An efficient stereoselective synthesis of a sulfur-bridged analogue of bosseopentaenoic acid as a potential antioxidant agent

An efficient approach to the stereoselective synthesis of a novel sulfur-bridged analogue of bosseopentaenoic acid (BPA) by employing the Z -selective modified Boland semi-reduction procedure as the key step is described. The free radical scavenging potential of the thiophene analogue of bosseopentaenoic acid is studied. The results showed that the thiophene ring led to increased antioxidant activity.

Once PUFAs are released from the membrane they can participate in signal transduction, either directly or after enzymatic conversion to a variety of important, bioactive lipid mediators. 4In several studies the consumption of polyunsaturated fatty acids have shown positive health effects like reducing the risk of cardiovascular diseases 5,6 and heart attack, 7 positively associated with cognitive and behavioral performances 4,5 as well as different types of cancer. 6,7he promising biological effects as well as the complex chemical structures of the polyunsaturated fatty acids have made them interesting synthetic targets as well as lead compounds for medicinal chemists.Several attempts have been made to modify the PUFA structures, to improve or modify their biological activities, as well as to simplify the chemical synthesis.In 1985, Corey et al. substituted the methylene group at 7-position in arachidonic acid with sulfur. 8The resulting compound 1 possessed inhibitory activity for 5-lipoxygenase (5-LO).Later, Hanko et al. synthesized and tested for 5-LO inhibition several analogues of arachidonic acid containing a sulfur atom at the 5-position. 9Structure-activity studies suggested the sulfur atom preferably should be attached to E-alkene as in compound 2 which was the most active 5-LO inhibitor of those tested in this study.In 2007, Skattebøl et al. 10 reported the synthesis of several thiophene-containing PUFAs such as compounds 3 and 4 (Figure 1).From the aforementioned results, it is worth noting that the introduction of sulfur-bridged atom to polyunsaturated fatty acids (PUFAs) is an approach which has received great interest in enhancement of biological effects.Hence, we envisaged to synthesize a rigidified analogue of bosseopenteanoic acid 6a by replacement the two conjugated E,E-double bonds existed in bosseopentaenoic acid 5a to thiophene moiety and testing their antioxidant activity (Figure 2).Bosseopentaenoic acid (BPA) 5a is a naturally occurring ω-6 fatty acid which containing four conjugated double bonds with Z,E,E,Z-configuration respectively and one skipped double bond with Z-configuration.The compound was isolated from the red alga Bossiella orbigniana by Burgess et al. in 1991, 11 and the first total synthesis of methyl bosseopentaenoate 5b was published in 2011. 12Herein, we report a synthetic strategy towards a thiophene analogue of BPA 6a as well as its biological activity evaluation in comparison with BPA 5a.

Results and Discussion
Chemistry A retrosynthetic analysis of the polyunsaturated thiophene is outlined in Figure 3.The triyne 7 is the key intermediate in the synthesis of the target molecule 6a.The compound 7 can be converted to 6a by Zstereoselective semi-reduction of the three triple bonds to corresponding three cis-double bonds followed by hydrolysis of ester group to acid.In the described retrosynthetic pathway, the commercially available 2iodothiophene 11 was selected to be as starting material in the synthesis of thiophene analogue of bosseopentaenoic acid 6a (Figure 3).
Our synthetic strategy started with the preparation of compound 13 from the reaction of 2-iodothiophene (15) with hept-1-yne (14) to produce compound 13 in 88% yield.This compound was reacted with lithium diisopropylamide (LDA) to form non-isolated intermediate 5-lithiothiophene 13 13' that was subsequently reacted with iodine to form 2-(hept-1-yn-1-yl)-5-iodothiophene (11) in 53% yield.A Sonogashira coupling 14 between compound 11 and propargyl alcohol 12 afforded compound 10 in 86% yield.Conversion of this alcohol 10 to corresponding bromide was performed using carbon tetrabromide and triphenyl phosphine at room temperature 15,16 to obtain compound 8 in 77% yield.The 1 H NMR analysis of compound 8 showed a singlet at δ 4.39 ppm, characteristic for CH2-Br.Triyne 7 was achieved in good yield (71%) through a skipped alkyne synthesis by coupling propargyl bromide 8 with methyl 5-hexynoate 9. 17,18 A Z-stereoselective semireduction of triyne 7, was carried out via a modified Boland protocol using Zn(Cu/Ag) in presence of trimethylsilyl chloride (TMSCl) 19,20 at room temperature, to produce compound 6b in 65% yield.The modified Boland protocol, using Zn(Cu/Ag) in presence of TMSCl, proved to be important for this reaction 12,[20][21][22][23] (Scheme 1); the same reduction carried out with a Lindlar catalyst resulted in a complex mixture of products.The hydrolysis of the methyl ester 6b using LiOH, provided the corresponding acid 6a in 62% isolated yield.An alternative milder and potentially higher yielding procedure for the hydrolysis of ester group existed in the sensitive substrate by the use of a lithium salt was considered. 25However, in our case the reaction did not proceed when the ester 6b was treated with 10 equivalents of LiI in THF/H2O (3:1) at room temperature for 24 h.In addition, the use of triethylamine with LiI gave a low conversion to the carboxylic acid 6a.From these results, we decided to investigate a mild and more efficient procedure for the hydrolysis of the methyl ester of the thiophene analogue 6b, as well as methyl bosseopentaenoate 5b that was prepared according to the reported literature procedures. 12Herein, an in situ formation of LiI in a two-step reaction was carried out.The first step is the formation of TMSI by mixing TMSCl with KI.Then LiOH was added subsequently, to produce lithium iodide (LiI) that mediated the hydrolysis of the methyl ester 6b under mild and efficient conditions to afford acid 6a after 6 hours in 87% isolated yield (Scheme 2).The correct configuration of the final compound was assigned by 1 H NMR and 13 C NMR data.The purity of the thiophene analogue 6b was determined by HPLC to be 99%.With this procedure, the hydrolysis of the ester group in methyl bosseopentaenoate 5b was executed.This method provided bosseopentaenoic acid 5a in 82% yield.The NMR data of BPA 5a were in good agreement with those previously reported. 11

Biological evaluation
The free radical-scavenging potential of both BPA 5a and the thiophene analogue 6a was investigated in a DPPH scavenging assay in comparison with ascorbic acid as standard antioxidant agent.The thiophene analogue 6a exhibited good radical scavenging activity with SC50 5.74 µM.However, bosseopentaenoic acid 5a exhibited SC50 6.82 µM (Table 2).Based on these results, both BPA and thiophene analogue have effect in scavenging superoxide.However, the thiophene analogue 6a has the higher susceptibility to oxidation more than BPA 5a dependent on the presence of sulfur-bridged atom in the structure that increased the stability of compound 6a.

Conclusions
An efficient stereoselective synthesis of a thiophene analogue of bosseopentaenoic acid 6a was achieved in 12% yield over 7 steps, as well as antioxidant activity were evaluated.The the two E,E-double bonds in the structure of bosseopentaenoic acid was replaced by thiophene moiety to act as a rigidified analogue of BPA.This replacement resulted in a less flexible structure with fewer rotational options.The key step in our synthetic approach was the Z-selective modified Boland-semi-reduction procedure to establish the three Zdouble bonds in one reaction.The biological evaluation revealed the thiophene analogue 6a exhibited an improved antioxidant activity compared to the lead bosseopentaenoic acid 5a.

Experimental Section
General.All reagents and solvents were used as purchased without further purification.Analytical TLC was performed on silica gel 60 F254 Aluminium sheets (Merck).Flash column chromatography was performed on silica gel 60 (40-60 μm, Fluka).NMR spectra were recorded on a Bruker Avance DPX spectrometer at 300 or at 400 MHz for 1 H NMR, 75 or 101 MHz for 13 C NMR respectively.Coupling constants (J) are reported in hertz, and chemical shifts are reported in ppm (δ) relative to CDCl3 (7.24 ppm for 1 H and 77.40 ppm for 13 C).The HPLC analyses were performed using a Agilent Technologies 1200 Series with an Eclipse XD 8-C18 5 μm, 4.6x150 mm column.High-resolution mass (ESI-MS) spectra were measured on (TOF) LC/MS; 6230 Series Accurate-Mass Time-of-Flight.

Table 2 .
Biological evaluation of the antioxidant activity of thiophene analogue using a DPPH radical scavenging method a Results of three experiments performed in triplicate.