Novel Pyridinone Derivatives As Non-Nucleoside Reverse

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Novel pyridinone derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs) with high potency against NNRTI-resistant HIV-1 strains Amin Li, Yabo Ouyang, Ziyun Wang, Yuanyuan Cao, Xiangyi Liu, Li Ran, Chao Li, Li Li, Liang Zhang, Kang Qiao, Weisi Xu, Yang Huang, Zhili Zhang, Chao Tian, Zhenming Liu, Shibo Jiang, Yiming Shao, Yansheng Du, Liying Ma, Xiaowei Wang, and Junyi Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400102x • Publication Date (Web): 29 Mar 2013 Downloaded from http://pubs.acs.org on March 30, 2013

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Journal of Medicinal Chemistry

Novel pyridinone derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs) with high potency against NNRTI-resistant HIV-1 strains

Amin Li,†,# Yabo Ouyang,§,# Ziyun Wang,‡ Yuanyuan Cao,† Xiangyi Liu,† Li Ran,‡ Chao Li,† Li Li,† Liang Zhang,† Kang Qiao,‡ Weisi Xu,§ Yang Huang,§ Zhili Zhang,† Chao Tian,† Zhenming Liu,‡ Shibo Jiang,┴ Yiming Shao,§ Yansheng Du,ǂ Liying Ma,*,§ Xiaowei Wang*,† and Junyi Liu*,†,‡

†

Department of Chemical Biology, School of Pharmaceutical Sciences; ‡State Key

Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191; §State Key Laboratory for Infection Disease Prevention and Control, National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention, Beijing 102206 and

┴

Laboratory of Medical Molecular Virology of

Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China. ǂ Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA

Keywords: HIV-1, non-nucleoside reverse transcriptase inhibitors (NNRTIs), drug resistance

#

These authors have contributed equally.

*To whom correspondence should be addressed: Phone/Fax: +86-10-82805203, e-mail: [email protected]

(X.W.);

Phone/Fax:

+86-10-82805203,

e-mail: [email protected] (J.L.); or Phone: +86-10-58900976, Fax: +86-10-58900980, e-mail: [email protected] (L.M.). 1 ACS Paragon Plus Environment


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Abstract Novel

6-substituted-4-cycloalkyloxy-pyridin-2(1H)-ones

were

synthesized

as

non-nucleoside reverse transcriptase inhibitors (NNRTIs), and their biological activity was evaluated. Most of the compounds, especially 26 and 22, bearing a 3-isopropyl and 3-iodine group, respectively, exhibited highly potent activity against wild-type HIV-1 strains and those resistant to reverse transcriptase inhibitors (RTIs). The diastereoisomers of 26-trans and 26-cis were synthesized separately, and confirmed with HPLC and NOESY spectra. The 26-trans isomers had an activity about 400-fold more potent than that of 26-cis. The pair of 26-trans enantiomers, one of the most potent inhibitors with EC50 of 4 nM and selectivity index (SI) of 75,000, was highly effective against a panel of RTIs-resistant strains with single (Y181C and K103N) or double (A17) mutations in reverse transcriptase. The results suggest that these novel pyridinone derivatives have the potential to be further developed as new antiretroviral drugs with improved antiviral efficacy and drug resistance profile.

Abbreviations: CC50, concentration for 50% cytotoxicity; EC50, effective concentration for 50% inhibition; ELISA, enzyme-linked immunosorbent assay; HAART, highly active antiretroviral therapy; HIV-1, human immunodeficiency virus type 1; NNBP, non-nucleoside binding pocket; NNRTI, non-nucleoside reverse transcriptase inhibitor; PDB, protein database; RT, reverse transcriptase; SI, selectivity index (ratio of CC50/EC50).

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Introduction The development of potent and effective antiviral drugs for the control of human immunodeficiency virus type 1 (HIV-1) infection is one of the most pressing goals of contemporary medicinal chemistry. Reverse transcriptase (RT) is an essential enzyme in the infectious life cycle of HIV and plays a multifunctional role in viral replication. RT inhibitors were used as important components of HAART and as a method of preventing sexual transmission or mother-to-child transmission of HIV-11, 2. Two functionally distinct classes of HIV-1 RT inhibitors (nucleoside and non-nucleoside) have been discovered and are being used clinically. Especially, non-nucleoside RT inhibitors (NNRTIs) have gained a definitive and important place in clinical use based on their favorable pharmacokinetic properties3, 4 and wide range of chemically diverse structures. To date, five NNRTIs have been approved for clinical use: nevirapine (NVP)5, delavirdine (DLV)6, efavirenz (EFV)7, etravirine (ETR)8 and rilpivirine (RPV)9. However, DLV is rarely used because of its poor pharmacokinetics10-13. Like other types of anti-HIV drugs, the therapeutic efficacy of NNRTIs is weakened by the very rapid development of drug-resistant mutations, in vitro and in vivo, both in monotherapy and in multidrug trials14-16. The commonly observed drug-resistant virus contains the single amino acid mutation Tyr181Cys and Lys103Asn, which are resistant to NVP, DLV, EFV17 and other NNRTIs. Therefore, it is important to develop new NNRTIs that can effectively inhibit commonly observed NNRTI-associated resistant viruses and have a higher genetic barrier to resistance.



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Crystallographic studies showed that NNRTIs bind in an allosteric pocket located about 10 Å from the polymerase active site18 and cause large conformational changes in HIV-1 RT, which could influence the geometry of the polymerase catalytic site19,

20

.

Recently, several compounds based on the 2-pyridinone scaffold, such as 1 (L-697,661)21, have been reported as having significant antiviral potency. In our previous study, we developed 2 (TNK-651)22 analogues as antiviral agents with better drug-resistance profiles23. Therefore, we designed novel pyridinone derivatives with respective hybrids of 2 analogues and 2-pyridinone, in which the central pyrimidinone ring of 2 is replaced with a pyridin-2-one ring, and the substituents on C-3, 4 positions are corresponding to the C-5, 6 positions of 2 analogues. Subsequently, modifications were performed on positions 3, 4 and 6 with different substituents (Figure 1). O HN

H N

O

Cl O

HN O

N Cl

N O

1 (L-697,661)

2 (TNK-651) O HN

R3 6

3 R2 4 O

R1 R1 cycloallkyl; R2 -X, -COOEt, -CH(CH3)2, H; R3 Aryl target compounds

Figure 1. Structures of typical NNRTIs and target compounds Structure and activity relationship (SAR) studies have revealed that the substituents on C-3 and C-4 of the pyridinone ring were very important for antiviral activity. Based on our previous results23 the optimization of position C-3 with the isopropyl and halogen


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substituent would have a twist effect on the conformation of position C-422. Moreover, the halogen substituent could act as a vehicle to incorporate diverse functionality at C-3 of the pyridinone ring24. Several studies have pointed out that compound flexibility could act as an important parameter for conserving potency against various mutant viral strains25. As we know, among the pyridinone compounds previously described, only the aromatic26 and heterocyclic27 substituents are included at position C-4 of the pyridinone ring. In our report, the modification on position C-4 by introducing saturated rings could increase the molecular flexibility and result in improving the potency against various mutant viral strains27. In addition, a methyl-bearing moiety on the cycle ring may result in inducing better contact with the conserved W229, while reducing aromatic stacking interactions with the highly mutable Y181 side chain28. Although considerable SAR information was available on the C-3 and C-4 positions, only a few studies have reported on the effect of 6-substituted pyridinones. The optimization we carried out was mainly to bind the side chain in a mode similar to the orientation of the N-1 side chain of 2, which extends into a tunnel leading toward the catalytic site comprised of Tyr318 and Pro236, thus providing a favourable interaction with HIV-1 RT, and then enhancing the potency against wild-type HIV-1 and several clinically relevant mutant strains with low cytotoxicity. To verify the relationship between structure and activity of the target compounds, we synthesized a series of novel pyridinone derivatives (Figure 1), and we detected their



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inhibitory activity on HIV-1 RT. The active compounds were further evaluated in vitro against a HIV-1 laboratory-adapted strain on both TZM-bl and MT4 cell lines, as well as against principal drug-resistant mutant strains, K103N or Y181C and both mutation, which confer resistance to the NNRTIs currently and commonly used in the clinical genotypic drug-resistant mutation. Chemistry Almost all the target compounds have been synthesized from the common intermediate 4, which is easily obtained from ethyl 3-oxo-4-phenylbutanoate 3 reacting with ammonia and diethylmalonate. The synthetic procedure used for the preparation of 7f-p is illustrated in Scheme 1. For preparation of 6 analogues, it was first necessary for O-benzylate to give 5a, which readily underwent Mitsunobu reaction with the corresponding cycloalkyl alcohol to yield 6f-p. Catalytic hydrogenation led to the target compounds 7f-p. Scheme 1. Synthesis of compounds 7f-pa


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a

Reagents and reaction conditions: (a) NH3/MeOH, diethyl malonate/EtONa, reflux, 4

d, yields 42~72%; (b) PhCH2Br, Ag2CO3, THF, 60 °C, 8 h, yields 68~88%; (c) corresponding cyclohexanol, DIAD, PPh3, THF, rt, 20 h, yields 27~94%; (d) H2, Pd/C, MeOH, rt, 8 h, yields 75~99%. Using a halogen atom replacement on position C-3, the halogenated compounds were synthesized as described in Scheme 2. The benzyl-protected ester 6e was heated in aqueous HCl to effect hydrolysis- decarboxylation to afford 8, which reacted with NBS or NIS to yield 9 and 10, respectively. Scheme 2. Synthesis of compounds 9-10a

a

Reagents and reaction conditions: (a) 1N HCl, reflux, 48 h, yield 49%; (b) NIS/NBS,

rt, 8 h, yields 90~99%. Further modifications were carried out according to Scheme 3, in which 5a-e were used as the starting materials. The ester intermediates 5a-e were subjected to reductive methylation, yielding the expected tertiary alcohols 11a-e. Dehydration was performed with thionylchloride to afford 12a-e. A Mitsunobu reaction with 2’-methyl cyclohexanol (mixture) was carried out, followed by hydrogenation to install the desired target compounds 14a-e with high yields. Scheme 3. Synthesis of derivatives 14a-ea 7 ACS Paragon Plus Environment


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OBn

OBn COOEt

N

OBn

OH

N

a

N

b

OH

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OH

R

OH

R

R

5 a-e

11 a-e

12 a-e

R : a = H, b = para-F , c = para-Cl, d = para-MeO, e = para-Me OBn

O

N

c

HN

d O

R

R 13 a-e

a

O

14 a-e

Reagents and reaction conditions: (a) MeLi, THF, -78 °C, yields 67~97%; (b) SOCl2,

yields 64~90%; (c) 2-methylcyclohexanol (mixture), DIAD, PPh3, THF, rt, 8 h, yields 32~36%; (d) H2, Pd/C, MeOH, rt, yields 85~96%. Using the similar strategy described in Schemes 1-3, we synthesized the derivatives 19-26 (Scheme 4). Structure assignments of these compounds were identified by NMR and mass spectral data. Scheme 4. Synthesis of derivatives 19-26a

a

Reagents and reaction conditions were consistent with Scheme 1-3. 8 ACS Paragon Plus Environment

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Results and Discussion For each rank of our compounds, their inhibition of HIV polymerase activity was initially assessed using purified recombinant wild-type HIV polymerase and a poly(rA)/oligo(dT)16 template primer. In the first step, the impact of the C-4 substituent was investigated, including the ring size and substituent on the cyclohexyl rings. As shown in Table 1, 7f-p, which contained rings ranging from cyclopentyl to cycloheptyl, were evaluated against HIV-1 RT. The biological result showed that the substituent with a cyclohexyl ring was superior to both that of cyclopentyl and cycloheptyl, especially 7j with the 2’-methylcyclohexyl substituent, which is a mixture of stereoisomers, had inhibitory activity superior to the other compounds. When more methyl moieties were introduced to the cyclohexyl ring, the activity of the compounds disappeared, notably 7m-p. The biological results showed that the cyclohexyl ring, with its optimized flexibility, could provide the better interaction with the NNBP. Structure-activity relationships on position C-3 of the pyridine-2(1H)-one ring showed that isopropyl or halo sustitutents could initiate the “trigger action” required for improved activity against mutant viral strains. The higher potency of 9, 10 and 14a against HIV-1 RT suggested that the overall steric and lipophilic characteristics were clearly beneficial for activity, as shown in Table 2. Completing the modifications on positions C-3 and C-4 of the pyridinone ring, we focused our attention on the modulation of the C-6 side chain with different length and 9 ACS Paragon Plus Environment


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aromatic rings. To verify our design, target compounds (14a-e, 19, 21, 22, 26, 26-cis and 26-trans) were initially tested for their inhibitory activity against HIV-1 RT with nevirapine and 2 as reference compounds29. As shown in Table 3, most compounds displayed promising anti-RT activities. Recently, the existence of a halogen bond with the Tyr188 carbonyl oxygen was reported. This was regarded as one of the important interactions leading to overall protein-ligand binding affinity and was credited with improving anti-HIV-1 activity 23, 30. Upon inspection of the 22-trans/1RT2 complex31 (Figure 2), we found that the distance between iodine atom with the carbonyl oxygen of Tyr188 are within the van der Waals contact, which could result in halogen bond.

Moreover, the large iodine atom may also

provide sufficient steric hindrance to modulate the conformation of cyclohexyl ring on the C-4 position.

Figure 2. Superimposed stereoview of the docked (±)-22-trans with 1RT2. The hydrogen bonds are shown as yellow dashed lines, and the halogen bonds are shown as purple dashed lines. In addition, the docking results revealed that the C-6 side chain could be twisted and 10 ACS Paragon Plus Environment

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located at the region similar to that of compound 2. Therefore, it was presumed that changing the chain length could modulate its flexibility and provide better interactions with Tyr318 and Pro236 (Figure 3), which would generate the compensation of entropic penalty by these favorable interactions and result in increased antivirus activity.

Figure 3. Superimposed stereoview of the docked (±)-26-trans and 2 (green) with 1RT2. The hydrogen bonds are shown as yellow dashed lines. These target compounds were synthesized as mixtures of diastereomers. From comparison of the antiviral activity of these compounds, the 26-trans enantiomers were prepared by using a stereoselective Mitsunobu reaction of cis-2-methylcyclohexanol, followed by hydrogenation. The diastereomers of 26-cis and 26-trans were synthesized by the corresponding trans- and cis- 2-methylcyclohexanol respectively, and confirmed by High-Performance Liquid Chromatography (HPLC) (Figure 4) and 1H-nuclear Overhauser effect (NOE) (Figure 5). Because the 1’-H of the trans isomers were close to the 2’-methyl, a spatial correlation would be established, as irradiation of 1’-H resulted in NOE in the 2’-CH3 groups. The NOE spectra proved the conclusion.



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26 (mixture)

(±)-26-trans

26 (mixture) + (±)-26-trans

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(±)-26-cis

26 (mixture) + (±)-26-cis

Figure 4. The HPLC results of 26 diastereomers.

(±)-26-trans

(±)-26-cis

(±)-26-trans

(±)-26-cis

Figure 5. NOE spectra of (±)-26-trans and (±)-26-cis. 12 ACS Paragon Plus Environment

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It can be observed that 26-trans enantiomers with the 2’-methyl substituent in the trans position had an antiviral potency (0.003 µM) which is 1483- and 29-fold more potent than Nevirapine and 2, respectively (Table 3). Thus, the configuration of substituted cyclohexyl would bring a significant impact on the antivirus activity, especially for the mutant strains. Subsequently, the anti-HIV-1 activity and cytotoxicity of target compounds, except 14a and 19, were determined using Nevirapine and 2 as reference compounds. As shown in Table 4, all these compounds were capable of inhibiting wild-type virus infection by HIV-1SF33 in TZM-bl and MT4 cells with EC50 values at 0.003 - 0.136µM (Table 4). In addition, 26-trans and 22, which had an excellent preliminary antiviral profile and higher selectivity index (SISF33/MT4 75000 and 17647, repectively), were selected for further profiling. Tests with a panel of NNRTI-resistant mutant viruses revealed that 26-trans and 22 strongly inhibit replication of the prevalent single mutations of K103N, Y181C, and double mutant A17 with EC50 nanomolar values, which compared to that of 2 (Table 5). Therefore, these compounds could be further developed as next-generation NNRTIs with improved antiviral efficacy and drug resistance profile. Moreover, docking studies displayed that the isopropyl or iodine group on C-3 located in a hydrophobic pocket, which could provide a favorable interaction with the NNBP. Meanwhile, they could regulate the conformation of the C-4 cyclohexyl, which is located in a binding pocket lined by the aromatic residues Tyr188, Phe227 and Trp229 (Figure 3). With the presence of 2’-methyl, the cyclohexyl ring could be close to Trp229, which



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would be conducive to forming π-σ interaction with the highly conserved amino residue Trp229, instead of the readily mutated residue Tyr181. Especially, the 26-trans enantiomers displayed the best profile against the mutant strains. Therefore, we docked 26-trans enantiomers into the clinically important K103N (PDB. 1FKP) mutant RT (Figure 6), as well as the Y181C (PDB. 1JLA) (Figure 7) respectively, to evaluate whether conformational flexibility and configuration would allow adaptation of the inhibitor to the mutant binding site.

Figure 6. Docking results of (±)-26-trans into the non-nucleoside binding-pocket 1FKP Lys103Asn. Hydrogen bonds are shown as dashed lines.

Figure 7 Docking results of (±)-26-trans into the non-nucleoside binding-pocket 14 ACS Paragon Plus Environment

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1JLA Tyr181Cys. Hydrogen bonds are shown as dashed lines. The docking results revealed that some changes took place with 26-trans/mutated RT complex. First, with RT-directed mutagenesis (1FKP and 1JLA), the binding mode toward NNBP produced a little change compared to the wild-type (Figure 3), especially 26-trans/1FKP complex (Figure 6). Second, for the 26-trans/1JLA complex (Figure 7), the mainly hydrogen bond was preserved with Lys101, while the hydrogen bond with Lys103 was disappeared. However, binding with 1FKP (Figure 6), the hydrogen bonds formed with not only Lys101, but also Asn103. Third, the cyclohexyl ring was buried deep in the hydrophobic pocket composed of Trp229 and Tyr188 for both of 1FKP and 1JLA, which increased π-σ interaction with the amino acid residues. Fourth, the C-6 side chain of 26-trans/1JLA complex could extend into a tunnel which would provide π-π and π-σ interactions with Tyr318 and Pro236. However, the substituent on C-6 of 26-trans/1FKP complex closed to Val179. Because the shape of NNBP changed as the K103N and Y181C mutated, some of the original interaction forces disappeared. Therefore, the inhibitory activity of 26-trans and 22 against the mutated strains was less than the wild strain. However, the affinity involved in these networks of van der Waals contacts stilled remained, which would be important for stabilizing both wild-type and mutant complexes. These results appear significant for understanding the resistance profiles of the target compounds. In addition, the docking results displayed similar binding modes of the enantiomers of 26-trans. Thus, we assume that the enantiomers may have the same contribution to the



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anti-HIV-1 activity. Conclusion In summary, we have identified a new series of 3-isopropyl-4-cyclohexyloxy pyridin-2(1H)-ones

and

3-iodo-4-cyclohexyloxypyridin-2(1H)-ones

derivatives

as

effective anti-HIV agents in comparison with nevirapine and 2. The biological results confirmed our hypothesis that the introduction of an isopropyl or iodine group on C-3 displayed a “trigger” action on the conformation of the C-4 cyclohexyl and that the C-6 side chain with two carbon linker could extend into the tunnel forming the π-π interaction with the Tyr318 and Pro236 residues. Additionally, the configuration of 2’-methyl on the cyclohexyl ring would significantly improve the potency against a panel of single and double mutant strains often observed in patients treated with NNRTIs. In particular, 26-trans and 22 were superior to 2 in that they displayed the best profile against the wild-type HIV, K103N, Y181C, and double mutant strains, making them potential lead compounds with improved resistance profiles and low toxicity. Further investigation is ongoing to characterize the pharmacokinetic profiles. Experimental Section Chemistry The structural characterization was performed with an NMR spectrometer and a high resolution mass spectrometer (HRMS). The purity of tested compounds was determined by HPLC (Dionex Ultimate 3000 HPLC system). All the assayed compounds displayed a purity of ≥95% (Table S1, Supporting Information). Melting points were determined on a WBS-1B type digital melting-point apparatus and are uncorrected. NMR 16 ACS Paragon Plus Environment

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spectra were recorded on a Bruker Avance 300 or Avance 500 with tetramethylsilane (TMS) as an internal standard, and chemical shifts are reported in δ (ppm). All the reactions were routinely monitored by TLC, which was performed on aluminum-backed silica gel plates (60 F254) with spots visualized by UV light. Silica gel (0.040 - 0.064 mm) was used for column chromatography and analytical silica gel for TLC plates. Unless otherwise stated, all reagents were purchased from commercial sources. When necessary, they were purified and dried by standard methods. Organic solutions were dried over anhydrous sodium sulfate. The cis- and trans- 2-methyl cyclohexanols were used as materials for synthesizing the compounds 26-trans and 26-cis, respectively. Other target compounds were prepared with the mixture of 2-methylcyclohexanol. Ethyl 6-aryl-4-alkyl-2-oxo-1,2-dihydropyridine-3-carboxylate (7f-p). General Procedure: Aryl acetyl chloride (37.81 mmol) was added dropwise to a solution of

Meldrum’s

acid (5.45 g, 37.81 mmol) and pyridine (6.2 mL) in CH2Cl2 (70 mL) at 0 °C. The solution was stirred for 30 min at 0 °C and then allowed to warm slowly to ambient temperature for 8 h. The reaction mixture was washed with 10% aqueous HCl (2 × 50 mL) and H2O (50 mL) separately. The organic layer was dried with MgSO4, filtered and concentrated under reduced pressure. The crude residue was dissolved in EtOH (100 mL) and heated to reflux for 4 h. The mixture was allowed to cool to room temperature and then concentrated under reduced pressure. The dark oily residue was purified by column to give required compounds 3a-e.



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A mixture of 3a-e (2.04 mmol) and 2 M NH3 in MeOH (100 mL) was heated to reflux for 8 h in a sealed tube. After cooling, the mixture was concentrated to dryness to provide 3-amino-4-arylbut-2-enoic acid ethyl ester which was used without further purification. To a solution of 1.2 g (48.0 mmol) of sodium in 60 mL of ethanol was added a solution of 7.4 mL (48.0 mmol) of diethyl malonate in 10 mL of ethanol. The resulting yellow mixture was stirred for 1 h, and another solution of 5 g (24.0 mmol) of 3-amino-4-arylbut-2-enoic acid ethyl ester in 24 mL of ethanol was added. The mixture was refluxed for 4 d, and a white precipitate was formed. After removing the solvent, the remainder was acidified with conc. HCl. The precipitate was filtered, washed with water and dried to yield 4a-e as a white, crystalline solid. Amounts of 4a-e (5.05 mmol), 0.77 g (2.78 mmol) of silver carbonate and 0.66 mL (5.56 mmol) of benzyl bromide were placed in a 20 mL THF and heated to reflux for 8 h. After cooling to room temperature, the mixture was filtered through a filter agent pad (Celite 521, Aldrich), and the solvent was evaporated. The crude product was purified by column chromatography to give 5a-e as a white solid. Diisopropyl azodicarboxylate (DIAD) (0.26 g, 1.27 mmol) was added dropwise at room temperature to a solution of 5a (0.23 g, 0.63 mmol), triphenylphosphine (PPh3) (0.33 g, 1.27 mmol) and the corresponding cyclohexanol (1.27 mmol) in 20mL of THF. After stirring overnight, the solvent was evaporated, and the crude product was purified by column chromatography to give 6f-p as a liquid. To a pressure reaction bottle were added 6f-p (0.23 mmol) and 0.02 g of Pd/C 10% in 18 ACS Paragon Plus Environment

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10 mL of methanol. The reaction was under three air/hydrogen exchange cycles and stirred at 15 psi for 8 h. After evaporating the solvent, the residue was purified by column chromatography to give 7 f-p as a white solid. Ethyl 3-oxo-4-phenylbutyrate (3a).32 pale yellow liquid, yield 57%; 1H NMR (300 MHz, CDCl3): δ 7.20-7.37 (m, 5H, Ph-H), 4.16 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.83 (s, 2H, CH2-Ph), 3.51 (s, 2H, CH2), 1.26 (t, 3H, J = 7.2 Hz, OCH2CH3); m/z (EI): 206 [M + H]+. Ethyl 4-(4-fluorophenyl)-3-oxobutanoate (3b).33 pale yellow liquid, yield 78%; 1H NMR (300 MHz, CDCl3): δ 6.98-7.25 (m, 4H, Ph-H), 4.18 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.81 (s, 2H, CH2Ar), 3.46 (s, 2H, CH2), 1.26 (t, 3H, J = 7.2 Hz, OCH2CH3). Ethyl 4-(4-chlorophenyl)-3-oxobutanoate (3c).32 pale yellow liquid, yield 77%; 1H NMR (300 MHz, CDCl3): δ 7.13-7.30 (m, 4H, Ph-H), 4.17 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.80 (s, 2H, CH2Ar), 3.45 (s, 2H, CH2), 1.26 (t, 3H, J = 7.2 Hz, OCH2CH3). Ethyl 4-(4-methoxylphenyl)-3-oxobutanoate (3d).32 pale yellow liquid, yield 84%; 1H NMR (400 MHz, CDCl3): δ 6.86-7.27 (m, 4H, Ph-H), 4.16 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.79 (s, 3H, CH3O-Ph), 3.75 (s, 2H, CH2Ar), 3.43 (s, 2H, CH2), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3). Ethyl 4-(4-methylphenyl)-3-oxobutanoate (3e).34 pale yellow liquid, yield 89%; 1H NMR (400 MHz, CDCl3): δ 7.10-7.14 (m, 4H, Ph-H), 4.62 (s, 1H, CH2), 4.10 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.41 (s, 3H, CH2), 2.33 (s, 3H, CH3-Ph), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3). Ethyl 3-oxo-5-phenylpentanoate (15)35 pale yellow liquid, yield 95%; 1H NMR (400 19 ACS Paragon Plus Environment


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MHz, CDCl3): δ 7.17-7.29 (m, 5H, Ph-H), 4.16 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.41 (s, 2H, CH2), 2.85-2.92 (m, 4H, CH2CH2Ph), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3). Ethyl 6-benzyl-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate (4a).36 white solid, yield 46%; m.p. 208-209 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.43 (s, 1H, OH), 11.57 (br s, 1H, NH), 7.24-7.36 (m, 5H, Ph-H), 5.72 (s, 1H, C5-H), 4.25 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.77 (s, 2H, CH2Ar), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, DMSO-d6): δ 172.22 (COOEt), 170.12 (C-4), 161.14 (C-2), 154.35 (C-6), 137.21, 129.39, 129.05, 127.37 (aryl), 99.12 (C-3), 97.84 (C-5), 61.06 (OCH2CH3), 38.37 (CH2Ar), 14.56 (OCH2CH3); m/z (EI): 274.2 [M + H]+. Ethyl

6-(4-fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate

(4b).

white solid, yield 42%; m.p. 221-222 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.43 (s, 1H, OH), 11.57 (br s, 1H, NH), 7.15-7.40 (m, 4H, aryl), 5.72 (s, 1H, C5-H), 4.24 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.76 (s, 2H, CH2Ar), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3); m/z (EI): 292.4[M + H]+ . Ethyl

6-(4-chlorobenzyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate

(4c).

white solid, yield 54%; m.p. 227-228 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.40 (s, 1H, OH), 11.57 (br s, 1H, NH), 7.35 (m, 4H, aryl), 5.71 (s, 1H, C5-H), 4.21 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.75 (s, 2H, CH2Ar), 1.22 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, DMSO-d6): δ 172.02 (COOEt), 169.96 (C-4), 161.14 (C-2), 153.75 (C-6), 136.23, 132.14, 131.28, 128.99 (aryl), 99.32 (C-3), 97.89 (C-5), 61.07 (OCH2CH3), 37.57 (CH2-Ph), 14.56 (OCH2CH3); m/z (EI): 307.9[M + H]+ . 20 ACS Paragon Plus Environment

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Ethyl 6-(4-methoxybenzyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate (4d). white solid, yield 46%; m.p. 209-210 °C; 1H NMR (400 MHz, CDCl3): δ 13.32 (s, 1H, OH), 12.51 (br s, 1H, NH), 6.83-7.30 (m, 4H, aryl), 5.78 (s, 1H, C5-H), 4.40 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.81 (s, 2H, CH2Ar), 3.78 (s, 3H, OCH3-Ph), 1.37 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 176.11 (COOEt), 172.00 (C-4), 163.50 (C-2), 158.95 (C-6), 155.18, 130.43, 127.47, 114.25 (aryl), 99.28 (C-3), 96.84 (C-5), 61.60 (OCH2CH3), 55.26 (OCH3), 38.46 (CH2Ar), 14.24 (OCH2CH3); m/z (EI): 304.2[M + H]+ . Ethyl 6-(4-methylbenzyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate

(4e).

white solid, yield 72%; m.p. 205-206 °C; 1H NMR (400 MHz, CDCl3): δ 13.32 (s, 1H, OH), 12.33 (br s, 1H, NH), 7.11-7.26 (m, 4H, aryl), 5.79 (s, 1H, C5-H), 4.40 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.83 (s, 2H, CH2Ar), 2.32 (s, 3H, CH3-Ph), 1.37 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 176.12 (COOEt), 172.01 (C-4), 163.37 (C-2), 154.89 (C-6), 137.16, 132.32, 129.56, 129.23 (aryl), 99.38 (C-3), 96.87 (C-5), 61.60 (OCH2CH3), 38.91 (CH2-Ph), 21.05 (CH3), 14.24 (OCH2CH3); m/z (EI): 287.7[M + H]+ . Ethyl 4-hydroxy-2-oxo-6-phenethyl-1,2-dihydropyridine-3-carboxylate (16). white solid, yield 33%; m.p. 192-193 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.58 (s, 1H, OH), 11.51 (br s, 1H, NH), 7.18-7.31 (m, 5H, aryl), 5.81 (s, 1H, C5-H), 4.26 (q, 2H, J = 7.2 Hz, OCH2CH3), 2.86-2.90 (m, 2H, CH2CH2-Ph), 2.69-2.73 (m, 2H, CH2CH2-Ph), 1.26 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, DMSO-d6): δ 172.82 (COOEt), 170.57 (C-4), 161.14 (C-2), 154.97 (C-6), 140.68, 128.78, 126.61 (aryl), 98.53 (C-3), 97.52 (C-5), 61.09 (OCH2CH3), 34.54 (CH2CH2-Ph), 34.09 (CH2CH2-Ph), 14.58 (OCH2CH3); m/z (EI): 288.5[M + H]+ . 21 ACS Paragon Plus Environment


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Ethyl 6-benzyl-2-(benzyloxy)-4-hydroxynicotinate (5a). white solid, yield 80%; m.p. 67-68 °C; 1H NMR (400 MHz, CDCl3): δ 12.25 (s, 1H, OH), 7.20-7.47 (m, 10H, aryl), 6.35 (s, 1H, C5-H), 5.44 (s, 2H, OCH2 Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.93 (s, 2H, CH2Ph), 1.33 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 171.56 (COOEt), 170.99 (C-4), 163.63 (C-2), 162.51 (C-6), 138.46, 137.41, 129.30, 128.50, 128.20, 127.62, 127.54, 126.52 (aryl), 106.23 (C-3), 95.35 (C-5), 68.20 (OCH2Ph), 61.74 ((OCH2CH3), 44.55 (CH2Ph), 14.06 (OCH2CH3); m/z (EI): 364.2 [M + H]+ . Ethyl 6-(4-fluorobenzyl)-2-(benzyloxy)-4-hydroxynicotinate (5b). white solid, yield 78%; m.p. 60-61 °C; 1H NMR (400 MHz, CDCl3): δ 12.29 (s, 1H, OH), 6.94-7.47 (m, 9H, Ar), 6.33 (s, 1H, C5-H), 5.43 (s, 2H, OCH2Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.88 (s, 2H, CH2Ar), 1.34 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 171.60 (COOEt), 170.97 (C-4), 163.31 (C-2), 162.55 (C-6), 160.49, 137.35, 134.15, 130.75, 130.67, 128.23, 127.58, 127.54, 115.36, 115.15 (aryl), 106.15 (C-3), 95.41 (C-5), 68.18 (OCH2Ph), 61.81 (OCH2CH3), 43.63 (CH2Ar), 14.07 (OCH2CH3); m/z (EI): 382.9 [M + H]+. Ethyl 6-(4-chlorobenzyl)-2-(benzyloxy)-4-hydroxynicotinate (5c). white solid, yield 74%; m.p. 81-82 °C; 1H NMR (400 MHz, CDCl3): δ 12.30 (s, 1H, OH), 7.16-7.45 (m, 9H, aryl), 6.33 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.87 (s, 2H, CH2Ar), 1.34 (t, 3H, J = 7.2 Hz, OCH2CH3);

13

C NMR (101 MHz, CDCl3): δ

171.63 (COOEt), 170.95 (C-4), 162.89 (C-2), 162.58 (C-6), 160.49, 137.34, 136.98, 130.65, 130.62, 128.63, 128.59, 128.54, 128.43, 128.24, 127.56 (aryl), 106.23 (C-3), 95.49 (C-5), 68.60 (OCH2Ph), 61.82 (OCH2CH3), 43.87 (CH2Ar), 14.08 (OCH2CH3); m/z (EI): 22 ACS Paragon Plus Environment

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398.9[M + H]+. Ethyl 6-(4-methoxybenzyl)-2-(benzyloxy)-4-hydroxynicotinate (5d). white solid, yield 68%; m.p. 54-55 °C; 1H NMR (400 MHz, CDCl3): δ 12.24 (s, 1H, OH), 76.81-7.48 (m, 9H, aryl), 6.32 (s, 1H, C5-H), 5.44 (s, 2H, OCH2Ph), 4.35 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.86 (s, 2H, CH2Ar), 3.77(s, 3H, CH3OAr), 1.33 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 171.56 (COOEt), 171.00 (C-4), 164.11 (C-2), 162.53 (C-6), 158.37, 137.48, 130.58, 130.25, 128.20, 127.60, 127.34, 114.01 (aryl), 106.05 (C-3), 95.29 (C-5), 68.16 (OCH2Ph), 61.89 (OCH2CH3), 55.25 (CH3OAr), 43.68 (CH2Ar), 14.06 (OCH2CH3); m/z (EI): 394.5 [M + H]+. Ethyl 6-(4-methylbenzyl)-2-(benzyloxy)-4-hydroxynicotinate (5e). white solid, yield 88%; m.p. 54-55 °C; 1H NMR (400 MHz, CDCl3): δ 12.23 (s, 1H, OH), 7.02-7.48 (m, 9H, aryl), 6.34 (s, 1H, C5-H), 5.44 (s, 2H, OCH2Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.89 (s, 2H, CH2Ar), 2.32 (s, 3H, CH3Ar), 1.33 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 171.55 (COOEt), 170.99 (C-4), 163.95 (C-2), 162.51 (C-6), 137.46, 136.03, 135.40 (CH3Ph), 129.18, 128.18, 127.62, 127.52 (aryl), 106.13 (C-3), 95.30 (C-5), 68.18 (OCH2Ph), 61.68 (OCH2CH3), 44.15 (CH2Ar), 21.03 (CH3Ar), 14.05 (OCH2CH3); m/z (EI): 377.7[M + H]+. Ethyl 2-(benzyloxy)-4-hydroxy-6-phenethylnicotinate (17). white solid, yield 82%; m.p. 52-53 °C; 1H NMR (400 MHz, CDCl3): δ 12.27 (s, 1H, OH), 7.15-7.52 (m, 10H, aryl), 6.35 (s, 1H, C5-H), 5.44 (s, 2H, OCH2Ph), 4.37 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.02 (t, 2H, J = 6.8 Hz, CH2CH2-Ph), 2.91 (t, 2H, J=6.8 Hz, CH2CH2-Ph), 1.35 (t, 3H, J = 7.2 Hz, 23 ACS Paragon Plus Environment


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OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 171.38 (COOEt), 171.09 (C-4), 164.03 (C-2), 162.50 (C-6), 141.44, 137.55, 128.45, 128.40, 128.26, 127.57, 127.50, 126.00 (aryl), 106.13 (C-3), 95.29 (C-5), 68.16 (OCH2-Ph), 61.78 (OCH2CH3), 39.71 (CH2CH2-Ph), 34.56 (CH2CH2-Ph), 14.06 (OCH2CH3); m/z (EI): 378.2 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(cyclopentyloxy)nicotinate (6f). pale yellow liquid, yield 71%; 1H NMR (400 MHz, CDCl3): δ 7.18-7.38 (m, 10H, aryl), 6.30 (s, 1H, C5-H), 5.42 (s, 2H, OCH2Ph), 4.70-4.72 (m, 1H, H1’),4.32 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.96 (s, 2H, CH2Ph), 1.54-1.80 (m, 8H, cyclopentyl), 1.29 (t, 3H, J = 7.2 Hz, OCH2CH3);

13

C

NMR (101 MHz, CDCl3): δ 165.54 (COOEt), 163.93 (C-4), 160.86 (C-2), 160.68 (C-6), 139.14, 137.73, 129.10, 128.43, 128.21, 127.59, 127.39, 126.37 (aryl), 104.40 (C-3), 102.46 (C-5), 80.59 (C1’), 67.52 (OCH2Ph), 61.08 (OCH2CH3), 44.67, 32.75, 23.84 (Ccyclopentyl, CH2Ph), 14.25 (OCH2CH3); m/z (EI): 432.3 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(cyclohexyloxy)nicotinate (6g). pale yellow liquid, yield 77%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.30 (s, 1H, C5-H), 5.42 (s, 2H, OCH2Ph), 4.28-4.36 (m, 3H, H1’, OCH2CH3), 3.95 (s, 2H, CH2Ph, 1.29-1.82 (m, 13H, Cyclohexyl, OCH2CH3);

13

C NMR (101 MHz, CDCl3): δ 165.62 (COOEt),

163.76 (C-4), 160.93 (C-2), 160.63 (C-6), 139.17, 137.73, 129.08, 128.42, 128.21, 127.61, 127.39, 126.35 (aryl), 106.68 (C-3), 102.26 (C-5), 75.71 (C1’), 67.52 (OCH2Ph), 61.13 (OCH2CH3), 44.63, 31.17, 25.44, 22.91 (Ccyclohexyl, CH2Ph), 14.24 (OCH2CH3); m/z (EI): 446.9 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(cycloheptyloxy)nicotinate (6h). pale yellow liquid,


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yield 75%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.24 (s, 1H, C5-H), 5.42 (s, 2H, OCH2Ph), 4.37-4.43 (m, 1H, H1’), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.95 (s, 2H, CH2Ph), 1.39-1.88 (m, 12H, cycloheptyl), 1.30 (t, 3H, J = 7.2 Hz, OCH2CH3);

13

C

NMR (101 MHz, CDCl3): δ 165.63 (COOEt), 163.79 (C-4), 160.93 (C-2), 160.70 (C-6), 139.14, 137.74, 129.12, 128.43, 128.21, 127.59, 127.38, 126.37 (aryl), 104.61 (C-3), 102.32 (C-5), 78.65 (C1’), 67.50 (OCH2Ph), 61.11 (OCH2CH3), 44.66, 39.40, 28.26, 22.67 (Ccycloheptyl, CH2Ph), 14.28 (OCH2CH3); m/z (EI): 459.8 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(2-chlorocyclohexyloxy)nicotinate (6i). pale yellow liquid, yield 81%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.30 (s, 1H, C5-H), 5.42 (s, 2H, OCH2Ph), 4.47-4.53 (m, 1H, H1’), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.95 (s, 2H, CH2Ph), 1.25-1.31 (m, 9H, cyclohexyl);

13


165.57 (COOEt), 163.90 (C-4), 160.92 (C-2), 160.78 (C-6), 139.14, 137.71, 129.09, 128.45, 128.22, 127.60, 127.41, 126.39 (aryl), 104.62 (C-3), 102.16 (C-5), 71.29 (C1’), 67.53 (OCH2Ph), 61.10 (OCH2CH3), 44.68, 33.71, 28.83, 23.23, 21.86 (Ccyclohexyl, CH2Ph), 14.24 (OCH2CH3); m/z (EI): 480.7 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(2-methylcyclohexyloxy)nicotinate (6j). pale yellow liquid, yield 80%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.29 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.95 (s, 2H, CH2Ph), 3.72-3.78 (m, 1H, H1’), 0.90-2.02 (m, 15H, OCH2CH3, 2-methylCyclohexyl);

13

C NMR

(101 MHz, CDCl3): δ 165.64 (COOEt), 164.39 (C-4), 160.96 (C-2), 160.58 (C-6), 139.17, 137.74, 129.11, 128.41, 128.22, 127.64, 127.40, 126.36 (aryl), 104.49 (C-3), 102.26 (C-5), 82.84 (C1’), 67.52 (OCH2Ph), 61.10 (OCH2CH3), 44.62, 37.68, 33.20, 30.79, 25.01, 24.35, 25 ACS Paragon Plus Environment


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Page 26 of 64

18.44 (2-methylcyclohexyl, CH2Ph), 14.27 (OCH2CH3); m/z (EI): 460.8 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(3-methylcyclohexyloxy)nicotinate (6k). pale yellow liquid, yield 97%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.39 (m, 10H, Ph-H), 6.31 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.11-4.16 (m, 1H, H1’), 3.95 (s, 2H, CH2Ph), 0.82-2.01 (m, 15H, OCH2CH3, 3-methylCyclohexyl); 13C NMR (101 MHz, CDCl3): δ 165.58 (COOEt), 163.92 (C-4), 160.93 (C-2), 160.63 (C-6), 139.21, 137.73, 129.11, 128.43, 128.23, 127.62, 127.41, 126.38 (aryl), 104.69 (C-3), 102.40 (C-5), 73.38 (C1’), 67.52 (OCH2Ph), 61.10 (OCH2CH3), 44.61, 40.45, 38.7, 33.98, 31.30, 23.81, 22.28 (3-methylcyclohexyl, CH2Ph), 14.29 (OCH2CH3); m/z (EI): 459.7 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(4-methylcyclohexyloxy)nicotinate (6l). pale yellow liquid, yield 94%; 1H NMR (400 MHz, CDCl3): δ 7.19-7.37 (m, 10H, aryl), 6.31 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.09-4.14 (m, 1H, H1’), 3.95 (s, 2H, CH2Ph), 0.88-2.01 (m, 15H, OCH2CH3, 4-methylCyclohexyl); 13C NMR (101 MHz, CDCl3): δ 165.58 (COOEt), 163.97 (C-4), 160.94 (C-2), 160.62 (C-6), 139.18, 137.72, 129.08, 128.43, 128.22, 127.61, 127.40, 126.37 (aryl), 104.69 (C-3), 102.34 (C-5), 73.68 (C1’), 67.53 (OCH2Ph), 61.10 (OCH2CH3), 44.62, 37.75, 31.53, 28.99, 21.75 (4-methylcyclohexyl, CH2Ph), 14.28 (OCH2CH3); m/z (EI): 459.5 [M + H]+. Ethyl

6-benzyl-2-(benzyloxy)-4-(2,6-dimethylcyclohexyloxy)nicotinate

(6m).

pale

yellow liquid, yield 54%; 1H NMR (400 MHz, CDCl3): δ 7.21-7.40 (m, 10H, Ph-H), 6.36 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.30-4.36 (m, 3H, OCH2CH3, H1’), 3.95 (s, 2H, CH2Ph), 0.80-1.72 (m, 17H, OCH2CH3, 2, 6-dimethylcyclohexyl); 26 ACS Paragon Plus Environment

13

C NMR (101 MHz,

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CDCl3): δ 166.38 (COOEt), 165.75 (C-4), 160.88 (C-2), 160.55 (C-6), 139.13, 137.74, 129.16, 128.42, 128.20, 127.63, 127.38, 126.35 (aryl), 104.20 (C-3), 101.93 (C-5), 82.06 (C1’), 67.54 (OCH2Ph), 61.14 (OCH2CH3), 44.70, 37.39, 28.19, 25.74, 18.71 (2, 6-dimethylcyclohexyl, CH2Ph), 14.28 (OCH2CH3); m/z (EI): 488.8 [M + H]+. Ethyl

6-benzyl-2-(benzyloxy)-4-(3,5-dimethylcyclohexyloxy)nicotinate

(6o).

pale

yellow liquid, yield 57%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.30 (s, 1H, C5-H), 5.41 (s, 2H, OCH2Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.12-4.21 (m, 1H, H1’), 3.96 (s, 2H, CH2Ph), 1.29 (t, 3H, J = 7.2 Hz, OCH2CH3), 0.52-2.00 (m, 14H, 3, 5-dimethylcyclohexyl);

13

C NMR (101 MHz, CDCl3): δ 165.53 (COOEt), 163.92 (C-4),

160.93 (C-2), 160.58 (C-6), 139.18, 137.69, 129.09, 128.39, 128.19, 127.59, 127.37, 126.34 (aryl), 104.67 (C-3), 102.42 (C-5), 80.79 (C1’), 67.53 (OCH2Ph), 61.08 (OCH2CH3), 44.56, 42.97, 39.96, 30.54, 22.10 (3, 5-dimethylcyclohexyl, CH2Ph), 14.26 (OCH2CH3); m/z (EI): 474.6 [M + H]+. Ethyl 6-benzyl-2-(benzyloxy)-4-(3,3,5-trimethylcyclohexyloxy)nicotinate (6p). pale yellow liquid, yield: 27%; 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.29 (s, 1H, C5-H), 5.42 (s, 2H, OCH2Ph), 4.61 (s, 1H, H1’), 4.29 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.95 (s, 2H, CH2Ph), 0.82-1.91 (m, 19H, OCH2CH3, 3,3,5-trimethylcyclohexyl); 13C NMR (101 MHz, CDCl3): δ 165.73 (COOEt), 163.56 (C-4), 160.87 (C-2), 160.66 (C-6), 139.18, 137.75, 129.07, 128.40, 128.16, 127.55, 127.35, 126.34 (aryl), 104.44 (C-3), 104.51 (C-5), 74.53 (C1’), 67.45 (OCH2Ph), 61.22 (OCH2CH3), 48.15, 44.71, 41.40, 34.1, 30.61, 27.09, 23.01, 22.46 (3,3,5-trimethylcyclohexyl, CH2Ph), 14.14 (OCH2CH3); m/z (EI): 487.8 [M + 1]+ . 27 ACS Paragon Plus Environment


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Ethyl

2-(benzyloxy)-4-(2-methylcyclohexyloxy)-6-phenethylnicotinate

Page 28 of 64

(18).

pale

yellow liquid, yield 76%; 1H NMR (400 MHz, CDCl3): δ 7.12-7.45 (m, 10H, aryl), 6.14 (s, 1H, C5-H), 5.45 (s, 2H, OCH2Ph), 4.34 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.69-3.70 (m, 1H, H1’), 2.91-3.00 (m, 4H, CH2CH2-Ph), 0.94-1.92 (m, 15H, OCH2CH3, 2-methylcyclohexyl); 13

C NMR (101 MHz, CDCl3): δ 165.70 (COOEt), 164.12 (C-4), 160.95 (C-2), 160.88

(C-6), 141.51, 137.86, 128.55, 128.31, 128.22, 127.48, 127.38, 125.88 (aryl), 104.40 (C-3), 101.99 (C-5), 82.83 (C1’), 67.48 (OCH2Ph), 61.06 (OCH2CH3), 39.99, 37.70, 35.13, 33.28, 30.75, 25.06, 24.44 18.44, (2-methylcyclohexyl, CH2CH2Ph), 14.26 (OCH2CH3); m/z (EI): 474.5 [M + H]+ . Ethyl

6-benzyl-4-(cyclopentyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate

(7f):

white solid, yield 81%, m.p. 176-177 °C; 1H NMR (400 MHz, CDCl3): δ 7.22-7.32 (m, 5H, aryl), 5.76 (s, 1H, C5-H), 4.71 (s, 1H, H1’), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.90 (s, 2H, CH2-Ph), 1.57-1.78 (m, 8H, cyclohexyl ), 1.32 (t, 3H, J = 7.2 Hz, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ 166.11 (COOEt), 16528 (C-4), 163.77 (C-2), 151.97 (C-6), 136.16, 129.34, 128.77, 127.17 (aryl), 106.76 (C-3), 95.62 (C-5), 81.21 (C1’), 60.85 (OCH2CH3), 39.50, 32.95, 23.79 (cyclohexyl, CH2-Ph), 14.29 (OCH2CH3); HRMS (m/z): calcd for C20H24NO4 [M + H]+ 342.16998, found 342.16983. Ethyl 6-benzyl-4-(cyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate (7g). white solid, yield 88%, m.p. 203-204 °C; 1H NMR (400 MHz, CDCl3): δ 7.24-7.32 (m, 5H, aryl), 5.75 (s, 1H, C5-H), 4.32-4.37 (q, 2H, OCH2CH3), 4.25-4.29 (m, 1H, H1’), 3.89 (s, 2H, CH2-Ph), 1.26-1.79 (m, 13H, OCH2CH3, cyclohexyl );

13


165.90 (COOEt), 165.34 (C-4), 163.80 (C-2), 151.83 (C-6), 136.16, 129.31, 128.77, 28 ACS Paragon Plus Environment

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127.18 (aryl), 107.17 (C-3), 95.38 (C-5), 76.29 (C1’), 60.92 (OCH2CH3), 39.50, 31.34, 25.30, 27.74 (cyclohexyl, CH2-Ph), 14.31 (OCH2CH3); HRMS (m/z): calcd for C21H26NO4 [M + H]+ 356.18503, found 356.18555. Ethyl

6-benzyl-4-(cycloheptyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate

(7h).

white solid, yield 88%, m.p. 206-207 °C; 1H NMR (400 MHz, CDCl3): δ 7.23-7.34 (m, 5H, aryl), 5.69 (s, 1H, C5-H), 4.31-4.39 (m, 3H, OCH2CH3, H1’), 3.89 (s, 2H, CH2-Ph), 1.30-1.89 (m, 15H, OCH2CH3, cycloheptyl );

13

C NMR (101 MHz, CDCl3): δ 165.92

(COOEt), 165.37 (C-4), 163.75 (C-2), 151.88 (C-6), 136.07, 129.36, 128.80, 127.21 (aryl), 107.10 (C-3), 95.50 (C-5), 79.31 (C1’), 60.90 (OCH2CH3), 39.50, 33.63, 28.15, 22.55 (cycloheptyl, CH2-Ph), 14.32 (OCH2CH3);

HRMS (m/z): calcd for C22H28NO4 [M + H]+

370.20128, found 370.20125. Ethyl

6-benzyl-4-(2-chlorocyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate

(7i). white solid, yield 75%, m.p. 185-186 °C; 1H NMR (400 MHz, CDCl3): δ 7.23-7.31 (m, 5H, aryl), 5.76 (s, 1H, C5-H), 4.47-4.48 (m, 1H, H1’), 4.38 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.11-4.13 (m, 1H, H2’), 3.89 (s, 2H, CH2-Ph), 1.33-2.05 (m, 11H, OCH2CH3, 2-chlorocyclohexyl );

13

C NMR (101 MHz, CDCl3): δ 165.48 (COOEt), 165.02 (C-4),

163.81 (C-2), 151.15 (C-6), 136.01, 129.35, 128.81, 127.23 (aryl), 107.82 (C-3), 95.30 (C-5),

61.13

(C1’),

60.31

(OCH2CH3),

39.48,

31.51,

28.66,

23.33,

20.21

(2-chlorocyclohexyl, CH2-Ph), 14.34 (OCH2CH3); HRMS (m/z): calcd for C22H25ClNO4 [M + H]+ 390.14666, found 390.14648. Ethyl6-benzyl-4-(2-methylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate



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Page 30 of 64

(7j). white solid, yield 95%, m.p. 158-159 °C; 1H NMR (400 MHz, CDCl3): δ 7.24-7.32 (m, 5H, aryl), 5.72 (s, 1H, C5-H), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 3.89 (s, 2H, CH2-Ph), 3.69-3.73 (m, 1H, H1’), 0.95-1.95 (m, 15H, OCH2CH3, 2-methylcyclohexyl ); 13


(C-6), 136.16, 129.34, 128.76, 127.16 (aryl), 106.91 (C-3), 95.26 (C-5), 83.34 (C1’), 60.86 (OCH2CH3), 39.49, 37.58, 33.01, 31.15, 24.80, 24.20, 18.35 (2-methylcyclohexyl, CH2-Ph), 14.31 (OCH2CH3); HRMS (m/z): calcd for C22H28NO4 [M + H]+ 370.20128, found 370.20139. Ethyl6-benzyl-4-(3-methylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate (7k). white solid, yield 82%, m.p. 153-154 °C; 1H NMR (400 MHz, CDCl3): δ 7.24-7.31 (m, 5H, aryl), 5.75 (s, 1H, C5-H), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.09-4.12 (m, 1H, H1’), 3.89 (s, 2H, CH2-Ph), 0.84-2.03 (m, 15H, OCH2CH3, 3-methylCyclohexyl );

13

C

NMR (101 MHz, CDCl3): δ 166.03 (COOEt), 165.27 (C-4), 163.87 (C-2), 151.82 (C-6), 136.25, 129.32, 128.72, 127.14 (aryl), 107.17 (C-3), 95.50 (C-5), 74.12 (C1’), 60.83 (OCH2CH3), 40.58, 39.42, 33.79, 31.21, 26.42, 23.68, 22.17, 20.17 (3-methylCyclohexyl, CH2-Ph), 14.33 (OCH2CH3); HRMS (m/z): calcd for C22H28NO4 [M + H]+ 370.20128, found 370.20130. Ethyl

6-benzyl-4-(4-methylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate

(7l). white solid, yield 99%, m.p. 166-167 °C; 1H NMR (400 MHz, CDCl3): δ 7.24-7.31 (m, 5H, aryl), 5.75 (s, 1H, C5-H), 4.32 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.06-4.11 (m, 1H, H1’), 3.88 (s, 2H, CH2-Ph), 0.86-1.99 (m, 15H, OCH2CH3, 3-methylcyclohexyl );

13

C

NMR (101 MHz, CDCl3): δ 166.10 (COOEt), 165.27 (C-4), 163.83 (C-2), 151.75 (C-6), 30 ACS Paragon Plus Environment

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136.16, 129.30, 128.77, 127.18 (aryl), 107.17 (C-3), 95.46 (C-5), 77.96 (C1’), 60.87 (OCH2CH3), 39.49, 32.59, 31.68, 29.68, 28.86, 21.63 (3-methylcyclohexyl, CH2-Ph), 14.32 (OCH2CH3); HRMS (m/z): calcd for C22H28NO4 [M + H]+ 370.20128, found 370.20132. Ethyl6-benzyl-4-(2,6-dimethylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylat e (7m). white solid, yield 99%, m.p. 193-194 °C; 1H NMR (400 MHz, CDCl3): δ 7.25-7.31 (m, 5H, aryl), 5.78 (s, 1H, C5-H), 4.34 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.26 (m, 1H, H1’), 3.89 (s, 2H, CH2-Ph), 0.81-1.72 (m, 17H, 2,6-dimethylcyclohexyl, OCH2CH3); 13


(C-6), 136.14, 129.40, 128.75, 127.15 (aryl), 106.58 (C-3), 95.13 (C-5), 82.70 (C1’), 60.94 (OCH2CH3), 39.48, 37.28, 28.07, 25.65, 18.57 (2,6-dimethylCyclohexyl, CH2-Ph), 14.34 (OCH2CH3); HRMS (m/z): calcd for C23H30NO4 [M + H]+ 384.21693, found 384.21700. Ethyl6-benzyl-4-(3,5-dimethylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylat e (7o). white solid, yield 88%, m.p. 196-197 °C; 1H NMR (400 MHz, CDCl3): δ 7.23-7.31 (m, 5H, aryl), 5.73 (s, 1H, C5-H), 4.33 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.10-4.16 (m, 1H, H1’), 3.89 (s, 2H, CH2-Ph), 1.32 (t, 3H, J = 7.2 Hz, OCH2CH3), 0.52-1.97 (m, 14H, 3,5-dimethylcyclohexyl ); 13C NMR (101 MHz, CDCl3): δ 166.08 (COOEt), 165.27 (C-4), 163.84 (C-2), 151.79 (C-6), 136.22, 129.32, 128.74, 127.17 (aryl), 107.13 (C-3), 95.58 (C-5),

77.62

(C1’),

60.86

(OCH2CH3),

42.82,

40.10,

39.43,

30.50,

22.04

(3,5-dimethylcyclohexyl, CH2-Ph), 14.33 (OCH2CH3); HRMS (m/z): calcd for C23H30NO4 [M + H]+ 384.21693, found 384.21676.



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Page 32 of 64

Ethyl6-benzyl-2-oxo-4-(3,3,5-trimethylcyclohexyloxy)-1,2-dihydropyridine-3-carboxyl ate (7p). white solid, yield 88%, m.p. 196-197 °C; 1H NMR (400 MHz, CDCl3):δ δ 7.24-7.32 (m, 5H, aryl), 5.75 (s, 1H, C5-H), 4.58 (m, 1H, H1’), 4.29 (q, 2H, J = 7.2 Hz, OCH2CH3),

3.89

(s,

3,3,5-trimethylcyclohexyl );

2H, 13

CH2-Ph),

0.82-1.93

(m,

19H,

OCH2CH3,

C NMR (101 MHz, CDCl3): δ 169.63 (COOEt), 165.59

(C-4), 163.75 (C-2), 151.79 (C-6), 136.15, 129.31, 128.80, 127.21 (aryl), 116.01 (C-3), 94.72 (C-5), 75.27 (C1’), 61.08 (OCH2CH3), 48.02, 41.22, 39.61, 38.23, 33.94, 30.56, 27.10, 22.96, 22.40 (3,3,5-trimethylcyclohexyl, CH2-Ph), 14.19 (OCH2CH3); HRMS (m/z): calcd for C24H32NO4 [M + H]+ 398.23258, found 398.23257. Ethyl6-benzyl-4-(2-methylcyclohexyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate (19). white solid, yield 93%, m.p. 91-92 °C; 1H NMR (400 MHz, CDCl3): δ 7.17-7.30 (m, 5H, aryl), 5.67 (s, 1H, C5-H), 4.30-4.35 (m, 2H, OCH2CH3), 3.66-3.71 (m, 1H, H1), 2.83-3.01 (m, 4H, CH2CH2Ar), 0.86-1.89 (m, 15H, OCH2CH3, 2-methylcyclohexyl ); 13C NMR (101 MHz, CDCl3): δ 166.42 (COOEt), 165.39 (C-4), 164,14 (C-2), 151.81 (C-6), 140.26, 128.69, 128.42, 126.23 (aryl), 106.91 (C-3), 95.26 (C-5), 83.39 (C1’), 60.83 (OCH2CH3), 37.86, 35.96, 35.07, 33.16, 31.14, 29.68, 29.33, 24.90, 24.38, 18.38 (2-methylcyclohexyl, CH2CH2-Ph), 14.25 (OCH2CH3); HRMS (m/z): calcd for C23H30NO4 [M + H]+ 384.21684, found 384.21693. 6-aryl-3-halogen-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one

9-10

(21-22).

General Procedure. Compound 6e (4.36 mol) was dissolved in 30mL 1N HCl and refluxed for 2 d; after


Page 33 of 64

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the reaction was completed, Na2CO3 was added to adjust the mixture to neutral. Next, the precipitate was formed and filtered, and the solid was recrystallized in petroleum ether : acetone = 3:1 to get 8 as a white solid. Compound 8 (2.22 mmol) was dissolved in 20 mL anhydrous THF, and NBS (NIS) (2.67 mmol) was added to the mixture, which was protected from light and stirred at r.t. overnight. After evaporating the solvent, the residue was purified by column chromatography to give 9 and 10 as a solid. 6-benzyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one (8). white solid, yield 49%, m.p. 153-154 °C; 1H NMR (400 MHz, CDCl3): δ 7.24-7.31 (m, 5H, aryl), 5.77 (s, 1H, C5-H), 5.58 (s, 1H, C3-H), 3.88 (s, 2H, CH2-Ph), 3.71-3.74 (m, 1H, H1’), 0.90-2.13 (m, 12H, 2-methylCyclohexyl); 13C NMR (101 MHz, CDCl3): δ 168.96 (C-4), 167.53 (C-2), 148.91 (C-6), 136.31, 129.44, 128.70, 126.99 (aryl), 100.76 (C-3), 95.14 (C-5), 82.13 (C1’), 38.88, 37.65, 33.46, 30.43, 25.17, 24.48, 18.48 (2-methylCyclohexyl, CH2-Ph); HRMS (m/z): calcd for C19H24NO2 [M + H]+ 298.18016, found 298.17991. 4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one (20). white solid, yield 53%, m.p. 159-160 °C; 1H NMR (400 MHz, CDCl3): δ 7.17-7.29 (m, 5H, aryl), 5.76 (s, 1H, C5-H), 5.68 (s, 1H, C3-H), 3.72-3.76 (m, 1H, H1’), 3.01 (t, 2H, J = 8 Hz, CH2CH2-Ph), 2.84 (t, 2H, J = 8 Hz, CH2CH2-Ph), 0.93-2.13 (m, 12H, 2-methylcyclohexyl);

13

C NMR (101

MHz, CDCl3): δ 168.84 (C-4), 167.70 (C-2), 143.95 (C-6), 140.38, 128.59, 128.40, 126.19 (aryl), 100.15 (C-3), 95.33 (C-5), 82.12 (C1’), 37.72, 34.80, 34.51, 33.51, 30.54, 25.23, 24.54, 18.43 (2-methylcyclohexyl,CH2CH2-Ph); HRMS (m/z): calcd for C20H26NO2 [M + 33 ACS Paragon Plus Environment


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Page 34 of 64

H]+ 312.18581, found 312.19569. 6-benzyl-3-bromo-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one (9). white solid, yield 90%, m.p. 178-179 °C; 1H NMR (400 MHz, CDCl3): δ 7.23-7.45 (m, 5H, aryl), 5.77 (s, 1H, C5-H), 4.56-4.62 (m, 1H, H1’), 4.15 (s, 2H, CH2-Ph), 1.11-1.91 (m, 12H, 2-methylcyclohexyl); 13C NMR (101 MHz, CDCl3): δ 168.96 (C-4), 167.53 (C-2), 148.91 (C-6), 136.31, 129.44, 128.70, 126.99 (aryl), 100.76 (C-3), 95.14 (C-5), 82.13 (C1’), 38.88, 37.65, 33.46, 30.43, 25.17, 24.48, 18.48 (2-methylcyclohexyl, CH2-Ph); HRMS (m/z): calcd for C19H22Br2NO2 [M + HBr]+ 454.00118, found 454.00089. 3-bromo-4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one (21). brown solid, yield 90%, m.p. 174-175 °C; 1H NMR (400 MHz, CDCl3): δ 7.13-7.35 (m, 5H, aryl), 5.63 (s, 1H, C5-H), 3.71-3.74 (m, 1H, H1’), 3.03 (t, 2H, J = 7.6 Hz, CH2CH2-Ph), 2.90 (t, 2H, J = 7.6 Hz, CH2CH2-Ph), 0.98-1.80 (m, 12H, 2-methylcyclohexyl);

13

C NMR (101 MHz,

CDCl3): δ 168.04 (C-4), 164.76 (C-2), 150.01 (C-6), 140.27, 128.90, 128.44, 126.23 (aryl), 95.45 (C-3), 84.16 (C-5), 72.54 (C1’), 37.56, 35.83, 35.44, 33.11, 31.26, 24.85, 24.45, 18.65 (2-methylcyclohexyl, CH2CH2-Ph); HRMS (m/z): calcd for C20H25BrNO2 [M + H]+ 390.10632, found 390.10586. 6-benzyl-3-iodo-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one (10). white solid, yield 99%, m.p. 178-179 °C; 1H NMR (400 MHz, CDCl3): δ 7.23-7.38 (m, 5H, aryl), 5.68 (s, 1H, C5-H), 3.93 (s, 2H, CH2-Ph), 3.75-3.80 (m, 1H, H1’), 0.98-1.97 (m, 12H, 2-methylcyclohexyl); 13C NMR (101 MHz, CDCl3): δ 168.05 (C-4), 164.37 (C-2), 149.78 (C-6), 136.18, 129.41, 128.79, 127.20 (aryl), 95.56 (C-3), 84.12 (C-5), 72.86 (C1’), 39.22,


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37.50, 32.95, 31.26, 24.76, 24.30, 18.62 (2-methylCyclohexyl, CH2-Ph); HRMS (m/z): calcd for C19H23INO2 [M + H]+ 424.07680, found 424.07642. 3-iodo-4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one (22). brown solid, yield 97%, m.p. 176-177 °C; 1H NMR (400 MHz, CDCl3): δ 7.17-7.39 (m, 5H, aryl), 5.64 (s, 1H, C5-H), 3.73-3.76 (m, 1H, H1’), 3.04 (t, 2H, J = 7.6 Hz, CH2CH2-Ph), 2.91 (t, 2H, J = 7.6 Hz, CH2CH2-Ph), 1.00-1.82 (m, 12H, 2-methylcyclohexyl);

13

C NMR (101 MHz,

CDCl3): δ 168.06 (C-4), 164.80 (C-2), 150.06 (C-6), 140.29, 128.93, 128.46, 126.25 (aryl), 95.47 (C-3), 84.18 (C-5), 72.57 (C1’), 37.59, 35.83, 35.49, 33.11, 31.28, 24.88, 24.47, 18.67 (2-methylCyclohexyl, CH2CH2-Ph); HRMS (m/z): calcd for C20H25INO2 [M + H]+ 438.09245, found 438.09186. 6-aryl-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one 14a-e (26). General Procedure. An amount of 5a-e (2.86 mmol) was dissolved in 20 mL of dry THF. After cooling to -78 °C, a total of 6 mL (9.42 mmol) of methyllithium (1.6 M in ether) was added dropwise by means of a syringe. The reaction mixture was kept at -78 °C for 1 h and at 0 °C for 3 h. Then the mixture was ended with 2 mL of cold water and slowly neutralized with cold 1 N HCl. The organic layer was separated, and the aqueous phase was extracted twice with 40 mL of ethyl acetate. The combined organic solution was subsequently washed with sodium bicarbonate solution and brine. After evaporation of the solvents, the residue was purified by column chromatography to give 11a-e as a liquid. An amount of 11a-e (0.8 mmol) was dissolved in 30 mL of anhydrous THF. The 35 ACS Paragon Plus Environment


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solution was cooled to 5 °C, and 0.1 mL of thionyl chloride (SOCl2, 1.12 mmol) was added dropwise. The mixture was stirred at room temperature for 2 h. Afterwards, the solvent was evaporated, and the residue was dissolved in 20 mL of dichloromethane. This solution was basified with a cold sodium bicarbonate solution, washed with brine, and dried over magnesium sulfate. After evaporation of the solvent, the residue was purified by column chromatography to give 12a-e as a liquid. Amounts of 12a-e (2.662 mmol), 1.4 g (5.323 mmol) of triphenylphosphine, and 0.7 mL (5.323 mmol) of 2’-methylcyclohexanol (mixture) were dissolved in 30 mL of dry THF. A total of 1.1mL of DIAD (diisopropyl azodicarboxylate, 5.323 mmol) dissolved in 5 mL of THF was added dropwise by means of a syringe. The reaction mixture was stirred at room temperature for 8h. After evaporation of the solvent, the residue was purified by column chromatography to give 13a-e as a liquid. To a pressure reaction bottle were added 13a-e (0.34 mmol) and 0.02 g of Pd/C in 20 mL of anhydrous methanol. The mixture was placed under hydrogen (1 atm) and stirred at room temperature for 8 h. After filtration and evaporation of the solvent, the resulting residue was purified by column chromatography to give 14a-e as a white solid. 6-benzyl-2-(benzyloxy)-3-(2-hydroxypropan-2-yl)pyridin-4-ol (11a). liquid, yield 92%, 1

H NMR (400 MHz, CDCl3): δ 7.26-7.45 (m, 10H, aryl), 6.33 (s, 1H, C5-H), 5.43 (s, 2H,

OCH2-Ph), 3.95 (s, 2H, CH2-Ph), 1.73 (s, 6H, C(CH3)2); 13C NMR (101 MHz, CDCl3): δ 164.57 (C-4), 159.73 (C-2), 156.95 (C-6), 139.58, 137.88, 129.36, 128.45, 128.40, 128.30, 127.64, 126.24 (aryl), 108.74 (C-3), 107.51 (C-5), 78.19 (OCH2-Ph), 67.82 (HOC(CH3)2), 36 ACS Paragon Plus Environment

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43.84 (CH2-Ph), 30.47 (CH3); m/z (EI): 350.3 [M + H]+. 6-(4-fluorobenzyl)-2-(benzyloxy)-3-(2-hydroxypropan-2-yl)pyridin-4-ol (11b). liquid, yield 94%, 1H NMR (400 MHz, CDCl3): δ 6.92-7.36 (m, 9H, aryl), 6.22 (s, 1H, C5-H), 5.34 (s, 2H, OCH2-Ph), 3.83 (s, 2H, CH2-Ph), 1.68 (s, 6H, C(CH3)2); 13C NMR (101 MHz, CDCl3): δ 164.57 (C-4), 159.73 (C-2), 156.65 (C-6), 137.81, 135.24, 130.72, 130.64, 128.37, 128.31, 128.16, 127.62, 115.17, 114.96 (aryl), 108.71 (C-3), 107.37 (C-5), 78.24 (OCH2-Ph), 67.74 (HOC(CH3)2), 42.88 (CH2-Ph), 30.46 (CH3); m/z (EI): 368.5 [M + H]+. 6-(4-chlorobenzyl)-2-(benzyloxy)-3-(2-hydroxypropan-2-yl)pyridin-4-ol (11c). liquid, yield 80%, 1H NMR (400 MHz, CDCl3): δ 7.16-7.33 (m, 9H, aryl), 6.23 (s, 1H, C5-H), 5.34 (s, 2H, OCH2-Ph), 3.82 (s, 2H, CH2-Ph), 1.68 (s, 6H, C(CH3)2); 13C NMR (101 MHz, CDCl3): δ 164.63 (C-4), 159.74 (C-2), 156.26 (C-6), 138.04, 137.76, 132.00, 130.63, 128.42, 128.36, 128.16, 127.63 (aryl), 108.83 (C-3), 107.46 (C-5), 78.23 (OCH2-Ph), 67.79 (HOC(CH3)2), 42.02 (CH2-Ph), 30.47 (CH3); m/z (EI): 384.7 [M + H]+. 6-(4-methoxybenzyl)-2-(benzyloxy)-3-(2-hydroxypropan-2-yl)pyridin-4-ol(11d). liquid, yield 67%, 1H NMR (400 MHz, CDCl3): δ 6.78-7.35(m, 9H, aryl), 6.190(s, 1H, C5-H), 5.33(s, 2H, OCH2-Ph), 3.79(s, 2H, CH2-Ph), 3.73(s, 3H, CH3OAr), 1.63(s, 6H, C(CH3)2); 13

C NMR (101 MHz, CDCl3): δ 164.72(C-4), 159.69(C-2), 158.08(C-6), 157.24, 137.89,

131.75, 130.27, 128.35, 128.25, 127.59, 113,84(aryl), 108.77(C-3), 107.31(C-5), 77.94(OCH2-Ph), 67.88(HOC(CH3)2),

55.26(CH3OPh), 42.86(CH2-Ph), 30.39(CH3); m/z

(EI): 380.8 [M + H]+. 6-(4-methylbenzyl)-2-(benzyloxy)-3-(2-hydroxypropan-2-yl)pyridin-4-ol (11e). liquid, 37 ACS Paragon Plus Environment


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yield 97%, 1H NMR (400 MHz, CDCl3): δ 7.04-7.34(m, 9H, aryl), 6.20(s, 1H, C5-H), 5.32(s, 2H, OCH2-Ph), 3.80(s, 2H, CH2-Ph), 2.37(s, 3H, CH3-Ph), 1.62(s, 6H, C(CH3)2); 13

C NMR

(101 MHz, CDCl3): δ 164.57 (C-4), 159.70(C-2), 157.22(C-6), 137.90, 136.52,

135.66, 129.21, 129.09, 128.36, 128.29, 127.61(aryl), 108.63 (C-3), 107.39(C-5), 78.19(OCH2-Ph), 67.81(HOC(CH3)2), 43.41(CH2-Ph), 30.48(CH3), 21.06(CH3-Ph); m/z (EI): 363.9 [M + H]+. 2-(benzyloxy)-3-(2-hydroxypropan-2-yl)-6-phenethylpyridin-4-ol (23). liquid, yield 92%, 1H NMR (400 MHz, CDCl3): δ 7.17-7.42 (m, 10H, aryl), 6.28 (s, 1H, C5-H), 5.37 (s, 2H, OCH2-Ph), 3.00 (t, 2H, CH2CH2Ph), 2.86 (t, 2H, CH2CH2Ph), 1.71 (s, 6H, C(CH3)2); 13

C NMR (101 MHz, CDCl3): δ 164.44 (C-4), 159.69 (C-2), 157.27 (C-6), 142.11 , 138.01,

128.44, 128.37, 128.32, 128.15, 127.60, 125.81 (aryl), 108.62 (C-3), 107.13 (C-5), 78.21 (OCH2-Ph), 67.77 (HOC(CH3)2), 38.91 (CH2CH2Ph), 35.02 (CH2CH2Ph), 30.52 (CH3); m/z (EI): 364.7 [M + H]+. 6-benzyl-2-(benzyloxy)-3-(prop-1-en-2-yl)pyridin-4-ol (12a). liquid, yield 86%, 1H NMR (400 MHz, CDCl3): δ 7.21-7.39 (m, 10H, aryl), 6.36 (s, 1H, C5-H), 6.10 (br s, 1H, OH), 5.43 (d, 1H, CH3C=CH2), 5.41 (s, 2H, OCH2-Ph), 5.04 (d, 1H, CH3C=CH2), 3.93 (s, 2H, CH2-Ph), 2.04 (s, 3H, CH3C=CH2);

13

C NMR (101 MHz, CDCl3): δ 160.28 (C-4),

160.27 (C-2), 157.89 (C-6), 139.46 (CH3C=CH2), 138.69, 137.95, 129.28, 128.40, 128.27, 127.80, 127.47, 126.26 (aryl), 117.94 (CH3C=CH2), 108.79 (C-3), 104.64 (C-5), 67.51 (OCH2-Ph), 44.22 (CH2-Ph), 22.81 (CH3C=CH2); m/z (EI): 332.5 [M + H]+ . 6-(4-fluorobenzyl)-2-(benzyloxy)-3-(prop-1-en-2-yl)pyridin-4-ol (12b). liquid, yield


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82%, 1H NMR (400 MHz, CDCl3): δ 6.93-7.38 (m, 9H, aryl), 6.35 (s, 1H, C5-H), 5.44 (d, 1H, CH3C=CH2), 5.39 (s, 2H, OCH2-Ph), 5.05 (d, 1H, CH3C=CH2), 3.88 (s, 2H, CH2-Ph), 2.04 (s, 3H, CH3C=CH2);

13

C NMR (101 MHz, CDCl3): δ 162.80 (C-4), 160.36 (C-2),

157.62 (C-6), 138.61 (CH3C=CH2), 137.92, 135.15, 130.68, 130.60, 128.36, 128.30, 127.80, 127.50 (aryl), 118.05 (CH3C=CH2), 108.88 (C-3), 104.57 (C-5), 67.49 (OCH2-Ph), 43.30 (CH2-Ph), 22.8 1(CH3C=CH2); m/z (EI): 349.8 [M + H]+ . 6-(4-chlorobenzyl)-2-(benzyloxy)-3-(prop-1-en-2-yl)pyridin-4-ol (12c). liquid, yield 90%, 1H NMR (400 MHz, CDCl3): δ 7.17-7.37 (m, 9H, aryl), 6.35 (s, 1H, C5-H), 6.15 (br s, 1H, OH), 5.44 (d, 1H, CH3C=CH2), 5.38 (s, 2H, OCH2-Ph), 5.05 (d, 1H, CH3C=CH2), 3.87 (s, 2H, CH2-Ph), 2.04 (s, 3H, CH3C=CH2); 13C NMR (101 MHz, CDCl3): δ 160.38 (C-4), 160.32 (C-2), 157.24 (C-6), 138.57 (CH3C=CH2), 137.96, 137.89, 132.08, 130.60, 128.48, 128.30, 127.81, 127.51 (aryl), 118.08 (CH3C=CH2), 108.95 (C-3), 104.65 (C-5), 67.50 (OCH2-Ph), 43.46 (CH2-Ph), 22.82 (CH3C=CH2); m/z (EI): 367.3 [M + H]+. 6-(4-methoxybenzyl)-2-(benzyloxy)-3-(prop-1-en-2-yl)pyridin-4-ol (12d). liquid, yield 64%, 1H NMR (400 MHz, CDCl3): δ 6.80-7.39 (m, 9H, aryl), 6.33 (s, 1H, C5-H), 5.41 (d, 1H, CH3C=CH2), 5.36 (s, 2H, OCH2-Ph), 5.02 (d, 1H, CH3C=CH2), 3.86 (s, 2H, CH2-Ph), 3.76 (s, 3H, OCH3), 2.03 (s, 3H, CH3C=CH2);

13

C NMR (101 MHz, CDCl3): δ 160.35

(C-4), 158.36 (C-2), 158.18 (C-6), 138.68 (CH3C=CH2), 138.01, 131.62, 130.25, 128.31, 127.91, 127.50 (aryl), 117.94 (CH3C=CH2), 108.79 (C-3), 104.56 (C-5), 67.55 (OCH2-Ph), 55.26 (OCH3), 43.33 (CH2-Ph), 22.86 (CH3C=CH2); m/z (EI): 362.7 [M + H]+ . 6-(4-methylbenzyl)-2-(benzyloxy)-3-(prop-1-en-2-yl)pyridin-4-ol (12e). liquid, yield



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67%, 1H NMR (400 MHz, CDCl3): δ 7.08-7.39 (m, 9H, aryl), 6.35 (s, 1H, C5-H), 5.41 (d, 2H, CH3C=CH2), 5.35 (s, 2H, OCH2-Ph), 5.03 (d, 1H, CH3C=CH2), 3.89 (s, 2H, CH2-Ph), 2.31 (s, 3H, CH3), 2.03 (s, 3H, CH3C=CH2); 13C NMR (101 MHz, CDCl3): δ 160.32 (C-4), 158.22 (C-2), 158.20 (C-6), 138.74 (CH3C=CH2), 138.01, 136.43, 135.74, 129.16, 129.12, 128.27, 127.91, 127.47 (aryl), 117.88 (CH3C=CH2), 108.76 (C-3), 104.57 (C-5), 67.53 (OCH2-Ph), 43.84 (CH2-Ph), 22.82 (CH3C=CH2), 21.05 (CH3); m/z (EI): 346.7 [M + H]+ . 2-(benzyloxy)-6-phenethyl-3-(prop-1-en-2-yl)pyridin-4-ol (24). liquid, yield 84%, 1H NMR (400 MHz, CDCl3): δ 7.17-7.44 (m, 10H, aryl), 6.38 (s, 1H, C5-H), 6.17 (br s, 1H, OH), 5.44 (d, 1H, CH3C=CH2), 5.40 (s, 2H, OCH2-Ph), 5.06 (d, 1H, CH3C=CH2), 2.88-3.03 (m, 4H, CH2CH2-Ph), 2.06 (s, 3H, CH3C=CH2); 13C NMR (101 MHz, CDCl3): δ 160.37 (C-4), 160.17 (C-2), 158.27 (C-6), 141.99 (CH3C=CH2), 138.80, 138.12, 128.50, 128.38, 128.35, 127.82, 127.53, 125.90 (aryl), 117.96 (CH3C=CH2), 108.76 (C-3), 104.44 (C-5), 67.52 (OCH2-Ph), 39.46 (CH2CH2-Ph), 35.17 (CH2CH2-Ph), 22.94 (CH3C=CH2); m/z (EI): 346.8 [M + H]+. 6-benzyl-2-(benzyloxy)-4-(2-methylcyclohexyloxy)-3-(prop-1-en-2-yl)pyridine

(13a).

liquid, yield 35%, 1H NMR (400 MHz, CDCl3): δ 7.20-7.38 (m, 10H, aryl), 6.28 (s, 1H, C5-H), 5.41 (s, 2H, OCH2-Ph), 5.26 (d, 1H, CH3C=CH2), 4.88 (d, 1H, CH3C=CH2), 3.94 (s, 2H, CH2-Ph), 3.69-3.73 (m, 1H, H1’), 1.98 (s, 3H, CH3C=CH2), 1.24-1.78 (m, 9H, cyclohexyl ), 0.92 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 163.29 (C-4), 161.01

(C-2), 157.02 (C-6), 139.83 (CH3C=CH2), 138.58, 137.42, 129.21, 128.34, 128.17, 127.63, 127.17, 126.18 (aryl), 116.38 (CH3C=CH2), 112.03 (C-3), 102.18 (C-5), 81.73 (C1’), 67.13 (CH2-Ph), 44.61, 37.71, 35.73, 33.34, 30.80, 25.13, 24.45 (Ccyclohexyl, CH2-Ph), 23.27 40 ACS Paragon Plus Environment

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(CH3C=CH2), 18.65 (CH32’); m/z (EI): 428.5 [M + H]+ . 6-(4-fluorobenzyl)-2-(benzyloxy)-4-(2-methylcyclohexyloxy)-3-(prop-1-en-2-yl)pyridin e (13b). liquid, yield 32%, 1H NMR (400 MHz, CDCl3): δ 6.92-7.37 (m, 9H, aryl), 6.27 (s, 1H, C5-H), 5.40 (s, 2H, OCH2-Ph), 5.27 (d, 1H, CH3C=CH2), 4.89 (d, 1H, CH3C=CH2), 3.89 (s, 2H, CH2-Ph), 3.70-3.75 (m, 1H, H1’), 1.99 (s, 3H, CH3C=CH2), 1.05-1.75 (m, 9H, cyclohexyl ), 0.93 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 163.32 (C-4), 161.02

(C-2), 156.76 (C-6), 138.54 (CH3C=CH2), 137.35, 135.49, 130.60, 128.18, 127.51, 127.19 (aryl), 116.43 (CH3C=CH2), 112.10 (C-3), 102.04 (C-5), 81.78 (C1’), 67.11 (OCH2-Ph), 43.67 (CH2-Ph), 37.71, 35.73, 33.34, 30.80, 25.13, 24.45 (Ccyclohexyl), 23.25 (CH3C=CH2), 18.67 (CH32’); m/z (EI): 446.7 [M + H]+ . 6-(4-chlorobenzyl)-2-(benzyloxy)-4-(2-methylcyclohexyloxy)-3-(prop-1-en-2yl)pyridin e (13c). liquid, yield 34%, 1H NMR (400 MHz, CDCl3): δ 7.17-7.36 (m, 9H, aryl), 6.28 (s, 1H, C5-H), 5.40 (s, 2H, OCH2-Ph), 5.27 (d, 1H, CH3C=CH2), 4.88 (d, 1H, CH3C=CH2), 3.89 (s, 2H, CH2-Ph), 3.71-3.76 (m, 1H, H1’), 1.98 (s, 3H, CH3C=CH2), 1.06-1.79 (m, 9H, cyclohexyl ), 0.93 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 163.32 (C-4), 160.99

(C-2), 156.34 (C-6), 138.28 (CH3C=CH2), 137.31, 131.95, 130.49, 128.37, 128.18, 127.50, 127.19 (aryl), 116.44 (CH3C=CH2), 112.16 (C-3), 102.08 (C-5), 81.79 (C1’), 67.12 (OCH2-Ph), 43.73 (CH2-Ph), 37.71, 35.72, 33.33, 30.79, 25.12, 24.46 (Ccyclohexyl), 23.24 (CH3C=CH2), 18.67 (CH32’); m/z (EI): 462.4 [M + H]+. 6-(4-methoxybenzyl)-2-(benzyloxy)-4-(2-methylcyclohexyloxy)-3-(prop-1-en-2-yl)pyrid ine (13d). liquid, yield 36%, 1H NMR (400 MHz, CDCl3): δ 6.79-7.39 (m, 9H, aryl), 6.26 41 ACS Paragon Plus Environment


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(s, 1H, C5-H), 5.40 (s, 2H, OCH2-Ph), 5.26 (d, 1H, CH3C=CH2), 4.86 (d, 1H, CH3C=CH2), 3.87 (s, 2H, CH2-Ph), 3.76 (s, 3H, CH3O-Ph), 3.68-3.72 (m, 1H, H1’), 2.30 (s, 3H, CH3C=CH2), 0.92-1.72 (m, 12H, cyclohexyl, CH32’);

13


163.32 (C-4), 161.09 (C-2), 157.33 (C-6), 138.65 (CH3C=CH2), 137.49, 136.84, 135.65, 129.07, 128.18, 127.67, 127.20 (aryl), 116.42 (CH3C=CH2), 111.98 (C-3), 102.11 (C-5), 81.73 (C1’), 67.17 (OCH2-Ph), 55.26 (CH3O-Ph), 44.16 (CH2-Ph), 37.74, 33.36, 30.86, 25.18, 24.48 (Ccyclohexyl), 23.32 (CH3C=CH2), 18.71 (CH32’); m/z (EI): 458.5 [M + H]+ . 6-(4-methylbenzyl)-2-(benzyloxy)-4-(2-methylcyclohexyloxy)-3-(prop-1-en-2-yl)pyridi ne (13e). liquid, yield 36%, 1H NMR (400 MHz, CDCl3): δ 7.07-7.39 (m, 9H, aryl), 6.28 (s, 1H, C5-H), 5.41 (s, 2H, OCH2-Ph), 5.25 (d, 1H, CH3C=CH2), 4.87 (d, 1H, CH3C=CH2), 3.90 (s, 2H, CH2-Ph), 3.70-3.73 (m, 1H, H1’), 2.32 (s, 3H, CH3C=CH2), 1.97 (s, 3H, CH3), 1.24-1.73 (m, 9H, cyclohexyl ), 0.92 (d, 3H, CH32’);

13


163.31 (C-4), 160.99 (C-2), 157.30 (C-6), 138.62 (CH3C=CH2), 137.47, 136.83, 135.65, 129.07, 128.18, 127.66, 127.19 (aryl), 116.38 (CH3C=CH2), 111.98 (C-3), 102.10 (C-5), 81.71 (C1’), 67.16 (OCH2-Ph), 44.15 (CH2-Ph), 37.73, 33.36, 30.83, 25.17, 24.48 (Ccyclohexyl), 23.30 (CH3C=CH2), 21.11 ( CH3Ph), 18.69 (CH32’); m/z (EI): 442.6 [M + H]+ . 2-(benzyloxy)-4-(2-methylcyclohexyloxy)-6-phenethyl-3-(prop-1-en-2-yl)pyridine (25). liquid, yield 54%, 1H NMR (400 MHz, CDCl3): δ 7.08-7.38 (m, 10H, aryl), 6.10 (s, 1H, C5-H), 5.36 (s, 2H, OCH2-Ph), 5.19-5.21 (d, 1H, CH3C=CH2), 4.82 (d, 1H, CH3C=CH2), 3.59-3.64 (m, 1H, H1’), 2.91-2.94 (m, 2H, CH2CH2Ph), 2.81-2.85 (m, 2H, CH2CH2Ph), 1.93 (s, 3H, CH3C=CH2), 1.13-1.71 (m, 9H, cyclohexyl ), 0.85 (d, 3H, CH32’); 13C NMR (101 MHz, CDCl3): δ 163.05 (C-4), 160.99 (C-2), 157.40 (C-6), 142.08 (CH3C=CH2), 42 ACS Paragon Plus Environment

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138.73, 137.55, 128.56, 128.30, 128.20, 127.52, 127.20, 125.80 (aryl), 116.35 (CH3C=CH2), 111.93 (C-3), 102.02 (C-5), 81.71 (C1’), 67.11 (OCH2-Ph), 39.90, 37.76, 35.38, 33.45, 30.80, 25.23, 24.58 (Ccyclohexyl, CH2CH2-Ph), 23.35 (CH3C=CH2), 18.72 (CH32’); m/z (EI): 442.5 [M + H]+. 6-benzyl-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one (14a). white solid, yield 90%, m.p. 149-150 °C; 1H NMR (400MHz, CDCl3): δ 7.21-7.38 (m, 5H, aryl), 5.72 (s, 1H, C5-H), 3.87 (s, 2H, CH2-Ph), 3.66-4.27 (m, 1H, H1’), 3.46-3.54 (m, 1H, HC(CH3)2), 1.24-1.92 (m, 6H, HC(CH3)2), 1.05-1.92 (m, 9H, cyclohexyl), 0.96 (d, 3H, CH32’);

13

C

NMR (101 MHz, CDCl3): δ 166.12 (C-2), 164.42 (C-4), 137.24 (C-6 ), 146.03, 129.33, 128.62, 126.87 (aryl), 117.38 (C-3), 95.87 (C-5), 81.72 (C1’), 39.21 (CH2-Ph), 37.78, 33.31, 31.31, 25.08, 24.41, 24.03 (Ccyclohexyl, HC(CH3)2), 20.28, 20.15 (CH(CH3)2), 18.71 (CH32’); HRMS (m/z): calcd for C22H30NO2 [M + H]+ 340.22711, found 340.22706. 6-(4-fluorobenzyl)-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one

(14b).

white solid, yield 96%, m.p. 154-155 °C; 1H NMR (400MHz, CDCl3): δ 6.98-7.35 (m, 4H, aryl), 5.73 (s, 1H, C5-H), 3.84 (s, 2H, CH2-Ph), 3.69-3.73 (m, 1H, H1’), 3.44-3.55 (m, 1H, HC(CH3)2), 1.26-1.96 (m, 15H, cyclohexyl, HC(CH3)2), 0.99 (d, 3H, CH32’);

13

C NMR

(101 MHz, CDCl3): δ 166.18 (aryl), 164.43 (C-2), 163.09 (C-4), 160.66 (C-6), 145.85, 133.05, 130.84, 115.47 (aryl), 117.47 (C-3), 95.81 (C-5), 81.81 (C1’), 38.39 (CH2-Ph), 37.79, 33.30, 31.31, 25.06, 24.42, 23.97 (Ccyclohexyl, HC(CH3)2), 20.26, 20.11 (CH(CH3)2), 18.71 (CH32’); HRMS (m/z): calcd for C22H29FNO2 [M + H]+ 358.21768, found 358.21759.



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6-(4-chlorobenzyl)-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one

(14c).

white solid, yield 85%, m.p. 139-140 °C; 1H NMR (400MHz, CDCl3): δ 7.28-7.37 (m, 4H, aryl), 5.72 (s, 1H, C5-H), 3.87 (s, 2H, CH2-Ph), 3.69-3.71 (m, 1H, H1’), 3.45-3.53 (m, 1H, HC(CH3)2), 1.24-1.95 (m, 15H, cyclohexyl, HC(CH3)2), 0.97 (d, 3H, CH32’);

13

C NMR

(101 MHz, CDCl3): δ 165.97 (C-2), 164.41 (C-4), 145.81 (C-6), 137.08, 129.30, 128.64, 126.91 (aryl), 117.43 (C-3), 95.91 (C-5), 81.73 (C1’), 39.21 (CH2-Ph), 37.76, 33.31, 31.29, 25.07, 24.40, 23.99 (Ccyclohexyl, HC(CH3)2), 20.29 (CH(CH3)2), 20.12 (CH(CH3)2), 18.71 (CH32’); HRMS (m/z): calcd for C22H30NO2 [M + H]+ 340.22711, found 340.22699. 6-(4-methoxybenzyl)-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one (14d). white solid, yield 95%, m.p. 138-139 °C; 1H NMR (400MHz, CDCl3): δ 6.85-7.24 (m, 4H, aryl), 5.74 (s, 1H, C5-H), 4.93-4.99 (m, 2H, CH2-Ph), 3.79 (s, 3H, OCH3), 3.71-3.74 (m, 1H, H1’), 3.45 (m, 1H, HC(CH3)2), 0.84-1.99 (m, 18H, cyclohexyl, HC(CH3)2, CH32’); 13C NMR (101 MHz, CDCl3): δ 164.79 (C-2), 163.43 (C-4), 142.78 (C-6), 128.18, 127.69, 126.26, 126.18 (aryl), 113.69 (C-3), 99.81 (C-5), 81.75 (C1’), 50.13(CH3O-Ph), 37.58 (CH2-Ph), 37.69, 33.41, 31.32, 25.17, 24.37, 23.66 (Ccyclohexyl, HC(CH3)2), 20.25, 20.10 (CH(CH3)2), 18.69(CH32’); HRMS (m/z): calcd for C23H32NO3 [M + H]+ 370.23767, found 370.23786. 6-(4-methylbenzyl)-3-isopropyl-4-(2-methylcyclohexyloxy)pyridin-2(1H)-one

(14e).

white solid, yield 94%, m.p. 138-139 °C; 1H NMR (400MHz, CDCl3): δ 7.11-7.24 (m, 4H, aryl), 5.75 (s, 1H, C5-H), 3.81 (s, 2H, CH2-Ph), 3.71-3.73 (m, 1H, H1’), 3.42-3.50 (m, 1H, HC(CH3)2), 2.32 (s, 3H, CH3), 1.24-1.99 (m, 15H, cyclohexyl+ HC(CH3)2), 0.98-1.00 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 165.79 (C-2), 164.39 (C-4), 154.51 (C-6), 44 ACS Paragon Plus Environment

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136.55, 133.93, 129.37, 129.15(aryl), 118.79 (C-3), 95.79 (C-5), 81.72 (C1’), 38.88 (CH2-Ph), 37.78, 33.32, 31.32, 25.09, 24.43, 24.01 (Ccyclohexyl, HC(CH3)2), 21.05（CH3-Ph）, 20.28, 20.12 (CH(CH3)2), 18.71 (CH32’); HRMS (m/z): calcd for C23H32NO2 [M + H]+ 354.24276, found 354.24324. 3-isopropyl-4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one

(26).

white

solid, yield 97%, m.p. 128-129 °C; 1H NMR (400 MHz, CDCl3): δ 7.36-7.14 (m, 5H, aryl), 5.71 (s, 1H, C5-H), 3.69-4.30 (m, 1H, H1’), 3.53-3.62 (m, 1H, HC(CH3)2), 3.02-3.06 (m, 2H, CH2CH2-Ph), 2.80-2.88 (m, 2H, CH2-CH2-Ph), 1.05-1.92 (m, 17H, cyclohexyl, HC(CH3)2), 0.97 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 166.60 (C-2), 164.48

(C-4), 146.47 (C-6), 141.04, 128.77, 128.37, 126.12 (aryl), 117.31 (C-3), 95.74 (C-5), 81.76 (C1’), 37.92, 35.95 (CH2CH2Ph), 35.56, 33.50, 31.36, 25.25, 24.62, 24.11 (Ccyclohexyl, HC(CH3)2), 20.43, 20.27 (HC(CH3)2), 18.81 (CH32’); HRMS (m/z): calcd for C23H32NO2 [M + H]+ 354.24276, found 354.24287. 3-isopropyl-4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one

(26-trans).

white solid, yield 97%, m.p. 149-150 °C; 1H NMR (400 MHz, CDCl3): δ7.30-7.19 (m, 5H, aryl), 5.72 (s, 1H, H-5), 4.29-4.30 (m, 1H, H1’), 3.53-3.55 (m, 1H, HC(CH3)2), 2.93-3.01 (m, 2H, CH2CH2-Ph), 2.78-2.81 (m, 2H, CH2-CH2-Ph), 1.24-1.85 (m, 17H, cyclohexyl, HC(CH3)2), 0.98 (d, 3H, CH32’);

13

C NMR (101 MHz, CDCl3): δ 166.42 (C-2), 165.39

(C-4), 146.43 (C-6), 141.02, 128.73, 128.32, 126.09 (aryl), 117.29 (C-3), 95.72 (C-5), 81.75 (C1’), 37.90, 35.93 (CH2CH2Ph), 35.54, 33.48, 31.32, 25.20, 24.59, 24.09 (Ccyclohexyl, HC(CH3)2), 20.40, 20.24 (HC(CH3)2), 18.80 (CH32’); HRMS (m/z): calcd for C23H32NO2 [M + H]+ 354.24276, found 354.24265. 45 ACS Paragon Plus Environment


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3-isopropyl-4-(2-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-one (26-cis). white solid, yield 97%, m.p. 123-125 °C; 1H NMR (400 MHz, CDCl3): δ7.30-7.19 (m, 5H, aryl), 5.69 (s, 1H, H-5), 3.68-3.69 (m, 1H, H1’), 3.44-3.51 (m, 1H, HC(CH3)2), 2.98-3.01 (m, 2H, CH2CH2-Ph), 2.78-2.82 (m, 2H, CH2-CH2-Ph), 1.09-1.89 (m, 17H, cyclohexyl, HC(CH3)2), 0.99 (d, 3H, CH32’); 13C NMR (101 MHz, CDCl3): δ 166.53 (C-2), 164.51 (C-4), 146.49 (C-6), 141.05, 128.78, 128.37, 126.16 (aryl), 117.36 (C-3), 95.73 (C-5), 81.76 (C1’), 37.95, 35.96 (CH2CH2Ph), 35.56, 33.51, 31.34, 25.23, 24.64 , 24.13 (Ccyclohexyl, HC(CH3)2), 20.43, 20.29 (HC(CH3)2), 18.84 (CH32’); HRMS (m/z): calcd for C23H32NO2 [M + H]+ 354.24276, found 354.24291. Biological assays Assay for Measuring the Inhibitory Activity of Compounds against HIV-1 RT Oligo(dT) (TaKaRa Co., Japan) was immobilized via its 5'-terminal phosphate to Covalink-NH microtiter plates (NUNC Co., Denmark). The biotin-dUTP was incorporated by reverse transcriptase (Sigma). Briefly, a serial concentration of inhibitor was added to the mixture, which contained 50 mmol/L Tris–HCl (pH 8.3), 3 mmol/L MgCl2, 75 mmol/L KCl, 5 mmol/L DTT (D,L,-dithiothreitol), 0.13 mg/mL BSA (Albumin Bovine V), 10 µg/mL poly (A), 0.75 µM biotin-11-dUTP, and 1.5 µM dTTP. The reaction mixture was incubated at 37 °C for 1 h and washed with a buffer containing 50 mmol/L Tris–HCl (pH7.5), 0.15 mol/L NaCl, 0.05 mmol/L MgCl2, and 0.02% Tween-20. After 100 µL of 1% BSA was added to each well and incubated at room temperature for another 30 min, the plate was washed with the same buffer. Before further incubation at 37 oC for 1 h, 50 µL of


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SA-ALP (Alkaline Phosphatase Streptavidin) solution (100 ng / mL) was added per well and washed again as above. Finally, 50µL of PNPP (p-nitrophenyl phosphate, disodium) (1 mg/mL, pH 9.5) was added and incubated at 37 °C for 30 min. The reaction was stopped by addition of 0.5 M NaOH. The inhibitory activity of the compounds was detected and quantified using a colorimetric streptavidin-alkaline phosphatase reporter system.

Infectious clone virus with Tyr181Cys and Lys103Asn mutation based on site-directed mutagenesis The mutations were introduced into the new T-vector containing HIV PR and RT regions by use of DNA polymerase (PrimerStar, Takara), and proper site mutation primers and then digested with BstE II and AgeI (NEB), purified by agarose gel electrophoresis, and ligated to BstE II- and AgeI-digested pNL4-3. DNA sequencing was performed in both directions across the entire RT-coding region to verify the absence of spurious mutations and the presence of the desired mutation. WT HIV-1 (HIV-1WT) and HIV-1 with the mutations (HIV-1MT) were generated by transfection of the plasmids into 293T/17 cells by using Fugene 6 Transfection Reagent (Roche Applied Science) according to the manufacturer’s instructions. The viral supernatants were harvested at 48h post-transfection, filtered through a 0.45-micron filter to remove the cellular debris, aliquoted, and stored at -80 °C. The 50% tissue culture infectious dose (TCID50) of a single thawed aliquot of each virus was determined in TZM-bl cells as described previously. Virus stock was quickly thawed and diluted with the 47 ACS Paragon Plus Environment


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DMEM medium (DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 2.9 mg/mL L-glutamine) and added to the 96-well tissue culture plate in quadruplicate. TZM-bl cells were resuspended in the medium (addition of 30 mg/mL DEAE to the complete medium) and dispensed into all wells at 1×104cells/well. After incubation for 48 hours at 37 °C and 5% CO2, the luciferase activity was measured using luciferase assay regent (Promega) and a Luminescence Counter (Perkin-Elmer) according to the manufacturer’s instructions. Assay for Measuring the Inhibitory Activity of Compounds on HIV-1SF33, HIV-1A17 and pNL4-3 infectious molecular clone

in both MT4 and TZM-bl cell lines

MT4 cells, HIV-1SF33, HIV-1IIIB, and HIV-1A17 were obtained from the NIH AIDS Research and Reference Reagent Program (USA). The inhibitory activity of the compounds on infection by a laboratory-adapted HIV-1 strain, SF33, and an NNRTI-resistant HIV-1 strain, A17, was tested in MT4 cells. Briefly, MT4 cells (4×104/well) were infected by addition of 200 TCID50 of HIV-1, followed by incubation for 2 hrs at 37 oC before addition of compounds at serial dilutions. After further incubation at 37 oC for 7 days, p24 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Vironostika® HIV-1 Plus O Microelisa System; BioMérieux, Marcy l’Etoile, France).The concentration of a compound for inhibiting 50% viral replication (EC50) was determined by nonlinear regression using GraphPad Prism 5.01. Assessment of in vitro Cytotoxicity in MT-4 Cells An XTT assay, as previously described36, was used to assess the cytotoxicity of 48 ACS Paragon Plus Environment

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target compounds to MT4 cells. Briefly, a compound at graded concentrations was added to MT4 cells (5×104/well), followed by incubation at 37 °C for 3 days. Ten microliters of CCK-8 reagent were added to the cells. After incubation at 37 °C for 4 h to allow color development of the XTT formazan product, the absorbance of each well was then read at 450 nm in a Victor2 1420 Multilabel Counter (Wallace-PerkinElmer Life and Analytical Sciences Inc., Boston, MA). The percent of cytotoxicity and CC50 (concentration causing 50% cytotocity) were calculated as previously described37. Molecular Modeling The AutoDock Vina program38 was uesd for docking investigations. Hydrogen atoms and the active torsions of ligands were assigned by using AutoDock tools (ADT). According to the binding energy, the best conformation of each ligand was chosen for binding pose analysis. The crystal structures of HIV-1 RT complexed with TNK-651 (PDB entry: 1RT2), NNRTI resistant K103N mutant HIV-1 RT in complex with Nevirapine (PDB entry: 1FKP) and Y181C mutant HIV-1 RT in complex with TNK-651 (PDB entry: 1JLA) were retrieved from the protein data bank39. The graphics above were generated using PyMOL program (http://www.pymol.org). Acknowledgment The research work was supported by the National Natural Science Foundation of China (21172014, 20972011, 21042009, 30872232, 812111023 and 81172733) and grants from the Ministry of Science and Technology of China (2009ZX09301-010) and SKLID development grant (2011SKLID102) for financial support. We also thank the NIH AIDS 49 ACS Paragon Plus Environment


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Research and Reference Reagent Program in the USA for providing MT4 cells, HIV-1SF33, and HIV-1A17.


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Table 1. In Vitro RT Assays, RT Inhibitory Activity Values of Pyridinone Compounds with Various Alkyl Ringsa

a

Compds

n

R1

IC50a(µM)

7f 7g 7h 7i 7j 7k 7l 7m 7o 7p NVPb 2b

1 2 3 2 2 2 2 2 2 2

H H H 2-Clc 2-methylc 3-methylc 4-methylc 2,6-dimethylc 3,5-dimethylc 3,3,5-trimethylc

> 100 80.99 > 100 > 100 5.29 19.81 32.88 37.07 73.54 > 100 4.45 0.088

Effective dose (µM) of the compounds required to inhibit HIV-1 RT activity by 50%.

Data represent mean values for three separate experiments; variation among triplicate samples was less than 15%. b

Nevirapine (NVP) and 2 were used as the reference compounds.

c

Mixture of stereoisomers.



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Table 2. In Vitro RT Assays, RT Inhibitory Activity Values of Pyridinone Compounds with Different Substitution on Position C-3a

Compdsc 8 9 10 14a 7j NVPb 2b a

R2

IC50a(µM)

H Br I i Pr COOEt

59.3 1.21 0.59 0.41 5.29 4.45 0.088




c

Mixture of stereoisomers.


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Table 3. In Vitro RT Assays, RT Inhibitory Activity Values of Pyridinone Compounds with Different Substitution on Positions C-3 and C-6a

Compds 14a 14b 14c 14d 14e 9 10 19 21 22 26 26-cis 26-trans NVPe 2e a

R2 i

Pr i Pr i Pr i Pr i Pr Br I COOEt Br I i Pr i Pr i Pr

R3

IC50a (µM)

PhCH2 p-F-PhCH2 p-Cl-PhCH2 p-MeO-PhCH2 p-Me-PhCH2 PhCH2 PhCH2 PhCH2CH2 PhCH2CH2 PhCH2CH2 PhCH2CH2 PhCH2CH2 PhCH2CH2

0.41b 0.13b 0.16b 0.033b 0.059b 1.21b 0.59b > 100b 0.65b 0.15b 0.01b 1.31c 0.003d 4.45 0.088



Mixture of stereoisomers. c cis-stereoisomer. d trans-stereoisomer.

e




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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Table 4. Antiviral activity of compounds on HIV-1SF33 and HIV-1A17 infection in TZM-bl and MT4 cell linesa Compds

MW

14b 14C 26 26-trans 26-cis 14d 14e 21 22 2c NVPc

357 339 353 353 353 369 353 389 437 364 265

CC50 (µM)

EC50 (µM) /SF33

EC50 (µM) /A17

TZM-Bl

MT4

TZM-Bl

MT4

74.457±0162 108.685±12.153 114.375±3.510 ＞300 189.359±7.808 132.395±2.539 98.954±3.259 83.088±7.946 ＞300 65.812±0.660 204.003±0.005

64.571±3.233 83.976±2.216 201.32±12.451 ＞300 87.937±4.186 74.462±1.152 112.339±1.451 100.312±1.548 ＞300 84.596±1.533 325.130 ± 0.02

0.026±0.001 0.023±0.001 0.036±0.010 0.009±0.001 0.136±0.038 0.005±0.003 0.057±0.011 0.092±0.073 0.053±0.055 0.022±0.002 0.190±0.02

0.023±0.004 0.010±0.003 0.007±0.001 0.004±0.001 0.068±0.015 0.006±0.001 0.006±0.002 0.003±0.001 0.017±0.002 0.022±0.004 0.068±0.230

TZM-Bl ＞10 ＞10 0.600±0.019 0.139±0.012 ＞10 ＞10 ＞10 ＞10 0.978±0.029 ＞10 ＞10

MT4

TZM-Bl

＞10 10.424±1.021 2.755±0.254 0.090±0.024 ＞10 0.479±0.021 ＞10 4.319±0.235 0.281±0.003 ＞10 ＞10

2,864 4,725 3,177 >33,333 1,392 26,479 1,736 3,885 >5,660 2991 1073

a

Each compound was tested in triplicate, and the data were presented as the mean ± SD.

b

SI was calculated based on the CC50 and EC50 for inhibiting infection of HIV-1SF33 and HIV-1A17, respectively.

c

Nevirapine (NVP) and 2 were used as the reference compounds

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SIb / SF33 MT4 2,807 8,398 28,760 >75,000 1,293 12,410 18,723 33,437 >17,647 3845 4781

SIb /A17 TZM-Bl

MT4

Novel Pyridinone Derivatives As Non-Nucleoside Reverse

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