Synthesis and structure–activity relationship studies of novel dihydropyridones as androgen receptor modulators

Antonella Pepe; Michael Pamment; Yeong Sang Kim; Sunmin Lee; Min-Jung Lee; Kristin Beebe; Anton Filikov; Len Neckers; Jane Trepel; Sanjay V Malhotra

doi:10.1021/jm301714s

. Author manuscript; available in PMC: 2020 Oct 22.

Published in final edited form as: J Med Chem. 2013 Oct 30;56(21):8280–8297. doi: 10.1021/jm301714s

Synthesis and structure–activity relationship studies of novel dihydropyridones as androgen receptor modulators

Antonella Pepe ^a, Michael Pamment ^a, Yeong Sang Kim ^b, Sunmin Lee ^b, Min-Jung Lee ^b, Kristin Beebe ^c, Anton Filikov ^a, Len Neckers ^c, Jane Trepel ^b, Sanjay V Malhotra ^a,^*

PMCID: PMC7581280 NIHMSID: NIHMS536714 PMID: 24044500

Abstract

A library of 3-hydroxy-2,3-dihyrdropyridones was synthesized and their activity as antiandrogens was tested in the human prostate cancer cell lines LNCaP. Structure–activity relationship studies resulted in the identification of a potent compound, whose activity is comparable to MDV3100. Homology modeling and molecular mechanics were used to build a structural model of the AR-ligand binding domain, and to investigate the structural basis of the antagonism. The model is qualitatively consistent with the SAR observed. Moreover, the enrichment plot shows that screening with the model performs significantly better than random screening. Therefore, the model probably represents a realistic conformation of the antagonist form and can be utilized for structure-based design of novel antiandrogens.

INTRODUCTION

The androgen receptor (AR) belongs to the steroid/nuclear receptor superfamily of intracellular ligand-dependent transcription factors.¹ It is expressed prevalently in the prostate, adrenal glands, epididymis, skeletal muscle, liver and central nervous system.² The transcriptional activity of AR is induced upon binding to one of its native ligands, testosterone (T) and dihydrotestosterone (DHT). Binding of ligand to the AR induces dissociation from heat-shock proteins (HSPs) and receptor phosphorylation. Subsequently, the AR dimerizes and can bind to androgen-response elements in the promoter regions of target genes. Activation (or repression) of target genes leads to biological responses including growth, survival and the production of prostate-specific antigen (PSA),³ Figure 1. AR-regulated gene expression is crucial for the development and maintenance of the male sexual phenotype, including maintaining spermatogenesis, muscle mass, strength and bone mineral density. The androgen/androgen receptor signaling is implicated in the normal growth regulation of the prostate, as well as in the pathogenesis of prostate cancer.⁴ Thus, hormonal therapy, or androgen deprivation is employed in various settings, including first-line treatment when prostate cancer requires systemic therapy, in combination with radiation therapy in high risk prostate cancer cases, and as neoadjuvant therapy prior to radiation⁵. Although surgical and medical castration drastically reduce testicular androgen levels and control the growth of prostate cancer in most men, after months or years many prostate tumors become resistant to first-line hormonal therapy and the disease progresses in a castrate-resistant state (castrate resistant prostate cancer, CRPC). It has been hypothesized that androgen levels in other tissues, such as the adrenal glands continues to circulate and stimulates tumor cell growth. Thus, hormonal therapy often includes a combination of castration and administration of antiandrogens.⁵

Structures of representative non-steroidal antiandrogen small molecules approved by the US FDA (flutamide^{6, 7} and its metabolite hydroxyflutamide, bicalutamide,^8–10 nicalutamide^11–13 and enzalutamide,^{14, 15} MDV3100) are shown in Figure 2. The reasons for de novo resistance to or progression on antiandrogens are not fully understood. However, it has become clear recently that, even in CRPC, prostate tumors are typically AR-driven, and although the AR may be mutated or overexpressed the AR remains an important target. Thus, it is critically important to develop novel strategies and new classes of compounds that target AR for the treatment of prostate cancer. Herein, we report our initial investigations on the design and synthesis of a novel series of AR-targeting compounds that are able to reduce the level of prostate specific antigen (PSA) in prostate cancer cells.

Antiandrogen molecules approved by the FDA

Dihydropyridones are heterocyclic compounds, often embedded in more complex molecules with important biological activity. As part of our ongoing research on the synthesis of heterocyclic molecules and their pharmaceutical application, we have discovered a new class of AR modulators bearing the dihydropyridone core. We have prepared a diverse library of dihydropyridones which showed activity on the prostate cancer cell line LNCaP and conducted an initial structure–activity relationship (SAR) study which allowed us to identify a potent analog. To develop a better understanding of experimental data, we have built a structural model of AR-ligand binding domain (LBD) using homology modeling and Monte Carlo global energy optimization in torsion angle coordinates. The model is qualitatively consistent with the SAR observed. Moreover, the enrichment plot shows that screening with the model performs significantly better than random screening. Therefore, the model could have a more general use and be applied to structure-based design of novel antiandrogens. We also computationally evaluated ability of binding of our compound series to the BF3 allosteric binding site of the AR. The docking scores suggest that our compounds do not bind to the allosteric region, rather to more specific site.

RESULTS AND DISCUSSION

Chemistry

A straightforward and short synthesis of trans 3-hydroxydihydropyridones, recently developed in our laboratories, was applied to the preparation of a screening library. 3-Hydroxydihydropyridone^16–20 is a scaffold particularly amenable to diversification and library preparation since it has a low molecular weight, and presents multiple handles for further functionalization. 2-Substituted 3-hydroxydihydropyridones (±)-1 a–h were prepared as racemic mixtures, starting from 4-methoxypyridine in a two steps protocol which includes alkylation with Grignard’s reagents of the in situ generated ammonium salt (imine), following Comin’s procedure,²¹ and osmium tetroxide oxidation of the resulting enol ether. The reaction is of general application and both aromatic ((±)-1 a,b, f–h) and aliphatic groups ((±)-1 c–e) can be introduced in position 2. Subsequent steps are simple functional group manipulations, whose order can be switched as needed for the preparation of diverse sub libraries. In our SAR study, we have also synthesized and tested compounds lacking the hydroxyl group (±)-3, prepared following the Comin’s protocol, and compounds (±)-4 lacking functionalization at the nitrogen, obtained from (±)-3 by cleavage of the phenyl carbamate. Functionalization of the hydroxyl group starting from compounds (±)-1 afforded the series (±)-2, with different substitutions in position 2. Compound (±)-1a was hydrolyzed to reveal the free nitrogen (±)-5, and compound (±)-1b was first functionalized at the hydroxyl group with N-methyl N-phenyl carbamate to give (±)-6, and then the phenyl carbamate on the nitrogen was replaced with the 6-(trifluoromethyl)nicotinoyl or the cyclopropyl group to give (±)-8a, b. Silyl ether protection of the hydroxyl group ((±)-9), cleavage of the phenyl carbamate ((±)-10) and functionalization with different acyl chlorides afforded amides (±)-11. Cleavage of the silyl ether in HF/pyridine gave compounds (±)-12 in good yields, and esterification of the hydroxyl group gave compounds (±)-13, (±)-14 and (±)-15.

Biology

With a method in hand for the synthesis of the 3-hydroxy dihydropyridones, and the preparation of screening libraries, we tested our compounds on the prostate cancer cell line LNCaP. The inhibitory activity of the compounds on the androgen receptor was measured based on the AR-mediated increase in transcription of prostate-specific antigen (PSA) and trans membrane protease serine 2 (TMPRSS2). Our first set of compounds (Figure 3) produced several hits, with inhibition up to 90% at 5 μM concentration, and valuable information on the SAR of this scaffold. Different substitutions at the 2-position did not affect greatly the activity, in fact biphenyl ((±)-4b), phenyl ((±)-5), 4-methylthiophenyl ((±)-7, (±)-8a, b), or 5-benzo[d][1,3]dioxolyl ((±)-10c, (±)-11b, c, d) could be introduced maintaining a moderate inhibitory activity. While functionalization on the nitrogen atom plays a more important role and a relevant increase of the activity is achieved with the introduction of phenyl carbamate (compare (±)-1a, (±)-1b, (±)-1f, (±)-1g, (±)-3a, (±)-3b, (±)-6, (±)-9a, (±)-9c). The phenyl carbamate stands out as the preferred substitution on the nitrogen. Comparing biological data for differently substituted compounds (±)-9c, with (±)-11b, (±)-11c, and (±)-11d, or (±)-6, with (±)-8a, and (±)-8b, it is evident that cyclopropanoyl, phenylacetyl, 6-(trifluoromethyl)nicotinoyl and benzoyl amides were visibly less active.

Structures of compounds tested and inhibition of PSA and TMPRSS2 mRNA expression at 5 μM.

Our initial SAR, also indicates that the hydroxyl group at the 3-position is not critical for activity, since compounds with free OH ((±)-1a), silylether ((±)-9a), or H ((±)-3a), show comparable activity. However, the comparison between (±)-12a and (±)-15 clearly shows that the presence of the right substituent on the hydroxyl group does affect activity.

Thus, we kept the 2-trifluoromethyl-4-fluorophenyl ester at position 3 and investigated the effect of alkyl, aryl and heteroaryl substituents at position 2 of the dihydropyridone scaffold (Figure 4). At a concentration of 5 μM, compounds bearing phenyl ((±)-2a), 4-methylthiophenyl, ((±)-2b), isopropyl, ((±)-2d), isobutenyl, ((±)-2e), and 3,5-difluorophenyl, ((±)-2h), showed comparable activities, while (±)-2c, bearing a methyl group resulted slightly less active. Similarly, (±)-2f, with a 5-benzo[d] [1, 3] dioxolyl and (±)-2g with a biphenyl group resulted considerably less potent. These data suggest that the C-2 substituent probably fits into a rather small hydrophobic pocket, which does not easily accommodate bulky substituents such as the biphenyl group. The most active compounds in this series are (±)-2a and (±)-2e.

In our last SAR study, we replaced the carboxyphenyl group with the less polar cinnamyl group, retained two representative substituents at C-2, namely phenyl and methyl and introduced different esters at C-3 (Figure 5). The compounds with the highest activity were (±)-13e, (±)-13h, and (±)-13p, while the entire data provide important information on the SAR of this scaffold. Compounds lacking the ester moiety and having only a free hydroxyl group ((±)-12b), or bearing instead a cyclopropyl ((±)-13a), or cyclobutyl ester ((±)-13b) demonstrated comparable activity. Less than ideal geometry is also demonstrated by (±)-13d that with the two cynnamyl substituents, does not easily fit into the binding pocket. Similarly modest activity is demonstrated by the para ((±)-13o) and meta ((±)-13n) cyano-, as well as the para methyl- ((±)-13f), para methoxy- ((±)-13q), meta trifluoromethyl ((±)-13p) and para chlorophenyl ((±)-13g) ester derivatives.

In our design of substituted phenyl esters, we followed also the Topliss’ decision tree and our observations are summarized below (Figure 6). The p-chlorophenyl analog (±)-13g is less active than unsubstituted phenyl (4-H (±)-12b), this could be due to the increased lipophilicity or increased electron withdrawing effect of chlorine. Both the p-methoxy (±)-13q) and p-methyl (±)-13f) analogs were equally potent with p-chlorophenyl ester.

This would suggest an unfavorable para steric effect, which is confirmed by the fact that among all the substituents we have introduced at the para position, the smallest, H and F, have shown the best activity, while electron donating, electro withdrawing or polar groups showed modest activity. The introduction of the trifluoromethyl group at the ortho position of the aromatic ring has a positive effect ((±)-13p), while moving the same group at the meta position is deleterious for activity ((±)-13m). Combining the two substitutions, o-trifluoromethyl and p-fluoro increased activity and resulted in our best compound ((±)-13h). Subsequently, (±)-13h was compared to enzalutamide (MDV3100), recently approved by the US FDA as androgen inhibitor. Both the PSA and TMPRSS2 are AR containing genes. Therefore, to demonstrate that our compounds interact with AR, we also performed a comparative study with E-cadherin a non-AR target gene. Results (Figure 7) showed that the E-cad is not impacted by either (±)-13h or MDV3100 which is known to interact with AR. Furthermore, the 18s rRNA used to normalize each of the gene expression is not impacted. This suggests that the compounds from our series are not cytotoxic but cause inhibition of the AR target gene. We also, carried out the concentration dependent binding assay with fluorescence polarization (FP) readout on 13h and MDV3100. As shown in figure 8, both compounds cause concentration dependent inhibition, and the IC₅₀ was 30.1μM and 5.6 μM respectively, for 13h and MDV3100. These results are comparable with those reported on MDV3100,²² and much more promising than similar investigation on different platform.²³ To test the cytotoxic effect, the LNCap cells were treated with 13h and MDV3100 for 48 hr. The viable cell count was determined by flow cytometric analysis. As seen from the growth curve (figure 9) 13h is not cytotoxic at the concentrations used for measurement of AR-driven gene expression, and thus the decrease in gene expression is not a non-specific effect of 13h on cell viability. Thus, all these experiments clearly establish that our lead compound 13h is comparable to MDV3100, and holds great potential for further development.

Inhibition of PSA and TMPRSS2 mRNA expression by **(±)-13h** or MDV3100 in the presence of R1881 at 20 h.

Molecular modeling

When the hormone binds to the AR-LBD a reorganization of the receptor occurs that results in the formation of an effective co-activator binding site. Analysis of X-ray structures of several nuclear receptor antagonist complexes suggests that the general mechanism involves perturbing helix 12, displacing it from its hormone-bound configuration, and distorting the co-activator binding site.^{24, 25} Studies suggest that the AR shares a similar mechanism, but this has not been confirmed by experimental structural data. Molecular dynamics has been used to elucidate the structural basis for antagonism of the AR LBD.^26–30 Replica-exchange molecular dynamics (REMD) has been used to model the effect of some mutations on the conformation of the AR LBD.³¹ The large conformational change that occurs in the transition between agonistic and antagonistic forms and it mainly involves the H12 helix, renders classic MD simulation not suitable for computational studies. On the contrary, REMD represents a valid alternative, and its application has resulted in the successful design of antiandrogens.³¹ We initiated our investigations from proteins with high homology to the AR, and for which both agonist and antagonist structures are known, namely estrogen (ER), progesterone (PR) and glucocorticoid receptors (GR). Our choice was based on the results published by Wilkinson et al.³² In their study, 35 compounds were tested for antagonistic activity on AR, ER, GR, PR and mineralocorticoid receptor (MR) and found that there is cross-talk between AR and other receptors. Three compounds showed low- or sub-nanomolar activity on both AR and GR, four compounds on AR and MR, one on AR and PR and no AR-actives cross-talk in low nM range with ER (but there is crosstalk in μM range). These data suggest that the antagonist conformation of MR and GR has the highest similarity to AR. Since the antagonistic structure of MR is not available we used the GR structure (PDB code 1NHZ)³³ as a template for homology modeling. Our model of AR in the antagonistic form is based on the X-ray structure of GR in the antagonistic form superimposed on the X-ray structure of AR in the agonistic form and is shown in Figure 10. A comparison with the agonistic structure (pdb code 2AMB³⁴) shows differences in the position and conformation of H11 and H12 helixes as well as H6 and the loop between H6 and H7.

The aligned X-ray structure of agonistic form (PDB code 2AMB) is shown in gray. H3 helix is colored blue, H6 green, H7 black, H11 cyan, H12 red. The RU-486 ligand from the template structure (PDB code 1NHZ) is shown in green. The docked conformation of **(±)-13h** is shown in colored stick representation.

Four new sub-pockets were identified in our LBD model (Figure 11). The first sub-pocket, SP1, is the result of the H11 helix (residues LHQFTFDLLIK according to Wurtz et al.³³) being unwound and unfolded, so that the side chain of residue T877 (2AMB structure) points out of the protein core and vacates the space of SP1 (Figure 11). Two more sub-pockets, SP2 and SP3 are created by the 14 Å movement of H12 (residues GMMAGIIS according to Wurtz et al.)³⁵ from the agonistic to the antagonistic structure. SP2 is the space occupied in 2AMB by V889 and N705, while SP3 is the space occupied by M895. SP2 is created when V889, which is in the loop connecting H11 and H12 helixes, moves out of the binding site together with H12 helix, and when N705, which is part of the H3 helix, rotates out of the binding site due to the slight rotation of H3.

The wire representation is the pocket surface detected by ICM PocketFinder. Sub-pockets that are formed in the antagonistic structure are designated SP1–SP4. The RU-486 ligand from the template structure (1NHZ antagonist form of GR) is shown in green. The colored molecule is the docked conformation of (±)-13h.

The fourth sub-pocket (SP4) in our model of the binding site (Figure 11) is the space occupied in 2AMD by M780 side chain and results from a slight movement of H6 and bending of the loop between H6 and H7, which move M780 side chain out of the binding site. Docking of our most active compound ((±)-13h) resulted in a model that places the dihydropyridone core and the 2-trifluoromethyl-4-fluorophenyl ester in the central part of the binding site, i.e. the same space present in the binding site of the agonistic structure 2AMB. SP1 is occupied by the phenyl ring on position 2, the amide-oxygen occupies the entrance to SP2, SP3 is occupied by the cinnamyl group and SP2 is empty. (±)-13h forms two hydrogen bonds, one from the amide oxygen to the NH on the side-chain of N705 and a second bond from the carbonyl of the pyridone core to the NH on the side-chain of Q783. The well stabilized binding mode of (±)-13h is consistent with its high activity. In addition to hydrogen bonding, (±)-13h forms extensive hydrophobic interactions. In fact, the phenyl ring on position 2 occupies the space (SP1) that in 2AMB structure is taken by the hydrophobic part of the T877 side chain, the cinnamyl group (hydrophobic) is in the space occupied in 2AMB by M895 side chain (hydrophobic), and the 2-trifluoromethyl-4-fluorophenyl moiety (hydrophobic) sits predominantly in the hydrophobic part of the binding site (i.e. the part that is available in the agonistic structure 2AMB).

Our model is qualitatively consistent with the SAR observed and explains the changes in activity resulting from even subtle structural modifications of the compounds. The biggest activity cliff in our series is (±)-13h (PSA level = 0.04) vs. (±)-13q (PSA level = 0.67), due to the replacement of 2-trifluoromethyl-4-fluorophenyl, with 4-methoxyphenyl. Our docking of (±)-13h indicates that the 2-trifluoromethyl-4-fluorophenyl moiety sits in the part of the binding site that is common to the agonistic structure 2AMB and our model of the AR-LBD in the antagonistic form and it occupies all the space available. Thus, there is not enough space for the methoxy group, which explains the drastic drop in activity. Another activity cliff is (±)-13h (PSA level = 0.04) vs. (±)-13a (PSA level = 0.62) and (±)-13b (PSA level = 0.55), and the comparison is between 2-trifluoromethyl-4-fluorophenyl, cyclopropyl and cyclobutyl. In our model 2-trifluoromethyl-4-fluorophenyl moiety forms extensive hydrophobic and van der Waals interactions with the binding site that surrounds it tightly. In addition fluorine in position 4 forms a hydrophilic interaction with the side chain amide of Q711. Cyclopropyl and cyclobutyl form only a fraction of these interactions. We next compared (±)-13h (PSA level = 0.04) to (±)-2g (PSA level = 0.63), that is a phenyl carbamate instead of cinnamyl and biphenyl instead of phenyl in position 2. The difference in activity is clearly explained by the fact that the carbamate can form fewer hydrophobic interactions than the cinnamyl group. Similarly, based on our model the replacement of phenyl with biphenyl results in clashes with H11. Along the same lines, the difference in activity measured for (±)-13h (PSA level = 0.04) and (±)-2f (PSA level = 0.46), with (±)-2f bearing a phenyl carbamate instead of cinnamyl and 5-benzo[d][1,3]dioxolyl instead of phenyl in position 2 can be explained by fewer hydrophobic interactions resulting from the exchange of the cinnamyl group with the phenyl carbamate. Replacing the phenyl group with 5-benzo[d][1,3]dioxolyl has no effect, and in our model, it does not result in clashes with H11. This is confirmed by the fact that the drop in activity is less pronounced for the pair (±)-13h, (±)-2f than for (±)-13h, (±)-2g. The analysis above validates our model and demonstrates its consistency with the SAR observed.

We also used enrichment to evaluate the ability of the model to rank the active compounds out of the whole set (Figure 12, supplementary information). The X-axis of the plot contains the fraction of records seen following sorting by the docking score. The Y-axis contains the fraction of good records found in the sorted list up to that point. The green line represents random screening. The red line represents ideal model. The plot shows that screening with the model performs significantly better than random screening.

Overall, a qualitative consistency of the model with the SAR observed and also a reasonable enrichment performance validate our model and merit its use for further design of novel androgen receptor modulators.

There was a recent study suggesting the Binding function 3 (BF3) as an alternative target of the Androgen Receptor.³⁶ Therefore, we also computationally evaluated ability of binding of our compound series to the BF3 allosteric binding site of the AR and concluded that docking scores of our compounds are inconsistent with binding to the BF3 site. In order to estimate docking scores consistent with binding to the BF3 site in micro molar range, we identified compounds similar to the BF3 binder from this literature study,³⁶ that have X-ray structures in complex with a protein and have Ki in the range of 1–1000 μM. Then we docked the compounds to their cognate protein binding sites and calculated their scores. The scores of these compounds were in the range from −38.3 to −29.3 (the average is −35.6±2.6). The BF3 docking scores of our compounds were in the range from −22.2 to −9.1 (the average is −14.7±4.5). Comparison of the score ranges further established that our compounds do not bind to the secondary i.e. allosteric region. But more likely to the specific AR site.

CONCLUSIONS

A new class of antiandrogen small molecules containing the 3-hydroxy-2,3-dihydropyridone scaffold has been identified. Up to 90% inhibition of cellular level of PSA in the prostate cancer cell line LNCaP at 5 μM concentrations has been achieved. SAR studies have identified a lead compound, with potency comparable with recently FDA approved drug MDV3100. A computational model has been developed based on homology study, derived from the X-ray structure of GR in the antagonistic form, and molecular dynamic. The resulting model of the AR-ligand binding domain has been used for docking studies of all the compounds synthesized and the docking scores were compared to the inhibitory activity of the compounds. The model is qualitatively consistent with the SAR observed. Also, the enrichment plot shows that screening with the model performs significantly better than random screening. This validates our model and supports its use as a tool for the discovery of novel antiandrogens.

EXPERIMENTAL SECTION

Chemistry

General

All reactions were carried out using round-bottom flasks, under positive nitrogen pressure when noted. All solvents were purchased anhydrous and used directly unless aqueous solutions were employed, or it is otherwise indicated. Analytical thin layer chromatography (TLC) was carried out on silica gel plates (silica gel 60 F254). Eluted plates were visualized by exposure to ultraviolet light and then by staining with an ethanolic solution of phosphomolybdic acid. The products were isolated and purified using a flash chromatography system, with a mixture of hexanes and ethyl acetate as the eluent. The purities of all of the tested compounds were >95% as estimated by HPLC. The peak area of the major product was ≥95% of the combined total peak areas when monitored by a UV detector at 254 nm. The ¹H (400 MHz), ¹³C (100 MHz) chemical shifts (δ) are expressed in ppm relative to chloroform or tetramethylsilane. ¹H NMR coupling constants (J) are expressed in Hz. High-resolution mass spectra and electrospray (ESI) experiments were performed with a time-of-flight (TOF) mass detector.

1.1.1.a. (2R,3R)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1a)

A solution of 4-methoxypyridine (0.800 mL, 7.88 mmol) in 25 mL of dry THF was cooled to 10 °C with a dry ice/acetone bath. Phenyl chloroformate (1.09 mL, 8.67 mmol) was added drop wise, and a white solid precipitate formed almost immediately. The reaction mixture was stirred for 25 minutes and cooled to −45 °C. Then a 2 M solution of phenyl magnesium chloride in THF (4.33 mL, 8.67 mmol) was added drop wise, keeping the internal temperature below −35 °C. Upon completion of the reaction (checked by TLC with a mixture of hexanes, ethyl acetate 2:1). KOtBu was added in THF at −45 °C. The reaction mixture was warmed to 10 °C and 10 ml of water were added followed by 15 ml of ethyl acetate. The organic layers were collected, and the aqueous layers were extracted with 2x15 ml of ethyl acetate. The combined organic layers were treated with brine and dried over anhydrous sodium sulfate. After evaporation of the solvent under reduced pressure, 20 ml of acetone, and 10 ml of water were added to give a slightly turbid solution. N-Methylmorpholine N-oxide (0.92 g, 7.88 mmol), was added, followed by a 2.5 wt. % solution of OsO₄ in tert-butanol (2.40 g, 0.236 mmol). The reaction mixture was stirred for 2 hours, then quenched with sodium sulfite /florisil. The solids were filtered, acetone was evaporated under reduced pressure, and the residue was diluted with 50 ml of ethyl acetate, and the aqueous layer separated. Florisil was added, and the residue was condensed to a solid. The crude was purified by silica gel column chromatography with a mixture of ethyl acetate in hexanes from 5 to 35% to afford 1.68 g (69% yield) of the product as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (dd, J = 8.5, 0.9 Hz, 1H), 7.52 – 7.14 (m, 8H), 6.96 (m, 2H), 5.65 (d, J = 3.5 Hz, 1H), 5.56 (dd, J = 8.5, 1.4 Hz, 1H), 4.21 (td, J = 3.7, 1.3 Hz, 1H), 3.36 (d, J = 3.9 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 192.0, 151.9, 150.4, 143.7, 135.4, 129.7, 129.4, 128.8, 126.6, 126.4, 121.3, 105.4, 73.4, 64.1. HRMS (ESI⁺) calculated for C₁₈H₁₅NNaO₄, [M+Na]⁺ 332.0893, found 332.0899.

1.1.1.b. (2S**, 3R)-phenyl 3-hydroxy-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1b)

To a solution of 4-methoxypyridine (0.465 mL, 4.58 mmol) in 25.0 mL of dry THF was added phenyl chloroformate (0.634 mL, 5.04 mmol), under nitrogen. A white precipitate formed almost immediately. and the reaction mixture was stirred for 25 minutes and then cooled to −30 °C in a dry ice/acetone bath. Then a 0.5 M solution of 3-methyl-2-thienylmagnesium bromide in THF (14.5 mL, 7.33 mmol) was added drop wise, keeping the internal temperature below −20 °C. Upon completion of the reaction (checked by tlc with a mixture of hexanes, ethyl acetate 2:1), 4-methylmorpholine (2.52 mL, 22.9 mmol) was added, followed by 8 mL of water. The reaction mixture was warmed to room temperature, and 25 mL of ethyl acetate were added. The organic layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were treated with brine and dried over anhydrous sodium sulfate. After evaporation of the solvent under reduced pressure, the crude was filtered through basic alumina with 80 ml of a 30% solution of ethyl acetate in hexanes. After evaporation of the solvent, the residue was diluted with 25.0 ml acetone and 10.0 ml water. A 2.5 wt. % solution of OsO₄ in tert-butanol (1.72 mL, 0.137 mmol) and 4-methylmorpholine N-oxide (0.537 g, 4.58 mmol) were added. The reaction was stirred overnight and quenched with solid sodium bisulfite followed by the addition of florisil. The black mixture was stirred for 45 minutes, then filtered, and after evaporation of the solvent, the crude was purified by silica gel column chromatography with a mixture of ethyl acetate in hexanes from 5 to 35% in 20 min, to afford 0.980 g of the title compound, 65% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.12 (dd, J = 8.5, 1.0 Hz, 1H), 7.37 (m, 2H), 7.26 (m, 1H), 7.11 (d, J = 5.1 Hz, 1H), 7.05 (m, 2H), 6.80 (d, J = 5.0 Hz, 1H), 5.98(d, J = 2.9 Hz, 1H), 5.60 (dd, J = 8.6, 1.5 Hz, 1H), 4.05 (s, 1H), 3.729 (bs, 1H), 2.31 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.6, 151.3, 150.3, 142.9, 137.0, 130.4, 130.0, 129.6, 126.5, 123.9, 121.2, 105.3, 72.7, 58.2, 14.0. HRMS (ESI⁺) calculated for C₁₇H₁₅NO₄SNa, [M+Na]⁺ 352.0614, found 352.0600.

1.1.1.c. (2R,3R)-phenyl 3-hydroxy-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1c)

The synthesis of 1c follows the method of synthesis for 1b using 4-methoxypyridine as starting material and methylmagnesium bromide as nucleophile. The title compound was isolated as a white solid in 50% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.96 (dd, J = 8.4, 1.4 Hz, 1H), 7.42 (m, 2H), 7.29 (m, 1H), 7.18 (m, 2H), 5.44 (dd, J = 8.4, 1.6 Hz, 1H), 4.78 (m, 1H), 3.81 (m, 1H), 3.77 (d, J = 8.4, 1.4 Hz, 1H), 1.33 (d, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 192.6, 151.3, 150.3, 141.8, 129.6, 126.5, 121.3, 104.3, 72.6, 55.7, 13.9. HRMS (ESI⁺) calculated for C₁₃H₁₃NO₄Na, [M+Na]⁺ 270.0737, found 270.0740.

1.1.1.d. (2R,3R)-phenyl 3-hydroxy-2-isopropyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1d)

The synthesis of 1d follows the method of synthesis for 1b using 4-methoxypyridine as starting material and isopropylmagnesium chloride lithium chloride complex solution as reagent. The title compound was isolated as a white solid in 58% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.04 (dd, J = 8.4, 1.5 Hz, 1H), 7.42 (m, 2H), 7.28 (s, 1H), 7.17 (m, 2H), 5.42 (dd, J = 8.4, 1.6 Hz, 1H), 4.50 (dd, J = 8.8, 1.8 Hz, 1H), 4.04 (s, 1H), 3.30 (s, 1H), 2.09 (m, 1H), 1.04 (dd, J = 11.4, 6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 192.7, 152.3, 150.4, 142.4, 129.6, 126.4, 121.2, 105.2, 69.9, 65.2, 27.36, 19.5, 19.3. HRMS (ESI⁺) calculated for C₁₅H₁₇NO₄Na, [M+Na]⁺ 298.1050, found 298.1055.

1.1.1.e. (2R,3R)-phenyl 3-hydroxy-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1e)

The synthesis of 1e follows the method of synthesis for 1b using 4-methoxypyridine as starting material and 2-methyl-1-propenylmagnesium bromide (0.5 M solution in THF) as reagent.

The title compound was isolated as a white solid in 41% yield, and a more polar compound was isolated in 13% yield, which corresponds to the external olefin being oxidized.

¹H NMR (400 MHz, CDCl₃) δ 8.00 (dd, J = 8.4, 1.4 Hz, 1H), 7.40 (m, 2H), 7.28 (m, 1H), 7.15 (dd, J = 8.7, 1.2 Hz, 2H), 5.44 (dd, J = 8.4, 1.6 Hz, 1H), 5.39 (ddd, J = 9.8, 2.6, 1.4 Hz, 1H), 5.15 (dt, J = 9.8, 1.4 Hz, 1H), 4.10 (d, J = 4.2 Hz, 1H), 3.78 (ddd, J = 4.2, 2.7, 1.7 Hz, 1H), 1.79 (d, J = 1.4 Hz, 3H), 1.74 (d, J = 1.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 192.0, 151.5, 150.4, 142.3, 140.0, 129.6, 126.4, 121.2, 115.8, 104.6, 72.1, 58.7, 25.8, 18.3. HRMS (ESI⁺) calculated for C₁₆H₁₇NO₄Na, [M+Na]⁺ 310.1050, found 310.1053.

1.1.1.f. (2R,3R)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1f)

The synthesis of 1f follows the method of synthesis for 1b using 4-methoxypyridine as starting material and 3, 4-(methylenedioxy) phenyl magnesium bromide solution as reagent. The title compound was isolated as a white foaming solid in 75% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 8.5 Hz, 1H), 7.36 (t, J = 7.7 Hz, 2H), 7.24 (t, J = 7.1 Hz, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.77 (s, 3H), 5.95 (m, 2H), 5.58 (d, J = 3.1 Hz, 1H), 5.54 (d, J = 8.5 Hz, 1H), 4.12 (s, 1H), 3.79 (d, J = 3.9 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 191.8, 151.6, 150.2, 148.3, 147.8, 143.4, 129.6, 128.9, 126.5, 121.2, 119.9, 108.8, 106.7, 105.1, 101.4, 73.3, 63.6. HRMS (ESI⁺) calculated for C₁₉H₁₅NO₆, [M+Na]⁺ 376.0792, found 376.0802.

1.1.1.g. (2R,3R)-phenyl 2-([1,1′-biphenyl]-3-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1g)

The synthesis of 1g follows the method of synthesis for 1b using 4-methoxypyridine as starting material and 3-biphenylmagnesium bromide (0.5 M solution in THF) as reagent. The title compound was isolated as an off-white solid in 73 % yield.

¹H NMR (400 MHz, CDCl₃) δ 8.27 (dd, J = 8.5, 1.0 Hz, 1H), 7.75 – 7.18 (m, 11H), 6.97 (m, 2H), 5.73 (d, J = 3.5 Hz, 1H), 5.58 (dd, J = 8.5, 1.3 Hz, 1H), 4.26 (d, J = 1.4 Hz, 1H), 3.66 (d, J = 3.7 Hz, 1H), 1.60 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 191.8, 151.9, 150.4, 143.8, 142.5, 140.6, 136.1, 129.9, 129.8, 129.1, 127.9, 127.7, 127.4, 126.7, 125.3, 125.2, 121.3, 105.4, 73.5, 64.2. HRMS (ESI⁺) calculated for C₂₄H₂₀NO₄, [M+H]⁺ 386.1387, found 386.1395.

1.1.1.h. (2R,3R)-phenyl 2-(3,5-difluorophenyl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1h)

The synthesis of 1h follows the method of synthesis for 1b using 4-methoxypyridine as starting material and (3,5-difluorophenyl)magnesium bromide (0.5 M solution in THF) as reagent. The title compound was isolated as an off-white solid in 62 % yield.

¹H NMR (400 MHz, CDCl₃) δ 8.23 (dd, J = 8.5, 0.9 Hz, 1H), 7.37 (m, 2H), 7.26 (s, 1H), 7.01 (m, 2H), 6.86 (m, 2H), 6.79 (tt, J = 8.7, 2.3 Hz, 1H), 5.58 (m, 2H), 4.16 (d, J = 1.3 Hz, 1H), 3.56 (d, J = 3.7 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 191.5, 163.6 (dd, J = 250.6, 12.7 Hz, CF), 151.5, 150.2, 143.6, 139.4 (t, J = 8.7 Hz CF), 129.9, 126.9, 121.2, 109.7 (m CF), 105.5, 104.4 (t, J = 25.1 Hz CF), 72.9, 63.3. HRMS (ESI⁺) calculated for C₁₈H₁₃F₂NO₄Na, [M+Na]⁺ 368.0705, found 368.0712.

1.1.2.a. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-2a)

(2R*,3R*)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1a) (40 mg, 0.129 mmol) was dissolved in 1.0 mL of THF. Triethyl amine (0.063 mL, 0.453 mmol) and DMAP (catalytic amount) were added to the reaction mixture, followed by 4-fluoro-2-(trifluoromethyl)benzoyl chloride (0.039 mL, 0.259 mmol). The reaction was stirred for four hours, then the solvent was removed under a stream of nitrogen and the crude material was purified by silica gel flash column chromatography to afford 56 mg (87% yield) of the title compound as a colorless foam. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (dd, J = 8.6, 1.3 Hz, 1H), 7.93 (dd, J = 8.7, 5.4 Hz, 1H), 7.50 (dd, J = 8.9, 2.6 Hz, 1H), 7.44 – 7.31 (m, 8H), 7.26 (m, 1H), 7.01 (d, J = 7.7 Hz, 2H), 6.02 (m, 1H), 5.64 (dd, J = 8.7, 1.5 Hz, 1H), 5.53 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.1, 164.6, 164.0 (d, J = 256.0 Hz), 151.3, 150.2, 143.6, 135.4, 134.0 (d, J = 8.8 Hz), 132.9, 129.6, 129.4, 129.0, 126.5, 126.2, 126.0 (m), 122.4 (dd, J = 274, 2.3 Hz), 121.1, 119.0, (d, J = 21.5 Hz), 115.0 (dq, J = 25.6, 5.7 Hz), 106.8, 74.0, 61.2. HRMS (ESI⁺) calculated for C₂₆H₁₈F₄NO₅, [M+H]⁺ 500.1116, found 500.1126.

1.1.2.b. (2S,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2b)

The synthesis of 2b follows the method of synthesis for 2a using (2S*,3R*)-phenyl 3-hydroxy-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1b) as starting material. The title compound is a colorless foam, isolated in 73% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.22 (dd, J = 8.6, 1.2 Hz, 1H), 7.91 (dd, J = 8.6, 5.4 Hz, 1H), 7.50 (dd, J = 8.8, 2.6 Hz, 1H), 7.44 – 7.31 (m, 3H), 7.34 – 7.20 (m, 1H), 7.16 (d, J = 5.1 Hz, 1H), 7.06 (d, J = 7.7 Hz, 2H), 6.86 (d, J = 5.1 Hz, 1H), 6.28 (s, 1H), 5.71 (dd, J = 8.6, 1.4 Hz, 1H), 5.33 (dd, J = 2.0, 1.4 Hz, 1H), 2.40 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 185.2, 164.6, 164.0 (d, J = 256 Hz), 150.9, 150.2, 143.1, 137. 5, 133.9 (d, J = 8.9 Hz), 131.4 (dd, J = 33.7, 8.1 Hz), 130.3, 129.7, 128.4, 126.6, 126.0 (m), 124.3, 122.4 (dd, J = 274, 2.4 Hz), 121.1, 119.0 (d, J = 21.2 Hz), 115.0 (dq, J = 25.6, 5.7 Hz), 106.8, 73.0, 56.0, 13.8. HRMS (ESI⁺) calculated for C₂₅H₁₈F₄NO₅S, [M+H]⁺ 520.0836, found 520.0825

1.1.2.c. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2c)

The synthesis of 2c follows the method of synthesis for 2a using (2R*,3R*)-phenyl 3-hydroxy-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1c) as starting material. The title compound is a colorless glass, isolated in 94% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.02 (dd, J = 8.4, 1.5 Hz, 1H), 7.89 (dd, J = 8.6, 5.4 Hz, 1H), 7.54 – 7.36 (m, 3H), 7.35 – 7.22 (m, 2H), 7.16 (m, 2H), 5.55 (d, J = 8.4 Hz, 1H), 5.20 (t, J = 1.8 Hz, 1H), 5.02 (s, 1H), 1.47 (d, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.0, 164.4, 164.0 (d, J = 255 Hz), 151.0, 150.3, 142.0, 134.0 (d, J = 8.9 Hz), 131.1 (m), 129.7, 126.6, 126.0 (dq, J = 3.9, 1.9 Hz), 122.5 (dd, J = 274.0, 2.4 Hz), 121.2, 119.0 (d, J = 21.3 Hz), 115.0 (dq, J = 25.7, 5.7 Hz), 105.4, 73.6, 53.7, 13.7. HRMS (ESI⁺) calculated for C₂₁H₁₅F₄NO₅Na, [M+Na]⁺ 460.0779, found 460.0785.

1.1.2.d. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-isobutyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2d)

The synthesis of 2d follows the method of synthesis for 2a using (2R*,3R*)-phenyl 3-hydroxy-2-isobutyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1d) as starting material. The title compound is a colorless glass, isolated in 95% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 8.3 Hz, 1H), 7.90 (bs, 1H), 7.54 – 7.20 (m, 5H), 7.13 (m, 2H), 5.61 – 5.36 (m, 2H), 4.70 (bs, 1H), 2.22 (s, 1H), 1.24 – 1.00 (bs, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 186.4, 164.0 (d, J = 256 Hz), 151.4, 150.3, 143.2, 142.5, 134.0 (m), 129.6, 126.5, 126.0, 123.8, 121.1, 118.9 (d, J = 21.1 Hz), 115.0, (dq, J = 25.7, 5.8 Hz), 105.9 (m), 74.0, 71.6, 63.0, 27.5, 19.5, 19.3. HRMS (ESI⁺) calculated for C₂₃H₁₉F₄NO₅Na, [M+Na]⁺ 488.1092, found 488.1101.

1.1.2.e. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2e)

The synthesis of 2e follows the method of synthesis for 2a using (2R*,3R*)-phenyl 3-hydroxy-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1e) as starting material. The title compound is a colorless foaming solid, isolated in 82% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (dd, J = 8.5, 1.4 Hz, 1H), 7.88 (dd, J = 8.6, 5.4 Hz, 1H), 7.62 – 7.18 (m, 5H), 7.13 (d, J = 7.9 Hz, 2H), 5.65 (d, J = 9.2 Hz, 1H), 5.55 (d, J = 8.4 Hz, 1H), 5.20 (d, J = 9.2 Hz, 1H), 5.12 (s, 1H), 1.88 (d, J = 1.4 Hz, 3H), 1.80 (d, J = 1.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.1, 164.6, 164.0 (d, J = 255 Hz), 151.0, 150.3, 142.8, 141.9, 133.8 (d, J = 8.9 Hz), 131.1 (m), 129.6, 126.4, 126.1 (m), 123.8 (m), 121.1, 118.9 (d, J = 21.3 Hz), 114.9 (dd, J = 25.8, 5.7 Hz), 114.5, 105.9, 72.9, 56.5, 25.9, 18.4. HRMS (ESI⁺) calculated for C₂₄H₁₉F₄NO₅Na, [M+Na]⁺ 500.1092, found 500.1049.

1.1.2.f. (2R,3R)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2f)

The synthesis of 2f follows the method of synthesis for 2a using (2R*,3R*)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1f) as starting material. The title compound is a colorless foaming solid, isolated in quantitative yield.

¹H NMR (400 MHz, CDCl₃) δ 8.31 (dd, J = 8.6, 1.2 Hz, 1H), 7.92 (dd, J = 8.7, 5.4 Hz, 1H), 7.50 (dd, J = 8.9, 2.5 Hz, 1H), 7.41 – 7.30 (m, 3H), 7.27 (m, 1H), 7.05 (d, J = 7.6 Hz, 2H), 6.94 – 6.78 (m, 3H), 5.99 (m, 2H), 5.91 (s, 1H), 5.64 (dd, J = 8.6, 1.4 Hz, 1H), 5.46 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.2, 164.6, 164.0 (d, J = 256.0 Hz), 150.2, 148.1, 143.5, 134.0 (d, J = 8.8 Hz), 131.4, (qd, J = 33.7, 8.2 Hz), 129.6, 126.6, 126.5, 126.0 (m), 123.9, 123.8, 121.2, 121.1, 119.9, 119.0 (d, J = 21.2 Hz), 115.0 (dq, J = 25.5, 5.7 Hz), 109.0, 106.7, 106.6, 101.5, 74.0, 61.0. HRMS (ESI⁺) calculated for C₂₇H₁₈F₄NO₇, [M+H]⁺ 544.1014, found 544.1006.

1.1.2.g. (2R,3R)-phenyl-2-([1,1′-biphenyl]-3-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2g)

The synthesis of 2g follows the method of synthesis for 2a using (2R*,3R*)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1g) as starting material. The title compound is isolated as a colorless foam in 58% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.37 (dd, J = 8.5, 1.3 Hz, 1H), 7.94 (dd, J = 8.6, 5.4 Hz, 1H), 7.67 – 7.17 (m, 14H), 7.03 (m, 2H), 6.09 (s, 1H), 5.76 (dd, J = 8.5, 1.4 Hz, 1H), 5.58 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.1, 164.6, 164.0 (d, J = 256 Hz), 151.3, 150.2, 143.6, 142.6, 140.3, 134.0 (d, J = 8.9 Hz), 133.5, 131.4 (d, J = 33.8, 8.0 Hz), 129.8, 129.6, 128.9, 127.9, 127.7, 127.2, 126.5, 125.9 (dd, J = 4.0, 2.1 Hz), 125.0, 124.9, 122.4 (dd, J = 274, 2.4 Hz), 121.1, 119.0 (d, J = 21.1 Hz), 115.0 (dq, J = 25.6, 5.7 Hz), 106.8, 74.0, 61.3. HRMS (ESI⁺) calculated for C₃₂H₂₂F₄NO₅, [M+H]⁺ 576.1429, found 576.1426.

1.1.2.h. (2R,3R)-phenyl-2-(3,5-difluorophenyl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2h)

The synthesis of 2h follows the method of synthesis for 2a using (2R*,3R*)-phenyl 2-(3,5-difluorophenyl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1h) as starting material. The title compound is isolated as a colorless foaming solid in 48% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.34 (dd, J = 8.6, 1.3 Hz, 1H), 7.93 (dd, J = 8.6, 5.4 Hz, 1H), 7.51 (dd, J = 8.9, 2.5 Hz, 1H), 7.43 – 7.30 (m, 3H), 7.28 (m, 1H), 7.05 (s, 2H), 6.94 (m, 2H), 6.84 (tt, J = 8.5, 2.1 Hz, 1H), 5.99 (s, 1H), 5.67 (d, J = 8.3 Hz, 1H), 5.45 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 184.4, 164.5, 164.1 (d, J = 256 Hz) 162.3 (dd, J = 251, 12.7 Hz), 150.9, 150.0, 143.2, 137.0 (m), 134.1, 134.0, 129.7, 126.7, 125.6 (m), 122.4 (dd, J = 275, 2.3 Hz), 121.0, 119.1 (d, J = 21.4 Hz), 115.1 (m), 109.5 (m), 106.9, 104.7 (t, J = 25.1 Hz), 73.3, 60.4. HRMS (ESI⁺) calculated for C₂₆H₁₆F₆NO₅, [M+H]⁺ 536.0927, found 536.0925.

1.1.3.a. Phenyl 4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate (3a)

The synthesis of 3a follows the method published by Comins et. al.^{21 1}H NMR (400 MHz, CDCl₃) δ 8.09 (d, J = 8.4 Hz, 1H), 7.50 – 7.20 (m, 7H), 7.08 (m, 2H), 5.88 (d, J = 7.5 Hz, 1H), 5.52 (d, J = 8.4 Hz, 1H), 3.27 (dd, J = 16.9, 7.5 Hz, 1H), 2.90 (dd, J = 16.7, 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 191.8, 150.3, 142.2, 138.2, 129.6, 129.0, 128.3, 126.4, 126.0, 121.2, 108.9, 105.0, 56.3, 41.8. HRMS (ESI⁺) calculated for C₁₈H₁₅NO₃, [M+H]⁺ 294.1125, found 294.1122

1.1.3.b. Phenyl 2-([1,1′-biphenyl]-4-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (3b)

The synthesis of 3b follows the method published by Comins et. al.^{21 1}H NMR (400 MHz, CDCl₃) δ 8.12 (dd, J = 8.5, 1.3 Hz, 1H), 7.56 – 7.49 (m, 4H), 7.47 – 7.32 (m, 6H), 7.32 – 7.21 (m, 2H), 7.08 (d, J = 7.9 Hz, 2H), 5.94 (d, J = 7.6 Hz, 1H), 5.54 (d, J = 8.4 Hz, 1H), 3.30 (dd, J = 16.6, 7.5 Hz, 1H), 2.95 (dt, J = 16.6, 1.5 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 191.7, 150.3, 142.0, 140.5, 138.8, 129.6, 129.4, 128.8, 127.5, 127.1, 127.1, 126.4, 124.8, 124.7, 121.2, 108.9, 69.5, 56.4. HRMS (ESI⁺) calculated for C₂₄H₁₉NO₃Na, [M+Na]⁺ 392.1254, found 392.1257.

1.1.4.a

Compound 4a was synthesized following the procedure published by Ege et. al.³⁹

1.1.4.b. 2-([1,1′-biphenyl]-3-yl)-2,3-dihydropyridin-4(1H)-one (4b)

To a solution of phenyl 2-([1,1′-biphenyl]-3-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (3b) (823 mg, 2.228 mmol) in 10 ml of dry MeOH, were added 0.51 ml (2.228 mmol) of a 25 % solution of sodium methanolate in methanol The solution was stirred for 30 min, then neutralized with 2 M HCl and concentrated in vacuo. The residue was dissolved in EtOAc, brine was added and the aqueous layer was extracted with EtOAc. The combined organic layers were dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude was purified on silica gel flash chromatography, with a mixture of ethyl acetate in hexanes from 10% to 25%, and the title compound was isolated in 74% yield (0.413 g) as a white powdery solid.

¹H NMR (400 MHz, CDCl₃) δ 7.64 – 7.55 (m, 4H), 7.50 – 7.42 (m, 3H), 7.38 (m, 2H), 7.28 (m, 1H), 5.16 (d, J = 7.5 Hz, 1H), 5.05 (bs, 1H), 4.83 (dd, J = 14.8, 4.8 Hz, 1H), 2.82 (dd, J = 16.3, 14.8 Hz, 1H), 2.60 (ddt, J = 16.2, 4.8, 1.2 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 192.2, 150.8, 142.2, 140.5, 140.5, 129.5, 128.9, 127.7, 127.4, 127.1, 125.4, 125.4, 100.2, 58.8, 44.7. HRMS (ESI⁺) calculated for C₁₇H₁₆NO, [M+H]⁺ 250.1226, found 250.1227

1.1.5. (2R,3R)-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (5)

The synthesis of 5 follows the method of synthesis for 4b using (2R*,3R*)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate (1a) as starting material. The title compound is a white solid, isolated in quantitative yield.

¹H NMR (400 MHz, DMSO) δ 7.92 (d, J = 6.6 Hz, 1H), 7.53 – 7.24 (m, 6H), 4.97 (d, J = 3.8 Hz, 1H), 4.80 (dd, J = 7.1, 1.5 Hz, 1H), 4.36 (d, J = 12.7 Hz, 1H), 3.97 (dd, J = 12.7, 3.8 Hz, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 192.8, 152.0, 137.4, 129.0, 129.0, 127.6, 96.1, 73.4, 64.6. HRMS (ESI⁺) calculated for C₁₁H₁₁NO₂Na, [M+Na]⁺ 212.0682, found 212.0686.

1.1.6. (2R,3R)-phenyl 3-((methyl(phenyl)carbamoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate ((±)-6)

(2S*,3R*)-phenyl 3-hydroxy-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1b) was dissolved in 2.0 ml of dichloromethane, triethyl amine (0.106 ml, 0.759 mmol), DMAP (7.40 mg, 0.061 mmol) and N-methyl-N-phenylcarbamoyl chloride (640 mg 0.380 mmol) were added and the reaction was stirred overnight. Then the solvent was evaporated under reduced pressure, the crude was purified by silica gel flash column chromatography with a gradient 0–15% of ethyl acetate in hexanes. Product isolated as a white foam (125 mg) in 89% yield. ¹H NMR (400 MHz, CDCl₃) δ 8. 05 (bs, 1H), 7.48 – 7.15 (m, 7H), 7.13 – 6.97 (m, 3H), 6.81 (d, J = 5.1 Hz, 1H), 6.21 (bs, 1H), 5.56 (bs, 1H), 5.05 (dd, J = 2.3, 1.5 Hz, 1H), 3.34 (s, 3H), 2.37 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.4, 153.9, 151.1, 150.3, 142.5, 137.4, 130.3, 129.7, 129.6, 128.9, 128.8, 126.5, 126.5, 125.6, 124.0, 121.1, 107.0, 73.0, 56.8, 38.0, 14.0. HRMS (ESI⁺) calculated for C₂₅H₂₂N₂NaO₅S, [M+Na]⁺ 485.1142, found 485.1145.

1.1.7. (2S,3R)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-7)

(2S*,3R*)-phenyl 3-((methyl(phenyl)carbamoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate ((±)-6) (92.0 mg, 0.199 mmol) was dissolved in 2 mL of MeOH, and 0.045 mL (0.199 mmol) of a 25 % methanolic solution of sodium methoxide was added drop wise. The reaction mixture was stirred for 1.0 hours, and upon completion, the solvent was removed under reduced pressure. The crude was purified by silica gel flash column chromatography with a gradient of EA/hexanes 5–45% to afford 62.0 mg (91% yield) of the title compound as a colorless foam. ¹H NMR (400 MHz, CDCl₃) δ 7.49 – 6.99 (m, 6H), 6.76 (d, J = 5.1 Hz, 1H), 5.42 (d, J = 13.6 Hz, 1H), 5.21 (d, J = 5.7 Hz, 1H), 5.12 (dd, J = 7.4, 1.6 Hz, 1H), 4.95 (d, J = 13.5 Hz, 1H), 3.22 (s, 3H), 1.87 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 188.8, 154.2, 150.1, 143.0, 137.3, 132.8, 130.0, 128.6, 126.1, 126.0, 124.6, 110.0, 99.1, 55.7, 38.0, 13.6. HRMS (ESI⁺) calculated for C₁₈H₁₈N₂O₃SNa, [M+Na]⁺ 365.0930, found 365.0926.

1.1.8.a. (2S,3R)-2-(4-methylthiophen-2-yl)-4-oxo-1-(6-(trifluoromethyl)nicotinoyl)-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-8a)

(2S*,3R*)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate (7)methyl(phenyl)carbamate ((±)-7) (100 mg, 0.029 mmol) was dissolved in 2 ml of THF. DMAP (7.85 mg, 0.064 mmol) and 6-(trifluoromethyl)nicotinoyl chloride (0.012 g, 0.058 mmol) were added. The reaction was stirred overnight, the solvent was removed, and the crude was purified by silica gel flash column chromatography with a gradient 0–15% of ethyl acetate in hexanes.. The title compound was isolated in quantitative yield. ¹H NMR (400 MHz, CDCl₃) δ 9.39 (bs, 1H), 8.55 (dd, J = 7.9, 2.0 Hz, 2H), 7.83 (dd, J = 8.1 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.35 (m, 2H), 7.29 (m, 1H), 7.29 (s, 2H), 7.10 (d, J = 5.1 Hz, 1H), 6.76 (d, J = 5.1 Hz, 1H), 6.18 (bs, 1H), 5.51 (bs, 1H), 5.16 (bs, 1H), 3.33 (s, 3H), 2.34 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.4, 167.2, 166.2, 151.5, 148.9, 142.4, 141.9, 139.3, 137.9, 137.5, 131.4, 130.5, 129.1, 127.7, 126.9, 126.0, 124.1, 120.4, 120.3 (m), 108.0, 72.7, 56.7, 38.2, 13.9. HRMS (ESI⁺) calculated for C₂₅H₂₀F₃N₃O₄S, [M+Na]⁺ 538.1019, found 538.1022.

1.1.8.b. (2S,3R)-1-(cyclopropanecarbonyl)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate (8b)

The synthesis of 8b follows the method of synthesis for 8a using (2S*,3R*)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate (7) as starting material and cyclopropanecarbonyl chloride as reagent. The title compound was isolated in quantitative yield.

¹H NMR (400 MHz, CDCl₃) δ 8.10 (bs, 1H), 7.48 – 6.96 (m, 6H), 6.79 (d, J = 5.1 Hz, 1H), 6.26 (bs, 1H), 5.49 (bs, 1H), 5.03 (m, 1H), 3.30 (s, 3H), 2.39 (s, 3H), 1.17 (m, 1H), 1.10 (m, 2H), 0.91 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 186.7, 172.3, 153.9, 142.5, 142.1, 136.8, 130.5, 128.9, 126.5, 125.6, 123.9, 106.3, 73.1, 56.0, 38.0, 29.7, 14.0, 11.8, 9.9, 8.9. HRMS (ESI⁺) calculated for C₂₂H₂₂N₂O₄SNa, [M+Na]⁺ 433.1192, found 433.1197

1.1.9.a. (2R,3R)-phenyl 3-((tert-butyldimethylsilyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-9a)

Imidazole (74.8 mg, 1.10 mmol) was added to a solution of (2R*,3R*)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1a) (170 mg, 0.55 mmol) and TBSCl (247 mg, 1.65 mmol) in CH₂Cl₂ (6 mL). The reaction mixture was stirred for 18 h at room temperature, diluted with dichloromethane, and quenched with a saturated aqueous solution of NH₄Cl. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography with a gradient 0–5% of ethyl acetate in hexanes to afford 215 mg (92% yield) of the title compound as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (dd, J = 8.6, 1.3 Hz, 1H), 7.21 (m, 8H), 6.95 (bs, 2H), 5.55 (s, 1H), 5.40 (dd, J = 8.4, 1.6 Hz, 1H), 4.02 (m, 1H), 0.85 (s, 9H), 0.14 (s, 3H), 0.05 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 190.8, 152.2, 150.5, 142.0, 134.5, 129.7, 129.3, 128.7, 126.6, 126.4, 121.4, 106.4, 73.8, 65.3, 25.8, 18.3, −4.5, −4.8.

1.1.9.b. (2R,3R)-phenyl-3-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9b)

The synthesis of 9b follows the method of synthesis for 9a using (2S*,3S*)-phenyl 3-hydroxy-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1c) as starting material. Colorless oil which solidified upon standing, 99% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 8.5, 1.6 Hz, 1H), 7.43 (m, 2H), 7.29 (m, 1H), 7.16 (m, 2H), 5.38 (d, J = 8.4 Hz, 1H), 4.65 (m, 1H), 3.72 (dd, J = 2.5, 1.5 Hz, 1H), 1.39 – 1.07 (m, 3H), 0.88 (s, 9H), 0.15 (s, 3H), 0.08 (s, 3H).

1.1.9.c. (2R,3R)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9c)

The synthesis of 9c follows the method of synthesis for 9a using (2R*,3R*)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1f) as starting material. The title compound was isolated as a colorless oil in 77% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.11 (dd, J = 8.5, 1.3 Hz, 1H), 7.38 (m, 2H), 7.26 (m, 1H), 7.06 (d, J = 7.7 Hz, 2H), 6.73 (m, 3H), 5.95 (dd, J = 3.5, 1.4 Hz, 2H), 5.52 (s, 1H), 5.47 (dd, J = 8.5, 1.4 Hz, 1H), 4.03 (dd, J = 2.4, 1.6 Hz, 1H), 0.92 (s, 9H), 0.21 (s, 3H), 0.12 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 190.9, 152.0, 150.6, 148.5, 147.9, 141.9, 129.8, 128.4, 126.6, 121.5, 120.1, 109.0, 107.0, 106.3, 101.6, 73.9, 65.1, 25.8, 18.3, −4.5, −4.8. HRMS (ESI⁺) calculated for C₂₅H₂₉NO₆SiNa, [M+Na]⁺ 490.1656, found 490.1660.

1.1.10.a. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-10a)

The synthesis of ((±)-10a) follows the method of synthesis for ((±)-7) using (2R,3R)-phenyl 3-((tert-butyldimethylsilyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate as starting material. The title compound was obtained as a white solid in 90% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.44 (m, 2H), 7.36 (m, 3H), 7.20 (m, 1H), 5.03 (dd, J = 7.4, 1.6 Hz, 1H), 5.00 (bs, 1H), 4.55 (d, J = 12.9 Hz, 1H), 4.26 (d, J = 12.9 Hz, 1H), 0.60 (s, 3H), 0.06 (s, 9H), −0.33 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 193.4, 150.3, 138.8, 128.9, 128.9, 128.4, 98.4, 77.0, 65.7, 25.7, 18.5, −4.0, −5.9. HRMS (ESI⁺) calculated for C₁₇H₂₆NO₂Si, [M+H]⁺ 304.1727, found 304.1729.

1.1.10.b. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-methyl-2,3-dihydropyridin-4(1H)-one (10b)

The synthesis of 10b follows the method of synthesis for 4b using (2R*,3R*)-phenyl 3-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9b) as starting material. The title compound was isolated as a colorless solid in 96% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.09 (dd, J = 7.3, 6.5 Hz, 1H), 4.96 (dd, J = 7.3, 1.5 Hz, 1H), 4.83 (s, 1H), 3.89 (d, J = 12.2 Hz, 1H), 3.61 (m, 1H), 1.36 (d, J = 6.4 Hz, 3H), 0.92 (m, 9H), 0.23 (s, 3H), 0.09 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 193.7, 150.0, 98.0, 77.1, 55.9, 26.2, 18.9, 18.5, −3.6, −5.2.

1.1.10.c. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (10c)

The synthesis of 10c follows the method of synthesis for 4b using (2R*,3R*)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9c) as starting material. The title compound was isolated as a powdery solid in 93% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.18 (ddd, J = 7.4, 6.5, 0.8 Hz, 1H), 6.92 (m, 2H), 6.80 (d, J = 7.9 Hz, 1H), 5.90 (m, 2H), 5.03 (dd, J = 7.4, 1.6 Hz, 1H), 4.93 (d, J = 6.3 Hz, 1H), 4.46 (d, J = 12.8 Hz, 1H), 4.19 (d, J = 12.8 Hz, 1H), 0.66 (s, 9H), 0.09 (s, 3H), −0.26 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 193.4, 150.2, 148.1, 148.0, 132.7, 122.0, 108.5, 108.4, 101.4, 98.5, 77.1, 65.5, 25.7, 18.5, −3.9, −5.8. HRMS (ESI⁺) calculated for C₁₈H₂₆NO₄Si [M+H]⁺ 348.1626, found 348.1629

1.1.11.a. (2R,3R)-1-benzoyl-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-11a)

A solution of ((±)-10a) (190 mg, 0.630 mmol) in THF (25.0 mL) was cooled to −78 °C in a dry ice/isopropanol bath, under nitrogen. NaHMDS (0.94 mL, 0.939 mmol) was added drop wise and the reaction mixture was stirred for one hour. Then benzoyl chloride (0.110 mL, 0.939 mmol) was added drop wise a t-78 °C. The mixture was stirred for one more hour and upon disappearance of the starting material, the reaction was quenched by addition of brine. The aqueous layer were collected and extracted with ethyl acetate. The combined organic layers were treated with brine and anhydrous sodium sulfate. After evaporation of the solvent, the crude was purified with silica gel flash column chromatography, with a gradient 0–30% of ethyl acetate in hexanes to yield 220 mg (86% yield) of the title compound as a foaming solid. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J = 8.3 Hz, 1H) 7.60 – 7.40 (m, 5H), 7.40 – 7.18 (m, 5H), 5.76 (s, 1H), 5.32 (d, J = 8.3 Hz, 1H), 4.16 (d, J = 1.9 Hz, 1H), 0.91 (m, 9H), 0.20 (s, 3H), 0.13 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.0, 170.7, 143.3, 133.9, 133.2, 131.7, 129.1, 128.8, 128.4, 128.3, 126.5, 105.6, 74.0, 64.3, 25.6, 18.1, −4.8, −5.0.

1.1.11.b. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-2,3-dihydropyridin-4(1H)-one (11b)

The synthesis of 11b follows the method of synthesis for 11a using (2R*,3R*)-2-(Benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (10c) as starting material. The title compound was isolated as a colorless oil in 26% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 8.3 Hz, 1H), 6.74 (m, 1H), 6.65 (m, 2H), 5.93 (q, J = 1.4 Hz, 2H), 5.55 (s, 1H), 5.40 (dd, J = 8.4, 1.6 Hz, 1H), 4.02 (dd, J = 2.5, 1.6 Hz, 1H), 1.82 (s, 1H), 1.30 – 1.15 (m, 2H), 1.05–0.95 (s, 2H), 0.88 (s, 9H), 0.18 (s, 3H), 0.09 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 190.9, 172.9, 148.3, 147.6, 141.3, 127.8, 119.7, 108.8, 106.6, 105.4, 101.3, 74.1, 64.3, 25.6, 18.0, 11.9, 9.80, 8.63, −4.76, −5.03. HRMS (ESI⁺) calculated for C₂₂H₂₉NO₅SiNa, [M+Na]⁺ 438.1707, found 438.1690.

1.1.11.c. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(2-phenylacetyl)-2,3-dihydropyridin-4(1H)-one (11c)

The synthesis of 11c follows the method of synthesis for 11a using (2R*,3R*)-2-(Benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (10c) as starting material. The title compound was isolated as a colorless oil, in 49% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.42 – 7.23 (m, 3H), 7.23 – 7.14 (m, 2H), 6.72 (m, 1H), 6.61 (m, 2H), 5.94 (s, 2H), 5.35 (m, 2H), 4.01 – 3.75 (s, 4H), 0.81 (s, 9H), 0.09 (s, 3H), 0.03 (s, 3H). HRMS (ESI⁺) calculated for C₂₆H₃₁NO₅SiNa, [M+Na]⁺ 488.1864, found 488.1863.

1.1.11.d. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(6-(trifluoromethyl)nicotinoyl)-2,3-dihydropyridin-4(1H)-one (11d)

(2R,3R)-2-(Benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (50 mg, 0.144 mmol) was dissolved in 1 ml of THF. Triethyl amine was added to the reaction mixture, followed by DMAP (40 mg, 0.317 mmol) and finally 6-(trifluoromethyl)nicotinoyl chloride (38 mg, 0.180 mmol). The reaction mixture was stirred overnight at room temperature, the solvent was removed under a nitrogen stream and the crude was purified by silica gel column chromatography to afford 61 mg (82% yields) of the title compound, as a colorless solid.

¹H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 6.80 – 6.63 (m, 3H), 5.96 (s, 2H), 5.60 (bs, 1H), 5.45 (d, J = 8.3 Hz, 1H), 4.12 (d, J = 2.1 Hz, 1H), 0.90 (s, 9H), 0.19 (s, 3H) 0.11 (s, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ −68.3. ¹³C NMR (100 MHz, CDCl₃) δ 190.8, 167.2, 151.7, 150.65 (q, J = 35.7 Hz), 149.1, 148.7, 148.1, 141.4, 139.4, 137.7, 132.4, 127.2, 120.8, 120.5 (q, J = 274.6 Hz), 120.3, 109.1, 107.6, 107.1, 101.7, 74.0, 65.0, 25.8, 18.2, −4.6, −4.8. HRMS (ESI⁺) calculated for C₂₅H₂₈F₃N₂O₅Si, [M+H]⁺ 521.1714, found 521.1713.

1.1.11.e. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (11e)

The synthesis of 11e follows the method of synthesis for 11a using (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one (10a) as starting material. The title compound was isolated in 98% yield, as a white foam.

¹H NMR (400 MHz, CDCl₃) δ 8.24 (bs, 1H), 7.83 (d, J = 15.3 Hz, 1H), 7.53 – 7.10 (m, 10H), 6.87 (bs, 1H), 5.65 (bs, 1H), 5.47 (d, J = 8.5 Hz, 1H), 4.13 (dd, J = 2.5, 1.5 Hz, 1H), 0.88 (s, 9H), 0.19 (s, 3H), 0.11 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.0, 165.9, 147.4, 141.6, 134.4, 134.2, 131.0, 129.5, 129.2, 128.7, 128.4, 126.4, 114.9, 106.2, 74.3, 64.5, 25.8, 18.3, −4.6, −4.7.

HRMS (ESI⁺) calculated for C₂₆H₃₂NO₃Si, [M+H]⁺ 434.2146, found 434.2149.

1.1.11.f. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-methyl-2,3-dihydropyridin-4(1H)-one (11f)

The synthesis of 11f follows the method of synthesis for 11a using (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-2-methyl-2,3-dihydropyridin-4(1H)-one (10b) as starting material. The title compound was isolated in 88% yield, as a colorless oil which solidifies upon standing.

¹H NMR (400 MHz, CDCl₃) δ 7.84 (d, J = 15.4 Hz, 2H), 7.59 (m, 2H), 7.43 (m, 3H), 6.95 (d, J = 15.3 Hz, 1H), 5.37 (dd, J = 8.4, 1.7 Hz, 1H), 4.75 (bs, 1H), 3.73 (dd, J = 2.5, 1.6 Hz, 1H), 1.24 (d, J = 7.1 Hz, 3H), 0.83 (s, 9H), 0.12 (s, 3H), 0.06 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.9, 165.4, 146.8, 140.0, 134.3, 130.7, 129.0, 128.2, 114.7, 104.5, 73.6, 60.4, 55.9, 25.6, 18.0, −4.8, −5.0. C₂₁H₂₉NO₃SiNa, [M+Na]⁺ 394.1809, found 394.1875.

1.1.12.a. (2R,3R)-1-benzoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-12a)

A solution of ((±)-11a) (220 mg, 0.540 mmol) in a 1:1 mixture of dry acetonitrile and pyridine (13 ml each) was cooled down in a water/ice bath and a 70% solution of HF in pyridine was added drop wise (2.0 mL). The reaction was stirred overnight and allowed to warm to room temperature. The mixture was transferred in a separatory funnel and quenched by addition of a saturated aqueous solution of NaHCO₃, solid K₂CO₃ was then added until no more effervescence was observed. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were treated with brine and anhydrous Na₂SO₄. After evaporation of the solvent the crude was purified on silica gel flash column chromatography with a mixture of hexanes ethyl acetate 4:1 to recover the starting material and 1:1 to collect the product. The title compound was isolated as a white solid (112 mg, 0.382 mmol) together with 20 mg of starting material. 78% based on starting material recovered. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 8.3 Hz, 1H), 7.67 – 7.50 (m, 3H), 7.45 (m, 2H), 7.48 – 7.13 (m, 5H), 5.69 (d, J = 4.7 Hz, 1H), 5.38 (dd, J = 8.3, 1.2 Hz, 1H), 4.29 (dd, J = 4.9, 1.2 Hz, 1H), 3.96 (bs, 1H).¹³C NMR (100 MHz, CDCl₃) δ 192.2, 170.9, 145.2, 135.2, 132.7, 132.1, 129.0, 128.9, 128.7, 128.3, 126.5, 104.2, 73.6, 63.6. HRMS (ESI+) calculated for C₁₈H₁₅NO₄Na [M+Na]+ 316.0944, found 316.0954.

1.1.12.b. (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b)

The synthesis of 12b follows the method of synthesis for 12a using (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (11e) as starting material. The title compound was isolated in 88% yield, as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 15.3 Hz, 1H), 7.53 – 7.18 (m, 10H), 6.81 (d, J = 15.3 Hz, 1H), 5.73 (d, J = 2.9 Hz, 1H), 5.50 (dd, J = 8.4, 1.5 Hz, 1H), 4.67 (bs, 1H), 4.19 (d, J = 3.1 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 192.1, 166.1, 147.8, 143.0, 134.6, 134.3, 131.0, 129.5, 129.1, 128.7, 128.5, 126.4, 115.0, 105.5, 73.7, 63.7. HRMS (ESI+) calculated for C₂₀H₁₇NO₃Na [M+Na]⁺ 342.1101, found 342.1102.

1.1.12.c. (2R,3R)-1-cinnamoyl-3-hydroxy-2-methyl-2,3-dihydropyridin-4(1H)-one (12c)

The silylether (11f) (132 mg, 0.355 mmol) was dissolved in 2 mL of dry THF, acetic acid (0.102 mL, 1.78 mmol) was added, and the solution was cooled down to 0°C in ice water bath. Tetrabutylammonium fluoride solution (1M in THF, 1.07 mL) was added drop wise and the reaction was stirred overnight, and allowed to warm up to room temperature. The solvent was evaporated under reduced pressure, and the crude was purified by silica gel column chromatography to afford 46 mg (50% yields) of the title compound, as colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.94 (dd, J = 8.4, 1.5 Hz, 1H), 7.82 (dd, J = 15.3, 1.1 Hz, 1H), 7.57 (m, 2H), 7.41 (m, 3H), 6.98 (d, J = 15.4 Hz, 1H), 5.41 (dd, J = 8.2, 1.5 Hz, 1H), 4.86 (dt, J = 7.0, 1.9 Hz, 1H), 4.65 (s, 1H), 3.81 (s, 1H)1.26 (dd, J = 7.1, 1.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 193.1, 165.8, 147.5, 141.5, 134.3, 131.0, 129.2, 128.5, 114.8, 104.2, 72.9, 55.1, 14.0. M+Na check mass

1.1.13.a. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclopropanecarboxylate ((±)-13a)

The synthesis of ((±)-13a) follows the method of synthesis for ((±)-2a) using (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-12b) as starting material. The title compound was isolated in 73% yield, as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.45 (s, 1H), 7.87 (d, J = 15.3 Hz, 1H), 7.50 – 7.15 (m, 10H), 6.77 (d, J = 15.1 Hz, 1H), 5.83 (s, 1H), 5.63 (dd, J = 8.5, 1.4 Hz, 1H), 5.32 (t, J = 1.8 Hz, 1H), 1.66 (m, 1H), 1.06 (m, 2H), 0.94 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 186.3, 174.0, 165.3, 148.2, 142.7, 134.0, 133.2, 131.1, 129.5, 129.0, 128.9, 128.4, 126.1, 114.1, 107.0, 73.2, 61.3, 12.9, 9.55, 9.46. HRMS (ESI⁺) calculated for C₂₄H₂₁NO₄Na, [M+Na]⁺ 410.1363, found 410.1348.

1.1.13.b. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclobutanecarboxylate (13b)

The synthesis of 13b follows the method of synthesis for 2a using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated as a colorless solid in 76% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.40 (bs, 1H), 7.85 (d, J = 15.3 Hz, 1H), 7.51 – 7.21 (m, 10H), 6.76 (d, J = 15.0 Hz, 1H), 5.83 (s, 1H), 5.61 (dd, J = 8.6, 1.4 Hz, 1H), 5.32 (t, J = 1.8 Hz, 1H), 3.19 (m, 1H), 2.41 – 2.12 (m, 4H), 2.11 – 1.79 (m, 2H). HRMS (ESI⁺) calculated for C₂₅H₂₃NO₄Na, [M+Na]⁺ 424.1519, found 424.1550.

1.1.13.c. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl benzoate (13c)

The synthesis of 13c follows the method of synthesis for 2a using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated as a colorless oil in 90% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.52 (d, J = 8.0 Hz, 1H), 8.02 (dd, J = 8.3, 1.3 Hz, 2H), 7.83 (d, J = 15.3 Hz, 1H), 7.57 (m, 1H), 7.51 – 7.30 (m, 12H), 6.75 (d, J = 15.1 Hz, 1H), 5.98 (s, 1H), 5.70 (dd, J = 8.6, 1.4 Hz, 1H), 5.56 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 186.0, 165.5, 165.3, 148.4, 142.9, 133.9, 133.7, 133.1, 131.1, 130.1, 130.0, 129.6, 129.0, 128.8, 128.5, 128.4, 126.2, 114.0, 107.1, 73.6, 61.5. HRMS (ESI⁺) calculated for C₂₇H₂₁NO₄Na, [M+Na]⁺ 446.1377, found 446.1378.

1.1.13.d. (E)- (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(3-(trifluoromethyl)phenyl)acrylate (13d)

(Racemic)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (80.0 mg, 0.251 mmol) and (E)-3-(3-(trifluoromethyl)phenyl)acryloyl chloride (88.0 mg, 0.376 mmol) were dissolved in 5 mL of dry CH₂Cl₂ under nitrogen atmosphere at room temperature. Triethyl amine (0.121 mL, 0.878 mmol) was added drop wise and the reaction was stirred for 1h. The reaction mixture was washed with a saturated aqueous solution of NH₄Cl, and the aqueous layer was extracted with methylene chloride. The combined organic layers were treated with brine and Na₂SO₄. The crude was purified on silica gel flash chromatography with a 10% mixture of ethyl acetate in hexanes. The title compound was isolated as a colorless film in 60% yield (78 mg).

¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 8.04 – 7.13 (m, 16H), 6.80 (d, J = 13.0 Hz, 1H), 6.53 (dd, J = 16.0, 1.0 Hz, 1H), 5.94 (s, 1H), 5.68 (d, J = 8.5 Hz, 1H), 5.55 – 5.14 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 186.2, 165.5, 148.6, 145.2, 143.1, 134.8, 134.1, 133.2, 131.8, 131.5, 131.3, 129.8, 129.7, 129.3, 129.2, 128.6, 127.31 (q, J = 3.7 Hz), 126.4, 125.2, 124.9 (q, J = 3.8 Hz), 122.5, 118.8, 114.1, 107.2, 73.5, 61.5. HRMS (ESI⁺) calculated for C₃₀H₂₂F₃NO₄, [M+Na]⁺, 540.1393, found 540.1389.

1.1.13.e. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 2-phenylacetate (13e)

The synthesis of 13e follows the method of synthesis for 2a using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one 12b as starting material. The title compound was isolated in 64% yield, as a colorless solid.

HRMS (ESI⁺) calculated for C₂₈H₂₃NO₄Na, [M+Na]⁺ 460.1519, found 460.1499

¹H NMR (400 MHz, CDCl₃) δ 8.50 (brs, 0.2H), 8.40 (brs, 0.8H), 7.85 (d, J = 15.3 Hz, 0.3H), 7.78 (d, J = 15.3 Hz, 0.7H), 7.16–7.50 (m, 13H), 6.95–7.14 (m, 1.4H), 6.78 (d, J = 15.2 Hz, 0.6H), 6.66–6.55 (m, 1H), 5.81 (brs, 0.3H), 5.73 (brs, 0.7H), 5.52–5.65 (m, 1H), 5.31–5.34 (m, 1H), 3.69 (s, 1.4H), 3.63 (s, 0.6H). The NMR spectrum is broad at room temperature due to the presence of rotamers.

1.1.13.f. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-methylbenzoate (13f)

The synthesis of 13f follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material and 4-methylbenzoyl chloride as reagent. The title compound was isolated in 85% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.53 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 15.3 Hz, 1H), 7.50 – 7.20 (m, 12H), 6.74 (d, J = 14.7 Hz, 1H), 5.96 (s, 1H), 5.70 (dd, J = 8.6, 1.4 Hz, 1H), 5.54 (m, 1H) 2.40 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.2, 165.6, 165.3, 148.3, 144.7, 142.8, 133.9, 133.1, 131.1, 130.0, 129.6, 129.2, 129.0, 129.0, 128.4, 126.2, 126.1, 114.0, 105.0, 73.4, 61.5, 21.8. HRMS (ESI+) calculated for C₂₈H₂₃NO₄Na, [M+Na]+ 460.1519, found, 460.1528.

1.1.13.g. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-chlorobenzoate (13g)

The synthesis of 13g follows the method of synthesis for 13d using (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material and 4-chlorobenzoyl chloride as a reagent. The title compound was isolated in 77% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.51 (bs, 1H), 7.95 (m, 2H), 7.84 (d, J = 15.3 Hz, 1H), 7.50 – 7.30 (m, 12H), 6.76 (d, J = 14.8 Hz, 1H), 5.97 (s, 1H), 5.69 (dd, J = 8.5, 1.0 Hz, 1H), 5.54 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.8, 165.2, 164.7, 148.5, 143.0, 140.3, 133.9, 133.0, 131.4, 131.1, 129.6, 129.1, 129.0, 128.9, 128.4, 127.3, 126.2, 113.8, 107.0, 73.7, 61.4. HRMS (ESI+) calculated for C₂₇H₂₁ClNO₄, [M+H]⁺ 458.1154, found, 458.1163.

1.1.13.h. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate (13h)

The synthesis of 13h follows the method of synthesis for 2a using (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in quantitative yield, as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 8.45 (d, J = 6.4 Hz, 1H), 7.90 (dd, J = 8.6, 5.3 Hz, 1H), 7.85 (d, J = 15.4 Hz, 1H), 7.60 – 7.20 (m, 12H), 6.79 (d, J = 15.5 Hz, 1H), 6.01 (s, 1H), 5.64 (d, J = 8.6 Hz, 1H), 5.56 (d, J = 1.7 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.2, 165.2, 164.51, 164.0 (d, J = 255 Hz), 148.3, 143.0, 134.0 134.0, 133.9, 132.6, 131.0, 129.5, 129.0, 129.0, 128.3, 126.2, 125.7 (m), 122.4 (dd, J = 273.7, 2.3 Hz), 118.9 (d, J = 21.4 Hz), 115.0 (dd, J = 25.7, 5.7 Hz), 113.9, 106.7, 74.3, 60.8. HRMS (ESI⁺) calculated for C₂₈H₁₉F₄NO₄Na, [M+Na]⁺, 532.1142, found 532.1155.

1.1.13.i. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3,4-dichlorobenzoate (13i)

The synthesis of 13i follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 84% yield, as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 7.85 (m, 1H), 7.83 (m, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.50 – 7.30 (m, 10H), 6.77 (d, J = 15.4 Hz, 1H), 6.96 (bs, 1H), 5.70 (dd, J = 8.5, 1.3 Hz, 1H), 5.53 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.5, 165.2, 163.8, 148.6, 143.1, 138.6, 133.8, 133.2, 132.9, 131.8, 131.2, 130.7, 129.6, 129.0, 129.0, 129.0, 128.6, 128.4, 126.2, 113.8, 106.9, 73.9, 61.2. HRMS (ESI+) calculated for C₂₇H₁₉Cl₂NO₄Na, [M+Na]⁺, 514.0583, found 514.0587.

1.1.13.j. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluorobenzoate (13j)

The synthesis of 13j follows the method of synthesis for 13d using (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12d) as starting material and 4-fluorobenzoyl chloride as reagent. The title compound was isolated in72% yield, as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.52 (bs, 1H), 8.04 (m, 2H), 7.84 (d, J = 15.3 Hz, 1H), 7.53 – 7.30 (m, 10H), 7.18 – 7.00 (m, 2H), 6.77 (bs, 1H), 5.96 (bs, 1H), 5.70 (dd, J = 8.5, 1.4 Hz, 1H), 5.53 (dd, J = 2.1, 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.9, 167.5, 165.3, 164.7 (d, J = 35.7 Hz), 148.5, 143.0, 133.9, 133.0, 132.7 (d, J = 9.5 Hz) 131.1, 129.6, 129.0, 128.4, 126.2, 125.1 (d, J = 3.0 Hz), 115.9, 115.7, 113.9, 107.0, 73.6, 61.4. HRMS (ESI+) calculated for C₂₇H₂₀FNO₄Na, [M+Na]⁺, 464.1269, found 464.1270.

1.1.13.k. (E)-(2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(3-(trifluoromethyl)phenyl)acrylate (13k)

The synthesis of 13k follows the method of synthesis for 2a using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in quantitative yield, as colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 8.4 Hz, 1H), 8.28 (m, 2H), 8.19 (m, 2H), 7.84 (d, J = 15.3 Hz, 1H), 7.50 – 7.31 (m, 8H), 6.79 (d, J = 15.4 Hz, 1H), 6.00 (s, 1H), 5.71 (dd, J = 8.5, 1.4 Hz, 1H), 5.58 (dd, J = 2.3, 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.3, 165.2, 163.7, 150.9, 148.6, 143.1, 134.2, 133.8, 132.8, 131.2, 131.1, 129.6, 129.1, 129.0, 128.4, 126.2, 123.7, 113.7, 106.8, 74.2, 61.2. HRMS (ESI⁺) calculated for C₂₇H₂₀N₂O₆Na, [M+Na]⁺ 491.1214, found 491.1213.

1.1.13.l. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-nitro-3-(trifluoromethyl)benzoate (13l)

The synthesis of 13l follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 30% yield, as a colorless film.

¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 8.1 Hz, 1H), 8.44 (d, J = 1.7 Hz, 1H), 8.35 (dd, J = 8.3, 1.8 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.57 – 7.30 (m, 10H), 6.79 (d, J = 15.3 Hz, 1H), 6.00 (s, 1H), 5.72 (dd, J = 8.6, 1.4 Hz, 1H), 5.58 (dd, J = 2.2, 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 184.9, 165.1, 162.6, 150.8, 148.8, 143.3, 134.7, 133.8, 132.7, 132.6, 131.3, 129.7 (m), 129.2, 129.1, 128.4, 126.2, 125.3, 124.2 (d, J = 35 Hz), 122.7, 120.0, 113.6, 106.7, 74.6, 61.1. HRMS (ESI⁺) calculated for C₂₈H₁₉F₃N₂O₆Na, [M+Na]⁺, 559.1087, found 559.1089.

1.1.13.m. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(trifluoromethyl)benzoate (13m)

The synthesis of 13m follows the method of synthesis for 13d using (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 83% yield, as a colorless film.

¹H NMR (400 MHz, CDCl₃) δ 8.53 (bs, 1H), 8.27 (s, 1H), 8.21(d, J = 7.8 Hz, 1H), 7.84 (m, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.51 – 7.31 (m, 10H), 6.77 (d, J = 15.1 Hz, 1H), 5.99 (s, 1H), 5.72 (dd, J = 8.6, 1.4 Hz, 1H), 5.57 (m, 1H).¹³C NMR (100 MHz, CDCl₃) δ 185.6, 165.3, 164.3, 148.5, 143.1, 133.8, 133.1, 132.9, 131.4, 131.2, 131.1, 130.3 (q, J = 3.3 Hz), 129.7, 129.4 (d, J = 34.4 Hz), 129.1, 129.0, 128.4, 126.9 (q, J = 4 Hz), 126.1, 123.4, (d, J = 272.6 Hz), 113.9, 107.0, 73.3, 61.3. HRMS (ESI+) calculated for C₂₈H₂₁F₃NO₄, [M+H]⁺, 492.1417, found 492.1418.

1.1.13.n. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-cyanobenzoate (13n)

The synthesis of 13n follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 83% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.52 (bs, 1H), 8.29 (m, 1H), 8.25 (d, J = 8.0 Hz, 1H) 7.88 – 7.78 (m, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.50 – 7.30 (m, 10H), 6.81 (d, J = 15 Hz, 1H), 5.99 (s, 1H), 5.71 (dd, J = 8.5, 1.4 Hz, 1H), 5.57 (t, J = 1.7 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.4, 165.2, 163.6, 148.6, 143.2, 136.7, 134.0, 133.8, 133.6, 132.8, 131.2, 130.2, 129.7, 129.6, 129.1, 129.0, 128.4, 126.2, 117.6, 113.7, 113.1, 106.8, 74.0, 61.1. HRMS (ESI+) calculated for C₂₈H₂₀N₂O₄Na, [M+Na]⁺, 471.1315, found 471.1317.

1.1.13.o. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-cyanobenzoate (13o)

The synthesis of 13o follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material and 4-cyanobenzoyl chloride as reagent. The title compound was isolated in 95% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 8.6 Hz, 1H), 8.11 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 15.3 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.50 – 7.30 (m, 10H), 6.77 (d, J = 15 Hz, 1H), 5.97 (bs, 1H), 5.70 (dd, J = 8.5, 1.4 Hz, 1H), 5.56 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.3, 165.2, 163.9, 148.6, 143.0, 133.8, 132.8, 132.6, 132.3, 131.2, 130.5, 129.6, 129.1, 129.0, 128.3, 126.1, 117.7, 117.1, 113.7, 106.9, 74.0, 61.2. HRMS (ESI+) calculated for C₂₈H₂₀N₂O₄Na, [M+Na]⁺, 471.1315, found 471.1315.

1.1.13.p. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 2-(trifluoromethyl)benzoate (13p)

The synthesis of 13q follows the method of synthesis for 13d using (2S*,3S*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 97% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.46 (bs, 1H), 7.80 (d, J = 15 Hz, 1H), 7.81 (dd, J = 3.5, 5.4 Hz, 1H), 7.74 (dd, J = 3.5, 5.4 Hz, 1H), 7.62 (m, 2H), 7.50–7.30 (m, 10H), 6.80 (bs, 1H), 6.02 (s, 1H), 5.63 (d, J = 8.5 Hz, 1H), 5.56 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.3, 165.8, 165.3, 148.2, 143.0, 134.0, 132.7, 131.9, 131.8, 131.0, 130.8, 129.8, 129.6, 129.0, 128.5, 128.3, 126.8 (q, J = 5.4 Hz), 126.3, 124.6, 121.9, 114.0, 106.7, 74.3, 60.8. HRMS (ESI+) calculated for C₂₈H₂₁F₃NO₄, [M+H]⁺, 492.1417, found 492.1331.

1.1.13.q. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-methoxybenzoate (13q)

The synthesis of 13r follows the method of synthesis for 13d using (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b) as starting material. The title compound was isolated in 88% yield, as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 8.54 (bs, 1H), 7.97 (m, 2H), 7.83 (d, J = 15.3 Hz, 1H), 7.50–7.30 (m, 10H), 6.90 (m, 2H), 6.79 (bs, 1H), 5.98 (bs, 1H), 5.70 (dd, J = 1.2, 8.5 Hz, 1H), 5.54 (m, 1H), 3.84 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 186.4, 165.3, 165.3, 164.0, 148.3, 142.9, 133.9, 133.2, 132.2, 131.1, 129.6, 129.0, 128.9, 128.4, 126.2, 121.1, 114.0, 113.8, 107.1, 73.4, 61.6, 55.5. HRMS (ESI⁺) calculated for C₂₈H₂₃NO₅Na, [M+Na]⁺, 476.1468, found 476.1464.

1.1.14. (2R,3R)-1-cinnamoyl-2-methyl-4-oxo-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate ((±)-14)

(2S*,3S*)-1-cinnamoyl-3-hydroxy-2-methyl-2,3-dihydropyridin-4(1H)-one (((±)-12c) 46 mg, 0.179 mmol) was dissolved in 2 ml of methylene chloride. Triethyl amine (0.087 mL, 0.626 mmol) was added to the reaction mixture, followed by 4-fluoro-2-(trifluoromethyl)benzoyl chloride (0.054 mL, 0.358 mmol). The reaction mixture was stirred overnight at room temperature, the solvent was removed under reduced pressure and the crude was purified by silica gel flash column chromatography to afford 80 mg (quantitative yield) of the title compound, as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (s, 1H), 7.92 – 7.81 (m, 2H), 7.58 (m, 2H), 7.43 (m, 4H), 7.30 (td, J = 8.4, 2.6 Hz, 1H), 6.92 (d, J = 15.3 Hz, 1H), 5.54 (dd, J = 8.4, 1.5 Hz, 1H), 5.21 (t, J = 1.8 Hz, 1H), 5.09 (m, 1H), 1.43 (d, J = 7.0 Hz, 3H). ¹³C NMR (101MHz, CDCl₃) δ 186.4, 165.5, 164.5, 164.0 (d, J = 256.0 Hz), 148.05, 141.8, 134.3, 134.2 (d, J = 8.9 Hz), 131.23, 129.30, 128.55, 122.4 (dd, J = 274.0, 2.6 Hz), 119.0 (d, J = 21.4 Hz), 115.2 (ddd, J = 25.6, 11.4, 5.7 Hz), 114.2, 105.35, 74.06, 52.91, 27.20, 14.02.

1.1.15. (2R,3R)-1-benzoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate ((±)-15)

The synthesis of ((±)-15) follows the method of synthesis for ((±)-14) using (2R*,3R*)-1-benzoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-12a) as starting material. The title compound was isolated in 81% yield, as a white foam. ¹H NMR (400 MHz, CDCl₃) δ 8.22 – 7.72 (m, 2H), 7.73 – 7.20 (m, 12H), 6.13 (s, 1H), 5.62 (dd, J = 2.2, 1.3 Hz, 1H), 5.50 (dd, J = 8.4, 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 185.5, 170.0, 164.4, 164.1 (d, J = 256.2 Hz), 145.0, 134.3, 134.2, 132.9, 132.3, 131.9, 129.3, 128.9, 128.8, 128.3, 126.4, 125.8 (dd, J = 4.1, 2.2 Hz), 122.4 (dd, J = 273.7, 2.3 Hz), 119.0 (d, J = 21 Hz), 115.0 (dq, J = 5.5, 25 Hz), 106.1, 74.2, 60.5. HRMS (ESI+) calculated for C₂₆H₁₇F₄NO₄Na [M+Na]⁺, 506.0986, found 506.0993.

Bioassay

Cell line and reagents

The human prostate cancer cell line LNCaP was obtained from the American Type Culture Collection (ATCC). LNCaP cells were cultured at 37°C in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% antimycotic-antibiotic solution. RPMI 1640, phenol-red free RPMI 1640, FBS, charcoal-stripped FBS and antimitotic-antibiotic solution were purchased from Mediatech, Inc. MDV3100 was purchased from Selleck Chemicals.

Quantitative real-time reverse transcription-polymerase chain reaction

PSA and TMPRSS2 mRNA levels were measured as described.³⁷ Briefly, total RNA was isolated using the RNeasy RNA Isolation Kit (Qiagen), and reverse transcribed (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, USA) using random primers included in the kit, according to the manufacturer’s instructions. Real-time RT-PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems) and data were analyzed by the Sequence Detection application. The threshold cycles (Ct) for the control (rRNA) and genes of interest were determined and relative RNA levels were calculated by the comparative Ct method. Real-time-PCR experiments were performed in triplicate with the following primers:

18S rRNA

forward 5′-AGTCCCTGCCCTTTGTACACA-3′
reverse 5′-CGATCCGAGGGCCTCACTA-3′

PSA

forward 5′-GCAGCATTGAACCAGAGGAGTT-3′
reverse 5′-CACGTCATTGGAAATAACATGGA-3′

TMPRSS2

forward 5′-AGCCTCTGACTTTCAACGACCTA-3′
reverse 5′-TGTTCTGGCTGCAGCATCAT-3′

1.2.3 Growth curve

LNCaP cells were treated with AT-65 or MDV3100 for 48h at the indicated concentrations in RPMI medium containing 10% fetal bovine serum (FBS). Cells treated with DMSO (1%) were used as vehicle control. The viable cell number was determined by flow cytometric analysis with 7-aminoactinomycin D (7-AAD) viability dye (BioLegend) using a MACSQuant flow cytometer (Miltenyi Biotec) and FlowJo software (TreeStar).

1.2.4 Fluorescence Polarization (FP)

Fluorescence polarization technique was used to analyze the binding of AT-65 and MDV3100 to the androgen receptor using the PolarScreen Androgen Receptor Competitor Assay Kit Green (Invitrogen) according to the manufacturer’s instructions. Briefly, the assay entails titration of test compound against a pre-formed complex of Fluormone AL green and the androgen receptor LBD. The assay mixture was allowed to equilibrate at room temperature in 96-well black plates for 4 hours after which the fluorescence polarization values were measured in a Victor³ (Perkin Elmer), using an excitation of 485 nm and an emission of 535 nm. Data analysis for the ligand binding assays was performed using Prism software from GraphPad Software, Inc.

Molecular modeling

Homology modeling of AR

The template for modeling was the structure of GR complexed with antagonist (PDB-id: 1NHZ).³³ The AR amino acid sequence (NCBI reference sequence NP_000035.2) was truncated from the N-terminus to match the N-terminus of the LBD sequence in the AR crystal structure with PDB code 2AMB. The alignment of this sequence with the sequence of 2AMB structure was derived from the alignment of 2AMB sequence with the GR sequence from 1NHZ structure, that was in turn obtained by optimally superimposing in 3D 2AMB (AR) and 3K23 (GR) structures using superimpose-align-minimize routine in ICM. These 2 structures are in agonistic form and are very similar in spite of low sequence identity – therefore they reliably superimpose in 3D and yield robust sequence alignment. Based on this alignment, the homology model of AR was constructed using the BuildModel macro of ICM.³⁸ Energy refinement of the homology models was performed using the ICM RefineModel macro.

Ligand docking

Compounds were constructed using the ChemDraw option of ICM. Default ECEPP/3 partial charges were assigned and the compounds were docked using a semi-flexible docking protocol where the receptor was kept rigid but the ligands flexible.

Evaluation of binding to the BF3 allosteric binding site

In order to estimate docking scores consistent with binding to the BF3 site in micro molar range, we identified compounds (using www.bindingdb.org) similar to the BF3 binder (compound 32 in ref 34) that have X-ray structures in complex with proteins and have Ki in the range of 1–1000 μM. There are 14 compounds within this Ki range with similarity to compound 32 ≥ 0.3. Their corresponding PDB codes are: 1GI9, 1GI5, 1O2K, 1GI8, 1O2O, 1GI5, 1O2K, 1GI1, 1GHX, 1O3P, 1O5C, 1O2O, 1O2P, 1GHX. Then we docked the compounds to their cognate protein binding sites and calculated their scores. The scores were in the range from −38.3 to −29.3 (the average is −35.6±2.6). The BF3 docking scores of our compounds were in the range from −22.2 to −9.1 (the average is −14.7±4.5). Comparison of the score ranges further established that our compounds do not bind to the secondary i.e. allosteric region. But more likely to the specific AR site.

Supplementary Material

1_si_001

NIHMS536714-supplement-1_si_001.pdf^{(196.6KB, pdf)}

Scheme 1 — i) PhCO₂Cl, THF; ii) R¹MgX; iii) OsO₄, NMO, acetone, H₂O; iv) 2-trifluoromethyl-4-fluorobenzoyl chloride, TEA, DMAP, THF; v) MeONa, MeOH; vi) N-Methyl-N-phenylcarbamoyl chloride, TEA, DMAP, THF; vii) R₂Cl, DMAP, THF; viii) TBSCl, imidazole, CH₂Cl₂; ix) HF/pyridine, CH₃CN, pyridine; x) R₃COCl, Et₃N, CH₂Cl₂.

Acknowledgments

A.P., A.F., M.P. and S.V.M. would like to thank the National Cancer Institute (NCI) Developmental Therapeutics Program. This project has been funded in whole or in part with federal funds from the NCI, National Institutes of Health, to the Intramural Research Program (Y.S.K., S.L., M.J.L., and J.B.T.), and to SAIC-Frederick, under Contract No. HSN261200800001E. Authors would also like to thank Dr. Len Neckers for help in experiments for growth curve and binding studies. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

ABBREVIATION USED

AR: Androgen Receptor
T: Testosterone
DHT: Dihydrotestosterone
HSPs: Heat-Shock Proteins
PSA: Prostate-Specific Antigen
LNCaP: Lymph Node Prostate Cancer
CRPC: castrate resistant prostate cancer
TMPRSS2: Trans membrane protease serine 2
REMD: Replica-exchange molecular dynamics, E-cad, E-cadherin

Footnotes

Supporting information

The NMR spectra of all compounds reported in this manuscript are available as supplementary material.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1_si_001

NIHMS536714-supplement-1_si_001.pdf^{(196.6KB, pdf)}

[R1] 1.Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988;240:889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Gao W, Bohl CE, Dalton JT. Chemistry and structural biology of androgen receptor. Chemical Reviews. 2005;105:3352–3370. doi: 10.1021/cr020456u. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Feldman BR, Feldman D. The development of androgen-independent prostate cancer. Nature Reviews Cancer. 2001;1:34–45. doi: 10.1038/35094009. [DOI] [PubMed] [Google Scholar]

[R4] 4.Denis LJ, Griffiths K. Endocrine treatment in prostate cancer. Seminars in Surgical Oncology. 2000;18:52–74. doi: 10.1002/(sici)1098-2388(200001/02)18:1<52::aid-ssu8>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]

[R5] 5.Leewansangtong S, Crawford ED. Maximal androgen withdrawal for prostate cancer therapy: current status and future potential. Endocrine-Related Cancer. 1998;5:325–339. [Google Scholar]

[R6] 6.Sogani PC, Whitmore WF., Jr Experience with flutamide in previously untreated patients with advanced prostatic cancer. J Urol. 1979;122:640–643. doi: 10.1016/s0022-5347(17)56539-8. [DOI] [PubMed] [Google Scholar]

[R7] 7.Delaere KP, Van TEL. Flutamide monotherapy as primary treatment in advanced prostatic carcinoma. Semin Oncol. 1991;18:13–18. [PubMed] [Google Scholar]

[R8] 8.Kolvenbag GJ, Blackledge GR, Gotting-Smith K. Bicalutamide (Casodex) in the treatment of prostate cancer: history of clinical development. Prostate. 1998;34:61–72. doi: 10.1002/(sici)1097-0045(19980101)34:1<61::aid-pros8>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]

[R9] 9.Iversen P, Roder MA. The Early Prostate Cancer program: bicalutamide in nonmetastatic prostate cancer. Expert Rev Anticancer Ther. 2008;8:361–369. doi: 10.1586/14737140.8.3.361. [DOI] [PubMed] [Google Scholar]

[R10] 10.Iversen P, McLeod DG, See WA, Morris T, Armstrong J, Wirth MP. Antiandrogen monotherapy in patients with localized or locally advanced prostate cancer: final results from the bicalutamide Early Prostate Cancer programme at a median follow-up of 9. 7 years. BJU Int. 2010;105:1074–1081. doi: 10.1111/j.1464-410X.2010.09319.x. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Synthesis and structure–activity relationship studies of novel dihydropyridones as androgen receptor modulators

Antonella Pepe

Michael Pamment

Yeong Sang Kim

Sunmin Lee

Min-Jung Lee

Kristin Beebe

Anton Filikov

Len Neckers

Jane Trepel

Sanjay V Malhotra

Abstract

INTRODUCTION

Figure 1.

Figure 2.

RESULTS AND DISCUSSION

Chemistry

Biology

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Molecular modeling

Figure 10. Androgen receptor model of antagonistic form (GR template), violet ribbon.

Figure 11. Binding site of androgen receptor model of antagonistic form based on the X-ray structure of the glucocorticoid receptor.

CONCLUSIONS

EXPERIMENTAL SECTION

Chemistry

General

1.1.1.a. (2R*,3R*)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1a)

1.1.1.b. (2S*, 3R*)-phenyl 3-hydroxy-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1b)

1.1.1.c. (2R*,3R*)-phenyl 3-hydroxy-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1c)

1.1.1.d. (2R*,3R*)-phenyl 3-hydroxy-2-isopropyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1d)

1.1.1.e. (2R*,3R*)-phenyl 3-hydroxy-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1e)

1.1.1.f. (2R*,3R*)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1f)

1.1.1.g. (2R*,3R*)-phenyl 2-([1,1′-biphenyl]-3-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1g)

1.1.1.h. (2R*,3R*)-phenyl 2-(3,5-difluorophenyl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1h)

1.1.2.a. (2R*,3R*)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-2a)

1.1.2.b. (2S*,3R*)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2b)

1.1.2.c. (2R*,3R*)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2c)

1.1.2.d. (2R*,3R*)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-isobutyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2d)

1.1.2.e. (2R*,3R*)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2e)

1.1.2.f. (2R*,3R*)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2f)

1.1.2.g. (2R*,3R*)-phenyl-2-([1,1′-biphenyl]-3-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2g)

1.1.2.h. (2R*,3R*)-phenyl-2-(3,5-difluorophenyl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2h)

1.1.3.a. Phenyl 4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate (3a)

1.1.3.b. Phenyl 2-([1,1′-biphenyl]-4-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (3b)

1.1.4.a

1.1.4.b. 2-([1,1′-biphenyl]-3-yl)-2,3-dihydropyridin-4(1H)-one (4b)

1.1.5. (2R*,3R*)-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (5)

1.1.6. (2R*,3R*)-phenyl 3-((methyl(phenyl)carbamoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate ((±)-6)

1.1.7. (2S*,3R*)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-7)

1.1.8.a. (2S*,3R*)-2-(4-methylthiophen-2-yl)-4-oxo-1-(6-(trifluoromethyl)nicotinoyl)-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-8a)

1.1.8.b. (2S*,3R*)-1-(cyclopropanecarbonyl)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate (8b)

1.1.9.a. (2R*,3R*)-phenyl 3-((tert-butyldimethylsilyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-9a)

1.1.9.b. (2R*,3R*)-phenyl-3-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9b)

1.1.9.c. (2R*,3R*)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9c)

1.1.10.a. (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-10a)

1.1.10.b. (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-2-methyl-2,3-dihydropyridin-4(1H)-one (10b)

1.1.10.c. (2R*,3R*)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (10c)

1.1.11.a. (2R*,3R*)-1-benzoyl-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-11a)

1.1.11.b. (2R*,3R*)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-2,3-dihydropyridin-4(1H)-one (11b)

1.1.11.c. (2R*,3R*)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(2-phenylacetyl)-2,3-dihydropyridin-4(1H)-one (11c)

1.1.11.d. (2R*,3R*)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(6-(trifluoromethyl)nicotinoyl)-2,3-dihydropyridin-4(1H)-one (11d)

1.1.11.e. (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (11e)

1.1.11.f. (2R*,3R*)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-methyl-2,3-dihydropyridin-4(1H)-one (11f)

1.1.12.a. (2R*,3R*)-1-benzoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-12a)

1.1.12.b. (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b)

1.1.12.c. (2R*,3R*)-1-cinnamoyl-3-hydroxy-2-methyl-2,3-dihydropyridin-4(1H)-one (12c)

1.1.13.a. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclopropanecarboxylate ((±)-13a)

1.1.13.b. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclobutanecarboxylate (13b)

1.1.13.c. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl benzoate (13c)

1.1.13.d. (E)- (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(3-(trifluoromethyl)phenyl)acrylate (13d)

1.1.13.e. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 2-phenylacetate (13e)

1.1.13.f. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-methylbenzoate (13f)

1.1.13.g. (2R*,3R*)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-chlorobenzoate (13g)

1.1.1.a. (2R,3R)-phenyl 3-hydroxy-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-1a)

1.1.1.b. (2S**, 3R)-phenyl 3-hydroxy-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1b)

1.1.1.c. (2R,3R)-phenyl 3-hydroxy-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1c)

1.1.1.d. (2R,3R)-phenyl 3-hydroxy-2-isopropyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1d)

1.1.1.e. (2R,3R)-phenyl 3-hydroxy-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1e)

1.1.1.f. (2R,3R)-phenyl 2-(benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1f)

1.1.1.g. (2R,3R)-phenyl 2-([1,1′-biphenyl]-3-yl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1g)

1.1.1.h. (2R,3R)-phenyl 2-(3,5-difluorophenyl)-3-hydroxy-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (1h)

1.1.2.a. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-2a)

1.1.2.b. (2S,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2b)

1.1.2.c. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2c)

1.1.2.d. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-isobutyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2d)

1.1.2.e. (2R,3R)-phenyl 3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-2-(2-methylprop-1-en-1-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2e)

1.1.2.f. (2R,3R)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2f)

1.1.2.g. (2R,3R)-phenyl-2-([1,1′-biphenyl]-3-yl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2g)

1.1.2.h. (2R,3R)-phenyl-2-(3,5-difluorophenyl)-3-((4-fluoro-2-(trifluoromethyl)benzoyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (2h)

1.1.5. (2R,3R)-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (5)

1.1.6. (2R,3R)-phenyl 3-((methyl(phenyl)carbamoyl)oxy)-2-(4-methylthiophen-2-yl)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate ((±)-6)

1.1.7. (2S,3R)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-7)

1.1.8.a. (2S,3R)-2-(4-methylthiophen-2-yl)-4-oxo-1-(6-(trifluoromethyl)nicotinoyl)-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate ((±)-8a)

1.1.8.b. (2S,3R)-1-(cyclopropanecarbonyl)-2-(4-methylthiophen-2-yl)-4-oxo-1,2,3,4-tetrahydropyridin-3-yl methyl(phenyl)carbamate (8b)

1.1.9.a. (2R,3R)-phenyl 3-((tert-butyldimethylsilyl)oxy)-4-oxo-2-phenyl-3,4-dihydropyridine-1(2H)-carboxylate ((±)-9a)

1.1.9.b. (2R,3R)-phenyl-3-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9b)

1.1.9.c. (2R,3R)-phenyl-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (9c)

1.1.10.a. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-10a)

1.1.10.b. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-methyl-2,3-dihydropyridin-4(1H)-one (10b)

1.1.10.c. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydropyridin-4(1H)-one (10c)

1.1.11.a. (2R,3R)-1-benzoyl-3-((tert-butyldimethylsilyl)oxy)-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-11a)

1.1.11.b. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-2,3-dihydropyridin-4(1H)-one (11b)

1.1.11.c. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(2-phenylacetyl)-2,3-dihydropyridin-4(1H)-one (11c)

1.1.11.d. (2R,3R)-2-(benzo[d][1,3]dioxol-5-yl)-3-((tert-butyldimethylsilyl)oxy)-1-(6-(trifluoromethyl)nicotinoyl)-2,3-dihydropyridin-4(1H)-one (11d)

1.1.11.e. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (11e)

1.1.11.f. (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-cinnamoyl-2-methyl-2,3-dihydropyridin-4(1H)-one (11f)

1.1.12.a. (2R,3R)-1-benzoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one ((±)-12a)

1.1.12.b. (2R,3R)-1-cinnamoyl-3-hydroxy-2-phenyl-2,3-dihydropyridin-4(1H)-one (12b)

1.1.12.c. (2R,3R)-1-cinnamoyl-3-hydroxy-2-methyl-2,3-dihydropyridin-4(1H)-one (12c)

1.1.13.a. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclopropanecarboxylate ((±)-13a)

1.1.13.b. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl cyclobutanecarboxylate (13b)

1.1.13.c. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl benzoate (13c)

1.1.13.d. (E)- (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(3-(trifluoromethyl)phenyl)acrylate (13d)

1.1.13.e. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 2-phenylacetate (13e)

1.1.13.f. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-methylbenzoate (13f)

1.1.13.g. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-chlorobenzoate (13g)

1.1.13.h. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate (13h)

1.1.13.i. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3,4-dichlorobenzoate (13i)

1.1.13.j. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluorobenzoate (13j)

1.1.13.k. (E)-(2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(3-(trifluoromethyl)phenyl)acrylate (13k)

1.1.13.l. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-nitro-3-(trifluoromethyl)benzoate (13l)

1.1.13.m. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-(trifluoromethyl)benzoate (13m)

1.1.13.n. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 3-cyanobenzoate (13n)

1.1.13.o. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-cyanobenzoate (13o)

1.1.13.p. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 2-(trifluoromethyl)benzoate (13p)

1.1.13.q. (2R,3R)-1-cinnamoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl-4-methoxybenzoate (13q)

1.1.14. (2R,3R)-1-cinnamoyl-2-methyl-4-oxo-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate ((±)-14)

1.1.15. (2R,3R)-1-benzoyl-4-oxo-2-phenyl-1,2,3,4-tetrahydropyridin-3-yl 4-fluoro-2-(trifluoromethyl)benzoate ((±)-15)