WO2011131619A1 - Method of synthesizing a 5-oxo-conjugated fatty acid - Google Patents

Abstract

There is provided a novel chemical synthetic method for the preparation of a 5-oxo-ETE and related compounds. The method is based on a short and efficient synthesis of 5-oxo-ETE from commercially available Arachidonic acid. The method is based on the surprising discovery that a 5-oxo-conjugated fatty acid may be efficiently converted into the corresponding 5-oxo-conjugated fatty acid by reaction with the Dess-Martin reagent (1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one)in the presence of pyridine or a substituted pyridine.

Description

Method of synthesizing a 5 -oxo -conjugated fatty acid

FIELD OF THE INVENTION

The present invention relates to 5-oxo-fatty acids. In particular, the present invention provides a method of synth esizing a 5-oxo-conjugated fatty acid, particularly (6£,8Z,1 1 Z,14Z)-5-oxoeicosatetraenoic acid (5-oxo-ETE).

BACKGROUND OF THE INVENTION

Several studies have established (6£,8Z,1 1 Z,14Z)-5-oxoicosatetraenoic acid (5-oxo- ETE) as a potent proinflammatory autacoid. 5-oxo-ETE is a 5-lipoxygenase pathway metabolite derived from (5S)-hydroxyicosatetraenoic acid (5(S)-HETE) by a stereospecific NADP⁺-dependent dehydrogenase. In neutrophils and eosinophils, it ind uces calci um mobi lization , degran u lation , actin polymerization, superoxide generation, chemotaxis, L-selectin shedding, and expression of adhesion molecules. Many of these effects are associated with G protein-linked receptors.

5-oxo-ETE is a potent inflammatory mediator and the endogenous agonist of the 7TM receptor known as TG1019 or OXE or R527, which is an interesting potential antiinflammatory target, in particular for treatment of asthma. 5-oxo-ETE is therefore a useful tool in research related to inflammation. The compound is commercially available, but only in small amounts and for a very high price.

US20050014826A1 discloses polyconjugated fatty acids, and methods for their synthesis and their use. It also provides a method of synthesizing a polyconjugated fatty acid product, comprising providing an iodolactone of a fatty acid, wherein the fatty acid is arachidonic, eicosapentaenoic, or eicosahexaenoic acid, reacting the fatty acid iodolactone with triethylamine to form a methyl ester of an epoxy fatty acid, reacting the methyl ester of an epoxy fatty acid with actylbromide to form a bromoacetate, and reacting the bromoacetate with dry DBU in dry benzene to form a methyl ester of a polyconjugated fatty acid. Khanapure et al (J. Org. C em. 1998, 63, 337-342) disclose a total synthesis of (6£,8Z,1 1 Z,14Z)-5-oxoicosatetraenoic acid (5-oxo-ETE) and its biotransformation product 6,7-dihydro-5-oxo-ETE. A convergent synthesis for the unstable title compounds is accomplished via two synthons, dithiolane aldehyde and bisdienyl phosphonium bromide. The total synthesis is completed in 7 linear (10 total) steps from commercial starting materials in 10% overall yield from a previously published alkyne which was available in two additional steps. The product required purification first by reverse phase HPLC, then by normal phase HPLC due to a by-product arising from cis-trans isomerization in the final deprotection step.

Mohapatra et al (Tetrahedron Lett., 2001 , 42, 4109-41 10) also disclose a method for preparing 5-oxo-ETE via a convergent aldol strategy that obviates the need for HPLC separation of olefinic isomers. The total synthesis was performed in 7 linear (9 total) steps in 27% overall yield from a previously published diyne which is was available in two additional steps

5-Oxo-ETE has also been produced in microgram amounts by oxidation of 5-HETE with DDQ (O'Flaherthy, Biochim. Biophys. Acta 1994, 1201, 505-515) or by biochemical oxidation of 5-HETE (Powell, et al., J. Biol. Chem. 1992, 267, 19233-19241 ; Powell, et al., J. Biol. Chem. 1993, 268, 9280-9286), but these methods are unsuitable for production. Furthermore, 5-oxo-ETE has been produced from the methyl ester of 5- HETE by Mn0₂-oxidation of the hydroxyl-group and hydrolysis of the ester (Kerdesky, et al. J. Med. Chem. 1987, 30, 1 177-1 186). All methods mentioned above are either unsuitable or less practical for production of 5-oxo-ETE than the procedure described here.

A major challenge in the synthesis of 5-oxo-ETE is the relatively unstable nature of the compound, which requires long-term storage in solution at low temperatures, typically -80 °C. The instability is associated with the conjugated enone (Khanapure et al., J. Org. Chem. 1998, 63, 337-342). This place high demands on the final step and isolation procedure. Direct oxidation of 5-HETE to 5-oxo-ETE on larger than microgram scale has not previously been described. The oxidation of the 5-HETE methyl ester with manganese dioxide (Kerdesky, et al., J. Org. Chem. 1987, 30, 1 177-1 186) does however not work with the unprotected 5-HETE. The Dess-Martin periodinane (DMP, 1 ,1 ,1 -triacetoxy-1 ,1 -dihydro-1 ,2-benziodoxol-3(1 H)-one) is well known as a reagent capable of efficiently oxidizing primary alcohols to aldehydes and secondary alcohols to ketones under mild conditions (Dess & Martin, J. Org. Chem. 1983, 48, 4155). Oxidation of 5-HETE by DMP by the standard protocol however leads to extensive formation of byproducts, primarily a result of the acetic acid developed in the reaction catalyzing lactone formation from 5-HETE. The oxidation of the related compound 4-OH-DHA

((5E,7Z,10Z,13Z,16Z,19Z)-4-Hydroxy-5,7,10,13,16,19-docosahexaenoic acid) with DMP in the presence of triethylamine resulted in the corresponding ketone with only 25% yield (Itoh, et al., Bioorg. Med. Chem. 2006, 14, 98-108). 5-oxo-ETE is the precursor to a variety of secondary metabolites of considerable current interest. To expedite continuing efforts to elucidate its pharmacology and metabolic fate, as well its involvement in chronic pathology, e.g. asthma, there is a need to provide an efficient synthesis of 5-oxo-ETE.

SUMMARY OF THE INVENTION

The present invention provides a novel chemical synthetic method for rapid and convenient preparation of a 5-oxo-ETE and related compounds. The method of the present invention is based on a short and efficient synthesis of 5-oxo-ETE from commercially available arachidonic acid via 5-HETE.

Herein is disclosed an efficient procedure for oxidation of 5-H ETE to 5-oxo-ETE with DMP as oxidant and pyridine as co-reagent. Furthermore is disclosed a procedure of removal of all side-products by precipitation and filtration to provide the pure 5-oxo-ETE.

The present invention provides a method of synthesizing a 5-oxo-conjugated fatty acid, wherein a 5-hydroxy- conjugated fatty acid is oxidised with Dess-Martin periodinane to the corresponding 5-oxo-conjugated fatty acid. Preferably, the Dess-Martin periodinane is dissolved in CH₂CI₂ and the oxidation is performed in the presence of pyridine.

The present invention describes a reaction sequence by which 5-oxo-conjugated fatty acid may be obtained from a iodolactone of a δ,ε-unsaturated fatty acid. Hence, specifically the present invention provides a method of synthesizing a 5-oxo-fatty acid, comprising: i) conversion of a δ,ε-unsaturated acid to the corresponding iodolactone;

ii) reacting the iodolactone of step i) with a base to eliminate the iodo-group;

iii) hydrolysing the lactone of step ii) to obtain a 5-hydroxy-fatty acid;

iv) oxidizing the 5-hydroxy-fatty acid of step iii) to obtain the corresponding 5-oxo- conjugated fatty acid.

Preferably, the iodolactone of the fatty acid is the iodolactone of arachidonic, eicosapentaenoic, or eicosahexaenoic acid. In a preferred embodiment of the present invention the iodolactone of the fatty acid is the iodolactone of arachidonic acid and the resulting 5-oxo-conjugated fatty acid is (6£,8Z,1 1 Z,14Z)-5-oxoicosatetraenoic acid (5- oxo-ETE). Preferably, the iodolactone of arachidonic is prepared by reacting arachidonic acid with iodine in the presence of collidine or lutidine.

In a particularly preferred embodiment the elimination agent is DBU, preferably dissolved in benzene. Also, preferably hydrolysis of the lactone is performed in an aqueous solution of LiOH.

In other embodiments, the present invention provides a method of purifying the 5-oxo- conjugated fatty acid products, comprising providing 5-oxo-conjugated fatty acid products in a high boiling solvent, evaporating the solvent in which a 5-oxo-conjugated fatty acid is dissolved, dissolving the residual 5-oxo-conjugated fatty acid in a water/alcohol/acid solution , and isolating 5-oxo-conjugated fatty acid product by ch romatography, such as solid phase extraction chromatography. In further embodiments, the method further comprises concentrating and purifying the 5-oxo- conjugated fatty acid by HPLC. In yet further embodiments, the method further comprising crystallizing or precipitating the 5-oxo-conjugated fatty acid at very low temperatures. In some embodiments of the method, the 5-oxo-conjugated fatty acid is a polyconj ugated fatty acid or an ester of a polyconjugated fatty acid. In other embodiments of the method, the high boiling solvent is dry diglyme, triglyme, tetraglyme or DMSO.

The method of the present invention may further comprise the steps of purifying the 5- oxo-conjugated fatty acid including some or all of the steps of:

I. diluting the reaction mixture with diethyl ether or tert-butylmethyl ether;

II. centrifuging or filtering the reaction mixture; adding diethyl ether or tert-butylmethyl ether and storing the mixture at low temperature for an extended period of time;

filtering the reaction mixture quickly through a pad of silica;

rinsing the silica pad with 20% chloroform in diethyl ether or tert-butylmethyl ether;

concentrating the combined solutions to give the pure product.

DETAILED DESCRIPTION OF THE INVENTION

An efficient and simple route of synthesis for the production of 5-oxo-ETE from arachidonic acid in 4 steps giving 50% overall yield has been invented. This approach is suitable for large-scale production of 5-oxo-ETE.

To facilitate an understanding of the present invention, a number of terms and phrases as used herein are defined below:

As used herein, the term "unsaturated" when used in reference to a fatty acid refers to the presence of at least one double bond in the fatty acid . Most double bonds in polyunsaturated fatty acids are unconjugated polyenes, or methylene-interrupted polyenes, in that the double bonds are separated by a methyl group.

As used herein, the term "polyunsaturated" when used in reference to a fatty acid refers to the presence of two or more double bonds in a fatty acid.

As used herein, the term "conjugated" when used in reference to an unsaturated fatty acid refers to two double bonds separated only by a single bond in a fatty acid.

The term "base" refers to a substance capable of taking up a proton. A "weak base" typically refers to a base where the negative logarithmic dissociation constant of the corresponding acid (pK_a) in water is lower than 9.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated , a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which is fully saturated, having the number of carbon atoms designated (e.g., C-I-C-IO means one to ten carbons). Examples of alkyl groups include methyl, ethyl, n- propyl, isopropyl, n-butyl , t-butyl, isobutyl , sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropyl, cyclopropylmethyl, and homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from alkyl, as exemplified by -CH2CH2CH2CH2-. The two valences may be on any carbon atom of the chain , including on the same carbon, resulting in an alkyl connected by a double bond. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. Similarly, the term dialkylamino refers to an amino group having two attached alkyl groups. The alkyl groups of a dialkylamino may be the same or different. In the following examples the present invention is described in more detail. These examples are merely provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. The synthesis of 5-oxo-ETE and related molecules can be achieved in four steps i.e. iodolactonization, elimination followed by hydrolysis and oxidation reaction as showed in Scheme 1 . The detailed discussion of each step is as follows;

5-oxo-ETE (5)

Scheme 1 lodolactonization of arachidonic acid by using iodine in presence of base in different organic solvents, as shown in Table 1 , including previously published protocols for iodolactionization of arachidonic acid (Entry 1 : Corey & Hasimoto, Tetrahedron Lett. 1981 , 22, 299-302; Kuklev, et al. Chem. Phys. Lipids 2004, 130, 1 45-158; US2005014826. Entry 2: US4599439). Under the reaction conditions with aqueous potassium bicarbonate (entry 1 , 2 & 3) a byproduct was detected by TLC which complicated the purification of the product. Using collidine (entry 4 & 5) under anhydrous conditions eliminated the byproduct and resulted in a moderately increased yield when acetonitrile was used as solvent (entry 5). Using dichloromethane as solvent gave a significant increased reaction yield and a significant decreased reaction time when collidine (entry 6) or lutidine (entry 7) was used as base. This was surprising, since dichloromethane resulted in a slightly decreased yield with aqueous KHC0₃ (entry 3).

Table 1

^a TLC indicated that the reaction did not reach to completion.

b TLC indicated presence of a by-product.

Compound 2 was converted to 3 by a published procedure (Kuklev, et al. Chem. Phys. Lipids 2004, 730, 145-158). The purified compound 3 was hydrolyzed without any delay due to instability of compound 3. Hydrolysis was conducted in THF by using aqueous LiOH (1.5 N), which was more efficient as compared to previously reported reaction conditions (Kuklev, et al. Chem. Phys. Lipids 2004, 730, 145-158). After neutralization compound 4 immediately starts reverting to lactone 3, and should be progressed to the next step without any delay. Addition of a small amount (one or a few drops) of pyridine to 4 inhibits lactone formation.

The oxidation of the 4 to 5-oxo-ETE (5) requires care because of the sensitive nature of the product 5. The oxidation of the corresponding methyl ester of compound 4 has been reported with Mn0₂ (Kerdesky et al., J. Med. Chem. 1987, 30, 1 177-1 186), but this method did not give the desired product with the free fatty acid 4. Dess-Martin periodinane (DMP) is well known as a mild and efficient reagent for oxidation of alcohols to ketones or aldehydes. Attempts to oxidize 4 with the standard protocol, using DMP in dichloromethane at room temperature, was unsuccessful , primarily because the developed acetic acid efficiently catalyze lactone formation of 4 back to 3 as a competing reaction. Strategies to trap the in situ generated acetic acid were explored. Eventually, by performing the oxidation with DMP in dichloromethane in the presence of pyridine, 4 was cleanly and efficiently converted to the target compound 5. All reagents and by-products could be removed by performing a repeated precipitation from cold diethyl ether and a quick filtration to provide the pure 5-oxo-ETE (5). Alternatively, 5-oxo- ETE (5) can be purified by chromatography on silica gel. The latter method is preferred if an excess of DMP has been used.

Experimental:

lodolactonization of arachidonic acid:

6-((3Z,6Z,92^-1 -lodopentadeca-3,6,9-trien-1 -yl)tetrahydro-2H-pyran-2-one (2). An ice cold solution of arachidonic acid 1 (400 mg, 1 .31 mmol) and γ-collidine (320 mg, 2.64 mmol) in CH₂CI₂ (20 mL) was added iodine (672 mg, 2.65 mmol). The reaction mixture was stirred for 14 h at 0 °C. The mixture was diluted with CH₂CI₂ (50 mL) and washed with 5% aqueous Na₂S₂0₃, aqueous KHS0₄ (0.5 M) and brine. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under vacuum. The residue was purified by flash chromatography (Si0₂, 6% EtOAc in petroleum ether) to yield 493 mg (83%) of 2 as a colorless oil: ¹H NMR (400 MHz, CDCI₃) δ 5.61-5.50 (m, 1 H, H-9'), 5.46-5.28 (m, 5H, H-3', 4', 6', 7' & 10'), 4.10 (td, J = 7.4, 2.6 Hz, 1 H, H-1 '), 3.96 (dt, J = 10.7, 3.1 Hz , 1 H, H-3), 2.88-2.76 (m, 6H, H-2', 5' & 8'), 2.69-2.44 (m, 2H, H-6), 2.10-1 .78 (m, 6H, H- 4, 5 & 1 1 '), 1 .41-1 .23 (m, 6H, H-12', 13' & 14'), 0.89 (t, J = 6.9 Hz, 3H, C-15'); ¹³C NMR (101 MHz, CDCIs) δ 170.4 (CO), 131 .5 (C-9'), 130.6, 129.2, 127.4, 127.2, 127.1 , 81 .0 (C-3'), 36.8 (C-1 '), 34.4, 31 .5 (C-14'), 29.6 (C-6), 29.3, 28.0, 27.3 (C-1 1 '). 25.9, 25.7, 22.6, 18.3, 14.1 (C-15'); HMDS m/z calcd for C₂oH₃i0₂INa (M + Na⁺) 453.1262, found 453.1256.

Elimination of hydrogen iodide from the iodolactone:

6-((1 E,3Z,6Z,9Z)-Pentadeca-1 ,3,6,9-tetraen-1 -yl)tetrahydro-2H-pyran-2-one (3). To a solution of iodolactone 2 (366 mg, 0.81 mmol) in of dry benzene (50 ml) was added DBU (149 mg, 0.98 mmol). The reaction mixture was stirred at room temperature under nitrogen for 12 h, then centrifuged for 5 min. The clear solution was separated from the precipitate and residue was stirred with dry benzene (10 mL) and centrifuged again, and the clear benzene solutions were combined. The resulting solution was evaporated under vacuum. The resid ue was pu rified by flash ch romatography (Si0₂, 6% ethylacetate in petroleum ether) to yield 21 1 mg (86%) of 3 as colorless oil: ¹H NMR (400 MHz, CDCI₃) δ 6.61 (ddt, J = 15.2, 1 1 .1 , 1 .2 Hz, 1 H, H-2'), 6.01 (t, J = 12 Hz, 1 H, H-3'), 5.69 (dd, J = 15.2, 6.3 Hz, 1 H, H-1 '), 5.52-5.47 (m, 2H, H-4'), 5.54-5.28 (m, 5H, H-6', 7', 9', & 10'), 4.94-4.84 (m, 1 H, H-3), 2.97 (t, J = 7.0 Hz, 2H, H-5'), 2.81 (t, J = 6.7 Hz, 2H, H-8'), 2.68-2.45 (m, 1 H, H-6), 2.08-1 .83 (m, 5H, H-1 1 ', 5 & H-4), 1.74-1 .65 (m, 1 H, H-4), 1.40-1 .25 (m, 6H, H-12', 13' & 14'), 0.89 (t, J = 6.9 Hz, 3H, H-15'); ¹³C NMR (101 MHz, CDCI₃) δ 171 .1 (CO), 132.1 (C-4'), 130.64, 130.61 , 129.2, 127.4 (3C), 127.1 , 80.3 (C-3'), 31 .5, 29.6 (C-6), 29.3 (C-12'), 28.4 (C-4), 27.2 (C-1 1 '), 26.1 (C-5'), 25.7 (C- 8'), 22.6, 18.3, 14.1 (C-15').

Hydrolysis of the iodolactone:

(6E,8Z,11Z,14Z)-5-Hydroxyicosa-6,8,11 ,14-tetraenoic acid (5-HETE, 4). To a solution of lactone 3 (200 mg, 0.66 mmol) in THF (7.5 mL) was added an aqueous solution of LiOH (23.9 mg, 1 .00 mmol) in water (3.6 mL). The homogeneous reaction mixture was stirred for 8 h at room temperature. Water (9 ml) was added, and the solution was carefully acidified with 1.5 M aqueous HCI to pH 5 and extracted diethyl ether (3 ^χ 20 ml_). The resulting organic extract was washed with brine and dried over Na₂S0₄. The dry extract was filtered and concentrated under vacuum to yield 200 mg (94%) of compound 4 which was quickly proceed for next step: ¹H NMR (400 MHz, CDCI₃) δ 6.53 (ddt, J = 15.1 , 1 1 .1 , 1.0 Hz, 1 H, H-7), 5.99 (t, J = 1 1 .1 Hz, 1 H, H-8), 5.69 (dd, J = 15.2,

6.9 Hz, 1 H, H-6), 5.59-5.25 (m, 5H, H-9, 1 1 , 12, 14 & 15), 4.20 (q, J = 6.1 Hz, 1 H, H-5), 2.96 (t, J = 6.8 Hz, 2H, H-10), 2.82 (t, J = 6.5 Hz, 2H, H-13), 2.41 (t, J = 7.2 Hz, 2H, H-2), 2.06 (q, J = 6.9 Hz, 2H, H-16), 1 .88-1.51 (m, 4H, H-3 & 4), 1.41-1 .21 (m, 6H, H-17, 18 &

19) , 0.89 (t, J = 6.9 Hz, 3H, H-20); ¹³C NMR (101 MHz, CDCI₃) δ 178.7 (CO), 135.7 (C- 6), 130.8, 130.6, 129.0, 127.8 (C-8), 127.4, 127.3, 125.9 (C-7), 72.4 (C-5), 36.4 (C-4),

33.6 (C-2), 31 .5, 29.3, 27.2 (C-16), 26.1 (C-10), 25.7 (C-13), 22.6, 20.6 (C-3), 14.1 (C-

20) .

Oxidation of 5-HETE and purification of 5-oxo-ETE by chromatography:

(6E,8Z,11Z,14Z)-5-Oxoicosa-6,8,11 ,14-tetraenoic acid (5-oxo-ETE, 5). Alcohol 4 (200 mg, 0.63 mmol) was added a solution of Dess-Martin periodinane (415 mg, 0.98 mmol) and pyridine (0.52 ml_, 6.49 mmol) in CH₂C1₂ (12 ml) in one portion and the reaction was stirred at room temperature. After 3 h, the reaction mixture was diluted with diethyl ether (70 ml_), the mixture was centrifuged and the clear solution was separated, dried (MgS0₄) and concentrated. The residue was purified by flash chromatography (Si0₂,

Et₂0 containing 2-4% CHCI₃) to yield 154 mg (77%) of 5 as a colorless oil: ¹H NMR (400 MHz, CDCIs) δ 7.54 (ddd, J = 15.3, 1 1 .5, 1.0 Hz, 1 H, H-7), 6.18 (d, J = 16 Hz, 1 H, H-6) 6.14 (t, J = 12 Hz, 1 H, H-8), 5.87 (dt, J = 10.6, 7.8 Hz, 1 H, H-9), 5.51-5.28 (m, 4H, H-1 1 , 12, 14 & 15), 3.09 (t, J = 7.4 Hz, 2H, H-10), 2.83 (t, J = 7.1 Hz, 2H, H-13), 2.67 (t, J = 7.2 Hz, 2H, H-4), 2.44 (t, J = 7.2 Hz, 2H, H-2), 2.05 (q, J = 6.8 Hz, 2H, H-16), 1 .98 (p, J = 7.2

Hz, 2H, H-3), 1.40-1.22 (m, 6H, H-17, 18 & 19), 0.89 (t, J = 6.9 Hz, 3H, H-20); ¹³C NMR (101 MHz, CDCI₃) δ 199.61 (C-5), 178.02 (C-1 ), 140.21 (C-9), 137.02 (C-7), 130.86 (C- 15), 130.04 (C-12/14), 129.57 (C-6), 127.10 (C-12/14), 126.96 (C-8), 126.06 (C-1 1 ), 39.64 (C-4), 32.86 (C-2), 31 .51 (C-18), 29.29 (C-17), 27.25 (C-16), 26.69 (C-10), 25.71 (C-13), 22.57 (C-19), 19.05 (C-3), 14.06 (C-20); HMDS m/z calcd for C₂₀H₃o0₂Na (M +

Na⁺) 341 .2088, found 341 .2090.

Oxidation of 5-HETE and purification of 5-oxo-ETE by precipitation of impurities:

In a flask containing the dry alcohol 4 (38.8 mg, 0.121 mmol) was added a solution of Dess-Martin periodinane (57 mg, 0.13 mmol, 1.14 equiv) and pyridine (68 μΙ, 0.84 mmol) in CH₂CI₂ (2.2 ml) in one portion. After 2 h, the reaction mixture was diluted with 10 mL of diethyl ether. Mixture was centrifuged for 5 min. The clear solution was separated from the precipitate which was then vigorously stirred with dry diethyl ether (10 mL) and centrifuged again, and the clear solutions were combined and stored at (-18 °C) for overnight. The cooled solution was filtered quickly through a pad of silica gel which was pre washed with cold ether. The silica was rinsed with a cold solution of 20% chloroform in diethylether. The filtered solutions were combined, concentrated and dried under vacuum to yield 26.6 mg (69%) of pure 5-oxo-ETE (5) as a colorless oil.

Claims

A method of synthesizing a 5-oxo-conjugated fatty acid, wherein a 5-hydroxy- conjugated fatty acid is oxidised with Dess-Martin periodinane (1 ,1 ,1 -triacetoxy-1 ,1 - dihydro-1 ,2-benziodoxol-3(1 H)-one) i n the presen ce of a weak base to the corresponding 5-oxo-conjugated fatty acid.

The method according to claim 1 , wherein the weak base is pyridine or a pyridine having 1 -5 substituents independently selected from the group consisting of (C-I-C-IO)- alkyl, (Ci-Ci₀)-alkylene and (Ci-Ci₀)-alkoxy.

The method according to claim 1 or 2, wherein the weak base is pyridine.

The method according to any one of the claims 1 to 3, wherein weak base is collidine or lutidine.

The method according to any one of the preceding claims, wherein the 5-hydroxy fatty acid is (6E,8Z,1 1 Z,14Z)-5-hydroxyicosatetraenoic acid (5-HETE) and the resulting 5-oxo-conjugated fatty acid is (6£,8Z,1 1 Z,14Z)-eicosatetraenoic acid (5- oxo-ETE).

A method for converting a 5,6-unsaturated fatty acid to the corresponding iodolactone with iodine in dichloromethane in the presence of collidine or lutidine.

The method according to claim 6, wherein the 5,6-unsaturated fatty acid is arachidonic acid and the resulting iodolactone is 6-((3Z,6Z,9Z)-1 -iodopentadeca- 3,6,9-trien-1 -yl)tetrahydro-2H-pyran-2-one.

A method of synthesizing a 5-oxo fatty acid, comprising:

I. providing a iodolactone of a alkane-5-enoic acid;

II. reacting the iodolactone of step I with a base to eliminate the iodo-group;

III. hydrolysing the lactone of step II to obtain a 5-hydroxy-fatty acid;

IV. oxidizing the 5-hydroxy-fatty acid of step III to obtain the corresponding 5-oxo fatty acid.

9. The method according to claim 8, wherein the iodolactone of the fatty acid is the iodolactone of arachidonic, eicosapentaenoic, or docosahexaenoic acid.

10. The method according to claim 8 or 9, wherein the iodolactone of the fatty acid is the iodolactone of arachidonic acid and the resulting 5-oxo-conjugated fatty acid is (6£,8Z,1 1 Z,14Z)-eicosatetraenoic acid (5-oxo-ETE).

1 1 . The method according to claim 10, wherein the iodolactone of arachidonic is prepared by reacting arachidonic acid with iodine in dichloromethane in the presence of collidine or lutidine.

12. The method according to any one of the claims 8 to 1 1 , wherein the elimination agent is DBU, preferably dissolved in benzene.

13. The method according to any one of the claims 8 to 12, wherein hydrolysing the lactone is performed in an aqueous solution of LiOH.

14. The method according to any one of the claims 8 to 13, wherein oxidizing the 5- hydroxy-fatty acid is performed with Dess-Martin periodinane (1 ,1 ,1 -triacetoxy-1 ,1 - dihydro-1 ,2-benziodoxol-3(1 H)-one) in the presence of a weak base.

15. The method according to claim 14, wherein the weak base is pyridine is a pyridine or a methyl-substituted pyridine, such as collidine or lutidine.

16. The methods of any one of the claims 1 to 15 further comprising the steps of purifying said 5-oxo-conjugated fatty acid including some or all of the steps of:

I. diluting the reaction mixture with diethyl ether or tert-butylmethyl ether;

II. centrifuging or filtering the reaction mixture;

III. adding diethyl ether or tert-butylmethyl ether and storing the mixture at low temperature for an extended period of time;

IV. filtering the reaction mixture quickly through a pad of silica;

V. rinsing the silica pad with 20% chloroform in diethyl ether or tert-butylmethyl ether;

VI. concentrating the combined solutions to give the pure product.