2-Oxoesters: A Novel Class of Potent and Selective Inhibitors of Cytosolic Group IVA Phospholipase A₂

Abstract

Cytosolic phospholipase A₂ (GIVA cPLA₂) is the only PLA₂ that exhibits a marked preference for hydrolysis of arachidonic acid containing phospholipid substrates releasing free arachidonic acid and lysophospholipids and giving rise to the generation of diverse lipid mediators involved in inflammatory conditions. Thus, the development of potent and selective GIVA cPLA₂ inhibitors is of great importance. We have developed a novel class of such inhibitors based on the 2-oxoester functionality. This functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA₂ inhibitors (X _I(50) values 0.00007–0.00008) and docking studies aid in understanding this selectivity. A methyl 2-oxoester, with a short chain carrying a naphthalene ring, was found to preferentially inhibit the other major intracellular PLA₂, the calcium-independent PLA₂. In RAW264.7 macrophages, treatment with the most potent 2-oxoester GIVA cPLA₂ inhibitor resulted in over 50% decrease in KLA-elicited prostaglandin D₂ production. The novel, highly potent and selective GIVA cPLA₂ inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of inflammatory diseases.

Introduction

In mammals, the phospholipase A₂ (PLA₂) superfamily consists of six types of diverse enzymes: GIV PLA₂ [cytosolic PLA₂ (cPLA₂)], GVI PLA₂ [calcium-independent PLA₂ (iPLA₂)], several groups of secreted PLA₂ (sPLA₂), two groups of platelet-activating factor-acetylhydrolases PLA₂ (PAF-AHs), GXV PLA₂ (lysosomal PLA₂), and GXVI PLA₂ (adipose PLA₂)¹. Among all these enzymes, cPLA₂ is the only PLA₂ that exhibits a marked preference for hydrolysis of arachidonic acid at the sn-2 position of phospholipid substrates². The activation of cPLA₂ results in the production of arachidonic acid and lysophospholipids giving rise to the generation of diverse lipid mediators, such as leukotrienes, prostaglandins, lysophosphatidic acid etc³. Since many of them are involved in the response to inflammation, the regulation of cPLA₂ is of great importance in chronic inflammatory conditions^{1, 4}. In a recent review article, Leslie has summarized the physiological function and the role of cPLA₂ in diseases⁵. The most recent studies on inherited GIVA cPLA₂ deficiency demonstrate the fundamental role of this enzyme in eicosanoid formation and cellular responses in human circulation⁶.

It was thirty years ago, when the first cytosolic PLA₂ activity (now attributed to GIVA cPLA₂ or cPLA₂ α) was reported in human neutrophils and platelets^{7, 8}. The purification, sequence, and cloning of the first human cPLA₂ was reported in 1991^{9, 10}. GIVA cPLA₂ contains 749 amino acids, is an 85 kDa protein, and consists of an N-terminal C2 domain and a C-terminal catalytic domain. The crystal structure of GIVA cPLA₂ was solved by Dessen et al. in 1999¹¹. The catalytic domain of GIVA cPLA₂ utilizes an unusual catalytic dyad, Ser-228/Asp-549, located in the α/β hydrolase domain, to catalyze the hydrolysis of the substrate phospholipid^{12, 13}.

The diverse bioactive lipids produced by the cPLA₂ activity regulate normal physiological processes and disease pathogenesis, and as a consequence, great attention has been given to the development of selective GIVA cPLA₂ inhibitors. The structural diversity of the synthetic inhibitors is summarized in a number of review articles^{1, 14,15,16}. The first synthetic inhibitor of GIVA cPLA₂ was an arachidonic acid derivative, arachidonoyl trifluoromethyl ketone, containing an activated carbonyl functionality¹⁷. Shionogi developed a series of pyrrolidine-based inhibitors, including pyrrophenone (1, Fig. 1), following a high throughput screening approach^{18, 19}. Wyeth has expended major efforts to develop novel indole-based inhibitors, for example, ecopladib (2a, Fig. 1), efipladib (2b, Fig. 1) and giripladib (2c, Fig. 1) as novel therapeutics for inflammatory diseases^20,21,22,23. Giripladib was the most promising among them as it was advanced into a Phase II clinical trial for osteoarthritis, however in 2007 the trial was terminated due to gastrointestinal side effects²⁴. A structurally related GIVA cPLA₂ inhibitor is currently on phase I/II clinical study in healthy volunteers and patients with moderate to severe dermatitis and the estimated date of completion is June 2017²⁵. Our groups have designed and developed long chain 2-oxoamides based on unnatural amino acids, for example compound 3, as GIVA cPLA₂ inhibitors^{26,27,28,29,30,31}. Lehr and coworkers studied a variety of activated carbonyl-based indol-1-yl-propan-2-ones, for example compound 4 (Fig. 1) containing a variety of substituents on the heterocyclic ring to optimize the enzyme-inhibitor binding^{32,33,34,35,36}. Recently, we have reported the new thiazolyl ketone GK470³⁷ (5, Fig. 1) as a GIVA cPLA₂ inhibitor, while Tomoo and colleagues demonstrated a new series of indole-based inhibitors, such as inhibitor 6 ³⁸.

Figure 1

Common inhibitors of phospholipases A₂.

To fully understand the role that each particular PLA₂ type plays in physiological and pathological conditions, and to develop new candidates for the treatment of various inflammatory diseases, potent and selective GIVA cPLA₂ inhibitors are needed. In this work, we present a novel class of potent and selective GIVA cPLA₂ inhibitors and our studies on their synthesis and study of their in vitro inhibitory potency and selectivity.

Results

Design and synthesis of inhibitors

Upon activation by intracellular calcium binding to the C2 domain of GIVA cPLA₂, the enzyme is translocated to the surface of the phospholipid membrane where it extracts a single phospholipid substrate into the active site^{39, 40}. Then, the catalytic active site serine attacks the ester bond of the phospholipid substrate initiating the hydrolysis step. A number of the existing potent GIVA cPLA₂ inhibitors, for example arachidonoyl trifluoromethyl ketone¹⁷, 2-oxoamides^{26,27,28,29,30,31}, indolyl-propanones^{32,33,34,35,36}, thiazolyl ketones³⁷ contain an activated carbonyl group able to interact with the active site serine. In our quest for novel potent and selective GIVA cPLA₂ inhibitors, we envisaged that the 2-oxoester (or α-keto ester) functionality could serve as such an activated carbonyl group. In 1990, it was demonstrated that α-keto ester derivatives of N-protected amino acids and peptides inhibit serine and cysteine proteinases⁴¹, while peptidyl α-keto esters inhibit the serine proteases porcine pancreatic elastase and human neutrophil elastase⁴². Later on, various peptide α-keto-esters and α-keto acids were reported as inhibitors of calpains and other cysteine proteases⁴³ and of hepatitis C virus NS3 protease⁴⁴. It is quite clear that a potential GIVA cPLA₂ inhibitor, in addition to a functionality targeting the active site serine, should contain a lipophilic chain able to mimic the interactions of the substrate arachidonoyl chain with the lipophilic binding site of the enzyme. In addition, a free carboxyl group may contribute significantly to the overall binding of the inhibitor to the enzyme. As we have proposed in the past²⁶, and according to the results of our mechanistic studies using a combination of hydrogen-deuterium exchange mass spectrometry with molecular dynamics simulations³¹, such a carboxyl group may interact with the side chain of the enzyme residue Arg-200. Taken together, we designed compounds containing a 2-oxoester functionality, a lipophilic chain and a free carboxyl group (Fig. 2).

Figure 2

Design of 2-oxoesters.

A variety of 2-hydroxy acids, required for the synthesis of 2-oxoesters, were synthesized as described in Fig. 3. Aldehydes 7a-d were converted into cyanohydrins 8a-d and consequently to 2-hydroxy methyl esters 9a-d by treatment with HCl in methanol. 2-Hydroxy acids 11a-d were obtained by alkaline hydrolysis of 9a-d. In addition, 2-hydroxy methyl esters 9a,b,e were oxidized to the corresponding 2-oxoesters 10a,b,e (Fig. 3). Free 2-oxohexadecanoic acid 12e was synthesized by mild alkaline hydrolysis of 10e using aqueous Cs₂CO₃ in methanol, as depicted in Fig. 3.

Figure 3

Synthesis of 2-hydroxy acids and 2-oxoacids. (a) (i) aq. sol. NaHSO₃, CH₂Cl₂, (ii) KCN, H₂O; (b) 4 N HCl/CH₃OH; (c) Dess-Martin periodinane reagent, dry CH₂Cl₂; (d) NaOH 1 N, CH₃OH; (e) 20% aq. sol. Cs₂CO₃, CH₃OH.

The general route for the synthesis of the designed 2-oxoesters carrying a free carboxyl group is quite straightforward and is depicted in Fig. 4. The key-step was the reaction between the cesium salt of the appropriate 2-hydroxy acids 11a, 11c, 11d and 13a,b with omega-bromo esters 14a,b. The resulting 2-hydroxy esters 15a-h ware then oxidized to the corresponding 2-oxoesters 16a-h using preferably the Dess-Martin periodinane reagent⁴⁵. Removal of the tert-butyl ester protecting group under acidic conditions led to the target compounds 17a-h.

Figure 4

Synthesis of 2-oxoesters. (a) i. 20% aq. sol. Cs₂CO₃, THF, H₂O, ii. Br(CH₂)_nCH₂CH₂COOBu^t, DMF, reflux overnight; (b) Dess-Martin periodinane reagent, dry CH₂Cl₂; (c) 50% CF₃COOH in CH₂Cl₂.

2-Oxoester 19 carrying an ethyl ester group and 2-hydroxyester 20 carrying a free carboxyl group were synthesized as depicted in Fig. 5.

Figure 5

Synthesis of compounds 19 and 20. (a) i. 20% aq. sol. Cs₂CO₃, THF, H₂O, ii. BrCH₂CH₂CH₂COOEt, DMF, reflux overnight; (b) Dess-Martin periodinane reagent, dry CH₂Cl₂; (c) 50% CF₃COOH in CH₂Cl₂.

In vitro inhibition of GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂

All synthesized 2-oxoesters were tested for their in vitro activity on recombinant human GIVA cPLA₂ using mixed micelle assays. In addition, their selectivity over human GVIA iPLA₂ and GV sPLA₂ was also studied using group specific mixed micelle assays_. The activity of these PLA₂s was tested on mixed-micelles containing 100 µM PAPC and 400 µM Triton-X.

The in vitro inhibition of human GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂ was carried out using previously described mixed micelle-based assays^{27, 28, 30}. The inhibition results are presented in Table 1, either as percent inhibition or as X _I(50) values. At first, the percent of inhibition for each PLA₂ enzyme at 0.091 mole fraction of each inhibitor was determined. Then, the X _I(50) values were measured for compounds that displayed greater than 95% inhibition of GIVA cPLA₂. The X _I(50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%.

Table 1 In vitro inhibitory potency and selectivity of 2-oxoesters.

Representative curves for the concentration dependence of the inhibition of GIVA cPLA₂ by 2-oxoesters 17a, 17b and 17d were fit to sigmoidal curves and are presented in Fig. 6.

Figure 6

Inhibition curves for 17a, 17b and 17d. The curves were generated using GraphPad Prism with a nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition versus log(inhibitor concentration). The reported X _I(50) values were calculated from the resultant plots.

Discussion

Methyl 2-oxopalmitate 10e (entry 1, Table 1) weakly inhibited, at a high concentration, both the intracellular enzymes GIVA cPLA₂ and GVIA iPLA₂. However, 2-oxopalmitic acid 12e (entry 2, Table 1) inhibited weakly, but selectively, GIVA cPLA₂. Interestingly, when the 2-oxoester functionality was combined with a long aliphatic chain together with a free carboxyl group at a distance of three carbon atoms, potent inhibition of GIVA cPLA₂ was observed and the inhibitor 17a (GK161) showed a X _I(50) value of 0.00008 (entry 3, Table 1). In addition, this inhibitor was selective and did not inhibit the activities of GVIA iPLA₂ and the secreted GV sPLA₂. This selectivity is in agreement with our previous observations that 2-oxoamides containing a free carboxyl group selectively inhibit GIVA cPLA₂ ^{28, 30}. Given that for the most potent 2-oxoamides present X _I(50) values are not lower than 0.003³⁰, the present 2-oxoester was proven to be a much more potent inhibitor of GIVA cPLA₂. The corresponding 2-hydroxy ester derivative 20 did not present any inhibition of either GIVA cPLA₂ or GVIA iPLA₂ (entry 4, Table 1), demonstrating the importance of the oxoester functionality for the inhibition.

When the long aliphatic chain was replaced by a chain of a similar size containing an aromatic ring, the inhibitory activity over GIVA cPLA₂ was considerably reduced (entry 5, Table 1). Compounds 16c and 19 containing a medium chain carrying an aromatic ring and a protected carboxyl group (either ethyl ester or tert-butyl ester) totally abolished any inhibitory activity (entries 6 and 7, Table 1). In accord with our expectation, the replacement of the long aliphatic chain by a more drug-like chain of four carbon atoms carrying a biphenyl system led again to a potent and selective inhibition of GIVA cPLA₂ (entry 8, Table 1). Inhibitor 17d (GK200) was found to be eight times less potent than 17a showing a X _I(50) value of 0.00068. To extend the structure-activity relationship studies, we either increased the distance between the free carboxyl group and the oxoester functionality or decreased the distance between the aromatic rings and the oxoester functionality. Inhibitor 17e (GK433) proved to be highly potent, slightly better than 17a, presenting a X _I(50) value of 0.00007 (entry 9, Table 1). The importance of the four-carbon atoms distance between the free carboxyl group and the oxoester functionality was clearly demonstrated by the inhibitor 17f (GK452), which presented highly potent inhibition of GIVA cPLA₂ with a X _I(50) value of 0.000078 (entry 10, Table 1). Decrease of the distance between the biphenyl aromatic system and the oxoester functionality (compounds 17g and 17h) resulted in considerable reduction of the potency (entries 11 and 12, Table 1). All the highly potent GIVA cPLA₂ inhibitors 17a, 17d, 17e and 17f presented selectivity, because none of them exhibited any appreciable inhibition of GVIA iPLA₂. In addition, none of the synthesized and tested 2-oxoesters inhibited GV sPLA₂.

Since both the intracellular enzymes GIVA cPLA₂ and GVIA iPLA₂ are serine hydrolases and both utilize a catalytic dyad in their catalytic mechanism, it is likely that cross reactivity may be observed for inhibitors designed to carry a functionality targeting the active site serine. Indeed, such cross reactivity has been observed for several inhibitors containing an activated carbonyl group initially developed to target GIVA cPLA₂. For example, arachidonoyl trifluoromethyl ketone was found to inhibit not only GIVA cPLA₂, but also GVIA iPLA₂. It is apparent that the presence of other groups able to develop appropriate hydrophobic and/or hydrophilic interactions contributes to the overall binding of the inhibitor to the enzyme, determining the inhibitory selectivity over GIVA cPLA₂ or GVIA iPLA₂. We have previously shown that pentafluoroethyl or trifluoromethyl ketones of a four-carbon atom chain carrying an aromatic ring are selective inhibitors of GVIA iPLA₂ ^46,47,48. Inspired by the structures of FKGK11⁴⁶ and FKGK18⁴⁷, we designed simple methyl 2-oxoesters with a linker of four methylene groups between the activated carbonyl group and the aromatic ring. Unfortunately, compound 10a (entry 13, Table 1) carrying a phenyl ring only weakly inhibited GVIA iPLA₂ at a high concentration. On the contrary, compound 10b (GK451) (entry 14, Table 1) carrying a naphthalene ring presented interesting inhibition of GVIA iPLA₂ with a X _I(50) value of 0.0052. At the same time, it presents selectivity, because it only weakly inhibits GIVA cPLA₂ at a high concentration (55% at 0.091 mole fraction), while it does not inhibit at all GV sPLA₂.

To better understand the interaction of 2-oxoesters with GIVA cPLA₂ and GVIA iPLA₂, the most potent GIVA cPLA₂ inhibitor 17f was docked in the active site of either GIVA cPLA₂ or GVIA iPLA₂. For the docking calculations, the structures of GIVA cPLA₂ and GVIA iPLA₂ with two different fluoroketone compounds in the active site were used (GK174: orange color in Fig. 7a and FKGK18: magenta color in Fig. 7b). The binding mode of these two fluoroketones was validated using H/D exchange and MD simulations in a previously published study⁴⁹. A theoretical score of 10.2 kcal/mol indicated that 17f is a tight binder for GIVA cPLA₂. The oxoester moiety forms hydrogen-bonding with the oxyanion hole (Gly197/Gly198), while the carboxylic moiety interacts with Arg200, which was found to stabilize the phosphate group of a phospholipid substrate molecule³⁹. Compared to GK174 (orange color in Fig. 7a) the addition of the carboxylic moiety is responsible for increasing the potency of 17f by 10-fold. This compound exhibits no activity towards GVIA iPLA₂ and it received a low theoretical binding score of 6.3 kcal/mol indicating that is a weak binder. Compared to fluoroketone FKGK18 (magenta color in Fig. 7b) the addition of the carboxylic moiety increases the size of the compound and it cannot be accommodated in the active site of GVIA iPLA₂.

Figure 7

Binding mode of inhibitor 17f in the active site of (a) GIVA cPLA₂ and (b) GVIA iPLA₂.

All the above data, clearly demonstrate that 2-oxoesters consisting of a quite long chain (aliphatic or incorporating aromatic systems like the biphenyl system) in combination with a free carboxyl group at a distance of four or three carbon-atoms from the oxoester functionality are highly potent and selective inhibitors of GIVA cPLA₂. Decreasing the size of the synthetic compound and eliminating the free carboxyl group may change the selectivity. Indeed, a methyl 2-oxoester based on a short chain carrying a naphthalene ring was found to inhibit preferentially GVIA iPLA₂. In other words, it seems that the selectivity of compounds based on the 2-oxoester functionality may be tuned choosing the structural features that ensure the appropriate interactions with each enzyme (either GIVA cPLA₂ or GVIA iPLA₂).

To compare our novel highly potent 2-oxoester inhibitors of GIVA cPLA₂ with the existing inhibitors, we studied the benchmark GIVA cPLA₂ inhibitor 4 in our mixed-micelle assay. This inhibitor, developed by Lehr³², is the most potent inhibitor in the literature presenting an IC₅₀ value of 4.3 nM in a vesicle assay³². In the mixed micelle assay, it was proved equipotent with oxoester 17a with a X _I(50) value of 0.00008 (entry 15, Table 1). In addition, several 2-oxoesters were found to be more potent than the other benchmark GIVA cPLA₂ inhibitor 1 (pyrrophenone), which presents an X _I(50) value of 0.002^{26, 27}. Another important property of a GIVA cPLA₂ inhibitor, is the ClogP value, which is a measure of the hydrophobicity. ClogP represents the calculated partition coefficient in octanol/water on a logarithmic scale. Usually, GIVA cPLA₂ inhibitors suffer from high lipophilicity. For example, the ClogP value of inhibitor 4 is 8.50, while pyrrophenone 1 and giripladib 2c present high lipophilicities too (ClogP 8.29 and 10.75, respectively). Inhibitors with such high values are not expected to present favorable ADME properties according to Lipinski’s rule of five⁵⁰. Although 2-oxoesters 17a and 17e contain a long aliphatic chain, they present lower lipophilicity (ClogP 6.76 and 6.68, respectively), while the 2-oxoesters 17d and 17f carrying the biphenyl system have considerably lower ClogP values (4.78 and 4.70, respectively). The logP value of 17f, measured by HPLC, was found 3.5. Thus, the lipophilicity of 17f is encouraging and this inhibitor is the first example of a highly potent GIVA cPLA₂ inhibitor, which presents a ClogP value lower than 5.

The cellular effect of the most potent GIVA cPLA₂ inhibitor 17f on eicosanoid biosynthesis was studied in macrophages. RAW264.7 macrophages were used as a model system to determine if 17f displays inhibitory activity toward GIVA cPLA₂ in vivo. It is well established that the toll-like receptor 4 (TLR4)-specific agonist Kdo2-lipid A (KLA) leads to GIVA cPLA₂ activation^{51, 52} and release of arachidonic acid in macrophages that is then converted into eicosanoids by cyclooxygenase-2^53,54,55. Previous work has demonstrated that the major eicosanoid produced by KLA stimulated RAW264.7 macrophages is prostaglandin D₂ (PGD₂)⁵⁶. The high levels of PGD₂ compared to background in culture supernatants following KLA stimulation makes it an ideal marker for GIVA cPLA₂ activity in macrophages. Inhibitor 17f did not show cellular toxicity at any concentrations tested as measured by trypan blue exclusion (data not shown). RAW264.7 macrophages were pre-treated with vehicle control, DMSO or 17f (5 μM) for one hour prior to stimulation with KLA (100 ng/mL). Culture supernatants were collected after 24 hours for eicosanoid quantification by LC-MS/MS. Treatment with inhibitor 17f resulted in over 50% decrease in KLA-elicited PGD₂ production by macrophages (Fig. 8). A similar reduction in other minor products including PGE₂, PGF_2α, 11-HETE and 15-HETE was observed (data not shown), suggesting that the inhibition was not specific to PGD₂. This data is consistent with 17f inhibition of GIVA cPLA₂ in living cells.

Figure 8

Inhibitor 17f inhibits KLA-elicited prostaglandin D₂ biosynthesis by macrophages. Macrophages were pre-treated with media (control), vehicle control (DMSO, 0.1%) or inhibitor 17f (5 μM) 1 hr before KLA (100 ng/mL, ■) or mock (□) treatment. Supernatants were collected 24 hr following stimulation for eicosanoid quantification. Graph displays the mean ± SEM of a single experiment containing technical duplicates that is representative of 2 independent experiments. * indicates statistical difference compared to KLA treatment (P ≤ 0.05).

In conclusion, we describe a novel class of GIVA cPLA₂ inhibitors based on the 2-oxoester functionality. This reactive functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA₂ inhibitors. Inhibitors 17a, 17e and 17f present X _I(50) values of 0.00007–0.00008 and are equipotent to the most potent known GIVA cPLA₂ inhibitor. In particular, inhibitors incorporating the biphenyl system, like 17f, present interesting favorable lipophilicity (ClogP values lower than 5). The novel highly potent and selective GIVA cPLA₂ inhibitors may be excellent tools for the study of the role of the enzyme in cells and in animals and may contribute to the development of novel medicinal agents for the treatment of inflammatory diseases.

Methods

General

Chromatographic purification of products was accomplished using Merck Silica Gel 60 (70–230 or 230–400 mesh). Thin-layer chromatography (TLC) was performed on Silica Gel 60 F254 aluminum plates. TLC pots were visualized with UV light and/or phosphomolybdic acid in EtOH. Melting points were determined using a Büchi 530 apparatus and were uncorrected. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury (200 MHz and 50 MHz respectively) in CDCl₃. Chemical shifts are given in ppm, and coupling constants (J) in Hz. Peak multiplicities are described as follows: s, singlet, d, doublet, t, triplet and m, multiplet. Electron spray ionization (ESI) mass spectra were recorded on a Finnigan, Surveyor MSQ Plus spectrometer. Dichloromethane was dried by standard procedures and stored over molecular sieves. All other solvents and chemicals were reagent grade and used without further purification. The purity of all compounds subjected to biological tests was determined by analytical HPLC, and was found to be ≥95%. HPLC analyses were carried out on a Shimadzu LC-2010AHT system and a Merck Chromolith Performance (100 × 4.6 mm) analytical column, using H₂O/MeOH 10/90 v/v, at a flow rate of 1.0 mL/min. HRMS spectra were recorded on a Bruker Maxis Impact QTOF Spectrometer.

Compounds 8a ⁵⁷, 8b ⁵⁷, 9e ⁵⁸, 11a ⁵⁷, 11b ⁵⁷, 13a ⁵⁹, 13b ⁶⁰ have been described elsewhere and their analytical data are in accordance with literature.

Synthesis of cyanohydrins 8c,d

To a stirred solution of aldehyde 7c,d (1.0 mmol) in CH₂Cl₂ (1.4 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 (1 mL) was added. The mixture was cooled to 0 °C and an aqueous solution of KCN (0.25 mL, 1.5 mmol) was added within 2 h under vigorous stirring. The reaction was stirred for 18 h at room temperature and then, water (10 mL) was added and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic phases was washed with brine (30 mL), dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by flash column chromatography [ethyl acetate (EtOAc)/petroleum ether (bp 40–60 °C), 2:8].

6-([1,1′-Biphenyl]-4-yl)-2-hydroxyhexanenitrile (8c)

Yield 80%; White solid; mp: 85–87 °C; ¹H NMR (200 MHz, CDCl₃): δ 7.67–7.14 (m, 9 H), 4.45 (t, J = 6.9 Hz, 1 H), 3.90 (br s, 1 H), 2.69 (t, J = 7.1 Hz, 2 H), 1.89 (q, J = 7.2 Hz, 2 H), 1.79–1.39 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 141.0, 140.9, 138.8, 129.0, 128.7, 127.0, 126.9, 126.5, 119.9, 61.1, 35.2, 34.9, 30.6, 24.2; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₈H₁₉NO, 283.2; found, 283.3; analysis (calcd., found for C₁₈H₁₉NO): C (81.47, 81.18), H (7.22, 7.41), N (5.28, 5.33).

5-([1,1′-Biphenyl]-4-yl)-2-hydroxypentanenitrile (8d)

Yield 76%; White solid; mp: 80–82 °C; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.23 (m, 9 H), 4.48 (t, J = 6.9 Hz, 1 H), 3.80 (br s, 1 H), 2.64 (t, J = 7.0 Hz, 2 H), 1.84 (q, J = 7.0 Hz, 2 H), 1.69–1.57 (m, 2 H); ¹³C NMR (50 MHz, CDCl₃): δ 141.3, 140.8, 138.4, 129.1, 128.7, 127.6, 127.2, 126.8, 118.5, 61.9, 35.1, 34.5, 23.8; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₇H₁₇NO 269.2; found, 269.2; analysis (calcd., found for C₁₇H₁₇NO): C (81.24, 81.02), H (6.82, 6.99), N (5.57, 5.69).

Synthesis of 2-hydroxy esters 9a-d

Cyanohydrin 8a-d (1 mmol) was dissolved in methanolic solution of HCl (10 mL, 4 N) and the reaction mixture was stirred for 24 h at room temperature. The organic solvent was evaporated in vacuo and the remaining solid was dissolved in diethyl ether (10 mL) and re-evaporated. Dilution and evaporation was repeated twice. Then, the product was purified by flash column chromatography [EtOAc-petroleum ether (bp 40–60 °C), 2:8].

Methyl 2-hydroxy-6-phenylhexanoate (9a)

Yield 61%; Yellow oil; ¹H NMR (200 MHz, CDCl₃): δ 7.37–7.04 (m, 5 H), 4.23–4.10 (m, 1 H), 3.77 (s, 3 H), 2.74 (br s, 1 H), 2.62 (t, J = 7.1 Hz, 2 H), 1.92–1.25 (m, 6 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.7, 142.3, 128.3, 128.2, 125.6, 70.3, 52.4, 35.7, 34.1, 31.1, 24.4; MS (m/,z ESI): [M + NH₄]⁺ calcd. for C₁₃H₁₈O₃ 240.2 found, 240.2; analysis (calcd., found for C₁₃H₁₈O₃): C (70.24, 70.01), H (8.16, 8.29).

Methyl 2-hydroxy-6-(naphthalen-2-yl)hexanoate (9b)

Yield 73%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.90–7.20 (m, 7 H), 4.30–4.02 (m, 1 H), 3.76 (s, 3 H), 3.35 (br s, 1 H), 2.97–2.75 (m, 2 H), 1.97–1.34 (m, 6 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.6, 139.8, 133.5, 127.7, 127.5, 127.3, 127.2, 126.2, 125.8, 125.0, 70.3, 52.4, 35.8, 34.1, 30.9, 24.4; MS (m/z, ESI): [M + Na]⁺ calcd. for C₁₇H₂₀O₃ 295.1, found, 295.2; analysis (calcd., found for C₁₇H₂₀O₃): C (74.97, 74.72), H (7.40, 7.62).

Methyl 6-([1,1′-biphenyl]-4-yl)-2-hydroxyhexanoate (9c)

Yield 69%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.70–7.06 (m, 9 H), 4.45 (t, J = 7.0 Hz, 1 H), 3.79 (s, 3 H), 3.00 (br s, 1 H), 2.69 (t, J = 7.1 Hz, 2 H), 1.89 (q, J = 7.5 Hz, 2 H), 1.79–1.36 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.7, 141.0, 140.9, 138.7, 129.0, 128.7, 127.0, 126.9, 70.4, 52.5, 35.2, 34.9, 30.7, 24.2; MS (m/z, ESI): [M + Na]⁺ calcd. for C₁₉H₂₂O₃ 321.1, found, 321.2; analysis (calcd., found for C₁₉H₂₂O₃): C (80.82, 80.61), H (7.85, 7.98).

Methyl 5-([1,1′-biphenyl]-4-yl)-2-hydroxypentanoate (9d)

Yield 71%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.69–7.18 (m, 9 H), 4.40 (t, J = 6.9 Hz, 1 H), 3.76 (s, 3 H), 3.54 (brs, 1 H), 2.65 (t, J = 7.1 Hz, 2 H), 1.84 (q, J = 7.1 Hz, 2 H), 1.64–1.36 (m, 2 H); ¹³C NMR (50 MHz, CDCl₃): δ 176.1, 141.3, 140.8, 138.2, 129.0, 128.9, 127.5, 127.0, 126.8, 70.3, 52.3, 35.5, 34.9, 24.4; MS (m/z, ESI): [M + Na]⁺ calcd. for C₁₈H₂₀O₃ 307.1, found, 307.2; analysis (calcd., found for C₁₈H₂₀O₃): C (76.03, 75.83), H (7.09, 7.27).

Synthesis of 2-oxoesters 10α, 10b, 10e, 16a-h, 19

To a stirred solution of 2-hydroxy esters 9a, 9b, 9e, 15a-h, 18 (1 mmol) in dry CH₂Cl₂ (10 mL) was added Dess-Martin periodinane (1.1 mmol, 0.47 g) and the reaction mixture was stirred for 1.5 h at room temperature. Then, CH₂Cl₂ (5 mL) was added and the organic phase was washed with a mixture of Na₂S₂O₃ 10% and NaHCO₃ 10% (15 mL, 1:1, v/v). Τhe aqueous phase was washed with CH₂Cl₂ (15 mL) and all the organic phases were collected, dried (Na₂SO₄) and evaporated under reduced pressure. The residue was purified by flash column chromatography [EtOAc-petroleum ether (bp 40–60 °C), 2:8].

Methyl 2-oxo-6-phenylhexanoate (10a, GK437)

Yield 66%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.40–7.08 (m, 5 H), 3.84 (s, 3 H), 2.85 (t, J = 6.4 Hz, 2 H), 2.62 (t, J = 6.5 Hz, 2 H), 1.78–1.58 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.0, 161.4, 141.8, 128.3, 128.1, 125.8, 52.9, 39.1, 35.5, 30.6, 22.5; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₃H₁₆O₃ 238.1, found, 238.2; HRMS (m/z, ESI): [M + Na]⁺ calcd. for C₁₃H₁₆O₃, 243.0992; found, 243.0994; analysis (calcd., found for C₁₃H₁₆O₃): C (70.89, 70.58), H (7.32, 7.46).

Methyl 6-(naphthalen-2-yl)-2-oxohexanoate (10b, GK451)

Yield 73%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.90–7.10 (m, 7 H), 3.85 (s, 3 H), 2.92–2.71 (m, 4 H), 1.83–1.49 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.0, 161.4, 139.2, 133.5, 131.9, 127.9, 127.5, 127.4, 127.2, 126.3, 125.9, 125.1, 52.9, 39.1, 35.6, 30.4, 22.5; MS (m/z, ESI): [(M + NH₄)⁺] calcd. for C₁₇H₁₈O₃ 288.2, found, 288.2; HRMS (m/z, ESI): [M + Na]⁺ calcd. for C₁₇H₁₈O₃, 293.1148; found, m/z 293.1149; analysis (calcd., found for C₁₇H₁₈O₃): C (75.53, 75.32), H (6.71, 6.95).

Methyl 2-oxohexadecanoate (10e)

Yield 73%; White solid; mp: 53–55 °C; ¹H NMR (200 MHz, CDCl₃): δ 3.85 (s, 3 H), 2.82 (t, J = 7.2 Hz, 2 H), 1.70–1.51 (m, 2 H), 1.37–1.16 (m, 22 H), 0.86 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.3, 161.5, 52.8, 39.3, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 22.9, 22.6, 14.1; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₇H₃₂O₃ 302.3; found, 302.3⁶¹.

4-(tert-Butoxy)-4-oxobutyl 2-oxohexadecanoate (16a)

Yield 87%; Colorless oil, ¹H NMR (200 MHz, CDCl₃): δ 4.27 (t, J = 6.0 Hz, 2 H), 2.81 (t, J = 7.8 Hz, 2 H), 2.33 (t, J = 6.0 Hz, 2 H), 2.04 (quint, J = 6.0 Hz, 2 H), 1.70–1.50 (m, 2 H), 1.44 (s, 9 H), 1.40–1.15 (m, 22 H), 0.86 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.5, 171.8, 161.2, 80.6, 65.2, 39.3, 31.9, 31.6, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 28.0, 23.8, 22.9, 22.6, 14.1; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₄H₄₄O₅ 430.4; found, 430.4; analysis (calcd., found for C₂₄H₄₄O₅): C (69.86, 69.6), H (10.75, 10.92).

Compound 16b was not isolated and used directly in the next step.

4-(tert-Butoxy)-4-oxobutyl 2-oxo-6-phenylhexanoate (16c, GK192)

Yield 77%; Yellow oil; ¹H NMR (200 MHz, CDCl₃): δ 7.35–7.10 (m, 5 H), 4.26 (t, J = 8.0 Hz, 2 H), 2.84 (t, J = 6.0 Hz, 2 H), 2.70–2.55 (m, 2 H), 2.32 (t, J = 6.0 Hz, 2 H), 2.00 (quint, J = 6.0 Hz, 2 H), 1.70–1.60 (m, 4 H), 1.44 (s, 9 H), ¹³C NMR (50 MHz, CDCl₃): δ 194.1, 171.7, 161.1, 141.8, 128.3, 128.2, 125.8, 80.6, 65.3, 39.1, 35.5, 31.6, 30.6, 28.0, 23.8, 22.5; HRMS (m/z, ESI): [M + Na]⁺calcd. for C₂₀H₂₈O₅ 371.1829; found, 371.1831; analysis (calcd., found for C₂₀H₂₈O₅): C (68.94, 68.66), H (8.10, 8.29).

4-tert-Butoxy-4-oxobutyl 6-(biphenyl-4-yl)-2-oxohexanoate (16d)

Yield 86%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.96–7.16 (m, 9 H), 4.28 (t, J = 6.4 Hz, 2 H), 2.97–2.83 (m, 2 H), 2.77–2.62 (m, 2 H), 2.42–2.28 (m, 2 H), 2.11–1.55 (m, 6 H), 1.45 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.1, 171.8, 161.0, 140.9, 138.7, 129.0, 128.9, 128.7, 128.6, 127.2, 127.0, 126.9, 80.6, 65.3, 39.1, 35.1, 31.6, 30.5, 28.0, 23.7, 22.5; MS (m/z, ESI): [M + Na]⁺ calcd. for C₂₆H₃₂O₅ 447.2; found, 447.0; analysis (calcd., found for C₂₆H₃₂O₅): C (73.56, 73.35), H (7.60, 7.78).

5-(tert-Butoxy)-5-oxopentyl 2-oxohexadecanoate (16e)

Yield 78%; White oil; ¹H NMR (200 MHz, CDCl₃): δ 4.23 (t, J = 6.9 Hz, 2 H), 2.79 (t, J = 7.3 Hz, 2 H), 2.24 (t, J = 6.9 Hz, 2 H), 1.80–1.45 (m, 4 H), 1.44 (s, 9 H), 1.30–1.15 (s, 24 H), 0.85 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.8, 172.6, 161.5, 80.5, 66.0, 39.5, 35.0, 32.1, 29.9, 29.8, 29.6, 29.5, 29.1, 28.3, 27.9, 23.1, 22.9, 21.6, 14.3; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₅H₄₆O₅ 444.4; found, 444.3; analysis (calcd., found for C₂₅H₄₆O₅): C (70.38, 70.17), H (10.87, 11.05).

5-(tert-Butoxy)-5-oxopentyl 6-([1,1′-biphenyl]-4-yl)-2-oxohexanoate (16f)

Yield 63%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.66–7.19 (m, 9 H), 4.25 (t, J = 6.0 Hz, 2 H), 2.87 (t, J = 6.3 Hz, 2 H), 2.68 (t, J = 6.1 Hz, 2 H), 2.26 (t, J = 7.0 Hz, 2 H), 1.86–1.53 (m, 8 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.3, 172.4, 161.1, 141.0, 138.8, 129.0, 128.8, 128.7, 127.2, 127.0, 126.9, 80.3, 65.9, 39.1, 35.2, 34.8, 30.6, 28.1, 27.7, 22.5, 21.3; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₇H₃₄O₅ 456.3; found, 456.3; analysis (calcd., found for C₂₇H₃₄O₅): C (73.95, 73.75), H (7.81, 7.99).

4-(tert-Butoxy)-4-oxobutyl 5-([1,1′-biphenyl]-4-yl)-2-oxopentanoate (16 g)

Yield 65%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.20 (m, 9 H), 4.27 (t, J = 6.4 Hz, 2 H), 2.88 (t, J = 7.2 Hz, 2 H), 2.70 (t, J = 7.5 Hz, 2 H), 2.33 (t, J = 7.2 Hz, 2 H), 2.10–1.90 (m, 4 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.3, 172.1, 161.3, 141.2, 140.5, 139.3, 129.1, 128.9, 127.4, 127.3, 127.2, 80.9, 65.6, 38.8, 34.6, 31.9, 28.3, 24.7, 24.0; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₅H₃₀O₅ 428.2; found, 428.3; analysis (calcd., found for C₂₅H₃₀O₅): C (73.15, 72.97), H (7.37, 7.56).

5-(tert-Butoxy)-5-oxopentyl 5-([1,1′-biphenyl]-4-yl)-2-oxopentanoate (16 h)

Yield 61%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.20 (m, 9 H), 4.24 (t, J = 6.0 Hz, 2 H), 2.87 (t, J = 6.3 Hz, 2 H), 2.69 (t, J = 7.5 Hz, 2 H), 2.25 (t, J = 5.9 Hz, 2 H), 2.20–1.90 (m, 2 H), 1.90–1.54 (m, 4 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃); δ 194.4, 172.7, 161.3, 141.2, 140.5, 139.3, 129.1, 128.9, 127.4, 127.3, 127.2, 80.6, 66.2, 38.8, 35.1, 34.6, 28.3, 27.9, 24.7, 21.6; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₆H₃₂O₅ 442.3; found, 442.3; analysis (calcd., found for C₂₆H₃₂O₅): C (73.56, 73.32), H (7.60, 7.82).

4-Ethoxy-4-oxobutyl 2-oxo-6-phenylhexanoate (19, GK194)

Yield 73%; Yellowish oil, ¹H NMR (200 MHz, CDCl₃): δ 7.30–7.10 (m, 5 H), 4.28 (t, J = 8.0 Hz, 2 H), 4.13 (q, J = 6.0 Hz, 2 H) 2.84 (t, J = 6.0 Hz, 2 H), 2.70–2.55 (m, 2 H), 2.41 (t, J = 8.0 Hz, 2 H), 2.15–1.95 (m, 2 H), 1.70–1.55 (m, 4 H), 1.24 (t, J = 6.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.0, 172.4, 161.0, 141.8, 128.3, 128.2, 125.7, 65.2, 60.5, 39.0, 35.5, 30.6, 30.5, 23.6, 22.5, 14.1; MS (m/z, ESI): [M + NH₄]⁺calcd. for C₁₈H₂₄O₅ 338.2; found, 338.2;HRMS (m/z, ESI): [M + Na]⁺ calcd for C₁₈H₂₄O₅ 343.1516; found, 343.1512; analysis (calcd., found for C₁₈H₂₄O₅): C (67.48, 67.19), H (7.55, 7.61).

Synthesis of 2-hydroxy acids 11α-e

To a stirred solution of 2-hydroxy ester 9a-e (1 mmol) in methanol (10 mL), aqueous NaOH (1.1 mL, 1 N) was added and the reaction mixture was stirred overnight at room temperature. The organic solvent was evaporated in vacuo to dryness and then aqueous HCl 1 N was added until acidic pH. The aqueous phase was washed with EtOAc (3 × 10 mL). Finally, the organic phase was dried (Na₂SO₄) and evaporated under reduced pressure.

6-([1,1′-Biphenyl]-4-yl)-2-hydroxyhexanoic acid (11c)

Yield 99%; White solid; mp: 143–145 °C; ¹H NMR (200 MHz, CDCl₃): δ 10.43 (s, 1 H), 7.67–7.02 (m, 9 H), 5.00 (br s, 1 H) 4.22 (t, J = 6.0 Hz, 1 H), 2.59 (t, J = 7.2 Hz, 2 H), 2.03–1.37 (m, 6 H); ¹³C NMR (50 MHz, CDCl₃): δ 176.6, 141.2, 140.6, 138.1, 129.1, 128.3, 128.2, 126.5, 126.4, 69.8, 34.9, 33.6, 30.8, 24.3; MS (m/z, ESI): [M-H]⁻ calcd. for C₁₈H₂₀O₃ 283.1; found, 283.1; analysis (calcd., found for C₁₈H₂₀O₃): C (76.03, 75.85), H (7.09, 7.25).

5-([1,1′-Biphenyl]-4-yl)-2-hydroxypentanoic acid (11d)

Yield 64% (over two steps); Light violet viscous oil; ¹H NMR (200 MHz, CDCl₃): δ 10.60 (s, 1 H), 7.66–7.06 (m, 9 H), 4.98 (s, 1 H), 4.29 (t, J = 6.0 Hz, 1 H), 2.68 (t, J = 6.4 Hz, 2 H), 1.99–1.68 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 178.4, 141.3, 141.2, 139.0, 129.1, 129.0, 128.9, 127.3, 127.2, 70.3, 35.3, 33.9, 26.8; MS (m/z, ESI): [M-H]⁻ calcd. for C₁₇H₁₈O₃ 269.1; found, 269.1; analysis (calcd., found for C₁₇H₁₈O₃): C (75.53, 75.31), H (6.71, 6.87).

2-Oxohexadecanoic acid (12e)

To a stirred solution of 9e (0.35 mmol, 100 mg) in MeOH (3.5 mL), aqueous Cs₂CO₃ 20% (w/v) (1.7 mL, 1.0 mmol) was added, and the reaction mixture was stirred at room temperature. The reaction progress was monitored by TLC, until completion. The organic solvent was evaporated in vacuo to dryness, water was added (10 mL) and then aqueous HCl 1 N was added until acidic pH. The aqueous phase was washed with EtOAc (3 × 10 mL). Finally, the organic phase was dried over Na₂SO₄ and evaporated under reduced pressure. Yield 32%; White solid; mp: 66–68 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.02 (br s, 1 H), 2.93 (t, J = 7.2 Hz, 2 H), 1.76–1.51 (m, 2 H), 1.43–1.05 (m, 22 H), 0.88 (t, J = 6.6 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.8, 162.5, 39.3, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 22.9, 22.7, 14.1; MS (m/z, ESI): [M-H]⁻ calcd. for C₁₆H₃₀O₃ 269.2; found, 269.2⁶².

Synthesis of 2-hydroxy esters 15a-h and 18

To a stirred solution of 2-hydroxy acids 11a, 11c, 11d, 13a,b (1 mmol) in tetrahydrofuran (THF) (6 mL), water (0.6 mL) and few drops of aqueous CsCO₃ 20% (w/v) were added in order to adjust pH in neutral value. The organic solvent was evaporated in vacuo and the residue was dissolved in N,N-dimethylformamide (DMF) (15 mL). Subsequently, tert-butyl 5-bromoalkanooate 14a,b or ethyl 4-bromobutyrate (1.2 mmol) was added and the reaction mixture was refluxed for 72 h. Water (20 mL) was then added and the reaction mixture was washed with EtOAc (2 × 20 mL). The organic phase was dried (Na₂SO₄) and evaporated under reduced pressure. The residue was purified by flash column chromatography [EtOAc-petroleum ether (bp 40–60 °C), 1:9 or 2:8].

4-(tert-Butoxy)-4-oxobutyl 2-hydroxyhexadecanoate (15a)

Yield 44%; Yellow oil, ¹H NMR (200 MHz, CDCl₃): δ 4.25–4.10 (m, 3 H), 2.73 (br s, 1 H), 2.30 (t, J = 6.0 Hz, 2 H), 1.94 (qu, J = 6.0 Hz, 2 H), 1.60–1.45 (m, 2 H), 1.43 (s, 9 H), 1.40–1.20 (m, 24 H), 0.86 (t, J = 6.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.3, 171.9, 80.6, 70.4, 64.5, 34.4, 31.9, 31.7, 29.6, 29.5, 29.4, 29.3, 28.0, 24.7, 24.0, 22.6, 14.1; MS (m/z, ESI): [M + NH₄]⁺ cald. for C₂₄H₄₆O₅ 432.4; found, 432.3; analysis (calcd., found for C₂₄H₄₆O₅): C (69.52, 69.36), H (11.18, 11.29).

4-(tert-Butoxy)-4-oxobutyl 6-(4-(hexyloxy)phenyl)-2-hydroxyhexanoate (15b)

Yield 35%; Yellowish oil, ¹H NMR (200 MHz, CDCl₃): δ 7.08 (d, J = 8.6 Hz, 2 H), 6.82 (d, J = 8.6 Hz, 2 H), 4.27–4.12 (m, 3 H), 3.93 (t, J = 6.0 Hz, 2 H), 2.78 (br s, 1 H), 2.58 (t, J = 7.0 Hz, 2 H), 2. 30 (t, J = 7.0 Hz, 2 H), 1.95 (q, J = 7.0 Hz, 2 H), 1.85–1.47 (m, 8 H), 1.46 (s, 9 H) 1.45–1.20 (m, 6 H), 0.91 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.1, 172.8, 157.1, 134.1, 129.1, 114.2, 80.3, 70.3, 67.9, 64.4, 34.7, 34.2, 31.5, 31.3, 30.3, 29.2, 28.0, 25.7, 24.4, 23.6, 22.5, 14.0; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₆H₄₂O₆ 468.3; found, 468.1; analysis (calcd., found for C₂₆H₄₂O₆): C (69.30, 69.08), H (9.40, 9.61).

4-(tert-Butoxy)-4-oxobutyl 2-hydroxy-6-phenylhexanoate (15c)

Yield 50%; Yellow oil, ¹H NMR (200 MHz, CDCl₃): δ 7.33–7.08 (m, 5 H), 4.23–4.10 (m, 3 H), 2.86 (br s, 1 H), 2.61 (t, J = 7.0 Hz, 2 H), 2.28 (t, J = 7.0 Hz, 2 H), 2.00–1.75 (m, 2 H), 1.70–1.45 (m, 6 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.1, 162.8, 142.3, 128.3, 128.1, 125.6, 80.3, 70.3, 64.5, 36.5, 35.7, 34.2, 31.5, 28.0, 24.4, 23.9; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₀H₃₀O₅ 368.2; found, 368.3; analysis (calcd., found for C₂₀H₃₀O₅): C (68.55, 68.34), H (8.63, 8.81).

4-tert-Butoxy-4-oxobutyl 6-(biphenyl-4-yl)-2-hydroxyhexanoate (15d)

Yield 61%; Oil, ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.19 (m, 9 H), 4.41–4.00 (m, 3 H), 2.75 (br s, 1 H), 2.67 (t, J = 7.4 Hz, 2 H), 2.30 (t, J = 7.4 Hz, 2 H), 2.03–1.48 (m, 8 H), 1.45 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.1, 171.8, 141.4, 138.5, 129.5, 128.7, 128.6, 126.9, 126.8, 80.5, 70.2, 64.5, 35.3, 34.2, 31.6, 31.1, 28.0, 24.4, 23.9; MS (m/z, ESI): [M + Na]⁺ calcd. for C₂₆H₃₄O₅ 449.2; found, 449.2; analysis (calcd., found for C₂₆H₃₄O₅): C (73.21, 73.00), H (8.03, 8.21).

5-(tert-Butoxy)-5-oxopentyl 2-hydroxyhexadecanoate (15e)

Yield 48%; Light yellow oil; ¹H NMR (200 MHz, CDCl₃): δ 4.19–4.05 (m, 3 H), 2.84 (br s, 1 H), 2.21 (t, J = 6.7 Hz, 2 H), 1.70–1.45 (m, 8 H), 1.40 (s, 9 H), 1.30–1.15 (m, 22 H), 0.83 (t, J = 6.3 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.6, 172.7, 80.5, 70.7, 65.3, 35.0, 34.6, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 28.2, 28.1, 25.0, 22.9, 21.6, 14.3; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₅H₄₈O₅ 446.4; found, 446.3; analysis (calcd., found for C₂₅H₄₈O₅): C (70.05, 69.89), H (11.29, 11.44).

5-(tert-Butoxy)-5-oxopentyl 6-([1,1′-biphenyl]-4-yl)-2-hydroxyhexanoate (15f)

Yield 70%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.14 (m, 9 H), 4.24–4.03 (m, 3 H), 2.70–2.54 (m, 3 H), 2.24 (t, J = 6.6 Hz, 2 H), 1.86–1.53 (m, 10 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.3, 172.5, 141.5, 138.6, 129.5, 128.8, 128.7, 127.0, 126.9, 80.3, 70.3, 65.2, 35.4, 34.8, 34.3, 31.1, 28.1, 27.9, 24.5, 21.4; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₇H₃₆O₅ 458.3; found, 458.2; analysis (calcd., found for C₂₇H₃₆O₅): C (73.61, 73.37), H (8.24, 8.39).

4-(tert-Butoxy)-4-oxobutyl 5-([1,1′-biphenyl]-4-yl)-2-hydroxypentanoate (15g)

Yield 26%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.20 (m, 9 H), 4.21 (m, 3 H), 2.85 (br s, 1 H), 2.69 (t, J = 7.0 Hz, 2 H), 2.29 (t, J = 7.2 Hz, 2 H), 2.06–1.66 (m, 6 H), 1.45 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.2, 172.6, 141.1, 139.2, 129.5, 128.8, 127.3, 127.2, 127.0, 80.8, 69.9, 64.8, 35.9, 34.8, 34.4, 28.4, 26.7, 21.5; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₅H₃₂O₅ 430.3; found, 430.3; analysis (calcd., found for C₂₅H₃₂O₅): C (72.79, 72.60), H (7.82, 7.92).

5-(tert-Butoxy)-5-oxopentyl 5-([1,1′-biphenyl]-4-yl)-2-hydroxypentanoate (15h)

Yield 54%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 7.64–7.18 (m, 9 H), 4.30–4.08 (m, 3 H), 2.87 (br s, 1 H), 2.68 (t, J = 6.0 Hz, 2 H), 2.23 (t, J = 5.9 Hz, 2 H), 1.90–1.50 (m, 8 H), 1.44 (s, 9 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.5, 172.8, 141.3, 139.0, 129.0, 128.9, 127.3, 127.2, 127.1, 80.6, 70.5, 65.5, 35.3, 35.1, 34.2, 28.3, 28.1, 26.8, 21.7; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₆H₃₄O₅ 444.3; found, 444.3; analysis (calcd., found for C₂₆H₃₄O₅): C (73.21, 73.07), H (8.03, 8.19).

4-Ethoxy-4-oxobutyl 2-hydroxy-6-phenylhexanoate (18)

Yield 57%; Yellow oil, ¹H NMR (200 MHz, CDCl₃): δ 7.30–7.05 (m, 5 H), 4.24–4.00 (m, 5 H), 2.92 (br s, 1 H), 2.61 (t, J = 7.1 Hz, 2 H), 2.36 (t, J = 6.0 Hz, 2 H), 1.96 (t, J = 7.1 Hz, 2 H), 1.80–1.60 (m, 4 H), 1.60–1.40 (m, 2 H), 1.24 (t, J = 6.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 175.1, 172.5, 142.2, 128.2, 128.1, 125.6, 70.2, 64.4, 60.5, 35.6, 34.1, 31.0, 30.5, 24.4, 23.8, 14.1; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₈H₂₆O₅ 340.2; found, 340.3; analysis (calcd., found for C₁₈H₂₆O₅): C (67.06, 66.93), H (8.13, 8.28).

Synthesis of compounds 17a-h and 20

A solution of tert-butyl ester 16a-h and 15a (1 mmol) in 50% trifluoroacetic acid (TFA) in CH₂Cl₂ (10 mL) was stirred for 1 h at room temperature. The organic solvent was evaporated under reduced pressure and then CH₂Cl₂ was added and re-evaporated twice. The product was purified by precipitation with a mixture of EtOAc and petroleum ether (5:95, v/v, 10 mL) or by column chromatography (CH₂Cl₂-MeOH, 95:5).

4-((2-Oxohexadecanoyl)oxy)butanoic acid (17a, GK161)

Yield 85%; White solid; mp: 76–78 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.25 (br s, 1 H), 4.32 (t, J = 6.0 Hz, 2 H), 2.82 (t, J = 6.0 Hz, 2 H), 2.51 (t, J = 6.0 Hz, 2 H), 2.15 (q, J = 6.0 Hz, 2 H), 1.80–1.50 (m, 2 H), 1.50–1.20 (m, 22 H), 0.88 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.3, 178.6, 161.1, 65.0, 39.3, 31.9, 30.3, 29.6, 29.6, 29.4, 29.3, 29.3, 28.9, 23.4, 22.9, 22.7, 14.1; HRMS (m/z, ESI): [M-H]⁻ calcd. for C₂₀H₃₆O₅ 355.2490; found, 355.2487; analysis (calcd., found for C₂₀H₃₆O₅): C (67.38, 67.12), H (10.18, 10.39).

4-((6-(4-(Hexyloxy)phenyl)-2-oxohexanoyl)oxy)butanoic acid (17b, GK186)

Yield 54%; Low melting point white solid; ¹H NMR (200 MHz, CDCl₃): δ 9.20 (br s, 1 H), 7.04 (d, J = 8.6 Hz, 2 H), 6.77 (d, J = 8.6 Hz, 2 H), 4.26 (t, J = 7.0 Hz, 2 H), 3.89 (t, J = 7.0 Hz, 2 H), 2.81 (t, J = 7.0 Hz, 2 H), 2.60–2.45 (m, 2 H), 2.39 (t, J = 7.0 Hz, 2 H), 2.00 (q, J = 7.0 Hz, 2 H), 1.72 (t, J = 7.0 Hz, 2 H), 1.65–1.50 (m, 4 H), 1.48–1.35 (m, 2 H), 1.35–1.20 (m, 4 H), 0.86 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.4, 178.1, 161.8, 157.1, 134.1, 129.1, 114.2, 67.9, 65.4, 34.7, 34.2, 31.5, 30.3, 29.2, 28.0, 25.7, 24.4, 23.6, 22.5, 14.0; MS (m/z, ESI): [M-H]⁻ calcd. for C₂₂H₃₂O₆ 391.2; found, 391.4; HRMS (m/z, ESI): [M-H]⁻ calcd. for C₂₂H₃₂O₆ 391.2126; found, 391.2122; analysis (calcd., found for C₂₂H₃₂O₆): C (67.32, 67.13), H (8.22, 8.39).

4-((2-Oxo-6-phenylhexanoyl)oxy)butanoic acid (17c)

Yield 60%; Colorless oil; ¹H NMR (200 MHz, CDCl₃): δ 9.23 (br s, 1 H), 7.32–7.05 (m, 5 H), 4.29 (t, J = 6.0 Hz, 2 H), 2.84 (t, J = 8.0 Hz, 2 H), 2.63 (t, J = 8.0 Hz, 2 H), 2.48 (t, J = 6.0 Hz, 2 H), 2.06 (q, J = 8.0 Hz, 2 H), 1.75–1.55 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.3, 178.9, 161.3, 142.1, 128.6, 128.1, 126.1, 65.3, 39.3, 35.8, 30.8, 30.5, 23.6, 22.7; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₁₆H₂₀O₅ 310.2; found, 310.1; analysis (calcd., found for C₁₆H₂₀O₅): C (65.74, 65.53), H (6.90, 7.08).

4-(6-(Biphenyl-4-yl)-2-oxohexanoyloxy)butanoic acid (17d, GK200)

Yield 94%; White solid; mp: 101–103 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.25 (br s, 1 H),7.63–7.17 (m, 9 H), 4.37–4.21 (m, 2 H), 2.93–2.79 (m, 2 H), 2.75–2.58 (m, 2 H), 2.55–2.40 (m, 2 H), 2.14–1.95 (m, 2 H), 1.81–1.59 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.0, 178.7, 160.9, 141.0, 140.2, 138.7, 128.8, 128.7, 127.2, 127.0, 126.9, 65.0, 39.1, 35.1, 30.5, 30.2, 23.3, 22.5; MS (m/z, ESI): [M-H]⁻ calcd. for C₂₂H₂₄O₅ 367.2; found, 367.3; HRMS (m/z, ESI): [M-H]⁻ calcd. for C₂₂H₂₄O₅ 367.1551; found, 367.1544; analysis (calcd., found for C₂₂H₂₄O₅): C (71.72, 71.49), H (6.57, 6.79).

5-((2-Oxohexadecanoyl)oxy)pentanoic acid (17e, GK433)

Yield 66%; White solid; mp: 77–79 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.28 (br s, 1 H), 4.25 (t, J = 6.0 Hz, 2 H), 2.80 (t, J = 7.3 Hz, 2 H), 2.40 (t, J = 6.8 Hz, 2 H), 1.88–1.46 (m, 6 H), 1.34–1.15 (m, 22 H), 0.85 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.8, 179.6, 161.4, 65.9, 39.6, 33.6, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.2, 27.9, 23.2, 22.9, 21.2, 14.4; HRMS (m/z, ESI): [M-H]⁻ calcd. for C₂₁H₃₈O₅ 369.2646; found, 369.2660; analysis (calcd., found for C₂₁H₃₈O₅): C (68.07, 67.82), H (10.34, 10.52).

5-((6-([1,1′-Biphenyl]-4-yl)-2-oxohexanoyl)oxy)pentanoic acid (17f, GK452)

Yield 91%; White solid; mp: 83–85 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.25 (bs, 1 H), 7.64–7.12 (m, 9 H), 4.25 (t, J = 7.0 Hz, 2 H), 2.87 (t, J = 6.0 Hz, 2 H), 2.67 (t, J = 5.9 Hz, 2 H), 2.40 (t, J = 6.3 Hz, 2 H), 1.89–1.54 (m, 8 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.2, 179.0, 161.1, 141.0, 138.7, 128.8, 128.7, 127.0, 126.9, 65.7, 39.1, 35.2, 33.2, 30.6, 27.6, 22.5, 20.9; MS (m/z, ESI): [M + NH₄]⁺ calcd. for C₂₃H₂₆O₅ 400.2; found, 400.2; HRMS (m/z, ESI): [M + Na]⁺ calcd. for C₂₃H₂₆O₅ 405.1672; found, 405.1677; analysis (calcd., found for C₂₃H₂₆O₅): C (72.23, 72.04), H (6.85, 6.99).

4-((5-([1,1′-Biphenyl]-4-yl)-2-oxopentanoyl)oxy)butanoic acid (17g, GK457)

Yield 47%; Light yellow solid; mp: 58–60 °C; ¹H NMR (200 MHz, CDCl₃): δ 10.06 (br s, 1 H), 7.66–7.18 (m, 9 H), 4.29 (t, J = 6.3 Hz, 2 H), 2.88 (t, J = 7.2 Hz, 2 H), 2.70 (t, J = 8.0 Hz, 2 H), 2.49 (t, J = 7.2 Hz, 2 H), 2.15–1.90 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.1, 178.2, 161.1, 141.2, 140.2, 139.3, 129.1, 128.9, 127.4, 127.3, 127.2, 65.3, 38.7, 34.5, 30.4, 24.6, 23.6; HRMS (m/z, ESI): [M + Na]⁺ calcd. for C₂₁H₂₂O₅ 377.1359; found, 377.1357; analysis (calcd., found for C₂₁H₂₂O₅): C (71.17, 71.02), H (6.26, 6.45).

5-((5-([1,1′-Biphenyl]-4-yl)-2-oxopentanoyl)oxy)pentanoic acid (17h, GK458)

Yield 65%; Light yellow solid; mp: 88–90 °C; ¹H NMR (200 MHz, CDCl₃): δ 9.81 (br s, 1 H), 7.65–7.18 (m, 9 H), 4.25 (t, J = 6.9 Hz, 2 H), 2.88 (t, J = 7.2 Hz, 2 H), 2.71 (t, J = 7.5 Hz, 2 H), 2.40 (t, J = 6.7 Hz, 2 H), 2.12–1.90 (m, 2 H), 1.81–1.65 (m, 4 H); ¹³C NMR (50 MHz, CDCl₃): δ 194.4, 179.5, 161.3, 141.2, 140.5, 139.3, 129.1, 128.9, 127.4, 127.3, 127.2, 66.0, 38.8, 34.6, 33.6, 27.9, 24.7, 21.2. HRMS (m/z, ESI): [M + Na]⁺ calcd. for C₂₂H₂₄O₅ 391.1516; found, 391.1504; analysis (calcd., found for C₂₂H₂₄O₅): C (71.72, 71.48), H (6.57, 6.71).

4-((2-Hydroxyhexadecanoyl)oxy)butanoic acid (20, GK515)

Yield 85%; Low melting point white solid; ¹H NMR (200 MHz, CDCl₃): δ 9.26 (br s, 1 H), 4.30–4.10 (m, 3 H), 2.78 (br s, 1 H), 2.46 (t, J = 6.0 Hz, 2 H), 2.10–1.90 (m, 2 H), 1.85–1.50 (m, 4 H), 1.50–1.10 (m, 22 H), 0.87 (t, J = 7.0 Hz, 3 H); ¹³C NMR (50 MHz, CDCl₃): δ 178.2, 175.4, 70.5, 64.4, 34.4, 31.9, 30.3, 29.7, 29.6, 29.5, 29.3, 24.8, 23.7, 22.7, 14.1; HRMS (m/z, ESI): [M-H]⁻ calcd. for C₂₀H₃₈O₅ 357.2646; found, 357.2639; analysis (calcd., found for C₂₀H₃₈O₅): C (67.00, 66.81), H (10.68, 10.89).

In vitro PLA₂ activity assay

The activities of human GVIA iPLA₂, GIV cPLA₂ and GV sPLA₂ were determine using a group-specific mixed micelle modified Dole assay^{27, 28, 30}. The substrate was prepared using slightly different conditions for each enzyme to achieve optimum activity: (i) GIVA cPLA₂ mixed micelle substrate consisted of 400 μM Triton X-100, 95.3 μM PAPC, 1.7 μM arachidonyl-1-¹⁴C PAPC, and 3 μM phosphatidyl inositol (4,5)-bisphosphate (PIP₂) in a buffer containing 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 90 μM CaCl₂, 2 mM dithiothreitol (DTT), and 0.1 mg/ml bovine serum albumin (BSA); (ii) GVIA iPLA₂ mixed micelle substrate consisted of 400 μM Triton X-100, 98.3 μM 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), and 1.7 μM arachidonyl-1-¹⁴C PAPC in a buffer containing 100 mM HEPES pH 7.5, 2 mM adenosine triphosphate (ATP), and 4 mM DTT; and (iii) GV sPLA₂ mixed micelles substrate consisted of 400 μM Triton X-100, 98.3 μM PAPC, and 1.7 μM arachidonyl-1-¹⁴C PAPC in a buffer containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0, and 5 mM CaCl₂. The compounds were initially screened at 0.091 mole fraction (5 μL of 5 mM inhibitor in dimethyl sulfoxide (DMSO)) in substrate (495 µL). X _I(50) was determined for compounds exhibiting greater than 95% inhibition. Inhibition curves were generated using GraphPad Prism 5.0 and the non-linear regression by plotting percentage of inhibition vs log (mole fraction) to calculate the reported X _I(50) and its associated error.

Docking Calculations

Enzyme structures were optimized using the PPW. The structures of the inhibitors were sketched using Maestro sketcher and they were optimized using LigPrep. Glide was used for the rigid-docking of the compounds into the enzyme active site. The grid required for the docking procedure was generated using a scaling factor of 1.0 and partial charge cutoff of 0.25, while X, Y, Z dimensions of the inner box were set to 12 Å. For the inhibitor docking a scaling factor of 0.8 and partial charge cutoff of 0.15 were used that allow complete flexibility of the structures. The poses were selected according to the binding mode and the XP GScore. The Glide Extra-Precision (XP) scoring function was used for the calculations⁶³.

Macrophage Eicosanoid Production

RAW264.7 murine macrophage cells (ATCC #TIB-71) were maintained at 37 °C, 5% CO₂ in DMEM (Life Technologies 11995–065) containing 10% FBS (Gemini), 100 U/mL penicillin/streptomycin, 1 mM sodium pyruvate and 4 mM L-glutamine. Macrophages were plated in 12-well tissue culture plates in 1 mL phenol red-free DMEM (Life Technologies) at a concentration of 5 × 10⁵ macrophages per well and were allowed to adhere for 24 hours. Wells receiving inhibitor treatment were spiked with 17f to a final concentration of 5 μM and incubated for 1 hour at 37 °C. Kdo2-Lipid A (KLA; Avanti Polar Lipids) was then added to a final concentration of 100 ng/mL. Supernatants were collected at 24 hours for eicosanoid quantification. Cells were washed 2 times with 1 mL PBS and then collected in 1 mL PBS for determination of total protein concentration using a Pierce BCA assay kit (ThermoFisher). Supernatants and cellular material were stored at −80 °C until analysis. Supernatants were thawed on ice, and then spiked with 100 μL of an internal standard mix in ethanol (100 pg/μL; Cayman). Samples were purified via solid-phase extraction (SPE) and prepared for eicosanoid analysis as described in detail previously⁶⁴. Briefly, following SPE, 10 μL of each sample was separated by reversed-phase liquid chromatography over 5.3 minutes using a gradient of the mobile phase A [water:acetonitrile:acetic acid (60:40:0.02; v/v/v)] and mobile phase B [acetonitrile:isopropanol (50:50; v/v)] on a 2.1 × 100 mm Acuity UPLC ® BEH Shield RP18 1.7 μm column. Online UPLC-electrospray ionization MS/MS quantitation of eicosanoids was performed on a QTRAP 6500 hybrid quadrupole/linear ion-trap mass spectrometer (AB Sciex) via multiple reaction monitoring (MRM) in negative ion mode. Eicosanoids were quantified by comparing the MRM signal and retention time to a pure standard. GraphPad Prism 7.0 was used for statistical analysis. Statistical significance was determined by one-way ANOVA analysis of variance and a Dunnett’s post-test comparing all columns to KLA treatment, P ≤ 0.05.