LIPID COMPOUNDS AND COMPOSITIONS AND THEIR OPTHALMIC USE

Abstract

The invention relates to lipid compounds of formula f(1) and their pharmaceutically acceptable salts for the prevention and/or treatment of ophthalmic disorders such as retinal degenerative disorders and ocular inflammatory diseases: (I) (wherein R.sup.1 is either a C.sub.9 to C.sub.22 alkyl group, or a C.sub.9 to C.sub.22 alkenyl group having from 1 to 6 double bonds; R.sup.2 is selected from the group consisting of a halogen atom, a hydroxy group, an alkyl group, an alkoxy group, an alkylthio group, a carboxy group, an acyl group, an amino group, and an alkylamino group; R.sup.3 is a hydrogen atom, or a group R.sup.2; R.sup.4 is a carboxylic acid or a derivative thereof; and X is methylene (CH.sub.2), or an oxygen or sulfur atom).

Claims

1-59. (canceled)

60. An omega-6 lipid compound of formula (II), or a pharmaceutically acceptable salt thereof: ##STR00060## wherein R.sup.12 is a C.sub.9 to C.sub.22 alkenyl group having from 1 to 5 double bonds in which: the first double bond counting from the -end is at carbon 6; and where two or more double bonds are present, at least one pair of consecutive double bonds is interrupted by a single methylene group; R.sup.2 is selected from the group consisting of a halogen atom, a hydroxy group, an alkyl group, an alkoxy group, an alkylthio group, a carboxy group, an acyl group, an amino group, and an alkylamino group; R.sup.3 is a hydrogen atom, or a group R.sup.2; R.sup.4 is a carboxylic acid or a derivative thereof selected from a carboxylic ester, a carboxylic anhydride, a carboxamide, a monoglyceride, a diglyceride, a triglyceride, and a phospholipid; and X is an oxygen or sulfur atom.

61. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.2, R.sup.3, or both R.sup.2 and R.sup.3 are an alkyl group.

62. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.2, R.sup.3, or both R.sup.2 and R.sup.3 are an unsubstituted, straight-chained or branched C.sub.1-6 alkyl.

63. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.2, R.sup.3, or both R.sup.2 and R.sup.3 is an unsubstituted, straight-chained or branched C.sub.1-3 alkyl.

64. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.3 is a hydrogen atom.

65. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.4 is a carboxylic acid.

65. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.4 is a derivative of a carboxylic acid which is a carboxylic ester.

67. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.12 is a straight-chained C.sub.9 to C.sub.22 alkenyl group.

68. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.12 is a C.sub.10 to C.sub.22 alkenyl group.

69. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.12 is a C.sub.20 alkenyl group.

70. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.12 is a C.sub.14 to C.sub.18 alkenyl group.

71. The omega-6 lipid compound as claimed in claim 60, wherein in formula (II), R.sup.12 is a C.sub.15 or C.sub.18 alkenyl group.

72. The omega-6 lipid compound as claimed in claim 60, wherein at least two double bonds are present in group R.sup.12.

73. The omega-6 lipid compound as claimed in claim 72, wherein at least one pair of successive double bonds is interrupted by no more than one methylene group.

74. The omega-6 lipid compound as claimed in claim 73, wherein each pair of consecutive double bonds is interrupted by no more than one methylene group.

75. The omega-6 lipid compound as claimed in claim 60, wherein all double bonds present in group R.sup.12 are in the Z-configuration.

76. The omega-6 lipid compound as claimed in claim 60, wherein said compound is derived from an -6 PUFA.

77. The omega-6 lipid compound as claimed in claim 76, wherein the -6 PUFA is (all-Z)-5,8,11,14-icosatetraenoic acid, (all-Z)-4,7,10,13,16-docosapentaenoic acid, (all-Z)-8,11,14-eicosatrienoic acid, (all-Z)-9,12-octadecadienoic acid, (all-Z)-6,9,12-octadecatrienoic acid, (all-Z)-11,14-eicosadienoic acid, (all-Z)-13,16-docosadienoic acid, (all-Z)-7,10,13,16-docosatetraenoic acid or (all-Z)-4,7,10,13,16-docosapentaenoic acid.

78. The omega-6 lipid compound as claimed in claim 60 which is selected from any of the following, or their pharmaceutically acceptable salts: ##STR00061## wherein Y is either hydrogen or an alkyl group.

79. The omega-6 lipid compound as claimed in claim 60 which is selected from the following compounds and their pharmaceutically acceptable salts: ##STR00062## ##STR00063## ##STR00064## ##STR00065##

80. The omega-6 lipid compound as claimed in claim 60 which is ##STR00066## or a pharmaceutically acceptable salt, or ester thereof.

81. The omega-6 lipid compound as claimed in claim 60, wherein R.sup.12 has 2 to 5 double bonds.

82. The omega-6 lipid compound as claimed in claim 60, wherein R.sup.12 is a C.sub.15 to C.sub.20 alkenyl group with 2 to 4 double bonds.

83. The omega-6 lipid compound as claimed in claim 60, wherein R.sup.12 has 2 to 4 double bonds and all of said double bonds are methylene-interrupted.

84. The omega-6 lipid compound as claimed in claim 60, wherein R.sup.12 has 2 to 4 double bonds which are methylene-interrupted and which are all in the Z-configuration.

Description

[0216] The invention will now be described in more detail by way of the following non-limiting Examples and with reference to the accompanying figures, in which:

[0217] a. FIG. 1illustrates the principle of cell based M1H assays in agonist mode used in Example 6;

[0218] b. FIG. 2illustrates the principle of cell based M1H assays in antagonist mode used in Example 6;

[0219] c. FIG. 3shows the results of the mitochondrial DNA study in Example 7;

[0220] d. FIG. 4shows the effects of compounds on the activity of the NF-B pathway using the NF-B reporter assay in Example 22;

[0221] e. FIG. 5shows the dose dependence of the inhibitory effect of dexamethasone, compound BIZ-110 and compound BIZ-101 on the NF-B pathway;

[0222] f. FIG. 6shows the PPAR activation of compounds at 5 M concentration;

[0223] g. FIG. 7shows the PPAR activation of compounds at 5 M concentration;

[0224] h. FIGS. 8A-8Cshow kinetic monitoring of cytolysis in ARPE-19 cells treated with 0.1% DMSO (FIG. 8A), Fenofibric acid (FIG. 8B), or BIZ-101 (FIG. 8C). FIG. 8D shows the dose response analysis of the tBHP cytotoxicity after 6, 24 and 96 hours; and

[0225] i. FIGS. 9A-9Cshow kinetic monitoring of cytolysis in ARPE-19 cells treated with compound BIZ-102. Cytotoxicity is induced by 10 M t-BHP (FIG. 9A), 30 M t-BHP (FIG. 9B), and 100 M t-BHP (FIG. 9C). In FIG. 9D the effects of BIZ-102 12 hrs after addition of tBHP is shown.

[0226] The NMR-spectra were recorded in CDC13, with a Bruker Avance DPX 200 or DPX 300 or DPX 400 instrument. Mass spectra were recorded at 70 eV with a Fision VG Pro spectrometer. All reactions were performed under nitrogen or argon atmosphere.

Example 1Preparation of Ethyl (All-Z) 2-ethyl-5, 8, 11,14-eicosatetraenoate

[0227] ##STR00032##

[0228] Butyl lithium (0.96 ml, 1.54 mmol in 1.6 M in hexane) was added dropwise to a stirred solution of diisopropyl amine (0.23 ml, 1.6 mmol) in dry THE (5 ml) under a nitrogen atmosphere at 0 C. The resulting solution was stirred at 0 C. for 20 minutes, cooled to and stirred an additional 10 minutes before dropwise addition of ethyl (all-Z)-5,8,11,14-eicosatetraenoate (466 mg, 1.4 mmol) in dry THE (5 ml). The mixture was stirred at 78 1 for 10 minutes before addition of ethyl iodide (170 l, 2.09 mmol). The mixture was allowed to warm to room temperature over 1 hour. The mixture was then poured into water, and extracted with heptane. The combined organic phase was washed with 1M HCl and then dried (Na.sub.2SO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography (2% EtOAc in hexane) gave compound (1) as a clear oil (450 mg, 89% yield).

Example 2Preparation of (All-Z) 2-ethyl-5,8,11,14-eicosatetraenoic Acid (BIZ 102)

[0229] ##STR00033##

[0230] Ethyl (all-Z) 2-ethyl-5,8,12,15-eicosatetraenoate produced in Example 1 (450 mg, 1.25 mmol) was dissolved in 30 ml ethanol and a solution of LiOH (420 mg) in water (10 ml) was added. The mixture was left stirring at 80 C. under an argon atmosphere for 18 hours. The mixture was cooled, then a 1M HCl solution (15 ml) was added and the mixture extracted with ether. The organic phase was washed with brine and dried (MgSO.sub.4). Filtration and evaporation gave the acid as a light yellow oil (416 mg) in 100% yield.

[0231] .sub.H (400 MHz, CDCl.sub.3) 0.89 (t, 3H, J=7.0,), 0.95 (t, 3H, J=7.4,), 1.16-1.45 (m, 6H), 1.45-1.83 (m, 4H), 1.87-2.20 (m, 4H), 2.34 (tt, 1H, J=8.4, 5.5), 2.62-2.96 (m, 6H) 5.56-5.13 (m, 8H), .sub.C (101 MHz, CDCl.sub.3) 11.83, 14.23, 22.74, 25.21, 25.31, 25.77, 25.80, 27.38, 29.49, 31.68, 46.37, 127.72, 128.05, 128.32, 128.37, 128.72, 128.86, 129.17, 130.66, 180.83

Example 3Preparation of 2-ethyleicosa-(all-Z)-5,8,11,14,17-pentaenoic Acid (-Ethyl EPA) (BIZ-101)

[0232] ##STR00034##

[0233] BIZ-101 was prepared based on the procedure described by Larsen et al., Biochemical Pharmacology, 1998, 405.

[0234] Butyl lithium (2.25 ml, 3.6 mmol in 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (594 p. 1, 4.2 mmol) in dry THF (5 ml) under a nitrogen atmosphere at 20 C. The resulting solution was stirred at 78 C. for 45 min before dropwise addition of ethyl (all-Z)-5,8,11,14J7-eicosapentaenoate (1.0 g, 3.0 mmol) in dry THF (20 ml). The mixture was stirred at 78 C. for 30 minutes before addition of ethyl iodide (388 l, 4.8 mmol). The mixture was stirred at 0 C. for 30 min before being poured into water (5 ml). The water phase was separated and extracted with hexane (210 ml). The combined organic phase was washed with 2M HCl (5 ml), water (25 ml) and then dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography (2% EtOAc in hexane) gave ethyl (all-Z)-2-ethyl-5,8,11,14,17-eicosapentaenoate as a clear oil (670 mg, 63% yield).

[0235] Ethyl (all-Z)-2-ethyl-5,8,11,14,17-eicosapentaenoate (670 mg, 1.9 mmol) was dissolved in a mixture of ethanol/THF (15 ml, 1:1) and a solution of LiOH (550 mg) in water (7.5 ml) was added. The mixture was left stirring at room temperature for 18 hours. Water and hexane were added and the organic phase was collected. The water phase was acidified with 5% HCl to pH 2 and extracted three times with hexane:ethylacetate (7:3). The organic phase was washed with water and brine and dried (MgSO.sub.4). Filtration and evaporation gave the acid as a light yellow oil (429 mg) in 68% yield.

[0236] .sub.H (200 MHz) 0.93 (t, 3H, J=7.3), 0.96 (t, 3H, J=7.5), 1.39-1.85 (m, 4H), 1.95-2.19 (m, 4H), 2.22-2.42 (m, 1H), 2.68-2.95 (m, 8H), 5.21-5.52 (m, 10H), .sub.C (50 MHz):

[0237] 12.4, 15.0, 21.2, 25.6, 25.7, 26.1, 26.2, 31.9, 46.8, 126.4, 127.2, 127.5, 127.6, 127.9, 128.0, 128.4, 131.3, 181.6

Example 4Preparation of 2-methyldocosa-(all-Z)-4,7,10,13,16,19-hexaenoic Acid (-Methyl DHA) (BIZ-105)

[0238] ##STR00035##

[0239] BIZ-105 was prepared based on the procedure described by Larsen et al., Lipids, Vol. 40, 2005.

[0240] Butyl lithium (1.12 ml, 1.7 mmol in 1.5 M in hexane) was added dropwise to a stirred solution of diisopropylamine (283 l, 2.0 mmol) in dry THF (4.2 ml) under nitrogen atmosphere at 20 C. The resulting solution was stirred at 78 C. for 45 min before dropwise addition of ethyl (all-Z)-4,7,10,13,16,19-docosahexaenoate (500 mg, 1.4 mmol) in dry THF (8.4 ml). The mixture was stirred at 78 C. for 30 minutes before addition of methyl iodide (140 l, 4.8 mmol). The mixture was stirred at 0 C. for 30 min before being poured into water (5 ml). The water phase was separated and extracted with hexane (210 ml). The combined organic phase was washed with 2M HCl (5 ml), water (25 ml) and then dried (MgSO.sub.4). Filtration and evaporation under reduced pressure gave ethyl (all-Z)-2-methyl-4,7,10,13,16,19-docosahexaenoate as a clear oil (520 mg, 100% yield).

[0241] Ethyl (all-Z)-2-methyl-4,7,10,13,16,19-docosahexaenoate (520 mg, 1.4 mmol) was dissolved in a mixture of ethanol/THF (9 ml, 2:1) and a solution of LiOH (437 mg) in water (6 ml) was added. The mixture was left stirring at room temperature for 18 hours. Water and hexane were added and the organic phase was collected. The water phase was acidified with 5% HCl to pH 2 and extracted three times with hexane:ethylacetate (7:3). The organic phase was washed with water and brine and dried (MgSO.sub.4). Filtration and evaporation gave the acid as a light yellow oil (390 mg) in 81% yield.

[0242] .sub.H (300 MHz): 0.96 (t, 3H, J=7.5 Hz), 1.18 (d, 3H, J=6.8 Hz), 2.06 (m, 2H), 2.20-2.30 (m, 1H), 2.35-2.55 (m, 2H), 2.75-2.95 (m, 10H), 5.25-5.55 (m, 12H); .sub.C (75 MHz) 14.25, 16.34, 20.55, 25.53, 26.63, 30.90, 39.41, 126.32, 127.01, 127.87, 127.98, 128.08, 128.11, 128.23, 128.56, 130.26, 132.03, 128.27, 182.37; m/z (CI) 343 (M+1, 1.65%), 215, 93, (100), HRMS: found M+1 343.262563.

Example 5Preparation of 2-methyl-tetradecylthioacetic Acid (-Methyl TTA) (BIZ-103)

[0243] ##STR00036##

[0244] BIZ-103 was prepared based on the procedure described in EP-A-0345038 and US 2004/0192908.

[0245] Potassium hydroxide (34.30 g, 0.611 mol), 2-mercapto propionic acid (31.2 g, 0.294 mol) and 1-bromotetradecane (50 ml, 0.184 mol) were added in that order to methanol (400 ml) and stirred overnight at room temperature. A concentrated hydrochloric acid solution (60 ml) dissolved in water (800 ml) was then added to the reaction mixture. Precipitation of 2 methyl tetradecylthioacetic acid occurred. The mixture was stirred overnight at room temperature. The precipitate was then filtered, washed five times with water and dried. The product was recrystallized from methanol and isolated as white flakes by filtration (yield 90%). TLC gave only one spot with iodine vapor.

[0246] .sub.H (400 MHz, CDCl.sub.3): 0.89 (t, 3H, J=6.8 Hz), 1.2-1.3 (m, 20H), 1.3-1.4 (m, 2H), 1.46 (d, 3H, J=7.2 Hz), 1.5-1.7 (m, 2H), 2.6-2.7 (m, 2H), 3.41 (q, 1H, J=7.2 Hz); .sub.C (101 MHz, CDCl.sub.3): 14.13, 16.90, 22.70, 28.88, 29.19, 29.21, 29.37, 29.50, 29.60, 29.66, 29.68, 29.70, 31.67, 31.93, 40.88, 179.27

Example 6PPAR Activity of Compounds

[0247] The compounds BIZ-102 (Example 2), BIZ-103 (Example 5) and BIZ-106 were tested at the human PPARalpha receptor in a cellular GAL4 Reporter gene assay. The assay was run in agonist and antagonist mode to detect agonistic as well as antagonistic activities of the tested compounds.

[0248] Materials and Methods:

[0249] Compounds Tested:

##STR00037##

[0250] Reference Compounds:

##STR00038##

[0251] GW7647 (a known PPAR agonist)

[0252] Fenofibric acid (a known PPAR agonist)

[0253] GAL4 Transactivation Assays:

[0254] FIG. 1 shows the principle of the cell based M1H assays in agonist mode.

[0255] FIG. 2 shows the principle of the cell based M1H assays in antagonist mode.

[0256] Performance of the GAL4 cellular reporter assay:

[0257] Day 1: Cells were seeded in 96 well plates in plating medium (MEM with serum), incubation overnight at 37 C., 5% CO.sub.2.

[0258] Day 2: Removal of plating medium

[0259] Addition of PEI-based transfection agent

[0260] Incubation for 4-6 hours at 37 C., 5% CO.sub.2

[0261] Addition of assay medium (MEM with serum)

[0262] Addition of compounds (dilution series was generated in MEM with serum)

[0263] Incubation overnight at 37 C., 5% CO.sub.2

[0264] Day 3: Removal of assay medium (16-20 hours after compound addition)

[0265] Addition of Passive Lysis Buffer (Promega)

[0266] 15 mins incubation at room temperature

[0267] Addition of luciferase buffers and measurement in a dual-flash procedure

[0268] The assays were done in HEK.sub.293 cells (DSMZ ACC 305). The plasmids used in the GAL4 assay system were derivatives of Stratagene's M2H plasmids: the reporter plasmid pFR-Luc (containing a synthetic promoter with five tandem repeats of the yeast GAL4 binding sites that control expression of the Photinus pyralis (American firefly) luciferase gene), and pCMV-BD (for fusions of nuclear receptor ligand binding domains to the DNA-binding domain of the yeast protein GAL4). In order to improve experimental accuracy, a second reporterRenilla reniformis luciferase, driven by a constitutive promoterwas included as an internal control. Using the control reporter (Renilla Luciferase) allowed corrections for variations in experimental handling, e.g. transfection efficacy, cell viability, pipetting errors, cell lysis efficiency and assay efficiency. For the antagonist mode experiments, the medium added after transfection contained intermediate concentrations of the reference compound GW7647 (2.5 nM).

Data Evaluation:

[0269] Primary read out of the assays was loaded into PhAST (Phenex Assay and Screening Tool) and checked for assay quality (generation of SB and Zprime values). These data were then loaded into the Analysis tool of PhAST to generate graphs and dose response curves. Within PhAST there are two measured and one calculated data layers:

[0270] LAYER1 (measured)contains the activity values of the firefly luciferase activities and is a direct measure for modulation of the cofactor binding properties of the Nuclear Receptors by the tested compounds.

[0271] LAYER2 (measured)contains the activity values of the renilla luciferase activities and is used as normalisation layer. As the renilla luciferase is expressed under control of the constitutively active CMV promoter, moderate well-to-well differences can be used to correct for variations in experimental handling.

[0272] LAYER3 (calculated)is calculated according to the following equation:

1000*Firefly luciferase value/Renilla luciferase value

[0273] These normalised values are, as well as LAYER1, a measure for the modulation of the cofactor binding properties of the nuclear receptors by the tested compounds.

Results and Discussion:

[0274] Results were obtained for the 10 compound concentration triplicate assays in direct Firefly and Renilla normalized measurement mode. Dose response curves were used to determine the EC.sub.50 values for BIZ-102 and BIZ-103 as 350 nM and 154 nM. Compared with the reference compound GW7647 the tested compounds showed an efficacy of 150 and 180%. Compared with fenofibric acid, the efficacies of the tested compounds are 45 and 55%, respectively.

[0275] The EC.sub.50 values for BIZ-106 and BIZ-107 were determined from dose response curves to be 5 M and 26 M, respectively. Compared with the reference compound GW7647 the tested compounds show an efficacy of 80 and 100%, respectively. Compared with fenofibric acid, the efficacies of the tested compounds are 25 and 30%, respectively.

[0276] All tested compounds showed agonistic effects at PPAR in antagonist mode. For evaluation of agonistic effects it is better to use agonist mode data as they are only influenced by the test compound. The table below shows the obtained potencies and efficacies in detail.

TABLE-US-00001 Compound Efficacy vs. Efficacy vs. tested EC.sub.50 value GW7647 Fenofibric Acid BIZ-102 350 nM 150% 45% BIZ-103 1.54 nM 180% 55% BIZ-106 5 M 80% 25% BIZ-107 26 M 100% 30%

[0277] It can be seen that BIZ-102 and BIZ-103 activated PPAR at nanomolar concentrations and their efficacy was significantly better than the reference compound GW7647. Although their efficacy is not as high as fenofibric acid, this compound is not active on PPAR at nanomolar concentrations (EC.sub.50 fenofibric acid measured as 10-18 M).

Example 7PPAR, and Activity of Compounds

[0278] The compounds BIZ-102 and BIZ-103 were tested at the human PPARbeta/delta and PPARgamma receptors using the same cellular GAL4 Reporter gene assays as in Example 6. The assays were run in agonist mode to detect agonistic activities of the tested compounds. Both tested compounds showed weak agonistic effects at PPARbeta/delta and PPARgamma.

[0279] The table below shows the obtained potencies and efficacies in detail.

TABLE-US-00002 PPARbeta/delta PPARgamma efficacy vs. efficacy vs. EC.sub.50 value GW501516 EC.sub.50 value Rosiglitazone BIZ-102 4.3 M 20% 1.5 M 23% BIZ-103 8.9 M 18% 1.6 M 20%

Example 8Effect on Mitochondrial DNA (mtDNA)

[0280] Mitochondrial DNA damage is a useful biomarker to evaluate the potential therapeutic effect of the compounds in relation to the treatment of retinal degenerative diseases.

Method:

[0281] In this study, neuroblastoma cells were cultivated at near confluency (50-75%) in DMEM/F12/10% serum high glucose (20 mM) supplied with 10 M BIZ-101 or BIZ-105 for 24 hours prior to analyses. These conditions readily induce mtDNA damage that is representative of that accumulating during aging as the result of age-associated oxidative stress.

[0282] For DNA damage analyses, cells were washed, and DNA isolated using Qiagen Blood&Tissue DNA isolation kit. DNA damage level was analyzed by a RT-qPCR method based on the ability of DNA lesions to inhibit restriction enzyme cleavage, as described previously (http://www.ncbi.nlm.nih.gov/pubmed/25631007) using primers 5-aaactgctcgccagaacact-3 and 5-catgggctacaccttgacct-3 (sense and anti sense, respectively). Briefly, genomic DNA (6 ng) was treated with 1 U TaqI for 15 min at 65 C. DNA damage frequency was calculated as 2exp(ctTaqlctnt), where ctTaql and ctnt represent CT values of TaqI-treated and non-treated genomic DNA, respectively.

Results:

[0283] The results from the experiment demonstrated reduced mtDNA damage in in vitro cultured neuroblastoma cells upon BIZ-101 administration and a slightly reduced level of mtDNA damage by BIZ-105 (see FIG. 3; NT=no treatment). The results indicate at least the potential for BIZ-101 to be used for treatment of retinal degenerative diseases like AMD.

Example 9Preparation of 2-ethyl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,11,10,13,16,19-hexaenoic Acid (BIZ 106)

[0284] ##STR00039##

[0285] Butyl lithium (0.96 ml, 1.54 mmol in 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (0.23 ml, 1.6 mmol) in dry THF (5 ml) under a nitrogen atmosphere at 0 C. The resulting solution was stirred at 0 C. for 20 minutes, cooled to 78 C. and stirred an additional 10 minutes before dropwise addition of ethyl ethyl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,11,10,13,16,19-hexaenoate (499 mg, 1.4 mmol) in dry THF (5 ml). The mixture was stirred at 78 C. for 10 minutes before addition of ethyl iodide (0.16 ml, 2.09 mmol). The mixture was allowed to warm to room temperature over 1 hour. The mixture was then poured into water, and extracted with heptane. The combined organic phase was washed with 1M HCl and then dried (Na.sub.2SO.sub.4). Filtration and evaporation under reduced pressure followed by filtration through a silica plug (hexane:EtOAC 98:2) gave the ethyl ester (330 mg):

##STR00040##

[0286] The ester (330 mg, 0.9 mmol) was dissolved in 20 ml ethanol and a solution of LiOH (320 mg) in water (10 ml) was added. The mixture was left stirring at 80 C. under an argon atmosphere for 18 hours. The mixture was cooled, then a 1M HCl solution (15 ml) was added and the mixture extracted with ether and dried (MgSO.sub.4). Filtration, evaporation followed by flash chromatography on silica gel (98:2 to 9:1 hexaene/ethylacetate) gave the acid as a light yellow oil (200 mg).

[0287] .sub.H (400 MHz): 0.97 (m, J=7.4, 6H), 1.55-1.75 (m, 2H), 2.06 (m, 2H), 2.25-2.50 (m, 2H), 2.752.95 (m, 10H), 5.25-5.50 (m, 12H);

[0288] .sub.C (100 MHz) 11.9, 14.4, 20.7, 24.8, 25.7, 25.79, 25.80, 29.5, 47.1, 126.6, 127.2, 128.0, 128.2, 128.25, 128.29, 128.38, 128.41, 128.42, 128.7, 130.2, 132.2, 181.1

Example 10Preparation of (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenol

[0289] ##STR00041##

[0290] A solution of arachidonic acid ethyl ester (3.0 g, 9.0 mmol) in MTBE (15 ml) was added dropwise to a suspension of lithium aluminium hydride (0.68 g, 18 mmol) in MTBE (6 ml) at 0 C. The solution was stirred for an additional 1 hour at 0 C. Water and HCl (2M) were added and the mixture was extracted with diethylether. The combined ether extract was washed with brine and dried (MgSO.sub.4). Evaporation under reduced pressure followed by dry flash on silica gel (50:50 diethylether/EtOAC) gave the pure alcohol (2.9 g) as an oil.

[0291] .sub.H (400 MHz): 0.86 (t, 3H, J=6.9 Hz), 1.2-1.5 (m, 9H), 1.5-1.6 (m, 2H), 1.95-2.15 (m, 2H), 2.75-2.85 (m, 6H), 3.62 (t, 2H, J=6.4), 5.2-5.5 (m, 8H);

[0292] .sub.C (100 MHz) 14.0 (2C), 22.5, 25.6 (2C), 25.7, 26.9, 27.2, 29.3, 31.5, 32.3, 62.8, 127.5, 127.9, 128.0 (2C), 128.3, 128.5, 129.8, 130.4

Example 11Preparation of 2-((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraen-1-yloxy) butanoic Acid (BIZ 114)

[0293] ##STR00042##

[0294] An aqueous solution of sodium hydroxide (50%, w/w, 3.5 ml) was added to a stirred solution of tetrabutylammonium bromide (133 mg, 0.41 mmol), t-butyl 2-bromobutyrate (930 mg, 4.1 mmol) and (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenol (566 mg, 1.8 mmol) in toluene (5 ml) at room temperature. The resulting mixture was heated to 30-40 C. and stirred for 3 hours. After cooling to room temperature, a saturated ammonium chloride (NH.sub.4Cl) solution was added and the organic phase was separated. The aqueous phase was extracted with hexane (325 ml). The combined organic layers were washed with NH.sub.4Cl solution, brine and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography on silica gel (98:2 to 95:5 hexaene/ethylacetate) gave the t-butylester as a light yellow oil:

##STR00043##

[0295] Tert-butyl 2-((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraen-1-yloxy)butanoate was dissolved in formic acid (95%, 6 ml) and stirred at room temperature under nitrogen atmosphere for 2.5 hours. The mixture was concentrated under vacuum and the residue purified by flash chromatography on silica gel (95:5 to 9:1 hexane/EtOAc containing 1% formic acid). Evaporation under reduced pressure gave the fatty acid (87 mg, 9%) as a light yellow oil.

[0296] .sub.H (400 MHz): 0.85 (t, 3H, J=6.9 Hz), 0.97 (t, 3H, J=7.4), 1.2-1.35 (m, 6H), 1.35-1.5 (m, 2H), 1.55-1.65 (m, 2H), 1.7-1.8 (m, 2H), 2.0-2.1 (m, 4H), 2.75-2.85 (m, 6H), 2.3-2.4 (m, 1H), 3.55-3.65 (m, 1H), 3.75-3.85 (m, 1H), 5.2-5.5 (m, 8H)

[0297] .sub.C (100 MHz) 9.5, 14.1, 22.6, 25.5, 25.7, 25.9, 26.9, 27.1, 29.2, 29.3, 31.5, 79.5, 127.4, 127.8, 128.0 (2C), 128.2, 128.4, 129.7, 130.4, 178.1.

Example 12Preparation of (3Z,6Z,9Z)-pentadeca-3,6,9-tetrien-1-ol

[0298] The alcohol was prepared from an algea oil containing arachidonic acid (40%) from Huatai Biopharm

##STR00044##

Step 1: Hydrolysis of Algea Oil

[0299] Algea oil (20 g) dissolved in ethanol (100 ml) was added to a stirred solution of

[0300] NaOH (16 g) in water (100 ml). The mixture was heated to 60 C. for 2 hrs and left stirring overnight at room temperature. Acetone (200 ml) was added to the mixture and the resulting slurry was filtrated. The filter cake was washed with acetone (2150 ml) and the filtrate evaporated under reduced pressure. Acidification with HCl solution (5%) and extraction with a mixture of hexane:EtOAc (1:1) gave a crude arachidonic acid product (10.5 g) as an oil. The oil was dissolved in hexane and filtered through a silica plug. Evaporation of solvents under reduced pressure yielded the arachidonic acid in a mixture (approx. 40%) of a light yellow oil (5.7 g) which was used without further purification.

Step 2: Iodolactonization

[0301] The crude arachidonic acid produced in step 1 was dissolved in ethanol (35 ml, 80%) and added to a saturated aqueous solution of NaHCO.sub.3 (15 ml). An ethanoic solution (80 ml, 95%) of iodine (2.59 g) was added dropwise under vigorous stirring within an hour. Additional iodine (2 g) was added after 1.5 hrs reaction time. The mixture was left stirring overnight and Na.sub.2S.sub.2O.sub.3 was added until the solution was colorless. The mixture was extracted with hexane (3100 ml) and the combined organic layer washed with water, brine and dried (MgSO.sub.4). Filtration and evaporation gave the crude iodolactone (5 g).

Step 3: Epoxidation

[0302] The crude iodolactone produced in step 3 was dissolved in methanol (100 ml), added to K.sub.2CO.sub.3 (2.7 g) and stirred for 4 hrs. Water was added to the mixture. The mixture was extracted with diethylether and the combined organic layers were washed with a saturated NH.sub.4Cl solution, dried (MgSO.sub.4), filtrated and evaporated under reduced pressure to give the crude epoxide (4 g).

Step 4: Cleavage of Epoxide [Procedure from Holmeide & Skattebol, Journal Chemical Society, Perkin Transactions 1, 2000, 2271]

[0303] A solution of crude epoxide (2.6 g) in formic acid (50 ml) and acetic anhydride (5 ml) was stirred at room temperature overnight. Volatile compounds were evaporated under reduced pressure, the residue was dissolved in methanol (65 ml) and K.sub.2CO.sub.3 (1.6 g) was added. After stirring for 3 hrs at ambient temperature water was added and the product extracted with ether. The extract was washed with water and the ether evaporated. The residue was dissolved in methanol (60 ml), cooled to 0 C. and a solution of sodium periodate (2.6 g) in water (20 ml) was added. The mixture was stirred for 1.5 hrs, diluted with water and the product extracted with hexane. The extract was washed with water, dried (MgSO.sub.4) and the solvents evaporated under reduced pressure giving the crude aldehyde (1.9 g).

Step 5: Reduction of Aldehyde

[0304] An ice-cooled solution of crude aldehyde (1.9 g) in methanol (100 ml) was added to a solution of NaBH.sub.4 (1.1 g) in methanol (30 ml). The reaction was stirred for 30 minutes before extraction with hexane (3150 ml). The extract was washed with a saturated aqueous NH.sub.4Cl solution and water. The extract was filtered through a plug of silica using a mixture of hexane:EtOAc (95:5). The filtrate was concentrated under reduced pressure to give the alcohol (910 mg) as a colorless oil.

[0305] .sub.H (400 MHz): 0.86 (t, J=6.9, 3H), 1.25-1.35 (m, 6H), 1.40 (s, 1H), 2.03 (q, J=6.8, 2H), 2.30-2.38 (m, 2H), 2.76-2.86 (m, 4H), 6.36 (bt, J=6.3, 2H), 5.23-5.43 (m, 5H), 5.49-5.57 (m, 1H)

[0306] .sub.C (100 MHz): 14.1, 22.6, 25.6, 25.7, 27.2, 29.3, 30.8, 31.5, 62.2, 125.6, 127.5, 127.7, 128.7, 130.5, 131.2.

Example 13Preparation of 2-((3Z,6Z,9Z)-pentadeca-3,6,9-tetrien-1-yloxy) butanoic Acid (BIZ 111)

[0307] ##STR00045##

[0308] An aqueous solution of sodium hydroxide (50%, w/w, 2 ml) was added to a stirred solution of tetrabutylammonium bromide (131 mg, 0.41 mmol), t-butyl 2-bromobutyrate (923 mg, 4.1 mmol) and (3Z,6Z,9Z)-pentadeca-3,6,9-trienol as prepared in Example 12 (400 mg, 1.8 mmol) in toluene (5 ml) at 30 C. The resulting mixture was stirred for 2 hours at 30 C. After cooling to room temperature, a saturated ammonium chloride (NH.sub.4Cl) solution was added and the organic phase was separated. The aqueous phase was extracted with hexane (325 ml). The combined organic layers were washed with NH.sub.4Cl solution, brine and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography on silica gel (98:2 to 9:1 hexaene/ethylacetate) afforded the ester (160 mg) containing small amounts of t-butyl-2-bromobutyrate and a pure ester (200 mg) in addition to recovery of the (3Z,6Z,9Z)-pentadeca-3,6,9-trienol (100 mg):

##STR00046##

[0309] The pure fraction of the ester (200 mg) was dissolved in formic acid (95%, 3.0 mL). The reaction mixture was left stirring at room temperature for 2 hours. The mixture was concentrated under vacuum, dissolved in hexane (60 ml) and extracted with a saturated NaCO.sub.3 solution. The aqueous phase was acidified using HCl solution and extracted with EtOAc (325 ml). The combined organic layers were washed with brine and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by filtration through a short plug of silica (9:1 Hexane/EtOAc+1% formic acid) afforded the title compound (130 mg) as a light yellow oil.

[0310] .sub.H (400 MHz): 0.86 (t, 3H, J=6.9 Hz), 0.98 (t, 3H, J=7.4), 1.20-1.40 (m, 6H), 1.70-1.90 (m, 2H), 2.03 (q, J=6.8, 2H), 2.35-2.45 (m, 2H), 2.75-2.85 (m, 4H), 3.45-3.50 (m, 1H), 3.553.65 (m, 1H), 3.83 (dd, J=6.7, J=4.9) 5.2-5.5 (m, 6H)

[0311] .sub.C (100 MHz) 9.3, 14.0, 22.5, 25.6, 25.3, 25.7, 27.2, 27.9, 29.3, 31.5, 70.3, 77.3, 125.4, 127.4, 127.6, 128.7, 130.50, 130.51, 176.9

Example 14Preparation of (2E,6Z,9Z,12Z)-Pentadeca-2,6,9,12-tetraen-1-ol

[0312] ##STR00047##

[0313] (2E,6Z,9Z,12Z)-Penradeca-2,6,9,12-tetraen-1-ol was prepared from eicosapentaenoic acid as described in the literature (see Flock et al., Acta Chemica Scandinavica, 1999, 53, 436Compound 24).

[0314] .sub.H (400 MHz): 0.95 (t, J=7.5, 3H), 1.3 (bs, 1H), 2.0-2.2 (m, 6H), 2.7-2.9 (m, 4H), 4.06 (bs, 2H), 5.20-5.45 (m, 6H), 5.6-5.7 (m, 2H)

[0315] .sub.C (100 MHz): 14.2, 20.5, 25.5, 25.6, 26.8, 32.1, 63.7, 127.0, 128.0, 128.35, 128.41, 129.1, 129.4, 132.0, 132.5

Example 15Preparation of 2-((2E,6Z,9Z,12Z)-pentadeca-2,6,9,12-tetraen-1-yloxy) butanoic Acid (BIZ 112)

[0316] ##STR00048##

[0317] To a stirred solution of (2E,6Z,9Z,12Z)-Pentadeca-2,6,9,12-tetraen-1-ol (240 mg, 1.1 mmol), t-butyl-2-bromobutyrate (585 mg, 2.6 mmol) and tetrabutylammoniumbromide (71 mg, 0.22 mmol) in toluene (2.5 mL) at room temperature was added an aqueous solution of NaOH (1 ml, 50% w/w). The mixture was stirred at room temperature for 2 hours before addition of a saturated NH.sub.4Cl solution. The organic phase was separated and the aqueous phase was extracted with hexane (325 ml). The combined organic phases were washed with brine water and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography on silica (hexane/EtOAc, 95:5) afforded the ester (190 mg):

##STR00049##

[0318] The ester (190 mg) was dissolved in formic acid (95%, 3.0 mL). The reaction mixture was left stirring at room temperature for 2 hours. The mixture was concentrated under vacuum and the residue purified by flash chromatography on silica gel (short column) (95:5 to 9:1 hexane/EtOAc+1% formic acid) to give the title compound (160 mg) as a light yellow oil.

[0319] .sub.H (400 MHz) 0.92-1.0 (dt, J=7.5 J=7.6, 6H), 1.70-1.85 (m, 2H), 2.0-2.2 (m, 6H), 2.75-2.85 (m, 4H), 3.85-3.95 (m, 2H), 4.08 (dd, J=6.1 J=11.7, 1H), 5.2-5.4 (m, 6H), 5.50-5.60 (m, 1H), 5.65-5.75 (m, 1H)

[0320] .sub.C (100 MHz) 9.4, 14.2, 20.5, 25.5, 25.6, 25.7, 26.6, 32.2, 71.3, 78.2, 125.7, 127.0, 127.9, 128.4, 128.4, 129.0, 132.0, 135.3, 177.57

Example 16Preparation of 2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaen-1-yloxy) butanoic Acid [Corresponding to Example 1 in WO 2010/128401] (BIZ 110)

[0321] ##STR00050##

[0322] To a stirred solution of (5Z,7Z,11Z,14Z,17Z)-icosa-5,7,11,14,17-icosa-1-ol (500 mg, 1.7 mmol), t-butyl-2-bromobutyrate (758 mg, 3.4 mmol) and tetrabutylammoniumbromide (110 mg, 0.34 mmol) in toluene (5 mL) at 30 C. was added an aqueous solution of NaOH (1.7 ml, 50% w/w). The mixture was stirred at 40-45 C. for 2.5 hours before additional amounts of t-butyl bromobutyrate (800 mg, 3.5 mmol) and NaOH (0.8 ml, 50% w/w) were added. The mixture was stirred for an additional 1.5 hours at 40-45 C. and cooled to room temperature before addition of a saturated NH.sub.4Cl solution. The organic phase was separated and the aqueous phase was extracted with hexane (325 ml). The combined organic phases were washed with NH.sub.4Cl, brine water and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography on silica (hexane/EtOAc, 97:3) afforded the ester (310 mg) containing small amounts of t-butyl-2-bromobutyrate and a pure ester (124 mg):

##STR00051##

[0323] .sub.H (400 MHz) 0.91-0.98 (dt, J=7.4 J=7.5, 6H), 1.35-1.5 (m, 11H), 1.55-1.75 (m, 4H), 2.02.1 (m, 4H), 2.75-2.85 (m, 8H), 3.25-3.35 (dt, J=6.6 and J=6.6, 1H), 3.55-3.65 (m, 2H), 3.753.85 (m, 1H) 5.2-5.4 (m, 10H)

[0324] .sub.C (100 MHz) 9.7, 14.3, 20.53, 25.51, 25.6, 26.2, 27.0, 28.1, 29.4, 70.2, 80.8, 81.0, 127.0, 127.86, 127.87, 127.9, 128.1, 128.2, 128.45, 128.52, 130.1, 132.0, 172.3

[0325] The tert-butylester (310 mg) was dissolved in formic acid (95%, 5.0 mL). The reaction mixture was left stirring at room temperature for 4 hours. The mixture was concentrated under vacuum and the residue purified by flash chromatography on silica gel (95:5 to 0:100 hexane/EtOAc) to give the title compound (120 mg) as a light yellow oil.

[0326] .sub.H (400 MHz) 0.92-0.98 (dt, J=7.4 J=7.5, 6H), 1.35-1.50 (m, 2H), 1.55-1.70 (m, 2H), 1.701.90 (m, 2H), 2.0-2.15 (m, 4H), 2.75-2.85 (m, 8H), 3.41-3.48 (m, 1H), 3.54-3.62 (m, 2H), 3.82 (bt, 1H) 5.2-5.4 (m, 10H)

[0327] .sub.C (100 MHz) 9.2, 14.3, 20.5, 25.4, 25.5, 25.6, 26.0, 26.9, 29.3, 70.8, 79.7, 127.0, 127.9, 128.05, 128.10, 128.19, 128.23, 128.3, 128.6, 129.7, 132.0, 177.6

Example 17Preparation of 2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaen-1-yloxy) butanoic Acid [Corresponding to Example 15 in WO 2010/128401] (BIZ 115)

[0328] ##STR00052##

[0329] To a stirred solution of (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaen-1-ol (835 mg, 2.6 mmol) in toluene (7 mL) at room temperature, was added tetrabutylammonium bromide (193 mg, 0.6 mmol, 0.23 equiv.) and t-butyl-2-bromobutyrate (1.34 g, 6.0 mmol, 2.3 equiv.). The reaction mixture was added to aq. 50% NaOH (2.45 mL). After 30 mins the mixture was added to aq. 50% NaOH (0.35 mL) dropwise. The reaction mixture was then heated to 30 C. for 3 hours. After cooling to room temperature, a saturated ammonium chloride (NH.sub.4Cl) solution was added and the organic phase was separated. The aqueous phase was extracted with hexane. The combined organic phases were washed with NH.sub.4Cl, then brine and dried (MgSO.sub.4). Filtration and evaporation under reduced pressure followed by flash chromatography on silica (hexane/EtOAc, 98:2) afforded tert-butyl-2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaen-1-yloxy)butanoate (552 mg) containing impurities of t-butyl-2-bromobutyrate:

##STR00053##

[0330] The tert-butylester (552 mg) was dissolved in formic acid (95%, 6.0 mL). The reaction mixture was left stirring at room temperature for 3 hours. The mixture was concentrated under vacuum and the residue purified by flash chromatography on silica gel (9:1 hexane/EtOAc containing 1% formic acid). Flash chromatography (hexane/EtOAc, 9:1, acidified with formic acid) eluted a mixture of product and the hydrolysed bromide. After evaporation the crude oil (291 mg) was dissolved in MTBE-ether (50 mL) and washed with sat. NaHCO.sub.3 (325 mL), sat. NH.sub.4Cl (25 mL) and brine (25 mL) and dried (MgSO.sub.4). Filtration and evaporation gave the pure acid as a colourless oil (202 mg) in 19% yield.

[0331] .sub.H (400 MHz) 0.9-1.0 (m, 6H), 1.6-1.9 (m, 4H), 2.0-2.1 (m, 2H), 2.1-2.2 (m, 2H), 2.7-2.9 (m, 10H), 2.35-3.45 (m, 1H), 3.55-3.65 (m, 1H), 3.75-3.85 (m, 1H) 5.2-5.54 (m, 12H)

[0332] .sub.C (100 MHz) 9.5, 14.3, 20.5, 23.6, 25.4, 25.5, 25.8, 29.4, 70.1, 79.6, 126.9, 127.7, 127.96, 127.97, 128.0, 128.07, 128.13, 128.2, 128.4, 128.5, 129.2, 132.0, 177.5.

Example 18Preparation of (3Z,6Z,9Z,12Z)-pentadeca-(3,6,9,12)-tetraen-1-thiol

[0333] ##STR00054##

[0334] Diisopropyl azodicarboxylate (DIAD) (1.97 ml, 10.01 mmol) was added to a stirred solution of triphenylphosphine (2.75 g, 10.50 mmol) in THF (30 ml) at 0 C., and the mixture was stirred at this temperature for 30 mins. A solution of (3Z,6Z,9Z,12Z)-pentadeca-(3,6,9,12)-tetraen-1-ol (Flock et al., Acta chemical scandinavica, 1999, 53, 436) (1.8 g, 9.1 mmol) and thioacetic acid (715 l, 10.01 mmol) in THF (10 ml) was added dropwise over 20 mins. The mixture was stirred for 1 hour at 0 C. and for an additional hour at ambient temperature. The mixture was concentrated and purified by flash chromatography on silica gel (95:5 hexane:EtOAc) to give thioacetic ester (1.2 g, 47%) as an oil.

[0335] The thioester (710 mg, 2.6 mmol) was dissolved in methanol (30 ml) and added to K.sub.2CO.sub.3 (1.06 g, 7.65 mmol). The mixture was stirred at room temperature for 2 hours before addition of 1M HCl, water and diethylether. The organic phase was separated and the aqueous phase extracted with diethylether (330 ml). The combined organic layers were washed with brine and dried (MgSO.sub.4). Filtration and evaporation gave the thiol as an oil (520 mg, 85% yield).

Example 19Preparation of 2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoic Acid [Corresponding to Example 9 in U.S. Pat. No. 8,759,558] (BIZ 109)

[0336] ##STR00055##

[0337] An ice-cooled solution of (3Z,6Z,9Z,12Z)-pentadeca-(3,6,9,12)-tetraen-1-thiol as prepared in Example 18 (480 mg, 2.03 mmol) in dry dimethylformamide (DMF) (10 ml) was added to NaH (89 mg, 60% in mineral oil). The mixture was stirred at 0 C. for an additional 10 minutes before addition of ethyl bromobutyrate (330 l, 2.3 mmol). The mixture was stirred at room temperature for 40 minutes, then the mixture was poured into a saturated NH.sub.4Cl solution and extracted with hexane. The extract was washed with a saturated NH.sub.4Cl solution, water and dried (MgSO.sub.4). Filtration, evaporation under reduced pressure and purification by flash chromatography (hexane:EtOAC 98:2) afforded the ethyl ester (450 mg, 70%) as an oil:

##STR00056##

[0338] The ester (270 mg, 0.85 mmol) was dissolved in ethanol (10 ml) and added to a solution of LiOH (267 mg, 6.4 mmol) in water (10 ml). The mixture was heated at 45 C. for 3 hours, cooled, added to water and 1M HCl until pH=2. The mixture was extracted with heptane (330 ml) and the extract was washed with brine, water and dried (MgSO.sub.4). Filtration and evaporation followed by purification by filtration through a short plug of silica gel (hexane:EtOAc 9:1) and concentration under reduced pressure afforded the acid (80 mg).

[0339] .sub.H (400 MHz) 0.95 (t, J=7.5, 3H), 1.02 (t, J=7.4, 31-1), 1.65-1.75 (m, 1H), 1.85-1.95 (m, 1H), 2.06 (quintett, J=7.3, 2H), 2.30-2.40 (m, 2H), 2.60-2.75 (m, 2H), 2.75-2.85 (m, 6H), 3.18 (t, J=7.5, 1H), 5.20-5.45 (m, 8H).

[0340] .sub.C (100 MHz) 11.9, 14.3, 20.6, 24.48, 25.53, 25.6, 25.7, 27.1, 31.3, 48.2, 127.0, 127.5, 127.8, 127.9, 128.4, 128.6, 129.7, 132.0, 178.7

Example 20Preparation of 2-methyl-2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)-propanoic Acid (BIZ 113)

[0341] ##STR00057##

[0342] An ice-cooled solution of (3Z,6Z,9Z,12Z)-pentadeca-(3,6,9,12)-tetraen-1-thiol (520 mg, 2.2 mmol) in dry dimethylformamide (DMF) (10 ml) was added to NaH (97 mg, 60% in mineral oil). The mixture was stirred at 0 C. for an additional 10 minutes before addition of 2-bromoisobutyrate (392 l, 2.6 mmol). The mixture was stirred at room temperature for 40 minutes, then the mixture was poured into a saturated NH.sub.4Cl solution and extracted with hexane. The extract was washed with a saturated NH.sub.4Cl solution, water and dried (MgSO.sub.4). Filtration, evaporation under reduced pressure and purification by flash chromatography (hexane:EtOAC 98:2) afforded the ester (270 mg) as an oil:

##STR00058##

[0343] The ester (270 mg) was dissolved in ethanol (10 ml) and added to a solution of LiOH (270 mg, 6.4 mmol) in water (10 ml). The mixture was heated at 65 C. for 4 hours, cooled, added to water and 1M HCl until pH=2. The mixture was extracted with hexane (330 ml) and the extract was washed with brine, water and dried (MgSO.sub.4). Filtration and evaporation followed by purification by filtration through a short plug of silica gel (hexane:EtOAc 9:1) and concentration under reduced pressure afforded the acid (200 mg).

[0344] .sub.H (400 MHz) 0.95 (t, J=7.5, 3H), 1.51 (s, 6H), 2.06 (quintet, J=7.3, 2H), 2.32 (quartet, J=7.0, 2H), 2.68 (t, J=7.3, 2H), 2.75-2.85 (m, 6H), 5.20-5-45 (m, 8H)

[0345] .sub.C (100 MHz) 14.3, 20.5, 25.4, 25.5, 25.6, 25.7, 26.9, 27.7, 46.6, 127.0, 127.6, 127.8, 127.8, 127.9, 128.4, 128.6, 129.6, 132.0, 180.3

Example 213-oxa-(6Z,9Z,12Z,15Z,18Z)-heneicosa-(6,9,12,15,18)-pentaenoic Acid (BIZ 108)

[0346] ##STR00059##

[0347] 3-oxa-(6Z,9Z,12Z,15Z,18Z)-heneicosa-(6,9,12,15,18)-pentaenoic acid was prepared as described in the literature (see Flock et al., Acta Chemica Scandinavica, 1999, 53, 436Compound 21b)

[0348] .sub.H (400 MHz) 0.95 (t, J=7.5, 3H), 2.05 (quintet, J=7.4, 2H), 2.40 (q, J=6.8, 2H), 2.7-2.9 (m, 8H), 3.56 (t, J=6.8, 2H), 4.14 (s, 2H), 5.20-5.60 (m, 10H)

[0349] .sub.C (100 MHz) 14.2, 20.5, 25.5, 25.6, 25.6, 25.7, 27.7, 67.7, 71.4, 125.2, 127.0, 127.8, 127.9, 128.0, 128.26, 128.31, 128.5, 130.4, 132.0, 175.0

Example 22Measuring the Effects of Compounds on the Activity of NF-B Pathway Using NF B Reporter Assay

[0350] Background:

[0351] Corticosteroids like dexamethasone have for many years been used for treatment of a broad spectrum of inflammatory conditions of the eye due to their potent anti-inflammatory effect. However, steroids may cause severe side effects like cataract and raised intraocular pressure after prolonged use, which limits their therapeutic use in chronic diseases. Corticosteroids exert their anti-inflammatory effects through influencing multiple signal transduction pathways. Inhibition of the NF-B pathway is central in their anti-inflammatory effect.

[0352] NF-B (Nuclear Factor-Kappa B, NF-KB) is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. The NF-B/Rel family of transcription factors (p50, p65, c-Rel, etc.) are involved in stress, immune, and inflammatory responses. In unstimulated cells, the NF-B dimers are sequestered in the cytoplasm by inhibitory IB proteins. Proinflammatory cytokines such as TNF-, LPS, growth factors, and antigen receptors activate I B kinase (IKK), which phosphorylates the IB proteins. Phosphorylation of IB leads to its degradation, freeing NF-B complexes to translocate to the nucleus, bind to NF-B DNA response elements, and induce the transcription of the target genes.

[0353] Description of Assay:

[0354] The NF-B reporter (luc)-HEK.sub.293 cell line is designed for monitoring the nuclear factor Kappa B (NF-B) signal transduction pathways. It contains a firefly luciferase gene driven by four copies of NF-B response element located upstream of the minimal TATA promoter. After activation by pro-inflammatory cytokines or stimulants of lymphokine receptors, endogenous NF-B transcription factors bind to the DNA response elements, inducing transcription of the luciferase reporter gene.

[0355] Cell Culture:

[0356] NF-B Reporter-HEK.sub.293 cells were cultured in MEM medium with 10% FBS, 1% non-essential amino acids, 1 mM Na-pyruvate, 1% Penn-strep, and 100 pg/ml of Hygromycin B.

[0357] Assay Conditions:

[0358] To perform the NF-B luciferase reporter assay, NF-B Reporter-HEK.sub.293 cells were seeded at 40,000 cells per well into white clear-bottom 96-well microplate in 45 l of growth medium without Hygromycin B. Cells were incubated at 37 C. and 5% CO.sub.2 overnight to allow them to recover and reattach. The following day a series of dilutions of compounds were made in assay medium (MEM medium with 0.5% FBS, 1% non-essential amino acids, 1 mM Na-pyruvate, 1% Penn-strep). The medium was removed from the wells and 45 l of diluted compound was added to the cells. Assay medium with DMSO was added to the untreated control wells and cell-free control wells. The final concentration of DMSO was 0.25%. The cells were incubated overnight at 37 C. in CO.sub.2 incubator. The next day 5 l of assay medium with TNFa was added to the wells. The final concentration of TNFa was 5 ng/ml. Cells were treated for 5 hours at 37 C. in a CO.sub.2 incubator.

[0359] After treatment, cells were then lysed and a luciferase assay was performed using the ONE-Step luciferase assay system. In brief, 50 l of One-Step Luciferase reagent was added per well and the plate rocked at room temperature for 30 minutes. Luminescence was measured using a luminometer (BioTek Synergy 2 microplate reader).

[0360] Data Analysis:

[0361] Reporter assays were performed in triplicate at each concentration. The luminescence intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the luminescence intensity (L.sub.t) in each data set was defined as 100%. In the absence of cells, the luminescence intensity (L.sub.b) in each data set was defined as 0%. The percent luminescence in the presence of each compound was calculated according to the following equation: % Luminescence=(LL.sub.b)/(LtL.sub.b), where L=the luminescence intensity in the presence of the compound, L.sub.b=the luminescence intensity in the absence of cells, and L.sub.t=the luminescence intensity in the absence of the compound. The values of % luminescence versus a series of compound concentrations were then plotted using non-linear regression analysis of a Sigmoidal dose-response curve generated with the equation Y=B+(T B)/1+10.sup.((Log EC50-X)Hill Slope), where Y=percent luminescence, B=minimum percent luminescence, T=maximum percent luminescence, X=logarithm of compound and Hill Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity.

[0362] Results:

[0363] The results from the NF-B assay clearly show that the tested compounds at 10 M concentration have a surprisingly potent inhibitory effect of the TNF- activated NF-B pathway as shown in FIG. 4. The compounds are more potent inhibitors of the NF-B pathway than dexamethasone.

[0364] The inhibitory effect of the tested compounds BIZ-110 and BIZ-101 on the Nf-B pathway is dose dependent as seen in FIG. 5. In fact the Nf-B pathway can be completely inhibited at high concentration using these two compounds which is not the case with dexamethasone.

Example 23Screening of PPAR Activity

[0365] To screen the human PPAR, PPAR and PPAR ligand activity of the compounds a stable reporter cell line was used (HeLa cell line). The stable reporter cell line express respectively a chimeric protein containing the ligand binding domain (LBD) of human PPAR, human PPAR and human PPAR fused to the yeast transactivator GAL4 DNA binding domain (DBD). The luciferase (Luc) reporter gene is driven by a pentamer of the GAL4 recognition sequence in front of a -globin promoter. The use of GAL4-PPAR, GAL4-PPAR and GAL4-PPAR chimeric receptors allows for elimination of background activity from endogenous receptors and quantification of relative activity across the three PPAR subtypes with the same reporter gene. The PPAR selectivity of the samples is determined by comparison to known drug references (GW7647) for PPAR, L-165041 for PPAR and BRL49653 for PPAR and a negative control (0.1% DMSO). Luciferase activity was measured by a luminometer and luciferase activity was expressed as relative light units (RLU).

[0366] Day 1: Seed 96well plate with PPAR cells. Cell appearance was checked using optical microscopy. Cells were cultivated at confluency (80-100%).

[0367] Day 2: The test articles dissolved in DMSO were added to the cells. The controls (positive controls and the solvent control) were included in each individual plate. The cells were incubated for an additional 24 hrs after addition of test articles before analysis.

[0368] Day 3: To determine the PPAR subtype activity of the tested compounds, the percentage of PPAR ligand activity was calculated for each tested compound as follows:

[0369] Percentage of PPAR activity for the tested compound in 5 M concentration=(RLUX.sub.comp100)/RLU.sub.GW7647 where RLUGW7647 is the luminescence measured from PPAR cells incubated with 1 M GW7647 and expressed as Relative Light Units. The activity of 1 M GW7647 is set to 100%

[0370] Percentage of PPAR activity for the tested compound in 5 M concentration=(RLUX.sub.comp100)/RLU.sub.L165041 where RLU.sub.L165041 is the luminescence measured from PPAR cells incubated with 1 M L and expressed as Relative Light Units. The activity of 1 M L165041 is set to 100%

[0371] Percentage of PPAR activity for the tested compound in 5 M concentration=(RLUX.sub.comp100)/RLU.sub.BRL49653 where RLU.sub.BRL49653 is the luminescence measured from PPAR cells incubated with 1 M L and expressed as Relative Light Units. The activity of 1 M BRL49653 is set to 100%

[0372] Results:

[0373] The compounds tested in this assay were: BIZ-101, BIZ-102, BIZ-108, BIZ-109, BIZ-111, BIZ-112 and BIZ-113. The results showed that the tested compounds had no activity on PPAR (results are not included). However, all compounds except BIZ-108 had potent activity on PPAR (FIG. 6) with response at the same level as GW7647. The results also show that most of the compounds tested activated PPAR except for the unsubstituted derivative BIZ-108 (FIG. 7). The PPAR activation is more potent in this stable reporter HeLA cell line than in the transient transfected HEK.sub.293 cell line described in Examples 6 and 7. Several differences in the assays can help to explain these results. BIZ-108 is unsubstituted in the a-position and was only added for comparison purposes. The results clearly show that a potent PPAR and PPAR activation requires one or two substituents in the a-position of fatty acid derivatives.

Example 24Assay for Measuring Effects of Compound BIZ-101 During Conditions of Oxidative Stress in a Retinal Pigment Epithelial Cell Line (ARPE-19)

[0374] In order to evaluate the potential of BIZ-101 to protective the eye from oxidative stress damage the compound was tested in a cell based assay. Oxidative stress was induced in an ARPE-19 cell line using tert-butyl hydroperoxide at different concentrations. The cellular viability was kinetically monitored and measured by analysing the plasma membrane integrity based on the incorporation of a non-permeant and fluorescent DNA intercalating agent that selectively stain cytolytic cells with comprised plasma membranes.

[0375] Procedure:

[0376] The ARPE-19 cell line was seeded and cultured in DMEM-F12 medium+10% SVF for 24 hrs in a 96 well plate. Cells were pre-treated or not with either fenofibric acid (25 M) or compound BIZ-101 (10 M) during this 24 hr period. After 24 hrs, the different concentrations of t-butyl hydroperoxide were added in the presence of a fluorescent DNA intercalating agent for the following monitoring. Live content time-lapse imaging was performed with a sampling rate of 1 image every 2 hr over a 96 hrs period. The number of fluorescent/cytolytic cells were counted and reported during the experiment. The treatment conditions were tested in one experiment session in a triplicate format.

[0377] Handling and Solubilisation of Compounds:

[0378] Fenofibric acid was used at 25 M and was ordered from Sigma Aldrich. The day of the pre-treatment, a 25 mM stock solution of fenofibric acid was prepared in DMSO and diluted in the complete culture medium at 25 M. 100 L of this solution or 100 L of complete culture medium+0.1% DMSO replaced the 100 L of the complete culture medium already present in the well. The day of the treatment with t-butyl hydroperoxide, a 2 times concentrated solution of fenofibric acid was freshly diluted in the complete culture medium and 50 L of this solution or 50 L of complete culture medium+0.2% DMSO were added to the 100 L of the culture medium already present in the well.

[0379] Compound BIZ-101 was tested at a final concentration of 10 M. A 10 mM predilution was prepared in DMSO from a 10 mM stock solution. The 10 mM solution of BIZ 101 was diluted in the complete culture medium at 10 M. 100 L, of this solution or 100 L of the complete culture medium+0.1% DMSO replaced the 100 L, of the complete culture medium already present in the well. The day of the treatment with t-butyl hydroperoxide, a 2 times concentrated solution of BIZ 101 was freshly diluted in the complete culture medium and 50 L of this solution or 50 L of complete culture medium+0.2% DMSO were added to the 100 L of the culture medium already present in the well.

[0380] Preparation of the tert-butyl hydroperoxide [tBHP] solution: 0, 0.01 mM, 0.03 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM and 10 mM. A 4 times concentrated solution of tBHP was freshly diluted in the complete culture medium for each of the concentrations to be tested. 50 L of the 4 times concentrated solution was added to the 150 L of the cultured medium.

[0381] Results:

[0382] The addition of tBHP induced a rapid, severe and dose dependent cytotoxic effect on the ARPE-19 cells as shown in FIG. 8A. A dose-dependent effect could be observed and 24 hrs after addition of tBHP the complete cytolysis of ARPE-19 cells was induced even with the lowest concentration of tBHP (FIGS. 8A and 8D).

[0383] Pre-treatment with either fenofibric acid or BIZ-101 slightly decreased the dose dependent cytotoxicity of tBHP 6 hrs after addition of tBHP. The protective effects of fenofibric acid and BIZ-101 were more efficient 24 hrs after addition of t-BHP, and remained stable until the end of the monitoring time (FIG. 8B, 8C, 8D).

[0384] The study clearly shows that BIZ-101 has a protective effect on retinal pigment epithelial cells (ARPE-19 cells) during conditions of oxidative stress. The effect is much more potent than the effect seen for fenofibric acid which is known to have therapeutic effects in treatment of diabetic retinopathy.

Example 25Assay for Measuring Effects of Compound BIZ-102 During Conditions of Oxidative Stress in a Retinal Pigment Epithelial Cell Line (ARPE-19)

[0385] In order to evaluate the potential of BIZ-102 to protect the eye from oxidative stress damage the compound was tested in a cell based assay similar to Example 24 with the following changes. The oxidative stress in the ARPE-19 cell line was induced by using 3 different concentrations of tBHP: 10 M, 30 M and 100 M and BIZ-102 was tested in 2 different concentrations: 1 M and 10 M.

[0386] Procedure:

[0387] The ARPE-19 cell line was seeded and cultured in DMEM-F12 medium+10% SVF for 24 hrs in a 96 well plate. Cells were pre-treated or not with compound BIZ-102 (1 M and 10 M) during this 24 hr period. After 24 hrs, the different concentrations of t-butyl hydroperoxide were added in the presence of a fluorescent DNA intercalating agent for the following monitoring. Live content time-lapse imaging was performed with a sampling rate of 1 image every 2 hr over a 96 hrs period. The number of fluorescent/cytolytic cells were counted and reported during the experiment. The treatment conditions were tested in one experiment session in a triplicate format.

[0388] Compound BIZ-102 was tested at a final concentration of 1 and 10 M. A 10 mM pre-dilution was prepared in DMSO from a 20 mM stock solution. The day of the pre-treatment with BIZ-102 and the day of the treatment with t-BHP, a 1000 times concentrated solution of BIZ-102 for each concentration to be tested was prepared in DMSO. The day of the pre-treatment, a one time concentrated solution for each concentration to be tested was prepared by diluting the 1000 times concentrated solution in complete culture medium. 100 L of those solutions replaced the 100 L of the culture medium already present in the well. The day of the treatment with t-BHP, for each concentration to be tested, a 2 times concentrated solution was freshly diluted in the complete culture medium and 50 L of this solution was added on the 100 L of the complete culture medium already present in the well.

[0389] Results:

[0390] The results show that BIZ-102 at the highest concentration (10 M) exhibited a significant inhibition of the cytotoxicity induced by both 10 M and 30 M of t-BHP but not for the highest concentration of t-BHP (FIG. 9A, 9B, 9C). The protective effect remained stable until the end of the monitoring time. In FIG. 9D the effects of BIZ-102 12 hrs after addition of tBHP is shown. The study clearly shows that BIZ-102 has a protective effect on retinal pigment epithelial cells (ARPE-19 cells) during conditions of oxidative stress.