RU2820995C2 - Structurally enhanced oxygen-containing fatty acids for treating non-alcoholic steatohepatitis - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: present group of inventions relates to pharmacology, specifically to the use of a compound of formula (II) and a composition thereof for preparing a drug and for the treatment of non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH), as well as to a method of slowing down hepatic fibrosis. Use of an unsaturated fatty acid with oxygen included in the β-position, and additionally containing α-substituent with the structure of a compound of formula (II), where R₁ is selected from C₁₀-C₂₂ alkenyl having 3–6 double bonds; R₂ and R₃ are identical or different and are selected from a group consisting of a hydrogen atom and an alkyl group; X is a carboxylic acid or derivative thereof, wherein the derivative is a carboxylate such as a carboxylic ester; glyceride; or carboxamide; or a pharmaceutically acceptable salt, is carried out in the therapeutic and/or preventive treatment of NASH or ASH. Application of the compound of formula (II) slows down or prevents development of hepatic fibrosis or reduces existing hepatic fibrosis.

EFFECT: providing a method of treating NASH or ASH to a subject in need thereof.

88 cl, 26 dwg, 13 tbl, 105 ex

Description

This application claims priority to Norwegian Patent Application No. 20171944, filed December 6, 2017; Norwegian Patent Application No. 20171945, filed December 6, 2017; and US Provisional Patent Application No. 62/734,013, filed October 9, 2018. All of the foregoing applications are incorporated herein by reference in their entirety.

Field of invention

The present disclosure relates to a method of treating non-alcoholic steatohepatitis-NASH (NASH), alcoholic steatohepatitis-ASH (ASH) and other liver diseases characterized by liver fibrosis and/or inflammation in a subject in need of treatment. In addition, the present disclosure relates to a compound and a composition containing the compound for use in the treatment of non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) and other liver diseases characterized by fibrosis and/or inflammation of the liver in a subject in need of treatment. The compound for use according to the invention is an unsaturated fatty acid with oxygen included in the β-position and additionally containing an α-substituent.

Prerequisites for creating the invention

Long chain omega-3 fatty acids, such as (5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenoic acid (EPA) and (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-docose-4,7, 10,13,16,19-hexaenoic acid (DHA) have broad-spectrum biological effects, affecting plasma lipids, cardiovascular and immune functions, insulin action, neuronal development and visual function . High doses of EPA/DHA are currently prescribed for the treatment of severe hypertriglyceridemia (HTG). These effects are mediated, at least in part, by their effects on hepatic fatty acid metabolism.

The use of omega-3 compounds such as EPA and DHA for the treatment of non-alcoholic steatohepatitis (NASH) has been proposed in the prior art. For example, WO 2014/057522 (Mochida) describes compositions containing ethyl cosapentate for use in the treatment or relief of symptoms of NASH.

Dignity Science LTD (WO2014/118097) proposes the use of modified omega-3 compounds such as 15-hydroxy-eicosapentaenoic acid (15-OHEPA) to treat fatty liver diseases such as non-alcoholic fatty liver disease (NAFLD) and NASH. Krisani Biosciences (WO2014/045293) also proposes the use of modified omega-3 compounds to treat various diseases, including NASH. More recently, Pronova Biopharma AS (WO2016173923 A1) proposed the use of sulfur-containing structurally modified fatty acids for the treatment of NASH.

The terms “fatty liver disease (NAFLD)” and “non-alcoholic steatohepatitis (NASH)” are often used interchangeably, although NAFLD covers a much broader spectrum of liver disease, including isolated hepatosteatosis (>5% of hepatocytes histologically). Hepatosteatosis is most likely a relatively benign disease unless it is accompanied by an inflammatory response and cellular damage. However, a subset of patients with NAFLD exhibit liver cell damage and inflammation in addition to hepatosteosis, i.e. There is a condition known as non-alcoholic steatohepatitis (NASH). NASH is histologically virtually indistinguishable from alcoholic steatohepatitis (ASH). While simple steatosis observed in NAFLD does not correlate with increased short-term morbidity or mortality, NASH dramatically increases the risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Liver cirrhosis due to NASH is an increasingly common reason for liver transplantation. While morbidity and mortality from liver disease are significantly increased in patients with NASH, liver disease is even more strongly correlated with morbidity and mortality from cardiovascular disease.

Uniform criteria for diagnosing and diagnosing NASH are still being debated (see later sections for details). The key histologic components of NASH are steatosis, hepatocellular ballooning, and lobular inflammation; fibrosis is not part of the histological diagnosis of NASH. However, the degree (stage) of fibrosis determined by liver biopsy is determinant of prognosis, whereas the degree of inflammation and necrosis (degree of damage) determined by liver biopsy is not.

Regarding the various histological components, treatment with omega-3 fatty acids has been shown to effectively reduce hepatosteatosis in patients with NAFLD (Scorletti E, et al., Effects of purified eicosapentaenoic and docosahexanoic acids in non-alcoholic fatty liver disease: Results from the *WELCOME stud, Hepatology, 2014 Oct;60(4):1211-21), and if treatment is started at an early stage of the disease, it can conceivably slow down the progression without leading to the final, more severe stages of the disease. However, it is unclear whether omega-3 fatty acids will be effective enough to treat and/or reverse NASH if significant histological/inflammatory changes have already developed (Sanyal AJ, et al; EPE-A Study Group, Gastroenterology. 2014 Aug; 147 (2) : 377-84.e1).

The moderate effectiveness of omega-3 fatty acids in treating NASH may be secondary to their mild effects on other cascades underlying the pathogenesis of NASH. Studies of NASH in both humans and animals convincingly demonstrate that multiple factors, not just isolated hepatosteatosis, are involved in the development of steatohepatitis and fibrosis. These factors include insulin resistance, oxidative stress, inflammation, intestinal endotoxins, and elevated levels of cholesterol and bile acids in the liver. All of these factors have been shown to play important contributing roles in genetically susceptible individuals, and accordingly, drugs targeting these pathways (response cascades) are being developed to treat NASH.

As with NASH, alcoholic liver disease (ALD) can be divided into histological stages representing a progression from fatty liver disease or simple steatosis to alcoholic hepatitis (i.e., ASH) and finally to chronic hepatitis with fibrosis or cirrhosis liver. Thus, although the origins of ASH and NASH may differ, the liver's response to the corresponding chronic insult has many similarities, including pro-inflammatory and pro-fibrotic cascades involving macrophage activation and cytokine production, and the resulting activated stellate cells, i.e. proliferating myofibroblasts. (See, for example, Friedman, SL; Alcoholism: Clinical and Experimental Research. 1999 May; 23(5):904-910.)

A challenge in developing drugs targeting NASH and ASH is the lack of preclinical models that replicate these diseases in a human-like manner. Rodent models that are more consistent with the pattern of metabolic abnormalities that typically accompany NASH, such as dyslipidemia and insulin resistance, are characterized by very mild liver inflammation and fibrosis. At the other end of the spectrum, models of chemically induced fibrosis, such as those induced by carbon tetrachloride (CCl ₄ ) or thioacetamide, develop more severe fibrosis, but may lack any correspondence with human patients regarding both metabolic components, such as insulin resistance and obesity. Thus, to identify potential drugs for the treatment of NASH and ASH, multiple preclinical models covering both ends of the etiopathogenic spectrum (metabolic disorders - inflammatory response - fibrosis) are needed.

Another element that must be considered when comparing liver fibrosis in rodents and humans is the location and functional significance of the induced fibrosis. For example, shorter duration models of severe fibrosis may only dismantle collagen-dense large portal tracts rather than parenchymal collagen deposits, which are functionally more relevant and often represent the majority of liver collagen. Thus, in the assessment of fibrosis in rodent models, the use of a biochemical component (eg, hydroxyproline assessment) in combination with a histological component (eg, Sirius Red morphometry) is optimal in terms of determining the amount, location and functional significance of collagen deposits in the liver.

Discovery of drugs that target the fibrotic component of NASH and ASH is critical as liver fibrosis can progress to cirrhosis, which in turn is associated with very high disease morbidity and mortality. Fibrosis also represents the primary hard endpoint (primary outcome) in clinical trials of chronic liver diseases. For example, emerging evidence suggests that fibrosis, rather than NASH per se, is the most important histological predictor of death in both liver and non-liver diseases. Additionally, cirrhosis is a major cofactor of primary liver cancer. Thus, it is critical that new drugs developed to treat NASH and ASH are effective enough to prevent the development and/or regression of established fibrosis.

WO2016173923A1, Pronova Biopharma Norge AS discloses that sulfur-containing structurally modified fatty acids such as 2-ethyl-2-((5Z,8Z,11Z,14Z,17Z)-eicose-5,8,11,14,17-pentaenylthio) butanoic acid (compound N), may be useful in the treatment of NASH. This is based on the finding that Compound N has greater effects than the PPAR-gamma agonist rosiglitazone in the prevention of diet-induced liver fibrosis. This also indicates that Compound N prevents the influx of inflammatory cells into the liver. Compound N was demonstrated to be effective in reducing fibrosis (as measured by the hydroxyproline/proline ratio) in APOE*3Leiden.CETP mice. It is important to note that APOE*3Leiden.CETP mice develop only a very mild hepatic fibrotic response (20-30% increase in extracellular matrix), as demonstrated by a biochemical assay measuring hydroxyproline (HYP). Additionally, in the specific model described in WO2016173923A1 (the contents of which are incorporated herein by reference), the extent (size) of liver fibrosis measured by Sirius Red (SR) morphometry was much lower than found in 5 previous studies using similar experiments . These studies found approximately 4-5% fibrosis at 20 weeks and 7-8% at 25-30 weeks as measured by SR morphometry, compared to 1.5% in the study reported in WO2016173923A1. For measuring fibrosis, SR morphometry is a more sensitive method than biochemical analysis (HYP), which may underestimate the more functionally relevant collagen, which is predominantly parenchymal, as opposed to the functionally less relevant but quantitatively predominant portal collagen.

Thus, based on the need for potent drugs for the treatment of NASH and ASH that can provide preventive treatment or regression of liver fibrosis, several new studies have been conducted to identify compounds that can be used to treat these aspects of liver disease, as well as to confirm the effect on based on new calculations/estimates and improved models demonstrating higher levels of fibrosis.

Based on the need for potent drugs for the treatment of NASH and ASH, for example, for the preventive treatment or regression of liver fibrosis, several new studies have been conducted to identify compounds that can be used in the treatment of these aspects of liver disease and to confirm the effect based on new calculations /estimates and improved models.

Brief description of the invention

The present disclosure provides a method of treating non-alcoholic steatohepatitis and/or alcoholic steatohepatitis in a subject in need of treatment, comprising administering to the subject a pharmaceutically effective amount of a compound of Formula (II):

where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds;

R ₂ and R ₃ are the same or different and are selected from the group of substituents consisting of a hydrogen atom, a hydroxy group, an alkyl group, a halogen atom, an alkoxy group, an acyloxy group, an acyl group, an alkenyl group, an alkynyl group, an aryl group, an alkylthio group, an alkoxycarbonyl group, a carboxy group , alkylsulfinyl group, alkylsulfonyl group, amino group and alkylamino group, with the proviso that R ₂ and R ₃ can be linked to form a cycloalkane such as cyclopropane, cyclobutane, cyclopentane or cyclohexane;

X represents: a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; anhydride; carboxamide; phospholipid; or hydroxymethyl; or a prodrug thereof;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt. The disclosure of the invention provides that the compound may be administered as monotherapy or in combination with one or more additional active agents.

An equivalent aspect of the disclosure relates to a compound of Formula (II)

where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds;

R ₂ and R ₃ are the same or different and are selected from the group of substituents consisting of a hydrogen atom, a hydroxy group, an alkyl group, a halogen atom, an alkoxy group, an acyloxy group, an acyl group, an alkenyl group, an alkynyl group, an aryl group, an alkylthio group, an alkoxycarbonyl group, a carboxy group , alkylsulfinyl group, alkylsulfonyl group, amino group and alkylamino group, with the proviso that R ₂ and R ₃ can be linked to form a cycloalkane such as cyclopropane, cyclobutane, cyclopentane or cyclohexane;

X represents: a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; anhydride; carboxamide; phospholipid; or hydroxymethyl; or a prodrug thereof;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt for use in the therapeutic and/or prophylactic treatment of non-alcoholic steatohepatitis and/or alcoholic steatohepatitis. The disclosure also provides that the compound for use may be administered either as monotherapy or in combination with one or more additional active agents.

In at least one embodiment, R ₂ and R ₃ are the same or different and can be selected from the group of substituents consisting of a hydrogen atom, an alkyl group, an alkoxy group, an alkenyl group; or R ₂ and R ₃ may be coupled to form a cycloalkane such as cyclopropane, cyclobutane, cyclopentane or cyclohexane;

X represents a carboxylic acid or a derivative thereof, where the derivative is a carboxylic acid ester, a glyceride or a phospholipid;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such a salt.

More specifically, the present disclosure relates to a method of treating non-alcoholic steatohepatitis and/or alcoholic steatohepatitis in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound of Formula (I):

where R ₂ and R ₃ and X are defined in the same way as for Formula II. In particular,

R ₂ and R ₃ are independently selected from hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups;

X represents a carboxylic acid or a derivative thereof, where the derivative is a carboxylic acid ester, a glyceride or a phospholipid;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such a salt.

Likewise, the present disclosure relates to a compound of Formula (I):

where R ₂ and R ₃ and X are defined in the same way as for Formula II. In particular,

R ₂ and R ₃ are independently selected from hydrogen atom or linear, branched and/or cyclic C1-C6 alkyl groups;

X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylic acid ester, glyceride or phospholipid;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt,

for use in the treatment of non-alcoholic steatohepatitis.

The present invention also provides a method of treating non-alcoholic steatohepatitis and/or alcoholic steatohepatitis in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8 ,11,14,17-pentaen-1-yl) hydroxy) butanoic acid (compound A):

or a pharmaceutically acceptable salt or ester thereof.

The present invention also provides 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (Compound A) or a pharmaceutically acceptable salt or an ester thereof for use in the treatment of non-alcoholic steatohepatitis and/or alcoholic steatohepatitis.

Brief description of charts

FIGS. 1A-1C illustrate the effects of Compound A on collagen fiber number ( FIG. 1A ), thickness ( FIG. 1B ) and length ( FIG. 1C ) in a mouse model of CDAA on a high-fat diet.

Fig. 2A-2D illustrate the effect of Compound A on hepatic lipid content for triglycerides (TG, Figure 2A ), diglycerides (DG, Figure 2B ), free fatty acids (FFA, Figure 2C ), and cholesteryl ester ( Figure 2D ) in CDAA mouse model on a high-fat diet.

Fig. 3A-3D show that Compound A had no significant effect on ob/ob mice fed the AMLN diet in terms of body weight ( Figure 3A ) or liver weight ( Figure 3B ) after 4 weeks of treatment, nor on body weight (Figure 3A). Fig. 3C ) or liver weight ( Fig. 3D ) after 8 weeks of treatment.

FIGS. 4A-4F show the effect of 4 weeks of Compound A treatment on the expression of canonical genes regulating liver fibrogenesis in ob/ob mice fed an AMLN diet.

FIGS. 5A-5D show the effect of 4 weeks of Compound A treatment on the expression of genes regulating extracellular matrix (ECM) stability or fibrolysis in ob/ob mice fed the AMLN diet.

In FIG. 6A-6F show that 8 weeks of Compound A treatment reduces hepatic α-SMA ( Figures 6A-6B ), col1a1 ( Figures 6C-6D ) and collagen ( Figures 6E-6F ) in ob/ob AMLN- mice. diet.

Figures 7A-7B show that after 8 weeks of treatment with Compound A in ob/ob mice on the AMLN diet, the number of activated liver stellate cells (myofibroblasts) decreases and fibrosis begins to regress, as demonstrated by a decrease in hepatic α-SMA ( Figure 7A ) and col1a1 ( Fig. 7B ), respectively, compared with pre-treatment levels.

Fig. 8A-8F show that 4 weeks of treatment with 112 mg/kg Compound A results in decreased expression of hepatic inflammation-related genes in ob/ob mice on the AMLN diet.

Fig. 9A-9B show the effect of 8 weeks of Compound A treatment on liver galectin-3 (Gal-3) levels in ob/ob mice on an AMLN diet.

FIGS. 10A-10B demonstrate the effect of 8 weeks of Compound A treatment on hepatocellular damage in ob/ob mice on an AMLN diet as measured by levels of the liver enzymes aminotransferase (ALT) ( FIG. 10A ) and aspartate transaminase (AST) ( FIG. 10B ) .

Fig. 11A-11E show the effect of 8 weeks of treatment with Compound A on hepatic steatosis, measured as a percentage of total area ( FIG. 11A ), based on total liver lipid content ( FIG. 11B ), cholesterol content ( FIG. 11C ), and triglyceride levels ( Figure 11D ) and plasma cholesterol ( Figure 11E ) in ob/ob mice on the AMLN diet.

Fig. 12A-12C depict the effect of 3 weeks of Compound A treatment on glycemic control in ob/ob mice on an AMLN diet.

Fig. 13A-13B show the effect of Compound A on human liver stellate cells (LX-2) in vitro .

Detailed description

It should be noted that embodiments and features described in the context of one aspect of the present disclosure are also applicable to other aspects of the invention. In particular, the embodiments applicable to the method of treating non-alcoholic steatohepatitis or alcoholic steatohepatitis in accordance with the present invention are also applicable to the aspect relating to a compound or composition containing a compound for use in treating non-alcoholic steatohepatitis or alcoholic steatohepatitis according to the present disclosure. In some embodiments, the compound or composition containing the compound is administered in combination with one or more additional active agents.

Specific aspects of the disclosure are described in more detail below. The terms and definitions used in this application and explained herein are given to define the concepts of this disclosure.

The singular forms "a", "an" and "the" also imply the use of the plural unless the context dictates otherwise.

The terms "about", "approximately" and "about" mean that the value is almost the same as the specified number or value. As used herein, the terms "about", "about" and "about" should generally be understood to cover a range of ±5% of a specified amount, frequency or value.

The terms “treat,” “treatment,” and “treatment” include any therapeutic or prophylactic application that may benefit a mammal, whether human or non-human. Both medical (human) and veterinary treatment are included within the scope of the present invention. Treatment can be given in response to an existing condition or prophylactically, that is, to prevent the occurrence of a disease.

The terms “administrate” and “administration” as used herein refer to (1) the provision, administration, dosage and/or administration by a physician or licensed medical personnel under the authority of a physician of a compound or composition in accordance with this disclosure and (2 ) administration, administration or consumption by a human or non-human mammal of a compound or composition in accordance with this disclosure.

The terms "prevention and/or treatment" and "therapeutic and/or prophylactic treatment" may be used interchangeably. Typically, compounds of Formula (I) or Formula (II) are used for the treatment, ie therapeutic cure, of NASH or ASH. However, it is also contemplated that in some cases the compounds of Formula (I) or Formula (II) will be used for the prophylactic treatment of NASH or ASH, for example, in cases where the patient has one or more risk factors associated with NASH or ASH.

The terms "administered in combination" and "co-administered" are used interchangeably and refer to the administration of (a) a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt; and (b) at least one additional active agent, in a jointly coordinated manner. For example, co-administration may be simultaneous administration, sequential administration, combined administration, interval administration, continuous administration, or a combination thereof. The route of administration may be different for the compounds and the additional agent(s), and co-administration means any route of administration such as oral, subcutaneous, sublingual, transmucosal, parenteral, intravenous, intra-arterial, intraperitoneal, buccal, sublingual, topical, vaginal, rectal, ophthalmic, otic, nasal, inhalant and transdermal, or a combination thereof. Examples of parenteral administration include, but are not limited to: intravenous (IV) administration, intraarterial administration, intramuscular administration, subcutaneous administration, intraosseous administration, intrathecal administration, or a combination thereof. The compound of Formula (I) or (II) and the additional active agent can be administered independently, for example, orally or parenterally. In one embodiment, a compound of Formula (I) or (II) is administered orally and the additional active agent is administered parenterally. Parenteral administration may be by injection or infusion. In some embodiments, the method and/or use of the present disclosure is directed to the therapeutic and/or prophylactic treatment of NASH or ASH using at least two different active agents: a compound of Formula (I) or (II) and an additional active agent, respectively. The at least two active agents may be considered a "combination product", where the agents are, for example, separately packaged, and where both agents are necessary to achieve the optimal intended effect.

The term "pharmaceutically effective amount" means an amount sufficient to achieve the desired pharmacological and/or therapeutic effects, that is, that amount of the disclosed compound that is effective to achieve the intended effect. Although the needs of individual subjects/patients may vary, determination of optimal ranges of effective amounts of the disclosed compound is within the skill of one skilled in the art. In general, a dosage regimen for treating a disease and/or condition with the presently disclosed compounds may be determined according to various parameters such as the type, age, weight, sex, diet and/or health status of the subject/patient.

The term "pharmaceutical composition" means a compound according to the present disclosure in any form suitable for medical use.

The compounds of Formula (I) and (II) may exist in various stereoisomeric forms, including enantiomers, diastereomers or mixtures thereof. It should be understood that the invention covers all optical isomers of the compounds of Formulas (I) and (II), as well as mixtures thereof. Therefore, the compounds of Formula (I) and (II) existing as diastereomers, racemates and/or enantiomers are included within the scope of the present invention.

In one aspect, a compound for use according to the invention is a compound of Formula (II)

where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds;

R ₂ and R ₃ are the same or different and can be selected from the group of substituents consisting of a hydrogen atom, a hydroxy group, an alkyl group, a halogen atom, an alkoxy group, an acyloxy group, an acyl group, an alkenyl group, an alkynyl group, an aryl group, an alkylthio group, an alkoxycarbonyl group , carboxy group, alkylsulfinyl group, alkylsulfonyl group, amino group and alkylamino group, and where R ₂ and R ₃ can be linked to form a cycloalkane such as cyclopropane, cyclobutane, cyclopentane or cyclohexane;

X represents: a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; anhydride; carboxamide; phospholipid; or hydroxymethyl; or a prodrug thereof;

or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt.

In one embodiment, R ₁ is a C ₁₈ -C ₂₂ alkenyl having 3-6 double bonds, such as 5 or 6 double bonds, where one double bond is preferably in the omega-3 position.

R ₂ and R ₃ are more preferably independently selected from a hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups. In one embodiment, at least one of R ₂ and R ₃ represents a hydrogen atom, a methyl group, an ethyl group, an n-propyl group or an isopropyl group, a butyl group or a pentyl group.

X is preferably a carboxylic acid or a carboxylic acid ester; or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt.

The compound of Formula (II) for use in treatment can be administered either as monotherapy or in combination with one or more additional active agents.

More specifically, the present disclosure relates to a method of treating non-alcoholic steatohepatitis or alcoholic steatohepatitis in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound of Formula (I):

where R ₂ , R ₃ and X are defined as for Formula (II).

Preferably, for compounds of Formula (I), R ₂ and R ₃ are independently selected from hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups; and X is a carboxylic acid or a carboxylic acid ester; or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt.

The compound of Formula (I) can be administered as monotherapy or in combination with one or more additional active agents.

In some embodiments, the present disclosure relates to a compound of Formula (I):

where R ₂ , R ₃ and X are defined as for Formula (II),

for use in the treatment of non-alcoholic steatohepatitis or alcoholic steatohepatitis.

Preferably, for compounds of Formula (I), R ₂ and R ₃ are independently selected from hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups;

X represents a carboxylic acid or a carboxylic acid ester; or a pharmaceutically acceptable salt thereof, solvate or solvate of such salt.

In cases where R ₂ and R ₃ are different, the compounds of Formula (I) and Formula (II) may exist in stereoisomeric forms. It should be understood that the invention covers all optical isomers of the compounds of Formula (I) and Formula (II) and mixtures thereof.

In at least one embodiment, R ₂ and R ₃ are independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a butyl group, and a pentyl group.

In at least one embodiment, R ₂ and R ₃ are independently selected from the group consisting of a hydrogen atom, a methyl group, and an ethyl group.

In at least one embodiment, one of R ₂ and R ₃ is a hydrogen atom and the other of R ₂ and R ₃ is selected from a C ₁ -C ₃ alkyl group. In one embodiment, one of R ₂ and R ₃ represents a hydrogen atom, and the other of R ₂ and R ₃ is selected from the list including a methyl group and an ethyl group, and most preferably, one of R ₂ and R ₃ represents a hydrogen, and the other is an ethyl group.

For compounds of both Formula I and Formula II, R ₂ and R ₃ in some embodiments independently represent C ₁ -C ₆ alkyl groups. In some embodiments, both R ₂ and R ₃ are C ₁ -C ₃ alkyl groups. In some embodiments, R ₂ and R ₃ are the same or different, and each is independently selected from a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. In some embodiments, R ₂ and R ₃ are the same and are selected from a pair of methyl groups, a pair of ethyl groups, a pair of n-propyl groups, or a pair of isopropyl groups. In at least one preferred embodiment, R ₂ and R ₃ are ethyl groups. In some embodiments, one of R ₂ and R ₃ is a methyl group and the other is an ethyl group. In some embodiments, one of R ₂ and R ₃ is an ethyl group and the other is an n-propyl group.

In at least one embodiment, the compound is present in its various stereoisomeric forms, such as an enantiomer (R or S), a diastereomer, or a mixture thereof. In at least one embodiment, the compound is present in racemic form.

In cases where the compound of Formula (I) is a counterion salt with at least one stereogenic center or an alcohol ester with at least one stereogenic center, the compound may have multiple stereocenters. In these situations, the compounds of the present invention may exist as diastereomers. Thus, in at least one embodiment, the compounds of the present invention are present as a compound where there is at least one diastereomer.

In at least one embodiment, the compound of the present invention is 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy ) butanoic acid (compound A):

In at least one embodiment, the compound of the present invention is 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoic acid (compound A), present in its S and/or R form, represented by the Formulas:

And .

In some embodiments, 2-((((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (compound A) is administered as monotherapy. In some embodiments, 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (compound A) is administered. combination with one or more additional active agents.

As previously described, multiple independent and interdependent metabolic, inflammatory, and ultimately fibrotic components act convergently in the development of human NASH. It is likely that any successful treatment will need to target multiple aspects of NASH, preferably through metabolic/inflammatory targets. However, since the development of fibrosis is associated with clinical manifestations, ideal therapy for NASH should target the inflammatory component, and ideally reduce or preventively treat both the development of fibrosis and promote regression of existing fibrosis. The accompanying Examples demonstrate the surprising and unexpectedly significant anti-inflammatory and anti-fibrotic effects of oxygenated structurally modified fatty acids such as Compound A. These results, which have been shown in several preclinical models of NASH, support the use of the oxygenated compounds of this disclosure in the therapeutic and prophylactic treatment of NASH and ASH in of people.

Surprisingly, Compound A was found to be remarkably active in slowing fibrosis, preventing the development of fibrosis, and reversing liver fibrosis, for example, as confirmed by a biochemical assay assessing hydroxyproline content in a mouse model of CDAA diet-induced fibrosis (methionine-choline-deficient diet) ( observed a 2-400% increase in extracellular matrix (ECM) compared to a 20-30% increase in ECM in the more benign APOE*3Leiden.CETP mouse model).

A new finding in the CDAA-induced NASH model was that Compound A also reduced liver fibrosis measured histologically using Sirius Red morphometry (SR morphometry). Unlike biochemical hydroxyproline (HYP) assessment, which measures collagen in large vessels, SR morphometry quantifies more functionally relevant sinusoidal collagen deposits.

Given the key importance of fibrosis in the morbidity and mortality associated with NASH, the results obtained in the CDAA-induced NASH model support the conclusion that oxygenated structurally modified fatty acids, such as compound A, are effective in treating complications associated with with NASH. This finding is also supported by changes in the expression of a liver gene associated with fibrosis (Col1a1) and a gene associated with inflammatory responses (TNF-a gene expression in the liver). The significant reduction in more functionally relevant sinusoidal collagen measured using SR morphometry also supports testing and use of Compound A in the treatment of human NASH.

Despite the critical importance of fibrosis in clinical presentation, regulatory approval of new effective drugs for the treatment of NASH is based on treating NASH without worsening fibrosis. It is also important that improvements in NASH assessment parameters be achieved with new compounds. Thus, improvements in steatosis, lobular inflammation, and hepatocellular ballooning parameters should ideally occur in addition to the desired improvement in fibrosis.

A new finding in another NASH model, the STAM mouse model, was that, in addition to improvements in steatosis and inflammation, Compound A improved hepatocellular ballooning. Hepatocellular ballooning is typically characterized as cell enlargement of 1.5 to 2 times the normal hepatocyte diameter due to rarefied cytoplasm and has been shown to correlate with fibrosis and be associated with liver injury.

Cellular bloat (defined as hepatocellular hypertrophy) was also prevented by Compound A in APOE*3L.CETP double transgenic mice. The definition of hypertrophy versus ballooning refers to the appearance of certain histological differences characteristic of rodent and human hepatocytes.

Combined with the reported anti-inflammatory effects, these new results associated with hepatocellular ballooning/hypertrophy highlight the potential utility of Compound A for improving all NASH assessment parameters, which in turn should contribute to a positive outcome in clinical development and subsequent regulatory approval. organ.

Compound A was surprisingly effective in modulating both inflammatory and fibrotic components in multiple rodent models of NASH of varying severity, as described above and as shown in the Examples.

The complex interplay of adaptive and maladaptive immune cell recruitment, activation, differentiation, and proliferation in NASH indicates that measurement (monitoring) of any one inflammatory parameter requires a concomitant measurement of fibrosis to interpret the functional significance of such measurements. Thus, the antifibrotic effects of Compound A in the CDAA model may enhance the clinical significance of the anti-inflammatory effects observed with oxygenated structurally modified fatty acids.

Since regulatory approval of new effective drugs for the treatment of NASH depends on improvement of NASH without worsening fibrosis, the improvements in hepatocellular ballooning/hypertrophy observed with Compound A treatment in two different rodent models of NASH are also an important and novel finding (ballooning/hypertrophy was not measured in the model mouse CDAA).

Combined with the reported anti-inflammatory effects, these new results associated with hepatocellular ballooning/hypertrophy highlight the potential utility of Compound A for improving all parameters of NASH assessment, which in turn should contribute to a positive outcome in clinical development and subsequent regulatory approval organs.

It is important to note that the ability of compounds such as Compound A to improve all parameters of NASH and fibrosis associated with both portal tracts and parenchyma in numerous preclinical models of NASH with different etiopathogenesis provides strong support for testing in human patients with NASH.

In addition, since a significant proportion of patients with NASH also have type 2 diabetes, it is important to study the effect of delayed initiation of treatment with a drug such as Compound A not only on hepatic steatosis, inflammation and fibrosis, but also its effect on glycemic control in a model of NASH-induced obesity. The above-described anti-inflammatory, anti-fibrotic and steatosis-lowering effects of Compound A were further demonstrated in an obesogenic diet-induced NASH model ( ob/ob mice on a high-fat AMLN diet). Compound A also had a positive effect on glycemic control in this model without affecting body weight, unlike treatment with thiazolidinediones (eg, pioglitazone), which have a negative effect on body weight. In addition, Compound A decreased plasma levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), indicating that hepatocellular injury and/or damage is reduced.

Based on these results, Compounds of Formula (II) or, preferably, Formula (I) can be administered for the treatment and/or regression of non-alcoholic steatohepatitis (NASH) or other liver diseases characterized by fibrosis, inflammation and/or hepatocellular ballooning. In some embodiments, treatment for NASH may be prophylactic. In addition, the compounds may be administered to treat at least one disease, condition or risk factor associated with NASH. In some embodiments, treatment of at least one disease, condition, or risk factor associated with NASH may be prophylactic.

Due to the similarities in the proinflammatory and profibrotic mechanisms of NASH and alcoholic steatohepatitis (ASH), the anti-inflammatory and antifibrotic effects of the compounds described here, described in NASH models and in vitro experiments, are also observed in the treatment and/or regression of ASH, in particular in preventing progression and induction regression of progressive ASH and associated fibrosis. For example, the inhibitory effect of Compound A on the proliferation of isolated LX-2 cells (human liver stellate cells) in vitro is independent of paracrine signaling from parenchymal cells and/or Kupffer cells, indicating that the antifibrotic effects of Compound A can be achieved independently of whether the preceding signals come from NASH- or ASH-associated liver lesions.

Thus, compounds of Formula (II) or preferably Formula (I) can be used for the treatment and/or reversal of ASH. In some embodiments, treatment of ASH may be prophylactic. In addition, the compounds may be administered to treat at least one disease, condition, or risk factor associated with ASH. In some embodiments, treatment of at least one disease, condition, or risk factor associated with ASH may be prophylactic.

Accordingly, the present disclosure covers a method of arresting the development or prophylactic treatment of those at risk of developing liver fibrosis and reducing pre-existing liver fibrosis. According to this method, fibrosis is cured by reducing the fibrotic area, fibrotic content/parameters or the severity of fibrosis. In at least one embodiment, the method provides a reduction, sometimes a significant reduction, in the percentage surface area of liver fibrosis. In at least one embodiment, the method provides a reduction in the total NASH score, for example, a significant reduction in the NASH score. In addition, the method includes reducing liver inflammation, such as lobular inflammation; reduction of hepatocellular ballooning; reduction of steatohepatitis; all in addition to improving fibrosis.

In some embodiments, a compound of the present invention reduces the fibrotic area of the liver by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as measured by Sirius Red morphometry . In some embodiments, a compound of the present invention reduces the fibrotic area of the liver by 20-30%, 30-40%, 10-40%, 40-50%, 40-60%, 50-60%, 50-70%, or 60 -70%. In some embodiments, a compound of the present invention reduces liver fibrotic content by 20%, 25%, 30%, 35%, or 40% as measured by liver hydroxyproline content. In some embodiments, use of the present invention reduced liver fibrotic content by 20-30%, 20-25%, 24-30%, or 30-40%, 30-35%, or 35-40%. In some embodiments, the compound of the present invention reduced hepatic collagen content by 20%, 25%, 30%, 35%, or 40%. In some embodiments, the compound of the present invention reduced hepatic collagen content by 20-30%, 20-25%, 25-30%, 30-40%, 30-35%, or 35-40%. In some embodiments, the compound of the present invention reduced the area of α-SMA in the liver compared to pre-treatment levels by 3%.

In some embodiments, a compound of the present invention reduces steatosis by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, a compound of the present invention reduces steatosis by 40-50%, 50-60%, 60-70%, 50-70%, 70-80%, 60-80%, 70-90 % or 80-90%. In some embodiments, a compound of the present invention reduces total liver lipid content by 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, a compound of the present invention reduces total liver lipid content by 20-30%, 30-40%, or 40-50%.

In some embodiments, a compound of the present invention reduces hepatocellular ballooning by 30%, 35%, 40%, 45%, or 50%. In some embodiments, a compound of the present invention reduces hepatocellular ballooning by 30-40%, 30-35%, 35-40%, 40-50%, 40-45%, or 45-50%. In some embodiments, a compound of the present invention reduces hepatocellular hypertrophy by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In some embodiments, a compound of the present invention reduces hepatocellular hypertrophy by 40-50%, 50-60%, 60-70%, or 70-80%. In some embodiments, a compound of the present invention reduces NASH scores by 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. In some embodiments, a compound of the present invention reduces NASH scores by 30-40%, 40-50%, 30-50%, 50-60%, 60-70%, or 50-70%.

In some embodiments, a compound of the present invention reduces liver inflammation by 20%, 30%, or 40%, as measured by galectin (Gal-3) levels. In some embodiments, a compound of the present invention reduces liver inflammation by 20-30%, 20-25%, 25-30%, 30-40%, 30-35%, or 35-40%. In some embodiments, a compound of the present invention reduces plasma alanine aminotransferase (ALT) levels by 20%, 35%, 30%, 35%, or 40%. In some embodiments, a compound of the present invention reduces ALT levels by 20-30%, 20-25%, 25-30%, 30-40%, 30-35%, or 35-40%. In some embodiments, a compound of the present invention reduces plasma aspartate transaminase (AST) levels by 10%, 15%, or 20%. In some embodiments, a compound of the present invention reduces AST levels by 10-15%, 10-20%, or 15-20%.

In some embodiments, a compound of the present disclosure and/or treatment with a compound of the present disclosure, e.g., Compound A, improves NASH scores on liver biopsy, LiverMultiScan PDFF and cT1 MRI scores, liver function test(s), HOMA-IR, and/or biomarkers of inflammation and fibrosis (including hsCRP, Pro-C3, ELF panel and other relevant biomarkers) compared to controls.

In some embodiments, a compound of the present disclosure and/or treatment with a compound of the present disclosure, e.g., Compound A, results in improvement, e.g., reduction in ballooning (e.g., score=0), e.g., with an associated lobular inflammation score of 0 or 1 and no worsening fibrosis compared to control. In some embodiments, a compound of the present disclosure and/or treatment with a compound of the present disclosure, e.g., Compound A, results in changes, e.g., improvements from baseline NASH scores on liver biopsy, selected histologic measures of steatosis, ballooning, inflammation and fibrosis, by liver enzymes, by imaging parameters and/or by biomarkers (including hsCRP, Pro-C3, ELF panel, cytokines and other appropriate biomarkers) compared with controls.

In some embodiments, the safety and tolerability of a compound of the present invention, such as Compound A, in patients can be assessed by monitoring patient side effects, monitoring laboratory parameters (hematology, chemistry, and urinalysis), vital signs (blood pressure, pulse rate, and temperature ), hsCRP and/or resting 12-lead ECG compared to controls. The pharmacokinetics of Compound A in patients can be studied by comparing mean trough plasma concentrations of Compound A at steady state, for example, using samples taken at regular intervals. For example, mean trough plasma concentrations can be taken at 8-week intervals to determine the pharmacokinetics of Compound A.

Patients meeting one or more of the following criteria may show an improved response to treatment with Compound A compared to patients not meeting one or more of the following criteria: histological diagnosis of NASH, fibrosis score 1-3 inclusive (F1 limited at 30%), PDFF > 10% by MRI, compensated liver disease with the following hematological and biochemical criteria on admission to treatment: ALT <5 x ULN, AST > 30, hemoglobin >11 g/dL for women and >12 g/dL for men, white blood cells (WBC) >2.5 thousand/µl, neutrophil count >1.5 thousand/µl, platelet count >100 thousand/µl, total bilirubin <35 µmol/l (although patients with bilirubin >35 µmol/l may be included subject to that it is unconjugated bilirubin in Gilbert syndrome), albumin >36 g/L, international normalized ratio (INR) <1.4, serum creatinine <1.3 mg/dL (men) or <1.1 (women) or estimated glomerular filtration rate ≥60 ml/min/1.73 ^m2 , in the absence of other causes of chronic liver disease (such as, for example, autoimmune, primary biliary cholangitis, HBV, HCV, Wilson's disease, α-1-antitrypsin deficiency, hemochromatosis ) and/or, if applicable, compensated type 2 diabetes (with HgbA1c <9.5% and fasting blood glucose <10 mmol/L, with no change in treatment during the previous 6 months, and/or with no new symptoms associated with decompensated diabetes within the previous 3 months).

Conversely, patients who meet one or more of the following criteria from the following list may not respond to treatment with Compound A compared to patients who do not meet one or more of the same criteria. The list of criteria includes: history of prolonged use of excess alcohol, unstable metabolic state (eg, weight gain or loss of more than 5 kg in the last three months, diabetes with poor glycemic control (HgbA1c> 9.5%), or use of an antidiabetic or drug antiobesity medications, malabsorption syndrome, or restrictive bariatric (weight loss) surgery within the last 6 months before screening); history of gastrointestinal malabsorption bariatric surgery less than 5 years prior to screening or use of medications known to cause hepatic steatosis, including corticosteroids, high-dose estrogens, methotrexate, tetracycline, or amiodarone in the previous 6 months; HB antigen>0, HCV PCR>0 (patients with a history of HCV infection may be included if HCV PCR negative for more than 3 years), or HIV infection, type 1 diabetes or type 2 diabetes with current treatment with insulin drugs, diabetic ketoacidosis, fasting triglycerides > 300 mg/dL, hemostatic disorders or current treatment with anticoagulants, current or presence of cardiac dysrhythmias and/or a history of cardiovascular disease, including myocardial infarction, except in patients with well-controlled hypertension; any clinically significant ECG abnormality and/or use of antidiabetic agents known to have anti-NASH activity, such as pioglitazone and GLP-1 receptor agonists.

The compounds of Formula (I) and Formula (II) can be prepared as described, for example, in PCT applications WO2009/061208, WO2010/128401, WO2011/089529, WO2016/156912 and in accordance with the Examples below. In addition, Compound A can be prepared as described, for example, in PCT WO2010/128401 and WO2014/132135 and in accordance with Example 2 below.

The Examples below are illustrative, and one skilled in the art will understand how to apply these general methods to prepare other compounds within Formula (I) and Formula (II). The compounds of the present invention may be in the form of a pharmaceutically acceptable salt or ester. For example, the compounds of Formula (I) and Formula (II) may be in the form of esters such as a phospholipid, glyceride or C ₁ -C ₆ alkyl ester. In at least one embodiment, the ester is selected from a glyceride or a C ₁ -C ₆ alkyl ester. In at least one embodiment, the ester is selected from triglyceride, 1,2-diglyceride, 1,3-diglyceride, 1-monoglyceride, 2-monoglyceride, methyl ester, ethyl ester, propyl ether, isopropyl ether, n-butyl ether and tert-butyl ether. In at least one embodiment, the Compound of Formula (I) is present in the form of methyl ester, ethyl ether, isopropyl ether, n-butyl ether or t-butyl ether, for example, methyl ether or ethyl ether. In general, the esters represented by Formula (I) (eg, ethyl esters) will hydrolyze in the gastrointestinal tract.

Salts suitable for the present disclosure include, but are not limited to, NH ₄ ⁺ salts; metal ions such as Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ or Ca ²⁺ ; protonated primary amines such as tert-butylammonium, (3S, 5S, 7S)-adamantane-1-ammonium, 1,3-dihydroxy-2-(hydroxymethyl)propane-2-ammonium, protonated aminopyridine (e.g. pyridine-2- ammonium); protonated secondary amines such as diethylammonium, 2,3,4,5,6-pentahydroxy-N-methylhexane-1-ammonium, N-ethylnaphthalene-1-ammonium, protonated tertiary amines such as 4-methylmorpholine-4-ium, protonated quaternary amines such as 2-hydroxy-N,N,N-trimethylethane-1-aminium and protonated guanidine such as amino((4-amino-4-carboxybutyl)amino)methaniminium, or protonated heterocyclic compounds such as 1H -imidazole-3-ium. Additional examples of suitable salts include salts of diprotonated diamines such as ethane-1,2-diammonium or piperazine-1,4-dium. Other salts according to the present disclosure may include protonated Chitosan:

At least in an embodiment, the salts are selected from sodium salt, calcium salt and choline salt. In one embodiment, the salt is a sodium or calcium salt.

The present invention relates to a method of treating NASH or ASH in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound of Formula (I) or Formula (II). The subject may be a human or a non-human mammal. The compounds currently disclosed may be administered in the form of a drug, such as a pharmaceutical composition. In one aspect, the invention relates to a pharmaceutical composition containing a compound of Formula (II), such as a compound of Formula (I), such as Compound A, for use in the treatment of non-alcoholic steatohepatitis. The composition currently disclosed may contain at least one compound disclosed herein and, optionally, at least one inactive pharmaceutical ingredient, that is, an excipient. The inactive ingredients may dissolve, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, and/or otherwise convert the active ingredients into a suitable and effective drug, such that the drug becomes safe, convenient, and/or, otherwise, acceptable for use. Examples of excipients include, but are not limited to, solvents, carriers, diluents, binders, fillers, sweeteners, flavors, pH modifiers, viscosity modifiers, antioxidants, fillers, humectants, disintegrating agents, dissolution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, dyes, dispersants and preservatives. Excipients may have more than one role or function or may be classified in more than one group; classifications are descriptive only and are not intended to limit the scope of the invention. In some embodiments, for example, the at least one excipient may be selected from corn starch, lactose, glucose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, ethanol, glycerin, sorbitol, polyethylene glycol, propylene glycol, cetyl stearyl alcohol , carboxymethylcellulose and fatty substances such as tallows, or suitable mixtures thereof. In some embodiments, the presently disclosed compositions contain at least one compound of Formula (II), such as one of the compounds of Formula (I), and at least one pharmaceutically acceptable antioxidant, such as a tocopherol, such as alpha -tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol or a mixture thereof, BHAs such as 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole, or mixtures thereof, and BHT (3 ,5-di-tert-butyl)-4-hydroxytoluene) or mixtures thereof.

The compositions disclosed herein may be formulated for oral administration, such as tablets or gelatin soft or hard capsules. The dosage form may be any shape suitable for oral administration, such as spherical, oval, ellipsoidal, cubic, regular and/or irregular. Conventional preparation techniques known in the art can be used to prepare the compounds of the present disclosure. In some embodiments, the composition may be in the form of a gelatin capsule or tablet.

A suitable daily dosage of a compound disclosed herein, such as a compound of Formula (I) or a compound of Formula (II), may be from about 5 mg to about 4 g, such as from about 5 mg to about 2 g. For example, in some embodiments implementation, the daily dose is from about 10 mg to about 1.5 g, from about 50 mg to about 1 g, from about 100 mg to about 1 g, from about 150 mg to about 900 mg, from about 50 mg to about 800 mg , from about 100 mg to about 800 mg, from about 100 mg to about 600 mg, from about 150 to about 550 mg, or from about 200 to about 500 mg. In at least one embodiment, the daily dose is from about 200 to about 600 mg. In at least one embodiment, the daily dose is about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg , about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg or about 900 mg. The compound(s) may be administered, for example, once, twice or three times daily.

In at least one embodiment, the compound of Formula (I) is administered in an amount of from about 200 to about 800 mg per dose. In at least one embodiment, the compound of Formula (I) is administered once daily. In at least one embodiment, the compound of Formula (I) is administered once daily at a dose of 750 mg. In some embodiments, a compound of Formula (I) is administered once daily at a dose of 600 mg. In some embodiments, a compound of Formula (I) is administered once daily at a dose of 500 mg. In some embodiments, a compound of Formula (I) is administered once daily at a dose of 300 mg. In some embodiments, a compound of Formula (I) is administered once daily at a dose of 250 mg. Preferably, the compound of Formula (I) is administered once daily at a dose of 300 or 600 mg.

In at least one embodiment, the compound of Formula (II) is administered in an amount of from about 200 to about 800 mg per dose. In at least one embodiment, the compound of Formula (II) is administered once daily. In at least one embodiment, the compound of Formula (II) is administered once daily at a dose of 750 mg. In some embodiments, a compound of Formula (II) is administered once daily at a dose of 600 mg. In some embodiments, a compound of Formula (II) is administered once daily at a dose of 500 mg. In some embodiments, a compound of Formula (II) is administered once daily at a dose of 300 mg. In some embodiments, a compound of Formula (II) is administered once daily at a dose of 250 mg. Preferably, the compound of Formula (II) is administered once daily at a dose of 300 or 600 mg.

According to the present disclosure, a compound of Formula (I) or (II) can be administered as monotherapy or in combination with one or more additional active agents. For co-administration, the additional active agent is preferably a therapeutically active agent, such as a drug, which preferably has a therapeutic effect on NASH or ASH, for example, affects one or more factors involved in the development and/or worsening of NASH or ASG. Preferably, the use of a combination product, ie a compound of Formula (I) or (II) in combination with one or more additional active agents, has a synergistic effect in the prevention and/or treatment of NASH or ASH. In one embodiment, one or more additional therapeutic agents are independently selected from the group of allosteric acetyl-CoA carboxylase (ACC) inhibitors, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, apoptosis signal kinase 1 (ASK1) inhibitors, inhibitors caspase inhibitors, cathepsin B inhibitors, CCR2 chemokine antagonists, CCR5 chemokine antagonists, chloride channel stimulators, cholesterol solubilizers, diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, dipeptidyl peptidase IV inhibitors (DPP IV inhibitor), fibroblast growth factor (FGF)-21 agonists, farnesoid X receptor (FXR) agonists, anti-CD3 mAbs, galectin-3 inhibitors, glucagon-like peptide 1 (GLP1) agonists, glutathione precursors, hepatitis C virus NS3 protease inhibitors, HMG inhibitors, CoA reductase inhibitors, 1-β-hydroxysteroid dehydrogenase inhibitors (I--HSD1), heat shock protein (Hsp) 47 inhibitors, IL-Iβ antagonists, IL-6 antagonists, IL-10 agonists, IL-17 antagonists, bile acid co-transporter inhibitors, leptin analogues, 5-lipoxygenase inhibitors , LPL gene stimulators, lysyl oxidase 2 homologue (LOXL2) inhibitors, lysophosphatidic acid 1 (LPA1) receptor antagonists, omega-3 fatty acids, PDE3 inhibitors, PDE4 inhibitors, phospholipase C (PLC) inhibitors, PPARa agonists, PPARy agonists, PPAR5 agonists, recombinant human protein pentraxin-2 (PRF-1), Rho-associated protein kinase 2 (ROCK2) inhibitors, semicarbazide-sensitive amine oxidase (SSAO) inhibitors, sodium-glucose transporter type 2 (SGLT2) inhibitors, stearoyl-CoA desaturase-1 inhibitors, thyroid hormone receptor agonists, tumor necrosis factor (TNFα) ligand inhibitors, transglutaminase inhibitors, transglutaminase inhibitor precursors and small activating RNAs (saRNAs).

Specifically, in some embodiments, the one or more additional active agents are selected from the group consisting of: glucagon-like peptide 1 (GLP-1) agonist, dipeptidyl peptidase inhibitor (DPP-4 antagonists), and omega-3 (n-3) fatty acids.

Glucagon-like peptide-1 receptor agonists, also known as GLP-1 receptor agonists or incretin mimetics, are agonists of the GLP-1 receptor. This class of medications is commonly used to treat type 2 diabetes. A non-limiting list of examples of GLP agonists includes: exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide and semaglutide.

In some embodiments, the additional active agent is a dipeptidyl peptidase inhibitor (DPP-4 antagonist). DPP-4 antagonists are a class of oral hypoglycemic agents that block DPP-4 (DPP-IV). They can be used to treat type 2 diabetes. Glucagon increases blood glucose levels, and DPP-4 inhibitors decrease glucagon and blood glucose levels. The mechanism of DPP-4 inhibitors is to increase incretin levels (GLP-1 and GIP) to inhibit glucagon release, which in turn increases insulin secretion, decreases gastric emptying, and decreases blood glucose levels. A non-limiting sample list of dipeptidyl peptidase inhibitors includes: Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Gemigliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Omarigliptin, Evogliptin, Eutogliptin, Dutogliptin.

In some embodiments, the additional agent is an omega-3 fatty acid. When the additional active agent is an omega-3 fatty acid, the omega-3 fatty acid is typically a long-chain polyunsaturated omega-3 fatty acid (LC n-3 PUFA). Preferably, this includes at least one of (all-Z omega-3)-5,8,11,14,17-eicosapentaenoic acids (EPA) and one of (all-Z omega-3)-4,7 ,10,13, 16,19-docosahexaenoic acids (DHA) or derivatives thereof. n-3 PUFAs, including EPA and DHA, can be in different forms and are at least one of the free forms of fatty acids; esterified form, represented by a C ₁ -C ₄ alkyl ether and, preferably, ethyl ether; phospholipids; mono/di/triglycerides; and salts thereof. The omega-3 fatty acid may be provided in the form of a composition, such as an oral composition. Such a composition may contain at least 40%, for example at least 50, 60, 70 or 80% active omega-3 fatty acid. In some embodiments, the additional active agent is a composition containing at least one of EPA and DHA, preferably in ethyl ester form, at a concentration of at least 70%.

In some embodiments, one or more additional active agents are independently selected from the group consisting of acetylsalicylic acid, alipogen tiparvovec, aramchol, atorvastatin, BI 1467335, BLX-1002, BMS-986036, BMS-986020, cenicriviroc, cobiprostone, colesevelam, emrican., enalapril, foramulab, GFT-505, GR-MD-02, GS-0976, GS-9674, hydrochlorothiazide, eicosapentaenoic acid ethyl ester (eicosapentaenoic acid ethyl ester, EPA ethyl ester), IMM-124E, IVA337, K-877 , KD-025, linagliptin, liraglutide, mercaptamine, MGL-3196, ND-L02-s0201, obeticholic acid, olesoxime, pegilodecakin, pioglitazone, PRM-151, PX-102, remogliflozin etabonate, SHE-S26TUSB 6 solithromycin, tipelukast, TRX -318, ursodeoxycholic acid and VBY-376. The first component of the combination product, ie the compound of Formula (I) or (II), can be administered or formulated in any manner as described above. The second component of the combination product, the additional active agent, can be formulated so that the method is suitable for the type of agent that it is depending on several factors, including the method of administration of the agent. The dosage of the additional active agent depends on the type of agent selected and should be consistent with the approved amounts for the specific agent. As stated in the Examples, the CDAA (methionine-choline-deficient) diet-induced NASH model is a model of both liver fibrosis and inflammation. As demonstrated by the Examples, the combination of a GLP-1 agonist with a compound of Formula (I) or (II), such as Compound A, has a superior effect compared to treatment with GLP-1 alone in the treatment of fibrosis and inflammation associated with NASH. Since liver fibrosis is largely a response to inflammatory responses, this effect is also supported by demonstrated changes in the expression of the liver gene associated with inflammation (TNF-α), thus confirming the superiority of oxygenated structurally modified fatty acids (Compound A) in combination with a GLP-1 agonist compared with GLP-1 agonist alone. Overall, the data suggest that the combination of a GLP-1 agonist (or alternatively DPP-4 antagonists) with a compound of the present invention may have a synergistic effect in the treatment of both liver inflammation and fibrosis.

Although hepatic steatosis itself may be a benign condition, it can sensitize the liver to a “second hit” when other factors cause an inflammatory response. Therefore, a reduction in steatosis is desirable, both in terms of its contribution to the NASH score and in identifying NASH as a potential “primary” factor for other existing or recurrent liver lesions. New significant improvements in liver lipids achieved using a combination of high doses of omega-3 ethyl esters and a low dose of oxygenated structurally modified fatty acid (compound A) suggest that a synergistic effect is achieved, especially since neither the compound does not significantly reduce hepatic triglyceride (TG) levels in ApoE*3L-CETP transgenic mice.

Notably, Compound A, at a fraction of the dose of omega-3 ethyl esters, significantly increased or decreased >1094 gene expression probes in the liver of APOE*3.CETP transgenic mice compared to <10 for omega-3 ethyl esters (data not available). shown). Without being bound by theory, this differential effect on the liver transcriptome may suggest that the observed synergistic effect is secondary to different reaction cascades rather than a dose accumulation effect. Its significant effect on liver cholesterol may also extend to inflammatory responses associated with the pathogenesis of NASH and ASH. Overall, the data obtained in APOE*3.CETP mice favor a combination of omega-3 PUFAs, such as a combination of EPA ethyl esters and an oxygenated structurally modified fatty acid according to Formula (I) or (II) as potent therapy to reduce hepatic steatosis and therefore treat NASH and/or ASH.

The compounds of Formula (II) or, preferably, Formula (I) can be administered as monotherapy or in combination with at least one additional active agent for the treatment and/or reversal of nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH). In some embodiments, the at least one additional active agent is a GLP-1 agonist. In addition, based on the results obtained in APOE*3.CETP transgenic mice, compounds of Formula (II) or, preferably, Formula (I) can be administered in combination with at least one additional active agent for therapeutic and/or prophylactic treatment and/or reversal of hepatic steatosis (triglycerides and/or cholesterol). In some embodiments, the at least one additional active agent is an omega-3 fatty acid, such as omega-3 PUFA ethyl ester.

The above Examples highlight the possibility of combining oxygen-containing structurally modified fatty acids with a number of other active agents for the treatment of NASH and/or ASH. These combinations may not only improve outcomes related to the effectiveness of the combination compared to monotherapy, but may also improve the safety of use. As an example of improved safety, Compound A was demonstrated to significantly reduce CETP expression in APOE*3.CETP mice and improve lipid profiles in both rodents and humans. It may be advantageous when used in combination with obeticholic acid (an FXR agonist), which, unlike Compound A, increases CETP expression and worsens lipid profiles in both APOE*3.CETP mice and humans.

The present inventors have discovered that compounds of Formula (I) such as 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoic acid have surprisingly good pharmaceutical activity. Surprisingly, the compounds of Formula (I) disclosed herein exhibit improved biological activity compared to a glucagon-like peptide 1 receptor (GLP-1R) agonist in the treatment of NASH-associated liver fibrosis and inflammation. The compounds of Formula (I) are also likely to exhibit a synergistic effect when administered in combination with additional active agents.

Examples

The present disclosure may be further described by the following non-limiting Examples, in which standard methods known to the skilled chemist and methods similar to those described in these Examples may be used where applicable. It will be understood that those skilled in the art will appreciate additional embodiments consistent with the description disclosed herein.

Unless otherwise stated, reactions were carried out at room temperature, typically between 18-25°C with HPLC grade solvents under anhydrous conditions. Evaporation was carried out by rotary evaporation in vacuum. Column chromatography was performed using a 40-63 μm silica gel flash chromatography procedure (Merck) or an Armen Spotflash using prepackaged MiniVarioFlash, SuperVarioFlash, SuperVarioPrep, or EasyVarioPrep silica gel columns (Merck). Nuclear magnetic resonance (NMR) shift values were recorded on a Bruker Avance DPX 200 or 300 instrument with peak folds described as follows: s, singlet; d, doublet; dd, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; br - broad spectrum. Mass spectra were recorded using an LC/MS spectrometer. Separation was performed using an Agilent 1100 series module on an Eclipse XDB-C18 2.1 x 150 mm gradient elution column. A gradient of 5-95% acetonitrile in buffers containing 0.01% trifluoroacetic acid or 0.005% sodium formate was used as an eluent. Mass spectra were recorded using a Gl956A mass spectrometer (electrospray, 3000 V), switching between positive and negative ionization modes. Yields reported are illustrative and do not necessarily reflect the maximum possible yield.

Preparation of compounds

Example 1. Preparation of tert-butyl-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-icoso-5,8,11,14,17-pentaen-1-yloxy) butanoate:

Tetrabutylammonium chloride (0.55 g, 1.98 mmol) was added to a solution of (5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-ol (3.50 g , 12.1) mmol) in toluene (35 ml) at room temperature under nitrogen atmosphere. Aqueous sodium hydroxide (50% (w/w), 11.7 mL) was added with vigorous stirring at room temperature, followed by tert-butyl 2-bromobutyrate (5.41 g, 24.3 mmol). The resulting mixture was heated to 50°C, and after 1.5 hours (2.70 g, 12.1 mmol), 3.5 hours (2.70 g, 12.1 mmol) and 4.5 hours (2.70 g, 12.1 mmol) additional tert-butyl 2-bromobutyrate was added and stirred for 12 hours. After cooling to room temperature, ice water (25 ml) was added and the resulting two phases were separated. The organic phase was washed with NaOH (5%) and brine, dried (MgSO ₄ ), filtered and concentrated. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (100:0 -> 95:5) as eluent. Concentration of appropriate fractions gave 1.87 g (36% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.10 (m, 6H), 1.35-1.54 (m, 11H), 1.53-1.87 (m, 4H) , 1.96-2.26 (m, 4H), 2.70-3.02 (m, 8H), 3.31 (dt, 1H), 3.51-3.67 (m, 2H), 5 ,10-5.58 (m, 10N).

Example 2. Preparation of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoic acid (compound A):

Tert-butyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate (19.6 g, 45.5 mmol) was dissolved in dichloromethane (200 ml) and placed in nitrogen. Trifluoroacetic acid (50 ml) was then added and the reaction mixture was stirred at room temperature for one hour. Water was added and the aqueous phase was extracted twice with dichloromethane. The combined organic extract was washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was flash chromatographed on silica gel using progressively polar mixtures of heptane, ethyl acetate and formic acid (90:10:1 -> 80:20:1) as eluent. Concentration of appropriate fractions gave 12.1 g (71% yield) of the title compound as an oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.90-1.00 (m, 6H), 1.50 (m, 2H), 1.70 (m, 2H), 1.80 (m, 2H), 2.10 (m, 4H), 2.80-2.90 (m, 8H), 3.50 (m, 1H), 3.60 (m, 1H), 3.75 (t, 1H ), 5.30-5.50 (m, 10H); MS (electrospray): 373.2 [M-H] -.

Example 3. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) calcium butanoate

2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-Eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (1.87 g, 4.99 mmol, 93 %) was mixed with CaCO ₃ (0.25 g, 2.50 mmol). Water (1 ml) was added and the mixture was stirred under mechanical stirring at room temperature for 1 hour. CO ₂ was formed. A dense, homogeneous paste was formed. Acetone (7 ml) was added while stirring. Solid material began to separate. The solid was filtered and dried over nitrogen, sealed and stored in a refrigerator at 4°C. Yield: 1.86 g (95%). The solid was not further characterized by analytical or spectroscopic methods, but several experiments were conducted showing that a calcium salt had formed.

• Solid material is melted on a hot plate at a temperature below 100°C. Specific melting point has not been determined

• The material does not release CO ₂ when acid is added, but is “dissolved” and precipitates as an oil.

Example 4. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)sodium butanoate

2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (1.87 g, 4.99 mmol, 93 %) was mixed with NaHCO ₃ (0.420 g, 5.00 mmol). Water (1 ml) was added and the mixture was stirred under mechanical stirring at room temperature for 1 hour. CO ₂ was released and a thick, homogeneous paste was formed. While stirring, ethanol (7 ml) was added to the reaction flask. The sodium salt, formed from 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-Eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid, goes into solution when ethanol is added (7 ml). Small amounts of unreacted NaHCO ₃ were filtered off and the solution was evaporated to dryness. The crude, slightly viscous oil was evaporated twice with 96% ethanol to remove traces of water.

Example 5. Preparation of 2-hydroxy-N, N, N-trimethylethane-1-aminium 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14,17-pentaene-1 -yl) hydroxy) butanoic acid

Choline hydroxide (327.7 µl) in water was pipetted into a scintillation vial containing approximately 2.5 ml MTBE and 7.5 ml n-heptane. Inside the nitrogen chamber 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (500 mg, 95.8%) was transferred to the bottle. In a nitrogen chamber, approximately 1.0 mL of water was added slowly and with stirring to the vial. The vial was then sealed. The reaction mixture was stirred for approximately 30 minutes. The resulting 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)choline salt of butanoic acid was a hard gel-like material, which was filtered on the Buchner funnel. The wet filter material was washed 3 times using 1 ml MTBE. The washed material was a hard gel-like solid.

Example 6. Preparation of (4S, 5R)-3 - ((S)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl) - 4 -methyl-5-phenyloxazolidin-2-one and (4S, 5R)-3 - ((R)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14, 17 -pentaenyloxy)butanoyl)-4-methyl-5-phenyloxazolidin-2-one:

DMAP (1.10 g, 8.90 mmol) and DCC (1.90 g, 9.30 mmol) were added to the mixture of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11 ,14,17-pentaenyloxy)butanoic acid (3.20 g, 8.50 mmol) in dry dichloromethane (100 ml), maintained at 0°C under a nitrogen atmosphere. The resulting mixture was stirred at 0°C for 20 minutes. (4S,5R)-4-methyl-5-phenyloxazolidin-2-one (1.50 g, 8.50 mmol) was added and the resulting cloudy mixture was stirred at ambient temperature for five days. The mixture was filtered and concentrated under reduced pressure to obtain a crude product containing the desired product as a mixture of two diastereomers. The residue was purified by flash chromatography on silica gel using 15% ethyl acetate in heptane as eluent. The two diastereomers were separated and the corresponding fractions were concentrated. (4S, 5R)-3 - ((S)-2- ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5 - phenyloxazolidin-2-one eluted first with a yield of 1.1 g (40% yield) as an oil. (4S, 5R)-3 - ((R)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5 -phenyloxazolidin-2-one was obtained as an oil in 0.95 g (34% yield).

(4S, 5R)-3 - ((S)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5 -phenyloxazolidin-2-one (E1): 1H-NMR (300 MHz, CDCl ₃ ): δ 0.90 (d, 3H), 1.00 (t, 3H), 1.07 (t, 3H), 1 .45-1.57 (m, 2H), 1.62-1.76 (m, 3H), 1.85-1.95 (m, 1H), 2.05-2.15 (m, 4H) , 2.87 (m, 8H), 3.39 (m, 1H), 3.57 (m, 1H), 4.85-4.92 (m, 2H), 5.30-5.45 (m , 10H), 5.75 (d, 1H), 7.32 (m, 2H), 7.43 (m, 3H).

(4S, 5R)-3 - ((R)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5 -phenyloxazolidin-2-one (E2): ^1H -NMR (300 MHz, CDCl ₃ ): δ 0.98 (d, 3H), 0.99 (t, 3H), 1.08 (t, 3H), 1.40-1.52 (m, 2H), 1.55-1.75 (m, 3H), 1.80-1.90 (m, 1H), 2.05-2.15 (m, 4H ), 2.84 (m, 8H), 3.39 (m, 1H), 3.56 (m, 1H), 4.79 (pent, 1H), 4.97 (dd, 1H), 5.30 -5.45 (m, 10H), 5.71 (d, 1H), 7.33 (m, 2H), 7.43 (m, 3H).

Example 7: Preparation of (S)-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaenyloxy)butanoic acid:

Hydrogen peroxide (35% in water, 0.75 ml, 8.54 mmol) and lithium hydroxide monohydrate (0.18 g, 4.27 mmol) were added to the solution of (4S, 5R)-3-((S)-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5-phenyloxazolidin-2-one (1.10 g, 2. 13 mmol) in tetrahydrofuran (12 ml) and water (4 ml) were kept at 0°C in a nitrogen atmosphere. The reaction mixture was stirred at 0°C for 30 minutes. 10% Na ₂ SO ₃ (aq) (30 ml) was added, the pH was adjusted to 2 with 2 M HCl and the mixture was extracted twice with heptane (30 ml). The combined organic extract was dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was subjected to flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (98:8 -> 1:1) as eluent. Concentration of appropriate fractions gave 0.48 g (60% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.00 (m, 6H), 1.48 (m, 2H), 1.65 (m, 2H), 1.85 (m, 2H ), 2.10 (m, 4H), 2.80-2.90 (m, 8H), 3.55 (m, 1H), 3.60 (m, 1H), 3.88 (t, 1H) , 5.35-5.45 (m, 10H); MS (electrospray): 373.3 [M-H]-; [α] _D -37°; (c=0.104, ethanol).

Example 8. Preparation of (R)-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoic acid:

Hydrogen peroxide (35% in water, 0.65 ml, 7.37 mmol) and lithium hydroxide monohydrate (0.15 g, 3.69 mmol) were added to the solution containing (4S, 5R)-3-((R) -2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)butanoyl)-4-methyl-5-phenyloxazolidin-2-one (0.95 g, 1.84 mmol) in tetrahydrofuran (12 ml) and water (4 ml), maintained at 0°C in a nitrogen atmosphere. The reaction mixture was stirred at 0°C for 30 minutes. 10% Na ₂ SO ₃ (aq) (30 ml) was added, the pH was adjusted to 2 with 2 M HCl and the mixture was extracted twice with heptane (30 ml). The combined organic extract was dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was subjected to flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (98:8 -> 50:50) as eluent. Concentration of the appropriate fractions gave 0.19 g (29% yield) of the title compound as an oil. 1H-NMR (300 MHz, CDCl ₃ ): δ 0.90-1.00 (m, 6H), 1.48 (m, 2H), 1.65 (m, 2H), 1.85 (m, 2H ), 2.10 (m, 4H), 2.80-2.90 (m, 8H), 3.55 (m, 1H), 3.60 (m, 1H), 3.88 (t, 1H) , 5.35-5.45 (m, 10H); MS (electrospray): 373.3 [M-H]-; [α] _D -31° (c=0.088, ethanol).

Example 9. Preparation of ethyl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoate:

Dicyclohexylmethanedimine (DCC) (304 mg, 1.47 mmol) and N,N-dimethylpyridin-4-amine (DMAP) (10 mg, 0.067 mmol) were added to the stirred solution of 2 - (((5Z, 8Z, 11Z, 14Z) , 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)hydroxy)butanoic acid (501.3 mg, 1.335 mmol) in dichloromethane (DCM) (4 ml) at 0°C atmosphere nitrogen. The mixture was stirred for 5 minutes before adding ethanol (EtOH) (0.16 ml, 2.67 mmol). The resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (100:0-99:1) as eluent. Concentration of the appropriate fractions gave 473 mg (88% yield) of the title compound as an oil. ^1H NMR (300 MHz, chloroform-d) δ 0.95 (2x, 6H), 1.37-1.48 (m, 2H), 1.54-1.79 (m, 4H), 2.01 -2.10 (m, 4H), 2.77-2.84 (m, 8H), 3.27-3.34 (m, 1H), 3.53-3.60 (m, 1H), 3 .69-3.73 (dd, 1H), 4.13-4.24 (m, 2H), 5.25-5.33 (m, 10H), MS (electrospray); 425.3 [M+Na]+; HRMS (electrospray): found 425.3021 [M+Na]+, calculated for [C ₂₆ H ₄₂ O ₃ +Na] ⁺ 425.3031.

Example 10. Preparation of isopropyl-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate

DCC (310 mg, 1.47 mmol) and DMAP (9 mg, 0.067 mmol) were added to a stirred solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17 -pentaen-1-yl)hydroxy)butanoic acid (501 mg, 1.335 mmol) in DCM (4 ml) at 0°C under nitrogen. The mixture was stirred for 5 minutes before isopropanol (iPrOH) (0.16 mL, 2.67 mmol) was added. The resulting mixture was stirred at room temperature for 20 hours. The mixture was filtered and concentrated in vacuo. Heptane (50 ml) was added to the residue, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 1% ethyl acetate in heptane as eluent. Concentration of the appropriate fractions gave 496 mg (89% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.97 (2x, 6H), 1.25 (m, 6H), 1.42-1.50 (m, 2H), 1.61-1.70 (m, 2H), 1.70-1.77 (m, 2H), 2.05-2.12 (m, 4H), 2.79-2.86 (m, 8H), 3.29-3 .34 (m, 1H), 3.54-3.59 (m, 1H), 3.67-3.71 (m, 1H), 5.06-5.10 (m, 1H), 5.31 -5.42 (m, 10N); MS (electrospray): 439.3 [M+Na]+.

Example 11. Preparation of methyl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoate:

Sulfuric acid (0.049 ml, 0.918 mmol) was added to the solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid ((385 mg, 1028 mmol) in methanol (20 ml) at room temperature under nitrogen and the resulting mixture was stirred at room temperature overnight. MS (electrospray): 389.3 [M+1]+.

Example 12. Preparation of butyl-2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoate:

Sulfuric acid (0.049 ml, 0.918 mmol) was added to the solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid ((33 g, 88 mmol) in butan-1-ol (400 ml, 4.37 mol) at room temperature under nitrogen and the reaction mixture was stirred for 120 hours. Heptane (400 ml) and ethyl acetate (400 ml) were added and the solution was washed with saturated aqueous NaHCO ₃ (3 x 300 ml) and water (2 x 300 ml), the combined aqueous phase was extracted with a mixture of heptane/ether (1: 1) (2 x 300 ml), the phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using progressively polar mixtures of heptane and ethyl acetate (99:1, 96:4) as the eluent. The concentrations of the corresponding fractions gave 26.3 g (67% yield) of the title compound as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 0.93-1.02 (m, 9H), 1.36-1.51 (m, 4H), 1.60-1.70 (m, 4H), 1.72-1.84 (m, 2H), 2.05-2.16 (m, 4H), 2.78-2.92 (m, 8H), 3.28-3.39 (m, 1H ), 3.54-3.65 (m, 1H), 3.70-3.82 (m, 1H), 4.08-4.24 (m, 2H), 5.27 -5.48 (m , 10H), MS (electrospray): 453.2 [M+Na]+.

Example 13: Preparation of 2,3-dihydroxypropyl 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate:

Step a) Preparation of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14,17- pentaen-1-yl) oxy) butanoate

2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (25 g, 66.7 mmol) and DMAP ( 8.15 g, 66.7 mmol) was added to a solution of 2,2-dimethyl-1,3-dioxolane-4-methanol (7.54 mL, 60.7 mmol) in chloroform (150 mL) under nitrogen. A solution of DCC (13.77 g, 66.7 mmol) in chloroform (65 ml) was then added dropwise at ambient temperature. The mixture was stirred overnight and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of 10% ethyl acetate in heptane as the eluent. Concentration of the appropriate fractions gave 19.6 g (66% yield) of the title product as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 0.99 (t, 6H), 1.37-1.40 (m, 3H), 1.41-1.53 (m, 5H), 1.59- 1.71 (m, 2H), 1.72-1.85 (m, 2H), 2.05-2.14 (m, 4H), 2.74-2.95 (m, 8H), 3. 31-3.38 (m, 1H), 3.57-3.65 (m, 1H), 3.72-3.86 (m, 2H), 4.07-4.12. (m, 1H), 4.15-4.27 (m, 2H), 4.29-4.40 (m, 1H), 5.23-5.50 (m, 10H). MS (electrospray): 511.3 [M+Na]+.

Step b) Preparation of 2,3-dihydroxypropyl 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate

To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl) methyl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaene- 1-yl)oxy)butanoate (27.5 g, 56.3 mmol) in dioxane (280 ml) was added at room temperature under nitrogen atmosphere with an aqueous solution of HCl (37% (w/w), 28 ml, 341 mmol) and the mixture was stirred for 60 minutes. The mixture was then carefully poured into saturated aqueous NaHCO ₃ (500 ml) and extracted with EtOAc (2 x 300 ml). The organic phase was washed with 1 M HCl (200 ml), brine (200 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using heptane and ethyl acetate (50:50) as eluent. Concentration of the appropriate fractions gave 19 g of the target product in the form of an oil contaminated with ~ 10% isomer 1,3-dihydroxypropan-2-yl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose -5,8,11, 14,17-pentaen-1-yl) oxy) butanoate. The material was mixed with 1.35 g of another batch before further purification by preparative HPLC. An isocratic mixture of water/acetonitrile (9:1) and acetonitrile (100%) in a ratio of 17:83 was used as the eluent. Concentration of the appropriate fractions gave 11.3 g (38% yield) of the title product as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.97-1.03 (m, 6H), 1.41-1.51 (m, 2H), 1.59-1.69 (m, 2H), 1.72-1.87 (m, 2H), 2.05-2.14 (m, 5H), 2.56 (s, 1H), 2.73-2.94 (m, 8H), 3 .33-3.40 (m, 1H), 3.55-3.68 (m, 2H), 3.69-3.77 (m, 1H), 3.79-3.85 (m, 1H) , 3.93-4.03 (m, 1H), 4.15-4.37 (m, 2H), 5.25-5.51 (m, 10H). MS (electrospray): 471.3 [M+Na] +.

Example 14. Preparation of 1,3-dihydroxypropan-2-yl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate

Step a) Preparation of oxiran-2-ylmethyl-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate

Mixture of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (800 mg, 2.14 mmol), glycidol (0.17 ml, 2.56 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC*HCl) (491 mg, 2.56 mmol) and 4-dimethylaminopyridine (DMAP) (313 mg, 2 .56 mmol) in dry DCM (7 ml) was stirred at room temperature under nitrogen atmosphere. The reaction mixture was concentrated in vacuo. The residue was purified by flash chromatography on silica gel using all progressively polar mixtures of heptane and ethyl acetate (99:1 ~ 95:5) as the eluent. Concentration of the appropriate fractions gave 527 mg (57% yield) of the title product as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 0.94-0.98 (m, 6H), 1.40-1.44 (m, 2H), 1.57-1.64 (m, 2H), 1.70-1.82 (m, 2H), 2.02-2.12 (m, 4H), 2.63 (b.s, 1H), 2.78-2.84 (m, 9H), 3.20 (b.s, 1H), 3.30-3.35 (m, 1H), 3.55-3.61 (m, 1H), 3.77-3.80 (m, 1H) , 3.94-4.01 (m, 1H), 4.42-4.48 (m, 1H), 5.36-5.26 (m, 10H). MS (electrospray): 453.3 [M+Na] +.

Step b) Preparation of 2 - ((2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoyl)oxy)propane - 1,3-diyl-bis(2,2,2-trifluoroacetate)

Trifluoroacetic anhydride (TFAA) (0.55 ml, 3.96 mmol) in dry DCM (3 ml) was added in portions to the pre-cooled solution of oxiran-2-ylmethyl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) - eicose -5,8,11,14,17-pentaen-1-yl) oxy)butanoate (286 mg, 0.66 mmol) in dry DCM (3 ml) at -20°C under nitrogen. The cooling bath was removed and the mixture was stirred for 19 hours at ambient temperature, after which the reaction mixture was concentrated in vacuo. The residue was dissolved in toluene (6 ml) and passed through a layer of silica (6.5 g), eluting with toluene (150 ml). Concentration in vacuo gave 357 mg (84% yield) of the title compound as an oil. ^1H NMR (400 MHz, _CDCl3 ) δ 0.95 (2x t, 6H), 1.38-1.45 (m, 2H), 1.57-1.63 (m, 2H), 1.66 -1.78 (m, 2H), 2.09-2.02 (m, 4H)), 2.78-2.84 (m, 8H), 3.27-3.33 (m, 1H), 3.51-3.56 (m, 1H), 3.77 (dd, 1H), 4.30-4.53 (m, 2H), 4.60-4.69 (m, 2H), 5. 17-5.43 (m, 10H), 5.43-5.55 (m, 1H). MS (electrospray): 661.1 [M+Na]+.

Step c) Preparation of 1,3-dihydroxypropan-2-yl 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate

A solution of pyridine (0.4 ml, 4.95 mmol) and methanol (0.3 ml, 7.41 mmol) in pentane/DCM (2:1) (4.5 ml) was added dropwise to the solution of 2 - (( 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoyl)oxy)propane-1,3-diyl-bis ( 2,2,2-trifluoroacetate) (354 mg, 0.552 mmol) in pentane/DCM (2:1) (5 ml), cooled to -20°C under nitrogen. The cooling bath was removed and the mixture was stirred for 3 hours at room temperature, then concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 90:10 80:20 50:50) as eluent. Concentration of the appropriate fractions gave 223 mg of the title product as crude oil. Preparative HPLC purification provided 58 mg (22% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.95 (t, 3H), 0.96 (t, 3H), 1.38-1.45 (m, 2H), 1.54-1.64 ( m, 2H), 1.67-1.84 (m, 2H), 2.01-2.09 (m, 4H), 2.45 (bs, 2H), 2.83-2.77 ( m, 8H), 3.36-3.30 (m, 1H), 3.60-3.55 (m, 1H), 3.84-3.78 (m, 5H), 4.98- 4.93 (m, 1H), 5.65-5.09 (m, 10H). MS (electrospray): 471.1 [M+Na] +.

Example 15. Preparation of 3-hydroxypropane-1,2-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl) hydroxy)butanoate)

Step a) Preparation of tert-butyl ((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)dimethylsilane

Tert-butylchlorodimethylsilane (14.41 g, 91 mmol) was added to a solution of (2,2-dimethyl-1,3-dioxolan-4-yl) methanol (10 g, 76 mmol) and imidazole (7.73 g, 114) . mmol) in THF (100 ml) at ambient temperature under nitrogen atmosphere. The mixture was stirred for 1.5 hours, poured into water (200 ml) and extracted with tert-butyl methyl ether (2 x 150 ml). The phases were separated and the organic layer was washed with water (100 ml), brine (100 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 3% ethyl acetate in heptane as eluent. Concentration of the appropriate fractions gave 18 g (97% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.02-0.05 (m, 6H), 0.85-0.89 (m, 9H), 1.31-1.35 (m, 3H), 1.35-1.40 (m, 3H), 3.50-3.60 (m, 1H), 3.63-3.72 (m, 1H), 3.75-3.85 (m, 1H ), 3.96-4.05 (m, 1H), 4.07-4.18 (m, 1H). MS (electrospray): 229.2 [M+Na] +.

Stage b). Preparation of 3 - ((tert-butyldimethylsilyl)oxy)propane-1,2-diol.

FeCl ₃ × 6H ₂ O absorbed on silica gel (30 g, 11.9 mmol), and the mixture was stirred overnight. The mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (50:50 ~ 25:75) as eluent. Concentration of appropriate fractions gave 760 mg (9% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.09-0.12 (m, 6H), 0.91-0.95 (m, 9H), 2.11-2.17 (m, 1H), 2.60 (d, 1H), 3.57-3.85 (m, 5H). MS (electrospray): 229.2 [M+Na] +.

Step c) Preparation of 3 - ((tert-butyldimethylsilyl)oxy)propane-1,2-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14, 17-pentaen-1-yl) oxy) butanoate)

To a solution of 3 - ((tert-butyldimethylsilyl)oxy)propane-1,2-diol (0.91 g, 4.41 mmol) in DMF (20 ml) under nitrogen atmosphere at ambient temperature was added 2 - (((5Z , 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl) oxy)butanoic acid (3.47 g, 9.3 mmol), DMAP (1.13 g , 9.3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DCI) (1.776 g, 9.26 mmol) and dry DCM (60 ml). The mixture was stirred overnight before the reaction mixture was diluted with diethyl ether (200 ml). The mixture was washed with 1 M HCl (100 ml) and brine (100 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 3% ethyl acetate in heptane as eluent. Concentration of appropriate fractions gave 2.26 g (56% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.08 (s, 6H), 0.90 (d, 9H), 0.95-1.03 (m, 12H), 1.40-1.52 ( m, 4H), 1.58-1.69 (m, 4H), 1.70-1.83 (m, 4H), 2.04-2.15 (m, 8H), 2.77-2. 92 (m, 16H), 3.27-3.37 (m, 2H), 3.57-3.67 (m, 2H), 3.72-3.80 (m, 4H), 4.14- 4.32 (m, 1H), 4.41-4.56 (m, 1H), 5.09-5.22 (m, 1H), 5.23-5.49 (m, 20H). MS (electrospray): 941.6 [M+Na]+.

Step d) Preparation of 3-hydroxypropane-1,2-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl) hydroxy)butanoate)

To a solution of 3 - ((tert-butyldimethylsilyl)oxy)propane-1,2-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14,17 - pentaen-1-yl) hydroxy) butanoate) (2.26 g, 2.46 mmol) in dioxane (100 ml). HCl (37% (w/w, 2 ml) and the mixture was stirred for 3 hours under nitrogen at ambient temperature, then concentrated in vacuo. The residue was purified by silica gel flash chromatography using 15% ethyl acetate in heptane as eluent. Concentration of appropriate fractions gave 0.83 g (42% yield) of the title compound as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 0.96-1.03 (m, 12H), 1.40-1. .53 (m, 4H), 1.58-1.68 (m, 4H), 1.70-1.85 (m, 4H), 1.87-2.01 (m, 1H), 2.05 -2.15 (m, 8H), 2.75-2.95 (m, 16H), 3.28-3.41 (m, 2H), 3.56-3.65 (m, 2H), 3.73-3.85 (m, 4H), 4.24-4.37 (m, 1H), 4.42-4.53 (m, 1H), 5.14-5.23 (m, 1H ), 5.26 -5.51 (m, 20H). MS (electrospray): 827.5 [M+Na]+.

Example 16. Preparation of 2-hydroxypropane-1,3-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl) hydroxy) butanoic acid):

Step a) 2-oxopropane-1,3-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy ) butanoate)

2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (5.0 g, 13.4 mmol) and DMAP (1.63 g, 13.4 mmol) was added to a solution of 1,3-dihydroxyacetone dimer (1.145 g, 6.36 mmol) in chloroform (25 mL) under nitrogen. A solution of DCC (2.75 g, 13.35 mmol) in chloroform (10 ml) was then added dropwise at ambient temperature. The mixture was stirred overnight at room temperature, then concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (90:10-88:12) as eluent. Concentration of appropriate fractions gave 2.4 g (47% yield) of the title compound as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 0.97-1.06 (m, 12H). 1.38-1.53 (m, 4H), 1.57-1.73 (m, 4H), 1.73-1.96 (m, 4H), 2.03-2.17 (m, 8H ), 2.76-2.92 (m, 16H), 3.35-3.42 (m, 2H), 3.63-3.70 (m, 2H), 3.89 (dd, 2H), 4.75-4.93 (m, 4H), 5.27-5.49 (m, 20H). MS (electrospray): 827.5 [M+Na]+.

Step b) 2-hydroxypropane-1,3-diyl -bis (2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy )butane)

.

NaBH ₄ (0.336 g, 8.87 mmol) was carefully added to the solution of 2-oxopropane-1,3-diyl-bis (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11 , 14,17-pentaen-1-yl) hydroxy) butanoate) (3.24 g, 4.03 mmol) in THF (55 ml) and water (4 ml) at 0°C. The mixture was stirred for 15 minutes at 0°C. Acetic acid (1 ml) was then carefully added followed by ethyl acetate (100 ml). The mixture was washed with water (100 ml), saturated aq. NaHCO ₃ (100 ml) and brine before drying (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was combined with another portion of the material before purification by flash chromatography on silica gel using 15% ethyl acetate in heptane as eluent. Concentration of appropriate fractions gave 1.62 g (50% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.97-1.03 (m, 12H), 1.41-1.52 (m, 4H), 1.58-1.69 (m, 6H), 1.71-1.87 (m, 4H), 2.05-2.14 (m, 8H), 2.38-2.42 (m, 1H), 2.78-2.92 (m, 16H ), 3.32-3.39 (m, 2H), 3.57-3.64 (m, 2H), 3.80-3.84 (m, 2H), 4.05 -4.34 (m , 5H), 5.26-5.49 (m, 20H). MS (electrospray): 827.5 [M+Na]+.

Example 17: Preparation of propan-1,2,3-triyl tris (2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy ) butanoate)

2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (4.0 g, 10.7 mmol), 4-dimethylaminopyridine (1.305 g, 10.7 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.047 g, 10.7 mmol) and dry DCM (30 ml) were added to the glycerol solution (0.173 ml, 2.373 mmol) in DMF (10 ml) under nitrogen atmosphere at room temperature. The mixture was stirred overnight, then the reaction mixture was diluted with diethyl ether (250 ml). The mixture was washed with aqueous 1 M HCl (100 ml) and brine (100 ml) before drying (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using 5% ethyl acetate in heptane as eluent. Concentration of the appropriate fractions gave 2.1 g (73% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 0.91-1.05 (m, 18H), 1.40-1.52 (m, 6H), 1.57-1.69 (m, 6H), 1.69-1.86 (m, 6H), 2.01-2.17 (m, 12H), 2.69-2.96 (m, 24H), 3.27-3.38 (m, 3H ), 3.53-3.67 (m, 3H), 3.73-3.81 (m, 3H), 4.17-4.27 (m, 2H), 4.37-4.54 (m , 2H), 5.28-5.47 (m, 30H). MS (electrospray): 1183.8 [M+Na]+.

Example 18: Preparation of tert-butyl 2 - ((2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoyl)oxy) benzoate:

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (316 mg, 1.65 mmol) and 4-dimethylaminopyridine (DMAP) (20 mg, 0.15 mmol) were added to the solution of 2-((5Z, 8Z , 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (561 mg, 1.5 mmol) in DCM (10 ml) and the reaction mixture was stirred for 10 minutes. Tert-butyl 2-hydroxybenzoate (291 mg, 1.5 mmol) was added and the mixture was stirred for 3 hours. Saturated saline solution was added and the resulting two phases were separated. The aqueous phase was extracted with DCM and the combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 5% EtOAc in heptane as eluent. Concentration of the appropriate fractions gave 500 mg (61% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.98 (t, 3H), 1.11 (t, 3H), 1.13 (t, 3H), 1.45-1.75 (m, 4H ), 1.56 (s, 9H), 1.90-2.20 (m, 6H), 2.80-2.90 (m, 8H), 3.45-3.60 (m, 1H), 3.80-3.90 (m, 1H), 4.05-4.15 (m, 1H), 5.25-5.50 (m, 10H) 7.06 (m, 1H), 7.28 (m, 1H), 7.52 (m, 1H), 7.89 (m, 1H). MS (ESI): 573 [M+Na]+.

Example 19: Preparation of 2-((2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoyl)oxy)benzoic acid:

.

Solution of tert-butyl 2 - ((2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoyl)oxy) benzoate (500 mg ( 0.9 mmol) in FA (10 ml) was stirred for 2 days. The solvent was removed under reduced pressure and the residue was flash chromatographed using a gradient of 1-5% EtOAc (containing 5% FA) in heptane (also containing 5% FA). ) as eluent. The appropriate fractions were combined and concentrated and the residue was further purified by a second flash chromatography using a gradient of 1-10% EtOAc in heptane as eluent. The concentration of the appropriate fractions gave 75 mg (17% yield) of the title compound ¹ . H NMR (300 MHz, CDCl ₃ ): δ 0.99 (t, 3H), 1.13 (t, 3H), 1.45-1.60 (m, 2H), 1.65-1.70 ( m, 2H), 1.90-2.15 (m, 6H) 2.80-2.90 (m, 8H), 3.45-3.55 (m, 1H), 3.80-3.90 (m, 1H), 4.05-4.15 (m, 1H), 5.30-5.45 (m, 10H), 7.14 (d, 1H), 7.35-7.45 (m , 1H), 7.60-7.70 (m, 1H), 8.10-8.15 (m, 1H): 517 [M+Na]+.

Example 20. Preparation of 2 - ((tert-butoxycarbonyl) amino) ethyl 2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate:

.

O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.1 mmol) and TEA (0.56 ml, 4 mmol) were added to solution 2 -((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (748 mg, 2 mmol) in DCM (10 ml) and reaction mixture stirred for 20 minutes. A solution of tert-butyl N-(2-hydroxyethyl)carbamate (340 mg, 2.1 mmol) in DCM (1 ml) was added and the resulting mixture was stirred at room temperature overnight. Et ₂ O (100 ml) was added and the mixture was washed with water, 1 M HCl (aq), saturated aq. NaHCO ₃ and brine, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 10-20% EtOAc in heptane as eluent. The concentration of the corresponding fractions gave 780 mg (yield 76%) of the title compound. ¹ H NMR (300 MHz, CDCl ₃ ): δ 0.98 (t, 6H), 1.40. -1.55 (m, 2H), 1.45 (s, 9H), 1.60-1.85 (m, 4H), 2.05-2.20 (m, 4H), 2.75-2 .90 (m, 8H), 3.30-3.45 (m, 3H), 3.55-3.65 (m, 1H), 3.75-3.80 (m, 1H), 4.20 -4.25 (m, 2H), 4.76 (b. C 1H) 5.30-5.45 (m, 10H) MS (ESI): 540 [M+Na]+.

Example 21. Preparation of 2-(2-acetoxybenzamido)ethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate:

.

Acetyl chloride (1 ml) was added to a solution of 2-((tert-butoxycarbonyl)amino)ethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene-1- yloxy)butanoate (300 mg, 0.58 mmol) in MeOH (5 ml) at 0°C and the reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo to obtain 2-aminoethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate HCl salt (286 mg) in as an unrefined product. TEA (109 μl, 0.78 mmol) was added to a solution of acetylsalicylic acid (137 mg, 0.76 mmol) in DCM (10 ml) and the mixture was cooled to 0°C. Ethyl chloroformate (75 μL, 0.78 mmol) was added dropwise and the reaction mixture was stirred for 2 hours. This solution was then added to a solution of the crude product HCl salt of 2-aminoethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate in the mixture DCM (10 ml) and TEA (2 ml). The resulting mixture was stirred at room temperature for 3.5 hours and then water was added. The resulting two phases were separated and the aqueous phase was extracted twice with DCM. The combined organic phases were washed with 1 M HCl (aq), saturated NaHCO ₃ (aq) and water, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 20% EtOAc in heptane as eluent. Concentration of the appropriate fractions gave 90 mg (yield 23%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.95-1.05 (m, 6H), 1.35-1.45 (m, 2H), 1.55-1.70 (m, 2H) , 1.70-1.85 (m, 2H), 2.05-2.15 (m, 4H), 2.35 (s, 3H), 2.80-2.90 (m, 8H), 3 ,30-3.40 (m, 1H), 3.55-3.65 (m, 1H), 3.70-3.85 (m, 3H), 4.33 (t, 2H), 5.30 -5.45 (m, 10H), 6.61 (broad s, 1H), 7.10-7.15 (m, 1H), 7.25-7.35 (m, 1H), 7.45 -7.50 (m, 1H), 7.70. -7.75 (m, 1H). MS (ESI); 602 [M+Na]+.

Example 22. Preparation of 2-(2-hydroxybenzamido)ethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate:

.

Ammonia (aq, 28%, 20 drops) was added dropwise to a solution of 2-(2-acetoxybenzamido)ethyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14, 17- pentaen-1-yloxy)butanoate (99 mg, 0.17 mmol) in 2-propanol (9 ml) and water (3 ml) and the reaction mixture was stirred for 10 minutes. Water was added and the resulting mixture was extracted twice with Et ₂ O. The combined organic phases were washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 0-40% EtOAc in heptane as eluent. Concentration of appropriate fractions gave 33 mg (36% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.95-1.05 (m, 6H), 1.35-1.45 (m, 2H), 1.60-1.70 (m, 2H) , 1.70-1.85 (m, 2H), 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.35-3.45 (m, 1H), 3.50-3.60 (m, 1H), 3.75-3.85 (m, 3H), 4.40-4.50 (m, 2H), 5.30-5.45 ( m, 10H), 6.80-6.90 (m, 2H), 6.95-7.05 (m, 1H), 7.35-7.45 (m, 1H), 12.22 (s, 1H). MS (ESI): 560 [M+Na]+.

Example 23. Preparation of N-benzyl-2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (302.1 mg, 0.807 mmol) and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium tetrafluoroborate (TBTU) (285 mg, 0.888 mmol) in DCM (10 ml) at room temperature under nitrogen was added triethylamine (123 μl) 0.887 mmol ). After stirring for 15 minutes, benzylamine (97 μL, 0.887 mmol) was added to the mixture and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of the appropriate fractions gave 0.253 g (66% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) d 0.97-0.90 (m, 6H), 1.41-1.35 (m, 2H), 1.59-1.52 (m, 2H), 1.75-1.68 (m, 1H), 1.86-1.80 (m, 1H), 2.09-2.03 (m, 4H), 2.82-2.77 (m, 8H ), 3.44 (t, J=6.4, 2H), 3.72-3.71 (m, 1H), 4.46 (d, J=5.8, 2H), 5.36-5 .28 (m, 10H), 6.83 (s, 1H), 7.26-7.24 (m, 3H), 7.33. - 7.30 (m, 2H).

Example 24. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)-N,N-dimethylbutanamide

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (300.2 mg, 0.801 mmol) and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium tetrafluoroborate (TBTU) (286.4 mg, 0.892 mmol) triethylamine (123 µl) was added to DCM (10 ml) at room temperature under nitrogen atmosphere 0.884 mmol). After stirring for 15 minutes, dimethylamine (2.0 M in THF) (0.80 mL, 1.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 0.268 g (81% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ) d 0.95 (t, 3H), 0.96 (t, 3H), 1.35-1.46 (m, 2H), 1.53-1.63 (m, 2H), 1.66-1.79 (m, 2H), 2.01-2.10 (m, 4H), 2.77-2.87 (m, 8H), 2.93. (s, 3H), 3.10 (s, 3H), 3.28 (dt, J=9.1, 6.5, 1H), 3.46 (dt, J=9.1, 6.3, 1H), 3.96 (dd, J=7.7, 6.1, 1H), 4.97-5.70 (m, 10H).

Example 25. Preparation of N-ethyl-2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (304.6 mg, 0.813 mmol) and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium tetrafluoroborate (TBTU) (286.9 mg, 0.894 mmol) triethylamine (123 µl) was added to DCM (10 ml) at room temperature under nitrogen atmosphere 0.884 mmol). After stirring for 15 minutes, ethylamine (2.0 M in THF) (0.80 mL, 1.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 0.186 g (56% yield) of the title compound as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) d 0.90 (t, 3H), 0.95 (t, 3H), 1.13 (t, 3H), 1.55-1.62 (m, 2H) , 1.64-1.66 (m, 2H), 1.69-1.69 (m, 1H), 1.74-1.83 (m, 1H), 2.01-2.12 (m, 4H), 2.78-2.84 (m, 8H), 3.25-3.34 (m, 2H), 3.44 (t, 2H), 3.64 (dd, 1H), 5.50 -5.13 (m, 10H), 6.50 (s, 1H).

Example 26: Preparation of tert-butyl (2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamido)ethyl ) carbamic acid:

Dicyclohexylmethanedimine (DCC) (1.73 g, 8.4 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol (HOBt) (1.14 g, 8.4 mmol) were added to stirred solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (3.14 g, 8.4 mmol ) in THF (25 ml) at 0°C under nitrogen atmosphere. The mixture was stirred for 20 minutes, after which a solution of N-Boc-ethylenediamine (1.12 g) in THF (1 ml) was added dropwise. The resulting mixture was stirred at room temperature for 1.5 hours. Diethyl ether (200 ml) was added and the mixture was washed with water, 1 M HCl, saturated NaHCO ₃ and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 25% ethyl acetate in heptane (added 0.5% HCOOH) as eluent. Concentration of appropriate fractions gave 3.0 g (83% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): d 0.90 (t, 3H), 0.96 (t, 3H), 1.43 (s, 9H), 1.40-1.80 (m, 6H ), 2.05-2.20 (m, 4H), 2.75 2.90 (m, 8H), 3.20-3.60 (m, 6H), 3.65-3.75 (m, 1H), 5.02 (br C, 1H), 5.30-5.45 (m, 10H), 7.01 (br C, 1H), MS (electrospray); 539 [M+Na]+.

Example 27: Preparation of N-(2-aminoethyl)-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

To a solution of tert-butyl (2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamido)ethyl)carbamate (774 mg, 1.5 mmol) in DCM (8 ml) under nitrogen at ambient temperature, TFA (2 ml) was added, the reaction mixture was stirred at room temperature for 30 minutes and then concentrated in vacuo. Diethyl ether (50 ml) and NaOH (aq. 1M, 50 ml) were added to the residue, and the mixture was stirred vigorously for 30 minutes. The phases were separated and the organic phase was washed with brine, dried (NaSO4), filtered and concentrated in vacuo to give 560 mg (90%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (2xt, 6H), 1.35-1.50 (m, 2H), 1.55-1.85 (m, 4H) , 2.05-2.20 (m, 4H), 2.75-2.95 (m, 8H), 3.10-3.20 (m, 1H), 3.30-3.80 (m, 6H), 4.05-4.20 (broad m, 1H), 4.25-4.75 (broad s, 2H), 5.30-5.50 (m, 10H). MS (electrospray); 417 [M+H]+, 439 [M+Na].

Example 28: Preparation of N-(2-hydroxyethyl)-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (4.5 g, 12 mmol) in DCM (100 ml) under nitrogen atmosphere at room temperature, oxalyl chloride (8.4 ml, 100 mmol) was added, followed by 2 drops of DMF. The mixture was stirred for 30 minutes at room temperature and concentrated in vacuo. The residue was dissolved in DCM (100 ml) under nitrogen atmosphere. Triethylamine (3.34 mL, 24 mmol) was added followed by ethanolamine (1.08 mL, 18 mmol). The reaction mixture was stirred for 2 hours at room temperature. Water (300 ml) was added and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (50:50 × 20:80) as eluent. Concentration of appropriate fractions gave 4.6 g (92% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (m, 6H), 1.40-1.55 (m, 2H), 1.60-1.90 (m, 4H ), 2.05-2.20 (m, 4H), 2.75-2.90 (m, 8H), 3.05 (br. s, 1H), 3.40-3.55 (m, 4H ), 3.70-3.80. (m, 3H), 5.30-5.45 (m, 10H), 7.04 (br s, 1H). MS (electrospray); 440 [M+Na]+

Example 29. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)-N-isopropylbutanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (300 mg, 0.801 mmol) and o- Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (284 mg, 0.884 mmol) was added to DCM (10 mL) at room temperature under nitrogen atmosphere with triethylamine (123 μL (0.884 mmol). After stirring for 15 minutes at room temperature under nitrogen, isopropylamine (76 μL, 0.884 mmol) was added and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 0.207 g (59% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.83-0.93 (m, 3H), 0.95 (t, J=7.5, 3H), 1.14 (t, J=6.1 , 6H), 1.39-1.47 (m, 2H), 1.56. 1.73 (m, 3H), 1.73-1.83 (m, 1H), 2.02-2.11 (m, 4H), 2.80-2.84 (m, 8H), 3, 43 (t, J=6.4, 2H), 3.60 (dd, J=6.6, 4.4, 1H), 4.02-4.11 (m, 1H), 5.66-5 .01 (m, 10H), 6.33 (d, J=5.9, 1H).

Example 30. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)-N-methylbutanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (303.6 mg, 0.811 mmol) and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium tetrafluoroborate (TBTU) (285.3 mg, 0.889 mmol) triethylamine (123 µl) was added to DCM (10 ml) at room temperature under nitrogen atmosphere 0.884 mmol). After stirring for 15 minutes at room temperature under nitrogen, methylamine 2.0 M in THF (0.8 mL, 1600 mmol) was added and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 0.235 g (72% yield) of the title compound as an oil.

¹ H NMR (300 MHz, CDCl ₃ ) d 0.89 (t, J=7.4, 3H), 0.95 (t, J=7.5, 3H), 1.37-1.47 (m , 2H), 1.56-1.72 (m, 3H), 1.74 1.84 (m, 1H), 2.01-2.15 (m, 4H), 2.78-2.84 ( m, 11H), 3.44 (t, J=6.4, 2H), 3.66 (dd, J=6.4, 4.4, 1H), 5.18-5.52 (m, 10H ), 6.51 (s, 1H).

Example 31: Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)-1-(piperidin-1-yl) butan-1-one:

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (302.2 mg, 0.807 mmol) and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium tetrafluoroborate (TBTU) (284 mg, 0.884 mmol) in DCM (8 ml) at room temperature under nitrogen was added triethylamine (123 μl) 0.884 mmol ). After stirring for 15 minutes at room temperature under nitrogen, piperidine (87 μL, 0.884 mmol) was added and the reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The organic phases were combined, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 0.270 g (73% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) d 0.93-0.98 (m, 6H) 1.37-1.46 (m, 2H), 1.51-1.62 (m, 8H), 1 .66-1.77 (m, 2H), 2.02-2.09 (m, 4H), 2.77-2.83 (m, 8H), 3.30 (dt, J=9.1, 6.6, 1H), 3.47-3.52 (m, 2H), 3.54-3.62 (m, 3H), 3.95 (dd, J=7.9 6.1, 1H) , 5.02-5.65 (m, 10N).

Example 32. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (320 g, 854 mmol) in toluene ( 2500 ml) oxalyl chloride (276 ml, 3.15 mol) was added. DMF (10 ml) was then carefully added dropwise, then the mixture was stirred at ambient temperature for 1 hour and concentrated in vacuo. The residue was dissolved in DCM (750 ml) and slowly added to a stirred, cooled (0°C) ammonium solution (28%, aq) (528 ml, 6.84 mol). The mixture was stirred for 30 minutes at ambient temperature. The phases were separated and the aqueous phase was extracted with methyl tert-butyl ether (700 ml). The combined organic phase was washed with 3M HCl (2x300 ml), saturated aq. NaHCO ₃ (500 ml), brine (2x300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (70:30 to 60:40) as eluent. Concentration of appropriate fractions gave 249.8 g (76% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) d 0.87-0.92 (m, 6H), 1.32-1.46 (m, 2H), 1.52-1.59 (m, 2H), 1.62-1.79 (m, 2H), 1.97-2.06 (m, 4H), 2.68-2.85 (m, 8H), 3.34-3.51 (m, 2H ), 3.60 (dd, J=6.5, 4.5, 1H), 5.18-5.42 (m, 10H), 5.61 (s, 1H) 6.42 (s, 1H) .

Example 33: Preparation of N-(tert-butyl)-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanamide:

.

To a solution of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (301.3 mg, 0.804 mmol) in DCM (10 ml) at room temperature under nitrogen, o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (285.6 mg, 0.889 mmol) was added followed by triethylamine (0.123 ). ml, 0.885 mmol). The mixture was stirred for 15 minutes at room temperature under nitrogen atmosphere, after which 2-amino-2-methylpropane (0.10 ml, 0.952 mmol) was added. The reaction mixture was stirred at room temperature overnight. Saturated NaHCO ₃ (25 mL) was added and the mixture was extracted with t-butyl methyl ether (2 x 50 mL). The combined organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (95:5 × 50:50) as eluent. Concentration of appropriate fractions gave 244.1 mg (70% yield) of the title compound as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.89 (t, J=7.4, 3H), 0.96 (t, J=7.5, 3H), 1.34 (s, 9H), 1.41-1.46 (m, 2H), 1.57-1.70 (m, 3H), 1.74-1.81 (m, 1H), 2.02-2.11 (m, 4H ), 2.80-2.83 (m, 8H), 3.27-3.48 (m, 2H), 3.48-3.56 (m, 1H) 4.93-5.55 (m, 10H), 6.38 (s, 1H).

Example 34: Preparation of (R)-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)-N-((R ) -1-phenylethyl) butanamide:

To a solution of (R)-2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (40 mg, 0. 11 mmol) and (R)-1-phenylethan-1-amine (14 μl, 0.11 mmol) in dry DMF (3 ml) at 0 ^o C under a nitrogen atmosphere was added N-methylmorpholine (14 μl, 0.13 mmol ) followed by the addition of o-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (45 mg, 0.14 mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. Water (10 ml) was added and the mixture was extracted with heptane (10 ml). The phases were separated and the aqueous phase was extracted with heptane (10 ml) and then with diethyl ether (10 ml). The organic phases were combined and washed with saturated aqueous NH ₄ Cl (10 ml) and brine (10 ml). The organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo to give the title compound (20 mg, 0.042 mmol, 38% yield) as an oil.

Example 35: Preparation of (2S)-ethyl 2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido) -4 - methyl pentanoate:

.

N,N'-dicyclohexylcarbodiimide (DCC) (1.13 g, 5.5 mmol), hydroxybenzotriazole (HOBt) (0.74 g, 5.5 mmol) and triethylamine (TEA) (1.58 ml, 11.4 mmol) was added to a solution of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (1.87 g, 5.0 mmol ) in tetrahydrofuran (THF) (20 ml) and the mixture was stirred for 10 minutes. L-leucine ethyl ester hydrochloride (0.89 g, 4.6 mmol) was added and the resulting mixture was stirred for 2 hours. The mixture was concentrated in vacuo, dissolved in Et2O (100 ml) and washed with 1M HCl (aq), saturated NaHCO ₃ (aq) and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 10-15% EtOAc in heptane as eluent. Concentration of appropriate fractions gave 1.88 g (80% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (m, 12H), 1.25-1.35 (m, 3H), 1.45-1.85 (m, 9H) , 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.40-3.70 (m, 3H), 4.18 (kv, 2H), 4 .55-4.70 (m, 1H), 5.30-5.45 (m, 10H), 6.80-6.95 (m, 1H). MS (ESI): 538 [M+Na]+.

Example 36. Preparation of (2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)-4-methylpentane acids:

.

A solution of LiOH (700 mg, 29 mmol) in water (10 ml) was added to a solution of (2S)-ethyl 2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5, 8,11,14 ,17-pentaen-1-yloxy)butanamido)-4-methylpentanoate (1.88 g, 3.65 mmol) in EtOH (20 ml) and the reaction mixture was stirred at 40°C for 1 hour. The mixture was cooled to ambient temperature and 3 M HCl (aq) was added to pH ~ 2. The resulting mixture was extracted twice with Et ₂ O and the combined organic phases were dried (NaSO ₄ ), filtered and concentrated under reduced pressure to give 1.55 g ( yield 87%) of the title compound. ^1H NMR (300 MHz, CDCl3): δ 0.90-1.05 (m, 12H), 1.45-1.55 (m, 2H), 1.65-1.85 (m, 7H), 2.05-2. 20 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.65 (m, 2H), 3.70-3.80 (m, 1H), 4.55- 4.70 (m, 1H), 5.30-5.45 (m, 10H), 6.85-7.00 (m, 1H), 10.30 (br s, 1H). MS (ESI); 510 [M+Na]+.

Example 37. Preparation of tert-butyl 2 - (((2S)-2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene-1-yloxy ) butanamido) -4-methylpentanoyl) oxy) benzoate:

2-Hydroxybenzoic acid tert-butyl ester (291 mg, 1.5 mmol), TEA (0.46 ml, 3.3 mmol) and O-(benzotriazol-1-yl)-N, N, N´, N´ -Tetramethyluronium hexafluorophosphate (TBTU) (500 mg, 1.65 mmol) was added to a solution of (2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14, 17-pentaene-1-yloxy)butanamido)-4-methylpentanoic acid (730 mg, 1.5 mmol) in dimethylformamide (DMF) (10 ml). The reaction mixture was heated for 2 hours at 65°C, cooled to room temperature and Et ₂ 0 (100 ml) was added. The resulting two phases were separated and the organic phase was washed with aqueous 10% NH ₄ Cl and brine, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 10% EtOAc in heptane as eluent. Concentration of the appropriate fractions gave 404 mg (41% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (m, 12H), 1.40-1.55 (m, 2H), 1.57 (s, 9H), 1.60 -1.85 (m, 6H), 2.00-2.20 (m, 5H), 2.80-2.90 (m, 8H), 3.40-3.60 (m, 2H), 3 .70-3.80 (m, 1H), 4.90-5.05 (m, 1H), 5.30-5.45 (m, 10H), 7.10 (d, 2H), 7.25 -7.35 (m, 1H), 7.50-7.55 (m, 1H), 7.85-7.95 (m, 1H). MS (ESI): 686 [M+Na]+.

Example 38. Preparation of 2 - (((2S)-2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido) -4-methylpentanoyl) hydroxy) benzoic acid:

.

TFA (1 ml) was added to the solution of tert-butyl 2-(((2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene -1-yloxy)butanamido)-4-methylpentanoyl)oxy)benzoate (150 mg, 0.23 mmol) in DCM (4 ml) at 0°C and the reaction mixture was stirred for 40 minutes. Toluene (10 ml) was added and the mixture was concentrated in vacuo. The residue was purified by flash chromatography using 13% EtOAc (containing 0.1% FA) in heptane (also containing 0.1% FA) as eluent. The appropriate fractions were concentrated and the residue (100 mg) was dissolved in Et2O (50 ml). The solution was washed with saturated aqueous NaHCO ₃ , dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give 60 mg of the desired product. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (m, 12H), 1.35-2.15 (m, 13H), 2.75-2.90 (m, 8H) , 3.40-3.60 (m, 2H), 3.70-3.80 (m, 1H), 4.85-5.05 (m, 1H), 5.30-5.45 (m, 10H), 7.0-7.20 (m, 2H), 7.25-7.40 (m, 1H), 7.50-7.65 (m, 1H), 8.00-8.15 ( m, 1H). MS (ESI); 630 [M+Na]+. HR-MS (ESI): calculated for C ₃₇ H ₅₃ NO ₆ +Na: 630.3770, found: 630.3786.

Example 39. Preparation of tert-butyl (2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)ethyl) carbamate:

DCC (1.73 g, 8.4 mmol) and HOBt (1.14 g, 8.4 mmol) were added to a solution of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11 ,14,17-pentaen-1-yloxy)butanoic acid (3.14 g, 8.4 mmol) in THF (25 ml) at 0°C and the mixture was stirred for 20 minutes. A solution of N-Boc-ethylenediamine in THF (1 ml) was added dropwise and the resulting mixture was stirred at room temperature for 1.5 hours. Et ₂ O (200 ml) was added and the mixture was washed with water, 1M HCl (aq), saturated NaHCO ₃ (aq) and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 25% EtOAc (containing 0.5% FA) in heptane (also containing 0.5% FA) as eluent. Concentration of appropriate fractions gave 3.0 g (83% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90 (t, 3H), 0.96 (t, 3H), 1.43 (s, 9H), 1.40-1.80 (m, 6H ), 2.05-2.20 (m, 4H), 2.75-2.90 (m, 8H), 3.20-3.60 (m, 6H), 3.65-3.75 (m , 1H), 5.02 (br s, 1H), 5.30-5.45 (m, 10H), 7.01 (b s, 1H), MS (ESI): 539 [M+Na] +.

Example 40: Preparation of 2-hydroxy-N-(2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)ethyl ) benzamide:

.

TFA (2 ml) was added to the tert-butyl (2-(2-((5Z, 8Z, 11Z, 14Z, 17Z))-eicosa-5,8,11,14,17-pentaen-1-yloxy))butanamido solution ) ethyl) carbamate (668 mg, 1.3 mmol) in DCM (8 ml) and the resulting mixture was stirred for 1.5 hours. The mixture was concentrated in vacuo to give the TFA salt of N-(2-aminoethyl)-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy) butanamide as crude product (800 mg, MS (ESI): 417 [M+H]+, 439 [M+Na]+). The residue was dissolved in DCM (10 ml) and DCC (320 mg, 1.55 mmol), TEA (0.36 ml, 2.6 mmol) and HOBt (210 mg, 1.55 mmol) were added. After 15 minutes, a solution of salicylic acid (214 mg, 1.55 mmol) in DCM (1 ml) was added dropwise and the resulting mixture was stirred overnight. The mixture was concentrated and the residue was dissolved in Et ₂ O (100 ml), washed with water, 1M HCl (aq), saturated aq. NaHCO ₃ and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The crude oil was purified by flash chromatography using a gradient of 4-15% EtOAc (containing 5% FA) in heptane (also containing 5% FA) as eluent. Concentration of the appropriate fractions gave 110 mg (16% yield over two steps) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.89 (t, 3H), 0.99 (t, 3H), 1.35-1.50 (m, 2H), 1.60-1.90 (m, 4H), 2.05-2.20 (m, 4H) 2.8-2.9 (m, 8H), 3.47 (t, 2H), 3.50-3.70 (m, 4H), 3.70-3.80 (m, 1H), 5.30-5.45 (m, 10H), 6.85-6.95 (m, 1H), 6.97 (dd, 1H) , 7.14 (br s, 1H), 7.35-7.45 (m, 1H), 7.48 (dd, 1H), 7.95 (br s, 1H), 12.51 (br .C, 1H). MS (ESI): 537 [M+H]+, 559 [M+Na]+.

Example 41: Preparation of 2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)ethyl 2-acetoxybenzoate:

.

TBTU (0.85 g, 2.64 mmol) and TEA (0.8 mL, 5.3 mmol) were added to a solution of acetylsalicylic acid (0.4 g, 2.4 mmol) in DCM (20 mL) and the mixture was stirred within 10 minutes. Solution of N-(2-hydroxyethyl)-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamide (1.0 g, 1 .4 mmol) in DCM (10 ml) and the reaction mixture was stirred at room temperature for 1 hour and then refluxed overnight. Water was added and the resulting mixture was extracted twice with DCM. The combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 0-20% EtOAc in heptane as eluent. Concentration of appropriate fractions gave 900 mg (65% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.00 (m, 6H), 1.35-1.50 (m, 2H), 1.50-1.85 (m, 4H) , 2.00-2.20 (m, 4H), 2.37 (s, 3H), 2.75-2.90 (m, 8H), 3.40-3.55 (m, 2H), 3 .55-3.75 (m, 3H), 4.15-4.45 (m, 2H), 5.30-5.50 (m, 10H), 6.80-95 (m, 1H), 7 ,10-7.15 (m, 1H), 7.30-7.40 (m, 1H), 7.55-7.65 (m, 1H), 8.00-8.05 (m, 1H) . MS (ESI): 602 [M+Na]+.

Example 42. Preparation of 2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)ethyl 2-hydroxybenzoate:

.

Ammonia (aq, 28%, 3 ml) was added dropwise to a solution of 2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy )butanamido)ethyl 2-acetoxybenzoate (390 mg, 0.67 mmol) in 2-propanol (27 ml) and water (9 ml), and the mixture was stirred for 10 minutes. Water was added and the resulting mixture was extracted twice with Et ₂ O. The combined organic phases were washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 0-20% EtOAc in heptane as eluent. Concentration of the appropriate fractions gave 63 mg (yield 18%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.91 (t, 3H), 0.97 (t, 3H), 1.35-1.45 (m, 2H), 1.55-1.90 (m, 4H), 2.00-2.15 (m, 4H) 2.80-2.90 (m, 8H), 3.44 (t, 2H), 3.65-3.80 (m, 3H), 4.40-4.50 (m, 2H), 5.30-5.45 (m, 10H), 6.85-6.95 (m, 2H), 6.95-7.05 ( m, 1H), 7.40-7.50 (m, 1H), 7.80-7.85 (m, 1H), 10.63 (s, 1H). MS (ESI): 560 [M+Na]+.

Example 43. Preparation of methyl 2-hydroxy-5-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido) benzoate:

.

2-((5Z, 8Z, 11Z, 14Z, 17Z)-Eicose-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (300 mg, 0.80 mmol), methyl 5-aminosalicylate ( 140 mg 0.83 mmol) and TEA (0.22 ml, 1.60 mmol) were dissolved in MeCN (3 ml). HATU (320 mg, 0.83 mmol) was added and the reaction mixture was stirred overnight. The mixture was then concentrated under reduced pressure and the residue was partitioned between Et ₂ O (30 ml) and brine (20 ml). The aqueous phase was extracted with Et ₂ O (20 ml) and the combined organic phases were washed with 2M HCl (aq, 15 ml), saturated NaHCO ₃ (aq, 15 ml) and brine (15 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 5% EtOAc in heptane as eluent. Concentration of the appropriate fractions gave 320 mg (77% yield) of the title compound. MS (ESI): 546 [M+Na]+.

Example 44. Preparation of 2-hydroxy-5-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)benzoic acid:

.

2M NaOH (aq, 6 ml) was added to the solution of methyl 2-hydroxy-5-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene -1 -yloxy)butanamido) benzoate (320 mg, 0.61 mmol) in MeOH (3 ml) and the reaction mixture was heated at 50°C overnight. The mixture was cooled to ambient temperature and acidified to pH ~2 with 5 M HCl (aqueous solution). The resulting mixture was extracted with EtOAc and the organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 1-2% MeOH in EtOAc as eluent. Concentration of the appropriate fractions gave 85 mg (yield 27%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (t, 3H), 1.20-1.30 (m, 1H), 1.45-1.60 (m, 2H) , 1.65-1.75 (m, 2H), 1.80-1.95 (m, 2H), 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.50-3.65 (m, 2H), 3.85-3.95 (m, 1H), 5.30-5.45 (m, 10H), 6.90-7.00 ( m, 1H), 7.58 (br s, 1H), 8.11 (br s, 1H), 8.40 (s, 1H). MS (ESI): 508 [M-N] -.

Example 45. Preparation of (2S)-ethyl-2-(2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)-4 -methyl pentanoate:

.

DCC (1.13 g, 5.5 mmol) and HOBt (0.74 g, 5.5 mmol) followed by TEA (1.58 mL, 11.4 mmol) were added to the solution of 2 - ((5Z, 8Z , 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (1.87 g, 5.0 mmol) in THF (20 ml) and the mixture was stirred for 10 minutes. L-leucine ethyl ester hydrochloride (0.89 g, 4.6 mmol) was added and the resulting mixture was stirred for 2 hours. EtOAc (100 ml) was added and the mixture was washed with water, 1 M HCl (aq), saturated NaHCO ₃ (aq) and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 10-15% EtOAc in heptane as eluent. Concentration of appropriate fractions gave 1.84 g (79% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (m, 12H), 1.25-1.35 (m, 3H), 1.40-1.85 (m, 9H) , 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.75 (m, 3H), 4.15-4.25 (m, 2H), 4.55-4.75 (m, 1H), 5.30-5.45 (m, 10H), 6.80-6.95 (m, 1H). MS (ESI): 538 [M+Na]+.

Example 46. Preparation of (2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido)-4-methylpentane acids:

.

1M LiOH (aq, 28 ml) was added to the solution of (2S)-ethyl 2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene -1 -yloxy)butanamido)-4-methylpentanoate (1.79 g, 3.5 mmol) in EtOH (50 ml) and the reaction mixture was heated to 50°C for 2 hours. The mixture was cooled to ambient temperature and 6 M HCl (aq) was added to pH ~ 2. The resulting mixture was extracted twice with Et ₂ O and the combined organic phases were dried (NaSO ₄ ), filtered and concentrated under reduced pressure to give 1.61 g (yield 95%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (m, 12H), 1.45-1.55 (m, 2H), 1.60-1.85 (m, 7H) , 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.65 (m, 2H), 3.70-3.80 (m, 1H), 4.60-4.75 (m, 1H), 5.30-5.45 (m, 10H), 6.90-7.05 (m, 1H), 10.15 (b. C, 1H). MS (ESI): 486 [M-N] -.

Example 47. Preparation of methyl 2-hydroxy-5 - ((2S)-2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene-1 yloxy ) butanamido) -4-methylpentanamido) benzoate:

.

DCC (248 mg, 1.2 mmol) and HOBt (163 mg, 1.2 mmol) were added to a solution of (2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5, 8 11,14,17-pentaen-1-yloxy)butanamido)-4-methylpentanoic acid (487 mg, 1.0 mmol) in THF (8 ml) at 0°C. A solution of methyl 5-aminosalicylate (201 mg, 1.2 mmol) in THF (1 ml) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. Et ₂ O (100 ml) was added and the mixture was washed with water, 1M HCl (aq), saturated NaHCO ₃ (aq) and brine. The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 3-15% EtOAc (containing 5% FA) in heptane (also containing 5% FA) as eluent. Concentration of the appropriate fractions gave 197 mg (31% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (m, 12H), 1.40-1.50 (m, 2H), 1.60-1.90 (m, 7H) , 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.55 (m, 2H), 3.75-3.80 (m, 1H), 3.94 (s, 1H), 4.55-4.65 (m, 1H), 5.30-5.45 (m, 10H), 6.90-7.00 (m, 2H) , 7.45-7.50 (m, 1H), 8.00-8.10 (m, 1H), 8.66 (s, 1H), 10.59 (s, 1H). MS (ESI): 659 [M+Na]+.

Example 48: Preparation of 2-hydroxy-5 - ((2S)-2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene-1-yloxy ) butanamido) -4-methylpentanamido) benzoic acid:

.

1M LiOH (aq 2.5 ml) was added to the solution of methyl 2-hydroxy-5-((2S)-2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8, 11, 14,17-pentaen-1-yloxy)butanamido)-4-methylpentanamido)benzoate (190 mg, 0.3 mmol) in MeOH (5 ml). The reaction mixture was heated at 50°C for 5 hours and then stirred at room temperature for 3 days. The mixture was acidified with 6 M HCl (aq) to pH ~ 2 and most of the solvent was removed in vacuo. The residue was extracted twice with Et ₂ O and the combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 5-25% EtOAc (containing 5% FA) in heptane (also containing 5% FA) as eluent. Concentration of appropriate fractions gave 100 mg (54% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (m, 12H), 1.40-1.50 (m, 2H), 1.60-1.90 (m, 7H) , 2.05-2.20 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.60 (m, 2H), 3.80-3.90 (m, 1H), 4.60-4.75 (m, 1H), 5.30-5.45 (m, 10H), 6.89 (d, 1H), 7.24 (d, 1H), 7.70 -7.90 (m, 2H), 9.02 (d, 1H), 10.37 (d, 1H). MS (ESI): 623 [M+H]+.

Example 49. Preparation of methyl 2-hydroxy-5 - (((2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z))-eicosa-5,8,11,14,17-pentaen-1-yloxy ) butanamido) ethoxy) carbonyl) amino) benzoate:

.

Diphosgene (0.16 ml, 1.3 mmol) and diisopropylamine (0.16 ml, 0.96 mmol) were added to the solution of N-(2-hydroxyethyl)-2-((5Z, 8Z, 11Z, 14Z, 17Z) -eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamide (0.4 g, 0.96 mmol) in DCM (15 ml) at 0°C. The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 2 hours. The mixture was concentrated under reduced pressure and the residue was suspended in THF (20 ml). The suspension was filtered and the filtrate was concentrated under reduced pressure. The residue was then suspended in Et ₂ O, filtered through a pad of silica and concentrated in vacuo to give 0.66 g of 2-(2-((5Z,8Z,11Z,14Z,17Z)-eicose-5,8,11,14 17-pentaen-1-yloxy)butanamido)ethylcarbonochloridate as a crude product. The residue was dissolved in DCM (15 ml) and methyl 5-aminosalicylate (0.13 g, 0.77 mmol) and TEA (0.2 ml, 1.54 mmol) were added. The reaction mixture was stirred for 2 hours and then water was added. The resulting mixture was extracted twice with DCM and the combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 30-50% EtOAc in heptane as eluent. Concentration of appropriate fractions gave 145 mg (30% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.00 (m, 6H), 1.35-1.50 (m, 2H), 1.50-1.85 (m, 4H) , 2.00-2.20 (m, 4H), 2.75-2.90 (m, 8H), 3.40-3.55 (m, 2H), 3.55-3.75 (m, 3H), 3.95 (s, 3H), 4.25-4.35 (m, 2H), 5.25-5.45 (m, 10H), 6.68 (broad s, 1H), 6 .85-7.00 (m, 2H), 7.35-7.45 (m, 1H), 7.91 (br s, 1H), 10.57 (s, 1H). MS (ESI): 633 [M+Na]+.

Example 50. Preparation of 2-hydroxy-5 - (((2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanamido ) ethoxy) carbonyl) amino) benzoic acid:

.

1M LiOH (aqueous solution, 1.9 ml) was added to the solution of methyl 2-hydroxy-5 - (((2- (2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11) 14,17-pentaen-1-yloxy) butanamido) ethoxy) carbonyl) amino) benzoate (145 mg, 0.24 mmol) in MeOH (20 ml) and the mixture was heated at 50°C overnight. The reaction mixture was acidified with 1 M HCl (aq) to pH ~ 2 and then extracted twice with Et ₂ O. The combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography using 20% EtOAc (containing 5% FA) in heptane (also containing 5% FA) as eluent. The appropriate fractions were combined and concentrated. The residue (46 mg) was further purified by preparative HPLC using a gradient of 30-95% MeCN in water (containing 5% MeCN and 0.01% TFA) as eluent. The appropriate fractions were combined and concentrated, and the residue was dissolved in toluene. Concentrating the solution gave 24 mg (17% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.91-1.01 (m, 6H), 1.35-1.50 (m, 2H), 1.55-1.90 (m, 4H) , 2.05-2.15 (m, 4H), 2.80-2.90 (m, 8H), 3.45-3.55 (m, 2H), 3.65-3.75 (m, 2H), 3.75-3.85 (m, 1H), 4.25-4.35 (m, 2H), 5.30-5.45 (m, 10H), 6.90-7.00 ( m, 2H), 7.00-7.10 (m, 1H), 7.63 (br. C, 1H), 7.83 (s, 1H), 10.43 (b. C, 1H). MS (ESI): 597 [M+H]+.

Example 51: Preparation of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy)-N-(2-isocyanatoethyl)butanamide:

.

1,8-bis(dimethylamino)naphthalene (577 mg, 2.7 mmol) was added to a solution of N-(2-aminoethyl)-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8 11 ,14,17-pentaen-1-yloxy)butanamide (560 mg, 1.35 mmol) in DCM (10 ml) and the mixture was cooled to 0°C. Trichloromethyl chloroformate (98 μL, 0.81 mmol) was added dropwise before the cooling bath was removed and the mixture was stirred for 15 minutes. 1 M HCl (aq, 30 ml) and DCM (30 ml) were added. The resulting two phases were separated and the organic phase was washed 4 times with 1 M HCl (aq) and once with 1 M NaOH (aq), dried (NaSO ₄ ), filtered and concentrated under reduced pressure to give 500 mg (yield 84%) of the title compound as a crude product. MS (ESI): 465 [M+Na]+.

Example 52: Preparation of methyl-2-hydroxy-5-(3-(2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaene-1- yloxy) butanamido) ethyl) ureido) benzoate:

.

Methyl 5-aminosalicyate (189 mg, 1.13 mmol) was added to a solution of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)- N-(2-isocyanatoethyl)butanamide (500 mg, 1.13 mmol) in DCM (5 ml). The reaction mixture was stirred for 2 hours and then concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 40-0% heptane in EtOAc as eluent. Concentration of the appropriate fractions gave 230 mg (yield 33%) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.85-1.05 (2x, 6H), 1.35-1.50 (m, 2H), 1.55-1.85 (m, 4H) , 2.05-2.20 (m, 4H), 2.75-2.95 (m, 8H), 3.35-3.50 (m, 6H), 3.65-3.75 (m, 1H), 3.94 (s, 3H), 5.30-5.50 (m, 10H), 6.95 (d, 1H), 7.10 (br s., 1H), 7.40 ( dd, 1H), 7.88 (d, 1H), 10.62 (s, 1H). MS (ESI); 632 [M+Na]+.

Example 53. Preparation of 2-hydroxy-5-(3-(2-(2-(5Z, 8Z, 11Z, 14Z, 17Z))-eicosa-5,8,11,14,17-pentaen-1-yloxy ) butanamido) ethyl) ureido) benzoic acid:

.

1M LiOH (aqueous solution, 2.9 ml) was added dropwise to the solution of methyl 2-hydroxy-5-(3-(2-(2-((5Z, 8Z, 11Z, 14Z, 17Z)))eicose-5,8 , 11,14,17-pentaen-1-yloxy) butanamido) ethyl) ureido) benzoate (220 mg, 0.36 mmol) in MeOH (10 ml) and the mixture was heated at 50°C overnight. The mixture was cooled to ambient temperature and then acidified to pH ~ 2 with 6 M HCl (aq). The resulting mixture was extracted twice with EtOAc and the combined organic phases were washed with brine, dried (NaSO ₄ ), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography using a gradient of 5-40% EtOAc (containing 5% FA) in heptane (also containing 5% FA) as eluent. Concentration of the appropriate fractions gave 92 mg (43% yield) of the title compound. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.91 (t, 3H), 0.98 (t, 3H), 1.40-1.50 (m, 2H), 1.60-1.90 (m, 4H), 2.05-2.20 (m, 4H) 2.80-2.90 (m, 8H), 3.40-3.60 (m, 6H), 3.75-3, 80 (m, 1H), 5.30-5.45 (m, 10H), 6.90 (d, 1H), 7.25-7.35 (m, 1H), 7.45-7.55 ( m, 1H), 7.80-7.95 (m, 2H), 10.56 (br s, 1H). MS (ESI): 596 [M+H]+, 618 [M+Na]+.

Example 54. Preparation of tert-butyl 2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy) propanoate:

.

Mixture of (5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-ol, (1.00 g, 3.47 mmol), tetrabutylammonium chloride (0.24 g , 0.87 mmol) and tert-butyl 2-bromopropionate (3.62 g, 17.3 mmol) were dissolved in toluene (36 ml) and placed under nitrogen atmosphere. With vigorous stirring, aqueous sodium hydroxide (50%, 8 ml) was slowly added and the resulting mixture was stirred at ambient temperature for twenty hours. Water was added and the mixture was extracted three times with ether. The combined organic extract was washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was purified by flash chromatography on silica gel using 2% ethyl acetate in heptane as eluent. Concentration of appropriate fractions gave 1.40 g (90% yield) of the title compound as an oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.95 (t, 3H), 1.41 (d, 3H), 1.48 (s, 9H), 1.48-1.66 (m, 4H), 2.05 (m, 4H), 2.83 (m, 8H), 3.35 (m, 1H), 3.55 (m, 1H), 3.79 (kv, 1H), 5, 32-5.44 (m, 10N).

Example 55. Preparation of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)propanoic acid:

.

Trifluoroacetic acid (2 ml) was added to a solution of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)propanoate (1.40 g, 3.36 mmol) in dichloromethane (10 ml), kept under nitrogen atmosphere and the reaction mixture was stirred at room temperature for three hours. Diethyl ether (50 ml) was added and the organic phase was washed with water (30 ml), dried (Na ₂ SO ₄ ) and concentrated. The residue was flash chromatographed on silica gel using progressively polar mixtures of heptane, ethyl acetate and formic acid (95:5:0.25 -> 80:20:1) as eluent. Concentration of the appropriate fractions gave 0.67 g of slightly contaminated product. This material was dissolved in heptane (15 ml), washed three times with water (5 ml), dried (Na ₂ SO ₄ ), filtered and concentrated to give 0.50 g (41% yield) of the title compound as an oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.99 (t, 3H), 1.40-1.48 (m, 5H), 1.67 (m, 2H), 2.09 (m, 4H), 2.80-2.60 (m, 8H), 3.53 (m, 2H), 4.01 (sq, 1H), 5.31-5.47 (m, 10H); MS (electrospray): 359.2 [M-H]-.

Example 56. Preparation of tert-butyl 2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)-2-methylpropanoate:

.

Mixture of (5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-ol (0.83 g, 3.14 mmol), tetrabutylammonium chloride (0.24 g, 0.85 mmol) and tert-butyl 2-bromoisobutyrate (3.50 g, 15.7 mmol) were dissolved in toluene (15 ml) and placed under nitrogen atmosphere. Aqueous sodium hydroxide solution (50%, 5 ml) was added slowly with vigorous stirring at room temperature. The resulting mixture was heated to 60°C and stirred for six hours. The mixture was cooled, water was added and extracted three times with ether. The combined organic extract was washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of 5-10% ethyl acetate in heptane as eluent. Concentration of appropriate fractions gave 0.60 g (44% yield) of the title compound as an oil. MS (electrospray): 453.3 [M+Na]+.

Example 57. Preparation of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaenyloxy)-2-methylpropanoic acid:

.

Trifluoroacetic acid (5 ml) was added to a solution of tert-butyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaenyloxy)-2-methylpropanoate (600 mg (1 .39 mmol) in dichloromethane (20 ml) under nitrogen and the reaction mixture was stirred at room temperature for two hours. Water was added and the aqueous phase was extracted twice with dichloromethane. The combined organic extract was washed with brine, dried (Na ₂ SO ₄ ). filtered and concentrated. The residue was purified by flash chromatography on silica gel using a mixture of heptane, ethyl acetate and formic acid (80:20:1) as eluent. The appropriate fractions were concentrated and the residue (135 mg) was further purified by flash chromatography on silica gel using a gradient. 5-10% mixture of ethyl acetate and formic acid (95:5) in heptane as eluent. The concentration of the corresponding fractions gave 80 mg of slightly contaminated product. This substance was dissolved in heptane (5 ml), washed twice with water (5 ml), dried ( Na ₂ SO ₄ ), filtered and concentrated to give 40 mg (yield 8%) of the title compound as an oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.99 (t, 3H), 1.47 (s, 6H), 1.64 (m, 2H), 2.07 (m, 4H), 2 .81-2.88 (m, 8H), 3.46 (t, 2H), 5. 29-5.44 (m, 10H); MS (electrospray): 373.3 [M-H] -.

Example 58. Preparation of tert-butyl-2-ethyl-2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy) butanoate:

.

Tert-butyl 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy)butanoate (480 mg, 1.11 mmol) was added dropwise to for 30 minutes to a solution of lithium diisopropylamine (LDA) (2.0 M, 750 µl, 1.50 mmol) in dry tetrahydrofuran (10 ml) maintained at -70°C under nitrogen atmosphere. The reaction mixture was stirred for 30 minutes. Ethyl iodide (312 mg, 2.00 mmol) was added in one portion and the resulting mixture was warmed to ambient temperature for 1 hour. The reaction mixture was stirred at ambient temperature for 17 hours. The mixture was poured into saturated NH ₄ Cl (aq) (50 ml) and extracted with heptane (2 x 50 ml). The combined organic phases were washed successively with brine (50 ml), 0.25 M HCl (50 ml) and again with brine (50 ml), dried (MgSO ₄ ), filtered and concentrated. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (100:0 -> 95:5) as eluent. Concentration of appropriate fractions gave 343 mg (67% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl ₃ ): δ 0.84 (t, 6H), 0.99 (td, 3H), 1.35-1.55 (m, 11H), 1.54-1.69 (m, 2H), 1.68-1.87 (m, 4H), 1.99-2.24 (m, 4H), 2.74-2.99 (m, 8H), 3.31 (t , 2H), 5.23-5.52 (m, 10H); MS (electrospray): 401.3 [M-1] -.

Example 59. Preparation of 2-ethyl-2-((5Z, 8Z, 11Z, 14Z, 17Z)-icoso-5,8,11,14,17-pentaen-1-yloxy)butanoic acid:

.

Mixture of formic acid (5 ml) and tert-butyl-2-ethyl-2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoate (250 mg, 0.55 mmol) was stirred vigorously under nitrogen atmosphere at room temperature for 4.5 hours. Formic acid was removed in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate (100:0 -> 80:20) as eluent. Concentration of the appropriate fractions gave 163 mg (74% yield) of the title compound as an oil. ^1H NMR (300 MHz, CDCl3): δ 0.86 (t, 6H), 0.99 (t, 3H), 1.36-1.57 (m, 2H), 1.68 (dd, 2H) , 1.73-1.98 (m, 4H), 2.11 (tt, 4H), 2.70-3.01 (m, 8H), 3.39 (t, 2H), 5.20-5 .56 (m, 10H). MS (electrospray): 481.4 [M+Na]+.

Example 60: Preparation of tert-butyl 2-(((3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-yl)oxy)butanoate

Step a) (8Z, 11Z, 14Z, 17Z)-5,6-dihydroxyeicose-8,11,14,17-tetraenoic acid

.

A solution containing 6-((3Z, 6Z, 9Z, 12Z)-1-iodopentadeca-3,6,9,12-tetraen-1-yl)tetrahydro-2H-pyran-2-one (12.5 g, 29 .2 mmol)) and KOH (5.73 g, 102 mmol) in a mixture of MeOH (150 ml) and water (7.5 ml) were stirred at 60°C under nitrogen atmosphere for 4 hours. The reaction mixture was cooled to room temperature, acidified with 2 M HCl (aq) and extracted with EtOAc (300 ml). The organic phase was washed with water (300 ml with a little brine), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give 9.8 g (100% yield) as an oil.

Step b) (3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-ol

.

Sodium periodate (9.34 g, 43.7 mmol) was added to a solution of (8Z, 11Z, 14Z, 17Z)-5,6-dihydroxyeicose-8,11,14,17-tetraenoic acid (9.8 g, 29, 1 mmol) in THF: water (150 ml) at 0°C under nitrogen and stirred for 1 hour. The reaction mixture was extracted with EtOAc (300 ml). The organic layer was separated and washed with dilute brine (200 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was dissolved in MeOH (100 ml) and cooled to 0°C. NaBH4 (3.31 g, 87 mmol) was added carefully in portions and the mixture was stirred for 30 minutes at 0°C. Water (300 ml) was carefully added and the mixture was extracted with EtOAc (2 x 200 ml). The organic phases were combined and washed with water (200 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 20% ethyl acetate in heptane as eluent. Concentration of the appropriate fractions gave 4.06 g (63% yield) of the title compound as an oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 0.91 (t, 3H), 1.97-2.05 (m, 2H), 2.27-2.32 (m, 2H), 2.73- 2.81 (m, 6H), 3.57-3.62 (m, 2H), 5.17-5.41 (m, 7H), 5.42-5.56 (m, 1H).

Step c) tert-butyl 2 - (((3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-yl)oxy)butanoate

.

Tetrabutylammonium chloride (0.10 g, 0.33 mmol) was added to a solution of ((3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-ol (0.22 g, 1. 00 mmol) in toluene (10 ml) at ambient temperature under nitrogen, followed by the addition of tert-butyl 2-bromobutyrate (1.11 g, 5.00 mmol) aqueous sodium hydroxide (50% (w/w). ), 4 ml) was added with vigorous stirring at room temperature. The resulting mixture was heated to 50°C and stirred for two hours, followed by stirring at ambient temperature overnight, water was added and the aqueous phase was extracted with diethyl ether. The phases were separated and the aqueous phase was extracted two more times with diethyl ether. The organic phases were combined and washed with water and then with brine, then dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel. 5% ethyl acetate in heptane as eluent. Concentration of the appropriate fractions gave 0.30 g (83%) of the title compound as an oil.

Example 61: Preparation of 2-(((3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-yl)oxy)butanoic acid:

.

Trifluoroacetic acid (2 ml) was added to a solution of tert-butyl 2-(((3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraen-1-yl)oxy)butanoate in dichloromethane (10 ml ) in a nitrogen atmosphere. The reaction mixture was stirred at room temperature for one hour, then water was added and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined and washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using 5% ethyl acetate in heptane (added 1% HCOOH) as eluent. Concentration of the appropriate fractions gave 0.18 g (71% yield) of the title compound as an oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.90-1.05 (2xt, 6H), 1.75-1.90 (m, 2H), 2.05-2.15 (m, 2H ), 2.30-2.50 (m, 2H), 2.85 (m, 6H), 3.60 (m, 2H), 3.85 (t, 1H), 5.25-5.60 ( m, 8H).

Example 62: Preparation of 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-nonadeca-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid:

.

Step 1:

A solution of deca-2,5,8-triin-1-ol (0.94 g, 6.43 mmol) in THF (100 ml), cooled to 0°C under nitrogen, was added to methanesulfonyl chloride (720 µl, 9. 24 mmol) followed by dropwise addition of TEA (1.97 mL, 14.13 mmol) over 15 minutes. The mixture was stirred at 0°C for 2 hours. Then the mixture was poured into water: ice 1:1 (200 grams) and extracted with diethyl ether (2x100). The combined organic phases were washed with saturated aqueous NaHCO ₃ (200 ml), water (200 ml) and brine (200 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo to give the crude product, which was then purified by dry evaporation on silica gel. Fractions 1-3 were collected using heptane, fractions 4-6 using EtOAc:heptane 2.5:100, fractions 7-10 using EtOAc:heptane (5:100), and fraction 11 using EtOAc. The appropriate fractions were combined and concentrated. 1.07 g (4.77 mmol, yield 74.1%) of nona-2,5,7-triin-1-yl methanesulfonate was obtained.

Step 2:

To a room temperature solution of ethylmagnesium bromide (4.00 mL, 12.00 mmol) diluted with dry THF (25 mL), a solution of propargyl alcohol (0.35 mL, 5.99 mmol) was added in portions over 20 minutes. The reaction mixture was refluxed for 90 minutes, then cooled to 0°C for 30 minutes. A catalytic amount of copper(I) bromo(dimethylsulfide) (330 mg, 1.605 mmol) was added and the cooling bath was removed. After stirring for 15 minutes, nona-2,5,7-triin-1-ylmethanesulfonate (1.05 g, 4.68 mmol) dissolved in THF (5 ml) was added dropwise over 5 minutes while heating. The reaction mixture was heated to reflux and stirred at reflux overnight. The reaction mixture was cooled to ambient temperature and the reaction was stopped by careful addition of 50 ml of saturated aqueous NH ₄ Cl solution. The resulting mixture was transferred to a separatory funnel containing diethyl ether (100 ml). The organic phase was separated and the aqueous phase was extracted twice with diethyl ether (2 x 100 ml). The combined organic phases were washed with brine (50 ml) and dried (Na ₂ SO ₄ ). The residue was concentrated in vacuo before purification by flash silica gel chromatography. The appropriate fractions were combined and concentrated to give 0.49 g (2.65 mmol, 56.6% yield) of dodeca-2,5,8,10-tetrain-1-ol.

Step 3:

A solution of dodeca-2,5,8,10-tetrain-1-ol (465 mg, 2.52 mmol) in THF (30 ml), cooled to 0°C under nitrogen, was added methanesulfonyl chloride (0.256 ml, 3.28 mmol) followed by dropwise addition of TEA (0.704 mL, 5.05 mmol) over 15 minutes. The mixture was stirred at 0°C for 1 hour. The mixture was then poured into a 1:1 water:ice mixture (100 grams) and extracted with diethyl ether (2 x 100 ml). The combined organic phases were washed with saturated aqueous NaHCO ₃ (200 ml), water (200 ml) and brine (200 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo to obtain 622 mg of crude product, which was then purified dry evaporation. Fractions 1-3 were collected using heptane, fractions 4-6 using EtOAc:heptane (2.5:100), fractions 7-10 using EtOAc:heptane (5:100) and fractions 11 using EtOAc. The appropriate fractions were combined and concentrated. 466 mg (1.78 mmol, yield 70.4%) of trideca-2,5,8,11-tetrain-1-yl methanesulfonate were obtained.

Step 4:

To a solution/suspension of cesium carbonate (553 mg, 1.696 mmol), sodium iodide (280 mg, 1.866 mmol) and copper(I) iodide (323 mg, 1.696 mmol) in DMF (dry) (15 ml), 5-inoate (262 mg, 1.866 mmol) in DMF (dry) (5 ml). The mixture was stirred at room temperature under nitrogen atmosphere for 40 minutes. Trideca-2,5,8,11-tetrain-1-yl methanesulfonate (445 mg, 1.696 mmol) in DMF (dry) (5 ml) was added and the mixture was stirred at room temperature overnight. Saturated NH ₄ Cl (200 ml) was added and the mixture was extracted with EtOAc:heptane (1:1, 3 x 150 ml). The combined organic phases were washed with brine (200 ml), dried (MgSO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and ethyl acetate as eluent, starting with a solution of 2% EtOAc in heptane. The appropriate fractions were combined and concentrated to give 51 mg (0.166 mmol, 9.8% yield) ethyl nonadec-5,8,11,14,17-pentayoate.

Step 5:

A solution of ethylnonadec-5,8,11,14,17-pentayoate (60 mg, 0.196 mmol) in heptane (10 ml) and toluene (15 ml) was bubbled with N ₂ gas before adding quinoline (10 μl, 0.084 mmol) and Lindlar catalyst (50 mg, 0.023 mmol), which were added under nitrogen atmosphere. The suspension was stirred under H ₂ atmosphere (1 atm) for 15 hours. The reaction mixture was filtered through celite and evaporated in vacuo. The crude material was purified by flash chromatography on silica gel using 2% EtOAc in heptane as eluent. The appropriate fractions were combined and concentrated to give (5Z, 8Z, 11Z, 14Z, 17Z)-ethylnonadeca-5,8,11,14,17-pentaenoate (24.6 mg, 0.078 mmol, 39.7% yield) as an oil . ^1H NMR (400 MHz, chloroform-d) δ 1.27 (t, 2H), 1.59-1.70 (m, 2H), 1.72 (s, 0H), 2.31 (d, 1H ), 2.72-2.95 (m, 3H), 4.15 (d, 1H), 5.40 (dq, 4H).

Step 6:

LAG ( LAH, lithium aluminum hydride) (8 mg, 0.211 mmol) in one serving. The reaction mixture was stirred for 30 minutes at 0°C and then 90 minutes at room temperature. The reaction mixture was poured into saturated NH ₄ Cl (aq) (10 ml) with ice (10 g) and the mixture was acidified using 10% HCl (aq) to pH ~ 1-2. The mixture was extracted twice with Et ₂ O (25 ml) and the combined organic phases were washed with water (25 ml) and brine (25 ml), then dried (MgSO ₄ ), filtered and concentrated in vacuo to give (5Z, 8Z, 11Z, 14Z, 17Z)-nonadeca-5,8,11,14,17-pentaen-1-ol (15.5 mg, 0.056 mmol, 81% yield) as an oil.

Step 7:

2-Bromobutyric acid (100 mg, 0.599 mmol) was added to a solution of (5Z, 8Z, 11Z, 14 Z, 17Z)-nonadeca-5,8,11,14,17-pentaen-1-ol (8 mg, 0.029 mmol ) in tetrahydrofuran (10 ml). The mixture was cooled to 5°C, after which a 17% (w/w) solution of sodium tert-butoxide (500 μl, 0.678 mmol) in tert-butyl methyl ether (30 ml) was added dropwise over 5 minutes. After 3 hours, additional sodium tert-butoxide (100 μl 0.136 mmol) was added and the reaction mixture was stirred for another 3 hours. The reaction was quenched by the addition of formic acid (0.100 ml, 2.61 mmol) and water (10 ml), and the resulting mixture was extracted with tert-butyl methyl ether (30 ml). The organic phase was washed four times with water (4 x 10 ml), dried (MgSO ₄ ), filtered and evaporated in vacuum. The concentrate was dissolved in Me-THF (50 ml) and washed with saturated aqueous NH ₄ Cl (20 ml) four times. The organic phase was dried (MgSO ₄ ), filtered and evaporated in vacuum, obtaining 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-nonadeca-5,8,11,14,17-pentaen-1-yl)oxy ) butanoic acid (6 mg, 0.017 mmol, 57.1%) as an oil. HRMS (electrospray), [MH] -: Calculated: 359.2586, found: 359.2578.

Example 63: Preparation of 2-(((6Z, 9Z, 12Z, 15Z)-octadeca-6,9,12,15-tetraen-1-yl)oxy)butanoic acid:

.

Tetrabutylammonium hydroxide (TBAOH) (2 mg, 0.008 mmol), tert-butyl 2-bromobutanoate (190 mg, 0.819 mmol) and NaOH (50 wt%, 1 ml) and the mixture was stirred for 3 hours at 45°C. More tert-butyl 2-bromobutanoate (190 mg, 0.819 mmol) was added to the reaction mixture and the reaction mixture was stirred for 24 hours at 45°C. The mixture was cooled to room temperature and extracted twice with Et ₂ O (2 x 30 ml). The combined organic extracts were washed with water (3 x 25 ml) and brine (25 ml), filtered and evaporated in vacuo to give the crude product tert-butyl 2-(((6Z, 9Z, 12Z, 15Z)-octadeca-6) ., 9,12,15-tetraen-1-yl) oxy) butanoate. The crude product tert-butyl 2-(((6Z, 9Z, 12Z, 15Z)-octadeca-6,9,12,15-tetraen-1-yl)oxy)butanoate was dissolved in formic acid (FA, 5 ml) and the resulting the solution was stirred at room temperature for 3 hours. The mixture was concentrated in vacuo at 25°C and the residue was dissolved in EtOAc (30 ml). The solution was washed with water (4x25 ml), diluted with EtOAc (20 ml) and dried (MgSO ₄ ). The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel using progressively polar mixtures of heptane and acetone (1:0 to 5:1) as eluent. The fractions were combined and evaporated in vacuum. The residue was dissolved in FA (5 ml) and stirred at room temperature for 3 hours, after which the solvent was evaporated in vacuo. EtOAc (30 ml) was added to the residue, washed with water (4 x 25 ml), brine (25 ml), dried (MgSO ₄ ) and evaporated in vacuo. The residue was purified by preparative HPLC using 85% MeCN in water as eluent. The appropriate fractions were combined and concentrated. The residue was dissolved in EtOAc, dried (MgSO ₄ ), filtered and concentrated in vacuo. The product was dissolved in Et ₂ O and concentrated in vacuo to give 10 mg (yield 8%) of the title compound. ^1H NMR (400 MHz, CDCl ₃ ) δ 1.09-0.81 (m, 6H), 1.41 (dt, 4H), 1.67 (d, 2H), 1.97-1.74 ( m, 2H), 2.10 (t, 4H), 2.84 (sq, 6H), 3.55-3.42 (m, 1H), 3.65-3.54 (m, 1H), 3 .88 (dd, 4.8 Hz, 1H), 5.53-5.25 (m, 8H). HRMS (electrospray), [MH] -: Calculated: 347.2592, detected: 347.2576.

Example 64: Preparation of 2-(((4Z, 7Z, 10Z, 13Z, 16Z)-nonadeca-4,7,10,13,16-pentaen-1-yl)oxy)butanoic acid

Step 1:

.

To a solution of (4Z, 7Z, 10Z, 13Z, 16Z)-nonadeca-4,7,10,13,16-pentaen-1-ol (446 mg, 1.625 mmol) and tert-butyl-2-bromobutyrate (501 mg) ., 2.246 mmol) in toluene (5 ml) and tetrabutylammonium hydrogen sulfate (102 mg, 0.300 mmol) was added. Sodium hydroxide (2.5 mL, 46.9 mmol) was added with stirring and the reaction mixture was heated directly to 50°C. After 1 hour, additional tert-butyl 2-bromobutyrate (533 mg, 2.389 mmol) was added and the reaction was left for a further 3 hours, then cooled with stirring overnight. Et ₂ O (50 ml) was added and emulated. Saturated NH ₄ Cl (aq) (50 ml) was added and the mixture was shaken. The phases were separated and the aqueous phase was extracted with Et ₂ O (50 ml). The combined organic phase was washed with 5% (wt%) NaOH (aq, 2 x 50 ml), dried (MgSO ₄ ), filtered and evaporated. The residue was dried under vacuum for 5 days, then diluted with heptane (1 ml) and purified by dry evaporation using silica gel (5 grams), eluting first with 2x50 ml heptane and then 6x50 ml heptane: EtOAc (95:5) for a total of 8 factions. Fraction 3-4 was collected to give tert-butyl 2-((4Z, 7Z, 10Z, 13Z, 16Z)-nonadeca-4,7,10,13,16-pentaenyloxy)butanoate (455 mg, 1.092 mmol, yield 67, 2%) after evaporation. ^1H NMR (300 MHz, chloroform-d) δ 0.98 (td, 6H), 1.49 (s, 9H), 1.57-1.84 (m, 4H), 2.00-2.28 (m, 4H), 2.74-2.94 (m, 8H), 3.33 (dt, 1H), 3.50-3.70 (m, 2H), 5.24-5.50 (m, 10H).

Step 2:

A mixture of tert-butyl 2-((4Z, 7Z, 10Z, 13Z, 16Z)-nonadeca-4,7,10,13,16-pentaenyloxy)butanoate (373 mg, 0.895 mmol) in HCOOH (10 ml) was stirred vigorously in for 2.5 hours. The mixture was concentrated after 3 hours of total reaction time, diluted with ethyl acetate (50 ml) and washed with water (3 x 50 ml) until the aqueous phase was pH neutral. The organic phase was dried (MgSO ₄ ), filtered and concentrated in vacuo. Purification was carried out by dry flash chromatography using silica gel (8 g) and eluting with heptane: EtoAc 90:10 (3 x 30 ml), then 80:20 (7 x 50 ml). Fractions 4-6 were collected and concentrated in vacuo to give 2-((4Z, 7Z, 10Z, 13Z, 16Z)-nonadeca-4,7,10,13,16-pentaenyloxy)butanoic acid (122 mg, 0.338 mmol, yield 37.8%). ^1H NMR (300 MHz, chloroform-d) δ 1.00 (td, 2.6 Hz, 6H), 1.62-1.79 (m, 2H), 1.79-1.92 (m, 2H ), 1.99-2.13 (m, 2H), 2.19 (dd, 2H), 2.85 (dtd, 8H), 3.47-3.67 (m, 2H), 3.88 ( dd, 1H), 5.31-5.47 (m, 10H). HRMS (electrospray), [MH] -: Calculated: 359.2586, found: 359.2590.

Example 65: Preparation of 2-(((8Z, 11Z, 14Z)-octadeca-8,11,14,17-tetraen-1-yl)oxy)butanoic acid:

.

Step 1:

To a solution of cesium carbonate (2.67 g, 8.20 mmol), NaI (1.23 g, 8.20 mmol) and copper(I) iodide (1.56 g, 8.20 mmol) in DMF (20 ml ) methyl non-8-inoate (1.38 g, 8.20 mmol) was added to DMF (5 ml). The mixture was stirred at room temperature under nitrogen atmosphere for 40 minutes. Nona-8-en-2,5-diin-1-yl methanesulfonate (1.74 g, 8.20 mmol) in DMF (5 ml) was added and the mixture was stirred at room temperature overnight. Saturated NH ₄ Cl (200 ml) was added and the mixture was extracted with EtOAc: heptane (1:1, 3 x 150 ml). The combined organic phase was washed with brine (100 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography (heptane/EtOAc 92/8) yielded methyloctadeca-17-ene-8,11,14-trinoate (1.43 g, 5.03 mmol, 61.3% yield) as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.80-5.67 (m, 1H), 5.29-5.18 (m, 1H), 5.08-4.99 (m, 1H), 3.60 (s, 3H), 3.15-3.09 (m, 2H)), 3.09-3.05 (m, 2H), 2.93-2.85 (m, 2H), 2 .24 (t, 2H), 2.11-2.05 (m, 2H), 1.60-1.52 (m, 2H), 1.46-1.38 (m, 2H), 1, 35-1.23 (m, 4H). MS (electrospray): 307.1 [M+Na]+.

Step 2:

A suspension of Lindlar Catalyst (1.063 g, 0.499 mmol) in heptane (15 ml) under nitrogen was added to quinoline (0.177 ml, 1.498 mmol) and a solution of methyloctadeca-17-ene-8,11,14-triinoate (1.42 g, 4.99 mmol) in heptane (5 ml). The mixture was stirred under H ₂ (1 atm) overnight. The mixture was filtered and concentrated in vacuo. Flash chromatography (heptane/EtOAc 98.5/1.5) gave (8Z, 11Z, 14Z)-methooctadeca-8,11,14,17-tetraenoate (0.830 g, 2.86 mmol, 57.2% yield) in the form of oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.84-5.65 (m, 1H), 5.43-5.19 (m, 6H), 5.05-4.85 (m, 2H), 3.60 (s, 3H), 2.81-2.68 (m, 6H)), 2.23 (t, 2H), 2.02-1.92 (m, 2H), 1.59-1 .52 (m, 2H), 1.34-1.18 (m, 6H). MS (electrospray): 313.2 [M+Na]+.

Step 3:

A solution of (8Z, 11Z, 14Z)-methyloctadeca-8,11,14,17-tetraenoate (830 mg, 2.86 mmol) in THF (2 ml) was added dropwise to a cooled (0°C) suspension of LAH (114 mg , 3.00 mmol) in THF (10 ml) under nitrogen. The mixture was stirred for 40 minutes at 0°C and carefully poured into cold saturated NH ₄ Cl (20 ml). The aqueous layer was acidified with HCl (2M) to pH ~ 2. The phases were separated and the aqueous phase was extracted with heptane (3 × 20 ml). The combined organic phase was washed with brine (20 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give (8Z, 11Z, 14Z)-octadeca-8,11,14,17-tetraen-1-ol (730 mg, 2.78 mmol, 97% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.84-5.66 (m, 1H), 5.43-5.20 (m, 6H), 5.03-4.82 (m, 2H), 3.57 (t, 2H), 2.84-2.62 (m, 6H), 2.06-1.90 (m, 4H), 1.58-1.42 (m, 3H), 1.33-1.20 (m, 6H). MS (electrospray): 285.1 [M+Na]+.

Step 4:

NaOH solution (2 mL, 37.9 mmol) was slowly added to a vigorously stirred solution of (8Z, 11Z, 14Z)-octadeca-8,11,14,17-tetraen-1-ol (720 mg, 2.74 mmol) tert -butyl-2-bromobutanoate (918 mg, 4.12 mmol) and tetrabutylammonium hydrogen sulfate (279 mg, 0.823 mmol) in toluene (8 ml) under nitrogen atmosphere. The mixture was heated at 40°C overnight. Additional tert-butyl 2-bromobutanoate (306 mg, 1.372 mmol) was added twice at 5 hours and 24 hours. After 48 hours the mixture was cooled to 5-10°C and saturated NH ₄ Cl (20 ml) was added. The phases were separated and the aqueous phase was extracted with EtOAc (50 ml). The combined organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography (heptane/EtOAc 99/1) gave tert-butyl 2-(((8Z,11Z,14Z)octadeca-8,11,14,17-tetraen-1-yl)oxy)butanoate (675 mg. 1.666 mmol, yield 60.8%) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.86-5.65 (m, 1H), 5.41-5.21 (m, 6H), 5.04-4.83 (m, 2H), 3.57-3.46 (m, 2H), 3.30-3.19 (m, 1H), 2.81-2.68 (m, 6H), 2.02-1.89 (m, 4H ), 1.71-1.60 (m, 2H), 1.54-1.50 (m, 2H), 1.41 (s, 12H), 1.32-1.20. (m, 8H), 0.89 (t, 3H).

Step 5:

A solution of tert-butyl 2-(((8Z,11Z,14Z)-octadeca-8,11,14,17-tetraen-1-yl)oxy)butanoate (675 mg, 1.666 mmol) in HCOOH (6.75 ml, 179 mmol) was stirred for 21 hours at 40°C under nitrogen atmosphere. The reaction mixture was evaporated under reduced pressure and purified by preparative HPLC, eluting with a mixture of CH ₃ CN: H ₂ O (90: 10) with 0.02% HCOOH. Fractions containing pure product were evaporated under reduced pressure to remove CH ₃ CN. Tert-butyl methyl ether and brine were added to the residue, and the phases were separated. The organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated under reduced pressure, yielding 2-((8Z,11Z,14Z)-octadeca-8,11,14,17-tetraen-1-yloxy)butanoic acid (163 mg , 0.464 mmol, yield 27.8%) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.89-5.70 (m, 1H), 5.51-5.22 (m, 6H), 5.00 (dd, 2H), 3.84 ( t, 1H), 3.62-3.38 (m, 2H), 2.79 (m, 6H), 2.12-1.95 (m, 2H), 1.88-1.75 (m, 2H), 1.64-1.58 (m, 2H), 1.31 (s, 8H), 0.96 (t, 3H). HRMS (electrospray) [MH] -: Calculated: 347.2586, detected: 347.2589.

Example 66: Preparation of 2-(((5Z, 8Z, 11Z, 14Z)-nonadeca-5,8,11,14-tetraen-1-yl)oxy)butanoic acid:

.

Step 1:

A solution of (5Z, 8Z, 11Z, 14Z)-ethylnonadeca-5,8,11,14-tetraenoate (21 g, 65.9 mmol) in THF (50 ml) was added dropwise over 15 minutes to cooled (0°C ) suspension of LAH (2.503 g, 65.9 mmol) in dry THF (300 ml) under nitrogen. The mixture was stirred for 1 hour at 0°C and carefully poured into cold saturated NH ₄ Cl (300 ml). The aqueous layer was acidified with HCl (2 M) to pH ~ 2. The phases were separated and the aqueous phase was extracted with heptane (2 × 250 ml). The combined organic phase was washed with brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give (5Z, 8Z, 11Z, 14Z)-nonadeca-5,8,11,14-tetraene-1 -ol. (17.9 g, 64.7 mmol, 98% yield) as an oil. ^1H NMR (300 MHz, CDCl3) δ 5.46-5.11 (m, 8H), 3.68-3.57 (m, 2H), 2.89-2.69 (m, 6H), 2 .12-1.98 (m, 4H), 1.62-1.51 (m, 2H), 1.47-1.23 (m, 6H), 0.91-0.85 (m, 3H ).

Step 2:

To a solution of (5Z, 8Z, 11Z, 14Z)-nonadeca-5,8,11,14-tetraen-1-ol (17.9 g, 64.7 mmol) in THF (350 ml) was added 2-bromobutanoic acid ( 10.35) ml, 97 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (17.42 g, 181 mmol) (17% in MTBE (120 ml)) was added over 25 minutes, maintaining the temperature between 8-10°C. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (2.489 g, 25.9 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutyric acid (6.90 ml, 64.7 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (4.98 g, 51.8 mmol) (17% in MTBE (40 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (2.489 g, 25.9 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. HCOOH (3 ml) and then 2 M HCl (aq) until pH ~ 2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 300 ml). The combined organic phase was washed with water (300 ml), saturated NaHCO ₃ (4x200 ml) and brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography (heptane/EtOAc/HCOOH 90/10/0.5), the appropriate fractions were combined and concentrated in vacuo. The residue was then further purified by preparative HPLC using 88% MeCN (containing 0.02% HCOOH) in water (containing 10% MeCN and 0.02% HCOOH) as eluent. After removing the solvents, 10.16 g of 2-(((5Z, 8Z, 11Z, 14Z)-nonadeca-5,8,11,14-tetraen-1-yl)oxy)butanoic acid (26.8 mmol, yield 41.5%) in the form of oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 9.93 (s, 1H), 5.51-5.21 (m, 8H), 3.84-3.80 (m, 1H), 3.62- 3.54 (m, 1H), 3.50-3.39 (m, 1H), 2.96-2.61 (m, 6H), 2.12-2.01 (m, 4H), 1. 90-1.71 (m, 2H), 1.69-1.57 (m, 2H), 1.49-1.37 (m, 2H), 1.36-1.23. (m, 4H), 0.97 (t, 3H), 0.91-0.84 (m, 3H). MS (electrospray): 361.2 [M-H] -.

Example 67: Preparation of 2-(((8Z, 11Z, 14Z)octadeca-8,11,14-trien-1-yl)oxy)butanoic acid

.

Step 1

To a stirred solution of Cs ₂ CO ₃ (106 g, 326 mmol), sodium iodide (44.8 g, 299 mmol) and copper(I) iodide (56.9 g, 299 mmol) in DMF (750 ml) under nitrogen atmosphere At ambient temperature, ethyl non-8-inoate (49.5 g, 272 mmol) was added followed by 1-bromomonone-2,5-diyne (54.1 g, 272 mmol). The mixture was stirred overnight at ambient temperature and poured into saturated NH ₄ Cl (600 ml). The mixture was extracted with MTBE (3x300 ml) and the combined organic phase was washed with brine (400 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography (heptane/EtOAc 95/5-92/8-90/10) provided ethyl octadeca-8,11,14-trinoate (52.8 g, 176 mmol, 64.7% yield) as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.09 (q, 2H), 3.15-3.06 (m, 4H), 2.26 (t, 2H), 2.15-2.06 ( m, 4H), 1.63-1.56 (m, 2H), 1.53-1.42 (m, 4H), 1.40-1.26 (m, 4H), 1.22 (t, 3H), 0.93 (t, 3H). MS (electrospray): 323.2 [M+Na]+.

Step 2:

To a stirred solution of nickel acetate tetrahydrate (7.67 mL, 55.4 mmol) in absolute EtOH (150 mL) at room temperature was added solid NaBH ₄ (2.097 g, 55.4 mmol) under H ₂ (1 atm). The resulting suspension was stirred for 30 minutes before adding ethylenediamine (12.38 ml, 185 mmol). After addition was complete, the suspension was stirred for a further 15 minutes, after which ethyl octadeca-8,11,14-triinoate (11.1 g, 36.9 mmol) in absolute EtOH (50 ml) was added. The reaction mixture was stirred at room temperature under H ₂ (1 atm) for 3 nights. Diethyl ether (500 ml) was added to dilute the reaction mixture, and the resulting mixture was then passed through a short silica gel column. The filtrate was concentrated in vacuo. The residue was purified by flash column chromatography (heptane/EtOAc 98/2) to give ethyl (8Z, 11Z, 14Z)-octadeca-8,11,14-trienoate (10 g, 32.6 mmol, 88% yield) as an oil . ^1H NMR (300 MHz, CDCl ₃ ) δ 5.46-2.23 (m, 6H), 4.10 (q, 2H), 2.87-2.67 (m, 3H), 2.26 ( t, 2H), 2.07-1.97 (m, 4H), 1.66-1.55 (m, 2H), 1.41-1.18 (m, 11H), 0.91-0, 84 (m, 3H).

Step 3:

A solution of ethyl (8Z, 11Z, 14Z)-octadeca-8,11,14-trienoate (24.6 g, 80 mmol) in THF (50 ml) was added dropwise over 15 minutes to the cooled (0°C) LAH suspension (3.05 g, 80 mmol) in dry THF (300 ml) under nitrogen. The mixture was stirred for 1 hour at 0°C. The mixture was carefully poured into cold saturated NH ₄ Cl (300 ml). The aqueous layer was acidified with HCl (2 M) to pH ~ 2. The phases were separated and the aqueous phase was extracted with heptane (2 × 250 ml). The combined organic phase was washed with brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give (8Z, 11Z, 14Z)-octadeca-8,11,14-trien-1-ol (20 .7 g, 78 mmol, yield 98%) as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 5.49-2.23 (m, 6H), 3.61 (t, 2H), 2.86-2.67 (m, 4H), 2.11- 1.92 (m, 4H), 1.59-1.49 (m, 2H), 1.43-1.21 (m, 10H), 0.94-0.83 (m, 3H).

Step 4:

To a solution of (8Z, 11Z, 14Z)-octadeca-8,11,14-trien-1-ol (20.7 g, 78 mmol) in THF (350 ml) was added 2-bromobutanoic acid (12.51 ml, 117 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (21.06 g, 219 mmol) (17% in MTBE (130 ml)) was added over 15 minutes, maintaining the temperature between 8-10°C. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (3.01 g, 31.3 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutyric acid (8.34 ml, 78 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (6.02 g, 62.6 mmol) (17% in MTBE (40 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (3.01 g, 31.3 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C.

HCOOH (3 mL) was added followed by 2 M HCl (aqueous solution) until pH ~ 2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 300 mL). The combined organic phase was washed with water (300 ml), saturated NaHCO ₃ (4 x 200 ml) and brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography (heptane/EtOAc/HCOOH 90/10/0.5) and the appropriate fractions were combined and concentrated in vacuo. The residue was then further purified by preparative HPLC using 88% MeCN (containing 0.02% HCOOH) in water (containing 10% MeCN and 0.02% HCOOH) as eluent. After removing the solvents, 10.55 g of 2-(((8Z,11Z,14Z)-octadeca-8,11,14-trien-1-yl)oxy)butanoic acid (28.8 mmol, 36.8% yield) was obtained ) in the form of oil. ^1H NMR (300 MHz, CDCl3) δ 9.70 (s, 1H), 5.47-5.25 (m, 6H), 3.85-3.81 (m, 1H), 3.60-3 .52 (m, 1H), 3.49-3.42 (m, 1H)), 2.87-2.68 (m, 4H), 2.10-1.94 (m, 4H), 1. 89-1.69 (m, 2H), 1.66-1.54 (m, 2H), 1.43-1.24 (m, 10H), 0.96 (t, 3H), 0.89 ( t, 3H). MS (electrospray): 349.2 [M-H] -.

Example 68. Preparation of 2 - (((9Z, 12Z, 15Z)-octadeca-9,12,15-trien-1-yl)oxy)butanoic acid:

.

To a solution of (9Z, 12Z, 15Z)-octadeca-9,12,15-trien-1-ol (29.6 g, 112 mmol) in THF (400 ml) was added 2-bromobutanoic acid (17.89 ml, 168 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (30.1 g, 313 mmol) (17% in MTBE (180 ml)) was added over 5 minutes, maintaining the temperature between 8-10°C. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (4.30 g, 44.8 mmol) (17% in MTBE (30 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutyric acid (11.93 ml, 112 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (8.61 g, 90 mmol) (17% in MTBE (55 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (4.30 g, 44.8 mmol) (17% in MTBE (30 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C.

HCOOH (3 mL) was added followed by 2 M HCl (aqueous solution) until pH ~ 2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 300 mL). The combined organic phase was washed with water (300 ml), saturated NaHCO ₃ (4 x 200 ml) and brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography (heptane/EtOAc/HCOOH 90/10/0.5) on silica gel. The appropriate fractions were combined and concentrated in vacuo to give 2-(((9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl)oxy)butanoic acid (28 g, 78 mmol, 69.9% output) in the form of oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 10.07 (s, 1H), 5.48-5.16 (m, 6H), 3.84-3.80 (m, 1H), 3.60- 3.53 (m, 1H), 3.48-3.40 (m, 1H)), 2.87-2.70 (m, 4H), 2.12-1.97 (m, 4H), 1 .91-1.70 (m, 2H), 1.66-1.53 (m, 2H), 1.38-1.27 (m, 10H), 0.99 0.93. (m, 6H). MS (electrospray): 373.3 [M+Na]+.

Example 69: Preparation of 2-(((6Z, 9Z, 12Z)octadeca-6,9,12-trien-1-yl)oxy)butanoic acid:

.

To a solution of (6Z, 9Z, 12Z)-octadeca-6,9,12-trien-1-ol (29.6 g, 112 mmol) in THF (400 ml) was added 2-bromobutanoic acid (17.89 ml, 168 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (30.1 g, 313 mmol) (17% in MTBE (180 ml)) was added over 25 minutes, maintaining the temperature between 8-10°C. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (4.30 g, 44.8 mmol) (17% in MTBE (30 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutyric acid (11.93 ml, 112 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (8.61 g, 90 mmol) (17% in MTBE (55 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (4.30 g, 44.8 mmol) (17% in MTBE (30 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C.

HCOOH (3 mL) was added followed by 2 M HCl (aqueous solution) until pH ~ 2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 300 mL). The combined organic phase was washed with water (300 ml), saturated NaHCO ₃ (4x200 ml) and brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography (heptane/EtOAc/HCOOH 90/10/0.5) on silica gel. The appropriate fractions were combined and concentrated in vacuo to give 2-((6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy)butanoic acid (30.2 g, 83 mmol, 74.1% yield ) in the form of oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 9.56 (s, 1H), 5.46-5.22 (m, 6H), 3.84-3.80 (m, 1H), 3.61- 3.54 (m, 1H), 3.47-3.40 (m, 1H), 2.86-2.71 (m, 4H), 2.10-1.99 (m, 4H), 1.91-1.70 (m, 2H), 1.65-1.56 (m, 2H), 1.45-1.18 (m, 10H), 0.97 (t, 3H), 0. 87 (t, 3H). MS (electrospray): 373.2 [M+Na]+.

Example 70. Preparation of 2 - (((6Z, 9Z, 12Z, 15Z, 18Z)-cheneicosa-6,9,12,15,18-pentaen-1-yl)oxy)butanoic acid:

.

To a solution of (6Z, 9Z, 12Z, 15Z, 18Z)-cheneicose-6,9,12,15,18-pentaen-1-ol (23 g, 76 mmol) in THF (350 ml) was added 2-bromobutanoic acid ( 12.15 ml, 114 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (20.46 g, 213 mmol) (17% in MTBE (140 ml)) was added over 25 minutes, maintaining the temperature between 8-10°C. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (2.92 g, 30.4 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutyric acid (8.10 ml, 76 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (5.85 g, 60.8 mmol) (17% in MTBE (45 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (2.92 g, 30.4 mmol) (17% in MTBE (25 ml)) was added over 5 minutes. The mixture was stirred for 30 minutes at 8-10°C. HCOOH (3 mL) was added followed by 2 M HCl (aq) until pH ~2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 300 mL). The combined organic phase was washed with water (300 ml), saturated NaHCO ₃ (4x200 ml) and brine (300 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography (heptane/EtOAc/HCOOH 90/10/0.5) on silica gel. The appropriate fractions were combined and concentrated in vacuo to give 2-(((6Z, 9Z, 12Z, 15Z, 18Z)-cheneicosa-6,9,12,15,18-pentaen-1-yl)oxy)butanoic acid (18, 9 g, 47.3 mmol, 62.2% yield) as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 9.85 (s, 1H), 5.48-5.21 (m, 10H), 3.84-3.81 (m, 1H), 3.61- 3.53 (m, 1H), 3.48-3.41 (m, 1H), 2.93-2.71 (m, 8H), 2.18-1.96 (m, 4H), 1. 91-1.70 (m, 2H), 1.66-1.57 (m, 2H), 1.45-1.30 (m, 4H), 0.99 0.93. (m, 6H). MS (electrospray): 411.4 [M+Na]+.

Example 71: Preparation of 2-(((4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-docosa-4,7,10,13,16,19-hexaen-1-yl)oxy)butanoic acid:

.

To a solution of (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-docosa-4,7,10,13,16,19-hexaen-1-ol (30 g, 95 mmol) in toluene (11 ml) and THF (400 ml) 2-bromoyric acid (17.28 ml, 162 mmol) was added followed by the addition of a solution of sodium tert-butoxide (25.7 g, 267 mmol) in THF (160 ml) over 35 minutes, maintaining the temperature at within 8-10°C. The reaction mixture was stirred for 40 minutes at 8-10°C. Sodium tert-butoxide (4.17 g, 43.4 mmol) in THF (60 ml) was added over 10 minutes. The resulting mixture was stirred at 8-10°C for 40 minutes, after which more 2-bromobutyric acid (10.17 ml, 95 mmol) was added in one portion. The mixture was stirred at 8-10°C for 1 hour, after which more sodium tert-butoxide (7.33 g, 76 mmol) in THF (100 ml) was added over 10 minutes, maintaining the temperature below 12°C. The reaction mixture was stirred at 10°C for 60 minutes. Sodium tert-butoxide (4.17 g, 43.4 mmol) was added in one portion and then stirred for an additional 1 hour. 2-bromobutyric acid (8.5 ml, 50 mmol) and sodium tert-butoxide (4.8 g, 50 mmol) were added and the mixture was stirred for 1 hour at 8-10°C. 2-bromobutyric acid (8.5 ml, 50 mmol) and sodium tert-butoxide (4.8 g, 50 mmol) were added and the mixture was stirred for 1 hour at 8-10°C. THF (200 ml) was added and the reaction mixture was stirred at room temperature overnight. 2-bromobutyric acid (17 ml, 100 mmol) and sodium tert-butoxide (9.6 g, 100 mmol) were added and the reaction mixture was stirred for 1 hour at 8-10°C. Formic acid (1 ml) was then added to the reaction mixture and the resulting mixture was stirred at 8-10°C for 10 minutes, then 6 M HCl was added to obtain pH ~ 2. Water (100 ml) was added, then aqueous and organic were separated phases. The aqueous phase was extracted twice with diethyl ether (2x500 ml). The combined organic phase was washed with water (3x1000 ml), saturated NaHCO ₃ (3x500 ml) and brine 1x100 ml), dried (Na ₂ SO ₄₎ , filtered and concentrated in vacuo. The residue was purified by flash chromatography (EtOAc/heptane/5% HCOOH) (90/10) on silica gel to give 2 - (((4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-dox-4,7, 10,13 ,16,19-hexaen-1-yl)hydroxy)butanoic acid (10.3 g, 25.07 mmol, 26.3% yield) as an oil. ^1H NMR (300 MHz, CDCl ₃ ) δ 5.49-5.20 (m, 12H), 3.90-3.75 (m, 1H), 3.65-3.52 (m, 1H), 3.51-3.35 (m, 1H), 2.91-2.69 (m 10H), 2.21-1.98 (m, 4H), 1.91-1.59 (m, 4H) , 1.05-0.88 (m, 6H). MS (electrospray): 423.3 [M+Na]+.

Example 72: Preparation of 2-(((8Z, 11Z, 14Z, 17Z)-eicosa-8,11,14,17-tetraen-1-yl)oxy)butanoic acid:

.

Step 1:

To a heated suspension of magnesium (0.217 g, 8.93 mmol) in THF (3 ml) was added 1,2-dibromoethane (2 drops), followed by dropwise addition of a solution of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran (2.07 g, 8.24 mmol) in THF (15 ml) under nitrogen. The mixture was refluxed for 1 hour and cooled to ambient temperature. A solution of (3Z, 6Z, 9Z, 12Z)-pentadeca-3,6,9,12-tetraenal (1.5 g, 6.87 mmol) in THF (10 ml) was added. The mixture was then stirred for 1 hour at ambient temperature. 1 M HCl was added and the mixture was extracted with EtOAc (2 x 50 ml). The combined organic phase was washed with brine (50 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography on silica gel (heptane/EtOAc 80/20-75/25) gave (8Z, 11Z, 14Z, 17Z)-1-((tetrahydro-2H-pyran-2-yl)oxy)eicosa-8, 11, 14,17-tetraen-6-ol (0.74 g, 1.895 mmol, 27.6% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.59-5.49 (m, 1H), 5.49-5.25 (m, 7H), 4.56-4.53 (m, 1H), 3.88-3.82 (m, 1H), 3.77-3.67 (m, 1H), 3.65-3.59 (m, 1H), 3.52-3.43 (m, 1H ), 3.40-3.34 (m, 1H), 2.85-2.77 (m, 6H), 2.23 (t, 2H), 2.13-1.97. (m, 2H), 1.84-1.77 (m, 1H), 1.73-1.67 (m, 1H), 1.63-1.34 (m, 12H), 0.96 (t , 3H). MS (electrospray): 413.3 [M+Na]+.

Step 2:

To a solution of (8Z, 11Z, 14Z, 17Z)-1-((tetrahydro-2H-pyran-2-yl)oxy)eicosa-8,11,14,17-tetraen-6-ol (0.74 g, 1.895 mmol) in THF (20 ml), TEA (0.528 ml, 3.79 mmol) was added, followed by methanesulfonyl chloride (0.192 ml, 2.446 mmol). The mixture was stirred under nitrogen atmosphere for 3 hours at ambient temperature. Citric acid (5%, aq, 50 ml) was added and the mixture was extracted with EtOAc (2 x 50 ml). The combined organic phase was washed with saturated Na-HCO3 (50 ml) and brine (50 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give 0.82 g of the mesylate intermediate. The mesylate intermediate was dissolved in Et ₂ O (10 ml) and added dropwise to a stirred suspension of LAH (0.288 g, 7.58 mmol) in Et ₂ O (25 ml). The mixture was stirred for 1 hour at ambient temperature. 1 M HCl (50 ml) was carefully added. The mixture was extracted with MTBE (2 x 50 ml). The combined organic phase was washed with saturated NaHCO ₃ (50 ml) and brine (50 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography on silica gel (heptane/EtOAc 97/3) gave 2-(((8Z, 11Z, 14Z, 17Z)-eicosa-8,11,14,17-tetraen-1-yl)oxy)tetrahydro-2H- pyran (0.46 g, 1.228 mmol, 64.8% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.46-5.20 (m, 8H), 4.57-4.54 (m, 1H), 3.88-3.82 (m, 1H), 3.74-3.68 (m, 1H), 3.51-3.44 (m, 1H), 3.39-3.33 (m, 1H), 2.84-2.77 (m, 6H ), 2.14-1.96 (m, 4H), 1.85-1.77 (m, 1H), 1.73-1.65 (m, 1H), 1.62. 1.46 (m, 6H), 1.37-1.24 (m, 8H), 0.96 (t, 3H). MS (electrospray): 397.3 [M+Na]+.

Step 3:

To a solution of 2-(((8Z, 11Z, 14Z, 17Z)-eicosa-8,11,14,17-tetraen-1-yl)oxy)tetrahydro-2H-pyran (0.46 g, 1.228 mmol) in EtOH (5 ml) pyridinium p-toluene sulfonate (PPTS, 0.309 g, 1.228 mmol) was added and the mixture was heated at 50°C for 2 hours. The mixture was cooled to ambient temperature. Saturated NaHCO ₃ (50 ml) was added and the mixture was extracted with EtOAc (2 x 50 ml). The combined organic phase was washed with brine (50 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Flash chromatography on silica gel (heptane/EtOAc 80/20) gave (8Z, 11Z, 14Z, 17Z)-eicose-8,11,14,17-tetraen-1-ol (0.26 g, 0.895 mmol, yield 72 .9%) in the form of oil. ^1H NMR (400 MHz, CDCl3) δ 5.41-5.17 (m, 8H), 3.57 (t, 2H), 2.84-2.68 (m, 6H), 2.06-1 .94 (m, 4H), 1.54-1.46 (m, 2H), 1.34-1.21 (m, 8H), 1.20-1.13 (m, 1H), 0.91 (t, 3H).

Step 4:

Tert-butyl 2-bromobutanoate ( 143 mg, 0.64 mmol), tetrabutylammonium hydroxide (TBAOH) (5 mg, 0.02 mmol) and NaOH (50 wt.%, 1 ml), and the mixture was heated to 45°C. 71.4 mg (0.32 mmol) tert-butyl 2-bromobutanoate was added to the reaction mixture after 1.5 hours of stirring, 71.4 mg (0.32 mmol) tert-butyl 2-bromobutanoate was added to the reaction mixture after 3 hours and 143 mg (0.64 mmol) tert-butyl 2-bromobutanoate was added to the reaction mixture after 8 hours at 45°C. After 24 hours, the mixture was cooled to room temperature and water (40 ml) was added. The resulting mixture was extracted twice with Et ₂ O (25+40 ml) and the combined organic phase was washed with water (4×25 ml) and brine (25 ml), dried (MgSO ₄ ), filtered and evaporated in vacuum. The residue was purified by flash chromatography on silica gel (heptane-heptate:acetone-5:1). The appropriate fractions were combined and concentrated to yield 15 mg of tert-butyl 2-(((8Z, 11Z, 14Z, 17Z)-eicosa-8,11,14,17-tetraen-1-yl)oxy)butanoate. This intermediate was dissolved in formic acid (5 ml) and the resulting mixture was stirred at room temperature for 1 hour and 45 minutes. The solvent was removed in vacuo and EtOAc (30 ml) was added to the residue. The solution was washed with water (4x25 ml) and brine (25 ml), dried (MgSO ₄ ), filtered and concentrated in vacuo. The residue was purified by preparative HPLC using 85% MeCN in water as eluent. The appropriate fractions were combined and concentrated to give 15 mg (0.04 mmol, 12% yield) of 2-(((8Z, 11Z, 14Z, 17Z)-eicosa-8,11,14,17-tetraen-1-yl)oxy ) butanoic acid. ^1H NMR (400 MHz, CDCl ₃ ) δ 1.00 (td, 6H), 1.49-1.20 (m, 8H), 1.64 (t, 2H), 1.96-1.73 ( m, 2H), 2.08 (dt, 4H), 2 0.84 (q, 6H), 3.55-3.43 (m, 1H), 3.65-3.54 (m, 1H), 3.87 (dd, 1H), 5.49-5.24 (m, 8H). HRMS (electrospray), [MH] -: Calculated: 375.2899, detected: 375.2892.

Example 73. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z)-eicosa-5,8,11,14-tetraen-1-yl)oxy)butanoic acid:

.

Step 1:

A solution of (5Z, 8Z, 11Z, 14Z)-ethaicose-5,8,11,14-tetraenoate (501.9 mg, 1.509 mmol) in dry THF (1 ml) was added dropwise to the cooled (0°C) LiAlH4 suspension (64.8 mg, 1.707 mmol) in dry THF (3 ml) under nitrogen. The mixture was stirred for 55 minutes at 0°C. Water (5 ml) was added dropwise and then 1 M HCl (10 ml) was added. The cooling bath was removed and the reaction mixture was stirred for 5 minutes. The reaction mixture was extracted with tert-butyl methyl ether (2×50 ml), the organic phase was washed with 1 M HCl (20 ml), dried (Na ₂ SO ₄ ), filtered and evaporated under reduced pressure to give (5Z, 8Z, 11Z). 14Z)-eicosa-5,8,11,14-tetraen-1-ol (397.7 mg, 1.369 mmol, 91% yield) as liquid. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.41-5.29 (m, 8H), 3.63 (t, 2H), 2.83-2.77 (m, 6H), 2.20- 1.91 (m, 4H), 1.61-1.54 (m, 2H), 1.46-1.41 (m, 2H), 1.35-1.28 (m, 8H), 0. 87 (t, 3H).

Step 2:

Tert-butyl 2-bromobutanoate ( 535 mg, 2.40 mmol), tetrabutylammonium hydroxide (TBAOH) (156 mg, 0.24 mmol, 40% w/w) and NaOH (50% wt, 2 ml), and the mixture was heated to 45°C . The mixture was cooled to room temperature after 12 hours and ice water (25 ml) and Et ₂ O (20 ml) were added. The phases were separated and the aqueous phase was extracted with Et ₂ O (20 ml). The combined organic phase was washed with water (25 ml) and brine (25 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo. The residue was dissolved in EtOH (20 ml) and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (heptane: acetone-1:20, heptane-acetone-1:10). The appropriate fractions were combined and concentrated to give 360 mg (0.83 mmol, 69% yield) of the intermediate tert-butyl 2 - (((5Z, 8Z, 11Z, 14Z)-eicose-5,8,11,14-tetraene-1 -yl) hydroxy) butanoic acid. 342 mg (0.79 mmol) of this intermediate were dissolved in formic acid (7 ml) and the resulting mixture was stirred at room temperature for 3 hours. The solvent was removed in vacuo and the residue was added to EtOAc (75 ml). The solution was washed with water (3×50 ml), dried (MgSO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (acetone:heptane:HOAc-10:10:1, acetone:heptane:HOAc-30:70:1). The appropriate fractions were combined and concentrated to give 181 mg (0.48 mmol, 58% yield) of 2 - (((5Z, 8Z, 11Z, 14Z)-eicosa-5,8,11,14-tetraen-1-yl ) hydroxy) butanoic acid.

Example 74: Preparation of 2-(((7Z, 10Z, 13Z, 16Z, 19Z)-docosa-7,10,13,16,19-pentaen-1-yl)oxy)butanoic acid:

.

Step 1:

A solution of (7Z, 10Z, 13Z, 16Z, 19Z)-ethyldocox-7,10,13,16,19-pentaenoate (1.5 g, 4.18 mmol) in THF (4 ml) was added dropwise to cooled (0 °C) suspension of LAH (0.159 g, 4.18 mmol) in dry THF (20 ml) under nitrogen. The mixture was stirred for 1 hour at 0°C. The mixture was carefully poured into 1 M HCl (100 ml). The phases were separated and the aqueous phase was extracted with heptane (100 ml). The combined organic phase was washed with brine (30 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give (7Z, 10Z, 13Z, 16Z, 19Z)-dokosa-7,10,13,16,19 -pentaene-1-ol (1.16 g, 3.66 mmol, 88% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.44-2.23 (m, 10H), 3.62 (t, 2H), 2.87-2.73 (m, 8H), 2.10- 1.99 (m, 4H), 1.60-1.51 (m, 2H)), 1.40-1.29 (m, 6H), 0.95 (t, 3H).

Step 2:

2 - bromobutanoic acid (0.918 g, 5.50 mmol). The reaction mixture was cooled to 8-10°C. A solution of sodium tert-butoxide (0.986 g, 10.26 mmol) in MTBE (15 ml) was added over 5 minutes. The reaction mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (0.141 g, 1.466 mmol) in MTBE (4 mL) was added. The mixture was stirred for 30 minutes at 8-10°C followed by the addition of 2-bromobutanoic acid (0.612 g, 3.66 mmol) over 5 minutes. The mixture was then stirred at 8-10°C for 30 minutes. Additional sodium tert-butoxide (0.282 g, 2.93 mmol) in MTBE (8 mL) was added. The mixture was stirred for 30 minutes at 8-10°C. Additional sodium tert-butoxide (0.141 g, 1.466 mmol) in MTBE (4 mL) was added. The mixture was stirred for 30 minutes at 8-10°C. HCOOH (0.5 mL) was added followed by 1 M HCl (aqueous solution) to give pH ~2. The phases were separated and the aqueous phase was extracted with MTBE (2 x 50 mL). The combined organic phase was washed with water (50 ml), saturated NaHCO ₃ (4 x 30 ml) and brine (30 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Preparative HPLC using 90% MeCN (containing 0.02% HCOOH) in water (containing 10% MeCN and 0.02% HCOOH) as eluent gave 2 - (((7Z, 10Z, 13Z, 16Z, 19Z) - dokosa-7,10 13,16,19-pentaen-1-yl) hydroxy) butanoic acid (1 g, 2.439 mmol, 66.6% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.24 (m, 10H), 3.84-3.81 (m, 1H), 3.59-3.64 (m, 1H), 3.47-3.41 (m, 1H), 2.90-2.73 (m, 8H), 2.11-1.98 (m, 4H), 1.90-1.71 (m, 2H ), 1.64-1.57 (m, 2H), 1.41-1.26 (m, 6H), 0.98-0.93 (m, 6H). HRMS (electrospray), [M]+: Calculated: 402.3134, detected: 402.3141.

Example 75. Preparation of (5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) - Eicosa -5,8,11,14,17-pentaen-1-yl) hydroxy) butanoate:

.

2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (14.28 g, 38.1 mmol) and (5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-ol (10.00 g, 34.7 mmol) was added to a mixture of D(+)-10-camphorsulfonic acid ( 0.530 g, 2.282 mmol) in cyclohexane (50 ml) and heated to reflux, removing water using a Dean-Stark apparatus. The reaction mixture was cooled to room temperature and purified by dry flash chromatography using silica gel (100 g) and eluting with cyclohexane (50 ml), then heptane (50 ml), 2% EtOAc in heptane (2 x 200 ml) and 4%. EtOAc in heptane (2x200 ml). The appropriate fractions were combined and concentrated in vacuo to give (5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl 2 - ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy) butanoate (20.3 g, 30.5 mmol, 88% yield) as an oil. ^1H NMR (300 MHz, chloroform-d) δ 0.99 (t, 9H), 1.45 (ddd, 4H), 1.56-1.93 (m, 6H), 2.09 (p, 8H ), 2.85 (dt, 16H), 3.34 (dt, 1H), 3.60 (dt, 1H), 3.76 (dd, 1H), 4.16 (dt, 2H), 5.05 -5.76 (m, 20H). MS (electro-spray): 645.5 [M+H]+.

Example 76. Preparation of (2R)-2-((2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoyl) hydroxy) butanoic acid:

.

Step 1:

To a solution of 2-((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yloxy)butanoic acid (400 mg, 1.07 mmol) in CDM (10 ) ml) N-methylmorpholine (235 µl, 2.15 mmol) and TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) (344 mg, 1.07 mmol) were added ). The reaction mixture was stirred for 30 minutes before tert-butyl(R)-2-hydroxybutanoate (250 mg, 2.14 mmol) was added. The resulting mixture was stirred at room temperature for 22 hours and then washed with water (10 ml), 1M HCl (10 ml), saturated NaHCO ₃ (10 ml) and brine (10 ml). The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. 161 mg (0.312 mmol, 29.2% yield) of intermediate (R) -1-(tert-butoxy)-1-oxobutan-2-yl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoate was isolated as an oil. MS (electrospray): 539.4 [M+Na]+.

Step 2:

(R) -1-(tert-butoxy)-1-oxobutan-2-yl 2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14,17-pentaene-1 yl)hydroxy)butanoate (161 mg, 0.312 mmol) was dissolved in formic acid (4 ml) and the reaction mixture was stirred at room temperature for 17 hours. EtOAc (10 ml) was added to the mixture, and the resulting mixture was washed with water (10 ml) and brine (10 ml). The organic phase was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. 60 mg (0.130 mmol, yield 41.8%) (2R) -2 - ((2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5,8,11,14,17- pentaen-1) yl) hydroxy) butanoyl) hydroxy) butanoic acid in the form of an oil. MS (electrospray): 459.3 [M-H] -.

Example 77: Preparation of (2S, 3S, 4S, 5R, 6S)-3,4,5-trihydroxy-6 - ((2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8, 11,14,17-pentaen-1-yl) oxy) butanoyl) oxy) tetrahydro-2H-pyran-2-carboxylic acid:

.

Step 1:

To a mixture of 4-methoxybenzyl (2S, 3S, 4S, 5R, 6R) -3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylate (2.47 g, 7.86 mmol), 2 - (( (5Z, 8Z, 11Z, 14Z, 17Z)-eicose-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (2.94 g, 7.85 mmol) and HATU (1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide) (3.00 g, 7.90 mmol) in acetonitrile (85 ml) was added 4- methylmorpholine (1.75 ml, 15.92 mmol). The reaction mixture was stirred for 23.5 hours under a nitrogen atmosphere at room temperature. Amberlyst-15 (4.7 mEq/g) (3.35 g) was added and the mixture was stirred for approximately 5 minutes. The reaction mixture was filtered and concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel, eluting with CH ₂ Cl ₂ -CH ₂ Cl ₂ : EtOH (95:5). The appropriate fractions were combined and concentrated in vacuo to give 2.64 g of contaminated mixture. The residue was purified by flash chromatography on silica gel, eluting with CH ₂ Cl ₂ - CH ₂ Cl ₂ : EtOH (95:5) and then CH ₂ Cl ₂ - CH ₂ Cl ₂ : EtOH (97: 3). The appropriate fractions were combined and concentrated in vacuo to give the intermediate (4-methoxycyclohexyl)methyl (2S, 3S, 4S, 5R, 6S)-3,4,5-trihydroxy-6 - ((2 - (((5Z, 8Z, 11Z , 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl) oxy) butanoyl) oxy) tetrahydro-2H-pyran-2-carboxylate (1.27 g, 1.78 mmol, 22.4% yield) as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 7.26-7.22 (m, 2H), 6.83-6.81 (m, 2H), 5.60-5.57 (m, 1H), 5.40-5.26 (m, 10H), 5.12-5.10 (m, 2H), 4.60-4.56 (m, 1H), 4.14-4.13 (m, 1H), 3.98 (t, 1H), 3.93-3.67 (m, 3H), 3.74 (s, 3H), 3.61-3.56 (m, 3H), 3.31 -3.25 (m, 1H), 2.82-2.77 (m, 8H), 2.07-2.02 (m, 4H), 1.80-1.67 (m, 2H), 1 .57-1.54 (s, 2H), 1.43-1.36 (m, 2H), 1.05-0.77 (m, 6H). MS (electrospray): 693.4 [M+Na]+.

Step 2:

(4-Methoxycyclohexyl)methyl (2S, 3S, 4S, 5R, 6S) -3,4,5-trihydroxy-6 - ((2 - (((5Z, 8Z, 11Z, 14Z, 17Z) -eicose-5, 8,11,14,17-pentaen-1-yl) oxy) butanoyl) oxy) tetrahydro-2H-pyran-2-carboxylate (1.20 g, 1.79 mmol) was treated with a solution of 10% TFA in DCM (8 ml )) at 0°C in a nitrogen atmosphere. After stirring for 3 hours at ambient temperature, the solvent was removed in vacuo. Flash chromatography (DCM/MeOH/HCOOH - 95/5/0.2) gave 500 mg of contaminated product. The product was further purified by preparative HPLC using 85% MeCN (containing 0.02% HCOOH) in water (containing 10% MeCN and 0.02% HCOOH) as eluent. The appropriate fractions were combined and concentrated to give (2S, 3S, 4S, 5R, 6S)-3,4,5-trihydroxy-6 - ((2 - ((5Z, 8Z, 11Z, 14Z, 17Z) -eicose -5 ,8,11,14,17-pentaen-1-yl) oxy) butanoyl) oxy) tetrahydro-2H-pyran-2-carboxylic acid (80 mg, 0.139 mmol, 7.8% yield) as a solid. ^1H NMR (400 MHz, MeOD) δ 5.60-5.56 (m, 1H), 5.51-5.27 (m, 10H), 3.99-3.88 (m, 2H), 3 .71-3.63 (m, 1H), 3.62-3.54 (m, 1H), 3.53-3.38 (m, 3H), 2.93-2.83 (m, 8H) , 2.19-2.07 (m, 4H), 1.93-1.83 (m, 1H), 1.81-1.72 (m, 1H), 1.69. 1.60 (m, 2H), 1.53-1.45 (m, 2H), 1.04-0.98 (m, 6H). MS (electrospray): 549.3 [M-H]-.

Example 78. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butan-1-ol:

.

A solution of butyl 2-(((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoate (1.008 g, 2.34 mmol) in dry THF (3 ml) was added dropwise to a suspension of LiAlH4 (94.9 mg, 2.50 mmol) in dry THF (10 ml) at 0°C under nitrogen atmosphere. The reaction mixture was stirred at 0°C for 1.5 hours, after which the cooling bath was removed and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was cooled in an ice bath before water (2.5 mL) and 1 M HCl (10 mL) were added dropwise. The cooling bath was removed and the mixture was stirred for 10 minutes at room temperature. The resulting mixture was extracted with heptane (2×50 ml), washed with 1 M HCl (10 ml), dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. Flash chromatography on silica gel eluting with heptane-EtOAc (90:10) gave 0.705 g (83% yield) of the title compound as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.71-4.93 (m, 10H), 3.63 (dd, 1H), 3.58-3.34 (m, 3H), 3.29- 3.23 (m, 1H), 2.95-2.65 (m, 8H), 2.18-1.98 (m, 4H), 1.88 (bs, 1H), 1.66- 1.51 (m, 3H), 1.45 (dt, 3H), 0.95 (t, 3H), 0.89 (t, 3H). MS (ESI, pos) 383 [M+Na]+.

Example 79. Preparation of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butyl acetate:

.

Mixture of 2 - (((5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butan-1-ol (300.9 mg, 0.834 mmol ), triethylamine (140 ml, 1.00 mmol) and DMAP (4.7 mg, 0.038 mmol) in dry CH ₂ Cl ₂ (5 ml) were added to acetic anhydride (95 ml, 1.00 mmol). The reaction mixture was stirred under nitrogen atmosphere at room temperature for 80 minutes. Water (10 ml) was added and the mixture was extracted with heptane (2 x 50 ml), washed with brine (20 ml), dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. Flash chromatography on silica gel eluting with heptane-EtOAc (100:1) gave 0.319 g (95%) of the title compound as an oil. ^1H NMR (400 MHz, CDCl ₃ ) δ 5.59-5.14 (m, 10H), 4.10 (dd, 1H), 4.02 (dd, 1H), 3.54 (dt, 1H) , 3.43 (dt, 1H), 3.38-3.29 (m, 1H), 2.92-2.65 (m, 8H), 2.14-1.95 (m, 4H), 2 .05 (s, 3H), 1.63-1.47 (m, 4H), 1.45-1.38 (m, 2H), 0.96 (t, 3H), 0.92 (t, 3H ). MS (ESI, pos) 425 [M+Na]+.

Biological Examples

Evaluation of Compound A in a Diet-Induced NASH Mouse Model (CDAA/High Fat Diet)

Methionine- and/choline-deficient mouse models (MCD and CDAA, respectively) are well established for studying the development and treatment of NASH in preclinical drug development. Dietary deficiency of methionine/choline, the precursors for phosphatidylcholine synthesis, results in an inability to synthesize hepatic lipoproteins for triglyceride export, resulting in severe hepatic steatosis, inflammation, and fibrosis.

Although the widely used methionine-choline-deficient diet (MCD) is capable of persistently inducing severe NASH-like liver inflammation and fibrosis in mice, it is also associated with severe weight loss (loss of both skeletal muscle and fat mass). This results in an increased risk of mortality, which poses significant challenges for long-term fibrogenesis experiments. When supplemented with a suboptimal dose of methionine (0.17%), the CDAA dietary model is able to overcome these problems and has been demonstrated to mimic human NASH in both mice and rats, consistently causing steatohepatitis, liver fibrosis, and liver cancer with less severe loss body weight.

In Biological Examples 1-7, studies were performed on male C56BL/6J mice (9 weeks old). Mice were fed either choline-rich (CS) or choline-deficient, high-fat diets (31% of total calories); "CDAA/high fat") The NASH-inducing diet was initiated 6 weeks before treatment to design the study as a treatment/reversion regimen rather than disease prevention.

In Biological Examples No. 1-5, mice were divided into 6 experimental groups (n=9 per group) and fed either CS or CDAA/high fat diet. After 6 weeks of CS- or CDAA diet, mice were orally administered Compound A or Compound N, both in 2 doses, 0.15 mmol/kg bw/day (low dose, LD) and 0.3 mmol/kg bw/day (high dose, HD). The test substances were administered orally as a supplement to a high-fat diet. Liver fibrosis was assessed by both hydroxyproline (HYP) measurements and Sirius Red (SR) morphometry. Levels of transcripts associated with fibrosis, inflammation and metabolism were measured using quantitative real-time polymerase chain reaction (qPCR).

In Biological Examples No. 6 and 7, mice were fed either CS or CDAA/high fat diet as described above. In Example #6, after 6 weeks of a CS or CDAA/high fat diet, mice were orally administered 56 mg/kg body weight per day of Compound A or Bydureon® (exenatide), which is a long-acting GLP-1R agonist that is approved for the treatment of diabetes. Type 2 and is administered subcutaneously at a dose of 0.4 mg/kg per week for six weeks. Effects on the number, thickness and length of collagen fibers in the liver were determined using a nonlinear multiphoton optical imaging system in isolated liver samples from the vehicle control (n=8) and Compound A (n=9) and GLP-1R (n) groups. =8).

In Biological Example 7, after 6 weeks of CS or CDAA/high fat diet, mice were orally administered 112 mg/kg body weight per day of Compound A or an equimolar dose of eicosapentaenoic acid (EPA). Liver lipidomic analysis was performed on isolated liver samples from the CS (n=9), Compound A (n=9) and EPA (n=8) groups.

Biological Examples Nos. 21-24 examined the effects of Compound A alone and in combination with the GLP-1 agonist exenatide. The studies were performed on male C56BL/6J mice (9 weeks old). Mice were divided into 6 experimental groups (n=9 per group) fed either CS or CDAA/high fat diet as described above. NASH-inducing CDAA/high-fat diet was started 6 weeks before treatment to evaluate treatment/reversal of the syndrome rather than prevention. Thus, after 6 weeks of CS or CDAA/high-fat diet, mice were treated with (A) oral Compound A at 0.3 mmol/kg body weight/day alone, (B) GLP-1 agonist alone, administered intraperitoneally, at dose of 0.4 mg/kg once weekly or (C) a combination of compound A at a dose of 0.3 mmol/kg body weight/day and a GLP-1 agonist administered intraperitoneally. at a dose of 0.4 mg/kg once weekly (reduced to 0.1 mg/kg for the last 3 weeks due to excessive weight loss). Compound A was administered orally as a supplement to a high-fat diet, while the GLP-1 agonist was delivered by intraperitoneal injection (ie, intraperitoneal). Levels of transcripts associated with fibrosis, inflammation and metabolism were measured using quantitative real-time polymerase chain reaction (qPCR) in isolated liver tissue.

Evaluation of Compound A in the Streptozotocin Injection and High Fat Diet Model of NASH (STAM Model)

In Biological Example No. 8, 15-day-old pathogen-free pregnant C57BL/6 mice were obtained from Charles River Laboratories in Japan Inc. (Kanagawa, Japan). NASH was induced in male mice by a single subcutaneous injection of streptozotocin (STZ) (Sigma, USA) after birth and feeding a high-fat diet (HFD; CLEA) Japan, Japan) ad libitum after 4 weeks of age (day 28 ±). 2). Mice were randomly divided into 3 groups of 8 mice at 6 weeks of age the day before treatment, into vehicle control (saline), Compound A and telmisartan (positive control) groups. Treatment with Compound A at a dose of 37 mg/kg body weight per day by oral gavage in combination with a high-fat diet was administered for 6 weeks before sacrifice. The NASH activity score (NAFLD) was calculated according to standardized criteria, and the area of fibrosis was assessed using Sirius Red staining.

Evaluation of the effect of Compound A in a mouse model of diet-induced NASH (APOE * 3Leiden.CETP, double transgenic mice)

The APOE*3Leiden.CETP double transgenic mouse expresses the human apolipoprotein E3 (APOE3) variant, APOE*3Leiden, in addition to human apolipoprotein C1 (APOC1) and CETP. APOE*3Leiden.CETP double transgenic mice exhibit elevated plasma cholesterol and triglyceride levels, mainly limited to the VLDL/LDL size lipoprotein fraction. By increasing dietary cholesterol in this model, all the characteristics of human NASH develop.

In Biological Example 9, studies were performed on APOE*3Leiden.CETP mice placed on a high fat diet (24% fat by weight) with varying cholesterol content (0.25-1% cholesterol by weight). In one study (using 1% cholesterol by weight), after a 3-week period on the diet, mice with a low response rate (20% of the total) were removed from the study and the remaining mice were divided into 4 groups of 12 mice each (+5 in control group), matched for plasma cholesterol, triglycerides, blood glucose, body weight and age (t=0), after which treatment began. The mice were given a gastric tube every day between 07:00 and 10:00. 00m. with Compound A at a dose of 0.3 mmol/kg body weight per day, Compound N (0.3 mmol/kg body weight per day), rosiglitazone (13 mg/kg body weight per day) or control (corn oil). After 20 weeks of treatment, mice were sacrificed by CO ₂ asphyxia, livers were collected, and the level of hepatocellular hypertrophy was assessed.

In Biological Example 25, the effects of Compound A alone and in combination with an omega-3 fatty acid were studied. APOE*3Leiden.CETP mice were placed on a high-fat semisynthetic diet (24% fat by weight) with 0.25 cholesterol by weight. After a 4-week period on the diet, mice with low levels of response were excluded from the study, and the remaining mice were divided into groups of 8 mice each, matched for plasma cholesterol, triglycerides, blood glucose, body weight and age (t=0) and started treatment. To facilitate mixing of compounds, sunflower oil was added to a volume of 10 ml/kg diet. Compound A was administered at 0.3 mmol/kg body weight/day, while omega-3 fatty acid ethyl esters (85% w/w of EPA/DHA ethyl esters) were administered at 3.0 mmol/kg body weight. body/day. Compound A and omega-3 ethyl esters were administered orally as a supplement to a high-fat diet. After 4 weeks of treatment, mice were sacrificed by CO ₂ asphyxia, livers were harvested, and liver free cholesterol, cholesterol esters, and triglycerides were measured after extraction and separation by high performance thin layer chromatography.

Evaluation of the effects of Compound A in a mouse model of obesity and diet-induced NASH (ob/ob AMLN model)

Obese B6.V-Lepob/Jrj mice are prone to fibrosis (liver) when cholesterol (2%), 40% fat (containing 18% trans fatty acids), and 20% fructose are added to a high-calorie diet (i.e. to an AMLN diet high in fat). Ob/ob mice on the AMLN diet (referred to as “ob/ob AMLN mice”) develop steatohepatitis and fibrosis in a shorter time frame (≤12 weeks) compared to wild-type C57BL/6 mice (AMLN mice) fed the same diet. AMLN ob/ob mice provide an obesity-induced model of NASH that also allows the study of effects on glycemic control. The development of NASH in the ob/ob AMLN model is confirmed by biopsy.

In Biological Examples 10-12, 15 and 19, studies were conducted on male ob/ob mice (5 weeks old) fed a high fat diet (AMLN diet) for 15 weeks, then randomized into 3 groups (n=10 per group) to administer either 112 mg/kg body weight per day of Compound A via diet, or pioglitazone via diet at a dose of 30 mg/kg body weight per day, or (vehicle) over the next 4 weeks. An oral glucose tolerance test (OGTT) was performed at 3 weeks. Liver fibrosis was assessed by measuring fibrogenic/fibrolytic gene expression in the liver by RNA sequencing after 4 weeks.

In Biological Examples 10, 13-14 and 16-18, studies were conducted on male ob/ob mice (5 weeks old) fed the high-fat AMLN diet for 18 weeks, then randomized into 6 groups (n=10 per group) to administer either Compound A (at 3 different daily doses, 45 mg/kg, 90 mg/kg, or 135 mg/kg) via diet, or obeticholic acid (OCA) at a dose of 30 mg/kg body weight per day with diet or vehicle (no treatment) for another 8 weeks. Assessment of NASH-related parameters and fibrosis was performed at the end of the study using quantitative immunohistochemistry (IHC) measurements.

According to medical parameters, the doses of Compound A used here (45 mg/kg, 90 mg/kg and 135 mg/kg in the 8-week study and 112 mg/kg in the 4-week study) correspond to human doses of approximately 250 mg/day , 500 mg/day and 750 mg/day (and 600 mg/day in the 4-week study) for a 70 kg person, adjusted for interspecies variation. See Nair, A.B., and Jacob, S., J Basic Clin Pharm, 2016; 7 (2): 27-31, for guidance on interspecies dose conversion.

Evaluation of the effect of Compound A on human liver stellate cells in vitro.

Stellate cells are resident cells of the liver and the main cell type involved in the initiation and progression of liver fibrosis. In Biological Example 20, in vitro studies on cultured human stellate cells (LX-2 cells) were conducted to evaluate the direct effects of Compound A on these cells. Cells were treated for 24 hours with Compound A (10 μM, 25 μM, 50 μM, or 75 μM) or oleic acid (OA; 75 μM), and bromodeoxyuridine (BrdU) incorporation was assessed to determine the effect of Compound A on proliferation. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to determine cell viability.

Biological Example 1: Effect of Compound A and Compound N on Liver Fibrosis as Based on Liver Total Hydroxyproline (HYP) Content

Compound A at both low (LD) and high (HD) doses administered to CDAA/fat-fed mice caused a significant reduction in liver fibrosis (liver HYP content) compared to control (p < 0.01 * and 0.001 ** respectively).). No significant effects were observed for the comparison compound N. The effect of Compound A on hepatic HYP is shown in Table 1.

Table 1. Hydroxyproline (µg HYP/liver) Compound Hydroxyline content (mcg) per liver CSAA (sufficient choline with high fat content) 310 CDAA (choline deficiency) 1250 CDAA+Connection A LD 800* CDAA+Connection A HD 750** CDAA+N LD connection 1100 CDAA+N HD connection 950

Biological Example 2. Effect of Compound A and Compound N on the state of liver fibrosis, as measured by relative hydroxyproline (HYP) content (μg HYP per 100 mg liver).

Compound A at both low (LD) and high (HD) doses administered to CDAA/fat-fed mice reduced liver fibrosis (liver HYP content), whereas no effects were observed for Compound N. The effect of compound A on the relative content of HYP is shown in Table 2.

Table 2. Hydroxyproline (µg HYP/per 100 mg liver)
Hydroxyproline compound content (µg) per 100 mg liver Compound Hydroxyline content per 100 mg of liver CSAA ((control diet high in fat and adequate in choline) 26 CDAA (choline deficiency) 52 CDAA+Connection A LD 38 CDAA+Connection A HD 37 CDAA+N LD connection 48 CDAA+N HD connection 54

Biological Example 3. Effect of compound A and compound N on the expression of the inflammatory process gene in the liver (TNF-a mRNA expression).

Compound A at both low (LD) and high (HD) doses administered to CDAA/fat-fed mice caused a significant reduction in liver inflammation (TNF-α mRNA) compared to control (both p<0.001***) . A significant reduction (p<0001) was also observed for compound N, but not for high dose (HD). The effect of Compound A on hepatic TNF-α is shown in Table 3.

Table 3. Hepatic gene expression during inflammation (TNF-α mRNA) Compound TNF-α (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency) 1.7 CDAA+Connection A LD 0.75*** CDAA+Connection A HD 0.65*** CDAA+N LD connection 1*** CDAA+N HD connection 1.2

Biological Example 4: Effect of Compound A and Compound N on Fibrosis-Related Gene Expression (Col1a1 mRNA Expression)

Compound A at both low (LD) and high (HD) doses administered to CDAA/fat-fed mice caused a significant reduction in liver fibrosis-associated gene expression (Col1a1 mRNA) compared to controls (both p< 0.001 *** respectively)). No significant effects were observed for Compound N. The effects of Compound A on hepatic Col1a1 are shown in Table 4.

Table 4. Expression of genes associated with liver fibrosis (Col1a1 mRNA) Compound Col1a1 (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency diet) 19 CDAA+Compound A LD (low dose) 10*** CDAA+Compound A HD (high dose) 6*** CDAA+Compound N LD (low dose) 15 CDAA+Compound N HD (high dose) 16

Biological Example 5: Effect of Compound A on Liver Fibrosis Content Measured Using Sirius Red Morphometry.

To more accurately quantify the location and functional significance of liver fibrosis, Sirius Red (SR) morphometry was performed. Unlike Examples 1 and 2, where biochemical hydroxyproline (HYP) assessment was used to measure collagen in large vessels, SR morphometry quantifies more functionally relevant sinusoidal collagen deposits. 10 areas on the slide were measured using 100x magnification. Compound A at both low (LD) and high (HD) doses administered to CDAA/fat-fed mice caused a significant reduction in surface area percentage of liver fibrosis compared to control (p < 0.001*** and 0.05 * respectively).). The effect of Compound A on liver fibrosis measured by SR morphometry is shown in Table 5.

Table 5. Liver fibrosis according to the results of SR morphometry Compound Area of fibrosis (average%) CSAA (control diet high in fat and adequate in choline) 0.9 CDAA (choline deficiency diet) 2.46 CDAA+Compound A LD (low dose) 0.76*** CDAA+Compound A HD (high dose) 1.56*

Biological Example 6: Effect of Compound A versus Bydureon® on Collagen Fibers

When administered at low doses (56 mg/kg) to CDAA/mice on a high fat diet, Compound A significantly reduced the number of collagen fibers (Figure 1A) by 51% (p=0.01). This effect was reflected in the almost complete disappearance of short (<8.5 μm) and thin (<3.5 μm) collagen fibers, and was reflected in a slight increase in fiber thickness and length by Compound A in FIG. 1B and Fig. 1C , respectively. Bydureon® (0.4 mg/kg administered subcutaneously weekly) had no significant effect.

Biological Example 7. Effect of compound A and EPA on lipid metabolism in the liver.

To further understand the effect of Compound A on hepatic lipid metabolism, lipidomic analysis of CS- and CDAA/high-fat (“CD”)-fed mice was performed on isolated liver tissue. When administered at a high dose (112 mg/kg) to CDAA/high-fat mice, Compound A significantly reduced the effects of choline deficiency as measured by hepatic triglycerides (TG) ( Figure 2A ), hepatic diglycerides (DG) ( Figure 2B ), liver free fatty acids (FFA) ( Figure 2C ), or cholesterol esters ( Figure 2D ). In contrast, treatment with equimolar amounts of EPA did not reduce the increases in hepatic TG, DG, FFA, or cholesteryl esters associated with a choline-deficient diet.

Biological Example 8. Effect of Compound A on Hepatocellular Ballooning, Lobular Inflammation, Steatosis, NAST Composite Score and Area of Fibrosis.

Compound A administered to mice with STAM reduced hepatocellular ballooning as well as steatosis and lobular inflammation. Both NAS composite score (p < 0.001*) and fibrosis area (positive area stained with Sirius Red) were reduced by Compound A.

Table 6. Hepatocellular ballooning, total NAS score and area of fibrosis Compound Steatosis Lobular inflammation Hepatocellular ballooning NAS score (mean ± standard deviation Area of fibrosis (%) Carrier n=6 (saline solution) 1 1.9 2 5.3 ±0.8 1.01 Compound A n=8 (37 mg/kg/day) 0.12 0.88 1 2.3±1.5* 0.79 Telmisartan n=6 0.7 0.45 0.8 1.8±0.8 0.44

Biological Example 9. Effect of Compound A and Compound N on Hepatocellular Hypertrophy.

Compound A administered to ApoE*3L-CETP mice (0.25% wt cholesterol diet) caused a significant reduction in hepatocellular hypertrophy (p<0.01*), which was 83% compared to control (p<0.01) . No effect was observed when Compound N was administered.

Table 7. Hepatocellular hypertrophy Compound Hepatocellular hypertrophy Decreased hypertrophy compared to control (%) Control 46 ± 23.9 - Connection A 8.9 ± 5.9* 83* Connection N 42.9 ± 29.7 7

Biological Example 10. Effect of Compound A, pioglitazone and OCA on liver weight and body weight.

As shown in Figure 3A , after 4 weeks, Compound A (112 mg/kg) had no effect on body weight of ob/ob AMLN mice, whereas pioglitazone caused a significant increase in body weight of 15% (p < 0.01). Liver weight was unchanged in all groups at week 4, as shown in Fig. 3B. After 8 weeks, neither Compound A (45 mg/kg, 90 mg/kg, and 135 mg/kg) nor OCA significantly affected body weight, as shown in FIG. 3C , and food intake did not change in either group. OCA significantly (p < 0.05) reduced liver weight at 8 weeks, while Compound A had no significant effect at any dose compared to vehicle, as shown in FIG. 3D

Biological Example 11. Effect of Compound A and pioglitazone on hepatic fibrogenic and fibrolytic gene expression.

The effect of 4 weeks of treatment with Compound A (112 mg/kg) or pioglitazone (30 mg/kg) on the expression of fibrogenic and fibrolytic genes in isolated liver samples from ob/ob AMLN- mice is shown in FIG. 4A-4F . Compound A significantly reduced the expression of canonical genes regulating liver fibrogenesis, including platelet-derived growth factor beta (PDGF-β) ( Figure 4A ), platelet-derived growth factor receptor-β (PDGFR-β) (Figure 4B), collagen-1 α1 (col1A1) ( Figure 4C ), col3A1 ( Figure 4D ), col4A1 ( Figure 4E ), and heat shock protein-47 (HSP47) ( Figure 4F ). Pioglitazone significantly reduced the expression of the tested collagen isoforms (col1A1, col3A1, col4A1) and HSP47, but had no effect on the expression of PDGF-β or PDGFR-β.

Biological Example 12. Effect of Compound A and pioglitazone on liver extracellular matrix stability and fibrolysis gene expression.

As shown in FIG. 5A-5D , for genes regulating extracellular matrix (ECM) stability or fibrolysis, 4 weeks of treatment with Compound A significantly reduced hepatic expression of lysyl oxidase (LOX) ( Figure 5A ), LOX-like 2 (LOXL2) ( Figure 5B ), and LOXL1 ( Figure 5C ) as well as tissue inhibitor of metalloproteinase (TIMP1) ( Figure 5D ) in ob/ob mice on an AMLN diet. Pioglitazone also significantly reduced the levels of these transcripts, although the effect was weaker than that obtained with compound A.

Biological Example 13. Effect of Compound A and OCA on the progression of liver fibrosis.

To evaluate the effect of 8 weeks of administration of Compound A (45 mg/kg, 90 mg/kg, or 135 mg/kg) and OCA (30 mg/kg) on the progression of liver fibrosis in ob/ob mice on an AMLN diet, quantitative immunohistochemistry (IHC) ) were carried out by measuring the content of col1A1 and α-smooth muscle actin (SMA). As shown in Figures 6A and 6B , at doses of 90 mg/kg and 135 mg/kg, Compound A markedly reduced the myofibroblast marker α-SMA (expressed as both total and percentage fraction), whereas OCA administration did not show any significant effect.

Similarly, compound A administered at 90 and 135 mg/kg decreased total col1A1 content ( Figure 6C ), whereas OCA had no significant effect. When considered as a percentage of total surface area ( Fig. 6D ), the 90 mg/kg dose caused a significant reduction in col1A1 levels, whereas the reduction at 45 and 135 mg/kg doses was not statistically significant. To confirm the antifibrotic effects observed with quantitative IHC, hepatic hydroxyproline (HYP) concentrations were measured as shown in FIG. 6E and FIG. 6F . Only Compound A reduced HYP content, expressed as either total (p < 0.01 for doses of 90 and 135 mg/kg) and relative (p < 0.05 for doses of 90 and 135 mg/kg) content.

Biological Example 14. Effect of Compounds A and OCA on regression of hepatic fibrosis.

Since baseline (pre-treatment) biopsies of ob/ob AMLN mice were taken in advance, including IHC measurements of both hepatic col1A1 and SM-SMA, final col1A1 and α-SMA values were compared with baseline (pre-treatment biopsy) values in all groups to to determine whether the significant reductions that were observed after treatment with Compound A, compared with vehicle at the end of the study, were associated with regression of fibrosis. As shown in Figure 7A , treatment with Compound A at a dose of 135 mg/kg reduced the percentage of α-SMA content assessed by immunohistochemical staining compared to the pre-dose biopsy α-SMA content. Doses of Compound A at 45 mg/kg and 90 mg/kg resulted in little difference in trends. OSA had no significant effect. In summary, all doses of Compound A showed a trend towards decreased col1A1 content compared to pre-biopsy content, although only the 90 mg/kg dose was statistically significant (p < 0.005) ( Figure 7B ). Both vehicle and OCA treatments showed a modest increase in hepatic col1A1 content after the 8-week treatment period, although the increase was significant only for OCA (p < 0.05).

Biological Example 15. Effect of Compound A and pioglitazone on the expression of inflammatory genes.

A particularly important finding for the progression of fibrosis in ob/ob mice on the AMLN diet was that 4 weeks of treatment with 112 mg/kg Compound A significantly reduced the expression of inflammatory genes compared to vehicle-fed control mice. Specifically, Compound A decreased transcript levels of both transforming growth factor beta 1 (TGF-β1) (p < 0.05) and TGF-β receptor 1 (TGFRβ (p < 0.01), as shown in Figure 8A and Figure 8B , respectively, Pioglitazone decreased the expression of TGFRβ (p < 0.05), but not TGF-β1, significant reductions in CCR2, MAC-2 (data not shown), CD68, and CD14 (all p < 0.001) were found after treatment. Compound A compared to control, as shown in Figures 8C , 8D, and 8F . Compound A treatment groups also significantly reduced galectin-3 (Gal-3) levels compared to vehicle control groups ( Figure 8E ). Pioglitazone significantly reduced CCR2 (p < 0.01) ( Figure 8C ), CD68 (p < 0.01) ( Figure 8D ), and CD14 (p < 0.001) ( Figure 8F ). interleukin (IL)-1β or MCP-1 transcript levels (data not shown).

Biological Example 16. Effect of Compound A and OCA on liver inflammation as measured by hepatic Gal-3.

To evaluate the effect of 8 weeks of treatment with Compound A (45 mg/kg, 90 mg/kg, or 135 mg/kg) or OCA (30 mg/kg) on liver inflammation in ob/ob mice on an AMLN diet, IHC was performed to assess levels of galectin-3 (Gal-3). As shown in FIG. 9A and 9B , treatment with Compound A resulted in a dose-dependent reduction in Gal-3 expression levels when measured as either total content or percent area, whereas OCA only reduced Gal-3 expression levels only when total content was measured.

Biological Example 17. Effect of Compound A and OCA on hepatocellular damage.

To evaluate the effect of Compound A and pioglitazone on hepatocellular damage or injury in ob/ob mice on the AMLN diet, plasma alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured after 8 weeks of administration. As shown in Figure 10A , a marked and significant reduction in ALT was achieved at Compound A doses of 90 mg/kg and 135 mg/kg (p < 0.01 and p < 0.001, respectively), whereas OCA had no effect. At doses of 90 mg/kg and 135 mg/kg, Compound A also significantly reduced AST (p < 0.05 and p < 0.01, respectively), whereas OCA had no effect on AST levels ( Figure 10B ).

Biological Example 18. Effect of Compound A and OCA on hepatic steatosis, liver lipid levels and plasma lipid levels.

As shown in FIG. 11A and FIG. 11B , both Compound A and OCA significantly reduced steatosis in ob/ob AMLN mice after 8 weeks of administration (as expressed as percent area size or total lipid content), with OCA demonstrating comparable efficacy to the highest dose (135 mg/kg) of Compound A (both p < 0.001). Likewise, both OCA and Compound A (at doses of 90 mg/kg and 135 mg/kg) demonstrated comparable reductions in plasma triglyceride and total cholesterol levels, as shown in Figure 11D and Figure 11E . Treatment with OCA, but not Compound A, showed a decrease in total liver cholesterol (p < 0.01) ( Figure 11C ).

Biological Example 19. Effect of Compound A and pioglitazone on glycemic control.

3 weeks after administration of Compound A (112 mg/kg), pioglitazone, or vehicle control, an oral glucose tolerance test (OGTT) was performed in ob/ob AMLN mice. As shown in FIG. 12A , both Compound A and pioglitazone significantly reduced glucose excursion during the first 240 minutes after glucose loading (AUC 0-240 minutes), although the effect was more pronounced with pioglitazone. Likewise, a significant reduction in fasting plasma glucose was achieved with both compounds, although the effect was more pronounced with pioglitazone compared to Compound A ( Figure 12B ). Pioglitazone, but not Compound A, reduced fasting insulin levels (p < 0.001) ( Figure 12C ).

Biological Example 20. Effect of Compound A and oleic acid on the proliferation and viability of stellate cells in vitro.

Cell proliferation of LX-2 human liver stellate cells treated for 24 hours with 10-75 µM Compound A or oleic acid (OA) (75 µM) was assessed by BrdU incorporation. Results are normalized means ± SEM of five independent experiments performed for eicosabutate and two independent experiments for OA. As shown in FIG. 13A , LX-2 cells treated with 25 μM (p < 0.005), 50 μM (p < 0.0001), and 75 μM (p < 0.005) Compound A were less proliferative compared to cells treated with OA or vehicle. The reduction in proliferation by Compound A was 25-34% after 24 hours, whereas OA had no effect. Comparisons were made by one-way ANOVA with Dunnett's correction for multiple comparisons. As shown in FIG. 13B , neither Compound A nor OA had a significant effect on cell viability as measured by MTS assay in two independent experiments.

Biological Example 21 Effect of Compound A and GLP-1 agonist (GLP-1a, exenatide), alone and in combination, on the expression of genes associated with liver fibrosis (Col1a1 mRNA expression).

Compound A, GLP-1a, and Compound A+GLP-1a in combination administered to CDAA/fat-fed mice caused a significant decrease in Col1a1 mRNA expression compared to CDAA-fed mice (all p<0.001***). The effect of Compound A alone and in combination with GLP-1a on hepatic Col1a1 mRNA is shown in Table 8.

Table 8 . Expression of genes associated with liver fibrosis (Col1a1 mRNA) Compound Col1a1 (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency diet) 19 CDAA+Compound A 6.2*** CDAA+GLP-1a 10.3*** CDAA+Compound A+GLP-1a 4.9***

Biological Example 22: Effect of Compound A and GLP-1 agonist (GLP-1a, exenatide), alone and in combination, on liver fibrosis-associated gene expression (on TIMP (tissue inhibitor of matrix metalloproteinase)-1a mRNA expression)

Compound A, GLP-1a, and Compound A+GLP-1a in combination administered to high-fat CDAA-fed mice caused a significant decrease in hepatic TIMP-1a mRNA expression compared to CDAA-fed mice (all p < 0.001*** ). The effect of Compound A alone and in combination with GLP-1a on hepatic TIMP-1a mRNA expression is shown in Table 9.

Table 9. Expression of genes associated with liver fibrosis (TIMP-1a mRNA) Compound TIMP-1a (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency diet) 6.7 CDAA+Compound A 2*** CDAA+GLP-1a 2.4*** CDAA+Compound A+GLP-1a 0.76***

Biological Example 23 Effect of Compound A and GLP-1 agonist (GLP-1a, exenatide), alone and in combination, on the expression of genes associated with liver fibrosis (MMP-13 (matrix metalloproteinase) mRNA expression)

Compound A, GLP-1a, and Compound A+GLP-1a in combination administered to mice fed a CDAA high-fat diet caused a significant reduction in hepatic MMP-13 mRNA expression compared to CDAA-fed mice (all p < 0.001** *). The effect of Compound A alone and in combination with GLP-1a on hepatic MMP-13 mRNA in the liver is shown in Table 10.

Table 10. Expression of genes associated with liver fibrosis (MMP-13 mRNA) Compound MMP-13 (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency diet) 3.2 CDAA+Compound A 1.6*** CDAA+GLP-1a 2.1*** CDAA+Compound A+GLP-1a 1.3***

Biological Example 24: Effect of Compound A and GLP-1 agonist (GLP-1a, exenatide), alone and in combination, on the expression of a gene associated with liver inflammation (TNF-α mRNA expression)

Compound A, GLP-1a, and Compound A+GLP-1a in combination administered to high-fat CDAAc-fed mice caused a significant decrease in hepatic TNF-α mRNA expression compared with CDAA-fed mice (p < 0.001 for Compound A alone or in combination with GLP-1a, p < 0.05 for GLP-1a only). The effect of Compound A alone and in combination with GLP-1a on hepatic TNF-α mRNA is shown in Table 11.

Table 11. Expression of genes associated with liver fibrosis (TNF-α mRNA expression) Compound TNF-α (relative mRNA expression) CSAA (control diet high in fat and adequate in choline) 1 CDAA (choline deficiency diet) 1.7 CDAA+Compound A 0.65*** CDAA+GLP-1a 1.05* CDAA+Compound A+GLP-1a 0.66***

Biological Example 25. Effect of Compound A and concentrated omega-3 ethyl esters, alone and in combination, on liver lipids (cholesterol ester-CE, free cholesterol-FC, triglycerides-TG).

Low dose (0.3 mmol/kg body weight/day) of Compound A (LD) administered to ApoE*3L-CETP transgenic mice fed a Western Diet with 0.25% cholesterol combined with a high dose (3 mmol/kg body weight per day) 85% EPA/DHA omega-3 ethyl esters (HD) caused significant reductions in FC (p<0.01), CE (p<0.001) and TG (p<0.01). Neither compound significantly reduced hepatic TG content as monotherapy in this model.

Table 12 . Liver lipids Compound FC in the liver CE in the liver TG in the liver Control 12.3 22.7* 95.4 Connection A LD 9.5 17* 58.8 EPA/DHA HD 8.9* 12** 65.4 Connection A LD+
EPA/DHA HD 8.1 8*** 39.1**

Biological Example 26: Multicenter, randomized, double-blind, placebo-controlled, parallel group study of the efficacy of Compound A in patients with non-alcoholic steatohepatitis (NASH)

The purpose of this clinical trial was to evaluate the effectiveness of different doses of Compound A in the treatment of NASH in patients without worsening fibrosis and to evaluate the safety and tolerability of Compound A in patients suffering from NASH.

Number of patients

600 patients with NASH were screened at approximately 30 centers to determine their eligibility for the study; It is expected that approximately 264 patients will be found meeting the eligibility criteria described below, of whom approximately 198 are predicted to complete the full trial (resulting in 66 subjects per treatment arm, and assuming that 25% of patients will not complete treatment).

Purpose of treatment

Patients deemed eligible for the study are randomly divided into 3 parallel groups of 88 patients each. Group 1 was administered Compound A placebo, Group 2 was administered 300 mg of Compound A, and Group 3 was administered 600 mg of Compound A. Compound A or placebo was administered once daily in one (300 mg) or two (600 mg) capsules for 52 weeks. . Distribution for F1 vs F2/F3.

The effectiveness of Compound A treatment is assessed, for example, by NAS liver biopsy scores, LiverMultiScan PDFF and cT1 MRI assessments, liver function tests, HOMA-IR, and biomarkers of inflammation and fibrosis (including hsCRP, Pro-C3, ELF panel, and other appropriate biomarkers).

Safety and tolerability are assessed by adverse event reports, vital sign monitoring, physical examination, hematology and biochemistry (kidney, liver), urinalysis, and resting 12-lead ECG.

Blood pressure is monitored by three blood pressure readings taken at each visit.

Study design

The study is conducted over 58 weeks and includes 10 clinical visits per patient, including screening, a double-blind treatment period and follow-up. The procedures performed at each visit are described below and summarized in Table 13.

Visit 1 (1 to 42 days before treatment): Patients are screened for study eligibility according to the criteria described below and patient consent is obtained. The suitability assessment may be carried out over more than one visit.

Visit 2: Patients are randomized into 3 treatment groups. Each treatment group received 300 or 600 mg of Compound A or placebo daily for 52 weeks. Basic assessments are performed, including liver imaging (MRI - PDFF and cT1), vital signs, treatment safety tests (basic hematology, biochemistry and urinalysis), resting 12-lead ECG and biomarker analysis ( HOMA-IR, hsCRP, Pro-C3 and ELF panel).

Visit 3 (after 4 weeks of treatment): Patients are assessed for side effects, vital signs, treatment safety tests (basic hematology, biochemistry and urinalysis), resting 12-lead ECG and detailed pharmacokinetics (PK) studies. samples).

Visit 4 (after 10 weeks of treatment): Patients are assessed for side effects, vital signs, treatment safety tests (basic hematology, biochemistry and urinalysis), resting 12-lead ECG and detailed pharmacokinetics (PK) studies. samples).

Visit 5 (after 16 weeks of treatment): Patients are assessed for side effects, liver imaging (MRI - PDFF and cT1), vital signs are taken, treatment safety tests are taken (basic hematology, biochemistry and urinalysis), ECG is performed in 12 leads at rest, biomarker analysis (HOMA-IR, hsCRP, Pro-C3 and ELF panel), and detailed pharmacokinetic studies (PK samples) are carried out.

Visits 6, 7, and 8 (after 24, 32, and 40 weeks of treatment): Patients are monitored for side effects, vital signs taken, treatment safety tests (basic hematology, biochemistry, and urinalysis), and 12-lead ECG performed. at rest, biomarker analysis (hsCRP) and detailed pharmacokinetic studies (PK samples) are carried out.

Visit 9 (after 52 weeks of treatment): Patients undergo liver biopsies, liver imaging (MRI - PDFF and cT1), vital signs are taken, treatment safety tests are taken (basic hematology, biochemistry and urinalysis), 12-lead ECG is performed at rest, biomarker analysis (HOMA-IR, hsCRP, Pro-C3 and ELF panel), and detailed pharmacokinetic studies (PK samples) are carried out.

Visit 10 (14-21 days after stopping the trial medication): Patients are assessed for treatment safety and are assessed for adverse events, vital signs are taken, treatment safety tests are taken (basic hematology, biochemistry and urinalysis), 12-lead ECG at rest and detailed pharmacokinetic studies (PK samples).

Patients who do not undergo studies to completion are assessed for side effects, have vital signs taken, treatment safety tests (basic hematology, biochemistry and urinalysis), resting 12-lead ECG, biomarker analysis performed (HOMA-IR, hsCRP, Pro-C3 and ELF panel) and detailed pharmacokinetic studies (PK samples).

Patient inclusion criteria :

Patients meet the following criteria to participate in the study.

Age: from 18 to 75 years.

Women are not of childbearing potential or are using acceptable contraception.

If female partners of male patients are of childbearing potential, male patients are willing to use contraception (eg, condoms). In addition, their partner must use contraception (eg, hormonal, intrauterine, etc.). Dual contraception is used from the first dose of the compound drug and for 90 days after the last dose of the study drug.

Written informed consent.

Optional criteria for fibrosis scanning before biopsy.

Histological diagnosis of NASH at screening or within 6 months after screening.

NAS score ≥ 4.

Fibrosis index: 1-3 inclusive (F1 is limited to 30%).

Additional PDFF > 10% on MRI

I agree to a liver biopsy after 52 weeks of treatment.

Compensated liver disease according to the following hematological and biochemical criteria upon entry into the protocol:

ALT <5 x ULN

AST>30

Hemoglobin >11 g/dL for women and >12 g/dL for men

white blood cells (WBC) > 2.5 K/µl

Neutrophil count > 1.5 K/µl

Platelets > 100 K/µl

Total bilirubin <35 µmol/l. Patients wit h bilirubin >35 µmol/L may be included if unconjugated bilirubin in Gilbert's syndrome.

Albumin > 36 g/l

International Normalized Ratio (INR) <1.4

serum creatinine <1.3 mg/dL (men) or <1.1 (women) or estimated glomerular filtration rate ≥ 60 ml/min/1.73 ^m2

Absence of other causes of chronic liver disease (autoimmune diseases, primary biliary cholangitis, HBV, HCV, Wilson's disease, α-1-antitrypsin deficiency, hemochromatosis, etc.)

If applicable, stable type 2 diabetes (defined as HgbA1c <9.5% and fasting blood glucose <10 mmol/L, with no change in medications in the previous 6 months and no new symptoms associated with decompensated diabetes in the previous 3 months) .

From the moment of liver biopsy, weight stability, and a loss of no more than 5% of initial body weight is considered stable.

Patient exclusion criteria

Patients do not meet any of the following exclusion criteria:

A history of persistent heavy drinking is assessed using an Alcohol Consumption Questionnaire.

Unstable metabolic state defined by: weight gain or loss of more than 5 kg in the last three months, diabetes with poor glycemic control (HgbA1c>9.5%) or administration of an antidiabetic or antiobesity drug/malabsorptive or restrictive bariatric (weight loss ) surgery within 6 months preceding screening.

History of gastrointestinal malabsorption bariatric surgery for less than 5 years or use of drugs known to cause hepatic steatosis, including corticosteroids, high-dose estrogens, methotrexate, tetracycline, or amiodarone in the previous 6 months.

Significant systemic or other serious diseases other than liver disease, including congestive heart failure (AHA class C and D), unstable coronary artery disease, cerebrovascular disease, pulmonary failure, renal failure, organ transplantation, serious mental illness, or malignancy that, in the opinion of the investigator, treatment with compound A is excluded.

Antigen HB>0, PCR HCV>0 (patients with a history of HCV infection can be included if the HCV PCR test has been negative for more than 3 years), or HIV infection.

Any other condition which, in the opinion of the investigator, may interfere with the conduct or compliance with the conditions of the study or may interfere with the completion of the study.

Body mass index (BMI) > 45 kg/ ^m2 .

Type 1 diabetes or type 2 diabetes, which are treated with insulin.

Diabetic ketoacidosis.

Fasting triglycerides >300 mg/dL.

Hemostasis disorders or current treatment with anticoagulants.

Contraindications to liver biopsy.

history or current cardiac dysrhythmias and/or history of cardiovascular disease, including myocardial infarction, excluding patients with well-controlled hypertension and any clinically significant ECG abnormality

Participation in any other pharmacological drug study within the previous 3 months or 5 half-lives of the specified drug.

Having hypersensitivity to any of the ingredients or excipients of IMP.

Taking antidiabetic agents that have activity against NASH, such as pioglitazone and GLP-1 receptor agonists.

Endpoint criteria

The primary efficacy endpoint of Compound A is the percentage of patients with NASH resolution, as defined by resolution of ballooning (score = 0) with a lobular inflammation score of "0 or 1" without worsening fibrosis.

Secondary efficacy endpoints for Compound A include change from baseline in NAS scores, changes in individual histology scores for steatosis, ballooning, inflammation and fibrosis, changes in liver enzymes, changes in imaging parameters, and changes in biomarkers (including hsCRP, Pro-C3, ELF panel and cytokines.

Secondary endpoints to assess safety/tolerability include reported adverse events, laboratory parameters (hematology, chemistry, and urinalysis), vital signs (blood pressure, pulse rate, and temperature), and hsCRP.

Secondary pharmacokinetic endpoints include comparison of mean steady state trough plasma concentrations for three doses of Compound A taken at Visits 4, 5, 6, 7 and 8.

Statistical methods

A sample size of 88 patients per group provides an 80% power to detect a 40% proportion of responders to the active compound vs. placebo, assuming a placebo response rate of 18% and a dropout rate of 25%. After the 16-week interim analysis, the sample size may be re-estimated. The effectiveness analysis is carried out according to the modified ITT-concept (intention to treat population). The population consists of patients with a baseline measurement and at least one post-baseline performance measurement. Comparisons are made between each individual dose and placebo.

Table 13. Survey schedule Survey schedule. Tests. Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 Visit 10 Elimination Screening Group breakdown and baseline After 4 weeks Rx After 10 weeks Rx After 16 weeks Rx After 24 weeks Rx After 32 weeks Rx After 40 weeks Rx After 52 weeks Rx Safety follow-up Abort before the end date Day -42--1 Day 0 Day 28 Day 70 Day 112 Day 168 Day 224 Day 280 Day 364 Day 378 Obtaining consent for research X Demographics + medical history X Related medications X X X X X X X X X X X Physical Exam ¹ X X X X X X X X X X X ECG with 12 leads X X X X X X X X X Biochemistry ² X X X X X X X X X X X Liver biopsy X X Confirmation of suitability for the study X MRI fibrosis tests - PDFF and cT1 X X X Biomarkers ³ X X X X X X X Taking samples for pharmacokinetics X X X X X X X X X Side effect analysis X X X X X X X X X X Dosing of drugs and their recording and control X X X X X X X X X ¹ Full examination at visits 1, 2, 9 and 10; abbreviated examination at all other visits;
² Hematology, biochemistry, coagulation, urinalysis, lipids;
³ hsCRP, Pro-c3, ELF panel, HOMA-IR, cytokines;
* The examination period of up to 6 weeks may include more than one actual visit if a biopsy is planned.

Claims (147)

1. Use of a compound of Formula (II) where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds; R ₂ and R ₃ are the same or different and selected from the group consisting of a hydrogen atom and an alkyl group; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, in the therapeutic and/or prophylactic treatment of non-alcoholic steatohepatitis or alcoholic steatohepatitis, where the use of the compound slows down or prophylactically prevents the development of liver fibrosis or reduces existing liver fibrosis. 2. Use according to claim 1, wherein the compound has Formula (I) and R ₂ , R ₃ and X have the meanings defined for Formula (II) 3. Application according to paragraph 1 or 2, where R ₂ and R ₃ are independently selected from a hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, for use in the treatment of non-alcoholic steatohepatitis. 4. Application according to any one of paragraphs. 1-3, wherein R ₂ and R ₃ are independently selected from a hydrogen atom, a methyl group, an ethyl group, an n -propyl group and an isopropyl group. 5. Use according to any one of claims 1 to 3, wherein both R ₂ and R ₃ independently represent C ₁ -C ₆ alkyl groups. 6. Use according to any one of claims 1 to 4, wherein one of R ₂ and R ₃ represents a hydrogen atom and the other represents an ethyl group. 7. Application according to any one of paragraphs. 1-6, where X represents a carboxylic acid. 8. Application according to any one of paragraphs. 1-6, where X is a C ₁ -C ₆ alkyl ether. 9. Use according to claim 8, wherein X is selected from methyl ether, ethyl ether, isopropyl ether, n -butyl ether and tert -butyl ether. 10. Application according to any one of paragraphs. 1, 8 and 9, where X is selected from the group consisting of methyl ether and ethyl ether. 11. Application according to any one of paragraphs. 1-6, where X is a glyceride selected from triglyceride, 1,2-diglyceride, 1,3-diglyceride, 1-monoglyceride and 2-monoglyceride. 12. Application according to any one of paragraphs. 1-11, where the compound is present in the form of an enantiomer, diastereomer or mixture thereof. 13. Use according to claim 12, wherein the compound is present in form R, form S or racemic form. 14. Use according to claim 1 or 2, where the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoic acid (Compound A) or a pharmaceutically acceptable salt or ester thereof, and the formula is 15. Application according to any one of paragraphs. 1-14, wherein said compound is administered at a dose ranging from about 5 mg to about 4 g per dose. 16. Application according to any one of paragraphs. 1-15, where the specified connection is administered once a day. 17. Application according to any one of paragraphs. 1-16, where the use leads to a cure or causes a decrease in non-alcoholic steatohepatitis (NASH). 18. Application according to any one of paragraphs. 1-17, where the use leads to: a decrease in liver inflammation, such as lobular inflammation; reduction of hepatocellular ballooning; and/or reduction of steatohepatitis. 19. Use of a pharmaceutical composition containing a compound of Formula (II) or a pharmaceutically acceptable salt thereof where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds; R ₂ and R ₃ are the same or different and are selected from the group of substituents consisting of a hydrogen atom and an alkyl group; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; or carboxamide, in the therapeutic and/or prophylactic treatment of non-alcoholic steatohepatitis and/or alcoholic steatohepatitis, where the use slows down or prevents the development of liver fibrosis or reduces existing liver fibrosis. 20. Use according to claim 19, where the compound has Formula (I) and where R ₂ and R ₃ are independently selected from a hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as a carboxylic acid ester, glyceride or carboxamide; or a pharmaceutically acceptable salt thereof. 21. Use according to claim 19 or 20, wherein said pharmaceutical composition is formulated for oral administration. 22. Application according to any one of paragraphs. 19-21, where the pharmaceutical composition further contains at least one binder, excipient, diluent or antioxidant or any combinations thereof. 23. Application according to any one of paragraphs. 19-22, where the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (compound A) , or a pharmaceutically acceptable salt or ester thereof, and is represented by the formula . 24. Application according to any one of paragraphs. 1-16 and 18, where the use is for the prophylactic treatment of non-alcoholic steatohepatitis (NASH) and/or alcoholic steatohepatitis. 25. A method of inhibiting liver fibrosis in a subject in need of treatment, comprising administering to the subject a pharmaceutically effective amount of a compound of Formula (II): where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds; R ₂ and R ₃ are the same or different and selected from the group consisting of a hydrogen atom and an alkyl group; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as an ester of a carboxylic acid; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, where the subject has non-alcoholic steatohepatitis or alcoholic steatohepatitis. 26. The method according to claim 25, in which the compound has Formula (I): . 27. Method according to paragraph 25 or 26, where R ₂ and R ₃ are independently selected from a hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups; and X represents a carboxylic acid or a derivative thereof; where the derivative is a carboxylate, such as a carboxylic acid ester; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof. 28. Method according to any one of paragraphs. 25-27, in which R ₂ and R ₃ are independently selected from a hydrogen atom, a methyl group, an ethyl group, an n -propyl group and an isopropyl group. 29. Method according to any one of paragraphs. 25-27, in which R ₂ and R ₃ are both independently C ₁ -C ₆ alkyl groups. 30. Method according to any one of paragraphs. 25-28, in which one of R ₂ and R ₃ represents a hydrogen atom and the other represents an ethyl group. 31. Method according to any one of paragraphs. 25-30, in which X represents a carboxylic acid. 32. Method according to any one of paragraphs. 25-30, in which X is a C ₁ -C ₆ alkyl ether. 33. The method of claim 32, wherein X is selected from methyl ether, ethyl ether, isopropyl ether, n -butyl ether and tert -butyl ether. 34. Method according to any one of paragraphs. 25, 32 or 33, wherein X is selected from methyl ether and ethyl ether. 35. Method according to any one of paragraphs. 25-30, wherein X is a glyceride selected from triglyceride, 1,2-diglyceride, 1,3-diglyceride, 1-monoglyceride and 2-monoglyceride. 36. Method according to any one of paragraphs. 25-35, in which the compound is present in the form of an enantiomer, a diastereomer or a mixture thereof. 37. The method of claim 36, wherein the compound is present in R form, S form or racemic form. 38. The method according to claim 25, in which the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butane acid (Compound A) or a pharmaceutically acceptable salt or ester thereof, and the formula is . 39. Method according to any one of paragraphs. 25-38, wherein said compound is administered at a dose ranging from about 5 mg to about 4 g per dose. 40. Method according to any one of paragraphs. 25-39, in which the compound is administered once daily. 41. Method according to any one of paragraphs. 25-40, in which the method cures or causes regression of non-alcoholic steatohepatitis and/or alcoholic steatohepatitis. 42. Method according to any one of paragraphs. 25-41, where the method includes prophylactic treatment of non-alcoholic steatohepatitis and/or alcoholic steatohepatitis. 43. Method according to any one of paragraphs. 25-42, in which the method slows or prophylactically prevents the development of liver fibrosis or reduces pre-existing liver fibrosis. 44. Method according to any one of paragraphs. 25-43, in which the method provides a reduction in liver inflammation, hepatocellular ballooning, steatohepatitis, fibrosis and/or biomarkers of inflammation and/or fibrosis, for example hsCRP, Pro-C3, ELF panel; and/or improvement in NAS scores based on liver biopsy, liver function test, LiverMultiScan PDFF and cT1 MRI analysis and/or HOMA-IR. 45. The method of claim 44, wherein the reduction in liver inflammation is a reduction in lobular inflammation. 46. Method according to any one of paragraphs. 25-45, in which the compound is formulated as a pharmaceutical composition. 47. The method according to claim 46, wherein the pharmaceutical composition is formulated for oral administration. 48. The method according to claim 46 or 47, wherein the pharmaceutical composition further contains at least one binder, excipient, diluent or antioxidant or any combination thereof. 49. Method according to any one of paragraphs. 46-48, where the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (compound A) , or a pharmaceutically acceptable salt or ester thereof, and is represented by the formula . 50. Method according to any one of paragraphs. 25-49, in which the treatment method is prophylactic. 51. Use of a compound of Formula (II) where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds; R ₂ and R ₃ are the same or different and selected from the group consisting of a hydrogen atom and an alkyl group; X represents a carboxylic acid or a derivative thereof; where the derivative is a carboxylate, such as a carboxylic acid ester; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, in obtaining a medicinal product for the therapeutic and/or prophylactic treatment of alcoholic steatohepatitis or non-alcoholic steatohepatitis, where the drug slows down or prophylactically prevents the development of liver fibrosis or reduces existing liver fibrosis. 52. Use according to claim 51, where the compound has Formula (I) and R ₂ , R ₃ and X have the meanings defined for Formula (II) 53. Application according to paragraph 51 or 52, where R ₂ and R ₃ are independently selected from a hydrogen atom or linear, branched and/or cyclic C ₁ -C ₆ alkyl groups; X represents a carboxylic acid or a derivative thereof; wherein the derivative is a carboxylate, such as a carboxylic acid ester; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of alcoholic steatohepatitis. 54. Application according to any one of paragraphs. 51-53, where R ₂ and R ₃ are independently selected from a hydrogen atom, a methyl group, an ethyl group, an n -propyl group and an isopropyl group. 55. Application according to any one of paragraphs. 51-53, where R ₂ and R ₃ are both independently C ₁ -C ₆ alkyl groups. 56. Application according to any one of paragraphs. 51-54, where one of R ₂ and R ₃ represents a hydrogen atom and the other represents an ethyl group. 57. Application according to any one of paragraphs. 51-56, where X represents a carboxylic acid. 58. Application according to any one of paragraphs. 51-56, where X is a C ₁ -C ₆ alkyl ether. 59. Use according to claim 58, wherein X is selected from methyl ether, ethyl ether, isopropyl ether, n -butyl ether and tert -butyl ether. 60. Application according to any one of paragraphs. 51-56, 58 and 59, where X is selected from the group consisting of methyl ether and ethyl ether. 61. Application according to any one of paragraphs. 51-56, where X is a glyceride selected from triglyceride, 1,2-diglyceride, 1,3-diglyceride, 1-monoglyceride and 2-monoglyceride. 62. Application according to any one of paragraphs. 51-61, where the compound is present in the form of an enantiomer, a diastereomer or a mixture thereof. 63. Use according to claim 62, wherein the compound is present in form R, form S or racemic form. 64. Use according to claim 51 or 52, where the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy) butanoic acid (Compound A) or a pharmaceutically acceptable salt or ester thereof, with the formula 65. Application according to any one of paragraphs. 51-64, wherein said compound is administered at a dose ranging from about 5 mg to about 4 g per dose. 66. Application according to any one of paragraphs. 51-65, where the specified connection is administered once a day. 67. Application according to any one of paragraphs. 51-66, the use of which treats or causes regression of non-alcoholic steatohepatitis. 68. Application according to any one of paragraphs. 51-67, which provides: reduction of liver inflammation, such as lobular inflammation; reduction of hepatocellular ballooning; and/or reduction of steatohepatitis. 69. Application and method according to any one of paragraphs. 1-50, where the compound is administered at a dose of 600 mg per day. 70. Application and method according to any one of paragraphs. 1-50, where the compound is administered at a dose of 300 mg per day. 71. Application and method according to any one of paragraphs. 1-50, 69 and 70, in which plasma alanine aminotransferase levels are reduced by 30-40%. 72. Application and method according to any one of paragraphs. 1-50 and 69-71, in which plasma aspartate transaminase levels are reduced by 10-20%. 73. Application and method according to any one of paragraphs. 1-50 and 69-72, in which liver steatosis is reduced by 70-90%. 74. Application and method according to any one of paragraphs. 1-50 and 69-73, in which NAS scale scores are reduced by 50-70%. 75. Application and method according to any one of paragraphs. 1-50 and 69-74, in which the fibrotic area in the liver is reduced by 40-60%. 76. Method according to any one of paragraphs. 25-50, in which the compound is administered as monotherapy. 77. Method according to any one of paragraphs. 25-50, further comprising co-administration of at least one additional active agent selected from the group consisting of a glucagon-like peptide 1 (GLP-1) agonist; dipeptidyl peptidase inhibitor (DPP IV) and omega-3 fatty acids (n-3 PUFA). 78. The method of claim 77, wherein coadministration is accomplished by simultaneous administration, sequential administration, coadministration, interval administration, continuous administration, or a combination thereof. 79. Application according to any one of paragraphs. 1-18, where the compound is administered as monotherapy. 80. Application according to any one of paragraphs. 1-18, wherein the compound is administered with at least one additional active agent selected from the group consisting of a glucagon-like peptide 1 (GLP-1) agonist; dipeptidyl peptidase inhibitor (DPP IV) and omega-3 fatty acids (n-3 PUFA). 81. Use according to claim 80, wherein co-administration is carried out by simultaneous administration, sequential administration, combined administration, interval administration, continuous administration, or a combination thereof. 82. Use of a compound of Formula (II) where R ₁ is selected from C ₁₀ -C ₂₂ alkenyl having 3-6 double bonds; R ₂ and R ₃ are the same or different and are selected from the group of substituents consisting of a hydrogen atom or an alkyl group; X is a carboxylic acid or a derivative thereof, where the derivative is a carboxylate, such as a carboxylic acid ester; glyceride; or carboxamide; or a pharmaceutically acceptable salt thereof, in the therapeutic and/or prophylactic treatment of non-alcoholic steatohepatitis or alcoholic steatohepatitis, wherein said compound is administered in conjunction with one or more additional active agents selected from the group consisting of a glucagon-like peptide 1 (GLP-1) agonist; dipeptidyl peptidase inhibitor (DPP IV) and omega-3 fatty acids (n-3 PUFA). 83. Use according to claim 82, wherein the compound has Formula (I) and R ₂ , R ₃ and X have the meanings defined for Formula (II) 84. Use according to claim 82 or 83, wherein R ₂ and R ₃ are independently selected from a hydrogen atom, a methyl group, an ethyl group, an n -propyl group and an isopropyl group. 85. Application according to any one of paragraphs. 82-84, where the compound is 2-(((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaen-1-yl)oxy)butanoic acid (Compound A) or a pharmaceutically acceptable salt or ester thereof, and the formula is 86. Application according to any one of paragraphs. 82-85, in which the specified joint administration is carried out by simultaneous administration, sequential administration, combined administration, interval administration, continuous administration, or a combination thereof. 87. Application according to any one of paragraphs. 82-85, where the use prevents, treats or causes reversal of non-alcoholic steatohepatitis (NASH). 88. Application according to any one of paragraphs. 82-86, where the use prevents the development of liver fibrosis or reduces pre-existing liver fibrosis.

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