TECHNICAL FIELD

[0001] The present invention provides methods for chemical synthesis of resolvin El (RvEl) and resolvin salts in general; and stable formulations comprising them.

[0002] Abbreviations: ACN, acetonitrile; CSA, camphorsulfonic acid; DCM, dichloromethane; DEA, diethylamine; DHA, docosahexaenoic acid; DIBAL-H, diisobutylaluminum hydride; DMAP, 4-dimethylaminopyridine; EA, ethyl acetate; EPA, eicosapentaenoic acid; HPLC, high-performance liquid chromatography; Im, imidazole; iPrOH, isopropanol; KHMDS, potassium bis(trimethylsilyl)amide (potassium hexamethyldisilazane); MTBE, methyl t-butyl ether; n-3 DPA, n-3 docosapentanoic acid; py, pyridine; RvEl, resolvin El; TBAF, tetra-ft-butylammonium fluoride; TBDMS, tert- butyldimethylsilyl; TBDPS, ieri-butyldiphenylsilyl; TBDPSCl, ieri-butyldiphenylsilyl chloride; TBS, ie/ -butyldimethylsilyl; TBSC1, ie/ -butschemeyldimethylsilyl chloride; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin-layer chromatography; TMS, trimethylsilyl.

BACKGROUND ART

[0003] Natural resolvins are endogenous picomolar-potent small molecules derived from cellular metabolism of dietary omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C22:6), that activate a complex intracellular mechanism by which tissue inflammation is modulated and ultimately resolved.

[0004] A particular such compound, resolvin El (RvEl; 5S,12R,18R-trihydroxy- 6Z,8E,10E,14Z,16E-EPA, is an oxidative metabolite of the omega-3 fatty acid EPA. RvEl is an endogenous lipid mediator and has been identified in local inflammation during the healing stage. RvEl reduces inflammation in several types of animal models including peritonitis and retinopathy, and blocks human neutrophil transendothelial cell migration.

[0005] In mammals, alpha-linolenic acid is converted via elongation and desaturation to EPA and subsequently to DHA. An intermediate in the conversion of EPA to DHA is n-3 docosapentaenoic acid (n-3 DPA), which carries 22 carbons and contains five double bonds, with the first double bond being found on carbon 7. [0006] Due to its limited availability in natural sources, it is of great importance to design methods for synthesis of RvEl so as to evaluate its pharmaceutical properties and potential in anti-inflammatory therapies. Such methods may also enable designing RvEl analogues.

[0007] Recent publications (Allard et al, Tetrahedron Letters 2011, 52, 2623-2626; Ogawa and Kobayashi, Tetrahedron Letters, 2009, 50(44), 6079-6082) describe total synthesis of RvEl; however, these methods are not applicable to commercial manufacture for pharmaceutical use.

[0008] International Publication No. WO 2017/064701 of the same applicant discloses methods for total chemical synthesis of RvEl.

SUMMARY OF INVENTION

[0009] In one aspect, the present invention relates to a method for the synthesis of a resolvin compound selected from a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt thereof, starting from an ester of said resolvin compound protected at one or more of its hydroxyl groups and optionally having a triple bond, said method comprising: (i) basic hydrolysis of the ester group of said resolvin compound to obtain a salt of said resolvin compound protected at one or more of its hydroxyl groups; (ii) reduction of said triple bond, if present, to a double bond; and (iii) removal of the hydroxyl-protecting groups with a deprotecting reagent to obtain said resolvin compound.

[0010] In particular such aspects exemplified herein, the invention provides synthetic routes for the preparation of RvEl, starting from compound 16 or compound 15 as depicted in Scheme 1 and Scheme 9, respectively. The procedures disclosed, including full chemical structures of all the compounds involved, are shown in the Appendix hereinafter, Schemes 1-12, wherein the various starting compounds, intermediates and products referred to are herein identified by the Arabic numbers 1-37 in bold. RvEl is referred to by its name and shown as its sodium salt.

[0011] Although most of the compounds involved in the syntheses disclosed herein are known, some of them are novel. In another aspect, the present invention thus provides a compound of the formula I, II or III, wherein R is H or Na, which are useful as intermediates in said syntheses.

III

[0012] In a further aspect, the present invention provides a composition comprising, as a sole active agent, a salt of a resolvin compound as defined above, i.e., a salt of a compound selected from a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, e.g., RvEl, wherein said composition is either (i) an aqueous solution having a basic pH, which is chemically stable for at least 1 week, e.g., for about 2 weeks, 3 weeks, 4 weeks, or more; or (ii) a solid that upon reconstitution with water produces said aqueous solution.

DETAILED DESCRIPTION

[0013] WO 2017/064701 discloses a procedure for the preparation of RvEl starting from an ethyl ester of RvEl derivative in which the hydroxyl groups at positions C12 and C18 are protected, the hydroxyl group at position C5 is acetylated, and a triple (rather than a double) bond exists at positions 6-7. The procedure disclosed comprises (i) selective removal of the protecting groups at positions C12 and C18 by treatment with TBAF in THF, and reduction of the triple bond to a double bond, thus obtaining an RvEl -ethyl ester derivative in which the hydroxyl group at position C5 is acetylated; and (ii) hydrolysis of said RvEl -ethyl ester derivative by treatment with sodium hydroxide, thus deacetylating the protected hydroxyl group at position C5 and consequently obtaining RvEl as its sodium salt (see Scheme 1 in said publication). According to another procedure disclosed in WO 2017/064701, RvEl -ethyl ester is first synthesized, and then hydrolyzed by treatment with sodium hydroxide to obtain RvEl as its sodium salt (see Scheme 15 in said publication).

[0014] It has now been found, in accordance with the present invention, that the procedures disclosed in WO 2017/064701 suffer from certain disadvantages that result in a mixture of RvEl isomers and relatively low yield of the particular desired isomer. [0015] As particularly found, the acetyl group at position C5 leads, after the hydrolysis of the RvEl -ethyl ester, to a mixture of RvEl isomers, whereas in order to obtain RvEl only, the hydroxyl group at position C5 should be free. Furthermore, while RvEl -ethyl ester is quite unstable during the synthesis and RvEl-ie/ -butyl ester is too stable, RvEl -isopropyl ester is robust, yet efficiently hydrolyzed when treated with sodium hydroxide.

[0016] As surprisingly further found, the sequence of the synthesis steps, more specifically removal of the hydroxyl -protecting groups from positions C12 and C18 and hydrolysis of the ester bond, is highly important and dramatically affects the yield of the process. In particular, while removal of the hydroxyl-protecting groups at positions C12 and C18 of the ethyl- or isopropyl esters (by treatment with TBAF) results in very unstable compounds, hydrolyzing the protected isopropyl ester first (by treatment with sodium hydroxide) provides the sodium salt of the protected RvEl, which is stable and effectively deprotected (by treatment with TBAF) to give RvEl sodium salt. With this new approach, the TBAF reaction on the -COONa salt provides only RvEl sodium salt and some deprotected silane as a side product, rendering the RvEl synthesis a lot easier and highly effective (yields are high due to the stability of the sodium salts in both steps). The pH range during the process is 8.0-13, and the RvEl sodium salt obtained is stored at pH 9.0- 9.5.

[0017] In one aspect, the present invention thus relates to methods for the synthesis of RvEl. The common feature linking the methods disclosed herein is that in sharp contrast to the teaching of the prior art, the starting compound in each one of these methods is isopropyl- rather than ethyl-ester of an RvEl derivative in which the hydroxyl groups at positions C12 and C 18 are protected, and said compound is first hydrolyzed with sodium hydroxide so as to obtain the stable sodium salt thereof, which is then effectively deprotected to give RvEl sodium salt only with high yield. Free acid RvEl is made by lowering pH (< 7.0) with acidic H ⁺ resins.

[0018] In one particular such aspect, the invention provides a method for the synthesis of RvEl starting from compound 16, wherein R is H or a hydroxyl-protecting group, said method is carried out as depicted in Scheme 1 and comprises: (i) basic hydrolysis of the ester group of compound 16 with an alkaline hydroxide to obtain an alkaline salt of compound 16; and (ii) removal of the hydroxyl-protecting groups with a deprotecting reagent to obtain RvEl. [0019] In certain embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is H, i.e., compound 16a; and in other embodiments, RvEl is synthesized starting from compound 16 wherein R is a hydroxyl-protecting group such as TBDPS or TBDMS, i.e., compound 16b.

[0020] The term "hydroxyl-protecting group" as used herein refers to a group capable of masking hydroxy! groups during chemical group transformations elsewhere in the molecule, i.e., to a group capable of replacing the hydrogen atom of a hydroxy group on a molecule that is stable and non-reactive to reaction conditions to which the protected molecule is to be exposed. Examples of hydroxyl-protecting groups include, without being limited to, groups that can be reacted with hydroxy! groups to form ethers, such as silyl ethers (e.g., trirnethylsilyl (T S), triethylsilyl (TES). ieri-butyldimethyl silyl (TBDMS; TBS), tert-butyldiphenylsilyl (TBDPS), or phenyldimethylsilyl ethers); substituted methyl ethers (e.g., methoxymethy! (MOM), henzyloxymethyl (BOM), tetrahydropyranyl (T P)); substituted ethyl ethers; benzyl ethers and substituted benzyl ethers; esters (e.g., acetate, formate, chloroacetate); and carbonates. Preferred hydroxyl-protecting groups for use in the syntheses disclosed herein are TBDPS and TBDMS. The removal of such groups to obtain the non-protected hydroxyl is carried out by using a deprotecting reagent, e.g., an acid, or a fluoride such as NaF, TBAF, HF-Py, or HF-NEt ₃ , as known to any person skilled in the art of organic chemistry.

[0021] The term "alkaline hydroxide" as used herein refers to any alkali or alkaline earth metal hydroxide, or any alkali or alkaline earth metal compound that produces a basic (i.e., pH>7) solution when mixed with or dissolved into water. Suitable alkaline hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide, barium hydroxide, calcium oxide, and barium oxide. A preferred alkaline hydroxide for use in the methods disclosed herein is sodium hydroxide.

[0022] In certain embodiments, step (i) of the synthesis illustrated in Scheme 1, regardless of whether R is H or a hydroxyl-protecting group, is carried out by reacting compound 16 with sodium hydroxide, optionally in the presence of THF or (Ci-C4)alkanol, to obtain the sodium salt of compound 16.

[0023] The term "(Ci-C4)alkanol" refers to a simple aliphatic alcohol having 1-4 carbon atoms, such as methanol, ethanol, propanol, isopropanol, rc-butanol, 2-butanol, isobutanol, and tert-butanol. In particular embodiments, the alkanol used in the various procedures disclosed herein is methanol.

[0024] In certain embodiments, the deprotecting reagent used in step (ii) of the synthesis illustrated in Scheme 1, regardless of whether R is H or a hydroxyl -protecting group, is TBAF.

[0025] In particular embodiments, step (i) of the synthesis illustrated in Scheme 1, regardless of whether R is H or a hydroxyl-protecting group, is carried out by reacting compound 16 with sodium hydroxide, in the presence of methanol or isopropanol, to obtain the sodium salt of compound 16; and said deprotecting reagent is TBAF.

[0026] In certain embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is H (i.e., compound 16a), and compound 16a is synthesized as depicted in Scheme 2, by (i) reaction of compound 12 with CrCb and CHI ₃ to obtain compound 13; (ii) reaction of compound 13 with compound 14, in the presence of DEA, Cul and Pd(PPh ₃ )4, to obtain compound 15; and (iii) reduction of the triple bond at position 6-7 of compound 15, e.g., by treatment with activated Zn, to a double bond.

[0027] In other embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is TBDPS (i.e., compound 16b), and compound 16b is synthesized as depicted in Scheme 3, by Wittig-Horner reaction of compound 12 and compound 17.

[0028] In still other embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is TBDPS, and compound 16b is synthesized as depicted in Scheme 4, by (i) reacting compound 18 with DIBAL-H followed by treatment with iodine and triphenylphosphine to make the iodo derivative and then a treatment with PPh ₃ , in the presence of a strong base, to obtain the triphenylphosphine salt 19; and (ii) Wittig reaction of said triphenylphosphine salt 19 with aldehyde 20.

[0029] In yet other embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is TBDPS, and compound 16b is synthesized as depicted in Scheme 5, by (i) reacting compound 21 with triphenylphosphine, in the presence of a strong base, to obtain the triphenylphosphine salt 22; and (ii) Wittig reaction of said triphenylphosphine salt 22 with aldehyde 23.

[0030] In still other embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is TBDPS, and compound 16b is synthesized as depicted in Scheme 6, by (i) reacting compound 24 with DIBAL-H, iodination and followed by treatment with triphenylphosphine, in the presence of a strong base, to obtain the triphenylphosphine salt 25; and (ii) Wittig reaction of said triphenylphosphine salt 25 with aldehyde 26.

[0031] In yet other embodiments, the synthesis illustrated in Scheme 1 starts from compound 16 wherein R is TBDPS, and compound 16b is synthesized as depicted in Scheme 7, by (i) reacting compound 27 with triphenylphosphine and iodine to obtain the iodo compound, then further treatment with PPh ₃ to produce triphenylphosphine salt 28; and (ii) Wittig reaction of said triphenylphosphine salt 28 with aldehyde 29.

[0032] Particular strong bases that can be used in step (i) of each one of the syntheses illustrated in Schemes 4-6 include, without being limited to, DIBAL-H, lithium aluminum hydride, and sodium bis(2-methoxyethoxy)aluminumhydride (Red-Al). In certain embodiments, the strong base used is DIBAL-H.

[0033] Schemes 3-7 illustrate various synthetic routes for the preparation of compound 16, wherein R is TBDPS. Yet, as will be understood to a person skilled in the art, hydroxyl-protecting groups other than TBDPS, e.g., TBDMS, might be used as well, following the same or very similar procedures.

[0034] Compound 12, used for the synthesis of compounds 16a and 16b, may be synthesized according to any method and using any technique known in the art, e.g., as depicted in Scheme 8, exemplified herein, and described in detail in the Experimental section.

[0035] In another particular such aspect, the invention provides a method for the synthesis of RvEl starting from compound 15, said method is carried out as depicted in Scheme 9 and comprises: (i) basic hydrolysis of the ester group of compound 15 with an alkaline hydroxide to obtain an alkaline salt of compound 15; (ii) removal of the hydroxyl- protecting groups at positions 12 and 18 with a deprotecting reagent to obtain the acetylene analog of RvEl; and (iii) reduction of the triple bond at position 6-7 to a cis double bond to thereby obtain RvEl. Alternatively, and as depicted in Scheme 10, the method comprises (i) basic hydrolysis of the ester group of compound 15 with an alkaline hydroxide to obtain an alkaline salt of compound 15; (ii) reduction of the triple bond at position 6-7 to a cis double bond; and (iii) removal of the hydroxyl-protecting groups with a deprotecting reagent to obtain RvEl. As shown in these schemes, the hydroxyl groups at positions 12 and 18 of compound 15 are each protected with TBDPS. Yet, as will be understood to a person skilled in the art, hydroxyl-protecting groups other than TBDPS might be used as well, following the same or a very similar synthetic procedure. [0036] In certain embodiments, step (i) of the synthesis illustrated in Schemes 9-10 is carried out by reacting compound 15 with sodium hydroxide, optionally in the presence of THF or (Ci-C4)alkanol, to obtain the sodium salt of compound 15. In particular such embodiments, compound 15 is reacted with sodium hydroxide, in the presence of methanol or isopropanol, but preferably isopropanol.

[0037] In certain embodiments, the reduction step of the synthesis illustrated in Schemes 9-10 is carried out by treatment with activated Zn.

[0038] In certain embodiments, the deprotecting reagent used in the synthesis illustrated in Schemes 9-10 is TBAF.

[0039] In particular embodiments, step (i) of the synthesis illustrated in Schemes 9-10 is carried out by reacting compound 15 with sodium hydroxide, in the presence of isopropanol or methanol, to obtain the sodium salt of compound 15; the reduction step is carried out by treatment with activated Zn; and said deprotecting reagent is TBAF.

[0040] Compound 15, used for the synthesis of RvEl, may be synthesized according to any method and using any technique known in the art, e.g., as depicted in Scheme 11. Compound 10, which is an intermediate in the synthesis of compound 15, may be synthesized according to any method and using any technique known in the art, e.g., as depicted in Scheme 12.

[0041] The procedures exemplified herein for the synthesis of RvEl may in fact be used for the preparation of any resolvin compound selected from mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt thereof.

[0042] The present invention thus generally relates to a method for the synthesis of a resolvin compound selected from a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt thereof, starting from an ester of said resolvin compound protected at one or more of its hydroxyl groups and optionally having a triple bond, said method comprising: (i) basic hydrolysis of the ester group of said resolvin compound to obtain a salt of said resolvin compound protected at one or more of its hydroxyl groups; (ii) reduction of said triple bond, if present, to a double bond; and (iii) removal of the hydroxyl-protecting groups with a deprotecting reagent to obtain said resolvin compound.

[0043] In certain embodiments, the resolvin compound synthesized by the method of the present invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, EPA, or a pharmaceutically acceptable salt thereof. [0044] In other embodiments, the resolvin compound synthesized by the method of the present invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, DHA, or a pharmaceutically acceptable salt thereof.

[0045] In further embodiments, the resolvin compound synthesized by the method of the present invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, n-3 DPA, or a pharmaceutically acceptable salt thereof.

[0046] The resolvin compound synthesized by the method of the invention has one or more asymmetric centers at each one of the hydroxyl groups thereof, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more, including the endpoints of the stated range of optical purity) and as diastereoisomers. Accordingly, each one of the hydroxyl groups of the resolvin compound or pharmaceutically acceptable salt thereof independently has either an R or S configuration, or is a racemic mixture.

[0047] Optically active forms of the resolvin compound may be obtained using any method known in the art, e.g., by resolution of the racemic form by recrystallization techniques; by chiral synthesis; by extraction with chiral solvents; or by chromatographic separation using a chiral stationary phase. A non-limiting example of a method for obtaining optically active materials is transport across chiral membranes, i.e., a technique whereby a racemate is placed in contact with a thin membrane barrier, the concentration or pressure differential causes preferential transport across the membrane barrier, and separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, can also be used. A wide variety of chiral stationary phases are commercially available.

[0048] The synthesis of the resolvin compound, as disclosed herein, starts from an ester of said resolvin compound protected at one or more of its hydroxyl groups and optionally having at least one triple bond. In certain embodiments, the ester of said resolvin compound is a linear or branched (Ci-Cs)alkyl-ester, (C3-Cio)cycloalkyl-ester, or phenyl- ester.

[0049] The term "alkyl" as used herein typically means a linear or branched saturated hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, ^-propyl, isopropyl, rc-butyl, sec-butyl, isobutyl, ieri-butyl, 2-methylpropyl, rc-pentyl, isopentyl, neopentyl, 2-methylbutyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, rc-hexyl, isohexyl, 2- methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, n- heptyl, 2-methylhexyl, 1,1-dimethylpentyl, 1,2-dimethylpentyl, 1,1,2-trimethylbutyl, n- octyl, 2-methylheptyl, 1,1-dimethylhexyl, 1,2-dimethylhexyl, 1,1,2-trimethylpentyl, and the like. Particular such alkyl groups are (Ci-C4)alkyl groups such as methyl, ethyl, isopropyl and tert-buty\.

[0050] The term "cycloalkyl" as used herein means a cyclic or bicyclic hydrocarbyl group having 3-10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. Particular such cycloalkyls are (C5-Cio)cycloalkyls, e.g., (C5-C7)cycloalkyls.

[0051] The synthesis of the resolvin compound, as disclosed herein, comprises two or three steps, wherein the first step is basic hydrolysis of the ester group of said resolvin compound to obtain a salt of said resolvin compound protected at one or more of its hydroxyl groups. The basic hydrolysis of the ester group of said resolvin compound may be carried out with any suitable basic compound as will be understood to any person skilled in the art. Particular such basic compounds include an organic amine such as triethylamine, ethanolamine, triethanolamine, meglumine, ethylenediamine, and choline; an insoluble salt such as procaine, or benzathine; an alkaline hydroxide as defined above, e.g., sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, or zinc hydroxide, but preferably sodium hydroxide; or a positively charged amino acid such as arginine, lysine, or histidine. In particular embodiments, the basic hydrolysis is carried out with an alkaline hydroxide, preferably sodium hydroxide, and the resolvin compound thus obtained by the method of the invention is in its alkaline salt form, preferably its sodium salt form.

[0052] The method of the present invention may be used for the synthesis of any resolvin compound selected from mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt thereof. In particular embodiments, the resolvin molecule synthesized according to this method is a poly-hydroxylated EPA selected from RvEl or RvE2 (5S,18R-dihydroxy-6E,8Z,l lZ,14Z,16E-EPA); or a poly-hydroxylated DHA selected from RvDl (7S,8R,17S-trihydroxy-4Z,9E,l lE,13Z,15E,19Z-DHA), RvD2 (7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-DHA), RvD3 (4S,l lR,17S-trihydroxy- 5Z,7E,9E,13Z,15E,19Z-DHA), RvD4 (4S,5,17S-trihydroxy-6E,8E,10E,13E,15Z,19Z- DHA), AT-RvDl (17(R)-RvDl; 7S,8R,17R-trihydroxy-4Z,9E,l lE,13Z,15E19Z-DHA), AT-RvD2 (7S, 16R, 17R-trihydroxy-4Z,8E, 10Z, 12E, 14E, 19Z-DHA), AT-RvD3 (4S , 11R, 17R-trihydroxy-5Z,7E,9E, 13Z, 15E, 19Z-DHA), AT-RvD4 (4S ,5, 17R-trihydroxy- 6E,8E,10E,13E,15Z,19Z-DHA), RvD5 (7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z- DHA), or RvD6 (4S,17S-dihydroxy-5E,7Z,10Z,13Z,15E,19Z-DHA) (see Table 1).

Table 1: Resolvin compounds (as their sodium salts) referred to herein

[0053] In another aspect, the present invention provides a compound of the formula I, II, or III, wherein R is H or Na, which are useful as intermediates in the syntheses of RvEl disclosed herein.

Table 2: Compounds of the formulae I, II and III disclosed herein

[0054] The compounds specifically included in this definition are (i) the compound of the formula I wherein R is Na, obtained following hydrolysis of the ester group of compound 16 according to the procedure depicted in Scheme 1 and herein identified compound 1-1; (ii) the compound of the formula I wherein R is H, herein identified compound 1-2; (iii) the compound of formula II wherein R is Na, obtained following hydrolysis of the ester group of compound 15 according to the procedure depicted in Scheme 9 and herein identified compound II- 1 (compound 30); (iv) the compound of the formula II wherein R is H, herein identified compound II-2; and (v) the compound of the formula III wherein R is Na or H, obtained following removal of the hydroxyl -protecting groups of compounds II- 1 and II-2, and herein identified compound III- 1 and III-2, respectively (see Table 2).

[0055] While compositions comprising resolvin compounds or their precursors in general, and RvEl in particular, are highly unstable due to rapid degradation of said compound, it has now been found that basic solutions comprising RvEl Na salt prepared by the method of the present invention, i.e., solutions containing RvEl Na salt and an excess of base, are chemically highly stable for at least several weeks. As specifically described in the Examples section, RvEl Na salt was dissolved in water and stock solutions at pH 7.0-8.0, 8.0-9.0 and > 10.0 were made by adjusting pH with H ⁺ resin and NaOH; and as surprisingly found, all solutions having pH >8.0 were highly stable when checked on HPLC, while only the solution kept at pH 7.0-8.0 developed degradation products.

[0056] In a further aspect, the present invention thus provides a formulation consisting of, or comprising as a sole active agent, a salt of a resolvin compound as defined above, i.e., a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, wherein said composition is in the form of (i) an aqueous solution having a basic pH, which is chemically stable, i.e., shows no degradation of the resolvin compound when checked on HPLC, for at least 1 week, e.g., for about 2 weeks, 3 weeks, 4 weeks, or more; or (ii) a solid that upon reconstitution with water produces an aqueous solution having a basic pH, which is chemically stable for at least 1 week.

[0057] In certain embodiments, the resolvin compound salt comprised within the composition of the invention is a salt of a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri-hydroxylated, EPA. In other embodiments, said resolvin compound salt is a salt of a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri-hydroxylated, DHA; and in further embodiments, said resolvin compound salt is a salt of a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri-hydroxylated, n-3 DPA.

[0058] The resolvin compound salt comprised within the composition of the invention has one or more asymmetric centers at each one of the hydroxyl groups thereof, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more, including the endpoints of the stated range of optical purity) and as diastereoisomers. Accordingly, each one of the hydroxyl groups of said resolvin compound salt has either an R or S configuration, or is a racemic mixture.

[0059] In particular embodiments, the resolvin compound salt comprised within the composition of the invention is a salt of a poly-hydroxylated EPA selected from RvEl or RvE2; or of a poly-hydroxylated DHA selected from RvDl, RvD2, RvD3, RvD4, AT- RvDl, AT-RvD2, AT-RvD3, AT-RvD4, RvD5, or RvD6 (see Table 1). In a preferred embodiment, the composition of the invention consists of RvEl.

[0060] The resolvin compound salt comprised within the composition of the present invention may be any pharmaceutically acceptable salt. Examples of such salts include, without limiting, organic amine salts such as the trimethylamine-, ethanolamine-, triethanolamine-, meglumine-, ethylenediamine-, or choline-salt of said resolvin compound; alkaline or alkaline-earth metal salts such as the sodium-, potassium-, calcium-, magnesium-, or zinc-salt of said resolvin compound; or positively charged amino acid salts such as the arginine-, lysine-, or histidine- salt of said resolvin compound. Preferred embodiments are those wherein said salt is an alkaline salt of said resolvin compound, e.g., the sodium salt thereof.

[0061] In certain embodiments, the composition of the invention as defined in any one of the embodiments above is in the form of an aqueous solution having a basic pH, i.e., a pH higher than 7.0. Particular such compositions have a pH of about >7.5, >8.0, >8.2, >8.4, >8.6, >8.8, >9.0, >9.2, >9.4, >9.6, >9.8, or >10.0. Preferred such composition have a pH of >8.0. In other embodiments, the composition of the invention as defined in any one of the embodiments above is in the form of a solid, e.g., a powder, that upon reconstitution with water produces an aqueous solution as defined above.

[0062] The invention will be now illustrated by the following non-limiting Examples.

EXAMPLES

Example 1. Synthesis of RvEl starting from compound 16a

[0063] RvEl, as its sodium salt, was synthesized as depicted in Scheme 1, starting from compound 16a and according to the following procedure.

[0064] Starting material 16 (760 mg) was dissolved in MeOH or THF or iPrOH and sodium hydroxide solution (20 equivalents) in water was added at 0°C. Reaction mixture turns turbid, more MeOH or THF or iPrOH was added accordingly. Get the reaction mixture to room temperature and stir it overnight. Concentrate the reaction mixture to give the sodium salt of compound 16a and take it to the next step. The salt was dissolved in THF-MeOH (3: 1) or THF solution and cooled to 0°C. TBAF in THF (10 equivalent) was added dropwise. Stir at 0°C for 1 hour and get to room temperature. Check the TLC and run HPLC until the reaction was complete. Water and ethyl acetate were added and aqueous layer was collected. Combine the aqueous layer and concentrate to l/3 ^rd volume. Purify through flash chromatography with a CI 8 column having a gradient of 1-100% ACN/water. RvEl Na salts elutes at 30-40% ACN/water. Collect the pure fractions and adjust pH to 9.0-9.5 with ion-exchange resin (Amberlite IR-120H). Retention time on HPLC matches with the reference standard purchased from Toronto Research Chemicals, Toronto, Canada. Concentrate the pure product, freeze and lyophilize it to achieve the RvEl sodium salt (160 mg).

Example 2. Synthesis of compound 16a starting from compound 12

[0065] Compound 16a was synthesized as depicted in Scheme 2, starting from compound 12 and according to the following procedure.

Synthesis of compound 12

[0066] Dry THF was added to a 25ml round bottom flask containing amide 11 (3g, 4.1 mmol). Nitrogen was purged through this solution and was stirred at -78°C. This was followed by addition of DIBAL-H solution (5.33ml, 5.33 mmol, 1M in THF) dropwise over 25 min. Let it stir for 1 hour at -78°C. Check the TLC and add more DIBAL-H if required. Get to room temperature and quench with potassium sodium tartare solution. Allow the slurry to stir at room temperature for 15 min and extract this solution with MTBE trice. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-10% Ethyl acetate/hexane. Concentrate the pure product 12 and use for next step. ^X H NMR (400 MHz, CDCb) d 0.76 (t, =8 Hz, 3H), 1.07(d, J=12, 18H), 1.45-1.50(m, 2H), 2.21-2.30(m, 2H), 4.08(q, J=6 Hz, 1H), 4.43(q, J=6 Hz, 1H), 5.21(q, =8Hz, 1H), 5.56(dd, J=8Hz, 1H), 5.82-5.94(m, 2H), 6.18(dd, J=8Hz, 1H), 6.58(dd, =8Hz, 1H), 7.28-7.41(m, 12H), 7.57-7.66(m, 8H).

Synthesis of compound 13

[0067] Take dry THF in a round bottom flask and add chromium(II) chloride (0.577g, 4.694 mmol), purge with nitrogen and stir in an ice bath. Take aldehyde 12 (0.537 g, 0.782 mmol) in another round bottom flask with dry THF, purge it with nitrogen and add iodoform (0.308 g, 0.782 mmol) to the solution. Add the aldehyde-iodoform solution to the chromium chloride solution with a glass syringe over 10 min. Stir the reaction at room temperature for 2 hours and check the TLC. Once starting material is consumed, cool the reaction mixture and then quench with sodium bicarbonate solution, extract with ethyl acetate trice. Combine the organic layer, wash with brine and dry over sodium sulfate. DEA was added to the organic layer and the resulting mixture was filtered through Ceilite- Silica bed with THF washing. Filtrate was concentrated and product 13 used for the next step.

Synthesis of compound 15

[0068] In a dry round bottom flask prepare a solution containing tetrakis(triphenylphosphine)palladium (0) (42mg, 0.037 mmol) and copper iodide (14mg, 0.074 mmol) with THF. Take the iodo compound 13 (600 mg, 0.740 mmol) and add THF and DEA (0.61ml, 5.92 mmol) in presence of nitrogen in another round bottom flask, stir for 5 min. To this reaction mixture add the tetrakis - copper iodide solution. Stir for 5 min and with a glass syringe add alkyne-THF solution to the iodo compound over 5 min. Stir the reaction mixture at room temperature overnight. Check the TLC and concentrate the reaction mixture. Purify it through flash chromatography with a gradient of 0-25% ethyl acetate/hexane. Concentrate the pure product and use for next step. ^l H NMR (400 MHz, CDCb) d 0.76 (t, =8Hz, 3H), 1.05(s, 18H), 1.23(q, =8Hz, 6H), 1.42-1.49(m, 3H), 1.75- 1.86(m, 5H), 2.16-2.24(m, 2H), 2.33(t, =8Hz, 2H), 4.06(q, =10Hz, 1H), 4.17(q, =8Hz, 1H), 4.52(s, 1H), 4.98-5.04(m, 1H), 5.22(q, =8Hz, 1H), 5.42-5.58(m, 3H), 5.84- 5.96(m,3H), 6.43(dd, =8Hz, 1H), 7.31-7.44(m, 12H), 7.58-7.67(m, 8H)

Synthesis of compound 16a

[0069] For activating the zinc dust, take HC1 (0.5-1.0 N) and add zinc dust to it, stir for 5 min, filter the mixture and wash it with DI water until neutral. To the dried zinc dust add water to form a suspension and then add acetic acid to it. To this suspension add alkyne 15 in iPrOH. The reaction mixture was stirred at room temperature for 2 hours. Check the TLC and filter through celite bed, rinse with ethyl acetate until no product is seen in aqueous layer. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify through flash chromatography. ^X H NMR (400 MHz, CDCb) d 0.76 (t, =8 Hz, 3H), 1.05(s, 18H), 1.23(d, =8Hz, 6H), 1.40-1.52(m, 2H), 1.63-1.75(m, 4H), 2.15-2.33(m, 4H), 4.06(q, =6Hz, IH), 4.18(q, =6Hz, IH), 4.57(t, =6Hz, IH), 4.96-5.03(m, IH), 5.23(q, =8Hz, IH), 5.38(t, =8Hz, IH), 5.49-5.55(m, IH), 5.64(dd, =8Hz, IH), 5.85(q, =10Hz, 2H), 5.93-6.14(m, 2H), 6.32(t, =14Hz, IH), 7.28-7.41(m, 12H), 7.60-7.67(m, 8H).

Example 3. Synthesis of compound 16b starting from compound 21

[0070] Compound 16b was synthesized as depicted in Scheme 5, starting from compound 21 and according to the following procedure.

[0071] Dry THF was added to a 25ml round bottom flask containing aldehyde. Nitrogen was purged through this solution and was stirred at -78°C. This was followed by addition of DIBAL-H solution dropwise over 15 min. Let it stir for 1 hour at -78°C. Check the TLC and add more DIBAL-H if required. Get to room temperature and quench with potassium sodium tartare solution. Allow the slurry to stir at room temperature for 15 min and extract this solution with MTBE trice. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-20% Ethyl acetate/hexane Concentrate the pure compound and use for next step.

[0072] To a flask add triphenylphosphine (1.1 eq), imidazole (1.1 eq) and anhydrous THF. Cool the mixture in ice bath. To this add iodine (1.1 eq) over the period of 20 min. Alcohol was dissolved in THF and the solution was added to the reaction mixture flask dropwise. Let the reaction stir at room temperature overnight. Concentrate the reaction mixture and dilute with 5% sodium bicarbonate. Extract this solution with ethyl acetate trice and combine the organic layer wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-5% Ethyl acetate/hexane. Concentrate the pure compound and use for next step.

[0073] The iodo compound obtained was taken in a dry round bottom flask and anhydrous acetonitrile was added. Nitrogen was flushed through this solution. Triphenylphosphine (1.2 eq) was then added under nitrogen. The reaction was stirred at room temperature until complete consumption of starting material. Concentrate the reaction mixture and purify it through flash chromatography with a gradient of 0-10% Methanol/ DCM. Concentrate the salt and use for next step.

[0074] The salt obtained was taken in a round bottom flask with anhydrous THF at -78. To this sodium hydride was added. The reaction mixture was got to -20 and aldehyde was added. Stir the reaction at room temperature overnight. Example 4. Synthesis of compound 16b starting from compound 24

[0075] Compound 16b was synthesized as depicted in Scheme 6, starting from compound 24 and according to the following procedure.

[0076] Dry THF was added to a 25ml round bottom flask containing ester 24 (4.4g, 6.05 mmol). Nitrogen was purged through this solution and was stirred at -78°C. This was followed by addition of DIBAL-H solution (4 eq, 1M in THF) dropwise over 15 min. Let it stir for 1 hour at -78°C. Check the TLC and add more DIBAL-H if required. Get to room temperature and quench with potassium sodium tartare solution. Allow the slurry to stir at room temperature for 15 min and extract this solution with MTBE trice. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-20% Ethyl acetate/hexane Concentrate the pure compound and use for next step.

[0077] To a flask add triphenyl phosphine (1.1 eq), imidazole (1.1 eq) and anhydrous THF. Cool the mixture in ice bath. To this add iodine (1.1 eq) over the period of 20 min. Alcohol was dissolved in THF and the solution was added to the reaction mixture flask dropwise. Let the reaction stir at room temperature overnight. Concentrate the reaction mixture and dilute with 5% sodium bicarbonate. Extract this solution with ethyl acetate trice and combine the organic layer wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-5% ethyl acetate/hexane. Concentrate the pure compound and use for next step.

[0078] The iodo compound obtained was taken in a dry round bottom flask and anhydrous acetonitrile was added. Nitrogen was flushed through this solution. Triphenylphosphine (1.2 eq) was then added under nitrogen. The reaction was stirred at room temperature until complete consumption of starting material. Concentrate the reaction mixture and purify it through flash chromatography with a gradient of 0-10% methanol/DCM. Concentrate the pure compound 25 and use for next step.

[0079] The salt 25 obtained above was taken in a round bottom flask with anhydrous THF at -78°C. To this sodium hydride was added. The reaction mixture was got to -20°C and aldehyde was added. Stir the reaction at room temperature overnight. Water added and extracted with ethyl acetate. The ethyl acetate layer was dried on sodium sulfate and concentrated. The crude compound 16b was dissolved in THF (10 ml). It was treated with TBAF in THF (1 M) for 2 days. The formation of isopropyl ester of RvEl (16b) was confirmed by MS.

Example 5. Synthesis of compound 16b starting from compound 27

[0080] Compound 16b was synthesized as depicted in Scheme 7, starting from compound 27 and according to the following procedure.

[0081] To a flask add triphenylphosphine (1.1 eq), imidazole (1.1 eq) and anhydrous THF. Cool the mixture in ice bath. To this add iodine (1.1 eq) over the period of 20 min. Alcohol was dissolved in THF and the solution was added to the reaction mixture flask dropwise. Let the reaction stir at room temperature overnight. Concentrate the reaction mixture and dilute with 5% sodium bicarbonate. Extract this solution with ethyl acetate trice and combine the organic layer wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-5% Ethyl acetate/hexane. Concentrate the pure compound and use for next step.

[0082] The iodo compound obtained was taken in a dry round bottom flask and anhydrous acetonitrile was added. Nitrogen was flushed through this solution. Triphenylphosphine (1.2 eq) was then added under nitrogen. The reaction was stirred at room temperature until complete consumption of starting material. Concentrate the reaction mixture and purify it through flash chromatography with a gradient of 0-10% Methanol/ DCM. Concentrate the salt and use for next step.

Example 6. Synthesis of compound 12 starting from compound 1

[0083] Compound 12 was synthesized as depicted in Scheme 8, according to the following procedure.

[0084] Triphenylphosphine (39.5 g) and imidazole (10.2 g) were dissolved in THF/ACN (200mL/60mL). Mixture was cooled with an ice/water bath and iodine (3.8 g, 15.1 mmol) was added with vigorous stirring over a 30 minute period. To the resulting slurry mixture was added dropwise a solution of compound 1 (20g) in THF/ACN(100 mL/30 mL) at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated, diluted with 5% sodium bicarbonate solution and extracted with hexane. The combined organic layer was dried, concentrated and purified by silica gel chromatography. Yield=24.7g of 2 as light brown oil. [0085] Iodo compound 2 (100 g) was dissolved in 80% acetic acid/water (500 mL). The solution was stirred at room temperature for 2-5 h. Concentrated and purified by silica gel chromatography to give a pure dihydroxy iodo deriavtive 3 (80 g), which was used for the next reaction.

[0086] A mixture of compound 3, imidazole (1 eq.) and DMAP (0.05 eq.) in DCM (40 mL) was stirred at 0°C. To the mixture was added TBSCl (1 eq.) in one portion at 0°C. The resulting was stirred at room temperature overnight. The reaction was quenched with water and extracted with DCM. Combined organic layer was dried, concentrated and purified by silica gel chromatography. Pure product 4 was dissolved in DCM. Imidazole (1 eq.) and DMAP (0.05 eq.) was added and the mixture was stirred at 0°C. TBDPSC1 (1 eq.) was added at 0°C. The resulting reaction mixture was stirred at room temperature overnight, then quenched with water and extracted with DCM. Combined organic layer was dried, concentrated and purified by silica gel chromatography to give disilylated compound 5 (7.8 g). The compound can be distilled under reduced pressure.

[0087] Compound 5 was dissolved in DCM and MeOH. To the solution was added CSA (0.5 eq.) at room temperature. The resulting reaction mixture was stirred at room temperature for 3 h. the reaction was quenched with Et ₃ N (2 eq.) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-20% EA/hexane). Pure fraction was concentrated providing pure monohydroxy TBDPS protected product 6. The product can be obtained by reacting compound 5 with 1% HC1- EtOH.

[0088] A solution of compound 6 in DCM was stirred at 0°C. Dess-Martin periodionane (1.1 eq.) was added in one portion. The reaction was stirred at room temperature for 3 h, then quenched with 10% sodium thiosulfate solution and extracted with DCM. Combined organic layer was dried, concentrated and purified by silica gel chromatography to give the desired aldehyde 7. Aldehyde 7 can also be prepared by oxidation reaction of compound 6 using TEMPO-NaOCl in ethyl acetate.

[0089] Aldehyde 7 was dissolved in DCM, and Ph ₃ P=CHCOOEt or Ph ₃ P=CHCONMeOMe was added at room temperature. The reaction mixture was stirred at room temperature overnight. Concentrated and purified by silica gel chromatography (10% EA in hexane) to give the desired product 8a or 8b. [0090] Product 8a was dissolved in anhydrous acetonitrile. Triphenylphosphine (1.2 eq) and sodium bicarbonate (1.2 eq.) was added. The mixture was refluxed overnight. Concentrated and purified by silica gel chromatography (0-10% methanol/DCM) to give the desired Wittig salt 9a. Compound 9b was also synthesized using similar procedure.

[0091] Compound 9a (5.3 g) was dissolved in dry THF. The solution was stirred at -78°C under nitrogen atmosphere and a KHMDS solution (1.1 eq., 0.5 M in toluene) was added in one portion. After 30 minutes, a solution of 10 (1.5 eq.) in THF was added and the resulting mixture was stirred at -78°C for 30 minutes. Work-up with water and EA. Concentrated and purified by silica gel chromatography (25% EA/hexane). 9.8 g of compound 11 was obtained (Y=89%). The reaction was also performed at -40°C.

[0092] The solution of compound 11 (9.8 g) in dry THF (50 mL) was stirred at -20°C. to the solution was added slowly a DIBAL-H solution (1 M in THF, 3 eq.). The resulting mixture was stirred at 0°C for 30 minutes. Work-up with ethyl acetate and water. Purification by silica gel chromatography (20% ethyl acetate/hexane) gave the desired alcohol 11a (9.1 g).

[0093] A solution of compound 11a (6.75 g) in DCM was stirred at 0°C. Dess-Martin periodionane (1.1 eq.) was added in one portion. The reaction was stirred at room temperature for 3 h, then quenched with 10% sodium thiosulfate solution and extracted with DCM. Combined organic layer was dried, concentrated and purified by silica gel chromatography (20% EA/hexane) to give the desired aldehyde 12 (6.47 g). Compound 12 was also prepared from 11 using 1.0-1.5 eq of DIBAL-H.

Example 7. Synthesis of RvEl starting from compound 15

[0094] RvEl, as its sodium salt, was synthesized as depicted in Scheme 9 or 10, starting from compound 15 and according to the following procedure.

Synthesis of compound 30

[0095] As depicted in Scheme 9, to a solution of compound 15 (2.980 g) in isopropanol (35 ml) was added NaOH (550 mg in 5 ml water) at 0°C. The mixture was stirred overnight. It was concentrated and diluted with 1 N HC1 (20 ml). Mixture was extracted in ethyl acetate, dried on Na ₂ S04, concentrated and dried under vacuum to give acid 30 (2.7 g). 1H NMR (CDCb): 0.78 (t, J=8 Hz, 3H), 1.43-1.50 (m, 2H), 1.75-1.87 (m, 4H), 2.05- 2.29 (m, 2H), 2.43 (t, =8 Hz, 2H), 4.05-4.08 (m, 1H), 4.15-4.20 (m, 1H), 4.52-4.55 (m, 1H), 5.19 -5.23 (dd, J=8 Hz and 16 Hz, 1H), 5.85-5.95 (m, 3H), 6.40-6.47 (dd, J=12 and 16 Hz, 1H), 7.28-7.42 (m, 12H), 7.59-7.68 (m, 8H).

Synthesis of acetylene analog ofRvEl

[0096] Compound 30 (2.7 g) was dissolved in THF (30 ml) and NaOH (1 N, 2 ml) and treated with TBAF in THF (5 eq) at 0°C. The mixture was stirred overnight at room temperature. Additional 5 eq of TBAF was added and stirred for 3 hours. THF was removed under vacuum. Aqueous HCl-ethyl acetate extraction gave crude acid. It was passed over silica bed using 5-10% MeOH to give pure product (1.1 g). 1H NMR (400 MHz, CDC1 ₃ ): 0.9 (t, =8 Hz, 3H), 1.37-1.42 (m, 2H), 1.65-1.79 (m, 4H), 2.31 (t, =8 Hz, 2H), 2.46-2.48 (m, 2H), 3.99-4.04 (m,l H), 4.14-4.19 (dd, =8 and 12 Hz, 1H), 4.43-4.46 (m, 1H), 5.41-5.48 (m, 1H), 5.63-5.68 (m, 2H), 5.80-5.85 (dd, J=8 and 16 Hz, 1H), 6.06- 6.13 (t, =8 Hz, 1H), 6.20-6.32 (m, 1H), 6.46-6.58 (m, 2H).

Synthesis ofRvEl

[0097] The acetylene analog of RvEl (350 mg) was dissolved in 20 ml isopropanol-water (1: 1) and treated with activated Zn (7 g) and AcOH (0.050 ml) in 20 ml isopropanol-water (1: 1). The reaction mixture was stirred at room temperature for 4 hours. Check the HPLC and filter through celite bed, rinse with water and isopropanol. pH of the filtrate was adjusted to 9.5. White precipitate formed was filtered. Isopropanol was removed by vacuum and water was removed by lyo. Silica gel column in 10% MeOH-CH ₂ Cl2 gave desired product (180 mg). Retention time on HPLC matches with the reference standard purchased from Toronto Research Chemicals, Toronto, Canada.

Synthesis of compound 32

[0098] Similarly, as depicted in Scheme 10, compound 32 was prepared from compound 30 using activated Zn in isopropanol-water and catalytic AcOH. The pH of the filtrate was adjusted to 9.5 and concentrated.

Synthesis of RvEl from compound 32

[0099] Compound 32 (100 mg) was dissolved in THF (20 ml) and NaOH (1 N, 1 ml) and treated with TBAF in THF (10 eq) at 0°C. The mixture was stirred overnight at room temperature. THF was removed under vacuum. Aqueous HCl-ethyl acetate extraction gave crude RvEl. It was passed over silica bed using 5-10% MeOH to give RvEl (30 mg). It was mixed with water solution (5 ml, pH 9.4 using NaOH) and lyophilized. Example 8. Synthesis of compound 15 starting from compound 1

[00100] Compound 15 was synthesized as depicted in Scheme 11. For steps 1 to 6, see experimental for Scheme 8 in Example 6.

[00101] Step 7. Dry THF was added to a 25ml round bottom flask containing aldehyde. Nitrogen was purged through this solution and was stirred at -78°C. This was followed by addition of DIBAL-H solution dropwise over 15 min. Let it stir for 1 hour at -78°C. Check the TLC and add more DIBAL-H if required. Get to room temperature and quench with potassium sodium tartare solution. Allow the slurry to stir at room temperature for 15 min and extract this solution with MTBE trice. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify through flash chromatography with a gradient of 0-20% ethyl acetate/hexane, concentrate the pure compound and use for next step.

[00102] Step 8. Take dry THF in a round bottom flask and add chromium(II) chloride (6 eq), purge with nitrogen and stir in an ice bath. Take aldehyde (2.2g) in another round bottom flask with dry THF, purge it with nitrogen and add iodoform (1 eq) to the solution. Add the aldehyde-iodoform solution to the chromium chloride solution with a glass syringe over 10 min. Stir the reaction at room temperature for 2 hours and check the TLC. Once starting material is consumed, cool the reaction mixture and then quench with sodium bicarbonate solution, extract with ethyl acetate trice. Combine the organic layer, wash with brine and dry over sodium sulfate. DEA was added to the organic layer and the resulting mixture was filtered through celite-silica bed with THF washing. Filtrate was concentrated and used for the next step.

[00103] Step 9. In a dry round bottom flask prepare a solution containing tetrakis(triphenylphosphine)palladium (0) (0.05eq) and copper iodide (O.leq) with THF. Take the iodo compound (1.7g) and add THF and DEA (8 eq) in presence of nitrogen in another round bottom flask, stir for 5 min. To this reaction mixture add the tetrakis -copper iodide solution. Stir for 5 min and with a glass syringe add alkyne-THF solution to the iodo compound over 5 min. Stir the reaction mixture at room temperature overnight. Check the TLC and concentrate the reaction mixture. Purify it through flash chromatography with a gradient of 0-25% ethyl acetate/hexane. Concentrate the pure product and use for next step.

[00104] Step 10. In the flask containing the alkyne add anhydrous acetonitrile. Purge with nitrogen followed by addition of triphenylphosphine (1.25 eq) and sodium bicarbonate (1.25 eq). Stir the reaction mixture overnight at 80°C. Stir the reaction until all starting material is consumed. Concentrate the reaction mixture and purify it through flash chromatography with a gradient of 0-10% methanol/DCM. Concentrate the salt and use for next step.

[00105] Step 11. The salt obtained was purged with nitrogen and anhydrous THF was added. Solution was cooled to -78°C for 10 min and KHMDS (2.2 eq, 1M in THF) was added dropwise over 5 minutes. The reaction mixture was stirred at -78°C for 1 hour. To another round bottom flask aldehyde was weighed (1 eq) and THF was added to it. This solution was then added to the reaction mixture containing salt. The reaction mixture was then stirred at -78 for 1 more hour. Continue to stir the reaction until all aldehyde has been consumed. Aqueous workup was done to the reaction and quenched with ethyl acetate. Combine the organic layer, wash with brine and dry over sodium sulfate. Purify it through flash chromatography. Concentrate the pure product and use for next step.

Example 9. Synthesis of compound 10 starting from compound 37

[00106] Compound 10 was synthesized as depicted in Scheme 12, according to the following procedure.

[00107] To a solution of imidazole (77 g), TBSC1 (81.5 g), and DMAP (7 g) in ethyl acetate (675 ml) at 0-5°C was added 1,2-butanediol 37 (48.6 g). The reaction was stirred and allowed to warm to ambient temperature overnight. The material is not UV active, but visible with vanillin in 10%EA/hexane). A solution of TBDPSC1 (leq), imidazole (leq), and DMAP (0.05eq) in 100 ml EtOAc (at 0-5°C added. The reaction was stirred and warmed to ambient temperature overnight. The reaction was quenched with ammonium chloride, the product extracted with DCM, the organic layer washed with sodium bicarbonate and brine. Dried over sodium sulfate and concentrated. The product was distilled under reduced pressure at 150°C (167 g).

[00108] The bis-silyl ether (8.3 g, 18.7mmol) was dissolved in 1: 1 DCM:MeOH (50mL) at rt. Camphorsulphonic acid (0.5eq) was added to the reaction mixture. The reaction was stirred for 2h at room temperature. Triethylamine (l.leq) was added to the reaction mixture to quench. The mixture was concentrated and purified by column chromatography. Product is UV active at 254nm. Chromatography with 0-20% EA/hexane gave 5.34g of product (87% yield). The deprotection can also be performed in 1% HCl-EtOH. The reaction was also performed in ethanol from 240 g disilane compound using 36-35% aq. HC1 for 2 hours and quenched with Et3. The product can be distilled under reduced pressure at 130°C.

[00109] The TBDPS protected alcohol (1.7g, leq) was dissolved in 20mL ethyl acetate or CH2C12 and TEMPO (O.leq) was added. To the stirring reaction mixture was added BAIB (1.2eq). The reaction was followed by TLC and complete after 3h. To the reaction mixture was added TEA (2mL), which was then concentrated and purified by column chromatography (0-20% EA: hexane). 1.3 g of product was isolated (77% yield). Aldehyde was also prepared from TEMPO/NaOCl combination on 220 g scale in presence of NaHC0 ₃ and KBr. The crude product was distilled under reduced pressure at 125°C/500 mtorr.

[00110] The aldehyde (99 g) was dissolved in 800 mL THF. The phosphoranylidene (122 g, CAS 129986-67-0) was added and the solution was stirred overnight. The solvent was evaporated, 100 mL hexanes added and evaporated. The mixture was stirred 30 minutes with 200 mL hexanes. The oil was extracted with 5x200 mL hexane. Evaporation of the solvent gave 136 grams of crude clear oil. The oil was purified on combi-flash starting from 100% hexane and then 10-40% EtOAc-hexane to give 70 grams of >98% clean amide.

[00111] The amide was dissolved in 160 mL THF under nitrogen and cooled to -30°C. The DIBAL-H solution is slowly added while maintaining a temperature below -25°C. When the starting material is below 1% (by GC), after 2-3 hours, a solution of the tartarate (in 200 mL water) was added followed by 200 mL MTBE. The organic phase washed with 100 mL water and 100 mL brine, dried over MgSC and the solvent evaporated under reduced pressure to give compound 10 as orange oil (16.6 g). 1 H NMR (CDC1 ₃ , 400 MHz): δ 0.84 (t, 3H, 7=8.0Hz), 1.08 (s, 9H), 1.51 (m, 2H), 4.43 (m, 1H), 6.17 (dd, 1H, 7=16.0, 8.0 Hz), 6.68 (dd, 1H 7=14.0, 6.0 Hz), 7.37 (m, 6H), 7.40 (m, 4H), 9.46 (d, 1H, 7=8.0 Hz).

Example 10. Basic solutions of RvEl Na salt are chemically stable

[00112] In this study, the chemical stability of the RvEl Na salt prepared as described in

Example 1 was evaluated.

[00113] RvEl Na salt was dissolved in water and stock solutions at pH 7.0-8.0, 8.0-9.0 and > 10.0 were made by adjusting pH with H ⁺ resin and NaOH, and compared with the stock solution of pH 9.0-9.5. The solutions were monitored for 4 weeks. All solutions that were kept at pH >8.0 were stable when checked on HPLC, and only the solution kept at pH 7.0-8.0 developed degradation products. Column: Kinetex C 18 (2.6μ, 100A). 100x4.6 mm (Phenomonex); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile; Gradient: 2 min (90% A), 10 min (10% A), 12 min (10% A), 14 min (90% A); Detection wavelengths: 214, 234, 254, 271, 340 nm, max: 271 nm; Retention time: RvEl=6.8 min.

APPENDIX

Scheme 1: Synthesis of RvEl starting from compound 16

16a: R = H

16b: R = protecting group

Scheme 2: S nthesis of compound 16a starting from compound 12

Scheme 3: Synthesis of compound 16b starting from compounds 12 and 17 Scheme 4: Synthesis of compound 16b starting from com ound 18

Scheme 5: Synthesis of compound 16b starting from compound 21

21 then Ph ₃ P, ACN

16b

Scheme 6: Synthesis of compound 16b starting from compound 24

Scheme 7: S nthesis of compound 16b starting from compound 27

Scheme 8: Synthesis of compound 12 starting from compound 1

1. PPh ₃ , 1 ₂ , Im, THF, ACN

2. 80% AcOH-water

6. DMP, CH ₂ C1 ₂ or TEMPO, NaOCl EtOAC

9a: -CONMeOMe 10

7. Ph ₃ P=CHCONMeOMe or

9b: -COOEt

Ph ₃ P=CHCOOEt

8. Ph ₃ P, ACN, NaHC0 ₃

Scheme 10: Synthesis of RvEl starting from compound 15

Scheme 11: Synthesis of compound 15 starting from compound 1

1. PPh ₃ , I ₂ , Im, THF, ACN

8. CrCl ₂ , CHI ₃ , THF, N ₂

6. TEMPO NaOCl or BAIB 33

7. Ph ₃ P=CHCONMeOMe

8. DIBAL-H

Scheme 12: Synthesis of compound 10

4. DMP, CH ₂ C1 ₂ or TEMPO, NaOCl, EtOAC

5. Ph ₃ P=CHCONMeOMe

6. DIBAL, THF