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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2017 April 15.
Published in final edited form as:
PMCID: PMC5292099
NIHMSID: NIHMS767981

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

Abstract

Inhibition of group IIA secreted phospholipase A2 (GIIA sPLA2) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA2s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC50 = 143 nM and 68 nM against human and mouse GIIA sPLA2, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity.

Keywords: Docking, Inhibitors, Molecular dynamics, 2-Oxoamides, Secreted phospholipase A2

1. Introduction

Phospholipases A2 (PLA2) are a superfamily of enzymes whose main role is the hydrolysis of the ester bond of the membrane glycerophospholipids at the sn-2 position.1 The products of this enzymatic activity are mainly unsaturated fatty acids which can act as bioactive mediators, correlating with numerous pathological conditions. PLA2 enzymes are categorized into six types according to their dependency on calcium, their amino acid sequence domain, their molecular weight and their evolutionary relationship.1

Approximately, one third of PLA2 enzymes belong to the secreted phospholipase (sPLA2) type, whose members are characterized by a low-molecular weight.24 They can act either by catalyzing reactions as enzymes or by binding to receptors and hence, they are involved in the activation of several biological pathways. To date, 11 gene products of sPLA2s (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA and XIIB) have been identified in mammals. Their domains are highly stabilized by six common disulfide bonds and additionally one or two that are typical of each enzyme. Their active site contains the His/Asp dyad that is utilized in the catalytic mechanism. Their activation may require concentration of Ca2+ in the mM range and thus these proteins are localized in the extracellular space, targeting both cellular glycerophospholipids and soluble lipids.

The up-regulation or down-regulation of the expression of these enzymes is related to pathological conditions, such as atherosclerosis, cardiovascular disease and cancer, as it has been summarized in a number of recent reviews.5,6 As a consequence, a variety of synthetic inhibitors of sPLA2 have been developed by pharmaceutical companies and research institutes.1,79 In particular, GIIA sPLA2 has been related to inflammation since it was identified in 1989.10 GIIA sPLA2 is highly expressed in synovial cells, while its concentration increases in the plasma of patients who suffer from coronary artery disease, making it a useful tool for prognostic purposes.

Many attempts for selective inhibition of GIIA sPLA2 have been made with many positive outcomes. Varespladib11 (1a, Fig. 1), which is a potent but not selective GIIA sPLA2 inhibitor, was advanced into clinical trials as an intravenously-administered therapy for sepsis-induced systemic inflammatory response syndrome. Although it was found to have an acceptable safety profile in patients with severe sepsis, its development was terminated because Phase II studies resulted in little efficacy. Also, Varespladib methyl (1b, Fig. 1) which functions as a prodrug and is rapidly converted in vivo to Varespladib, was synthesized as an attempt to improve Varespladib efficacy. Both inhibitors were claimed by Anthera Pharmaceuticals as new agents for the treatment of cardiovascular diseases and underwent clinical trials from 2006 to 2012. However, on 2012 phase III clinical studies were terminated due to lack of efficacy.

Figure 1
Common inhibitors of secreted PLA2.

The expression of the full set of human and mouse groups I, II, V, X, and XII sPLA2s in Escherichia coli and insect cells provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies.12 Among the class of inhibitors of sPLA2s based on substituted indoles, 6,7-benzoindoles and indolizines,13,14 it was found that compound 2 (Fig. 1) was a selectively potent inhibitor against hGX over all other human and mouse sPLA2 enzymes, while compound 3 (Fig. 1) inhibited nearly all human and mouse sPLA2s in the low nanomolar range. Another important class of sPLA2 inhibitors is amides based on non-natural amino acids. Compound FPL67047XX15 (4, Fig. 1) was found to be a potent inhibitor of human platelet sPLA2, and its precise binding interactions with the human nonpancreatic sPLA2 were determined by high-resolution X-ray crystallography. The structurally related compound 5 (Fig. 1) was co-crystallized with GIIA hnpsPLA2 and the crystal structure revealed a chelation to a Ca2+ ion through its carboxylate and amide oxygen atoms, H-bonding through an amide NH group to His48, multiple hydrophobic contacts and a T-shaped aromatic group—His6 interaction.16 In addition, compound GK11517 (6, Fig. 1) was found to inhibit GV sPLA2 (XI(50) = 0.003 ± 0.0004) without affecting the activities of intracellular GIVA cPLA2 and GVIA iPLA2. In a continuation of our studies on a novel class of cPLA2 inhibitors,18,19 the 2-oxoamide based on the natural α-amino acid (S)-leucine GK126,20 (7, Fig. 1), demonstrated a promising IC50 value of 0.30 μM against GIIA sPLA2.20

Computer-aided drug design has recently offered powerful tools for the rational design of PLA2 inhibitors, raising the potential for the development of new compounds with improved inhibitor properties. A recent review article demonstrates various applications of rational design on PLA2 inhibitors.21 For example, docking calculations have led to the design of new indole inhibitors of sPLA2.22 The aim of this work was to develop improved inhibitors of GIIA sPLA2 by keeping the 2-oxoamide functionality of the lead 2-oxoamide inhibitor 7 and altering its other structural features. Using molecular docking simulations, we designed new 2-oxoamides based on α-amino acids. Herein, the synthesis of new 2-oxoamides derivatives and their in vitro activities against various sPLA2s are presented. Moreover, we report our results from docking, molecular dynamics simulations and free energy calculations of the complexes, in an attempt to understand their structure–activity relationships.

2. Results and discussion

2.1. Design and docking of new 2-oxoamides

New 2-oxoamides were designed based on the structure of the lead inhibitor 7 (Fig. 2). Proteinogenic α-amino acids of both S and R configuration were used to replace the (S)-leucine part of 7 and the resulted derivatives were docked in the active site of GIIA sPLA2 using GOLD v5.223 program by CCDC. The evaluation of the generated poses was mainly based on the number of interactions they formed with the residues of the active site upon binding. The ΔEClash, ΔEInternal, Chemscore.Internal.Correction.Weighted and Chemscore.Rot.Weighted penalty terms of the ChemScore scoring function were also considered. The highly scored penalty terms usually indicate poor geometry of the bound inhibitors.

Figure 2
The workflow of the development of new potential inhibitors based on the inhibitor 7 guided by molecular docking calculations.

According to the docking results, the derivatives containing α-amino acids with R configuration did not generate favorable conformations. This, in combination with the low inhibitory activity of similar derivatives previously reported20 led us to discard these structures.

The derivatives containing non-polar α-amino acids with S configuration frequently generated favorable binding modes with comparatively good scoring values. Therefore, in order to better understand this behavior, simulated annealing was employed to generate 30 possible conformations for each of these molecules, which subsequently were docked in GOLD. By docking multiple conformations of each ligand, improved statistical analysis of the docking results was achieved.

Indeed, the recurrence statistics of the desirable binding motif of (S)-valine, (S)-alanine and (S)-proline derivatives was high, according to their docking solutions. More specifically, the carboxyl group of the derivatives chelates the calcium ion and also forms a hydrogen bond with either Gly31 or Lys62 residues. In addition, the 2-carbonyl group or the oxygen atom of the amide group point to Ca2+ ion. A hydrogen bond is formed between Gly29 and the 2-carbonyl group in most cases (Supporting info). Similar results were generated when derivatives of two non-natural amino acids ((S)-tert-leucine and 2-aminoisobutyric acid) were docked. Moreover, some 2-hydroxy amides were also docked and no significant differences between the generated poses and the corresponding 2-oxoamides ones were observed. The two (S)-Glu derivatives were docked for comparison.

Synthesis and biological evaluation of the docked ligands were accomplished. According to the experimental data, which are discussed later, the (S)-valine derivative 16a (GK241) demonstrated inhibitory activity against GIIA sPLA2. Therefore, an effort to modify its structure, in order to succeed a higher potency, was made. It is well established that when a ligand has multiple conformations while unbound, then narrowing down this number to the only favorable conformations in the binding pocket, is entropically expensive. Thus, the replacement of 16a long aliphatic chain by a shorter, carrying an aromatic system (phenyl, biphenyl or naphthyl), was a reasonable concept. New analogs were designed and some of the most promising structures, according to the docking simulations, were synthesized and tested.

2.2. Synthesis of inhibitors

The synthesis of 2-oxoamides and 2-hydroxyamides is presented in Schemes 13. The protected (S)-α-amino acids 9ad and 13ac were coupled with 2-hydroxyhexadecanoic acid using 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (WSCI·HCl) as a condensing agent in the presence of 1-hydroxybenzotriazole (HOBt). The α-hydroxyamide methyl esters 10ad were saponified to produce the acids 11ad. Compounds 11bd were then oxidized to the corresponding 2-oxoamide acids 12bd using Dess–Martin periodinane. In addition, α-hydroxyamide tert-butyl esters 14ac were first oxidized using Dess–Martin reagent to afford 15ac and subsequently, the protecting group was removed by trifluoroacetic acid to afford the desired 2-oxoamides 16a,c,d. The synthesis of compound 21 is described in Scheme 3.

Scheme 1
Reagents and conditions: (a) WSCI·HCl, HOBt, Et3N, CH2Cl2; (b) 1 N aq NaOH, MeOH; (c) Dess–Martin periodinane, CH2Cl2.
Scheme 3
Reagents and conditions: (a) CH3(CH2)13CHOHCOOH (8), WSCI·HCl, HOBt, Et3N, CH2Cl2; (b) Dess–Martin periodinane, CH2Cl2; (c) CF3COOH, CH2Cl2.

The synthesis of 2-oxoamide (S)-valine analogs, where the long chain has been replaced by a medium one carrying an aromatic system, is depicted in Scheme 4. Horner–Wadsworth–Emmons olefination of 1,1′-biphenyl-4-carbaldehyde and 2-naphthaldehyde 22a,b led to esters 23a,b and subsequently catalytic hydrogenation to saturated products 24a,b. The ethyl esters were then converted to alcohols 25a,b using the di-isobutyl aluminum hydride (DIBALH) reducing agent, followed by the NaOCl/AcNH-TEMPO oxidation method, to form the corresponding aldehydes 26a,b. The synthesis of 31c started from 5-phenyl-1-pentanol 25c, which was converted to the corresponding aldehyde 26c, as it described above. Cyanohydrin reaction followed by acid and then alkaline conditions afforded the desirable α-hydroxy acids 28ac. These acids were coupled with (S)-valine, followed by oxidation and removal of the protecting group to afford the final products 31ac. The synthesis of 37 was accomplished by similar methods (Scheme 5).

Scheme 4
Reagents and conditions: (a) C2H5OOCH═CHCH2P(═O)(OC2H5)2, LiOH·H2O, dry THF; (b) H2/Pd, THF; (c) DIBAL-H, Et2O; (d) NaBr/H2O, toluene/EtOAc, AcNH-TEMPO, 0.5 M NaOCl/NaHCO3/H2O; (e) (i) NaHSO3, CHCl3 (ii) H2O, 6 M KCN; (f) (i) HCl ...
Scheme 5
Reagents and conditions: (a) EtOAc/THF/80% CH3COOH, 6 M KCN; (b) (i) HCl conc. (ii) EtOH/H2O, KOH; (c) (S)-Val-OtBu, WSCI·HCl, HOBt, Et3N, CH2Cl2; (d) Dess–Martin periodinane, CH2Cl2; (e) CF3COOH, CH2Cl2.

2.3. In vitro inhibition of GIIA sPLA2

The new 2-oxoamide derivatives were tested for inhibitory activity against a panel of human and mouse sPLA2 enzymes, using a previously described continuous fluorometric assay.24 None of the compounds 12bd, 16c,d and 21 presented inhibition of GIIA sPLA2 higher than 25% at 1 μM concentration and thus we did not further study them. However, the (S)-valine derivative 16a, exhibited high and selective potency for inhibition of GIIA sPLA2 and the IC50 values are summarized in Table 1. Compound 16a demonstrates a two times higher potency for GIIA sPLA2 than the previously reported inhibitor 7 and it is ten times more selective for GIIA than GV sPLA2. The IC50 values for Varespladib (1a, Fig. 1) using the same assay conditions, are included in Table 1 for comparison.

Table 1
IC50 values of 1a and 16a on hGIIA, hGV, and mGIIA

In addition, 16a does not exhibit any appreciable inhibition against various other human and mouse sPLA2 (Table 2).

Table 2
Inhibitory activity of 16a on other sPLA2 enzymes

Moreover, the lack of inhibitory activity of 2-hydroxyamide derivatives (data not shown) indicates that indeed the 2-oxoamide functionality has a crucial contribution to the binding affinity of these compounds.

The analogs of 16a were also tested against several sPLA2s and the results are summarized in Table 3. The in vitro activities of 31ac and 37 failed to meet our docking predictions. Molecular dynamics simulations were applied on 16a and on one of its analogs in an attempt to understand the in vitro data.

Table 3
Inhibitory activity of analogs of 16a on sPLA2 enzymes

2.4. Molecular dynamics simulations

The lowest energy docking poses of 16a and 31c were subjected to MD simulations in AMBER v12 molecular dynamics package.25 Poses were selected so that the key interactions between the ligands and the binding site to be present. The total simulation time was 200 ns in explicit solvent and RMSD calculations were performed with respect to the starting coordinates of each complex.

In the initial poses, the inhibitor interacts with the calcium ion via its carboxyl group and the 2-carbonyl group of the amide (Figs. 3 and and4).4). During the first 20 ns of the simulation, both molecules were reoriented within the active site in such a way that the carboxylate oxygen atoms chelated the metal in bidentate fashion (Figs. 3 and and4).4). This interaction lasted until the end of the simulation of both complexes.

Figure 3
Conformational changes of the complex 16a–GIIA sPLA2 at the beginning (up) and at the end (down) of the MD simulation.
Figure 4
Conformational changes of the complex 31c–GIIA sPLA2 at the beginning (up) and at the end (down) of the MD simulation.

During the MD simulation of 16a complex, two hydrogen bonds are formed between the amide group and the residues His47 and Gly29 (Table 4), resulting in the stable binding of 16a to the enzyme. The RMSD distribution further supports the low mobility of the 16a functional groups, which allows the formation of this H-bonds network (Fig. 5). However, the 2-oxoamide group of 31c does not form any H-bond with the residues of the catalytic centre, during the simulation time (Table 4). Due to conformational changes of 31c, the 2-oxoamide group moves away from the catalytic centre and periodically interacts only with the solvent molecules.

Figure 5
RMSD for the 16a and 31c.
Table 4
Main H-bond interactions between the ligands and the residues of the GIIA sPLA2 active site

The fluctuations of the RMSD values manifest the high mobility of the 31c in the active site (Fig. 5). The RMSD values were calculated for the part of the molecules which form the key interactions and consists of the Cα atom of (S)-valine, the two carbon atoms of the 2-oxoamide moiety and the nitrogen atom of the amide.

Using the MD trajectories, free energy calculations were performed with the Molecular Mechanics/Poisson–Boltzmann Surface Area method (MM–PBSA) (Table 5). Frames from the second half of the simulation were used for the calculations. Analysis of the energetic terms for each complex, revealed a high contribution from van der Waals forces.

Table 5
Energetic analysis for 16a–GIIA sPLA2 and 31c–GIIA sPLA2 complexes

16a creates two additional H-bonds within the cavity of the enzyme compared to 31c, which is reflected on the difference between the ΔE electrostatic term of the two complexes (Table 5). Moreover, the entropic term does not greatly differ between the two complexes. Partly, this could be due to the fact that both molecules are similarly flexible. Interestingly, in the case of 16a the overall flexibility of the compound does not increase the mobility of the 2-oxoamide group, which creates the main interactions with the residues. The difference in binding energies between 16a and 31c is 10.96 kcal mol−1. This indicates that 16a complex formation is more favored over 31c complex formation, in agreement with the in vitro results.

3. Conclusion

Structural modifications were applied on the GIIA sPLA2 inhibitor 7 and the new compounds were docked using GOLD. The results suggested that the non-polar α-amino acids derivatives with S configuration could generate favorable conformations. Their in vitro evaluation revealed that the 2-oxoamide inhibitor based on (S)-valine 16a is a potent and selective inhibitor of GIIA sPLA2. Further attempts to optimize the 16a activity by altering its structure, did not lead to any desirable outcomes.

Thus, molecular dynamics simulations were used in order to understand how the replacement of the long aliphatic chain of 16a resulted in the decrease of its potency. It was suggested that the aliphatic chain, which creates van der Waals interactions, adopts a conformation that keeps the 2-oxoamide moiety close to the key residues of the catalytic centre. Subsequently, the 2-oxoamide moiety forms a permanent H-bond network which further stabilize the 16a binding. The replacement of the long aliphatic chain by a four C atoms chain carrying a phenyl ring led to an increase of 2-oxoamide group’s mobility and the loss of the H-bonding interactions it crated with the active side. Although its carboxylic group chelates the Ca2+ ion and creates an H-bond with Gly29, these interactions seems to be not enough to keep the derivative bound to the enzyme.

Further work will be needed to test the in vivo potency of 16a. Molecular dynamics simulations are a useful tool for explaining and evaluating the experimental data and should be used towards this direction.

4. Experimental section

4.1. Computational methods

4.1.1. Preparation of ligands’ structures

The preparation of the structures was accomplished using SYBYL v8.0.26 The charges were defined at physiological pH and the S or R configuration was assigned. Several minimization steps were performed to accomplish optimization of the structures, using initially the steepest descent algorithm for 300 iteration steps followed by the conjugate gradients method for another 300 iteration steps and finally the Powell algorithm for 700 iteration steps.27 The Tripos force field28 was applied during optimization and each method was terminated when a convergence of 0.05 kcal mol−1 Å−1 was reached. For the Powell algorithm, the simplex method29 was used for its initial optimization.

The simulated annealing method30 was used in order to sample 30 relevant conformations for each structure. Molecules were heated at 600 K for 1500 fs and annealed at 250 K for 2000 fs. The Tripos force field was used and the non-bonded cutoff was set to 8.0 Å. The structures produced were optimized using 200 iteration steps with the Powell algorithm with a gradient of 0.05 kcal mol−1 Å−1. The simplex method was used as well.

4.1.2. Docking

The atomic coordinates of the human secreted phospholipase group IIA protein were downloaded from Protein Data Bank31 (PDB code: 1KQU).16 Missing loops were added with the Prime module of Schrödinger Suite.32 Hydrogen atoms were added in GOLD and atoms types were automatically assigned by the program.

Docking simulations were performed in GOLD. The cavity of the catalytic centre of GIIA sPLA2 was oriented within 5 Å around the crystallographic ligand including Leu2, Phe5, His6, Gly29, Gly31, His47, Asp48, Lys52, Lys62 and Asp91 amino acids. Eight crystal water molecules in the spatial vicinity to the catalytic center were kept and assigned as toggle and spin, while the rest were deleted. His6 was assigned to have flexible side chain (1 rotamer). H-bond donors/acceptors were treated as solvent accessible. The options ‘flip amide bonds and ring corners’ were set on for ligand flexibility. The ChemScore scoring function (1) along with chemscore. p450_csd.params parameters file were used to rank the generated binding modes.

ChemScore=-(ΔG+ΔE(clash)+ΔE(int))
(1)

where ΔG is the free energy change upon ligand binding, ΔE (clash) is the protein–ligand clash penalty and ΔE (int) is the ligand internal torsion strain penalty. It was established via additional computational experiments that this combination of scoring function and parameters were suitable for the docking. Internal ligand energy offset was enabled and the preset genetic algorithm parameter settings were used and set to 100,000 operations.

4.1.3. Molecular dynamics simulations

MD simulations in explicit solvent were performed using AMBER.25 The Antechamber tool was used to generate the partial atomic charges of each ligand with the AM1-BCC33 method. Modification of the force field parameters of each structure was made by Parmchk program. The AMBER force field ff12SB34 and the general AMBER force field (GAFF)35 were loaded in tLEaP module to create the parameters and topologies of the protein and the organic molecule, respectively. The complex was loaded in tLEaP along with the parameters file of calcium ions, which were created by a combination of crystallographic data,16 GAFF force field parameters35 and the Aqvist et al. method.36 Disulfide bonds between Cys83-Cys59, Cys124-Cys49, Cys117-Cys26, Cys50-Cys90, Cys28-Cys44, Cys43-Cys97 and Cys88-Cys77 were assigned, as well as the coordination between the metal ion of the catalytic centre and the residues His27, Gly29 and Gly31. The complex was then immersed in a truncated octahedral water box using the TIP3P water model with 12.0 Å periodic boundary conditions. Finally, the system was neutralized by adding 18 Cl counter ions using the Joung and Cheatham ion parameters37 and its coordinate and parameter topology files were saved.

Prior to the MD simulation, an initial minimization was performed using 1500 iteration steps of steepest descent and 1500 iteration steps of conjugate gradient in SANDER,38 keeping the complex almost fixed with a restraint set to 400 kcal mol−1 Å−1. In addition, four sets of steepest descent and conjugate gradient steps were applied to the system, with the strength of the restraint being gradually reduced to 8 kcal mol−1 Å−1. The system was then smoothly heated from 0 to 300 K in three steps under constant volume. During these steps of total time 100 ps, the restraint was retained to 8 kcal mol−1 Å−1, hydrogen atoms were constrained using the SHAKE algorithm and the Langevin thermostat was used for temperature control. Two steps of equilibration were applied keeping the restraint to 8 kcal mol−1 Å−1 for the first 50 ps and then removed it for the next 50 ps, under constant pressure. MD simulations were performed with the pmemd.cuda39 implementation for 200 ns and a 8 Å nonbonded cutoff was applied. SHAKE was kept during the production runs to allow the use of 2 fs time step, and the long-range electrostatic interactions were accounted for using the particle mesh Ewald (PME) method.40 Analysis on the produced trajectories (RMSD, distances, H-bond interactions) was performed with the ptraj analysis tool available in the AmberTools13.41

The relative binding energies of each system (Table 5) were estimated using the MM–PBSA.42 The ΔEelect term is the electrostatic interaction energy and the ΔEvdW term is the van der Waals interaction energy.

The Poisson–Boltzmann (PB) model43 was used for the estimation of the electrostatic contribution to solvation energies (ΔGPB) and the solvent-accessible surface area (SASA) was used for the estimation of the non electrostatic contributions (ΔGNP). The SASA is calculated by the following equation:

ΔGNP=γSASA+β
(2)

where the SASA term is the surface area of the solute, the γ term is 0.00542 kcal mol−1 Å−2 while β is −1.008000 kcal mol−1.44 The γ and β are constants related with the surface tension coefficient and the offset, respectively.

The entropic term T·ΔS was calculated using the nmode program available in AMBER. The metal ions were removed during the calculation (strip_mask) to avoid misleading results.

4.2. Chemistry

Merck Silica Gel 60 (70–230 or 230–400 mesh) was used for the chromatographic purification of products and Silica Gel 60 F254 aluminum plates for the thin-layer chromatography (TLC). UV light and/or phosphomolybdic acid in EtOH was employed for visualizing spots. A Büchi 530 apparatus was used to estimate melting points and they were uncorrected. 1H and 13C NMR spectra were recorded on Varian Mercury at 200 MHz or 300 MHz and 50 MHz respectively. Samples were diluted in CDCl3, CD3OD or DMSO. Chemical shifts are given in ppm, and coupling constants (J) in Hz. Peak multiplicities are typified as: s, singlet, d, doublet, t, triplet and m, multiplet. Electron spray ionization (ESI) mass spectra were recorded on a Finnigan Surveyor MSQ Plus spectrometer. Specific rotations of the compounds were measured at 25 °C on a Perkin-Elmer 343 polarimeter using a 10 cm cell. Dichloromethane, diethylether and toluene were dried by standard procedures and stored over molecular sieves. No further purification of other solvents and chemicals needed as they were reagent grade. HRMS spectra were recorded on a Bruker Maxis Impact QTOF Spectrometer.

Compounds 23b,45 24b,45 25a,46 26a,46 25b,47 26b48 have been described elsewhere and their analytical data are in accordance with literature.

4.2.1. Coupling method

To a stirred solution of hydrochloride amino component (1.0 mmol) in CH2Cl2 (10 mL), Et3N (0.3 mL, 2.2 mmol) and subsequently 1-(3-dimethyl-aminopropyl)-3-ethyl carbodiimide hydrochloride (WSCI·HCl) (0.21 g, 1.1 mmol) and 1-hydroxybenzotriazole (HOBt) (0.14 g, 1.0 mmol) were added at 0 °C. The acid reactant (1.0 mmol) was added and the reaction mixture was stirred for 1 h at 0 °C and then overnight at room temperature. After the completion of the reaction, the solvent was evaporated under reduced pressure and EtOAc (20 mL) was added. The organic layer was washed consecutively with brine, 1 N HCl, brine, 5% NaHCO3 and brine, dried over Na2SO4 and evaporated under reduced pressure. Purification by flash column chromatography eluting with the appropriate mixture of EtOAc/petroleum ether (bp 40–60 °C) afforded the product.

4.2.1.1. (2S)-Methyl 2-(2-hydroxyhexadecanamido)-3-(1Hindol-3-yl)propanoate (mixture of diastereomers) (10a)

Yield 52%; White solid; mp 78–85 °C; [α]D20 41.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 8.22 (s, 1H, NH), 7.61–7.50 (m, 1H, arom), 7.43–6.88 (m, 5H, arom & NH), 5.08–4.88 (m, 1H, NHCH), 4.14–3.97 (m, 1H, CHOH), 3.81–3.67 (m, 3H, COOCH3), 3.34 (d, J = 5.6 Hz, 2H, NHCHCH2), 1.87–1.64 (m, 2H, CH2CHOH), 1.62–1.15 (m, 24H, CH2), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.6, 172.3, 169.0, 168.4, 135.9, 122.7, 122.2, 119.6, 119.5, 118.5, 118.3, 111.3, 109.8, 80.6, 72.0, 52.4, 34.6, 31.9, 29.6, 29.5, 29.4, 29.3, 27.6, 24.9, 22.6, 14.1; MS (ESI) m/z (%): 473.3 (100) [M+H]+; Anal. Calcd for C28H44N2O4: C, 71.15; H, 9.38; N, 5.93. Found: C, 71.03; H, 9.45; N, 5.87.

4.2.1.2. (2S)-Methyl 1-(2-hydroxyhexadecanoyl)pyrrolidine-2-carboxylate (mixture of diastereomers) (10b)

Yield 64%; White solid; mp 42–55 °C; [α]D20 −50.0 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 4.75–4.43 (m, 1H, NCH), 4.29–4.14 (m, 1H, CHOH), 3.71 (s, 3H, CH3), 3.62–3.44 (m, 2H, NCH2), 3.06 (br, 1H, OH), 2.57–1.74 (m, 6H, NCH2CH2CH2 & CH2CHOH), 1.73–1.42 (m, 4H, CH2), 1.23 (s, 20H, CH2), 0.85 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.6, 172.4, 81.9, 69.5, 59.4, 59.1, 56.7, 52.5, 46.7, 45.6, 34.4, 32.7, 32.1, 29.9, 29.7, 29.6, 29.2, 28.9, 25.3, 24.9, 22.9, 14.3; MS (ESI) m/z (%): 384.4 (100) [M+H]+; Anal. Calcd for C22H41NO4: C, 68.89; H, 10.77; N, 3.65. Found: C, 68.63; H, 10.84; N, 3.56.

4.2.1.3. (2S)-Methyl 2-(2-hydroxyhexadecanamido)-3,3-dimethylbutanoate (mixture of diastereomers) (10c)

Yield 84%; White solid; mp 73–75 °C; [α]D20 2.30 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.29–7.07 (m, 1H, NH), 4.47–4.37 (m, 1H, NHCH), 4.21–4.04 (m, 1H, CHOH), 3.70 (s, 3H, CH3), 1.93–1.70 (m, 2H, CH2CHOH), 1.52–1.16 (m, 24H, CH2), 0.96 (s, 9H, CH3), 0.85 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.0, 172.1, 171.7, 72.2, 71.9, 59.5, 59.4, 51.7, 34.7, 31.8, 29.6, 29.3, 29.2, 26.4, 25.0, 24.9, 22.6, 14.0; MS (ESI) m/z (%): 400.2 (100) [M+H]+; Anal. Calcd for C23H45NO4: C, 69.13; H, 11.35; N, 3.51. Found: C, 69.05; H, 11.51; N, 3.32.

4.2.1.4. Methyl 2-(2-hydroxyhexadecanamido)-2-methylpropanoate (racemic mixture) (10d)

Yield 54%; White solid; mp 53–56 °C; [α]D20 2.35 (c 1.02, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.13 (s, 1H, NH), 4.09–3.93 (m, 1H, CHOH), 3.72 (s, 3H, COOCH3), 3.53 (br, 1H, OH), 1.84–1.58 (m, 2H, CH2CHOH), 1.53 (s, 6H, CH3), 1.34–1.10 (m, 24H, CH2), 0.86 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.9, 173.7, 71.8, 55.9, 52.5, 34.5, 31.8, 29.6, 29.4, 29.3, 24.8, 24.6, 22.6, 14.0; MS (ESI) m/z (%): 372.2 (100) [M+H]+; Anal. Calcd for C21H41NO4: C, 67.88; H, 11.12; N, 3.77. Found: C, 67.66; H, 11.29; N, 3.72.

4.2.1.5. (2S)-tert-Butyl 2-(2-hydroxyhexadecanamido)-3-methylbutanoate (mixture of diastereomers) (14a)

Yield 62%; White solid; mp 49–53 °C; [α]D20 9.80 (c 1.01, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.17 (d, J = 9.0 Hz, ½H, NH), 7.05 (d, J = 9.0 Hz, ½H, NH), 4.46–4.31 (m, 1H, CH), 4.18–4.03 (m, 1H, CHOH), 4.00 (d, J = 4.8 Hz, ½H, OH), 3.83 (d, J = 4.8 Hz, ½H, OH), 2.24–2.03 (m, 1H, NHCHCH), 1.90–1.52 (m, 2H, CH2CHOH), 1.44 (s, 9H, CH3), 1.34–1.13 (m, 24H, CH2), 0.97–0.75 (m, 9H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.1, 174.0, 171.3, 170.9, 82.0, 81.9, 72.2, 71.9, 57.0, 56.8, 34.8, 31.8, 31.3, 31.2, 29.6, 29.5, 29.3, 27.9, 25.0, 24.8, 22.6, 18.9, 18.8, 17.5, 17.4, 14.0; MS (ESI) m/z (%): 428.3 (95) [M+H]+; Anal. Calcd for C25H49NO4: C, 70.21; H, 11.55; N, 3.28. Found: C, 70.03; H, 11.70; N, 3.19

4.2.1.6. (2S)-Di-tert-butyl 2-(2-hydroxyhexadecanamido)pentanedioate (mixture of diastereomers) (14b)

Yield 76%; Yellowish oil; [α]D20 8.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.24–7.06 (m, 1H, NH), 4.61–4.38 (m, 1H, NHCH), 4.22–4.02 (m, 1H, CHOH), 3.61 (br, 1H, OH), 2.44–1.71 (m, 6H, CH2), 1.53–1.38 (m, 18H, CH3), 1.36–1.17 (m, 24H, CH2), 0.97–0.78 (m, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.2, 174.0, 172.0, 170.8, 82.3, 82.2, 80.7, 71.9, 51.7, 34.7, 31.8, 31.5, 29.6, 29.3, 29.2, 27.9, 27.6, 24.9, 22.6, 14.0; MS (ESI) m/z (%): 514.6 (90) [M+H]+; Anal. Calcd for C29H55NO6: C, 67.80; H, 10.79; N, 2.73. Found: C, 67.62; H, 10.88; N, 2.67.

4.2.1.7. (2S)-tert-Butyl 2-(2-hydroxyhexadecanamido)propanoate (mixture of diastereomers) (14c)

Yield 40%; Colorless oil; [α]D20 7.7 (c 0.99, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.22–7.04 (m, 1H, NH), 4.56–4.35 (m, 1H, NHCH), 4.17–4.02 (m, 1H, CHOH), 3.45 (br, 1H, OH), 1.93–1.55 (m, 2H, CH2CHOH), 1.45 (s, 9H, CH3), 1.36 (d, J = 7.0 Hz, 3H, NHCHCH3), 1.32–1.18 (s, 24H, CH2), 0.86 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.9, 173.7, 172.3, 172.0, 81.9, 71.9, 48.1, 34.7, 34.6, 31.8, 29.6, 29.5, 29.4, 29.3, 29.2, 27.8, 24.9, 22.6, 18.4, 14.0; MS (ESI) m/z (%): 400.4 (100) [M+H]+; Anal. Calcd for C23H45NO4: C, 69.13; H, 11.35; N, 3.51. Found: C, 68.91; H, 11.46; N, 3.43.

4.2.1.8. (2S)-5-tert-Butyl 1-ethyl-2-(2-hydroxyhexadecanamido) pentanedioate (mixture of diastereomers) (19)

Yield 53%; Yellow oil; [α]D20 5.80 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.33–7.19 (m, 1H, NH), 4.55–4.38 (m, 1H, NHCH), 4.18–3.95 (m, 3H, CHOH & COOCH2), 2.33–1.74 (m, 6H, CH2 & CH2CHOH), 1.34 (s, 9H, CH3), 1.29–1.07 (m, 27H, CH2 & CH3), 0.76 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.6, 174.4, 171.8, 171.5, 80.6, 71.8, 61.4, 51.1, 34.5, 34.5, 34.4, 31.7, 31.2, 29.4, 29.2, 29.1, 27.8, 27.1, 24.8, 24.7, 22.4, 13.9; MS (ESI) m/z (%): 486.4 (100) [M+H]+; Anal. Calcd for C27H51NO6: C, 66.77; H, 10.58; N, 2.88. Found: C, 66.48; H, 10.72; N, 2.78.

4.2.1.9. (2S)-tert-Butyl 2-(2-hydroxy-6-(naphthalen-2-yl)hexanamido)-3-methylbutanoate (mixture of diastereomers) (29a)

Yield 73%; Colorless oil; [α]D20 10.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.86–7.68 (m, 3H, arom), 7.63–7.54 (m, 1H, arom), 7.51–7.24 (m, 3H, arom), 7.08 (d, J = 8.8 Hz, ½H, NH), 6.91 (d, J = 8.8 Hz, ½H, NH), 4.51–4.39 (m, 1H, NHCH), 4.21–4.06 (m, 1H, CHOH), 3.02 (br, 1H, OH), 2.78 (t, J = 7.4 Hz, 2H, CH2), 2.27–2.06 (m, 1H, NHCHCH), 2.01–1.64 (m, 4H, CH2), 1.62–1.40 (m, 11H, CH2 & CH3), 0.98–0.80 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.9, 173.8, 171.3, 170.9, 139.8, 133.5, 131.8, 127.7, 127.5, 127.3, 127.2, 126.2, 125.7, 124.9, 82.1, 82.0, 72.0, 71.8, 57.0, 56.8, 35.8, 35.8, 34.7, 34.6, 31.3, 31.2, 31.0, 27.9, 24.6, 18.9, 18.8, 17.5, 17.3; MS (ESI) m/z (%): 412.2 (100) [M−H]; Anal. Calcd for C25H35NO4: C, 72.61; H, 8.53; N, 3.39. Found: C, 72.38; H, 8.69; N, 3.20.

4.2.1.10. (2S)-tert-Butyl 2-(6-([1,1′-biphenyl]-4-yl)-2-hydroxyhexanamido)-3-methylbutanoate (mixture of diastereomers) (29b)

Yield 68%; Colorless oil; [α]D20 10.8 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.64–6.90 (m, 10H, arom & NH), 4.47 (dd, J1 = 9.0 Hz, J2 = 4.6 Hz, 1H, NHCH), 4.25–4.05 (m, 1H, CHOH), 3.72–3.33 (m, 1H, OH), 2.67 (t, J = 7.4 Hz, 2H, CH2), 2.31–2.08 (m, 1H, NHCHCH), 1.98–1.34 (m, 15H, CH2 & CH3), 1.00–0.81 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.0, 171.3, 170.9, 141.4, 141.0, 138.5, 128.8, 128.6, 126.8, 82.1, 82.0, 72.0, 71.8, 57.0, 56.8, 35.3, 34.6, 31.3, 31.2, 31.1, 27.9, 24.6, 18.9, 18.8, 17.5, 17.4; MS (ESI) m/z (%): 438.4 (100) [M−H]; Anal. Calcd for C27H37NO4: C, 73.77; H, 8.48; N, 3.19. Found: C, 73.55; H, 8.65; N, 3.14.

4.2.1.11. (2S)-tert-Butyl 2-(2-hydroxy-6-phenylhexanamido)-3-methylbutanoate (mixture of diastereomers) (29c)

Yield 92%; Colorless oil; [α]D20 8.38 (c 0.99, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.33–6.96 (m, 6H, arom & NH), 4.42 (dd, J1 = 9.0 Hz, J2 = 4.6 Hz, 1H, NHCH), 4.21–4.03 (m, 1H, CHOH), 3.72 (br, 1H, OH), 2.61 (t, J = 7.4 Hz, 2H, CH2), 2.30–2.06 (m, 1H, NHCHCH), 1.96–1.52 (m, 6H, CH2), 1.47 (s, 9H, CH3), 0.97–0.82 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.1, 174.0, 171.3, 170.8, 142.3, 128.2, 128.1, 125.5, 82.0, 81.9, 71.9, 71.8, 56.9, 56.7, 35.7, 34.5, 31.1, 27.9, 24.6, 18.8, 17.5, 17.4; MS (ESI) m/z (%): 362.4 (100) [M−H]; Anal. Calcd for C21H33NO4: C, 69.39; H, 9.15; N, 3.85. Found: C, 69.27; H, 9.27; N, 3.75.

4.2.1.12. (2S)-tert-Butyl 2-(2-hydroxy-2-(naphthalen-2-yl)acetamido)-3-methylbutanoate (mixture of diastereomers) (35)

Yield 66%; Yellow oil; [α]D20 7.9 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.88–7.68 (m, 4H, arom), 7.55–7.38 (m, 3H, arom), 7.29–6.77 (m, 1H, NH), 5.20 (d, J = 3.0 Hz, 1H, CHOH), 4.47–4.33 (m, 1H, NHCH), 2.20–1.97 (m, 1H, NHCHCH), 1.46–1.33 (m, 9H, CH3), 0.93–0.66 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 172.1, 172.0, 170.7, 170.6, 136.7, 133.1, 133.0, 128.5, 128.3, 128.0, 127.5, 126.2, 126.1, 126.0, 125.9, 124.0, 123.9, 82.1, 74.3, 74.1, 57.3, 57.1, 31.3, 27.8, 18.7, 17.4, 17.2; MS (ESI) m/z (%): 356.2 (100) [M−H]; Anal. Calcd for C21H27NO4: C, 70.56; H, 7.61; N, 3.92. Found: C, 70.38; H, 7.70; N, 3.86.

4.2.2. Saponification of methyl esters

To a stirred solution of a methyl ester (1.0 mmol) in water, 1 N NaOH (1 mL, 1.0 mmol) was added and the mixture was left overnight at room temperature. After the completion of the reaction, the mixture was washed with EtOAc, acidified with 1 N HCl to pH 1 and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated under reduced pressure.

4.2.2.1. (2S)-2-(2-Hydroxyhexadecanamido)-3-(1H-indol-3-yl) propanoic acid (mixture of diastereomers) (11a)

Yield 80%; White solid; mp 144–146 °C; [α]D20 18.7 (c 1.00, MeOH); 1H NMR (CD3OD, 200 MHz): δ 7.63–7.51 (m, 1H, NH), 7.36–7.26 (m, 1H, arom), 7.15–6.92 (m, 3H, arom), 4.82–4.67 (m, 1H, NHCH), 4.01–3.88 (m, 1H, CHOH), 3.44–3.19 (m, 2H, NHCHCH2), 1.77–1.50 (m, 2H, CH2CHOH), 1.48–1.12 (m, 24H, CH2), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CD3OD, 50 MHz): δ 177.0, 175.0, 142.0, 128.9, 124.5, 122.4, 119.9, 119.4, 112.2, 110.4, 72.5, 53.3, 35.5, 33.1, 30.8, 30.5, 28.5, 25.9, 23.8, 14.5; HRMS (ESI) calcd for C27H42N2NaO4 [M+Na]+: 481.3037. Found: 481.3057; Anal. Calcd for C27H42N2O4: C, 70.71; H, 9.23; N, 6.11. Found: C, 70.59; H, 9.35; N, 6.01.

4.2.2.2. (2S)-1-(2-Hydroxyhexadecanoyl)pyrrolidine-2-carboxylic acid (mixture of diastereomers) (11b)

Yield 80%; White solid; mp 71–74 °C; [α]D20 −5.2 (c 1.02, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 4.82–4.38 (m, 1H, NCH), 4.37–4.08 (m, 1H, CHOH), 3.82–3.30 (m, 2H, NCH2), 2.56–1.76 (m, 4H, NCHCH2CH2), 1.73–1.00 (m, 26H, CH2), 0.86 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 174.6, 167.8, 81.5, 69.5, 59.5, 56.4, 46.7, 45.4, 33.9, 32.3, 31.8, 29.5, 29.4, 29.3, 28.9, 28.4, 25.0, 24.5, 22.6, 22.1, 14.0; HRMS (ESI) calcd for C21H39NNaO4 [M+Na]+: 392.2771. Found: 392.2785; Anal. Calcd for C21H39NO4: C, 68.25; H, 10.64; N, 3.79. Found: C, 68.14; H, 10.73; N, 3.65.

4.2.2.3. (2S)-2-(2-Hydroxyhexadecanamido)-3,3-dimethylbutanoic acid (mixture of diastereomers) (11c)

Yield 80%; White solid; mp 99–101 °C; [α]D20 1.8 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.52–7.01 (m, 1H, NH), 4.51–4.25 (m, 1H, NHCH), 4.24–4.03 (m, 1H, CHOH), 1.92–1.11 (m, 26H, CH2), 0.96 (s, 9H, CH3), 0.89–0.72 (m, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 175.5, 174.6, 174.4, 74.5, 59.8, 34.6, 34.5, 31.9, 29.7, 29.6, 29.5, 29.3, 26.5, 25.1, 24.5, 22.6, 14.1; MS (ESI) m/z (%): 386.2 (100) [M+H]+; Anal. Calcd for C22H43NO4: C, 68.53; H, 11.24; N, 3.63. Found: C, 68.44; H, 11.35; N, 3.52.

4.2.2.4. 2-(2-Hydroxyhexadecanoylamino)-2-methylpropanoic acid (racemic mixture) (11d)

Yield 72%; White solid; mp 81– 83 °C; [α]D20 1.7 (c 1.02, MeOH); 1H NMR (CDCl3, 200 MHz): δ 7.20 (br s, 1H, NH), 4.21–3.92 (m, 1H, CHOH), 1.94–1.03 (m, 32H, CH2 & CH3), 0.88 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.1, 96.9, 96.4, 72.0, 34.3, 31.9, 29.8, 29.6, 29.5, 29.3, 25.1, 24.7, 24.5, 22.7, 14.1; MS (ESI) m/z (%): 358.3 (100) [M+H]+; Anal. Calcd for C20H39NO4: C, 67.19; H, 10.99; N, 3.92. Found: C, 67.02; H, 11.08; N, 3.86.

4.2.3. Oxidation of 2-hydroxyamides

To a solution of 2-hydroxyamide (1.0 mmol) in dry CH2Cl2 (10 mL) Dess–Martin periodinane was added (0.64 g, 1.5 mmol) and the mixture was stirred for 1–3 h at room temperature. The organic solvent was evaporated under reduce pressure and Et2O (30 mL) was added. The organic phase was washed with saturated aqueous NaHCO3 (20 mL) containing Na2S2O3 (1.5 g, 9.5 mmol), H2O (20 mL), dried over Na2SO4 and the organic solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography [EtOAc/petroleum ether (bp 40–60 °C), 2:8 or CHCl3/MeOH, 9:1].

4.2.3.1. (S)-1-(2-Oxohexadecanoyl)pyrrolidine-2-carboxylic acid (rotamers) (12b)

Yield 80%; Colorless semi solid; [α]D20 −3.2 (c 1.01, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.40 (br, 1H, COOH), 4.86–4.31 (m, 1H, NCH), 3.95–3.41 (m, 2H, NCH2), 3.06–2.60 (m, 2H, CH2CO), 2.46–1.12 (m, 28H, NCHCH2CH2 & CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 200.0, 177.4, 163.0, 61.3, 48.3, 47.4, 39.1, 38.5, 31.9, 29.7, 29.5, 29.3, 29.1, 25.1, 23.1, 22.7, 22.6, 14.0; HRMS (ESI) calcd for C21H38NO4 [M +H]+: 368.2795. Found: 368.2794; Anal. Calcd for C21H37NO4: C, 68.63; H, 10.15; N, 3.81. Found: C, 68.51; H, 10.22; N, 3.74.

4.2.3.2. (S)-3,3-Dimethyl 2-(2-oxohexadecanamido)butanoic acid (12c)

Yield 62%; Colorless oil; [α]D20 1.8 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.50 (d, J = 9.2 Hz, 1H, NH), 4.31 (d, J = 9.2 Hz, 1H, NHCH), 2.91 (t, J = 6.8 Hz, 2H, CH2CO), 1.74–1.49 (m, 2H, CH2CH2CO), 1.43–1.18 (m, 22H, CH2), 1.01 (s, 9H, CH3), 0.88 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.5, 175.7, 159.8, 60.8, 36.8, 34.7, 31.9, 29.6, 29.4, 29.3, 29.0, 26.5, 23.0, 22.6, 14.1; HRMS (ESI) calcd for C22H41NNaO4 [M+Na]+: 406.2928. Found: 406.2945; Anal. Calcd for C22H41NO4: C, 68.89; H, 10.77; N, 3.65. Found: C, 69.11; H, 10.88; N, 3.59.

4.2.3.3. 2-Methyl 2-(2-oxo-hexadecanoylamino)propanoic acid (12d)

Yield 95%; Colorless oil; 1H NMR (CDCl3, 200 MHz): δ 7.77 (br s, 1H, COOH), 2.88 (t, J = 6.8 Hz, 2H, CH2CO), 1.66–1.40 (m, 8H, CH3 & CH2CH2CO), 1.39–1.15 (m, 22H, CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 199.0, 198.8, 180.6, 160.0, 57.2, 36.4, 31.9, 29.6, 29.5, 29.4, 29.3, 29.1, 24.2, 23.0, 22.6, 14.1; HRMS (ESI) calcd for C20H37NNaO4 [M+Na]+: 378.2615. Found: 378.2627; Anal. Calcd for C20H37NO4: C, 67.57; H, 10.49; N, 3.94. Found: C, 67.36; H, 10.61; N, 3.87.

4.2.3.4. (S)-tert-Butyl 3-methyl-2-(2-oxohexadecanamido)butanoate (15a)

Yield 92%; Yellow oil; [α]D20 12.7 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.40 (d, J = 9.0 Hz, 1H, NH), 4.40 (dd, J1 = 9.2 Hz, J2 = 4.4 Hz, 1H, NHCH), 2.91 (t, J = 7.2 Hz, 2H, CH2COCO), 2.33–2.10 (m, 1H, NHCHCH), 1.71–1.53 (m, 2H, CH2CH2CO), 1.48 (s, 9H, CH3) 1.40–1.16 (m, 22H, CH2), 1.01–0.81 (m, 9H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.2, 169.6, 159.5, 81.9, 57.1, 36.4, 31.5, 31.1, 29.2, 29.0, 28.7, 27.6, 22.8, 22.3, 18.6, 17.1, 13.7; MS (ESI) m/z (%): 387.2 (100) [M−tBu+NH4]+; Anal. Calcd for C25H47NO4: C, 70.54; H, 11.13; N, 3.29. Found: C, 70.43; H, 11.24; N, 3.21.

4.2.3.5. (S)-Di-tert-butyl 2-(2-oxohexadecanamido)pentanedioate (15b)

Yield 84%; Yellow oil; [α]D20 9.18 (c 1.02, CHCl3); 1H NMR (CDCl3, 300 MHz): δ 7.47 (d, J = 8.4 Hz, 1H, NH), 4.49– 4.37 (m, 1H, NHCH), 2.88 (t, J = 7.2 Hz, 2H, CH2COCO), 2.38–2.08 (m, 3H, CH2CHH), 2.05–1.87 (m, 1H, CHH), 1.66–1.51 (m, 2H, CH2-CH2CO), 1.46 (s, 9H, CH3), 1.42 (s, 9H, CH3), 1.35–1.16 (m, 22H, CH2), 0.86 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.3, 171.7, 170.0, 159.8, 82.7, 80.8, 52.1, 36.7, 31.8, 31.3, 29.6, 29.5, 29.4, 29.3, 29.2, 29.0, 28.0, 27.9, 27.4, 23.0, 22.6, 14.0; MS (ESI) m/z (%): 529.5 (100) [M+NH4]+; Anal. Calcd for C29H53NO6: C, 68.06; H, 10.44; N, 2.74. Found: C, 67.90; H, 10.55; N, 2.68.

4.2.3.6. (S)-tert-Butyl 2-(2-oxohexadecanamido)propanoate (15c)

Yield 95%; White solid; mp 35–36 °C; [α]D20 5.0 (c 1.02, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.44 (d, J = 7.0 Hz, 1H, NH), 4.52–4.33 (m, 1H, NHCH), 2.90 (t, J = 7.4 Hz, 2H, CH2CO), 1.76–1.38 (m, 14H, CH3 & CH2CH2CO), 1.38–1.17 (m, 22H, CH2), 0.86 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.5, 171.1, 159.4, 82.4, 48.3, 36.6, 31.9, 29.6, 29.0, 27.9, 23.1, 22.6, 18.2, 14.1; MS (ESI) m/z (%): 415.3 (100) [M+NH4]+; Anal. Calcd for C23H43NO4: C, 69.48; H, 10.90; N, 3.52. Found: C, 69.34; H, 10.96; N, 3.43.

4.2.3.7. (S)-5-tert-Butyl 1-ethyl 2-(2-oxohexadecanamido)pentanedioate (20)

Yield 85%; Colorless oil; [α]D20 11.6 (c 1.00, CHCl3); 1H NMR (CDCl3, 300 MHz): δ 7.52 (d, J = 8.2 Hz, 1H, NH), 4.60–4.49 (m, 1H, NHCH), 4.21 (q, J = 7.2 Hz, 2H, COOCH2), 2.89 (t, J = 7.2 Hz, 2H, CH2COCO), 2.41–2.17 (m, 3H, CH2CHH), 2.08–1.92 (m, 1H, CHH), 1.66–1.52 (m, 2H, CH2CH2CO), 1.43 (s, 9H, CH3), 1.37–1.17 (m, 25H, CH2 & CH3), 0.85 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.2, 171.6, 170.8, 159.9, 80.9, 61.7, 51.7, 36.7, 31.8, 31.2, 29.6, 29.4, 29.3, 29.0, 28.0, 27.2, 23.0, 22.6, 14.0; MS (ESI) m/z (%): 428.2 (100) [M−tBu+H]+; Anal. Calcd for C27H49NO6: C, 67.05; H, 10.21; N, 2.90. Found: C, 66.96; H, 10.29; N, 2.81.

4.2.3.8. (S)-tert-Butyl 3-methyl-2-(6-(naphthalen-2-yl)-2-oxohexanamido) butanoate (30a)

Yield 91%; Colorless oil; [α]D20 11.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.86–7.68 (m, 3H, arom), 7.65–7.53 (m, 1H, arom), 7.52–7.18 (m, 4H, arom & NH), 4.39 (dd, J1 = 9.2 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.99 (t, J = 7.2 Hz, 2H, CH2), 2.80 (t, J = 7.0 Hz, 2H, CH2CO), 2.32–2.08 (m, 1H, NHCHCH), 1.86–1.58 (m, 4H, CH2), 1.48 (s, 9H, CH3), 1.00– 0.82 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.3, 169.9, 159.8, 139.4, 133.5, 131.8, 127.8, 127.5, 127.3, 127.2, 126.3, 125.8, 125.0, 82.3, 57.4, 36.5, 35.6, 31.4, 30.6, 27.9, 22.7, 18.9, 17.4; MS (ESI) m/z (%): 356.2 (100) [M−tBu+H]+; Anal. Calcd for C25H33NO4: C, 72.96; H, 8.08; N, 3.40. Found: C, 72.85; H, 8.17; N, 3.26.

4.2.3.9. (S)-tert-Butyl 2-(6-([1,1′-biphenyl]-4-yl)-2-oxohexanamido)-3-methylbutanoate (30b)

Yield 82%; Yellow oil; [α]D20 12.2 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.75–7.16 (m, 10H, arom & NH), 4.48–4.29 (m, 1H, NHCH), 3.11–2.87 (m, 2H, CH2), 2.80–2.55 (m, 2H, CH2CO), 2.34–2.09 (m, 1H, NHCHCH), 1.85–1.60 (m, 4H, CH2), 1.48 (s, 9H, CH3) 1.04–0.82 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.3, 169.9, 159.8, 141.1, 141.0, 138.6, 128.7, 128.6, 127.0, 126.9, 82.3, 57.4, 36.5, 35.1, 31.4, 30.7, 27.9, 22.7, 18.9, 17.4; MS (ESI) m/z (%): 436.4 (100) [M−H]; Anal. Calcd for C27H35NO4: C, 74.11; H, 8.06; N, 3.20. Found: C, 74.02; H, 8.17; N, 3.09.

4.2.3.10. (S)-tert-Butyl 3-methyl-2-(2-oxo-6-phenylhexanamido) butanoate (30c)

Yield 74%; Colorless oil; [α]D20 11.3 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.50–6.94 (m, 6H, arom & NH), 4.36 (dd, J1 = 9.2 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.93 (t, J = 6.8 Hz, 2H, CH2), 2.62 (t, J = 6.8 Hz, 2H, CH2CO), 2.31–2.06 (m, 1H, NHCHCH), 1.73–1.55 (m, 4H, CH2), 1.46 (s, 9H, CH3), 1.00–0.74 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 197.9, 169.7, 159.8, 142.0, 128.3, 125.6, 82.3, 57.1, 36.5, 35.5, 31.4, 30.7, 27.9, 22.6, 18.9, 17.4; MS (ESI) m/z (%): 360.4 (100) [M−H]; Anal. Calcd for C21H31NO4: C, 69.78; H, 8.64; N, 3.87. Found: C, 69.61; H, 8.73; N, 3.81.

4.2.3.11. (S)-tert-Butyl 3-methyl-2-(2-(naphthalen-2-yl)-2-oxoacetamido) butanoate (36)

Yield 95%; Yellow oil; [α]D20 7.03 (c 1.01, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 9.21–8.96 (m, 1H, arom), 8.23–7.05 (m, 7H, arom & NH), 4.55 (dd, J1 = 9.2 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.43–2.08 (m, 1H, NHCHCH), 1.50 (s, 9H, CH3), 1.09–0.79 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 186.6, 170.0, 161.6, 136.0, 134.8, 134.7, 132.2, 130.4, 130.2, 129.2, 128.2, 127.6, 126.7, 125.1, 82.3, 57.6, 31.5, 27.9, 19.0, 18.9, 17.6; MS (ESI) m/z (%): 354.1 (100) [M−H]; Anal. Calcd for C21H25NO4: C, 70.96; H, 7.09; N, 3.94. Found: C, 70.74; H, 7.20; N, 3.86.

4.2.4. Cleavage of tert-butyl protecting group

A solution of the tert-butyl ester derivative (1.0 mmol) in 50% TFA/CH2Cl2 (0.5 M) was stirred for 1–3 h at room temperature. After the completion of the reaction, the organic solvent was evaporated under reduced pressure and the residue was purified by recrystallization.

4.2.4.1. (S)-3-Methyl 2-(2-oxohexadecanamido)butanoic acid (16a, GK241)

Yield 72%; White solid; mp 55–58 °C; [α]D20 5.04 (c 1.03, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.43 (d, J = 8.0 Hz, 1H, NH), 4.59–4.35 (m, 1H, NHCH), 2.91 (t, J = 6.8 Hz, 2H, CH2CO), 2.42–2.16 (m, 1H, NHCHCH), 1.74–1.50 (m, 2H, CH2CH2CO), 1.47– 1.15 (m, 22H, CH2), 1.08–0.79 (m, 9H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.4, 176.0, 160.2, 57.4, 36.8, 31.9, 31.0, 29.7, 29.4, 29.3, 29.1, 23.1, 22.7, 19.1, 17.5, 14.1; HRMS (ESI) calcd for C21H40NO4 [M+H]+: 370.2952. Found: 370.2938; Anal. Calcd for C21H39NO4: C, 68.25; H, 10.64; N, 3.79. Found: C, 68.09; H, 10.76; N, 3.70.

4.2.4.2. (S)-2-(2-Oxohexadecanamido)propanoic acid (16c)

Yield 90%; White solid; mp 98–101 °C; [α]D20 5.8 (c 1.03, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 8.92 (br, 1H, COOH), 7.43 (d, J = 7.6 Hz, 1H, NH), 4.69–4.50 (m, 1H, NHCH), 2.91 (t, J = 7.2 Hz, 2H, CH2CO), 1.70–1.48 (m, 5H, CH2CH2CO & CH3), 1.44–1.19 (s, 22H, CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.3, 176.8, 159.6, 47.8, 36.7, 31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 23.0, 22.6, 17.7, 14.1; HRMS (ESI) calcd for C19H36NO4 [M+H]+: 342.2639. Found: 342.2650; Anal. Calcd for C19H35NO4: C, 66.83; H, 10.33; N, 4.10. Found: C, 66.61; H, 10.45; N, 4.01.

4.2.4.3. (S)-2-(2-Oxohexadecanamido)pentanedioic acid (16d)

Yield 90%; White solid; mp 100–103 °C; [α]D20 30.5 (c 1.02, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 9.67 (br, 1H, COOH), 7.63 (d, J = 8.0 Hz, 1H, NH), 4.72–4.56 (m, 1H, NHCH), 2.91 (t, J = 7.0 Hz, 2H, CH2CO), 2.64–2.43 (m, 2H, NHCHCH2CH2), 2.38–2.20 (m, 2H, NHCHCH2), 1.72–1.51 (m, 2H, CH2CH2CO), 1.44–1.18 (m, 22H, CH2), 0.88 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.1, 178.5, 176.2, 159.8, 51.2, 36.7, 31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 23.0, 22.6, 14.1; HRMS (ESI) calcd for C21H37NNaO6 [M+Na]+: 422.2513. Found: 422.2513; Anal. Calcd for C21H37NO6: C, 63.13; H, 9.33; N, 3.51. Found: C, 62.99; H, 9.44; N, 3.43.

4.2.4.4. (2S)-2-(2-Hydroxyhexadecanamido)-3-methylbutanoic acid (mixture of diastereomers) (17)

Yield 93%; White solid; mp 95–99 °C; [α]D20 17.1 (c 1.00, MeOH); 1H NMR (CDCl3, 200 MHz): δ 7.01 (d, J = 9.2 Hz, 1H, NH), 4.57–4.42 (m, 1H, NHCH), 4.24–4.10 (m, 1H, CHOH), 3.68 (br, 1H, OH), 2.39–2.15 (m, 1H, NHCHCH), 1.95–1.53 (m, 2H, CH2CHOH), 1.51–1.12 (s, 24H, CH2), 1.10–0.76 (m, 9H, 3xCH3); 13C NMR (CDCl3, 50 MHz): δ 176.2, 175.3, 174.4, 72.2, 57.0, 56.7, 34.5, 34.4, 31.8, 30.6, 30.5, 29.6, 29.3, 25.0, 22.6, 19.0, 17.4, 14.1; HRMS (ESI) calcd for C21H41NNaO4 [M+Na]+: 394.2928. Found: 394.2947; Anal. Calcd for C21H41NO4: C, 67.88; H, 11.12; N, 3.77. Found: C, 67.73; H, 11.23; N, 3.69.

4.2.4.5. (S)-5-Ethoxy-5-oxo-4-(2-oxohexadecanamido)pentanoic acid (21)

Yield 93%; White solid; mp 64–66 °C; [α]D20 13.4 (c 1.03, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 9.75 (br, 1H, COOH), 7.57 (d, J = 8.2 Hz, 1H, NH), 4.67–4.49 (m, 1H, NHCH), 4.22 (q, J = 7.0 Hz, 2H, COOCH2), 2.89 (t, J = 7.2Hz, 2H, CH2CO), 2.36–1.94 (m, 2H, NHCHCH2CH2), 1.70–1.48 (m, 2H, NHCHCH2), 1.46–1.08 (m, 24H, CH2), 0.87 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 50MHz): δ 198.1, 177.8, 170.7, 160.0, 61.9, 51.5, 36.7, 31.8, 29.9, 29.6, 29.3, 29.2, 29.0, 26.9, 23.0, 22.6, 14.0; HRMS (ESI) calcd for C23H41NNaO6 [M+Na]+: 450.2826. Found: 450.2826; Anal. Calcd for C23H41NO6: C, 64.61; H, 9.67; N, 3.28. Found: C, 64.49; H, 9.74; N, 3.22.

4.2.4.6. (S)-3-Methyl 2-(6-(naphthalen-2-yl)-2-oxohexanamido) butanoic acid (31a)

Yield 83%; Colorless solid; [α]D20 −2.8 (c 0.5, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.85–7.69 (m, 3H, arom), 7.64–7.58 (m, 1H, arom), 7.51–7.28 (m, 4H, arom & NH), 5.94 (br, 1H, COOH), 4.52 (dd, J1 = 9.2 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.98 (t, J = 6.8 Hz, 2H, CH2), 2.81 (t, J = 7.0 Hz, 2H, CH2CO), 2.41–2.21 (m, 1H, NHCHCH), 1.88–1.59 (m, 4H, CH2), 1.08–0.89 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.1, 175.4, 159.9, 139.4, 133.5, 131.9, 127.8, 127.5, 127.3, 127.2, 126.3, 125.8, 125.0, 56.9, 36.5, 35.6, 31.0, 30.5, 22.7, 19.0, 17.4; HRMS (ESI) calcd for C21H26NO4 [M +H]+: 356.1856. Found: 356.1841; Anal. Calcd for C21H25NO4: C, 70.96; H, 7.09; N, 3.94. Found: C, 70.84; H, 7.16; N, 3.87.

4.2.4.7. (S)-2-(6-([1,1′-Biphenyl]-4-yl)-2-oxohexanamido)-3-methylbutanoic acid (31b)

Yield 92%; White solid; mp 123–124 °C; [α]D20 −6.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 7.70–7.08 (m, 10H, arom & NH), 4.56–4.26 (m, 1H, NHCH), 3.12–2.80 (m, 2H, CH2), 2.76–2.46 (m, 2H, CH2CO), 2.39–2.08 (m, 1H, NHCHCH), 1.83–1.44 (m, 4H, CH2), 1.10–0.68 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.1, 176.2, 160.1, 141.0, 140.9, 138.6, 128.7, 128.6, 127.0, 126.9, 57.5, 36.6, 35.2, 30.7, 27.3, 22.6, 19.1, 17.5; HRMS (ESI) calcd for C23H28NO4 [M+H]+: 382.2013. Found: 382.1995; Anal. Calcd for C23H27NO4: C, 72.42; H, 7.13; N, 3.67. Found: C, 72.64; H, 7.24; N, 3.25.

4.2.4.8. (S)-3-Methyl 2-(2-oxo-6-phenylhexanamido)butanoic acid (31c)

Yield 50%; Colorless oil; [α]D20 5.8 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 9.43 (br, 1H, COOH), 7.37 (d, J = 9.0 Hz, 1H, NH), 7.32–7.03 (m, 5H, arom), 4.50 (dd, J1 = 9.0 Hz, J2 = 4.6 Hz, 1H, NHCH), 3.04–2.86 (m, 2H, CH2), 2.72–2.50 (m, 2H, CH2CO), 2.41–2.16 (m, 1H, NHCHCH), 1.79–1.50 (m, 4H, CH2), 1.11–0.81 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 198.0, 175.8, 159.9, 141.9, 128.2, 125.7, 57.1, 36.5, 35.5, 30.9, 30.6, 22.5, 19.0, 17.4; HRMS (ESI) calcd for C17H24NO4 [M+H]+: 306.1700. Found: 306.1695; Anal. Calcd for C17H23NO4: C, 66.86; H, 7.59; N, 4.59. Found: C, 66.65; H, 7.71; N, 4.52.

4.2.4.9. (S)-3-Methyl 2-(2-(naphthalen-2-yl)-2-oxoacetamido) butanoic acid (37)

Yield 80%; Yellow solid; mp 122–124 °C; [α]D20 −25.3 (c 1.03, CHCl3); 1H NMR (CDCl3, 200 MHz): δ 9.58 (br, 1H, COOH), 9.15 (s, 1H, arom), 8.17 (d, J = 8.6 Hz, 1H, NH), 8.00 (d, J = 7.8 Hz, 1H, arom), 7.94–7.76 (m, 2H, arom), 7.75–7.44 (m, 3H, arom), 4.71 (dd, J1 = 9.0 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.54–2.26 (m, 1H, NHCHCH), 1.18–0.95 (m, 6H, CH3); 13C NMR (CDCl3, 50 MHz): δ 186.4, 176.4, 161.9, 136.0, 135.0, 132.2, 130.3, 130.2, 129.4, 128.4, 127.6, 126.8, 125.0, 57.1, 31.1, 19.1, 17.5; HRMS (ESI) calcd for C17H18NO4 [M+H]+: 300.1230. Found: 300.1216; Anal. Calcd for C17H17NO4: C, 68.21; H, 5.72; N, 4.68. Found: C, 68.12; H, 5.80; N, 4.59.

4.2.4.10. (2E,4E)-Ethyl 5-(naphthalen-2-yl)penta-2,4-dienoate (23a)

To a stirred solution of aldehyde (1.0 mmol) in dry THF (10 mL) and molecular sieves (1.5 g/mmol aldehyde) under Ar, C2H5OOCH═CHCH2P(═O)(OC2H5)2 (1.5 mmol) was added at 0 °C. Then, LiOH·H2O (1.5 mmol) was added dropwise and the reaction mixture heated at reflux for 24 h. The reaction was filtered over celite and the organic solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography [EtOAc/petroleum ether (bp 40–60 °C), 5:95]. Yield 50%; White solid; mp 100–102 °C; 1H NMR (CDCl3, 200 MHz): δ 7.90–7.60 (m, 5H, arom), 7.55–7.45 (m, 3H, arom & CH), 7.10–6.95 (m, 2H, CH), 6.04 (s, 1H, CH), 4.26 (m, 2H, CH2), 1.28 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 167.1, 144.6, 140.4, 133.5, 133.4, 128.5, 128.2, 128.1, 127.7, 126.6, 126.5, 126.4, 123.3, 121.3, 60.4, 14.3; MS (ESI) m/z (%): 253 (100) [M+H]+; Anal. Calcd for C17H16O2: C, 80.93; H, 6.39. Found: C, 80.85; H, 6.55.

4.2.6. Ethyl 5-(naphthalen-2-yl)pentanoate (24a)

To a stirred solution of the alkene (1.0 mmol) in absolute EtOH (10 mL), Pd/C 10% was added. The reaction left overnight under H2 at room temperature. The reaction was filtered over celite and the organic solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography [EtOAc/petroleum ether (bp 40–60 °C), 5:95]. Yield 80%; Colorless oil; 1H NMR (CDCl3, 200 MHz): δ 8.03–7.18 (m, 7H, arom), 4.34–4.06 (m, 2H, COOCH2CH3), 3.03–2.69 (m, 2H, CH2), 2.51–2.31 (m, 2H, CH2), 1.91–1.69 (m, 4H, CH2), 1.41–1.22 (m, 3H, CH3); 13C NMR (CDCl3, 50 MHz): δ 173.4, 139.4, 133.4, 131.8, 127.6, 127.4, 127.2, 127.1, 126.2, 125.7, 124.9, 60.0, 35.5, 34.0, 30.5, 24.4, 14.1; MS (ESI) m/z (%): 274.1 (100) [M+NH4]+; Anal. Calcd for C17H20O2: C, 79.65; H, 7.86. Found: C, 79.39; H, 8.00.

4.2.7. General method of oxidation of primary alcohols to aldehydes and then to cyanohydrins

To a solution of primary alcohol (1.0 mmol) in a mixture of toluene (3 mL) and EtOAc (3 mL), a solution of NaBr (0.11 g, 1.1 mmol) in water (0.5 mL) was added and the mixture was cooled down to −5 °C. To the resulting biphasic system, AcNH-TEMPO (2.2 mg, 0.01 mmol) was added, followed by the drop-wise addition of an aqueous solution of 0.5 M NaOCl (2.2 mL, 1.1 mmol) and NaHCO3 (0.24 g, 3.0 mmol) over a period of 1 h under vigorous stirring. After the mixture was stirred for a further 15 min at 0 °C, EtOAc (10 mL) and H2O (10 mL) were added. The aqueous layer was separated and washed with EtOAc (2 × 10 mL). The combined organic layers were washed consecutively with 5% aqueous citric acid (10 mL) containing KI (0.04 g), 10% aqueous Na2S2O3 (10 mL), and brine and dried over Na2SO4. The solvents were evaporated under reduced pressure and the aldehyde was used straight to the next reaction without further purification.

To a solution of the aldehyde (1.0 mmol) in CH2Cl2 (1.3 mL), an aqueous solution of NaHSO3 (0.25 mL, 1.5 mmol) was added and the mixture was stirred for 30 min at room temperature. The organic solvent was evaporated under reduced pressure and H2O (5 mL) was added. The mixture was cooled down to 0 °C and KCN 6 M (0.25 mL, 1.5 mmol) was added drop-wise under vigorous stirring. The reaction was left stirring for 18 h at room temperature. After the completion of the reaction, the organic layer was extracted with CH2Cl2 (2 × 20 mL), washed with brine and dried over Na2SO4. The organic solvent was evaporated under reduced pressure and the compound was purified with flash column chromatography [EtOAc/petroleum ether (bp 40–60 °C), 2:8].

4.2.8. 2-Hydroxy-6-(naphthalen-2-yl)hexanenitrile (27a)

Yield 56%; Yellow oil; 1H NMR (CDCl3, 200 MHz): δ 7.95–7.19 (m, 7H, arom), 4.41 (t, J = 6.6 Hz, 1H, CHOH), 2.99 (br, 1H, OH), 2.88–2.66 (m, 2H, CH2), 1.98–1.42 (m, 6H, CH2); 13C NMR (CDCl3, 50 MHz): δ 139.3, 133.4, 131.8, 127.8, 127.5, 127.3, 127.1, 126.3, 125.9, 125.1, 119.9, 61.0, 35.6, 34.9, 30.5, 24.1; MS (ESI) m/z (%): 257.1 (100) [M+NH4]+; Anal. Calcd for C16H17NO: C, 80.30; H, 7.16; N, 5.85. Found: C, 80.07; H, 7.32; N, 5.66.

4.2.9. 6-([1,1′-Biphenyl]-4-yl)-2-hydroxyhexanenitrile (27b)

Yield 80%; Pink solid; mp 78–82 °C; 1H NMR (CDCl3, 200 MHz): δ 7.69–7.25 (m, 9H, arom), 4.50–4.46 (m, 1H, CHOH), 3.64 (br, 1H, OH), 2.83–2.70 (m, 2H, CH2), 1.97–1.90 (m, 2H, CH2), 1.80–1.61 (m, 4H, CH2); 13C NMR (CDCl3, 50 MHz): δ 140.9, 140.7, 138.5, 128.6, 126.9, 126.7, 120.0, 60.8, 35.0, 34.7, 30.5, 24.1; MS (ESI) m/z (%): 282.9 (100) [M+NH4]+; Anal. Calcd for C18H19NO: C, 81.47; H, 7.22; N, 5.28. Found: C, 81.25; H, 7.39; N, 5.23.

4.2.9.1. 2-Hydroxy-6-phenylhexanenitrile (27c)

Yield 78%; Colorless oil; 1H NMR (CDCl3, 200 MHz): δ 7.44–7.15 (m, 5H, arom), 4.56–4.32 (m, 1H, CHOH), 4.03 (br, 1H, OH), 2.81–2.59 (m, 2H, CH2), 2.01–1.48 (m, 6H, CH2); 13C NMR (CDCl3, 50 MHz): δ 141.7, 128.1, 125.5, 119.9, 60.7, 35.3, 34.6, 30.4, 23.9; MS (ESI) m/z (%): 207.3 (90) [M+NH4]+; Anal. Calcd for C12H15NO: C, 76.16; H, 7.99; N, 7.40. Found: C, 76.04; H, 8.11; N, 7.30.

4.2.10. 2-Hydroxy-2-(naphthalen-2-yl)acetonitrile (33)

To a stirring solution of naphthalene-2-carbaldehyde (1.0 mmol) in EtOAc/THF/AcOH 4:4:2, aqueous solution of NaCN 6 M (0.25 mL, 1.5 mmol) was added. After the completion of the reaction, the organic layer was extracted with CH2Cl2 (2 ×20 mL), washed with brine and dried over Na2SO4. The organic solvent was evaporated under reduced pressure and the compound was purified with flash column chromatography [EtOAc/petroleum ether (bp 40–60 °C), 2:8]. Yield 95%; White solid; mp 110–113 °C; 1H NMR (CDCl3, 200 MHz): δ 7.99–7.24 (m, 7H, arom), 5.78–5.59 (m, 1H, CHOH), 2.08 (d, J = 2.4 Hz, 1H, OH); 13C NMR (CDCl3, 50 MHz): δ 133.6, 132.8, 132.3, 129.4, 128.3, 127.7, 127.2, 123.6, 63.8; MS (ESI) m/z (%): 157.1 (73) [M–CN]+; Anal. Calcd for C12H9NO: C, 78.67; H, 4.95; N, 7.65. Found: C, 78.49; H, 5.10; N, 7.56.

4.2.11. Converting the nitriles into corresponding carboxylic acids

The nitrile (1.0 mmol) was dissolved in HCl conc. (2.5 mL) and stirred for 18 h at room temperature. H2O (5 mL) was then added and the organic layer was extracted with CH2Cl2, washed with brine and dried over Na2SO4. The organic solvent was evaporated under reduced pressure and the amide derivative was crystallized over cold petroleum ether.

To a solution of the amide derivative (0.8 mmol) in MeOH/H2O (2:1, 6 mL), KOH (8.0 mmol) was added and the reaction mixture was heated under reflux for 2 h. Then, methanol was evaporated under reduced pressure, H2O was added and the aqueous layer was acidified with 1 N HCl to pH 1. The organic layer was extracted with EtOAc (3 × 15 mL), dried over Na2SO4 and the solvent was evaporated under reduced pressure. The compound was purified by recrystallization [CH2Cl2/petroleum ether (bp 40–60 °C)].

4.2.11.1. 2-Hydroxy-6-(naphthalen-2-yl)hexanoic acid (28a)

Yield 98%; White solid; mp 115–118 °C; 1H NMR (CDCl3, 200 MHz): δ 7.81–7.25 (m, 7H, arom), 4.35–4.10 (m, 1H, CHOH), 2.78 (t, J = 7.4 Hz, 2H, CH2), 2.07–1.31 (m, 6H, CH2); 13C NMR (CDCl3, 50 MHz): δ 177.1, 139.8, 133.4, 131.7, 127.6, 127.4, 127.2, 127.2, 126.1, 125.7, 124.9, 69.9, 49.7, 49.2, 48.8, 35.7, 33.9, 30.9, 24.5; MS (ESI) m/z (%): 257.2 (100) [M−H]; Anal. Calcd for C16H18O3: C, 74.39; H, 7.02. Found: C, 74.31; H, 7.18.

4.2.11.2. 6-([1,1′-Biphenyl]-4-yl)-2-hydroxyhexanoic acid (28b)

Yield 93%; White solid; 1H NMR (DMSO, 200 MHz): δ 7.48–7.11 (m, 9H, arom), 4.06–3.94 (m, 1H, CHOH), 2.58–2.50 (m, 2H, CH2), 1.74–1.37 (m, 6H, CH2); 13C NMR (CDCl3, 50 MHz): δ 177.1, 141.4, 140.9, 138.4, 128.6, 128.5, 126.8, 126.8, 69.9, 35.2, 33.9, 31.0, 24.5; MS (ESI) m/z (%): 282.9 (100) [M−H]; Anal. Calcd for C18H20O3: C, 76.03; H, 7.09. Found: C, 75.77; H, 7.23.

4.2.11.3. 2-Hydroxy-6-phenylhexanoic acid (28c)

Yield 63%; White solid; mp 100–102 °C; 1H NMR (CDCl3, 200 MHz): δ 7.41–7.10 (m, 5H, arom), 4.36–4.20 (m, 1H, CHOH), 2.64 (t, J = 7.2 Hz, 2H, CH2), 2.02–1.37 (m, 6H, CH2); 13C NMR (CDCl3, 50 MHz): δ 179.5, 142.3, 128.4, 128.3, 125.7, 70.1, 35.7, 34.0, 31.1, 24.4; MS (ESI) m/z (%): 208.1 (100) [M]; Anal. Calcd for C12H16O3: C, 69.21; H, 7.74. Found: C, 69.04; H, 7.89.

4.2.11.4. 2-Hydroxy-2-(naphthalen-2-yl)acetic acid (34)

Yield 90%; White solid; mp 57–60 °C; 1H NMR (CDCl3, 200 MHz): δ 7.89–7.24 (m, 7H, arom), 5.28 (s, 1H, CHOH), 3.32–3.08 (m, 1H, OH); 13C NMR (CDCl3, 50 MHz): δ 175.1, 135.8, 133.1, 128.2, 127.9, 127.5, 126.2, 126.1, 126.1, 125.8, 124.1, 72.6; MS (ESI) m/z (%): 202.21 (100) [M+H]+; Anal. Calcd for C12H10O3: C, 71.28; H, 4.98. Found: C, 71.11; H, 5.13.

Scheme 2
Reagents and conditions: (a) WSCI·HCl, HOBt, Et3N, CH2Cl2; (b) Dess–Martin periodinane, CH2Cl2; (c) CF3COOH, CH2Cl2.

Supplementary Material

Acknowledgments

This research has been co-financed by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework (NSRF)—Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.

This work was supported in part by Grant HL36235 from the National Institutes of Health (to M.H.G.).

Partial support was offered by LinkSCEEM-2 project, funded by the European Commission under the 7th Framework Program through Capacities Research Infrastructure, INFRA-2010-1.2.3 Virtual Research Communities, Combination of Collaborative Project and Coordination and Support Actions (CP-CSA) under grant agreement no. RI-261600.

Footnotes

Supplementary data

Supplementary data (code numbers of tested compounds, 1H, 13C NMR and HRMS spectra of inhibitor 16a are given. Docking results of the synthesized compounds and MD simulations of 16a and 31c) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2016.02.040.

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