Asymmetric Synthesis of Saturated and Unsaturated Hydroxy Fatty Acids (HFAs) and Study of Their Antiproliferative Activity

Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-(S)-Hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA) and 7-(R)- and (S)-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of (R)- and (S)-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-(S)-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression.


Introduction
Among the diverse fatty acids (FAs), hydroxy fatty acids (HFAs) constitute a unique class, which is characterized by the presence of a hydroxyl group attached to a long aliphatic chain.Although they comprise a relatively small class of lipids, HFAs have attracted attention, due to their importance as components of animal and plant tissues, engaging in diverse biological functions [1].They are classified by the position of the hydroxyl functionality (defined by the number of the carbon atom, which carries the hydroxyl, or a Greek letter such as α, β, or ω, ω-1, etc.), the number of hydroxyl groups (mono, di-or poly) and the nature of the long chain.Special attention has been paid to 2-hydroxy fatty acids (2HFAs) and 3-hydroxy fatty acids (3HFAs) (Figure 1), which are widespread in nature, being involved in oxidative bio-transformations in animal and plant organisms.2HFAs are found as components of sphingolipids, produced by the 2-hydroxylation of FAs [2], whereas 3HFAs are present in mitochondria, generated by fatty acid β-oxidation [3], and are components of inflammatory lipopolysaccharides [4].
activities against human cancer cell lines (A549, Caco-2, and SF268 cells) [5].Furthermore, 7HSA and 9HSA were demonstrated to suppress β-cell apoptosis induced by proinflammatory cytokines [5].As a matter of fact, 9HSA was first identified in Lewis lung carcinoma cells, in 1991, as an oxidation product [8].Later on, it was demonstrated that 9HSA was able to upregulate p21 WAF1 in HT29 cancer cells [9], to inhibit the cell growth in human colon cancer through histone deacetylase 1 [10], and to interfere with EGF signaling in a human colon adenocarcinoma [11].In our previous work, we synthesized 7-, 9-, 10-HSAs and 7-, 9-, 10-HPAs and studied their inhibitory effect on cancer cell growth [5].At the same time, Calonghi et al. synthesized derivatives of 9HSA and studied their antiproliferative activity on HT29 cancer cells [12].Most recently, Calonghi et al. studied the effect of regioisomerism on the antiproliferative activity of HSAs on human cancer cell lines and reported that 5HSA not only presented antiproliferative activity, but also induced changes in cell displacement, directionality and speed [13].To extend our knowledge on HFA bioactivities and to complete structure-activity relationship studies, we decided to synthesize chiral HPAs and HSAs carrying a hydroxyl group at the 6-, 8-and 11-positions and (R)-and (S)-7-hydroxymargaric acids (7HMAs), as well as unsaturated 7-hydroxyoleic acid and 7-hydroxypalmitoleic acid, which have not previously been prepared and tested.In this work, we present the synthesis of various regioisomers of HPAs, HSAs, HMAs and unsaturated HFAs with 16C and 18C carbon chains, as well as the study of their antiproliferative activity on cancer cell lines.
In our previous work, we synthesized 7-, 9-, 10-HSAs and 7-, 9-, 10-HPAs and studied their inhibitory effect on cancer cell growth [5].At the same time, Calonghi et al. synthesized derivatives of 9HSA and studied their antiproliferative activity on HT29 cancer cells [12].Most recently, Calonghi et al. studied the effect of regioisomerism on the antiproliferative activity of HSAs on human cancer cell lines and reported that 5HSA not only presented antiproliferative activity, but also induced changes in cell displacement, directionality and speed [13].To extend our knowledge on HFA bioactivities and to complete structure-activity relationship studies, we decided to synthesize chiral HPAs and HSAs carrying a hydroxyl group at the 6-, 8-and 11-positions and (R)-and (S)-7-hydroxymargaric acids (7HMAs), as well as unsaturated 7-hydroxyoleic acid and 7-hydroxypalmitoleic acid, which have not previously been prepared and tested.In this work, we present the synthesis of various regioisomers of HPAs, HSAs, HMAs and unsaturated HFAs with 16C and 18C carbon chains, as well as the study of their antiproliferative activity on cancer cell lines.

General Remarks
All commercially available products and solvents were purchased from Fluorochem (Fluorochem Ltd., Hadfield, Glossop, UK) Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), Fluka (Fluka Chemicals Ltd., Gillingham, Dorset, UK), Merck (Merck, Darmstadt, Germany), and Alfa Aesar (Alfa Aesar, Ward Hill, MA, USA).Solvents were used as received or dried over molecular sieves (4 Å).All water-or air-sensitive reactions were performed under an argon atmosphere, employing dry solvents and anhydrous conditions.Chromatographic purification of products was accomplished using forced-flow chromatography on Merck ® (Merck, Darmstadt, Germany) Kieselgel 60 F254 230-400 mesh.Thin-layer chromatography (TLC) was performed on aluminum-backed silica plates (0.2 mm, 60 F254).Visualization of the developed chromatogram was performed by fluorescence quenching using phosphomolybdic acid.Melting points were measured on a Buchi 530 apparatus (Buchi, Flawil, Switzerland) and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on a Varian Mercury (Varian, Palo Alto, CA, USA) (200 and 50 MHz, respectively) or an Avance III HD Bruker 400 MHz (Bruker, Fällanden, Switzerland) (400 MHz and 100 MHz, respectively) and are internally referenced to residual solvent signals.Data for 1 H NMR are reported as follows: chemical shift (δ ppm), integration, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br s = broad signal), coupling constant and assignment.Data for 13 C NMR are reported in terms of chemical shift (δ ppm).Optical rotations were measured using a PerkinElmer 343 (PerkinElmer, Shelton, Connecticut, USA) or an AA-65 series polarimeter (Optical Activity Ltd., Bury, UK) in a 10 cm cell at room temperature.Mass spectra (ESI) were recorded on a Finningan ® Surveyor MSQ LC-MS spectrometer (Thermo Finnigan, Co. Ltd., San Jose, CA, USA).High-resolution mass spectra were obtained on a Bruker Maxis Impact QTOF spectrometer (Bruker Daltonics, Bremen, Germany) or an AB Sciex 4600 Triple TOF mass spectrometer (AB Sciex, Singapore).The enantiomeric excess (ee) of compounds (S)-and (R)-11a-d was determined by HPLC analysis performed on an Agilent 1100 Series (Agilent Co., Santa Clara, CA, USA) with a DAD UV detector, and the peak intensities were measured in the UV range between 206 and 280 nm.A Daicel Chiralpak OD-H chromatography column (250 × 4.6 mm ID) was used.HPLC-grade hexane and i PrOH were used as solvents in a n-hexane: i PrOH 99:1 ratio and a flow rate of 1 mL•min -1 .

General Procedure for the Synthesis of Secondary Alcohols from Terminal Epoxides Using Alkynes
To a flame-dried flask under an argon atmosphere, a solution of 1-decyne or 1-octyne or 1-undecyne (0.72 mL or 0.59 mL or 0.79 mL, 4.00 mmol) in anhydrous THF (1.1 mL) was added and the mixture was cooled at −78 • C.Then, the slow addition of n-BuLi (1.6 M solution in hexanes, 2.2 mL, 3.50 mmol) over 10 min followed and the reaction mixture was left stirring at −78 • C for 25 min, before BF 3 •OEt 2 (0.38 mL, 3.00 mmol) and epoxide 3, 18 or 25 (1.00 mmol) in anhydrous THF (3 mL) were added dropwise.The reaction mixture was left stirring for another 1 h at −78 • C, and then warmed to room temperature.Subsequently, a saturated aqueous solution of NaHCO 3 (10 mL) was added, and the aqueous layer was extracted with Et 2 O (3 × 20 mL).The combined organic layers were washed with brine (1 × 50 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.The crude residue was purified by flash column chromatography eluting with petroleum ether (bp 40−60 Lindlar's catalyst (50 mg) was added to a round bottom flask containing alkyne 4a,b (1.00 mmol) in MeOH (10 mL), followed by the addition of quinoline (6 µL, 0.05 mmol), and the reaction mixture was left stirring under a hydrogen atmosphere for 16 h.After filtration through a celite pad, the solvent was evaporated and the crude reaction mixture was purified by flash silica column chromatography eluting with petroleum ether (bp 40−60 • C):ethyl acetate (100:0-95:5) to give the desired hydroxy alkene.

General Procedure for the Deprotection of the Tert-Butyldimethylsilyl (TBDMS) Group
To a flame-dried flask under an argon atmosphere, the appropriate TBDMS-protected alcohol (1.00 mmol) in dry THF (5 mL) was added and, then, a solution of tetra-Nbutylammonium fluoride (1M in THF, 1 mL, 1.00 mmol) was added dropwise at 0 • C. The reaction mixture was then left to reach room temperature, and stirring was continued for 1 h.The solvent was removed under reduced pressure and the crude reaction mixture was purified by flash chromatography on silica gel eluting with petroleum ether (bp 40−60 • C):ethyl acetate (80:20-70:30) to give the desired product.

General Procedure for the Oxidation of Alcohols to Acids Using Jones Reagent
To a round-bottomed flask containing an alcohol (1.00 mmol) in acetone (10 mL), the Jones reagent (2 M, 1.5 mL, 3.00 mmol) was added dropwise at 0 • C and the reaction mixture was left under stirring at this temperature for 1 h.Then, the reaction mixture was quenched with a saturated solution of NaHSO 3 (10 mL), which was added at room temperature.The aqueous layer was extracted with Et 2 O (3 × 20 mL), washed with brine (1 × 50 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a crude mixture, which was then purified by flash chromatography eluting with petroleum ether (bp 40−60 • C):ethyl acetate (60:40) to afford the desired acid.

General Procedure for the Removal of the Acetyl Group
LiOH•H 2 O (168 mg, 4.00 mmol) was added to a solution containing acid 8a,b, 15a-f, 23, or 30 (1.00 mmol) in THF:H 2 O (1:1, 5 mL).The resulting reaction mixture was stirred at room temperature for 16 h.Subsequently, the pH of the reaction mixture was adjusted to one by addition of an aqueous solution of HCl 1 N (10 mL).The aqueous layer was then subjected to extraction with EtOAc (3 × 10 mL), and the combined organic layers were washed with brine (1 × 30 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.Lastly, the isolation of the desired hydroxy fatty acids followed, using silica gel flash chromatography eluting with petroleum ether (bp 40−60 • C):ethyl acetate (20:80).

General Procedure for the Synthesis of Secondary Alcohols Using Grignard Reagents
Nonylmagnesium bromide (or heptylmagnesium bromide or butylmagnesium chloride or hexylmagnesium bromide) (2 M solution in diethyl ether, 1 mL, 2.00 mmol) was introduced to a flame-dried flask containing copper(I) iodide (38 mg, 0.20 mmol) under an argon atmosphere.After cooling the reaction mixture at −40 • C and stirring it for 10 min, the appropriate epoxide (1.00 mmol) in dry THF (10 mL) was added dropwise.Stirred at −40 • C for 1 h, the reaction mixture was then brought to room temperature.Subsequently, 10 mL of a saturated aqueous NH 4 Cl solution were added, and the resulting aqueous layer underwent extraction with Et 2 O (3 × 20 mL).The combined organic layers were washed with brine (1 × 50 mL), dried over Na 2 SO 4 , filtered, and then concentrated under reduced pressure.The desired alcohol was isolated through silica gel flash chromatography using petroleum ether (bp 40−60 • C):ethyl acetate (80:20-70:30) as the elution system.

General Procedure for the Removal of Benzyl Group
To a round bottom flask containing the benzyl-protected alcohol (1.00 mmol) in MeOH (10 mL), 10% palladium on activated charcoal was added and the reaction mixture was left stirring under a hydrogen atmosphere for 16 h.After filtration through a celite pad, the solvent was removed in vacuo, leading to the isolation of the desired alcohol without further purification.
Cell growth inhibition analysis: Microsoft Excel was used for data analysis (Office Professional Plus 2016).The background absorbance at 690 nm was subtracted from the corresponding values at 570 nm and the average of three repeats for each condition was determined.The resulting dataset was normalized to DMSO control cells as the 100% survival value.Inhibition curves were generated with the use of GraphPad Prism version 6.01 and the corresponding IC 50 values were calculated from the resultant plot.Two-way ANOVA statistical analyses with multiple comparisons were performed on the dataset, in order to compare the different concentrations of the compounds investigated to the DMSO control.
As depicted in Scheme 1, mono-tert-butyldimethylsilyl (TBDMS)-protected α,ω-diol 1 underwent oxidation using pyridinium chlorochromate (PCC), for the formation of aldehyde 2, which was converted to a chiral terminal epoxide following a simple, one-pot, three-step organocatalytic protocol.The aldehyde was converted to an α-chloro aldehyde by utilization of (2S,5R)-2-(tert-butyl)-3,5-dimethylimidazolidin-4-one trifluoroacetate as the catalyst and 2,3,4,5,6,6-hexachlorocyclohexa-2,4-dien-1-one as the chlorinating agent.Then, reduction by NaBH 4 was carried out and the subsequent epoxide-ring formation occurred through treatment with an aqueous KOH solution in ethanol, following a S N 2 mechanism.The succeeding step, consisting of the alkynylation of epoxide 3 using commercially available alkynes, 1-decyne or 1-octyne, in the presence of n-BuLi and Lewis acid, boron trifluoride diethyl etherate [30], was inspired by Durand and co-workers' work [31], enabling the synthesis of hydroxy alkynes 4a,b.Then, hydrogenation was carried out using Lindlar's catalyst and quinoline under an H 2 atmosphere, for the conversion of The asymmetric synthesis of saturated (R)-HFAs is presented in Scheme 2. The asymmetric epoxidation of mono-benzyl-protected aldehydes 10a-d led to the formation of (S)epoxides 11a-d in ee's varying from 90% to 95%, which were then treated with the appropriate Grignard reagent in the presence of CuI for the formation of 16-, 17-or 18-carbon atom aliphatic chain secondary alcohols 12a-f, with the hydroxyl group at positions 6-, 7-, 8-and 11-.The acetylation of compounds 12a-f, the hydrogenation of 13a-f for the deprotection of the benzyl group and the subsequent Jones oxidation enabled the isolation of acetoxy-palmitic, margaric or oleic acids 15a-f, the hydrolysis of which with lithium hydroxide afforded the desired HFAs 16a-f.The same procedure was followed for the synthesis of (S)-HFAs, using (2R,5S)-2-(tert-butyl)-3,5-dimethylimidazolidin-4-one trifluoroacetate as the chlorination catalyst, permitting the synthesis of (R)-epoxides in ee s varying The asymmetric synthesis of saturated (R)-HFAs is presented in Scheme 2. The asymmetric epoxidation of mono-benzyl-protected aldehydes 10a-d led to the formation of (S)-epoxides 11a-d in ee's varying from 90% to 95%, which were then treated with the appropriate Grignard reagent in the presence of CuI for the formation of 16-, 17-or 18carbon atom aliphatic chain secondary alcohols 12a-f, with the hydroxyl group at positions 6-, 7-, 8-and 11-.The acetylation of compounds 12a-f, the hydrogenation of 13a-f for the deprotection of the benzyl group and the subsequent Jones oxidation enabled the isolation of acetoxy-palmitic, margaric or oleic acids 15a-f, the hydrolysis of which with lithium hydroxide afforded the desired HFAs 16a-f.The same procedure was followed for the synthesis of (S)-HFAs, using (2R,5S)-2-(tert-butyl)-3,5-dimethylimidazolidin-4-one trifluoroacetate as the chlorination catalyst, permitting the synthesis of (R)-epoxides in ee's varying from 90% to 93%.In case the above-mentioned synthetic methodology of saturated HFAs was not feasible, due to the poor availability of the starting materials, another synthetic route to obtain HFAs was developed, which constituted a combination of the previous two.This route was used to synthesize (S)-and (R)-6HSAs (Scheme 3).Chiral epoxides 18 and 25, derived from the previously described organocatalytic protocol, were submitted to alkynylation after the addition of commercially available 1-undecyne, alongside with n-BuLi and BF3•OEt2.Hydroxy alkynes 19 and 26 were then hydrogenated over Rosenmund catalyst, without the addition of any amine, as described by Balas et al. [31], to form the fully saturated compounds 20 and 27.Then, a similar synthetic pathway was followed (including the acetylation of the secondary hydroxyl group, TBDMS-deprotection with TBAF reagent, Jones oxidation and acetyl deprotection using LiOH), allowing the preparation of HFAs 24 and 31.In case the above-mentioned synthetic methodology of saturated HFAs was not feasible, due to the poor availability of the starting materials, another synthetic route to obtain HFAs was developed, which constituted a combination of the previous two.This route was used to synthesize (S)-and (R)-6HSAs (Scheme 3).Chiral epoxides 18 and 25, derived from the previously described organocatalytic protocol, were submitted to alkynylation after the addition of commercially available 1-undecyne, alongside with n-BuLi and BF 3 •OEt 2 .Hydroxy alkynes 19 and 26 were then hydrogenated over Rosenmund catalyst, without the addition of any amine, as described by Balas et al. [31], to form the fully saturated compounds 20 and 27.Then, a similar synthetic pathway was followed (including the acetylation of the secondary hydroxyl group, TBDMS-deprotection with TBAF reagent, Jones oxidation and acetyl deprotection using LiOH), allowing the preparation of HFAs 24 and 31.

Antiproliferative Activity
The antiproliferative activity of six enantiomers of HSAs (6-, 8-, 11-HSAs), six enantiomers of HPAs (6-, 8-, 11-HPAs), two enantiomers of hydroxymargaric acid (7HMA), 7-(S)-hydroxyoleic acid (7SHOA) and 7-(S)-hydroxypalmitoleic acid (7SHPOA) was studied on A549 cells and the results are summarized in Figure 2. Data on 7RHSA, palmitic acid (PA) and stearic acid (SA) were included for comparison purposes.The A549 cell line was selected because it had been used in our previous work [5], thus permitting direct comparison of the effect of the newly synthesized analogs with the previous results.In addition, the antitumor drug 2-hydroxyoleic acid (2HOA, trade name Minerval), possessing a similar structure, has at concentrations over 35 µM, while 7HMAs, 8HPAs and 8HSAs exhibited moderate antiproliferative activity at concentrations over 50 µM.When the hydroxyl group was at position 11-of the aliphatic chain, no activity was observed for the corresponding HPAs, while the presence of a double bond in the aliphatic chain resulted in very weak activity.All statistical comparisons for the antiproliferative activity of the investigated HFAs are included in Table S1 (Supplementary Materials).For A549 cancer cells, the IC50 values for the enantiomers 6SHPA, 6RHSA and 6SHSA were determined from the curves depicted in Figure 3 and vary from 35 to 62 µM.The position of the hydroxyl group in the fatty acid chain drastically influences the in vitro potency.Interestingly, 6HFAs exhibited the most potent antiproliferative activity at concentrations over 35 µM, while 7HMAs, 8HPAs and 8HSAs exhibited moderate antiproliferative activity at concentrations over 50 µM.When the hydroxyl group was at position 11-of the aliphatic chain, no activity was observed for the corresponding HPAs, while the presence of a double bond in the aliphatic chain resulted in very weak activity.All statistical comparisons for the antiproliferative activity of the investigated HFAs are included in Table S1 (Supplementary Materials).
For A549 cancer cells, the IC 50 values for the enantiomers 6SHPA, 6RHSA and 6SHSA were determined from the curves depicted in Figure 3  Based on these results, the enantiomers of 6HPA, 6HSA, 8HPA and 8HSA were selected for evaluation on an SF268 human astrocytoma cell line.The results are presented in Figure 4, and 7RHSA, PA and SA were included for comparison purposes.The IC50 values for the enantiomers 6SHPA and 6SHSA were determined from the curves depicted Based on these results, the enantiomers of 6HPA, 6HSA, 8HPA and 8HSA were selected for evaluation on an SF268 human astrocytoma cell line.The results are presented in Figure 4, and 7RHSA, PA and SA were included for comparison purposes.The IC 50 values for the enantiomers 6SHPA and 6SHSA were determined from the curves depicted in Figure 5. Based on these results, the enantiomers of 6HPA, 6HSA, 8HPA and 8HSA were selected for evaluation on an SF268 human astrocytoma cell line.The results are presented in Figure 4, and 7RHSA, PA and SA were included for comparison purposes.The IC50 values for the enantiomers 6SHPA and 6SHSA were determined from the curves depicted in Figure 5.For both cell lines, (S)-6-hydroxypalmitic acid (6SHPA) was found to exhibit the most potent antiproliferative activity, with IC50 values of 35 and 63 µΜ for A549 and SF268 cells, respectively.The (R)-enantiomers of 6HPA and 6HSA were found to be considerably less potent than the corresponding (S)-enantiomers and, in the case of 6RHPA, very weak po- For both cell lines, (S)-6-hydroxypalmitic acid (6SHPA) was found to exhibit the most potent antiproliferative activity, with IC 50 values of 35 and 63 µM for A549 and SF268 cells, respectively.The (R)-enantiomers of 6HPA and 6HSA were found to be considerably less potent than the corresponding (S)-enantiomers and, in the case of 6RHPA, very weak potency was observed in A549 cells and no potency in SF268 cells.PA and SA were also studied for their potential antiproliferative activity against A549 and SF268 cells for comparison purposes.As shown in Figure 2, both compounds did not inhibit the proliferation of A549 cells.In addition, PA did not exhibit any activity against SF268 cells (Figure 4), while SA exhibited a very weak effect at the highest concentration (100 µM).These findings indicate that the presence of the hydroxyl group is crucial for the antiproliferative effect of HFAs.

Western Blot Analysis
As we have demonstrated in our previous work, saturated HFAs can lead to cell cycle arrest and do not promote apoptosis, as reflected by the levels of phosphorylated H3 and activated caspase 3 [5].Additionally, through real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western Blot analysis, 7RHSA was previously found to considerably reduce the levels of transcription regulator signal transducer and activator of transcription 3 (STAT3), a highly important regulator of tumor development and its resistance to treatment [33].To corroborate these findings, the most potent compound (6SHPA) in the current study was tested for its ability to inhibit STAT3 expression, using Western Blot analysis (Figure 6).Importantly, 6SHPA led to a significant reduction in STAT3 expression, as depicted in Figure 6A,D.Subsequently, 6SHPA was tested for its ability to induce the acetylation of histone H3 and α-tubulin, using Western Blot analysis (Figure 6).Evidently, 6SHPA increased the levels of acetylated histone H3 (Figure 6A,C), with no significant changes found in acetylated α-tubulin levels (Figure 6A,B).These results indicate that 6SHPA may act as a class I Histone Deacetylase (HDAC) inhibitor and are in accordance with a previous study reporting that another HFA (9RHSA) was found to be an inhibitor of HDAC1, 2 and 3 of class I HDACs [33].Moreover, its potential action as a class I HDAC inhibitor may also explain the antiproliferative action attributed to this compound.

Discussion
In our previous work [5], we synthesized and tested a series of HPA and HSA regioisomers with the hydroxyl functionality at positions 7-, 9-and 10-, as well as 2RHSA and 12RHSA.We demonstrated that the (R)-enantiomer of 7HSA exhibited the highest potency (IC50 values of 38 and 27 µM against human cancer cells A549 and SF268, respectively), followed by 7RHPA (IC50 values of 42 and 49 µM against human cancer cells A549 and SF268, respectively).The corresponding (S)-enantiomers were slightly less potent, followed by the 9HSA and HPA isomers.10HPA and HSA isomers exhibited very weak po-

Discussion
In our previous work [5], we synthesized and tested a series of HPA and HSA regioisomers with the hydroxyl functionality at positions 7-, 9-and 10-, as well as 2RHSA and 12RHSA.We demonstrated that the (R)-enantiomer of 7HSA exhibited the highest potency (IC 50 values of 38 and 27 µM against human cancer cells A549 and SF268, respectively), followed by 7RHPA (IC 50 values of 42 and 49 µM against human cancer cells A549 and SF268, respectively).The corresponding (S)-enantiomers were slightly less potent, followed by the 9HSA and HPA isomers.10HPA and HSA isomers exhibited very weak potency, while 2RHSA and 12RHSA exhibited no potency against these cell lines [5].Calonghi et al. previously identified 9HSA as an antiproliferative agent against HT29 adenocarcinoma cells and demonstrated that the antiproliferative effect brought about by the (R)-enantiomer is more pronounced than the (S)-enantiomer [34], in accordance with our findings for 7-and 9-HSAs and HPAs [5].In a recent study, Calonghi et al. evaluated the growth inhibitory effects of a series of HSAs on a panel of cancer cell lines.They found that 5HSA, 7HSA and 9HSA exhibited the highest inhibitory potency (7HSA was the most potent), while 10HSA and 11HSA exhibited a very weak effect and 8HSA showed no inhibitory activity in all cell lines [13].Our current findings are in agreement with these results, regarding the weak effect of 11HSA enantiomers, while we found that 8HSAs exhibited moderate antiproliferative activity against A549 and SF268 cell lines, which were not utilized by Calogni et al.In addition, the 7HMA enantiomers, which were synthesized and tested for the first time, also exhibited moderate antiproliferative activity against A549 cells, indicating that an odd-numbered long chain is not favored.It seems that shortening the aliphatic chain by just one carbon atom significantly reduces the antiproliferative activity of 7HMA in comparison to 7HSA.
Interestingly, in this work, we found that 6-HPAs and HSAs, which have not been studied so far for their antiproliferative activity, can potently inhibit the proliferation of A549 and SF268 cells, with the (S)-enantiomers being the most potent, in particular 6SHPA.These findings are not in accordance with the trend previously observed for 7-and 9-HFAs, where the (R)-enantiomers were found to be more potent than the (S), though 6SHPA was found to inhibit the expression of the STAT3 protein in the same manner as 7RHSA [5] and to induce the acetylation of histone H3, indicating class I HDAC inhibition.
Regarding unsaturated HFAs, limited derivatives have been studied for their antiproliferative activity so far.Interestingly, 2-hydroxyoleic acid (2HOA) has been investigated as an anticancer drug.It has been reported that it induces glioma cell differentiation and autophagy [35] and that it stimulates signaling and retrograde transport [36].It seems that 2HOA exerts its antitumor action through membrane fatty acid remodeling [37].In an effort to extend the knowledge on the bioactivities of the corresponding unsaturated HFAs, we synthesized, for the first time, 7HOA and 7HPOA and tested them against A549 cells.Both compounds showed weak inhibitory activity at concentrations up to 100 µM.Consequently, if we compare the activity of 7HOA and 7HPOA with that of 7HSA, we may conclude that the unsaturation of the long chain reduces the antiproliferative activity.In comparison, 2HOA was previously reported to exhibit weak inhibitory activity against A549 cells with an IC 50 of 90 µM [32].

Conclusions
The interesting antiproliferative properties of saturated HFAs, reported so far, have prompted us to explore various synthetic methods leading to chiral unsaturated and saturated HFAs, in an effort to extend and complete the structure-activity relationship studies.Herein, we present diverse synthetic routes for producing HFAs, using an organocatalytic process for crafting asymmetric terminal epoxides and employing MacMillan's third-generation imidazolidinone catalyst.By these methods, 16 HFAs, namely 7-(S)hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA), two enantiomers of hydroxymargaric acid (7HMA), six enantiomers of HSAs (6-, 8-, 11-HSAs) and six enantiomers of HPAs (6-, 8-, 11-HPAs), were synthesized.The antiproliferative activities of the compounds synthesized were evaluated on A549 and SF268 cancer cell lines, showing that 6SHPA presented the most promising results, with IC 50 values of 35 and 63 µM, respectively.Western blot analysis in A549 cells revealed that 6SHPA exhibited selective inhibition of HDAC class I enzymes, since it induced the acetylation of histone 3 in A549 cells, without affecting the levels of acetylated α-tubulin.Furthermore, 6SHPA was found to inhibit STAT3 expression in A549 cells.Overall, the antiproliferative activity of HFAs depends not only on the position of the hydroxyl functionality, but also on the configuration of the asymmetric center and the saturation or unsaturation of the long chain.Further studies are needed to better understand the biological roles of saturated and unsaturated HFAs.

Figure 5 .
Figure 5. Inhibition curves of enantiomers 6SHSA and 6SHPA in SF268 cells.IC 50 values were derived from the dose-response relationship for a minimum of six experiments (95% CI log IC 50 −4.15 to −4.06 and −4.23 to −4.17, respectively).

Figure 6 .
Figure 6.6SHPA induces the acetylation of histone 3 and inhibits STAT3 expression in A549 cells.Cells treated with 6SHPA (50 µM) versus DMSO and assessed after 48 h.(A,B) Western blot analysis for acetylated α-tubulin upon 6SHPA treatment.(A,C) Western blot analysis for acetylated histone 3 upon 6SHPA treatment.(A,D) Western blot analysis for STAT3 expression upon 6SHPA treatment.Protein expression levels quantitated by using the ImageJ software.All experiments were repeated three times (n = 3).For all cases, *** p < 0.001.Original images can be found in Supplementary File S1.

Figure 6 .
Figure 6.6SHPA induces the acetylation of histone 3 and inhibits STAT3 expression in A549 cells.Cells treated with 6SHPA (50 µM) versus DMSO and assessed after 48 h.(A,B) Western blot analysis for acetylated α-tubulin upon 6SHPA treatment.(A,C) Western blot analysis for acetylated histone 3 upon 6SHPA treatment.(A,D) Western blot analysis for STAT3 expression upon 6SHPA treatment.Protein expression levels quantitated by using the ImageJ software.All experiments were repeated three times (n = 3).For all cases, *** p < 0.001.Original images can be found in Supplementary File S1.