Eicosanoid derivatives

Abstract

The present invention provides compounds (n-3 PUFA derivatives) of formula (I): ##STR00001##
that modulate conditions associated with cardiac damage, especially cardiac arrhythmias.

Claims

1. A compound of the general formula (I): ##STR00100## or a pharmacologically acceptable salt, solvate, hydrate or a pharmacologically acceptable formulation thereof, wherein R.sup.1 is selected from ##STR00101## R.sup.2 is hydroxy, heteroalkyl, alkoxy, polyalkoxyalkyl, NR.sup.3R.sup.4, (NHS(O).sub.2-m-(C.sub.6H4)N.sub.3, or Xaa.sub.o; R.sup.3 and R.sup.4 are each and independently of each other selected from hydrogen atom, hydroxy, alkyl, heteroalkyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl, or heteroaralkyl; Xaa is Gly, a conventional D, L-, D- or L-amino acid, a non-conventional D, L-, D- or L-amino acid, or a 2- to 10-mer peptide, wherein Xaa is joined to —C(O) by an amide bond; o is an integer selected from 1 to 10; B is CH.sub.2 or S; m is an integer from 1 to 6; T, U, and W are each —CH.sub.2CH.sub.2—; V is cis or trans —CH═CH—; X is absent or NR.sup.5 Z is selected from CH.sub.2, and NR.sup.5′; R.sup.5 and R.sup.5′ are each and independently of each other selected from a hydrogen atom, a hydroxy, alkyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl, heteroaralkyl group; Y is —C(O)—, —C(O)—C(O)—, —O—, or —S—; and n is an integer from 0 to 6.

2. The compound according to claim 1, wherein R.sup.1 is —COR.sup.2.

3. The compound according to claim 1, wherein m is 1.

4. The compound according to claim 1, wherein n is 0 or 1.

5. The compound according to claim 1, wherein Y is —C(O)— or —C(O)—C(O)—.

6. The compound according to claim 1, wherein X is NR.sup.5 with R.sup.5 being a hydrogen atom, a methyl, ethyl, propyl or iso-propyl group.

7. The compound according to claim 1, wherein Z is NR.sup.5′ with R.sup.5′ being a hydrogen atom, a methyl, ethyl, propyl or iso-propyl group.

8. A pharmaceutical composition that comprises at least one compound according to claim 1 and, optionally, a carrier substance and/or an adjuvant.

9. A method for treatment of conditions and/or diseases associated with inflammation, hypertension, coagulation, immune function, heart failure and cardiac damage comprising administering to a patient in need thereof the pharmaceutical composition of claim 8 in a treatment of conditions and/or diseases associated with inflammation, hypertension, coagulation, immune function, heart failure and cardiac damage effective amount.

10. The method of claim 9, wherein the conditions and/or diseases are associated with cardiac damage.

11. The method of claim 9, wherein the conditions and/or diseases are associated with cardiac arrhythmias.

12. The compound according to claim 2, wherein m is 1.

13. The compound according to claim 2, wherein n is 0 or 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the structures of compounds tested and the values for the chronotropic effects (Δ beats/min) based on the difference between the basal and compound-induced beating rate of the individual clusters.

(2) FIG. 2 shows the structures and chronotropic effects of compounds tested as potential antagonists of EPA and 17,18-EETeTr.

(3) FIG. 3 shows that compound C3 is a highly potent antagonist not only of C4 but also of EPA, 17,18-EETeTr, C2 and C13.

(4) FIG. 4 shows the negative chronotropic effects of EPA (01), 17,18-EETeTr (02) and of the synthetic agonist C13 are blocked by 11,12-EET, compound C3, AH6089 (unselective prostanoid receptor antagonist) and calphostin C (PKC-inhibitor) but not by H89 (PKA inhibitor). The positive chronotropic effect of butaprost (EP2 agonist) is blocked by AH6089 and H89 but not by C3 and caplphostin C.

(5) FIGS. 5A, B show the synthetic agonist C11 suppresses the response of NRCMs to ß-adrenergic stimulation (isoptroterenol, FIG. 5A) and increased extracellular Ca.sup.2-concentrations (FIG. 5B).

(6) FIGS. 6A, B show treatment with compound C17, a synthetic agonist of 17, 18-EETeTr, ameliorates the frequency (FIG. 6A) and severity (FIG. 6B) of cardiac arrhythmias in a rat model of myocardial infarction.

DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

(7) The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

Example 1

Synthesis of (5Z,14Z)-16-(3-Ethyloxirane-2-yl)hexadeca-5,14-dienoic acid (1)

(8) ##STR00007##

(9) 1,4-Butanediol (32 g, 35.55 mmol; Alfa Aesar) and aq. 48% HBr (45 mL) were heated under reflux in benzene (380 mL) with water removal using a Dean-Stark apparatus. After 12 h, all volatiles were removed in vacuo and the residue was purified by SiO.sub.2 column chromatography using a gradient of 10-30% EtOAc/hexanes as eluent to give 4-bromobutan-1-ol (29.20 g, 68%). TLC: 30% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 3.70 (t, J=6.1 Hz, 2H), 3.45 (t, J=6.1 Hz, 2H), 1.92-2.04 (m, 2H), 1.68-1.78 (m, 2H).

(10) ##STR00008##

(11) 3,4-Dihydro-2H-pyran (8.0 g, 95.36 mmol) was added to a 0° C. solution of 4-bromobutan-1-ol (12.0 g, 79.47 mmol) in dichloromethane (150 mL) followed by p-toulenesulphonic acid (20 mg). After 1 h, the reaction was carefully quenched with sat. aq. NaHCO.sub.3 solution (5 mL), washed with water (100 mL), brine (70 mL), and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 2% EtOAc/hexanes as eluent to give 2-(4-bromobutoxy)tetrahydro-2H-pyran (16.57 g, 88%) as colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.50; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 4.58 (t, J=2.5 Hz, 1H), 3.90-3.72 (m, 2H), 3.38-3.50 (m, 4H), 1.92-2.04 (m, 2H), 1.65-1.80 (m, 4H), 1.60-1.50 (m, 4H). Lit. ref: G. L. Kad; I. Kaur; M. Bhandari; J. Singh; J. Kaur Organic Process Research &Development 2003: 7, 339.

(12) ##STR00009##

(13) A solution of 1,7-dibromoheptane (13.5 g, 52.32 mmol) in anhydrous dimethylsulfoxide (25 mL) was added dropwise to a stirring, 0° C. solution of lithium acetylide ethylenediamine complex (12.04 g, 130.8 mmol) in anhydrous dimethylsulfoxide (125 mL) under an argon atmosphere. After stirring at 5-8° C. for 2 h, the reaction mixture was diluted with ether (100 mL) and washed with water (2×40 mL). The aqueous washes were extracted with ether (2×50 mL). The combined ethereal fractions were dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using hexanes as eluent to give undec-1,10-diyne as a colorless oil (5.3 g, 68%) (lit. ref: Hellbach, Björn; Gleiter, Rolf; Rominger, Frank Synthesis 2003, 2535-2541). TLC: SiO.sub.2, hexane (100%), R.sub.f≈0.8; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 2.14-2.18 (m, 4H), 1.92 (t, J=2.55 Hz, 2H), 1.50-1.53 (m, 4H), 1.40-1.42 (m, 4H), 1.23-1.25 (m, 2H).

(14) ##STR00010##

(15) n-BuLi (4.86 mL of 2.5 M in hexanes, 12.16 mmol) was added dropwise to a −78° C. solution of undec-1,10-diyne (2.0 g, 13.51 mmol) in dry tetrahydrofuran/HMPA (105 mL, 6:1) under an argon atmosphere. After 30 min, the reaction mixture was warmed to −10° C. over 2 h and maintained at this temperature for 20 min, then re-cooled to −75° C. To this was added a solution of 2-(4-bromobutoxy)-tetrahydropyran (2.4 g, 10.14 mmol) in dry THF (15 mL). The resulting mixture was warmed to room temperature over 3 h, maintained at this temperature for 12 h, then quenched with sat. aq. NH.sub.4Cl (25 mL). After 20 min, the mixture was extracted with ether (2×125 mL). The combined ethereal extracts were washed with water (2×100 mL), brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to give 2-(pentadeca-5,14-diynyloxy)tetrahydropyran (1.97 g, 64%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.58 (t, J=2.5 Hz, 1H), 3.82-3.89 (m, 1H), 3.71-3.78 (m, 1H), 3.43-3.53 (m, 1H), 3.36-3.47 (m, 1H), 2.01-2.20 (m, 6H), 1.93 (t, J=2.5 Hz, 1H), 1.27-1.81 (m, 20H). Lit. ref: F. Slowinski; C. Aubert; M. Malacria Eur. J. Org. Chem. 2001: 3491.

(16) The reaction also produced approximately 10% of the dialkylated adduct. TLC: 10% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 4.58 (t, J=2.5 Hz, 2H), 3.82-3.89 (m, 2H), 3.71-3.78 (m, 2H), 3.43-3.53 (m, 2H), 3.36-3.47 (m, 2H), 2.01-2.20 (m, 8H), 1.27-1.81 (m, 30H).

(17) ##STR00011##

(18) A solution of the 2-(pentadeca-5,14-diynyloxy)tetrahydropyran (4.05 g, 13.27 mmol) and p-toluenesulphonic acid (42 mg) in MeOH (100 mL) was stirred at room temperature for 4 h. All volatiles were then removed in vacuo and the residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give pentadeca-5,14-diyn-1-ol (2.77 g, 95%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 3.85 (t, 2H, J=7.0 Hz), 2.03-2.30 (m, 6H), 1.93 (t, 1H, J=2.6 Hz), 1.26-1.83 (m, 14H).

(19) Jones reagent (10 mL of a 10 N solution in water) in acetone (25 mL) was added to a stirring, −40° C. solution of above alcohol (1.9 g, 4.55 mmol) in acetone (75 mL). After 1 h, the reaction mixture was warmed to −10° C. and maintained for another 2 h, then quenched with excess (5 equiv) of isopropanol. The green chromium salts were removed by filtration and the filter cake was washed with acetone. The combined filtrates were concentrated in vacuo and the obtained residue was dissolved in EtOAc (100 mL), washed with water (50 mL) and again concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give pentadeca-5,14-diynoic acid (2.42 g, 82%) as a white solid. TLC: 40% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.48 (t, 2H, J=7.3 Hz), 2.10-2.17 (m, 6H), 1.93 (t, 1H, J=2.6 Hz), 1.75-1.86 (m, 2H), 1.25-1.55 (m, 10H).

(20) ##STR00012##

(21) tert-Butyl hydroperoxide (15.72 g, 33 mL of a 5.2 M solution in decane) was added to a stirring solution of pent-2(Z)-en-1-ol (5.00 g, 58.14 mmol) and vanadium(III) acetylacetonate (150 mg) in dry benzene (200 mL) under an argon atmosphere. The initial pale green solution turned pink. After 3 h, the reaction was quenched with dimethylsulfide (52 g, 87.33 mmol, 5 equiv). After an additional 1 h, the reaction was diluted with an equal volume of Et.sub.2O (250 mL), washed with water (2×250 mL), brine (200 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 30% EtOAc/hexanes as eluent to give (Z)-(3-ethyloxiranyl)-methanol (4.86 g, 82%) as a pale yellow oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.86 (dd, 1H, J=12.1 Hz, 4.0 Hz), 3.67 (dd, 1H, J=6.8 Hz, 4.0 Hz), 3.17 (ddd, 1H, J=4.1 Hz, 4.3 Hz, 6.8 Hz), 3.01 (ddd, 1H, J=4.3 Hz, 6.4 Hz, 6.4 Hz) 1.46-1.71 (m, 2H), 1.04 (t, 3H, J=7.6 Hz). Lit. ref: C. Arnold; W. Stefan; Y. A. Yse; S. H. Dieter Liebigs Annalen der Chemie 1987: 7, 629.

(22) ##STR00013##

(23) A solution of carbon tetrabromide (10.8 g, 32.64 mmol) in CH.sub.2Cl.sub.2 (25 mL) was stirred into a −10° C. solution of triphenylphosphine (8.6 g, 32.94 mmol) and the above epoxy alcohol (2.8 g, 27.45 mmol) in dry CH.sub.2Cl.sub.2 (100 mL) under an argon atmosphere. After 30 min, the reaction mixture was washed with water (75 mL), brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, and all volatiles were removed under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to give (Z)-2-bromomethyl-3-ethyloxirane (2.92 g, 65%) as colorless oil. TLC: 20% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.49-3.53 (dd, 1H, J=4.9, 9.3 Hz), 3.22-3.31 (m, 2H), 3.01-3.06 (m, 1H), 1.54-1.62 (m, 2H), 1.08 (t, 3H, J=7.6 Hz).

(24) ##STR00014##

(25) n-BuLi (1.8 mL of a 2.5 M hexanes solution, 4.48 mmol) was added slowly to a −70° C. solution of pentadeca-5,14-diynoic acid (0.5 g, 2.14 mmol) in dry tetrahydrofuran (30 mL) and HMPA (8 mL) under an argon atmosphere. The resulting mixture was stirred at −75° C. for 30 min, then allowed to warm to 0° C. over 2 h. After 1 h at 0° C., the reaction mixture was re-cooled −72° C. and a solution of (Z)-2-bromomethyl-3-ethyloxirane (0.46 g, 2.56 mmol) in dry THF (10 mL) was introduced. The resulting mixture was warmed to room temperature over 3 h. After stirring at room temperature for 12 h, the reaction was quenched with sat. aq. NH.sub.4Cl (10 mL), stirred for 20 min, and then extracted with ether (3×75 mL). The combined ethereal extracts were washed with water (2×100 mL), brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was dissolved in 5% MeOH/ether, cooled to 0° C., and treated with an excess of ethereal diazomethane until the yellow color persisted for 10 min. After 1 h, all volatiles were removed under reduced pressure and the residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5,14-diynoate (0.39 g, 56%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.65 (s, 3H), 3.07-3.12 (m, 1H), 2.88-2.92 (m, 1H), 2.51-2.58 (m, 1H), 2.41 (t, 2H, J=7.3), 2.08-2.26 (m, 7H), 1.74-1.81 (m, 2H), 1.22-1.64 (m, 12H), 1.05 (t, 3H, J=7.6 Hz). Lit. ref: J. R. Falck; P. S. Kumar; Y. K. Reddy; G. Zou; J. H. Capdevila Tetrahedron Lett. 2001: 42, 7211.

(26) ##STR00015##

(27) NaBH.sub.4 (33 mg, 0.88 mmol) was added portionwise to a stirring solution of nickel(II) acetate tetrahydrate (190 mg, 0.76 mmol) in absolute ethanol (5 mL) under a hydrogen blanket (1 atm). After 15 min, freshly distilled ethylenediamine (200 mg, 3.24 mmol) was added followed by a solution of methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5,14-diynoate (360 mg, 1.08 mmol) in absolute ethanol (5 mL). The heterogeneous mixture was maintained at room temperature for 90 min, then diluted with ether (15 mL), and filtered through a short pad of silica gel.

(28) The filter cake was washed with ether (3×5 mL). The combined ethereal filtrates were dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),14(Z)-dienoate (0.35 g, 97%) as a colorless oil sufficiently pure to be used in the next step without purification. TLC: 20% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.24-5.54 (m, 4H), 3.62 (s, 3H), 2.82-2.92 (m, 2H), 2.26-2.38 (m, 1H), 2.29 (t, 2H, J=7.3 Hz), 2.10-2.18 (m, 1H), 1.93-2.06 (m, 6H), 1.60-1.69 (m, 2H), 1.46-1.59 (m, 2H), 1.20-1.34 (m, 10H), 1.01 (t, 3H, J=7.3 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 174.24, 133.12, 130.16, 128.62, 124.12, 58.6, 56.8, 51.96, 33.72, 29.91, 29.84, 29.58, 29.46, 27.54, 27.48, 26.84, 26.43, 25.06, 21.21, 10.08.

(29) ##STR00016##

(30) LiOH (1 mL, 2 M aqueous solution) was added to a 0° C. solution of methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),14(Z)-dienoate (90 mg, 0.266 mmol) in THF (8 mL) and deionized H.sub.2O (2 mL). After stirring at room temperature overnight, the reaction mixture was cooled to 0° C., the pH was adjusted to 4 with 1 M aq. oxalic acid, and extracted with ethyl acetate (2×20 mL). The combined extracts were washed with water (30 mL), brine (25 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 25% EtOAc/hexanes as eluent to give 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),14(Z)-dienoic acid (82 mg, 92%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.26-5.51 (m, 4H), 2.88-2.98 (m, 2H), 2.31-2.44 (m, 1H), 2.35 (t, 2H, J=7.7 Hz), 2.13-2.20 (m, 1H), 1.96-2.11 (m, 6H), 1.64-1.70 (m, 2H), 1.48-1.61 (m, 2H), 1.22-1.37 (m, 10H), 1.05 (t, 3H, J=7.51); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 179.96, 133.02, 131.87, 128.40, 123.97, 58.85, 57.73, 33.86, 30.04, 29.96, 29.94, 29.88, 29.81, 27.64, 27.42, 26.81, 26.24, 24.86, 21.28, 10.81.

Example 2

Synthesis of (5Z,11Z)-16-(3-Ethyloxirane-2-yl)hexadeca-5,11-dienoic acid (2)

(31) ##STR00017##

(32) Oct-1,7-diyne (9.0 g, 84.9 mmol; G F Smith) was alkylated with 2-(4-bromobutoxy)-tetrahydropyran (15 g, 63.68 mmol) as described above for the synthesis of 2-(pentadeca-5,14-diynyloxy)tetrahydropyran to give 2-(dodeca-5,11-diynyloxy)tetrahydropyran (10.85 g, 65%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.57 (t, J=2.5 Hz, 1H), 3.82-3.87 (m, 1H), 3.70-3.77 (m, 1H), 3.46-3.51 (m, 1H), 3.36-3.42 (m, 1H), 2.14-2.20 (m, 6H), 1.93 (t, 1H, J=2.5 Hz), 1.46-1.72 (m, 14H).

(33) ##STR00018##

(34) A solution of carbon tetrabromide (10.8 g, 32.94 mmol) in CH.sub.2Cl.sub.2 (25 mL) was stirred into a 0° C. solution of triphenylphosphine (8.6 g, 32.94 mmol) and oct-5(Z)-en-1-ol (2.8 g, 14.06 mmol) in dry CH.sub.2Cl.sub.2 (100 mL) under an argon atmosphere. After 30 min, the reaction mixture was washed with water (75 mL), brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, and all volatiles were removed under reduced pressure. The residue was purified by fractional distillation to afford 8-bromo-oct-3(Z)-ene (2.01 g, 75%) as a light yellow oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.7; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.26-5.45 (m, 2H), 3.42 (t, 2H, J=7.6 Hz), 1.98-2.22 (m, 4H), 1.63-1.82 (m, 2H), 1.46-1.54 (m, 2H), 0.95 (t, 3H, J=7.3 Hz). Lit. ref: R. M. Seifert J. Agric. Food Chem. 1981: 29, 647.

(35) ##STR00019##

(36) n-BuLi (2.5 M solution in hexanes, 20.65 mmol), 2-(dodeca-5,11-diynyloxy)tetrahydropyran (4.5 g, 17.2 mmol), and 8-bromo-oct-3(Z)-ene (4.1 g, 21.5 mmol) were reacted as described above for the synthesis of 2-(pentadeca-5,14-diynyloxy)tetrahydropyran to give 2-[eicos-17 (Z)-ene-5,11-diynyloxy]tetrahydropyran (4.15 g, 65%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.26-5.41 (m, 2H), 4.58 (t, J=2.5 Hz, 1H), 3.82-3.87 (m, 1H), 3.70-3.77 (m, 1H), 3.46-3.51 (m, 1H), 3.36-3.42 (m, 1H), 2.11-2.20 (m, 8H), 1.92-2.04 (m, 4H), 1.62-1.86 (m, 4H), 1.39-1.69 (m, 14H), 0.94 (t, 3H, J=7.5 Hz).

(37) ##STR00020##

(38) A solution of 2-[eicos-17(Z)-ene-5,11-diynyloxy]tetrahydropyran (1.3 g, 3.49 mmol) and p-toluenesulphonic acid (50 mg; PTSA) in MeOH (50 mL) was stirred at room temperature for 4 h, then concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give eicosa-17(Z)-ene-5,11-diyn-1-ol (925 mg, 92%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.35; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.27-5.42 (m, 2H), 3.66 (t, 2H, J=6.8 Hz), 2.00-2.19 (m, 12H), 1.43-1.72 (m, 12H), 0.95 (t, 3H, J=7.7 Hz).

(39) Jones reagent (5 mL of a 10 N aq. solution) in acetone (10 mL) was added slowly to a stirring, −40° C. solution of eicosa-17(Z)-ene-5,11-diyn-1-ol (1.0 g, 3.47 mmol) in acetone (50 mL). After 1 h, the reaction mixture was warmed to −10° C., maintained at this temperature for 3 h, then quenched with excess (5 equiv) isopropanol. The green chromium salts were removed by filtration, the filter cake was washed with acetone, and the combined filtrates were concentrated in vacuo. The residue was dissolved in ethyl acetate (100 mL), washed with water (50 mL), and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give eicosa-17(Z)-ene-5,11-diynoic acid (920 mg, 88%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.35; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.24-5.41 (m, 2H), 2.41 (t, 3H, J=6.9 Hz), 2.10-2.19 (m, 8H), 1.98-2.09 (m, 4H), 1.75-1.81 (m, 2H), 0.96 (t, 3H, J=7.7 Hz).

(40) ##STR00021##

(41) A solution of eicosa-17(Z)-ene-5,11-diynoic acid (0.8 g, 2.63 mmol) and PTSA (20 mg) in MeOH (30 mL) was stirred at room temperature for 10 h, then concentrated in vacuo and the residue was purified by SiO.sub.2 column chromatography using 3% EtOAc/hexanes as eluent to give methyl eicos-17(Z)-ene-5,11-diynoate (682 mg, 82%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.60; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.27-5.42 (m, 2H), 3.67 (s, 3H), 2.43 (t, 2H, J=7.6 Hz), 2.12-2.21 (m, 8H), 1.99-2.09 (m, 4H), 1.76-1.82 (m, 2H), 1.42-1.58 (m, 8H), 0.95 (t, 3H, J=7.7 Hz).

(42) ##STR00022##

(43) m-Chloroperbenzoic acid (1.6 g, 4.76 mmol; m-CPBA) was added to a 0° C. solution of methyl eicosa-17(Z)-ene-5,11-diynoate (1.15 g, 3.66 mmol) in CH.sub.2Cl.sub.2 (50 mL). After 2 h at room temperature, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (25 mL), washed with sat. aq. NaHCO.sub.3 (2×25 mL), brine (2×25 mL), water (50 mL), dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5,11-diynoate (990 mg, 82%) as colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.67 (s, 3H), 2.84-2.94 (m, 2H), 2.42 (t, 2H, J=7.3 Hz), 2.14-2.23 (m, 8H), 1.74-1.83 (m, 2H), 1.42-1.61 (m, 12H), 1.03 (t, 3H, J=7.6 Hz).

(44) ##STR00023##

(45) Methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5,11-diynoate (250 mg, 0.75 mmol) was subjected to semi-hydrogenation as described above for the synthesis of methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),14(Z)-dienoate to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),11(Z)-dienoate (246 mg, 98%) as a colorless oil. TLC: 20% EtOAc/hexanes, R.sub.f≈0.65; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.27-5.42 (m, 4H), 3.66 (s, 3H), 2.83-2.93 (m, 2H), 2.30 (t, 2H, J=7.3 Hz), 1.92-2.09 (m, 8H), 1.63-1.72 (m, 2H), 1.25-1.58 (m, 12H), 1.03 (t, 3H, J=7.7 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 174.45, 131.24, 130.04, 129.68, 128.88, 58.30, 56.75, 51.65, 33.63, 29.92, 29.76, 27.94, 29.74, 27.36, 26.86, 26.52, 25.54, 21.36, 10.89. Lit. ref: J. R. Falck; L. M. Reddy; Y. K. Reddy; M. Bondlela; U. M. Krishna; Y. Ji; J. Sun.; J. K. Liao Bioorg. Med. Chem. Lett. 2003: 13, 4011.

(46) ##STR00024##

(47) Methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),11(Z)-dienoate (0.25 g, 0.74 mmol) was hydrolyzed as described above for 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),14(Z)-dienoic acid to give 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),11(Z)-dienoic acid (222 mg, 93%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.28-5.40 (m, 4H), 2.87-2.97 (m, 2H), 2.34 (t, 3H, J=7.0 Hz), 1.97-2.12 (m, 8H), 1.63-1.74 (m, 2H), 1.30-1.60 (m, 12H), 1.02 (t, 3H, J=7.4 Hz); .sup.13C NMR (300 MHz, CDCl.sub.3) δ 180.06, 131.75, 130.03, 129.77, 128.66, 58.86, 57.87, 33.93, 29.93, 29.84, 29.81, 27.89, 27.68, 26.41, 26.36, 24.83, 21.26, 10.84.

Example 3

Synthesis of (8Z,14Z)-16-(3-Ethyloxirane-2-yl)hexadeca-8,14-dienoic Acid (3)

(48) ##STR00025##

(49) Heptane-1,7-diol (36.0 g, 272 mmol; Alfa Aesar) and aq. 48% HBr (38 mL) were heated under reflux in benzene (400 mL) with water removal using a Dean-Stark apparatus. After 12 h, all volatiles were removed in vacuo and the residue was purified by SiO.sub.2 column chromatography using a gradient of 10-30% EtOAc/hexanes as eluent to give 7-bromoheptan-1-ol (26.22 g, 62%) as colorless oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.4; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.61 (t, 2H, J=7.1 Hz), 3.39 (t, 2H, J=6.8 Hz), 1.80-1.88 (m, 2H), 1.52-1.58 (m, 2H), 1.30-1.46 (m, 6H).

(50) ##STR00026##

(51) 7-Bromoheptane-1-ol (11.0 g, 56.7 mmol) from above was protected as its THP ether as described previously to give 2-(7-bromoheptyloxy)tetrahydro-2H-pyran (14.50 g, 92%) as colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.58 (m, J=2.5 Hz, 1H), 3.84-3.88 (m, 1H), 3.68-3.77 (m, 1H), 3.46-3.3.51 (m, 1H), 3.33-3.43 (m, 3H), 1.80-1.81 (m, 2H), 1.30-1.62 (m, 14H).

(52) ##STR00027##

(53) Oct-1,7-diyne (6.3 g, 59.3 mmol) was alkylated with 2-(7-bromoheptyloxy)tetrahydro-2H-pyran (11 g, 39.56 mmol) as described above to give 2-(pentadeca-8,14-diynyloxy)tetrahydro-2H-pyran (7.82 g, 64%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.57 (t, J=2.5 Hz, 1H), 3.82-3.87 (m, 1H), 3.70-3.77 (m, 1H), 3.46-3.51 (m, 1H), 3.36-3.42 (m, 1H), 2.14-2.20 (m, 6H), 1.93 (t, J=2.6 Hz, 1H), 1.46-1.72 (m, 20H)

(54) ##STR00028##

(55) 2-(Pentadeca-8,14-diynyloxy)tetrahydro-2H-pyran (5 g, 16.45 mmol) was cleaved using p-toluenesulphonic acid (60 mg) in MeOH (100 mL) as described above and the product was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give pentadeca-8,14-diyn-1-ol (3.26 g, 90%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.35; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.63 (t, 2H, J=5.5 Hz), 2.10-2.18 (m, 6H), 1.93 (t, 1H, J=2.6 Hz), 1.24-1.62 (m, 14H).

(56) ##STR00029##

(57) Oxidation of pentadeca-8,14-diyn-1-ol (3.0 g, 13.69 mmol) using Jones reagent as described above and purification by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent gave pentadeca-8,14-diynoic acid (2.80 g, 87%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.33; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.34 (t, J=7.0 Hz, 2H), 2.10-2.18 (m, 6H), 1.93 (t, J=2.6 Hz, 1H), 1.55-1.67 (m, 6H), 1.33-1.49 (m, 6H).

(58) ##STR00030##

(59) Pentadeca-8,14-diynoic acid (0.80 g, 3.42 mmol) was alkylated with (Z)-2-(bromomethyl)-3-ethyloxirane (0.74 g, 4.10 mmol) and esterified using diazomethane as described above to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5,14-diynoate to give methyl 16-[(Z)-3-ethyloxiran-2-yl]hexadeca-8,14-diynoate (658 mg, 58%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5; 1H NMR (400 MHz, CDCl.sub.3) δ 3.65 (s, 3H), 3.07-3.12 (m, 1H), 2.88-2.92 (m, 1H), 2.51-2.61 (m, 1H), 2.32-2.50 (m, 1H), 2.30 (t, J=7.5 Hz, 3H), 2.08-2.25 (m, 6H), 1.25-1.65 (m, 14H), 1.06 (t, J=7.3 Hz, 3H)

(60) ##STR00031##

(61) Methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-8,14-diynoate was subjected to the semi-hydrogenation procedure above to give methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-8(Z),14(Z)-dienoate (97%) as a colorless oil. TLC: 20% EtOAc/hexanes, R.sub.f≈0.55; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.31-5.56 (m, 4H), 3.66 (s, 3H), 2.86-2.96 (m, 2H), 2.25-2.42 (m, 1H), 2.28 (t, 2H, J=7.33 Hz), 2.12-2.20 (m, 1H), 1.96-2.08 (m, 6H), 1.52-1.64 (m, 4H), 1.26-1.39 (m, 10H), 1.03 (t, 3H, J=7.3 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 174.30, 132.60, 129.99, 129.84, 124.13, 58.40, 56.73, 51.51, 34.17, 29.66, 29.47, 29.30, 29.18, 29.03, 27.46, 27.27, 27.20, 26.28, 25.05, 21.21, 10.76.

(62) ##STR00032##

(63) Methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-8(Z),14(Z)-dienoate was hydrolyzed as described above to give 16-[(Z)-3-ethyloxiranyl]hexadeca-8(Z), 14(Z)-dienoic acid (93%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.31-5.53 (m, 4H), 2.87-2.98 (m, 2H), 2.33-2.43 (m, 1H), 2.33 (t, J=7.3 Hz, 2H), 2.13-2.22 (m, 1H), 1.94-2.08 (m, 6H), 1.52-1.64 (m, 4H), 1.30-1.38 (m, 10H), 1.04 (t, J=7.4 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) δ 180.06, 132.54, 130.03, 130.01, 125.03, 58.87, 57.73, 34.16, 29.86, 29.74, 29.71, 29.52, 29.45, 27.84, 27.67, 27.42, 26.33, 24.75, 21.48, 10.82.

Example 4

Synthesis of 16-[(Z)-3-Ethyloxiranyl]hexadec-11(Z)-enoic Acid (4), 16-[(Z)-3-Ethyloxiranyl]hexadec-5(Z)-enoic Acid (7), and 16-[(Z)-3-Ethyloxiranyl]hexadecanoic Acid (8)

(64) ##STR00033##

(65) A stream of dry air was passed through a stirring solution of hydrazine hydrate (400 mg, 12 mmol, 20 equiv), methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),11(Z)-dienoate (200 mg, 0.60 mmol), and CuSO.sub.4.5H.sub.2O (10 mg) in ethanol (5 mL). The stream of air was passed through EtOH to saturated it with ethanol and help maintain the reaction volume. After 12 h, the reaction mixture was passed through a short pad of silica gel and the filter cake was washed with dichloromethane (3×10 mL). The combined filtrates were dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was resolved into its components by AgNO.sub.3-impregnated PTLC using 2% CH.sub.2Cl.sub.2/benzene: R.sub.f≈0.2, 0.4, 0.55, and 0.85 for methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-5(Z),11(Z)-dienoate, methyl 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoate, methyl 16-[(Z)-3-ethyloxiranyl]hexadec-5(Z)-enoate, and methyl 16-[(Z)-3-ethyloxiranyl]hexadecanoate, respectively, isolated in a ratio of 2:3:3:2, respectively. Lit. ref: E. J. Corey; T. M. Eckrich Tetrahedron Lett. 1984: 25, 2415.

(66) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-5(Z)-enoate: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.27-5.42 (m, 2H), 3.66 (s, 3H), 2.84-2.92 (m, 2H), 2.30 (t, J=7.4 Hz, 2H), 1.96-2.08 (m, 4H), 1.64-1.71 (m, 2H), 1.45-1.58 (m, 4H), 1.21-1.36 (m, 16H), 1.03 (t, J=7.3 Hz, 3H); .sup.13 C NMR (100 MHz, CDCl.sub.3) δ 174.45, 131.88, 128.63, 58.64, 57.87, 51.96, 33.88, 29.99, 29.86, 29.74, 29.46, 27.98, 27.76, 26.88, 26.72, 25.88, 21.32, 10.48.

(67) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoate: .sup.1H NMR (300 MHz, CDCl.sub.3) 5.25-5.35 (m, 2H), 3.61 (s, 3H), 2.79-2.89 (m, 2H), 2.25 (t, J=7.3 Hz, 2H), 1.93-2.04 (m, 4H), 1.19-1.60 (m, 22H), 1.00 (t, J=7.2 Hz, 3H); .sup.13 C NMR (100 MHz, CDCl.sub.3) δ 174.48, 130.41, 129.54, 58.54, 57.45, 51.62, 34.27, 29.92, 29.81, 29.67, 29.63, 29.47, 29.46, 29.34, 27.80, 27.42, 27.27, 26.42, 25.14, 10.82.

(68) Methyl 16-[(Z)-3-ethyloxiranyl]hexadecanoate: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.67 (s, 3H), 2.84-2.94 (m, 2H), 2.31 (t, 2H, J=7.4 Hz), 1.42-1.65 (m, 6H), 1.22-1.34 (m, 24H), 1.04 (t, 3H, J=7.3 Hz).

(69) ##STR00034##

(70) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-5(Z)-enoate was hydrolyzed as described above to afford 16-[(Z)-3-ethyloxiranyl]hexadec-5(Z)-enoic acid (7, 92%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.27-5.43 (m, 2H), 2.85-2.93 (m, 2H), 2.34 (t, J=7.6 Hz, 2H), 1.95-2.11 (m, 4H), 1.64-1.72 (m, 2H), 1.49-1.60 (m, 4H), 1.22-1.36 (m, 16H), 1.03 (t, J=7.4 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) δ 179.42, 131.54, 128.40, 60.08, 58.75, 57.73, 34.59, 31.86, 29.86, 29.74, 29.71, 29.45, 27.84, 27.42, 26.81, 26.64, 24.85, 21.28, 15.47, 10.81.

(71) ##STR00035##

(72) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoate was hydrolyzed as described above to afford 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoic acid (4, 92%) as a colorless oil. TLC: SiO.sub.2, 30% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.28-5.40 (m, 2H), 2.84-2.94 (m, 2H), 2.31 (t, J=7.6 Hz, 2H), 1.96-2.04 (m, 4H), 1.02-1.62 (m, 22H), 1.01 (t, 3H, J=7.4 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) δ 180.10, 130.45, 129.57, 58.74, 57.67, 34.27, 29.92, 29.81, 29.66, 29.60, 29.46, 29.43, 29.25, 27.76, 27.43, 27.28, 26.41, 24.89, 21.27, 10.81.

(73) ##STR00036##

(74) Methyl 16-[(Z)-3-ethyloxiranyl] hexadecanoate was hydrolyzed as described above to afford 16-[(Z)-3-ethyloxiranyl]hexadecanoic acid (8, 94%) as white solid. M.P.: 62.1-62.5° C., TLC: 30% EtOAc/hexanes, R.sub.f≈0.35; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.86-2.94 (m, 2H), 2.34 (t, 2H, J=7.3 Hz), 1.46-1.65 (m, 30H), 1.04 (t, 3H, J=7.35 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 180.04, 58.83, 57.47, 34.24, 30.06, 30.03, 29.92, 29.81, 29.66, 29.60, 29.46, 29.43, 29.25, 27.76, 27.43, 27.28, 26.41, 24.89, 21.27, 10.89.

Enantiomeric Resolution of Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoate by Chiral HPLC

(75) Chromatography of methyl 16-[(Z)-3-ethyloxiranyl]hexadec-11(Z)-enoate using a Chiralcel® OJ-H column (250×4.6 mm) with hexane/iPrOH (99.7:0.3) at a flow rate of 1 mL/min, uv detector at 195 nm, furnished the R,S-enantiomer (R.sub.t=15.17 min) and S,R-enantiomer (R.sub.t=17.68 min). Preparative separation: Chiralcel® OJ-H column (250×20 mm) using hexane/iPrOH (99.5:0.5) at a flow rate of 8 mL/min, uv detector at 195 nm, injecting 7 mg/100 μL in mobile phase.

Example 5

Synthesis of 16-[(Z)-3-Ethyloxiranyl]hexadec-14(Z)-enoic Acid (5), 16-[(Z)-3-Ethyloxiranyl]hexadec-8(Z)-enoic Acid (6) and 16-[(Z)-Ethyloxiranyl]hexadec-14 (Z)-enoic Acid (8)

(76) ##STR00037##

(77) Methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-8(Z),14(Z)-dienoate was partially reduced using diimide as described above. AgNO.sub.3-impregnated PTLC using 2% CH.sub.2Cl.sub.2/benzene: R.sub.f≈0.2, 0.5, 0.6, and 0.85 for methyl 16-[(Z)-3-ethyloxiranyl]hexadeca-8(Z),14(Z)-dienoate, methyl (Z)-16-(3-ethyloxiranyl)hexadec-14(Z)-enoate, methyl 16-[(Z)-3-ethyloxiranyl]hexadec-8(Z)-enoate, and methyl 16-[(Z)-3-ethyloxiranyl]hexadecanoate, respectively, isolated in a ratio of 2:3:3:2, respectively.

(78) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-8(Z)-enoate: .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.31-5.35 (m, 2H), 3.66 (s, 3H), 2.84-2.91 (m, 2H), 2.27 (t, J=7.3 Hz, 2H), 1.97-2.08 (m, 4H), 1.47-1.64 (m, 4H), 1.22-1.39 (m, 18H), 1.03 (t, J=7.3 Hz, 3H).

(79) Methyl 16-[(Z)-ethyloxiranyl]hexadec-14(Z)-enoate: .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.35-5.53 (m, 2H), 3.63 (s, 3H), 2.84-2.95 (m, 2H), 2.32-2.39 (m, 1H), 2.27 (t, J=7.3 Hz, 2H), 2.12-2.95 (m, 1H), 1.98-2.04 (m, 2H), 1.48-1.64 (m, 4H), 1.20-1.34 (m, 18H), 1.04 (t, J=7.4 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) 174.62, 132.86, 123.86, 58.84, 56.92, 51.76, 34.48, 29.96, 29.89, 29.84, 29.79, 29.74, 29.68, 29.66, 29.59, 29.57, 27.76, 26.36, 25.17, 21.33, 10.07.

(80) ##STR00038##

(81) Methyl 16-[(Z)-3-ethyloxiranyl]hexadec-8(Z)-enoate was hydrolyzed as described above to afford 16-[(Z)-3-ethyloxiranyl]hexadec-8(Z)-enoic acid (6, 91%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.33; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.34-5.40 (m, 2H), 2.90-2.96 (m, 2H), 2.36 (t, 2H, J=7.7 Hz), 2.01-2.05 (m, 4H), 1.22-1.65 (m, 22H), 1.07 (t, 3H, J=7.4 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) δ 180.08, 130.52, 129.66, 58.54, 57.47, 34.23, 29.81, 29.61, 29.56, 29.36, 29.16, 29.13, 29.07, 28.86, 27.53, 26.78, 26.61, 24.49, 21.46, 10.78.

(82) ##STR00039##

(83) Methyl 16-[(Z)-3-ethyloxiranyl] hexadec-14 (Z)-enoate was hydrolyzed as described above to afford 16-[(Z)-3-ethyloxiranyl] hexadec-14(Z)-enoic acid (5, 90%). TLC: 30% EtOAc/hexanes, R.sub.f≈0.32; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 5.36-5.59 (m, 2H), 2.87-2.98 (m, 2H), 2.34 (t, J=7.6 Hz, 2H), 2.31-2.43 (m, 1H), 2.12-2.22 (m, 1H), 1.99-2.06 (m, 2H), 1.50-1.64 (m, 4H), 1.20-1.35 (m, 18H), 1.04 (t, J=7.3 Hz, 3H); .sup.13 C NMR (75 MHz, CDCl.sub.3) δ 180.04, 133.06, 123.96, 58.46, 57.42, 34.12, 30.04, 30.01, 30.00, 29.98, 29.84, 29.96, 29.92, 29.89, 29.87, 27.88, 26.38, 25.01, 21.27, 10.92.

Example 6

Synthesis of 16-(3-Ethylureido)hexadec-11(Z)-enoic Acid (11)

(84) ##STR00040##

(85) NaH (7.5 g, 60% oil dispersion, 326 mmol) was added portionwise to a stirring, 0° C. solution of dodec-3-yn-1-ol (10.0 g, 54.95 mmol; GF Smith) in ethylenediamine (40 mL). After 1 h, the temperature was raised to 70° C. After another 8 h, the reaction mixture was cooled to 0° C., carefully quenched with ice cold water (100 mL), and extracted with ether (3×60 mL). The combined ethereal extracts were washed with water (100 mL). The aqueous wash was back-extracted with ether (3×60 mL). The combined organic extracts were concentrated in vacuo and the residue subjected to column chromatography using 10% EtOAc/hexanes afforded dodec-10-yn-1-ol (7.4 g, 74%) contaminated with 3-5% of other regioisomers. TLC: 30% EtOAc/hexane, R.sub.f≈0.4; .sup.1H NMR (300 MHz, CDCl.sub.3) δ 3.66 (t, 2H, J=7.3 Hz), 2.14-2.21 (m, 2H), 1.93 (t, J=1.9 Hz, 1H), 1.20-1.63 (m, 16H). Lit. ref: R. V. Novikov; A. A. Vasil'ev; I. A. Balova Russ. Chem. Bull., Internat. Ed. 2005: 54, 1043-1045.

(86) ##STR00041##

(87) tert-Butyldiphenylsilyl chloride (TBDPSCl, 8.70 g, 31.65 mmol) was slowly added to a 0° C. solution of dodec-11-yn-1-ol (4.80 g, 26.37 mmol) and imidazole (3.23 g, 47.47 mmol) in anhydrous dichloromethane (100 mL). After stirring at room temperature for 3 h, the reaction mixture was washed with water (75 mL), brine (50 mL), and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 3% EtOAc/hexanes as eluent to give 12-(tert-butyldiphenylsilyloxy)dodec-1-yne (9.75 g, 88%) as a colorless oil. TLC: 6% EtOAc/hexanes, R.sub.f≈0.7; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.65-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.65 (t, J=7.3 Hz, 2H), 2.18 (dt, J=7.0, 2.4 Hz, 2H), 1.94 (t, J=1.9 Hz, 1H), 1.20-1.60 (m, 16H), 1.04 (s, 9H).

(88) ##STR00042##

(89) Alkylation of 12-(tert-butyldiphenylsilyloxy)dodec-1-yne with 2-(4-bromobutoxy)tetrahydropyran as described above gave tert-butyldiphenyl-[16-(tetrahydropyran-2-yloxy)hexadec-11-ynyloxy]silane (66%) as a colorless oil which was used in the next reaction without further purification. TLC: 10% EtOAc/hexane, R.sub.f≈0.5.

(90) ##STR00043##

(91) Tetra-n-butylammonium fluoride (3.14 g, 12.5 mL of a 1 M soln in THF, 12.50 mmol) was added to a solution of the above crude tert-butyldiphenyl-[16-(tetrahydropyran-2-yloxy)hexadec-11-ynyloxy]silane (6 g, 10.42 mmol) in THF (150 mL) under an argon atmosphere. After 5 h, the reaction mixture was quenched with sat. aq. NH.sub.4Cl (5 mL), washed with water (100 mL), and brine (75 mL). The aqueous layer was back-extracted with ether (2×75 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, concentrated under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography using 5-10% EtOAc/hexanes as eluent to give 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11-yn-1-ol (3.17 g, 80% overall) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.4; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 4.57-4.59 (m, 1H), 3.82-3.90 (m, 1H), 3.71-3.79 (m, 1H), 3.64 (t, 2H, J=6.8 Hz), 3.46-3.53 (m, 1H), 3.36-3.44 (m, 1H), 2.10-2.22 (m, 4H), 1.20-1.80 (m, 26H).

(92) ##STR00044##

(93) Semi-hydrogenation of 16-(tetrahydro-2H-pyran-2-yloxy) hexadec-11-yn-1-ol as described above gave 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11(Z)-en-1-ol (99%) as a colorless oil. TLC: 20% EtOAc/hexane, R.sub.f=0.30; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.33-5.37 (m, 2H), 4.58 (m, 1H), 3.83-3.90 (m, 1H), 3.73-3.77 (m, 1H), 3.65 (t, 2H, J=6.7 Hz), 3.46-3.53 (m, 1H), 3.34-3.44 (m, 1H), 1.97-2.09 (m, 4H), 1.20-1.83 (m, 26H).

(94) ##STR00045##

(95) Jones oxidation of 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11(Z)-en-1-ol as described above gave 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11(Z)-enoic acid (68%) as a colorless oil. TLC: SiO.sub.2, 40% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.33-5.37 (m, 2H), 4.56-4.58 (m, 1H), 3.83-3.88 (m, 1H), 3.73-3.78 (m, 1H), 3.49-3.53 (m, 1H), 3.35-3.43 (m, 1H), 2.34 (t, J=7.0 Hz, 2H) 1.97-2.09 (m, 4H), 1.20-1.84 (m, 24H).

(96) ##STR00046##

(97) A solution of 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11(Z)-enoic acid (2.1 g, 5.93 mmol) and PTSA (50 mg) in MeOH (30 mL) was stirred at room temperature for 10 h, then concentrated in vacuo and the residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to give methyl 16-hydroxyhexadec-11(Z)-enoate (1.42 g, 83%) as a colorless oil. TLC: 20% EtOAc/hexanes, R.sub.f≈0.35; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.33-5.37 (m, 2H), 3.65 (s, 3H), 3.63 (t, J=7.3 Hz, 2H), 2.29 (t, J=7.0 Hz, 2H), 1.97-2.08 (m, 4H), 1.21-1.64 (m, 18H).

(98) ##STR00047##

(99) Diisopropyl azodicaboxylate (DIAD; 1.15 g, 5.70 mmol) was added dropwise to a −20° C. solution of triphenylphosphine (1.49 g, 5.70 mmol) in dry THF (30 mL) under an argon atmosphere. After stirring for 10 min, a solution of methyl 16-hydroxyhexadec-11(Z)-enoate (1.35 g, 4.75 mmol) in anhydrous THF (5 mL) was added dropwise. After 30 min at −20° C., the reaction mixture was warmed to 0° C. and diphenylphosphoryl azide (DPPA, 1.38 g, 5.70 mmol) was added dropwise. After stirring at room temperature for 6 h, the reaction was quenched with water (3 mL), diluted with ether (50 mL), and washed with brine (40 mL). The aqueous layer was back-extracted with ether (2×30 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to afford methyl 16-azidohexadec-11(Z)-enoate (1.14 g, 78%) as a light yellow oil (contaminated with a little DIAD impurity). TLC: 10% EtOAc/hexanes, R.sub.f≈0.45; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.31-5.43 (m, 2H), 3.66 (s, 3H), 3.26 (t, J=6.7 Hz, 2H), 2.30 (t, J=7.1 Hz, 2H), 1.97-2.10 (m, 4H), 1.50-1.64 (m, 4H), 1.15-1.48 (m, 14H). Lit. ref.: C. M. Afonso; M. T. Barros; L. S. Godinhoa; C. D. Maycock Tetrahedron 1994: 50, 9671.

(100) ##STR00048##

(101) Triphenylphosphine (1.15 g, 4.41 mmol) was added to a room temperature solution of methyl 16-azidohexadec-11(Z)-enoate (1.05 g, 3.4 mmol) in THF (25 mL). After 2 h, water (200 □L) was added and the stirring was continued for another 8 h. The reaction mixture was then diluted with EtOAc (20 mL), washed with water (20 mL) and brine (25 mL). Aqueous layers were back-extracted with EtOAc (2×30 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, concentrated under reduced pressure and further dried under high vacuum for 4 h. The crude methyl 16-aminohexadec-11(Z)-enoate was used in the next step without additional purification. Lit. ref.: S. Chandrasekhar; S. S. Sultana; N. Kiranmai; Ch. Narsihmulu Tetrahedron Lett. 2007: 48, 2373.

(102) Ethyl isocyanate (60 mg, 0.85 mmol) was added to a room temperature solution of the above crude methyl 16-aminohexadec-11(Z)-enoate (200 mg. 0.71 mmol) in dry THF (20 mL). After 6 h, reaction mixture was concentrated under reduced pressure and the residue was purified by SiO.sub.2 column chromatography using 30% EtOAc/hexanes as eluent to give methyl 16-(3-ethylureido)hexadec-11(Z)-enoate (223 mg, 86%) as a colorless, thick oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.23-5.38 (m, 2H), 5.08 (br s, 2H), 3.63 (s, 3H), 3.09-3.20 (m, 4H), 2.27 (t, J=7.1 Hz, 2H), 1.93-2.04 (m, 4H), 1.20-1.62 (m, 18H), 1.08 (t, J=7.3 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.72, 130.53, 129.45, 51.70, 40.47, 35.26, 34.32, 30.24, 29.91, 29.66, 29.60, 29.46, 29.34, 27.43, 27.27, 27.12, 25.15, 15.80. Lit. ref.: V. Papesch; E. F. Schroeded J. Org. Chem. 1951: 16, 1879.

(103) ##STR00049##

(104) Methyl 16-(3-ethylureido)hexadec-11(Z)-enoate was hydrolyzed as described above to give 16-(3-ethylureido)hexadec-11(Z)-enoic acid (82%) obtained as a white powder. M.P.: 83.1-83.3° C. TLC: SiO.sub.2, 75% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.26-5.42 (m, 2H), 4.89 (br s, 1H), 3.06-3.24 (m, 4H), 2.32 (t, J=7.1 Hz, 2H), 1.97-2.08 (m, 4H), 1.22-1.64 (m, 18H), 1.14 (t, J=7.3 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 179.72, 130.79, 129.35, 40.99, 35.66, 34.45, 29.70, 29.67, 29.24, 29.12, 28.99, 27.26, 27.14, 27.04, 24.97, 15.50.

Example 7

Synthesis of 16-(Butyrylamino)hexadec-11(Z)-enoic acid (12)

(105) ##STR00050##

(106) Butyric acid (100 mg, 1.10 mmol), 1-hydroxybenzotriazole (145 mg, 1.10 mmol; HOBt) and diisopropylethylamine (150 mg, 1.10 mmol; DIPEA) were added to a stirring solution of the previously described crude methyl 16-aminohexadec-11(Z)-enoate (240 mg, 0.85 mmol) in anhydrous DMF (20 mL) under an argon atmosphere. After 5 min, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (210 mg, 1.10 mmol; EDCI) was added as a solid. After stirring for 12 h at room temperature, the reaction mixture was diluted with EtOAc (30 mL), washed with water (30 mL), and brine (20 mL). The combined aqueous layers were back-extracted with EtOAc (3×30 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, concentrated under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography using 30% EtOAc/hexanes as eluent to give methyl 16-(butyrylamino)hexadec-11(Z)-enoate (246 mg, 82%) as a viscous oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.58 (br s, 1H), 5.26-5.40 (m, 2H), 3.65 (s, 3H), 3.19-3.26 (m, 2H), 2.25-2.31 (m, 2H), 2.12 (t, J=7.1 Hz, 2H), 1.95-2.08 (m, 4H), 1.22-1.66 (m, 18H), 0.92 (t, J=7.1 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.61, 173.26, 130.71, 129.31, 51.67, 39.60, 38.99, 34.32, 29.90, 29.66, 29.60, 29.50, 29.45, 29.34, 27.43, 27.21, 27.01, 25.15, 19.46, 13.98. Lit. ref.: J. Cesar; M. S. Dolenc Tetrahedron Lett. 2001, 42, 7099.

(107) ##STR00051##

(108) Methyl 16-(butyrylamino)hexadec-11(Z)-enoate was hydrolyzed as described above to give 16-(butyrylamino)hexadec-11(Z)-enoic acid (88%) as a white solid. M.P. 99.2-99.6° C. TLC: 75% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 5.28-5.41 (m, 2H), 3.15 (t, 2H, J=7.3 Hz), 2.01-2.21 (m, 8H), 1.22-1.64 (m, 20H), 0.93 (t, 3H, J=7.1 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.89, 130.10, 129.17, 39.07, 37.88, 29.67, 29.55, 29.49, 29.20, 28.89, 27.00, 26.95, 26.66, 26.52, 22.96, 19.31, 12.85.

Example 8

Synthesis of 16-(2-(methylamino)-2-oxoacetamido)hexadac-11(Z)-enoic acid (13)

(109) ##STR00052##

(110) Methylamine (1.5 g, 23 mL of a 1 M THF solution, 48.38 mmol) solution was added dropwise to a −10° C. solution of ethyl chlorooxoacetate (5.0 g, 36.76 mmol) and triethylamine (5.6 g, 7.6 mL, 55.44 mmol) in dry THF (100 mL) under an argon atmosphere. After stirring at 0° C. for 1 h, then reaction was quenched with water (5 mL). Following another 20 min, the reaction mixture was extracted into ethyl acetate (2×30 mL) and the combined organic extracts were washed with water (2×100 mL), dried and concentrated in vacuo. The residue was purified by column chromatography using 40% EtOAc/hexanes to give monoethyl N-methyloxalamic acid (3.95 g, 82%) as a white powder. TLC: 75% EtOAc/hexane, R.sub.f≈0.4; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 4.35 (q, 2H, J=7.0 Hz), 2.92 (d, 3H, J=5.2 Hz), 1.37 (t, 3H, J=7.3 Hz).

(111) ##STR00053##

(112) The obtained mass (2 g, 15.26 mmol) was subjected to hydolize in the presence of lithium hydroxide (2.0 M) solution in aqueous tetrahydrofuran. After completion of the reaction (as per TLC), the whole mass was acidify with IN HCl (15 mL) to bring P.sup.H=1 and then diluted with ethyl acetate (50 mL) and washed with water (50 mL). The aqueou layer was back extracted with ethyl acetate (3×40 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure and the obtained mass was washed with hexanes/ether (1/1) to give white solid which was used for next reaction without further purification.

(113) ##STR00054##

(114) Methyl 16-aminohexadec-11(Z)-enoate (180 mg, 0.64 mmol) was condensed with 2-(methylamino)-2-oxoacetic acid (mg, 0.77 mmol) as described above to give methyl 16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoate (160 mg, 68%) as a white solid. TLC: 100% EtOAc, R.sub.f≈0.4; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.45 (br s, 1H), 5.26-5.42 (m, 2H), 3.66 (s, 3H), 3.27-3.35 (m, 2H), 2.90 (d, 3H, J=5.2 Hz), 2.30 (t, 2H, J=7.3 Hz), 1.96-2.08 (m, 4H), 1.24-1.66 (m, 18H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.60, 160.81, 159.94, 130.87, 129.08, 51.68, 39.79, 34.33, 29.91, 29.68, 29.63, 29.50, 29.46, 29.36, 29.02, 27.46, 27.08, 26.91, 26.40, 25.17.

(115) ##STR00055##

(116) Methyl 16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoate (150 mg, 0.40 mmol) was hydrolyzed using LiOH as described above to afford 16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoic acid (126 mg, 89%) as a white powder. M.P.: 110.2-110.6° C. TLC: 5% MeOH/CH.sub.2Cl.sub.2, R.sub.f≈0.4; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.80 (br s, 1H), 7.66 (br s, 1H), 5.26-5.42 (m, 2H), 3.28-3.35 (m, 2H), 2.90 (s, 3H), 2.36 (t, 2H, J=7.3 Hz), 1.97-2.08 (m, 4H), 1.51-1.64 (m, 4H), 1.22-1.42 (m, 14H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 177.98, 160.96, 159.93, 130.83, 129.22, 39.91, 33.91, 29.58, 29.25, 29.12, 29.01, 28.95, 27.21, 27.09, 26.93, 26.46, 24.89.

(117) ##STR00056##

(118) 16-(2-(Methylamino)-2-oxoacetamido)hexadec-11(Z)-enoic acid (30 mg) was dissolved in deionized water (30 mL) and NaHCO.sub.3 (2 g, 10 equiv) was added with stirring. After 1 h at room temperature, pre-washed Bio-Rad® Bio-Beads (SM-2, 20-50 mesh, 15 g) were added. After gently stirring for 1 h, the beads were collected on a sintered glass funnel and washed with water (150 mL), and then the salt was stripped from the beads by washing with 99% ethanol (200 mL). The ethanol washings were concentrated under reduced pressure to give sodium 16-(2-(Methylamino)-2-oxoacetamido)hexadec-11(Z)-enoate as a white amorphous solid. .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 7.52-7.64 (m, 2H), 5.27-5.38 (m, 2H), 3.27 (t, 2H, J=7.4 Hz), 2.82 (s, 3H), 2.25 (t, 2H, J=7.5 Hz), 1.97-2.05 (m, 4H), 1.52-1.65 (m, 4H), 1.20-1.41 (m, 14H);

Example 9

Synthesis of 16-(N-Isopropylbutyramido)hexadec-11(Z)-enoic acid (15)

(119) ##STR00057##

(120) Triphenylphosphine (730 mg, 2.78 mmol) and imidazole (190 mg, 2.78 mmol) were added to a 0° C. solution of methyl 16-hydroxyhexadec-11(Z)-enoate (660 mg, 2.32 mmol) in dry THF (50 mL) under an argon atmosphere. After 10 min, solid iodine (700 mg, 1.2 equiv) was added portionwise. After stirring at room temperature for 3 h, the reaction mixture was quenched with sat. aq. sodium bisulfite solution (10 mL). After an additional 1 h, the solution was washed with water (2×30 mL), concentrated under reduced pressure, and the residue was purified by flash column chromatography using 10% EtOAc/hexanes as eluent to give methyl 16-iodohexadec-11(Z)-enoate (505 mg, 76%). TLC: 10% EtOAc/hexanes, R.sub.f≈0.55; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.28-5.42 (m, 2H), 3.66 (s, 3H), 3.18 (t, J=7.0 Hz, 2H), 2.30 (t, J=7.6 Hz, 2H), 1.98-2.08 (m, 4H), 1.24-1.85 (m, 18H).

(121) ##STR00058##

(122) Isopropylamine (220 mg, 3.8 mmol) was added to a solution of methyl 16-iodohexadec-11(Z)-enoate (300 mg, 0.76 mmol) from above and potassium carbonate (320 mg) in THF (20 mL) under an argon atmosphere in a sealed tube. After heating at 90° C. for 10 h, the reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), washed with water (20 mL), dried, and concentrated under high vacuum for 5 h. The crude methyl 16-(N-isopropylamino) hexadec-11(Z)-enoate was used in the next reaction without further purification. TLC: 20% MeOH/CH.sub.2Cl.sub.2, R.sub.f≈0.20; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.28-5.40 (m, 2H), 3.66 (s, 3H), 2.72-2.84 (m, 1H), 2.58 (t, J=7.2 Hz, 2H), 2.29 (t, J=7.6 Hz, 2H), 1.98-2.08 (m, 4H), 1.22-1.62 (m, 18H), 1.05 (d, 6H, J=6.4 Hz).

(123) ##STR00059##

(124) Methyl 16-(N-isopropylamino)hexadec-11(Z)-enoate (400 mg, 1.2 mmol) was acylated with n-butyric acid (130 mg, 1.47 mmol) as described above to give methyl 16-(N-isopropylbutyramido)hexadec-11(Z)-enoate (348 mg, 74%). TLC: 50% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR (CDCl.sub.3, 300 MHz, rotamers) δ 5.28-5.42 (m, 2H), 4.61-4.67 and 3.99-4.10 (m, 1H for two rotamers 60/40 ratio), 3.66 (s, 3H), 3.06-3.16 (m, 2H), 2.21-2.36 (m, 4H), 1.95-2.10 (m, 4H), 1.20-1.72 (m, 20H), 1.17 and 1.12 (d, J=6.6 Hz, 3H for two rotamers in 60/40 ratio), 0.96 and 0.95 (t, 3H, J=7.3 Hz for two rotamers in 60/40 ratio).

(125) ##STR00060##

(126) Methyl 16-(N-isopropylbutyramido)hexadec-11(Z)-enoate (320 mg, 0.81 mmol) was hydrolyzed as described above to give 16-(N-isopropylbutyramido)hexadec-11(Z)-enoic acid (254 mg, 83%) as a thick, colorless oil. TLC: 75% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz, rotamers) δ 5.26-5.41 (m, 2H), 4.63-4.69 and 4.00-4.10 (m, 1H for two rotamers in 60/40 ratio), 3.06-3.17 (m, 2H), 2.22-2.37 (m, 4H), 1.98-2.12 (m, 4H), 1.50-1.72 (m, 4H), 1.22-1.40 (m, 16H), 1.18 and 1.12 (d, J=7.0 Hz, 6H for two rotamers in 60/40 ratio), 0.96 and 0.95 (t, J=7.3 Hz, 3H for two rotamers in 60/40 ratio); .sup.13C NMR (CDCl.sub.3, 75 MHz, rotamers) δ 179.07, 178.95, 173.42, 172.89, 131.03, 130.35, 129.70, 128.99, 48.51, 45.70, 43.58, 41.22, 35.98, 35.83, 34.37, 31.20, 29.90, 29.86, 29.67, 29.61, 29.53, 29.48, 29.39, 28.37, 29.28, 27.84, 27.50, 27.46, 27.35, 27.19, 26.90, 25.00, 21.54, 20.75, 19.35, 19.22, 14.23; MS: m/z 380 (M-H).sup.+.

Example 10

Synthesis of Methyl 16-(3-ethyl-1,3-dimethylureido)hexadec-11(Z)-enoic Acid (16)

(127) ##STR00061##

(128) Methylamine (1 mL of a 1.0 M THF soln, 33 mg) was added to a solution of methyl 16-iodohexadec-11(Z)-enoate (300 mg, 0.76 mmol) from above and potassium carbonate (320 mg, 2.28 mmol, 3 equiv) in THF (20 mL) under an argon atmosphere in a sealed tube. After heating at 90° C. for 12 h, the reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), washed with water (20 mL), dried, and concentrated under high vacuum for 5 h. The crude methyl 16-(methylamino)hexadec-11(Z)-enoate was used in the next reaction without further purification. TLC: 10% MeOH/CH.sub.2Cl.sub.2, R.sub.f≈0.2; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.28-5.40 (m, 2H), 3.66 (s, 3H), 2.56 (t, J=6.8 Hz, 2H), 2.42 (s, 3H), 2.29 (t, J=7.6 Hz, 2H), 1.96-2.06 (m, 4H), 1.24-1.64 (m, 18H).

(129) ##STR00062##

(130) Triethylamine (12.84 g, 127.11 mmol) and p-nitrophenyl chloroformate (63.56 mmol, 12.8 g) were added to a room temperature solution of N-ethylmethylamine (2.50 g, 42.37 mmol) in dry DMF (70 mL) under an argon atmosphere. After 2 h, the reaction mixture was quenched with water, diluted with EtOAc (200 mL), washed with water (2×100 mL), and brine (75 mL). All volatiles were removed under reduced pressure and the residue was purified by SiO.sub.2 column chromatography using 10% EtOAc/hexanes to afford compound 4-nitrophenyl ethyl(methyl)carbamate (5.8 g, 76%) as a yellow oil. TLC: 20% EtOAc/hexanes, R.sub.f≈0.50; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 8.18-8.21 (m, 2H), 7.25-7.29 (m, 2H), 3.37-3.46 (m, 2H), 3.05 and 2.97 (s, 3H for two rotamers in 60/40 ratio), 1.17-1.22 (m, 3H).

(131) ##STR00063##

(132) A solution of crude methyl 16-(methylamino)hexadec-11(Z)-enoate from above (150 mg, 0.51 mmol) in anhydrous acetonitrile (20 mL) was added to a mixture of p-nitrophenyl chloroformate (130 mg, 0.72 mmol) and K.sub.2CO.sub.3 (230 mg, 1.5 mmol.) in dry acetonitrile (20 mL) at room temperature. After heating under reflux for 36 h, the solvent was removed under reduced pressure and the residue was diluted with water (30 mL) and then extracted into EtOAc (2×30 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 15% EtOAc/hexanes as eluent to afford methyl 16-(3-ethyl-1,3-dimethylureido) hexadec-11 (Z)-enoate (65 mg, 34%) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.40; 1H NMR (CDCl.sub.3, 300 MHz) δ 5.27-5.40 (m, 2H), 3.66 (s, 3H), 3.10-3.18 (m, 4H), 2.77 (s, 3H), 2.75 (s, 3H), 2.29 (t, J=7.2 Hz, 2H), 1.97-2.05 (m, 4H), 1.50-1.68 (m, 4H), 1.20-1.42 (m, 14H), 1.12 (t, J=6.9 Hz, 3H).

(133) ##STR00064##

(134) Methyl 16-(3-ethyl-1,3-dimethylureido) hexadec-11 (Z)-enoate (30 mg, 0.08 mmol) was hydrolyzed as described above to give 16-(3-ethyl-1,3-dimethylureido)hexadec-11(Z)-enoic acid (15 mg, 75%) as colorless oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 5.33-5.41 (m, 2H), 3.12-3.19 (m, 4H), 2.79 (s, 3H), 2.76 (s, 3H), 2.31-2.38 (m, 2H), 1.98-2.06 (m, 4H), 1.20-1.68 (m, 18H), 1.13 (t, J=6.9 Hz, 3H); 13C NMR (CDCl.sub.3, 75 MHz) δ 177.52, 166.83, 130.61, 129.567, 51.58, 45.38, 37.91, 36.93, 34.12, 29.74, 29.67, 28.72, 28.42, 27.43, 26.68, 24.99, 22.64, 15.34.

Example 11

Synthesis of Sodium 17-Oxo-17-(propylamino)heptadec-11(Z)-enoate (14)

(135) ##STR00065##

(136) Jones oxidation of methyl 16-hydroxyhexadec-11(Z)-enoate (2.0 g, 7.04 mmol) as described above gave 16-methoxy-16-oxohexadec-5(Z)-enoic acid (1.72 g, 83%) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.27-5.45 (m, 2H), 3.66 (s, 3H), 2.36 (t, 2H, J=7.7 Hz), 2.30 (t, 2H, J=7.4 Hz), 1.98-2.12 (m, 4H), 1.57-1.72 (m, 4H), 1.20-1.41 (m, 12H).

(137) ##STR00066##

(138) Triethylamine (122 mg, 1.18 mmol) and ethyl chloroformate (130 mg, 1.13 mmol) were added to a −15° C. solution of 16-methoxy-16-oxohexadec-5(Z)-enoic acid (300 mg, 1.06 mmol) in dry THF (50 mL) under an argon atmosphere. After 15 min, the reaction mixture was warmed to −5° C. and an ethereal solution of diazomethane was added slowly until the yellow color of diazomethane persisted for 15 min. Afterwards, the reaction mixture was stirred at room temperature for an additional 3 h, then the excess diazomethane was evaporated under a stream of argon. The reaction solution was washed with sat. aq. NaHCO.sub.3 (50 mL), sat. aq. NH.sub.4Cl (50 mL), brine (50 mL), dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was rapidly purified by SiO.sub.2 column chromatography using 20% EtOAc/hexanes as eluent to give methyl 17-diazo-16-oxoheptadec-11(Z)-enoate (180 mg, 55%) as a light yellow oil that was used immediately in the next step. TLC: 40% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (C.sub.6D.sub.6, 300 MHz) δ 5.25-5.48 (m, 2H), 4.13 (s, 1H), 3.32 (s, 3H), 2.07 (t, 2H, J=7.4 Hz), 1.85-2.04 (m, 6H), 1.44-1.61 (m, 4H), 1.15-1.38 (m, 12H). Lit. ref.: J. Cesar; M. S. Dolenc Tetrahedron Lett. 2001: 42, 7099.

(139) A solution of silver benzoate (5 mg, 10 mol %) in triethylamine (68 mg, 100 μL, 0.66 mmol) was added to a −25° C. solution of methyl 17-diazo-16-oxoheptadec-11(Z)-enoate (70 mg, 0.22 mmol) and n-propylamine (40 mg, 10 equiv) in dry THF (20 mL) under an argon atmosphere with exclusion of light. The reaction mixture was warmed to room temperature over 3 h, diluted with ether (10 mL), quenched with 0.2 N HCl (5 mL), washed with brine (30 mL), sat. aq. NaHCO.sub.3 (10 mL), dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 20% EtOAc/hexanes as eluent to give methyl 17-oxo-17-(propylamino) heptadec-11(Z)-enoate (49 mg, 64%) as a pale yellow oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.47 (br s, 1H), 5.27-5.40 (m, 2H), 3.66 (s, 3H), 3.17-3.24 (m, 2H), 2.29 (t, 2H, J=7.1 Hz), 2.16 (t, 2H, J=7.1 Hz), 1.96-2.07 (m, 4H), 1.24-1.67 (m, 20H), 0.91 (t, 3H, J=7.3 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.62, 173.22, 130.59, 129.41, 51.68, 41.40, 37.05, 34.33, 29.93, 29.67, 29.63, 29.48, 29.36, 27.44, 27.16, 25.73, 25.17, 23.14, 11.60. Lit. ref.: J. Podlech; D. Seebach Angew. Chem., Int. Ed. 1995: 34, 471.

(140) ##STR00067##

(141) Methyl 17-oxo-17-(propylamino)heptadec-11(Z)-enoate (48 mg, 0.14 mmol) was converted to its sodium salt as described above to give sodium 17-oxo-17-(propylamino)heptadec-11(Z)-enoate as a white solid. M.P.: 84.8-85.2° C. TLC (free acid): 75% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR for sodium salt (CD.sub.3OD, 300 MHz) δ 5.30-5.42 (m, 2H), 3.16 (t, 2H, J=7.0 Hz), 2.00-2.22 (m, 8H), 1.22-1.68 (m, 20H), 0.93 (t, 3H, J=7.2 Hz); .sup.13C NMR for sodium salt (CD.sub.3OD, 75 MHz) δ 180.33, 174.88, 130.08, 129.22, 39.07, 37.88, 36.80, 29.70, 29.53, 29.49, 29.45, 29.21, 28.90, 27.02, 26.96, 26.68, 26.12, 19.32, 12.88.

Example 12

Synthesis of 16-(Butylamino)-16-oxohexadec-11 (Z)-enoic acid

(142) ##STR00068##

(143) 16-Methoxy-16-oxohexadec-5(Z)-enoic acid (230 mg, 0.77 mmol) was condensed with n-butylamine (70 mg, 1.08 mmol) using EDCI as described to give methyl 16-(butylamino)-16-oxohexadec-11(Z)-enoate (185 mg, 68%) as a colorless oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.26-5.42 (m, 2H), 3.66 (s, 3H), 3.21-3.29 (m, 2H), 2.30 (t, 2H, J=7.2 Hz), 2.16 (t, 2H, J=7.1 Hz), 1.97-2.08 (m, 4H), 1.55-1.74 (m, 4H), 1.24-1.54 (m, 14H), 0.92 (t, 3H, J=7.3 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.60, 173.1, 131.18, 128.83, 51.67, 39.42, 36.44, 34.32, 31.98, 29.91, 29.66, 29.60, 29.49, 29.45, 29.34, 27.47, 26.87, 25.95, 25.15, 20.30, 13.98.

(144) ##STR00069##

(145) Methyl 16-(butylamino)-16-oxohexadec-11(Z)-enoate (150 mg, 0.44 mmol) was hydrolyzed to give 16-(butylamino)-16-oxohexadec-11(Z)-enoic acid (114 mg, 82%) as a white solid. M.P.: 78.2-78.8 OC. TLC: 75% EtOAc/hexanes, R.sub.f≈0.3; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.81 (br s, 1H), 5.24-5.40 (m, 2H), 3.18-3.24 (m, 2H), 2.30 (t, 2H, J=7.3 Hz), 2.16 (t, 2H, J=7.2 Hz), 1.93-2.06 (m, 4H), 1.19-1.70 (m, 20H), 0.88 (t, 3H, J=7.4 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 178.98, 173.78, 131.19, 128.74, 39.54, 36.36, 34.37, 31.84, 29.84, 29.56, 29.53, 29.40, 29.38, 29.22, 27.42, 26.85, 25.99, 24.98, 20.26, 13.96.

Example 13

Synthesis of 2-(2-(2-Hydroxyethoxy) ethoxy)ethyl 16-(3-ethylureido)hexadec-11 (Z)-enoate (18)

(146) ##STR00070##

(147) Triethyleneglycol (42 mg, 0.29 mmol; dried over molecular sieves) was added to a solution of 16-(3-ethyl-1,3-dimethylureido)hexadec-11(Z)-enoic acid (10 mg, 0.029 mmol) and N,N-dimethylaminopyridine (DMAP, 4.2 mg, 0.034 mmol) in anhydrous DMF (3 mL) under an argon atmosphere at room temperature. After 3 min, solid EDCI (6.4 mg, 0.034 mmol) was added. After 12 h, the reaction mixture was diluted with EtOAc (10 mL), washed with water (5 mL), and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using EtOAc to give 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl 16-(3-ethylureido)hexadec-11(Z)-enoate (11 mg, 85%) as a viscous, colorless oil. TLC: 100% EtOAc, R.sub.f≈0.20; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.27-5.42 (m, 2H), 4.34 (br s, 1H), 4.23 (t, 2H, J=5.8 Hz), 3.59-3.74 (m, 10H), 3.12-3.24 (m, 4H), 2.46 (br s, 1H), 2.33 (t, 2H, J=7.3 Hz), 1.96-2.07 (m, 4H), 1.22-1.64 (m, 18H), 1.13 (t, 3H, J=7.3 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.21, 158.42, 130.66, 129.44, 72.70, 70.79, 70.57, 69.44, 63.46, 61.98, 40.74, 35.59, 34.40, 30.11, 29.88, 29.63, 29.60, 29.44, 29.42, 29.31, 27.41, 27.22, 27.08, 25.10, 15.73.

Example 14

Synthesis of Sodium (Z)-2-(16-(3-Ethylureido) hexadec-11-enamido)acetate (17)

(148) ##STR00071##

(149) 16-(3-Ethyl-1,3-dimethylureido)hexadec-11(Z)-enoic acid (50 mg, 0.15 mmol) was condensed with glycine methyl ester (96 mg, 0.38 mmol) as described above to give methyl 2-(16-(3-ethylureido)hexadec-11(Z)-enamido)acetate (51 mg, 84%) as a colorless oil. TLC: 75% EtOAc/hexanes, R.sub.f≈0.50; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 6.28 (br s, 1H), 5.26-5.42 (m, 2H), 4.89 (br s, 1H), 4.03 (d, 2H, J=5.2 Hz), 3.10-3.22 (m, 4H), 2.24 (t, 2H, J=7.1 Hz), 1.96-2.08 (m, 4H), 1.22-1.67 (m, 18H), 1.12 (t, 3H, J=7.3 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 173.84, 170.86, 158.68, 130.61, 129.50, 52.58, 41.40, 40.67, 36.58, 35.49, 30.18, 29.92, 29.73, 29.53, 29.46, 29.41, 29.22, 27.28, 27.25, 27.10, 25.78, 15.73.

(150) ##STR00072##

(151) Methyl 2-(16-(3-ethylureido)hexadec-11(Z)-enamido)acetate was hydrolyzed as described above to give sodium 2-(16-(3-ethylureido)hexadec-11(Z)-enamido)acetate as a white solid. M.P.: 152.4-152.8° C. .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 7.57-7.65 (m, 1H), 5.32-5.42 (m, 2H), 3.73 (s, 2H), 3.07-3.18 (m, 4H), 2.36 (t, 2H, J=7.3 Hz), 1.98-2.09 (m, 4H), 1.22-1.65 (m, 18H), 1.08 (t, 3H, J=7.1 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 175.41, 174.67, 160.72, 129.98, 129.31, 43.32, 39.70, 35.98, 34.58, 29.83, 29.66, 29.42, 29.31, 29.21, 29.14, 26.96, 26.91, 26.72, 25.70, 14.66.

Example 15

Synthesis of 16-[(1S,2R)-3-Ethyl-oxiranyl]hexadec-11(Z)-enoic acid (10)

(152) ##STR00073##

(153) 2-(Prop-2-ynyloxy)tetrahydro-2H-pyran (5.6 g, 36.36 mmol) was alkylated with (4-bromobutoxy) (tert-butyl)diphenylsilane (18.5 g, 47.2 mmol) as described above to give tert-butyldiphenyl(7-(tetrahydro-2H-pyran-2-yloxy)hept-5-ynyloxy) silane (10.64 g, 65%) and used after extractive isolation without further purification. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5.

(154) Removal of the THP ether from tert-butyldiphenyl(7-(tetrahydro-2H-pyran-2-yloxy)hept-5-ynyloxy)silane (10 g, 22.22 mmol) as described above furnished 7-(tert-butyldiphenylsilyloxy)hept-2-yn-1-ol (7.15 g, 88%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.65-7.67 (m, 4H), 7.33-7.42 (m, 6H), 4.22-4.26 (m, 2H), 3.64 (t, 2H, J=6.4 Hz), 2.12-2.16 (m, 2H), 1.40-1.46 (m, 4H), 1.03 (s, 9H).

(155) ##STR00074##

(156) Semi-hydrogenation of 7-(tert-butyldiphenylsilyloxy) hept-2-yn-1-ol (7.4 g, 20.22 mmol) as described above furnished 7-(tert-butyldiphenylsilyloxy)hept-2(Z)-en-1-ol (7.3 g, 98%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 7.65-7.69 (m, 4H), 7.40-7.44 (m, 6H), 5.44-5.64 (m, 2H), 4.16 (d, 2H, J=6.1 Hz), 3.65 (t, 2H, J=6.1 Hz), 2.03-2.10 (m, 2H), 1.42-1.60 (m, 4H), 1.04 (s, 9H).

(157) ##STR00075##

(158) (−)-Diethyl tartrate (570 mg, DET) and titanium tetra(isopropoxide) (775 mg) were added sequentially to a stirring, −20° C. suspension of activated, powdered type 4 Å molecular sieves (2 g) in dry CH.sub.2Cl.sub.2 (50 mL) under an argon atmosphere. After 30 min, a solution of 7-(tert-butyldiphenylsilyloxy)hept-2(Z)-en-1-ol in (5 g, 13.58 mmol) dry CH.sub.2Cl.sub.2 (20 mL) was added slowly and the resulting mixture was stirred for 2 h at the same temperature. tert-butyl hydroperoxide (2.5 g, 5.1 mL of a 5.5 M solution in decane; TBHP) was added very slowly. After stirring at −20° C. for 2 d, water (2 mL) was added and the mixture was allowed to stir at 0° C. for 1 h. A solution of 1 M aq. NaOH (5 mL) was added and stirred for 30 min. The reaction mixture was then washed with water (100 mL) and concentrated under reduced pressure.

(159) Purification of the residue by SiO.sub.2 column chromotography using 10% EtOAc/hexanes as eluent gave ((2R,3S)-3-(4-(tert-butyldiphenylsilyloxy)butyl)oxiran-2-yl)methanol (3.23 g, 62%) as a colorless oil. Chiral HPLC analysis as described above revealed the sample was 60% ee. TLC: 30% EtOAc/hexanes, R.sub.f0.4; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 7.64-7.68 (m, 4H), 7.35-7.44 (m, 6H), 3.79-3.88 (m, 1H), 3.61-3.69 (m, 3H), 3.12-3.17 (m, 1H), 2.98-3.04 (m, 1H), 1.53-1.65 (m, 4H), 1.03 (s, 9H). Lit. ref.: T. Katsuki; K. B. Sharpless J. Am. Chem. Soc. 1980: 102, 5974.

(160) ##STR00076##

(161) Dry DMSO (114 mg, 0.4 mmol) was added dropwise to a stirring, −80° C. solution of oxalyl chloride (110 mg, 0.3 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) under an argon atmosphere. After 20 min, a solution of ((2R,3S)-3-(4-(tert-butyldiphenylsilyloxy)butyl)oxiran-2-yl)methanol (200 mg, 0.1 mmol) in dry CH.sub.2Cl.sub.2 (50 mL) was added slowly. After 45 min, triethylamine (200 mg, 0.5 mmol) was added and the reaction mixture was warmed to 0° C. After 0.5 h, the reaction mixture was quenched with water (50 mL). The aqueous layer was separated and back-extracted with CH.sub.2Cl.sub.2 (2×10 mL). The combined organic extracts were washed with water, brine, and dried over anhydrous Na.sub.2SO.sub.4, and evaporated in vacuo. The residue was purified via SiO.sub.2 column chromatography using 5% EtOAc/hexanes to give (2S,3S)-3-[4-(tert-butyldiphenylsilanyloxy)-butyl]-oxirane-2-carbaldehyde. The crude aldehyde was used for the next reaction without further purification.

(162) ##STR00077##

(163) Sodium bis(trimethylsilyl)amide (2.4 g, 13.08 mmol, 13.1 mL, 1.0 M in THF) was added to a stirring, 0° C. solution of methyl triphenylphosphonium bromide (4.68 g, 13.08 mmol) in dry THF (10 mL). After 30 min, the reaction mixture was cooled to −50° C. and a solution of (2S,3S)-3-[4-(tert-butyldiphenylsilanyloxy)-butyl]-oxirane-2-carbaldehyde (2.5 g, 6.55 mmol) in THF (10 mL) was added over 5 min. The solution was warmed to room temperature over 1 h. After an additional 2 h at room temperature, the reaction mixture was quenched with water (30 mL) and extracted with ether (3×60 mL). The combined ethereal extracts were washed with water (2×100 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes to give (3R,4S)-tert-butyldiphenyl-[4-(3-vinyl-oxiranyl)-butoxy]-silane (1.84 g, 76%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.4; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 7.65-7.69 (m, 4H), 7.35-7.44 (m, 6H), 5.64-5.76 (m, 1H), 5.32-5.50 (m, 2H), 3.67 (t, 2H, J=7.06 Hz), 3.38-3.42 (m, 1H), 3.02-3.11 (m, 1H), 1.44-1.68 (m, 4H), 1.05 (s, 9H).

(164) ##STR00078##

(165) Desilylation of (3R,4S)-tert-butyldiphenyl-[4-(3-vinyl-oxiranyl)-butoxy]-silane as described above gave (3R,4S)-4-(3-vinyl-oxiranyl)-butan-1-ol (92%) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 5.65-5.77 (m, 1H), 5.33-5.50 (m, 2H), 3.65 (t, 2H, J=6.1 Hz), 3.38-3.43 (m, 1H), 3.06-3.11 (m, 1H), 1.44-1.66 (m, 6H).

(166) ##STR00079##

(167) 4(S)-(3(R)-Vinyloxiranyl)-butan-1-ol was reduced with in situ generated diimide as described above to give 4(S)-[3(R)-ethyloxiranyl]butan-1-ol (92%) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 3.66 (t, 2H, J=6.1 Hz), 2.85-2.94 (m, 2H), 1.49-1.65 (m, 8H), 1.03 (t, J=7.2 Hz, 3H).

(168) ##STR00080##

(169) Treatment of 4(S)-[3(R)-ethyloxiranyl]butan-1-ol with Ph.sub.3P/CBr.sub.4 as described above gave 2(S)-(4-bromobutyl)-3(R)-ethyloxirane (64%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.7.

(170) ##STR00081##

(171) Jones oxidation of dodec-10-yn-1-ol (2.5 g, 13.73 mmol) as described above afforded dodec-11-ynoic acid (2.3 g, 86%). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 2.34 (t, 2H, J=7.0 Hz), 2.14-2.21 (m, 2H), 1.93 (t, 1H, J=2.75 Hz), 1.21-1.64 (m, 22H).

(172) ##STR00082##

(173) Alkylation of dodec-11-ynoic acid (580 mg) with 2(S)-(4-bromobutyl)-3(R)-ethyloxirane (500 mg) as described above furnished 16(S)-[3(R)-ethyloxiranyl]-hexadec-11-ynoic acid (64%) which was esterified with diazomethane to give methyl 16(S)-[3(R)-ethyloxiranyl]-hexadec-11-ynoate as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 3.66 (s, 3H), 2.82-2.88 (m, 2H), 2.29 (t, 2H, J=7.3 Hz), 2.10-2.17 (m, 4H), 1.28-1.63 (m, 22H), 1.03 (t, 3H, J=7.1 Hz).

(174) ##STR00083##

(175) Semi-hydrogenation of methyl 16(S)-[3(R)-ethyloxiranyl]-hexadec-11-ynoate as described above gave methyl 16(S)-[3(R)-ethyloxiranyl]-hexadec-11(Z)-enoate (96%) as a colorless oil. TLC: 10% EtOAc/hexanes, R.sub.f≈0.55; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.31-5.36 (m, 2H), 3.64 (s, 3H), 2.84-2.91 (m, 2H), 2.28 (t, 2H, J=7.3 Hz), 1.96-2.06 (m, 4H), 1.36-1.61 (m, 6H), 1.21-1.35 (m, 16H), 1.03 (t, 3H, J=7.3 Hz).

(176) ##STR00084##

(177) Column: Chiracel OJ-H preparative.

(178) Wavelength: 210 nm

(179) Mobil phase: 99.97:0.03 (Hex/IPA)

(180) Flow rate 8 mL/min.

(181) 1st. Fraction is: PN-III-191-18. (Acid)

(182) 2nd fraction is: PN-III-192-13. (Acid)

Example 16

Synthesis of 16-(3-Ethylureido)hexadec-14-enoic Acid (21)

(183) ##STR00085##

(184) Alkylation of 2-(prop-2-ynyloxy)tetrahydro-2H-pyran (15.5 g, 110.71 mmol) with 1-bromododecane (34.0 g, 132.04 mmol) as described above gave 2-(pentadec-2-ynyloxy)tetrahydro-2H-pyran (27.2 g, 80%) which was used without further purification. TLC: 10% EtOAc/hexanes, R.sub.f≈0.5.

(185) Cleavage of the THP ether from crude 2-(pentadec-2-ynyloxy)tetrahydro-2H-pyran (30 g) using PTSA as described above gave pentadec-2-yn-1-ol (18.6 g, 85%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 4.25 (s, 2H), 2.17-2.23 (m, 2H), 1.70 (br s, 1H), 1.40-1.53 (m, 2H), 1.20-1.48 (m, 18H), 0.87 (t, 3H, J=7.3 Hz).

(186) ##STR00086##

(187) Isomerization of pentadec-2-yn-1-ol (12.5 g, 54.95 mmol) using NaH/ethylenediamine as described above furnished pentadec-14-yn-1-ol (9.4 g, 76%) as a white solid. M.P.: 54.2-54.8 OC. TLC: 30% EtOAc/hexanes, R.sub.f≈0.45; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.60-3.65 (m, 2H), 2.16 (dt, 2H, J=7.1 Hz, 2.4 Hz), 1.92 (t, 1H, J=2.4 Hz), 1.47-1.60 (m, 4H), 1.22-1.35 (m, 18H).

(188) ##STR00087##

(189) Silylation of pentadec-14-yn-1-ol (8.80 g, 39.28 mmol) using TBDPSCl (12.92 g, 47.14 mmol) as described above gave tert-butyl(pentadec-14-ynyloxy)diphenylsilane (16.7 g, 87%) as a colorless oil. TLC: 6% EtOAc/hexanes, R.sub.f≈0.6; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.65-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.65 (t, J=7.3 Hz, 2H), 2.15-2.21 (m, 2H), 1.94 (t, J=1.9 Hz, 1H), 1.20-1.60 (m, 22H), 1.04 (s, 9H).

(190) ##STR00088##

(191) n-BuLi (2.5 M solution in hexanes, 1.29 g, 8 mL, 20.24 mmol) was added to a stirring, −40° C. solution of tert-butyl(pentadec-14-ynyloxy)diphenylsilane (8.5 g, 18.40 mmol) in THF (175 mL) under an argon atmosphere. After 30 min, the reaction mixture was gradually warmed over 3 h to −10° C., held at this temperature for 20 min, then re-cooled to −50° C. Then, a solution of paraformaldehyde (3.05 g, 92.2 mmol) in THF (30 mL) was cannulated into the stirring reaction mixture. After 30 min, the temperature was gradually warmed over 3 h to room temperature. Following 1 h at room temperature, the reaction mixture was quenched with sat. aq. NH.sub.4Cl (10 mL), diluted with ether (100 mL), and washed with water (2×75 mL). The combined aqueous washes were back-extracted with ether (2×50 mL). The combined All of the organic extracts were combined, dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromatography using 5% EtOAc/hexanes as eluent to give 16-(tert-butyldiphenylsilyloxy) hexadec-2-yn-1-ol (6.12 g, 68%). TLC: 30% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.70-7.74 (m, 4H), 7.34-7.44 (m, 6H), 4.3 (t, 2H, J=2.1 Hz), 3.65 (t, 2H, J=7.3 Hz), 2.12-2.17 (m, 2H), 1.20-1.61 (m, 22H), 1.04 (s, 9H).

(192) ##STR00089##

(193) 16-(tert-Butyldiphenylsilyloxy) hexadec-2-yn-1-ol (6.0 g, 12.5 mmol) was converted to the corresponding THP ether as described above to give tert-butyldiphenyl(16-(tetrahydro-2H-pyran-2-yloxy)hexadec-14-ynyloxy)silane (6.12 g, 87%). TLC: 10% EtOAc/hexanes, R.sub.f≈0.5; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 7.70-7.73 (m, 4H), 7.35-7.43 (m, 6H), 4.82 (t, 1H, J=3.1 Hz), 4.16-4.32 (m, 2H), 3.80-3.88 (m, 1H), 3.64 (t, 2H, J=6.6 Hz), 3.50-3.56 (m, 1H), 2.17-2.23 (m, 2H), 1.22-1.81 (m, 28H), 1.05 (s, 9H).

(194) ##STR00090##

(195) Desilylation of tert-butyldiphenyl (16-(tetrahydro-2H-pyran-2-yloxy) hexadec-14-ynyloxy) silane (6.1 g, 10.6 mmol) as described above furnished 16-(tetrahydro-2H-pyran-2-yloxy) hexadec-14-yn-1-ol (3.26 g, 91%) as a colorless oil. TLC: 40% EtOAc/hexanes, R.sub.f≈0.4; 4.83 (t, 1H, J=3.0 Hz), 4.17-4.31 (m, 2H), 3.82-3.87 (m, 1H), 3.66 (t, 2H, J=7.2 Hz), 3.51-3.57 (m, 1H), 2.18-2.24 (m, 2H), 1.20-1.82 (m, 28H).

(196) ##STR00091##

(197) RuCl.sub.3 (10 mg) and potassium persulphate (2.8 g, 10.2 mmol) were added to a solution of 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-14-yn-1-ol (1.2 g, 3.55 mmol) in acetonitrile (20 mL). After 10 min, KOH (30 mL of a 2 M soln) was added. After an additional 3 h, the reaction mixture was neutralized to pH 7, diluted with EtOAc (100 mL), and washed with water (3×75 mL). The combined aqueous extracts were back-extracted with EtOAc (3×75 mL). All of the organic extracts were combined, dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. The residue was purified by SiO.sub.2 column chromotography using 20% EtOAc/hexanes as eluent to give 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-14-ynoic acid (1.05 g, 91%) as a colorless oil that was used without further purification. TLC: 50% EtOAc/hexanes, R.sub.f≈0.35. Lit. ref.: R. S. Varma; M. Hogan Tetrahedron Lett. 1992: 33, 719.

(198) Concomitant esterification of the carboxylic acid and cleavage of the THP ether in 16-(tetrahydro-2H-pyran-2-yloxy)hexadec-14-ynoic acid (1.0 g, 2.84 mmol) as described above furnished methyl 16-hydroxyhexadec-14-ynoate (665 mg, 83%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 4.22-4.26 (m, 2H), 3.66 (s, 3H), 2.29 (t, 2H, J=7.3 Hz), 2.20 (tt, 2H, J=2.1 Hz, 6.8 Hz), 1.21-1.66 (m, 20H).

(199) ##STR00092##

(200) Semi-hydrogenation of methyl 16-hydroxyhexadec-14-ynoate (650 mg, 2.30 mmol) as described above furnished methyl 16-hydroxyhexadec-14(Z)-enoate (640 mg, 98%) as a colorless oil. TLC: 30% EtOAc/hexanes, R.sub.f≈0.45; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.49-5.62 (m, 2H), 4.17-4.21 (m, 2H), 3.66 (s, 3H), 2.30 (t, 2H, J=7.6 Hz), 2.02-2.09 (m, 2H), 1.42-1.68 (m, 4H), 1.20-1.41 (m, 16H).

(201) ##STR00093##

(202) Conversion of methyl 16-hydroxyhexadec-14(Z)-enoate (0.6 g, 2.11 mmol) to the corresponding azide as described above gave methyl 16-azidohexadec-14(Z)-enoate (510 mg, 78%) as white solid. M.P.: 42.5-42.8° C. TLC: 10% EtOAc/hexanes, R.sub.f≈0.50; 1H NMR (CDCl.sub.3, 300 MHz) δ 5.66-5.82 (m, 1H), 5.46-5.55 (m, 1H), 3.80 (d, 2H, J=7.4 Hz), 3.66 (s, 3H), 2.30 (t, 2H, J=7.3 Hz), 2.02-2.14 (m, 2H), 1.21-1.40 (m, 20H).

(203) ##STR00094##

(204) Starting with methyl 16-azidohexadec-14(Z)-enoate (150 mg, 0.48 mmol), the azide was reduced using Ph.sub.3P and the resultant amine reacted with ethyl isocyanate as described above to give methyl 16-(3-ethylureido)hexadec-14(Z)-enoate (118 mg, 70% over two steps) as a white solid. M.P.: 63.4-63.6° C. TLC: 50% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.31-5.52 (m, 2H), 5.08-5.22 (br s, 2H), 3.76 (t, 2H, J=5.2 Hz), 3.63 (s, 3H), 3.15 (q, 2H, J=6.7 Hz), 2.27 (t, 2H, J=7.3 Hz), 1.95-2.04 (m, 2H), 1.54-1.64 (m, 2H), 1.18-1.38 (m, 18H), 1.07 (t, 3H, J=6.9 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.69, 159.03, 132.91, 126.75, 51.67, 37.67, 35.27, 34.32, 29.81, 29.74, 29.64, 29.50, 29.46, 29.34, 27.57, 25.15, 15.76.

(205) ##STR00095##

(206) Hydrolysis of methyl 16-(3-ethylureido)hexadec-14(Z)-enoate as described above furnished 16-(3-ethylureido)hexadec-14(Z)-enoic acid (92%) as a white solid. M.P.: 59-60° C. TLC: 75% EtOAc/hexanes, R.sub.f≈0.30; .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 5.33-5.56 (m, 2H), 3.74 (d, 2H, J=6.3 Hz), 3.13 (q, 2H, J=7.0 Hz), 2.26 (t, 2H, J=7.2 Hz), 1.98-2.12 (m, 2H), 1.52-1.64 (m, 2H), 1.18-1.38 (m, 18H), 1.06 (t, 3H, J=7.0 Hz); .sup.13C NMR (CD.sub.3OD, 75 MHz) δ 176.69, 159.94, 132.31, 126.57, 36.98, 34.70, 33.96, 29.63, 29.61, 29.54, 29.50, 29.34, 29.26, 29.15, 27.20, 24.98, 14.52.

Example 17

Synthesis of 16-Butyramidohexadec-14(Z)-enoic acid (22)

(207) ##STR00096##

(208) Crude methyl 16-aminohexadec-14(Z)-enoate (crude 150 mg) was condensed with n-butyric acid (48 mg, 0.55 mmol) as described above to give methyl 16-butyramidohexadec-14(Z)-enoate (100 mg, 71%) as a colorless oil. TLC: 50% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.28-5.64 (m, 2H), 3.78-3.90 (m, 2H), 3.65 (s, 3H), 2.30 (t, 2H, J=7.2 Hz), 2.14 (t, 2H, J=7.6 Hz), 1.97-2.08 (m, 2H), 1.54-1.65 (m, 4H), 1.20-1.38 (m, 18H), 0.93 (t, 3H, J=7.2 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 174.62, 173.18, 134.12, 125.84, 51.67, 41.65, 38.94, 38.88, 36.82, 34.32, 32.45, 29.79, 29.72, 29.65, 29.46, 29.36, 27.58, 25.16, 19.40, 13.99.

(209) ##STR00097##

(210) Hydrolysis of methyl 16-butyramidohexadec-14(Z)-enoate (96 mg, 0.27 mmol) as described above gave 16-butyramidohexadec-14(Z)-enoic acid (82 mg, 91%) as a white solid. M.P.: 72.7-73.1° C. TLC: 75% EtOAc/hexanes, R.sub.f≈0.40; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.28-5.70 (m, 4H), 3.76-3.90 (m, 2H), 2.31 (t, 2H, J=7.4 Hz), 2.15 (t, 2H, J=6.9 Hz), 1.97-2.18 (m, 2H), 1.56-1.68 (m, 4H), 1.20-1.40 (m, 18H), 0.92 (t, 3H, J=7.3 Hz); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 179.27, 173.58, 134.23, 125.66, 41.75, 38.86, 38.80, 36.91, 34.37, 32.44, 29.76, 29.70, 29.66, 29.62, 29.44, 29.37, 29.29, 24.98, 19.42, 13.98.

Example 18

Synthesis of 16-(2-(Methylamino)-2-oxoacetamido) hexadec-14(Z)-enoic acid (23)

(211) ##STR00098##

(212) Condensation of methyl 16-aminohexadec-14(Z)-enoate (crude 140 mg) with 2-(methylamino)-2-oxoacetic acid (54 mg, 0.52 mmol) as described above gave methyl 16-(2-(methylamino)-2-oxoacetamido)hexadec-14(Z)-enoate (92 mg, 72%) as a white solid. M.P.: 104.5-1.4.8° C. TLC: 75% EtOAc/hexanes, R.sub.f≈0.40. .sup.1H NMR (CDCl.sub.3, 300 MHz) δ δ 7.80 (br s, 2H), 5.32-5.71 (m, 2H), 3.82-3.96 (m, 2H), 3.62 (s, 3H), 2.82 (s, 3H), 2.28 (t, 3H, J=7.1 Hz), 1.93-2.08 (m, 2H), 1.56-1.64 (m, 2H), 1.22-1.36 (m, 18).

(213) ##STR00099##

(214) Hydrolysis of methyl 16-(2-(methylamino)-2-oxoacetamido)hexadec-14(Z)-enoate (75 mg, 0.20 mmol) as described above gave 16-(2-(methylamino)-2-oxoacetamido)hexadec-14(Z)-enoic acid (63 mg, 88%) as a white solid. 118.9-119.3° C. TLC: 100% EtOAc, R.sub.f≈0.30; .sup.1H NMR (CDCl.sub.3, 300 MHz) δ 5.12-5.47 (m, 2H), 3.58-3.72 (m, 2H), 2.66 (s, 3H), 2.05 (t, 3H, J=7.2 Hz), 1.76-1.86 (m, 2H), 0.99-1.41 (m, 20H). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ 182.04, 163.77, 160.03, 134.06, 124.53, 41.28, 36.54, 34.11, 32.22, 29.62, 29.50, 29.35, 29.17, 29.08, 27.33, 27.56, 25.03.

Example 19

(215) Identification of Agonists

(216) This example shows the identification of compounds which act as agonists of EPA and 17,18-EETeTr and thus mimic the physiological effects of n-3 PUFAs and their CYP-dependent omega-3 epoxy-metabolites. The agonistic effects determined in this example consist in a reduction of the spontaneous beating rate of cultured neonatal rat cardiomyocytes (NRCMs). This negative chronotropic effect reflects the capacity of the analogs to interact with and to activate a G-protein coupled receptor or other primary cellular targets that reduce the contractility of cardiomyocytes under basal and stress-induced conditions.

(217) Materials and Methods

(218) The structures of all compounds tested are given in FIG. 1. The compounds included EPA and 17,18-EETeTr (compounds 01 and 02; purchased from Cayman Chemical) as well as all but one (compound 16) of the analogs synthesized as described in examples 1-24. The R,S— and S,R-enantiomers of 17,18-EETeTr (compounds 03 and 04) were prepared resolving the racemic mixture (compound 02) by means of chiral-phase HPLC as described previously (Barbosa-Sicard E, Markovic M, Honeck H, Christ B, Muller D N, Schunck W H. Biochem Biophys Res Commun. 2005 Apr. 22; 329(4):1275-81). Before use, the compounds to be tested were prepared as 1000-fold stock solutions in ethanol.

(219) Isolation and cultivation of NRCMs were performed as described previously (Wallukat, G; Wollenberger, A. Biomed Biochim Acta. 1987; 78:634-639; Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen J W, Haller H, Luft F C. J Clin Invest. 1999; 103: 945-952). Briefly, neonatal Wistar rats (1-2 days old) were killed in conformity to the recommendations of the Community of Health Service of the City of Berlin and the cardiomyocytes were dissociated from the minced ventricles with a 0.2% solution of crude trypsin. The isolated cells were then cultured as monolayers on the bottom (12.5 cm.sup.2) of Falcon flasks in 2.5 ml of Halle S M 20-I medium equilibrated with humidified air. The medium contained 10% heat-inactivated FCS and 2 μmol/l fluoro-deoxyuridine (Serva, Heidelberg, Germany), the latter to prevent proliferation of non-muscle cells. The NRCMs (2.4×10.sup.6 cells/flask) were cultured at 37° C. in an incubator. After 5 to 7 days, the NRCMs formed spontaneously beating cell clusters. The cells in each cluster showed synchronized contraction with a beating rate of 120 to 140 beats per minute. On the day of the experiment, the culture medium was replaced by 2.0 ml fresh serum-containing medium. Two hours later, the beating rates were monitored at 37° C. using an inverted microscope equipped with a heating stage. To determine the basal rate, 6 to 8 individual clusters were selected and the number of contractions was counted for 15 sec. After that, the compound to be tested was added to the culture and the beating rate of the same clusters was monitored 5 min later again. Based on the difference between the basal and compound-induced beating rate of the individual clusters, the chronotropic effects (Δ beats/min) were calculated and are given as mean±SE values. N refers to the number of clusters monitored which originated, in general, from at least three independent NRCM cultures.

(220) Results

(221) The results of these experiments are presented in FIG. 1.

(222) Addition of EPA (C01) in concentrations above 1 μM to the NRCM cultures resulted in a progressive reduction of the beating rate. This effect was fully expressed using an EPA concentration of 3.3 μM and an incubation time of 30 min. In contrast, 17,18-EETeTr (C02) produced the same effect almost immediately and already in the low nanomolar range (EC50 of 1-2 nM, data not shown). To compare the activity of 17,18-EETeTr with that of its synthetic analogs, all these compounds were tested at a final concentration of 30 nM and using an incubation time of 5 min. Under the same conditions, the vehicle control (0.1% ethanol) showed no effect on the spontaneous beating rate.

(223) As summarized in FIG. 1, various synthetic analogs showed a negative chronotropic effect similar to that of EPA and 17,18-EETeTr. These analogs are therefore designated as agonists.

(224) Agonists included: (i) analogs containing a double bond in 11,12-position in combination with an epoxy-group in 17,18-position, whereby the epoxy-group is racemic or in R,S-configuration (C03, C2, C4 and C9) (ii) analogs containing an 11,12-double bond in combination with a suitable substitute of the 17,18-epoxy-group (C11, C13 and C24) (iii) analogs belonging to category ii but modified at the carboxy-group (C17 and C18)

(225) In contrast, most of the analogs not carrying an 11,12-double bond showed no significant agonistic effects (i.e. their addition altered the beating rate of NRCMs by less than 5 beats per min). To this group belong C1, C3, C5, C6, C7, C8, C19 and C23. A shift of the double bond from the 11,12- to 14,15-position abolished the agonistic properties of some compounds; compare C9-C5 and C11-C23. Moreover, with some compounds the same shift of the double bond inversed the effect from a negative to positive chronotropic response of the NRCMs (compare C11-C21) or conferred a positive chronotropic effect to a compound that was largely inactive (compare C12 and C22).

(226) A comparison of the effects of compounds C03-C04 shows that the 17,18-epoxy-group conferred agonistic properties if present in the R,S-configuration whereas the corresponding S,R-enantiomer was inactive. The respective racemic mixture (C02) acted as agonist indicating that the effect of the R,S-enantiomer was predominating. Exactly the same stereochemical conditions applied to the 17,18-EETeTr analogs which carry only one double-bond in 11,12-position: the racemate (C4) and the R,S-enantiomer (C9) exerted agonistic effects and the S,R-enantiomer was inactive. In contrast, the analog containing only one double bond in 14,15-position showed no effect as racemate (C5) and S,R-enantiomer (C19) but an agonistic effect as R,S-enantiomer (C20). Thus, in this case the agonistic effect of the R,S-enantiomer was abolished when the S,R-enantiomer was simultaneously present.

(227) The effects of compounds C11, C13 and C24 demonstrate that the 17,18-epoxy-group can be replaced by residues carrying a suitable oxygen-functionality. These types of substitution did not only maintain (C24) but even significantly increased the agonistic effect: p<0.05 for the comparisons of the agonistic effects between C11 (−27.0±1.2; n=27) or C13 (−33.7±1.3; n=24) with 17,18-EETeTr (−22.5±0.8; n=60) and C4 (−18.3±1.5; n=21).

Example 20

(228) Identification of Antagonists

(229) This example shows the identification of compounds that act as antagonists of EPA and 17,18-EETeTr and thus block the physiological effects of n-3 PUFAs and their CYP-dependent omega-3 epoxy-metabolites. These antagonists were selected based on their capacity to abolish the negative chronotropic effects of EPA, 17,18-EETeTr and their synthetic agonists on the contractility of neonatal rat cardiomyocytes.

(230) Materials and Methods

(231) The structures of the compounds tested are presented in FIG. 2. Potential antagonists included compounds C1, C3, C5, C6, C7, and C8, which were synthesized as described above in the corresponding examples.

(232) The bioassay was performed with NRCMs as described in example 25. In the first series of experiments, compound C4 was used as the agonist and its effect was determined after preincubating the cultured NRCMc for 5 min with one of the potential antagonists. Both C4 and the potential antagonist were used at a final concentration of 30 nM. In the second series of experiments, compound C3 (30 nM) was tested for its antagonistic effect against EPA (3.3 μM) and 17,18-EETeTr (30 nM) as well as against the agonistic analogs C2, C4 and C13 (30 nM each).

(233) Results

(234) The results are presented in FIGS. 2 and 3. The data summarized in FIG. 2 show that the agonistic effect of compound C4 was significantly inhibited by compounds C3 and C5. This antagonistic capacity of C3 and C5 became only obvious in combination with the agonist since both compounds did not exert any significant effect when added alone to the cultured NRCMs (compare example 25, FIG. 1). The other compounds (C1, C6, C7 and C8) did not inhibit the agonistic effect of C4 (FIG. 2) and were also inactive when tested alone (compare example 25, FIG. 1). The structural feature which distinguishes the active antagonists (C3 and C5) from the completely inactive analogs (C1, C6, C7 and C8) consisted in the presence of a 14,15-double bond.

(235) The data summarized in FIG. 3 show that compound C3 is a highly potent antagonist not only of C4 but also of EPA, 17,18-EETeTr, C2 and C13. At a concentration of 30 nM, C3 abolished the negative chronotropic effect of EPA which was applied at a concentration of 3.3 μM. Even the effect of the most potent agonist (C13) was almost completely blocked by C3 when both analogs were present in equimolar concentrations (30 nM).

Example 21

EPA and its Agonistic Analogs Act Via the Sam Cellular Mechanisms

(236) This example shows that EPA, 17,18-EETeTr and their most potent synthetic agonist (C13) share the same mechanism of cellular action as judged by identical responses to several pharmacological interventions.

(237) Materials and Methods

(238) The bioassay with NRCMs was performed as described in examples 25 and 26. Compounds used as putative inhibitors of agonistic effects were: 11,12-epoxyeicosatrienoic acid (11,12-EET from Cayman Chemicals; used at a final concentration of 30 nM), AH6089 (unspecific antagonist of EP2 and related prostanoid receptors from Cayman Chemical; used at a final concentration of 10 μM), calphostin C (PKC-epsilon inhibitor from Sigma-Aldrich; used at a final concentration of 100 nM), and H89 (PKA-inhibitor from Sigma-Aldrich; used at a final concentration of 1 μM). The cultured NRCMs were preincubated without or with one of the compounds indicated in FIG. 4 for 5 min before the effect of the following agonists was determined: EPA (3.3 μM), 17,18-EETeTr (30 nM) or C13 (30 nM). In some experiments, the NRCMs were stimulated with a selective EP2 prostanoid receptor agonist (butaprost from Sigma-Aldrich; used at a final concentration of 100 nM) to provide a control for the effect of certain inhibitors.

(239) Results

(240) The results are presented in FIG. 4. The negative chronotropic effects of EPA, 17,18-EETeTr and compound C13 were strongly inhibited by 11,12-EET, C3, AH6089 and calphostin C but were not affected by H89. These results show that EPA, 17,18-EETeTr and their most potent synthetic analog share the same inhibitory profile and thus confirm that these compounds exert their biological effect via identical cellular mechanisms. More specifically, the results indicate that the three agonists compete with 11,12-EET, C3 and AH6089 for binding and activation of the same primary target (the putative omega-3 epoxyeicosanoid receptor) and that the subsequent signaling pathway includes the activation of a protein kinase C isoform as essential component. In contrast, to EPA, 17,18-EETeTr and C3, butaprost exerted a positive chronotropic effect. The butaprost effect was blocked by AH6089 and H89 but not by C3 and calphostin C. Thus, both the primary target of butaprost (EP2 receptor) and the butaprost-induced signaling pathway (involvement of PKA instead of PKC) are different from that of EPA, 17,18-EETeTr and their synthetic analog.

(241) FIG. 4: The negative chronotropic effects of EPA (01), 17,18-EETeTr (02) and of the synthetic agonist C13 are blocked by 11,12-EET, compound C3, AH6089 (unselective prostanoid receptor antagonist) and calphostin C (PKC-inhibitor) but not by H89 (PKA inhibitor). The positive chronotropic effect of butaprost (EP2 agonist) is blocked by AH6089 and H89 but not by C3 and caplphostin C.

Example 22

17,18-EETeT Agonists Protect Against Calcium Overload and ß-Adrenergic Stimulation

(242) This example shows that stress-induced responses of cardiomyocytes such as to increased extracellular Ca.sup.2+-concentrations or to ß-adrenergic stimulation are suppressed by the 17,18-EETeTr agonist C11.

(243) Materials and Methods

(244) Compound C11 was synthesized as described above (example 11). NRCMs were isolated and cultured as in Example 19. The basal Ca.sup.2+-concentration of the medium was 1.2 mM. Increased extracellular Ca.sup.2+-concentrations (2.2, 5.2 and 8.2 mM) were adjusted by adding appropriate amounts of a 1 M CaCl.sub.2 solution to the cultures. Isoproterenol (from Sigma-Aldrich) was used as ß-adrenoreceptor agonist and added to the cultures to give final concentrations of 0.1, 1 or 10 μM. C11 was used at a final concentration of 30 nM and added to the cultures 5 min before changing the Ca.sup.2+-concentration or adding isoproterenol. Controls were performed in the absence of C11.

(245) Results

(246) The results are presented in FIG. 5. In control experiments, the NRCMs responded to enhanced extracellular Ca.sup.2+-concentrations with massively increased beating rates. Preincubation with C11 significantly reduced the beating rate of NRCMs not only under basal conditions (1.2 mM Ca.sup.2+) but also at higher Ca.sup.2+-concentrations up to 8.2 mM (FIG. 5A). Similarly, C11 reduced the response to increasing concentrations of isoproterenol which acts as an adrenoreceptor agonist and thereby enhances the contractility and beating rate of NRCMs (FIG. 5B).

(247) FIG. 5: The synthetic agonist C11 suppresses the response of NRCMs to ß-adrenergic stimulation (isoptroterenol, FIG. 5A) and increased extracellular Ca.sup.2+-concentrations (FIG. 5B).

Example 23

Anti-Arrhythmic Effect of 17,18-EETeTr Agonists Under In Vivo Conditions

(248) This example shows that the agonistic analog C17 ameliorates arrhythmias as induced by myocardial infarction.

(249) Materials and Methods

(250) Study design: To gain insight into the in-vivo effects of synthetic 17,18-EETeTr-agonists, myocardial infarction studies were performed in male Wistar rats. Briefly, rats weighing 220-250 g were randomized to receive an i.v. bolus of compound C17 (100 μg in 300 μl 0.9% NaCl) or only 300 μl 0.9% NaCl as vehicle control two hours before induction of myocardial infarction. For safe bolus application, animals were mildly anesthetized using isoflorane. Two hours after bolus application, animals were re-anesthetized with a mixture of ketamine and xylazine (i.v.). Continuous monitoring of the surface-ECG was started (EPTracer, Netherlands) and maintained until the end of the study. After recording of the basal ECG, myocardial infarction was induced by ligation of the left anterior descending artery (LAD). One hour after myocardial infarction animals were sacrificed and organ harvested. Samples from urine, blood, liver, kidney and heart were stored for further analysis.

(251) Method of arrhythmia analysis: Ventricular tachycardia burden was calculated as the sum of all arrhythmic events originating from the ventricular myocardium, which were observed within the first hour after induction of myocardial infarction. In order to quantify not only the frequency but also the severity of the ventricular arrhythmias, an arrhythmia-severity-score was calculated. This score was calculated as the sum of the number of different arrhythmia events (PVC, couplet, triplet, VT<1.5 sec, VT>=1.5 sec), each class factorized by an increasing severity index of 1-5 (e.g. PVC×1, couplets×2, . . . , VT>=1.5 sec×5).

(252) Results

(253) The results are presented in FIG. 6. Bolus injection of the synthetic 17,18-EETeTr agonist (compound C17) did not induce any obvious negative side effects. Ventricular arrhythmias occurred after coronary artery ligation and were observed as single premature ventricular contractions (PVC), short runs of non-sustained ventricular tachycardia (VT) and ventricular tachycardia/fibrillation. Rats treated with the synthetic 17,18-EETeTr-agonist showed a significantly reduced ventricular tachycardia burden compared to controls (7526.2±5664.3 vs. 56377.4±17749.9 ms/h, p<0.05, n=5 per group); FIG. 6A. Moreover, the arrhythmia severity score was lower (125±25 vs. 336±93 arbitrary units, n=5 per group) in the 17,18-EETeTr-agonist group; FIG. 6B.

(254) FIG. 6: Treatment with compound C17, a synthetic agonist of 17,18-EETeTr, ameliorates the frequency (A) and severity (B) of cardiac arrhythmias in a rat model of myocardial infarction.