FIELD

[0001] The specification relates to a process for preparation of an intermediate for eribulin synthesis, and compounds prepared therein for preparation of the intermediate.

BACKGROUND

[0002] Halichondrins have been disclosed as having anti-cancer and antimitotic activity (Chem. Rev. 2009, 109, 3044-3079, incorporated herein by reference). In particular, Halichondrin B has been reported as a potent anticancer agent that was first isolated from the marine sponge Halichondra okadai (US 5,436,238; Tetrahedron Lett. 1994, 35, 9435 and WO 1993/017690 Al, all incorporated herein by reference). It was further reported that analogs of Halichondrin B bearing only macrocyclic fragment of its molecule (Cl - C30 fragment) and having a ketone function instead of ester at Cl position demonstrate anticancer activity similar to Halichondrin B (Bioorg. Med. Chem. Lett., 2000, 10, 1029 and Bioorg. Med .Chem. Lett., 2004, 14, 5551). It was established that such macrocyclic fragment is responsible for induction of mitotic blocks in cancer cells via disruption of tubulin polymerization process that triggers apoptosis of cancerous cells and stops their proliferation (Cancer Res., 2004, 64, 5760 and Mol. Cane. Then, 2008, 7, 2003).

[0003] Eribulin mesylate, a macrocyclic C1-keto analog of Halichondrin B, has been reported as having potent anticancer properties (WO 1999/065894 A1, incorporated herein by reference). Eribulin is marketed under the trade name Halaven, and it is also known as E7389, B1939 and ER-086526. [0004] US 8,975,422 B2 (incorporated herein by reference) discloses a synthetic route for preparation of eribulin by assembly of three fragments F-l, F-2 and F-3, as shown in Scheme I below.

Scheme I : Synthetic route in the preparation of eribulin.

[0005] There is a need in the art for an alternate process for preparation of fragments, used in the process for preparation of eribulin and other macrocyclic Cl-keto analogs of Halichondrin B and their salts. In addition, there is a need in the art for an alternate process for preparation of the F-3 fragment, shown in Scheme I.

SUMMARY [0006] In one aspect, the specification relates to a process for preparation of a compound of formula 13, the process including the steps of: [0007] - protecting the vicinal diol of the compound of formula 1 to form the compound of formula 2,

[0008] - converting the compound of formula 2 to form the compound of formula 4,

[0009] - protecting the primary hydroxyl group of the compound of formula 4 to form the compound of formula 5, [0010] - converting the compound of formula 5 to form the compound of formula 6,

[0011] - converting the compound of formula 6 to form the compound of formula 7, [0012] - converting the compound of formula 7 to form the compound of formula 8,

[0013] - protecting the alcohol functional groups of the compound of formula

8 to form the compound of formula 9,

8 9

[0014] - converting the compound of formula 9 to form the compound of formula 10, [0015] - converting the compound of formula 10 to form the compound of formula 11,

[0016] - deprotecting the compound of formula 11 to form the compound of formula 12, and

[0017] - oxidizing the compound of formula 12 to form the compound of formula 13, [0018] wherein PG ¹ , PG ² , PG ³ and R are as disclosed herein.

[0019] In a second aspect, the specification relates to a process for preparation of a compound of formula 13, the process having the steps of:

[0020] - converting the compound of formula 10 to form the compound of formula 11,

[0021] - deprotecting the compound of formula 11 to form the compound of formula 12, and

[0022] - oxidizing the compound of formula 12 to form the compound of formula 13,

[0023] wherein PG ¹ , PG ² , PG ³ and R. are as disclosed herein.

[0024] In a third aspect, the specification relates to the compound of formula

5a

[0025] In a fourth aspect, the specification relates to the compound of formula 6a

[0026] In a fifth aspect, the specification relates to the compound of formula

7a [0027] In a sixth aspect, the specification relates to the compound of formula

8a

[0028] In a sixth aspect, the specification relates to the compound of formula

[0029] In a seventh aspect, the specification relates to the compound of formula 10a [0030] In an eighth aspect, the specification relates to the compound of formula 11a

DESCRIPTION OF EXAMPLE EMBODIMENTS [0031] As noted above, in one aspect, the specification relates to a process for preparation of a compound of formula 13, the process including the steps of:

[0032] - protecting vicinal diol of the compound of formula 1 to form the compound of formula 2,

[0033] - converting the compound of formula 2 to form the compound of formula 4, [0034] - protecting the primary hydroxyl group of the compound of formula 4 to form the compound of formula 5,

[0035] - converting the compound of formula 5 to form the compound of formula 6, [0036] - converting the compound of formula 6 to form the compound of formula 7,

[0037] - converting the compound of formula 7 to form the compound of formula 8,

[0038] - converting the compound of formula 8 to form the compound of formula 9, [0039] - converting the compound of formula 9 to form the compound of formula 10,

[0040] - converting the compound of formula 10 to form the compound of formula 11, [0041] - deprotecting the compound of formula 11 to form the compound of formula 12, and

[0042] - oxidizing the compound of formula 12 to form the compound of formula 13,

[0043] wherein PG ¹ , PG ² and PG ³ are protecting groups, and R. is a hydrocarbon.

[0044] The step of protecting vicinal diol of the compound of formula 1 to form the compound of formula 2, as shown below, is not particularly limited and should be known to a person of skill in the art.

[0045] A protecting group PG, as used herein, is not particularly limited, and should be known to a person of skill in the art. A protecting group PG is introduced into a molecule by chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction, and as such, prevent reactivity of the functional group in one or more subsequent chemical reactions.

[0046] A diol protecting group for protecting the vicinal diol is not particularly limited and should be known to a person of skill in the art. In one embodiment, for example and without limitation, the diol in the compound of formula 1 can be protected by forming an acetal or ketal. In a further embodiment, for example and without limitation, the diol in the compound of formula 1 can be protected by reaction with acetone, benzaldehyde or cyclohexanone to form an acetonide, benzylidene acetal or a ketal with cyclohexanone to protect the diol in the compound of formula 1. [0047] The step of converting the compound of formula 2 to form the compound of formula 4 is not particularly limited and should be known a person of skill in the art or can be determined. The conversion of the compound of formula 2 to the compound of formula 4 results in an inversion of stereochemistry at the C ⁴ carbon (shown with the arrow below).

[0048] In one embodiment, for example and without limitation, the hydroxyl group in the compound of formula 2 is converted into a leaving group. Without being bound by any theory, it is believed that ring opening of the lactone can lead to formation of an epoxide, which can undergo an intramolecular ring closure reaction, along with inversion of stereochemistry at the C ⁴ carbon (noted with an arrow) to form the lactone, as shown as the compound of formula 4.

[0049] A leaving group, as disclosed herein, is a molecular fragment or stable species that can be detached from a molecule in a bond-breaking step. The leaving group, in accordance with the specification, is not particularly limited and should be known to a person of skill in the art or can be determined. The ability of a leaving group to depart is correlated with the pK _a of the conjugate acid, with lower pK _a being associated with better leaving group ability. Examples of leaving group include, without limitation, halide or a sulfonate. Halides can include, for example, Cl, Br or I. Examples of sulfonates can include, without limitation, nonaflate, triflate, fluorosulfonate, tosylate, mesylate or besylate. In one embodiment, for example and without limitation, the leaving group is tosylate. In another embodiment, for example and without limitation, the leaving group is mesylate.

[0050] The process for the conversion of the hydroxyl group of the compound of formula 2 to a leaving group (LG), as described herein, to form the compound of formula 3, is not particularly limited, and should be known to a person of skill in the art or can be determined. In one embodiment, for example and without limitation, the hydroxyl group is converted into a leaving group by formation of, for example and without limitation, a sulfonate group. In a further embodiment, for example and without limitation, the hydroxyl group is converted into a leaving group by formation of a mesylate.

[0051] The method for ring opening of a lactone is not particularly limited and should be known to a person of skill in the art. In one embodiment, for example and without limitation, ring opening of the lactone is carried out in the presence of a basic solution. The basic solution as used herein is not particularly limited and should be known to a person of skill in the art. In a further embodiment, for example and without limitation, ring opening of the lactone is carried out in the presence of an aqueous basic solution, such as, for example and without limitation, sodium hydroxide, potassium hydroxide or lithium hydroxide.

[0052] Without being bound to any theory, it is believed that reaction of the compound of formula 3 with an aqueous base can lead to ring opening of the lactone, followed by formation of an epoxide at the C ⁴ -C ⁵ position, along with leaving of the leaving group LG. Upon formation of the epoxide, ring closure of the reaction can be carried out in the presence of an acid. The acid as used herein is not particularly limited and should be known to a person of skill in the art. In one embodiment, for example and without limitation, the ring closure and inversion of stereochemistry at the C ⁴ position is carried out in the presence of an aqueous acidic solution. In a further embodiment, for example and without limitation, ring opening of the lactone is carried out in the presence of an aqueous acidic solution, such as, for example and without limitation, hydrochloric acid (HCI), hydrobromic acid (HBr) or triflic acid.

[0053] The step of protecting the primary hydroxyl group of the compound of formula 4 to form the compound of formula 5 is not particularly limited and should be known to a person of skill in the art or can be determined.

[0054] The protecting group PG ² used for protecting the primary hydroxyl group is not particularly limited and should be known to a person of ordinary skill in the art or can be determined. The primary hydroxyl protecting group can be selected to allow selective removal of the primary hydroxyl group PG ² over the vicinal diol protecting group PG ¹ . In one embodiment, for example and without limitation, the primary hydroxyl protecting group is an ether-based or a silyl-based protecting group.

[0055] In a further embodiment, the ether-based protecting group is, for example and without limitation, benzyl (Bn), 2-methoxyethoxymethyl (MEM), trityl (Tr), monomethoxytrityl (MMT), dimethoxytrityl (DMT), methoxymethyl (MOM), p- methoxybenzyl (PMB) or tetrahydropyranyl (THP). Process for removing ether- based protecting groups is not particularly limited, and should be known to a person of skill in the art or can be determined. In one embodiment, for example and without limitation, ether-based protecting groups can be removed by use of an acid-deprotection step or by hydrogenation. As noted above, the protecting group should be selected to allow for selective removal of one protecting group PG ¹ over a second protecting group PG ² .

[0056] In another embodiment, the silyl-based protecting group is, for example and without limitation, tert- butyldi methylsilyl (TBDMS), tri-/so- propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS) or tert-butyldiphenylsilyl (TBDPS). Process for removing silyl-based protecting groups is not particularly limited, and should be known to a person of skill in the art or can be determined. In one embodiment, for example and without limitation, silyl-based protecting groups are removed by use of a fluoride source. The fluoride source is not particularly limited, and should be known to a person of skill in the art or can be determined. In a further embodiment, the fluoride source is, for example and without limitation, sodium fluoride (NaF), tetra-n-butylammonium fluoride (TBAF), pyridinium hydrofluoride (HF-Py) or triethylammonium fluoride (HF-NEts).

[0057] The step of converting of the compound of formula 5 to form the compound of formula 6 is not particularly limited.

[0058] In one embodiment, for example and without limitation, the conversion of compound of formula 5 to the compound of formula 6 is carried out by reducing the carbonyl of the lactone in the compound of formula 5. The process and reagent for carrying out the reduction is not particularly limited and should be known to a person of skill in the art or can be determined. In one embodiment, for example and without limitation, the reduction of the carbonyl is carried out by using a metal hydride as a reducing agent. Metal hydrides of boron and aluminum should be known to a person of skill in the art. In one embodiment, for example and without limitation, the reduction of the compound of formula 5 to the compound of formula 6 is carried out using di-isobutyl aluminum hydride (DIBAL), lithium aluminum hydride, lithium borohydride or lithium triethylborohydride.

[0059] The step of converting the compound of formula 6 to the compound of formula 7 is not particularly limited. In one embodiment, for example and without limitation, the step of converting the compound of formula 6 to the compound of formula 7 involves reacting the compound of formula 6 under Wittig reaction conditions to form the compound of formula 7.

[0060] A Wittig reaction, and variations thereof, should be known to a person of skill in the art. In a Wittig reaction, an aldehyde or ketone is reacted with an ylide (which can be generated from a phosphonium salt), such as triphenyl phosphonium ylide (often called a Wittig reagent), to form an alkene.

[0061] Without being bound to a particular theory, in the subject application, ring opening of the five-membered furan-type moiety, in the compound of formula 6, leads to an aldehyde and hydroxyl group, and where the aldehyde can react with the ylide to form the alkene, shown in the compound of formula 7. The Wittig reagent used to carry out the reaction is not particularly limited and can be determined. In one embodiment, for example and without limitation, the Wittig reagent used is (methoxymethyl)triphenylphosphonium chloride (Ph3P(CI)CH2OMe). In another embodiment, for example and without limitation, another reagent can be used to carry out the conversion of the compound of formula 6 to the compound of formula 7; such as, for example and without limitation, another ylide that can carry -CH ₂ OMe group and form the respective alkene of formula 7 can be used. For instance, and without limitation, any tri-arylphosphonium salt can be used.

[0062] The step of converting the compound of formula 7 to form the compound of formula 8 is not particularly limited. In one embodiment, for example and without limitation, the conversion involves oxidation of the alkene of the compound of formula 7, followed by intramolecular cyclization to form the compound of formula 8.

[0063] The process and reagents used for oxidation of an alkene to a diol is not particularly limited and should be known to a person of skill in the art, or can be determined. In one embodiment, for example and without limitation, an oxidizing agent is used for oxidizing the alkene. In another embodiment, for example and without limitation, osmium tetraoxide can be used for oxidation of the alkene to a diol. The diol formed can undergo intramolecular cyclization to form the compound of formula 8.

[0064] The step of protecting the alcohol functional groups of the compound of formula 8 to form the compound of formula 9 is not particularly limited.

[0065] Alcohol protecting groups have been described herein, and orthogonal protecting groups can be selected to allow for protection and selective deprotection of the diol from the other diol and primary hydroxyl group in the compound of formula 8 and 9. In one embodiment, for example and without limitation, the protecting group PG ³ is an ester. In another embodiment, for example and without limitation, the protecting group PG ³ is an acetyl group (CH ₃ C(=O)-).

[0066] The step of converting the compound of formula 9 to form the compound of formula 10 is not particularly limited.

[0067] In one embodiment, for example and without limitation, the compound of formula 9 is reacted with a hydrocarbon silyl pentenoate in the presence of a Lewis acid to form the compound of formula 10. In another embodiment, for example and without limitation, the compound of formula 9 is reacted with methyl 3-(trimethylsilyl)-4-pentenoate in the presence of boron trifluoride diethyl etherate to form a compound of formula 10. Without being bound to a particular theory, use of boron trifluoride can lead to activation of the anomeric carbon and increased reactivity at the anomeric position.

[0068] The R group in the compound of formula 10 is not particularly limited, and should be known to a person of skill in the art or can be determined. In one embodiment, the R group is an alkyl group or an aryl group. The length of the alkyl group or the number of atoms in the alkyl group or the aryl group are not particularly limited, and should be known to a person of skill in the art or can be determined. In one embodiment, for example and without limitation, the alkyl group is a Ci-6 alkyl. In another embodiment, for example and without limitation, the aryl group is a Ce-i4 aryl.

[0069] The term Ci-6 alkyl in accordance with the specification is not particularly limited and should be known to a person of skill in the art. The Ci-6 alkyl may be, for example, and without limitation, any straight or branched alkyl, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n- pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1,2-dimethylpropyl, 2- ethylpropyl, l-methyl-2-ethylpropyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2-triethylpropyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3- dimethylbutyl, 2-methylpentyl or 3-methylpentyl.

[0070] The term aryl in accordance with the specification is not particularly limited and should be known to a person of skill in the art. The term "aryl" refers to aromatic groups which have at least one ring having a conjugated □-electron system and includes carbocyclic aryl, heterocyclic aryl (also known as heteroaryl groups) and biaryl groups, all of which may be optionally substituted. The aryl groups can include, for example and without limitation, six to fourteen atoms. Examples of aryl group can include, without limitation, phenyl, pyridyl or naphthyl.

[0071] The step of converting the compound of formula 10 to form the compound of formula 11 is also not particularly limited.

10 11

[0072] In one embodiment, for example and without limitation, the compound of formula 10 is deprotected and allowed to undergo an intramolecular cyclization to form the compound of formula 11. In another embodiment, for example and without limitation, the protecting group PG ³ is selected to allow for selective removal over the other protecting groups PG ¹ and PG ² . For instance, and without limitation, protecting group PG ¹ can be selected to be acid labile, protecting group PG ² can be selected to be removable by hydrogenation and protecting group PG ³ can be selected to be base labile.

[0073] In a further embodiment, for example and without limitation, PG ¹ can be a benzylidene ketal, PG ² can be benzyl and PG ³ can be acetyl. In such an embodiment, for example and without limitation, the compound of formula 10 can be reacted with a base for removal of the protecting group PG ³ , which can undergo an intramolecular cyclization to form the compound of formula 11.

[0074] The step of deprotecting the compound of formula 11 to form the compound of formula 12 is not particularly limited. Removal of protecting group vary depending upon the protecting group used and should be known to a skilled worker or can be determined. In one embodiment, for example and without limitation, the protecting group PG ² can be a benzyl protecting group and can be removed by hydrogenation. [0075] The step of oxidizing the compound of formula 12 to form the compound of formula 13 is not particularly limited and involved oxidation of the primary alcohol to an aldehyde. The process and reagents used for oxidation of an alcohol to an aldehyde is not particularly limited and should be known to a person of skill in the art or can be determined.

[0076] In one embodiment, for example and without limitation, the reagent for oxidation of an alcohol to an aldehyde can be (i) Chromium-based reagents, such as Collin's reagent (CrO ₃ -Py ₂ ), pyridinium dichromate (PDC), pyridinium chlorochromate (PCC), (ii) Sulfonium species known as "activated DMSO" which can result from reaction of DMSO with electrophiles, such as oxalyl chloride (Swern oxidation), a carbodiimide (Pfitzn er- Moffatt oxidation) or the complex SO ₃ -Py (Parikh-Doering oxidation), or (iii) Hypervalent iodine compounds, such as Dess- Martin periodinane or 2-Iodoxybenzoic acid. In a further embodiment, for example and without limitation, the reagent used for oxidation of an alcohol to an aldehyde is Dess-Martin periodinane.

[0077] Scheme II discloses an embodiment of a process for preparation of compound 13.

Scheme II: embodiment of a process for preparation of a compound of formula

13a. [0078] The compound of formula 1 is reacted with cyclohexanone to protect the vicinal diol to form the compound of formula 2a. The primary hydroxyl of the compound of formula 2a is converted into a leaving group, followed by ring opening and closing of the lactone, along with removal of the leaving group to form the compound of formula 4a. The primary hydroxyl group in the compound of formula 4a is protected with a benyl protecting group to form the compound of formula 5a, followed by reduction of the carbonyl group in the lactone to form the compound of formula 6a. Wittig reaction of the compound of formula 6a with (methoxymethyl)triphenylphosphonium chloride leads to formation of the compound of formula 7a, which is then oxidized using osmium tetraoxide, followed by an intramolecular cyclization leading to the compound of formula 8a.

Acetylation of the compound of formula 8a leads to the compound of formula 9a, which is reacted with methyl 3-(trimethylsilyl)-4-pentenoate in the presence of boron trifluoride diethyl etherate to form the compound of formula 10a. Removal of the acetyl protecting group from the hydroxyl group leads to intramolecular cyclization and formation of the compound of formula 11a. The benzyl protecting group is removed by hydrogenation to form the compound of formula 12a, followed by oxidation of the primary hydroxyl group to the aldehyde to form the compound of formula 13a.

[0079] Certain adaptations and modifications of the described embodiments can be made. Therefore, the above discussed embodiments are considered to be illustrative and not restrictive.

EXAMPLES

[0080] The following examples are illustrative and non-limiting, and represent specific embodiments of the present invention.

[0081] EXAMPLE 1 : Preparation of the compound of formula 2a

[0082] To a round bottom flask was added 1 (500 g, 1.0 eq), copper sulphate (1616 g, 3.0 eq), cyclohexanone (663 g, 2 eq) and dichloromethane (5 L, 10 parts v/w). The mixture was agitated at 40-45°C for 48 h under nitrogen. ¹ H NMR.

(CD3OD) analysis indicated reaction completion. The reaction mixture was cooled to 20-24°C and the insoluble material was removed by filtration and rinsed with dichloromethane (1 L, 2 parts v/w). The filtrate was washed with 50% saturated NaCI (2 L, 4 parts v/w) solution (2 L, 4 parts v/w) and then concentrated to 3 parts v/w under reduced pressure. Heptane (2 L, 4 parts v/w) was charged slowly over 30 min at 20-24°C and the resulting slurry was agitated for 16 h at this temperature. The mixture was filtered, washed with heptane (2 x IL, 2 x 2 parts v/w), and dried under vacuum under nitrogen to give 2a (466 g) as a white solid in 60% yield. ¹ H NMR. (300 MHz, Chloroform-d) 5 4.89 - 4.75 (m, 2H), 4.66 (t, J = 2.1 Hz, 1H), 4.01 (ddd, J = 12.2, 5.4, 2.3 Hz, 1H), 3.82 (ddd, J = 12.3, 5.7, 1.8 Hz, 1H), 2.62 (d, J = 6.2 Hz, 1H), 1.80 - 1.29 (m, 13H).

[0083] EXAMPLE 2: Preparation of the compound of formula 3a

[0084] To a round bottom flask was added 2a (100 g, 1.0 eq) and dichloromethane (1 L, 10 parts v/w) under nitrogen. The resulting solution was agitated and cooled to 0-5°C. Methanesulfonyl chloride (60 g, 1.2 eq) and triethylamine (53 g, 1.2 eq) were added and the reaction was maintained at 0-5°C. HPLC analysis indicated reaction completion. Water (300 ml, 3 parts v/w) was added while maintaining the internal temperature less than 25°C. The mixture was agitated for 30 min and then the layers were separated. The organic layer was washed with 50% saturated NaCI (aq) solution (300 ml, 3 parts v/w) and concentrated to 3-4 parts v/w. Ethyl acetate (800 ml, 8 parts v/w) was charged and the solution was concentrated to 3-4 parts v/w. Heptane (400 ml, 4 parts v/w) was charged and the solution was concentrated to 3-4 parts v/w. The mixture was filtered and washed with heptane (2 x 100 ml, l x l parts v/w) to give to give the 3a intermediate (124 g) as a light yellow solid in quantitative yield.

[0085] EXAMPLE 3: Preparation of the compound of formula 4a

[0086] To a round bottom flask was added 3a (123 g, 1.0 eq) and water (246 ml, 2 parts v/w). The mixture was cooled to 10-15°C forming a slurry mixture.

45% w/v Aqueous solution of KOH (150 ml, 2.4 eq) ¹ was slowly added at a rate maintaining an internal reaction temperature less than 30°C. The mixture was agitated at 20-24°C for 1-2 h. HPLC analysis indicated conversion of D825 to epoxide intermediate. A 30% w/v aqueous solution of KCI (134 g, 1 part v/w) was added. 3M HCI (aq) (190 ml, 1.4 eq) was added to pH=3 at a rate maintaining an internal reaction temperature of less than 30°C. The reaction mixture was heated at 40°C over 24-48 h. ² HPLC analysis indicated reaction completion from epoxide intermediate to 4a. The reaction mixture was cooled to 20-24°C and extracted with methyl tert-butyl ether (2 x 500 ml, 2 x 5 parts v/w). The organic extracts were washed with water (500 ml, 5 parts v/w) and then concentrated under vacuum to 3-4 parts v/w. Heptane (1 L, 10 parts v/w) was charged and the mixture was concentrated under vacuum to 5 parts v/w. The slurry was cooled and then filtered and dried in vacuum oven to afford 52 g of crude 4a as a white solid. The solid was purified by column chromatography using EtOAc/Hept gradient to afford pure 4a (43 g) in 46% yield. ¹ H NMR (300 MHz, Chloroform-d) 5 4.93 - 4.74 (m, 2H), 4.66 (t, J = 2.1 Hz, 1H), 4.01 (ddd, J = 12.3, 5.3, 2.4 Hz, 1H), 3.82 (ddd, J = 12.3, 5.7, 1.8 Hz, 1H), 2.67 (t, J = 5.5 Hz, 1H), 1.88 - 1.28 (m, 10H).

[0087] EXAMPLE 4: Preparation of the compound of formula 5a

[0088] 2,4,6-Tris(benzyloxy)-l,3,5-triazine (TriBOT) is prepared according to the procedure reported in Org. Lett. 2012, 14, 5026, incorporated herein by reference.

[0089] To a round-bottom flask, charge 4a (4.6 g, 1.0 eq), TriBOT (3.2 g, 0.4 eq) and 4A molecular sieves (3.0 g, 50 mg/mL). Anhydrous 1,4-dioxane (60 mL, 13 part v/w) was added under nitrogen. Trifluoromethanesulfonic acid (350 pL, 0.2 eq) was added at ambient temperature. The reaction mixture was monitored by HPLC. After stirring overnight, HPLC shows 8% of 4a remaining. Additional TriBOT (400 mg, 0.05 eq) was added and stirred for additional 3 hours. HPLC shows only 2% of 4a remaining. The reaction mixture was quenched with NEts (1.0 g, 0.5 eq) and the resulting mixture was diluted with EtOAc (68 mL, 15 parts v/w). To the suspension was added 50% sat. NaHCO ₃ aq (46 mL, 10 parts v/w) and stirred at ambient temperature for 1 hour. The mixture was filtered through a Celite pad (9 g, 2 parts) and the wet cake was washed with EtOAc (15 mL, 3 parts v/w). The filtrate was separated and the aqueous layer was extracted with EtOAc (15 mL, 3 parts v/w). The combined organic layers were washed with IM HCI solution (15 mL, 3 parts v/w) then 50% brine 15 mL, 3 parts v/w). The solvent was swapped to EtOAc (to remove excess dioxane) (9 mL, 2 parts v/w) and heptane (68 mL, 15 parts v/w) was added dropwise over 40 minutes. A solid formed and the suspension was stirred at RT for 2 hours and filtered. The wet cake was washed with heptane twice (2 x 18 mL, 4 parts v/w) and vacuum dried to afford 3.6 g 5a. More solid formed in the filtrate and the mixture was filtered and washed with heptane to get 0.8 g 5a as a second crop. The overall yield is 68%. ¹ H NMR (300 MHz, Chloroform-d) 5 7.35 (m, 5H), 4.84 (m, 2H), 4.65 (m, 3H), 3.93 (dd, J = 10.7, 5.2 Hz, 1H), 3.83 (dd, J = 10.6, 7.0 Hz, 1H), 1.65-1.42 (m, 10H).

[0090] EXAMPLE 5: Preparation of the compound of formula 6a

[0091] To a round bottom flask was added 5a (3 g, 1.0 eq) followed by anhydrous toluene (30 ml, 10 parts v/w). The resulting suspension was agitated at ambient temperature under nitrogen until the solids dissolved. The clear solution was then cooled to -20 to -25°C and 1.0 M diisobutyl aluminum hydride (DIBAL) in toluene (11.5 ml, 1.2 eq) was added slowly while maintaining an internal temperature of -20 to -25°C. The mixture was agitated at -20 to -25°C for 2 h. HPLC analysis indicated reaction completion. Methanol (6 ml, 2 parts v/w) was slowly added (at -20 to -25°C). The mixture was agitated for 1-2 h at -20 to -10°C. A 50% w/w aqueous solution of K2HPO4 (6 g, 2 parts w/w) was charged at a rate maintaining the internal temperature below 22°C. The resulting slurry was agitated at 20-24°C for 16 h. MgSO4 (3 g, 1 part w/w) was then added and the slurry was agitated for 2 h. The mixture was filtered and the filtrate was concentrated to dryness to give 3.4 g of crude 6a as an oil. The crude oil was purified by column chromatography (SiO ₂ , 10 parts w/w, methyl tert-butyl ether /heptane gradient) to give 2.5 g of 6a as colorless oil in 80% yield. ¹ H NMR (300 MHz, Chloroform-d) 5 7.34 (m, 5H), 5.44 (s, 1H), 4.76 (dd, J = 5.9, 3.7 Hz, 1H), 4.62 (m, 3H), 4.40 (dt, J = 7.6, 3.8 Hz, 1H), 3.83 (dd, J = 10.4, 3.9 Hz, 1H), 3.71 (dd, J = 10.4, 7.7 Hz, 1H), 3.24 (s, 1H), 1.59 (m, 9H), 1.39 (q, J = 5.4, 4.9 Hz, 3H).

[0092] EXAMPLE 6: Preparation of the compound of formula 7a

[0093] To a round bottom flask was added (methoxymethyl)triphenylphosphonium chloride (5.4 g, 2.5 eq) and anhydrous tetrahydrofuran (10 ml, 5 parts v/w). The suspension was agitated and cooled to 0- 5°C under nitrogen. 20% w/v potassium t-butoxide in tetra hydrofuran solution (8.6 g, 2.5 eq) was slowly added while maintaining an internal temperature below 10°C. The reaction mixture was allowed to warm to ca. 20-24°C and agitation was continued for 1 h. The resulting deep red solution was cooled to 0-5°C and a solution of 6a (2 g, 1.0 eq,) dissolved in anhydrous tetra hydrofuran (7.5 ml, 3.7 part v/w) was added while maintain a temperature below 10°C. The reaction was warmed to 20-24°C and the mixture was agitated over 16 h. HPLC analysis indicated reaction completion. The reaction mixture was cooled to 0-5°C and saturated NH ₄ CI (aq) (20 ml, 10 parts v/w) was slowly charged while maintaining the temperature at 5-10°C. The mixture was warmed to room temperature and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 ml, 2 x 5 parts v/w). The organic layers were combined, washed with saturated NH ₄ CI (20 ml, 10 parts v/w), dried over MgSO ₄ (5 g, 2.5 parts w/w), filtered, and concentrated to an oil. A 2: 1 mixture of heptane/methyl t-butyl ether (20 ml, 10 parts v/w) was added the mixture was concentrated under vacuum at 35-45°C. This step was repeated twice. A final portion of 2: 1 heptane/methyl t-butyl ether (20 ml, 10 parts v/w) was added. The mixture was agitated until a mobile suspension was obtained, cooled to 0-5°C and then agitated for a 1 h. The resulting precipitate was filtered and the filtrate was concentrated under reduced pressure to afford 3.6 g of crude 7a as an oil. The crude material was purified by column chromatography (SiO ₂ , 10 parts w/w, using methyl t-butyl ether /heptane gradient) to afford 2 g of 7a as a clear colorless oil in 55% yield. ¹ H NMR (300 MHz, Chloroform-d) 5 7.35 (m, 6H), 6.54 (m, 0.43H), 6.05 (d, J = 6.3 Hz, 0.46H), 5.20 (dd, J = 9.4, 6.7 Hz, 0.44H), 5.00 (m, 0.5H), 4.60 (m, 3H), 4.18 (ddt, J = 6.8, 4.7, 2.7 Hz, 1H), 3.82 (m, 1H), 3.59 (t, J = 3.3 Hz, 3H), 3.53 (m, 2H), 2.40 (t, J = 5.8 Hz, 1H), 1.67 (m, 9H), 1.44 (dt, J = 10.4, 4.3 Hz, 2H).

[0094] EXAMPLE 7: Preparation of the compound of formula 8a

[0095] To a round bottom flask was added compound 7a (10 g, 1.0 eq) and acetone (40 ml, 4 parts v/w). The clear solution was cooled to 0-5°C. N- Methylmorpholine /V-oxide mono hydrate (6.4 g, 1.6 eq) and 4% w/w aqueous OsO4 (0.65 ml, 0.0035 eq) were added in succession while maintaining a temperature of 0-5°C. The resulting light yellow suspension was slowly warmed to ambient temperature and agitated at 20-24°C for 16-20 h. HPLC analysis indicated reaction completion. The reaction mixture was cooled to 0-5°C and 10% w/v NazSzCh (aq) (100 ml, 10 parts v/w) was added followed by tert-butyl methyl ether (50 ml 5 parts v/w). The mixture was agitated for 20 min. The layers were separated and the aqueous layer was further extracted with tert-butyl methyl ether (2 x 50 ml, 2 x 5 parts v/w). The combined organic layers were concentrated under vacuum to give crude oil. The crude product was purified by column chromatography (SiO ₂ , 20 parts w/w, using heptane/methyl t-butyl ether gradient) to give 3.8 g of 8a (30 % yield, >85: 15 mixture of diastereomers) as a white solid. The product was recrystallized from 2: 1 water/acetone to give 3 g of a >95:5 mixture of diastereomers. ¹ H NMR (300 MHz, Chloroform-d) 5 7.34 (m, 7H), 5.18 (dd, J = 5.9, 3.6 Hz, 1H), 4.60 (ddd, J = 37.4, 12.4, 4.1 Hz, 2H), 4.43 (m, 2H), 4.18 (dd, J = 7.5, 2.0 Hz, 1H), 4.01 (m, 1H), 3.67 (d, J = 5.8 Hz, 4H), 3.03 (d, J = 6.8 Hz, 1H), 1.63 (m, 10H).

[0096] EXAMPLE 8: Preparation of the compound of formula 9a

[0097] To a round bottom flask was added 8a (10 g, 1.0 eq), 4- (dimethylamino)pyridine (DMAP) (0.37 g, 0.1 eq), and dichloromethane (100 ml, 10 parts v/w). The mixture was agitated and cooled to 0-5°C under nitrogen. Triethylamine (10 ml, 2.5 eq) and acetic anhydride (13.5 ml, 5.0 eq) was slowly charged in succession while maintaining a temperature of 0-10°C. The mixture was agitated at 0-5°C for 2 h. TLC analysis (1 : 1 methyl tert-butyl ether/heptane) indicated reaction completion. The reaction mixture was washed with 1 M HCI (aq) (100 ml, 10 parts v/w), saturated NaHCO ₃ (aq) (100 ml, 10 parts v/w), and half saturated brine (100 ml, 10 parts v/w). The organic layer was then concentrated under vacuum to afford crude 17.7 g of 9a as an oil. The crude oil was purified by column chromatography (SiO ₂ 10 parts w/w, heptane/methyl tert-butyl ether gradient) to remove the mono-acetylated by-product resulting in 9 g of 9a as a pale yellow oil in 78% yield. ¹ H NMR. (300 MHz, Chloroform-d) 5 7.34 (m, 5H), 6.21 (d, J = 7.0 Hz, 1H), 5.12 (dd, J = 7.0, 2.8 Hz, 1H), 4.61 (m, 3H), 4.43 (dd, J = 7.7, 1.9 Hz, 1H), 3.98 (d, J = 6.5 Hz, 1H), 3.68 (m, 2H), 2.17 (s, 2H), 2.11 (t, J = 2.8 Hz, 1H), 2.08 (s, 2H), 1.66 (m, 10H). [0098] EXAMPLE 9: Preparation of the compound of formula 11a

[0099] To a round bottom flask was added 9a (5 g, 1.0 eq) and anhydrous acetonitrile (50 ml, 10 parts v/w). The solution was cooled to 0-5°C under nitrogen. Methyl 3-(trimethylsilyl)-4-pentenoate (5.5 ml, 2.3 eq) was charged follow by boron trifluoride diethyl etherate (2.8 ml, 2.0 eq) at a rate maintaining an internal reaction temperature of 5-8°C. The reaction was maintained at 0-8°C for 16-20 h. HPLC analysis indicated reaction completion. A 1 : 1 mixture of saturated brine and saturated NaHCO ₃ (aq) (30 ml, 6 parts v/w) was added maintaining the temperature at 0-10°C. After continued agitation and warming to ambient temperature, the layers were separated and the aqueous layer was extracted with toluene (10 ml, 2 parts v/w). The combined organic layers were dried over MgSC , filtered, and concentrated under vacuum to afford the crude 10a as an oil. The crude oil was purified by column chromatography (SiO ₂ , 20 parts w/w, heptane/methyl tert-butyl ether gradient) to give 3.4 g of 10a as a colorless oil in 61% yield.

[00100] To a round bottom flask was added 10a (3 g, 1 eq) and anhydrous tetrahydrofuran (30 ml, 10 parts v/w). The solution was cooled to 0-5°C and 25 % w/w NaOMe in methanol solution (3 g, 2.0 eq) was charged in one portion. The reaction mixture was agitated at 0-5°C for 2 h. HPLC analysis indicated reaction completion. The reaction mixture was diluted with t-butyl methyl ether (15 ml, 5 parts v/w). Acetic acid (0.8 ml, 2.0 eq) was charged followed by a 1 : 1 mixture of saturated brine and saturated NaHCO ₃ (aq) (30 ml, 10 parts v/w). After continued agitation and warming to ambient temperature, the layers are separated. The aqueous layer was extracted with tert-butyl methyl ether (2 x 15 ml, 2 x 5 parts v/w). The combined organic layers were washed with half saturated brine (15 ml, 5 parts v/w) and concentrated under vacuum to an oil. The crude oil was then purified by chromatography (SiO ₂ , 8 parts w/w, tert-butyl methyl ether/heptane gradient) to afford 1.9 g of Ila as a pale yellow oil in 62% yield. ¹ H NMR (300 MHz, Chloroform-d) 5 7.34 (m, 6H), 4.67 (dd, J = 12.2, 1.6 Hz, 1H), 4.54 (m, 2H), 4.44 (dt, J = 8.4, 1.8 Hz, 1H), 3.90 (m, 3H), 3.71-3.57 (m, 2H), 3.69 (s, 3H), 3.52 (dt, J = 10.3, 2.3 Hz, 1H), 2.74 (ddd, J = 16.1, 6.9, 1.7 Hz, 1H), 2.44 (ddd, J = 16.1, 6.2, 1.6 Hz, 1H), 2.11 (m, 1H), 1.62 (m, 7H).

[00101] EXAMPLE 10: Preparation of the compound of formula 12a

[00102] To a round bottom flask was added Ila (12.1 g, 1.0 eq) followed by 10 % w/w Pd/C (2.4 g, 0.2 parts w/w) and tetra hydrofuran (60 ml, 5 parts v/w). The mixture was agitated and the headspace of the reactor was purged with hydrogen, then heated at 45-50°C for 16 h. NMR. analysis indicated reaction completion The reaction mixture was purged with nitrogen, filtered through a pad of Celite, and rinsed with tert-butyl methyl ether (100 ml, 8 parts v/w), The filtrate was concentrated under vacuum to give 9 g of 12a as a colorless oil in 93% yield. ¹ H NMR (300 MHz, Chloroform-d) 5 4.57 (dd, J = 8.3, 3.0 Hz, 1H), 4.41 (dd, J = 8.4, 1.4 Hz, 1H), 3.85 (m, 5H), 3.70 (s, 3H), 3.54 (dd, J = 10.2, 3.0 Hz, 1H), 2.74 (dd, J = 16.1, 6.9 Hz, 1H), 2.45 (dd, J = 16.1, 6.0 Hz, 1H), 2.25 (d, J = 8.5 Hz, 1H), 2.14 (m, 1H), 1.67 (m, 9H).

[00103] EXAMPLE 11 : Preparation of the compound of formula 13a

[00104] To a round bottom flask was added 12a (8.7 g, 1.0 eq) and dichloromethane (100 ml, 11 parts v/w). The solution was agitated and cooled to 0- 5°C under nitrogen. Dess-Martin periodinane (15.6 g, 1.5 eq) was added and the reaction was warm to 20 - 24°C and agitated for 2 h. NMR analysis indicated reaction completion. , The reaction mixture was cooled to 0-5°C and a 10% w/v solution of Na ₂ S ₂ O ₃ (50 ml, 6 parts v/w) was slowly charged. After warming to ambient temperature with agitation, the layers were separated and the organic layer was washed with saturated NaHCO ₃ (aq) (50 ml, 6 parts v/w) and brine (50 ml, 6 parts v/w). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to a semi-solid. Crude 13a was triturated with tert-butyl methyl ether (50 ml, 6 parts v/w) to remove insoluble by-products by filtration and the filtrate was concentrated under vacuum to an oil. Crude 13a was purified by chromatography (SiO ₂ , 6 parts w/w, using 1 : 1 heptane/ethyl acetate eluent) to afford 5.2 g of 13a as a pale yellow oil in 59% yield. ¹ H NMR.

(300 MHz, Chloroform-d) 5 9.63 (d, J = 0.9 Hz, 1H), 4.71 (dd, J = 8.2, 2.2 Hz, 1H), 4.59 (dd, J = 8.2, 3.0 Hz, 1H), 4.11 (d, J = 2.2 Hz, 1H), 4.01 (td, J = 10.5, 4.8 Hz, 1H), 3.88 (m, 1H), 3.69 (s, 3H), 3.44 (dd, J = 10.2, 3.1 Hz, 1H), 2.74 (dd, J = 16.1, 7.0 Hz, 1H), 2.45 (dd, J = 16.2, 6.0 Hz, 1H), 2.21 (dt, J = 11.1, 3.7 Hz, 1H), 1.63 (m, 13H), 1.26 (td, J = 8.6, 6.9, 4.5 Hz, 1H).