7.05 (1H, s, H5-pyridone), 7.63 (2H, d, Ar–H, J = 8.82 Hz), 7.90 (2H, d, Ar–H, J = 8.82 Hz); 13C-NMR (DMSO-d6) δ 18.80, 20.67, 24.10, 25.15, 28.84, 30.62, 60.32, 63.32, 66.91, 99.63, 116.0, 120.2, 127.9, 128.1, 129.3, 130.8, 133.2, 134.5, 159.4, 170.3; Anal. Calcd for C24H28N2O5 (424.49): C, 67.91; H, 6.65; N, 6.60. Found: C, 67.94; H, 6.63; N, 6.60. Spectral and analytical data for 14: IR (KBr)2222 cm−1 (CN) and 1740 cm−1 (C=O, acetoxy); 1H-NMR (DMSO-d6) δ 1.47–1.76 (10H, m, cyclohexyl), 1.98 (3H, s, COCH3), 2.65 (1H, t, cyclohexyl-H-1, J = 2.85 Hz), 3.83 (3H, s, OCH3), 4.05 (2H, t, CH2(c), J = 6.42 Hz), 4.44 (2H, t, CH2d, J = 5.91 Hz), 4.64 (2H, s, CH2(a)), 7.03 (1H, s, pyridone-H-5), 7.08 (2H, d, Ar–H, J = 8.83 Hz), 7.62 (2H, d, Ar–H, J = 8.83 Hz); Anal. Calcd for C24H28N2O5 (424.49): C, 67.91; H, 6.65; N, 6.60. Found: C, 67.89; H, 6.64; N, 6.60.

6-Cyclohexyl-1-((2-hydroxyethoxy)methyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (13). A mixture of 12 (0.42 g, 1.0 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was crystallized from 95% ethanol to give 13 (0.32 g, 84% yield) as a pale yellow powder: m.p. 60–62 °C; IR (KBr) 3420 cm−1 (OH), 2222 cm−1 (CN) and 1644 cm−1 (C=O, amide); 1H-NMR (DMSO-d6/D2O) δ 1.55–1.86 (10H, m, cyclohexyl), 2.26 (1H, d, H1-cyclohexyl, J = 2.62 Hz), 3.31 (3H, s, OCH3), 3.47 (2H, t, CH2d, J = 5.60 Hz), 4.14 (2H, t, CH2c, J = 6.52 Hz), 4.49 (2H, s, CH2a), 7.03–7.34 (5H, m, Ar–H); Anal. Calcd for C22H26N2O4 (382.45): C, 69.09; H, 6.85; N, 7.32. Found: C, 69.06; H, 6.87; N, 7.32.

6-Cyclohexyl-2-((2-hydroxyethoxy)methoxy)-4-(4-methoxyphenyl)-nicotinonitrile (15). A mixture of 14 (0.42 g, 1.0 mmol), Et3N (1 mL), and H2O (0.1 mL) in MeOH (5 mL) was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure. The residue was repeatedly co-evaporated with MeOH until complete removal of Et3N, and the residue was purified by a silica gel column chromatography (eluate: 6% MeOH in CH2Cl2) to give 15 (0.33 g, 87% yield) as a colorless syrup: IR (KBr) 3428 cm−1 (OH) and 2222 cm−1 (CN); 1H-NMR (DMSO-d6) δ1.24–1.89 (10H, m, cyclohexyl), 2.69 (1H, t, cyclohexyl-H-1, J = 2.83 Hz), 3.45 (2H, t, CH2(d), J = 5.6 Hz), 3.75 (2H, t, CH2(c), J = 4.43 Hz), 3.83 (3H, s, OCH3), 4.45 (2H, s, CH2(a)), 4.87 (1H, t, OH, J = 4.65 Hz, exchange with D2O), 7.06 (1H, s, H5-pyridine-), 7.09 (2H, d, Ar–H, J = 8.00 Hz), 7.61 (2H, d, Ar–H, J = 7.69 Hz); Anal. Calcd. for C22H26N2O4 (382.45): C, 69.09; H, 6.85; N, 7.32. Found: C, 69.11; H, 6.88; N, 7.30.

6-Cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1-(prop-2-yn-1-yl)-1,2-dihydropyridine-3-carbonitrile (16) and 6-Cyclohexyl-4-(4-methoxyphenyl)-2-(prop-2-yn-1-yloxy)nicotinonitrile (17). A mixture of 3 (2 g, 6.48 mmol) with propargyl bromide (0.7 g, 6.5 mmol) was added to dry acetone (20 mL) in the presence anhydrous K2CO3 (0.9 g, 6.5 mmol); the reaction mixture was heated under reflux for 15 h. The inorganic residue was removed by filtration; the solvent was evaporated under reduced pressure and then the formed solid product was separated by silica gel column chromatography (eluate: CH2Cl2 to 1% MeOH/CH2Cl2) to give 16 (0.67 g, 30% yield) as a yellow oil and 17 (0.65 g, 29% yield) as pale yellow oil. Spectral and analytical data for 16: IR (KBr) 2216 cm−1 (CN) and 1641 cm−1 (C=O, amide); 1H-NMR (DMSO-d6) δ 1.14–1.96 (10 H, m, cyclohexyl), 2.90 (1H, t, H-1-cyclohexyl, J = 2.81 Hz), 3.34 (1H, s, ≡ C-H), 3.84 (3H, s, OCH3), 4.99 (2H, d, N-CH2, J = 2.41 Hz), 6.46 (1H, s, H-5-pyridone), 7.09 (2H, d, Ar–H, J = 8.69 Hz), 7.65 (2H, d, Ar–H, J = 9.01 Hz); Anal. Calcd for C22H22N2O2 (346.42): C, 76.28; H, 6.40; N, 8.09. Found: C, 76.26; H, 6.44; N, 8.08. Spectral and analytical data for 17: IR (KBr) 2222 cm−1 (CN); 1H-NMR (DMSO-d6) δ 1.22–1.93 (10 H, m, cyclohexyl), 2.72 (1H, t, H-1-cyclohexyl, J = 2.78 Hz), 3.51 (1H, s, ≡ C-H), 3.84 (3H, s, OCH3), 5.14 (2H, d, OCH2, J = 2.42 Hz), 7.09–7.12 (3H, m, Ar–H and H5-pyridone), 7.61 (2H, d, Ar–H, J = 8.69 Hz); Anal. Calcd for C22H22N2O2 (346.42): C, 76.28; H, 6.40; N, 8.09. Found: C, 76.25; H, 6.45; N, 8.06.

1-Allyl-6-cyclohexyl-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (18) and 2-(Allyloxy)-6-cyclohexyl-4-(4-methoxyphenyl)nicotinonitrile (19): A mixture of 3 (1 g, 3.24 mmol) with allyl bromide (0.36 g, 3.0 mmol) was added to dry acetone (10 mL) in the presence anhydrous K2CO3 (0.45 g, 3.27 mmol); the reaction mixture was heated under reflux for 15 h. The inorganic residue was removed by filtration; the solvent was evaporated under reduced pressure and then the formed solid product was separated by silica gel column chromatography (eluate: CH2Cl2 to 5% EtOAc/CH2Cl2) to give 18 (0.37 g, 32% yield) as a yellow powder and 19 (0.39 g, 39%yield) as a white powder. Spectral and analytical data for 19: m.p. 90–92 °C; IR (KBr) 2213 cm−1 (CN) and 1644 cm−1 (C=O, amide); 1H-NMR (DMSO-d6) δ 1.22–1.91 (10H, m, cyclohexyl), 2.70 (1H, t, cyclohexyl-H-1, J = 2.39 Hz), 2.84 (3H, s, OCH3), 4.98 (2H, d, CH2(a), J = 4.49 Hz), 5.31 (2H, d, Hc’, J = 10.49 Hz), 5.43 (2H, d, Hc, J = 17.41 Hz), 6.12 (1H, m, Hb), 7.06 (1H, s, pyridone-H-5), 7.09 (2H, d, Ar–H, J = 8.69), 7.60 (2H, d, Ar–H, J = 8.69); Anal. Calcd for C22H24N2O2 (348.44): C, 75.83; H, 6.94; N, 8.04. Found: C, 75.85; H, 6.95; N, 8.03. Spectral and analytical data for 18: m.p. 60–62 °C; IR (KBr) 2220 cm−1 (CN); 1H-NMR (DMSO-d6) δ 1.15–1.86 (10H, m, cyclohexyl), 2.72 (1H, t, cyclohexyl-H-1, J = 2.49 Hz), 3.85 (3H, s, OCH3), 4.79 (2H, d, CH2(a), J = 4.49 Hz), 5.06 (1H, d, Hc, J = 17.40 Hz), 5.19 (1H, d, Hc’, J = 10.79 Hz), 5.92–6.05 (1H, m, Hb), 6.42 (1H, s, pyridone-H-5), 7.09 (2H, d, Ar–H, J = 8.69 Hz), 7.64 (2H, d, Ar–H, J = 9.01 Hz); Anal. Calcd for C22H24N2O2 (348.44): C, 75.83; H, 6.94; N, 8.04. Found: C, 75.80; H, 6.92; N, 8.07.

6-Cyclohexyl-4-(4-methoxyphenyl)-1-(oxiran-2-ylmethyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (20). A mixture of 3 (1 g, 3.24 mmol) and anhydrous K2CO3 (0.45 g, 3.27 mmol) in dry acetone (10 mL) was added to epichlorohydrin (0.27 g, 3.0 mmol), and the mixture was heated under reflux for 15 h. The inorganic residue was removed by filtration; the solvent was evaporated under reduced pressure and then the formed solid product was purified by silica gel column chromatography (eluate: 1% MeOH in CH2Cl2) to give 20 (0.47 g, 42%) as a colorless syrup, IR (KBr) 2222 cm−1 (CN) and 1608 cm−1 (C=O, amide); 1H-NMR (DMSO-d6) δ 1.14–1.84 (10H, m, cyclohexyl), 2.51 (1H, t, cyclohexyl, J = 2.81 Hz), 3.83 (3H, s, OCH3), 4.33–4.36 (1H, m, Hc’-oxiran ring), 4.44 (1H, t, Hc-oxiran ring, J = 4.82 Hz), 4.49–4.60 (2H, m, Ha, and Hb), 5.44 (1H, m, Ha’), 7.04 (1H, s, H5-pyridone), 7.11 (2H, d, Ar–H, J = 9.29 Hz), 7.57 (2H, d, Ar–H, 8.69 Hz); Anal. Calcd for C22H24N2O3 (364.44): C, 72.50; H, 6.64; N, 7.69. Found: C, 72.55; H, 6.66; N, 7.68.