Process for preparing di-, TRI-, or tetra-saccharides

Abstract

Disclosed is a process for preparing di-, tri-, or tetrasaccharides useful as intermediates for the synthesis of vaccine adjuvants. The process comprises reacting in the presence of a dehydrating agent and a sugar catalyst characterized by hydroxy groups converted to trichloroacetimidate or N-phenyl trifluoroacetimidate.

Description

[0001] PROCESS FOR PREPARING DI-, TRI-, OR TETRA-SACCHARIDES

[0002] The invention relates to a process for preparing di-, tri-, or tetrasaccharides, which comprises the reaction in the presence of a dehydrating agent and a sugar catalyst characterized by hydroxy groups converted to trichloroacetimidate or N-phenyl trifluoroacetimidate.

[0003] STATE OF THE ART

[0004] Adjuvants are substances capable of enhancing the immune response of vaccines. They play a crucial role in improving efficacy, especially in those cases where vaccine antigens alone are not sufficiently immunogenic. Adjuvants can act in several ways: by prolonging the duration of the antigen in the body, by stimulating a more robust and specific immune response, or by modulating the type of immune response (for example, by promoting the production of antibodies or the activation of T cells). Some of the most commonly used adjuvants include aluminium compounds, oil emulsions, saponins, and liposomes.

[0005] Thanks to the adjuvants, it is therefore possible to obtain a more powerful and persistent immune response, the antigens can be administered in smaller quantities and fewer administrations are required (Phytomedicine 2019 60 152905).

[0006] One of the most potent and widely used vaccine adjuvants is a mixture of triterpene glycosides isolated from the bark of Quillaja saponaria Molina and known as saponin QS-21. Such a mixture comprises two isomeric molecules, QS-21 apiosium (QS-21 Api) and QS-21 xylose (QS-21 Xyl), each comprising four moieties: the triterpene quillajic acid, a branched trisaccharide linked to the C-3 of quillajic acid via an O-glycosidic bond, a linear tetrasaccharide linked to the C-28 of quillajic acid via an ester bond, and an acyl chain linked to the 0-4 of the fucose unit present in the tetrasaccharide. The two isomers QS-21 Api and QS- 21 Xyl differ in the tetrasaccharide portion containing a P-D-Xyl-1,3-P-D-Xyl disaccharide in QS-21 Xyl (depicted in the figure below), or P-D-Api-1,3-P-D-Xyl in QS-21 Api.

[0007] The methods for isolating and preparing triterpene glycosides and their constituent portions are known. For example, D. Gin et al (Angew. Chem. Int. Ed. 2008, 47, 6395 -6398) describes a method for the preparation of the linear tetrasaccharide of QS-21 Xyl that provides for the sequential addition of each individual sugar, using different strategies that provide for the use of reagents that are difficult to manage on a large scale, such as DIBAL-H, and sensitive to humidity. An alternative tetrasaccharide preparation process is described in Wang (J. Org. Chem. 2013, 78, 11525-11534), where the preparation of the tetrasaccharide is implemented through a 2+2 strategy activated using N-iodo- succinimide (NIS) and triflic acid (TfOH). This approach is more convergent than the one described by Gin. However, the use of NIS / TfOH has several disadvantages such as poor reproducibility, due to the sensitivity of the reagents to moisture, and the difficulty of use on an industrial scale.

[0008] Considering the usefulness of the QS-21 mixture, it is necessary to identify new methods of preparing its constituent isomers and the four portions that compose them.

[0009] DESCRIPTION OF THE INVENTION

[0010] The Applicant has developed a process for preparing the sugar moieties constituting the isomers present in the mixture QS-21, in which at least one hydroxyl of a sugar is converted into a trichloroacetimidate (-OC(NH)CC13) or into an N-phenyl trifluoroacetimidate (- OC(NPh)CF3) and the reaction is carried out in the presence of a dehydrating agent and a catalyst; in particular, a glycosidic bond is created between a hydroxyl group converted into trichloro- or N-phenyl trifluoroacetimidate of a first sugar, defined as a donor, and a free hydroxyl group of a second sugar, defined as an acceptor.

[0011] In a first aspect, the invention therefore relates to a process for preparing a di-, tri- or tetrasaccharide P by reaction of a donor mono- or disaccharide D having free or protected hydroxyls of which at least one hydroxyl is activated as trichloroacetimidate (-OC(NH)CC13) or as N-phenyl trifluoroacetimidate (-OC(NPh)CF3) with a mono- or disaccharide acceptor A bearing free or protected hydroxyls of which at least one free hydroxyl, in the presence of a dehydrating agent and a catalyst to give the compound of formula P (0= oxygen) compound P wherein D and A, the same or different, are a monosaccharide selected from DE- xylose, E-xylose, D-xylose, DL-galactose, E-galactose, D-galactose, D-glucuronic acid, DL- glucuronic acid, E-glucuronic acid, D-fucose, E-fucose. DL-fucose, DL-rhamnose, D-rhamnose, L-rhamnose, DL-apiosium L-apiosium and D-apiosium or a disaccharide consisting of two of said monosaccharides, the same or different.

[0012] Preferably, the compound D is a free or protected hydroxyl-bearing monosaccharide or disaccharide of which at least one hydroxyl activated as trichloroacetimidate (-0(NH)CC13) or as A-phenyl trifluoroacetimidate (-O(NPh)CF3), wherein the monosaccharide is selected from D-xylose, D-galactose, -D-glucuronic acid, L-fucose, L-rhamnose, D-apiose or wherein the disaccharide comprises pairs of the same or different monosaccharides selected from D- xylose, D-galactose, -D-glucuronic acid, L-fucose, L-rhamnose, D-apiosium.

[0013] Preferably, A is a free or protected hydroxyl bearing monosaccharide or disaccharide of which at least one free hydroxyl, wherein the monosaccharide is selected from D-xylose, D-galactose, -D-glucuronic acid, L-fucose, L-rhamnose, D-apiosium or wherein the disaccharide comprises pairs of the same or different monosaccharides selected from D-xylose, D-galactose, -D-glucuronic acid, L-fucose, L-rhamnose, D-apiosium.

[0014] Typically, the reaction between compound D and compound A is carried out using an equivalent ratio of the two compounds comprised between 2: 1 and 1:2, preferably, between 1.5: 1 and 1: 1.5; more preferably, between 1.2: 1 and 1: 1.2.

[0015] Typically, the reaction of compound D and compound A is carried out in the presence of a dehydrating agent selected from molecular sieves, acid-washed molecular sieves, sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride, preferably, the dehydrating agent is represented by acid-washed molecular sieves. For the sake of clarity, "acid-washed molecular sieves" are a commercially available product, such as, for example, "Fluka Molecular sieve UOP Type AW 300" marketed by Honeywell.

[0016] Typically, the reaction between compound D and compound A is carried out in the presence of a catalyst selected from BFs EtiO, TI'iO, In(OTf)3, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably, TMSOTf; typically, the equivalent ratio of the compound of formula D to the catalyst is comprised between 1:0.005 and 1:0.25. Before adding the catalyst, the reaction mixture is cooled to a temperature comprised between -78 °C and 0 °C.

[0017] In a preferred aspect thereof, the invention relates to a process for preparing the compound of formula 1

[0018] 1 by reaction of a compound of formula 2 and a compound of formula 3

[0019] 2 3 in the presence of a dehydrating agent and a catalyst wherein:

[0020] R is selected from hydrogen, benzoyl, acetyl, levulin, fluorenyhnethyloxycarbonyl and pivaloyl; preferably, pivaloyl;

[0021] Ri, R2, R3, and R4 are hydrogen or Ri and R2 and / or R3 and R4 form together with the OHs that they protect a 5-membered ring by reaction with a ketone selected from acetone, cyclopentanone and cyclohexanone; preferably acetone;

[0022] R5 is hydrogen or allyl, with the provision that, in formula 3, R5 is allyl;

[0023] X is selected from -C(NH)CC13and - C(NPh)CF3

[0024] Typically, the reaction between compound 2 and compound 3 is carried out using an equivalent ratio of the two compounds comprised between 1: 1 and 1:2, preferably comprised between 1: 1 and 1: 1.5.

[0025] Typically, compounds 2 and 3, before being used in the process of the invention, are anhydrified by dissolution in a first solvent capable of forming an azeotropic mixture with water; preferably the solvent is selected from toluene, benzene, chloroform, ethyl acetate, pentane, cyclohexane, cyclohexene, 1,2-dimethoxy ethane and hexane. Upon completion of azeotropic distillation, the solvent is removed and compounds 2 and 3 are maintained under vacuum until use.

[0026] Typically, the reaction between compound 2 and compound 3 is carried out using a solvent selected from anhydrous tetrahydrofuran, 2-methyl-tetrahydrofuran, dichloromethane and chloroform; preferably anhydrous dichloromethane.

[0027] Typically, the reaction of compound 2 and compound 3 is carried out in the presence of a dehydrating agent selected from molecular sieves, acid washed molecular sieves, sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride; preferably acid washed molecular sieves.

[0028] Typically, the reaction between compound 2 and compound 3 is carried out in the presence of a catalyst selected from BFs-EtiO, TI'iO, In(OTf)3, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably, TMSOTf; typically, the equivalent ratio of the compound of formula 2 to the catalyst is comprised between 1:0.005 and 1:0.25.

[0029] Preferably, the process of the invention is applied to the preparation of the compound of formula la

[0030] 1a by reaction of a compound 2a with a compound 3 a or by reaction of a compound 2b with a compound 3a in the presence of a dehydrating agent and a catalyst

[0031]

[0032] 3a under the conditions described above for the preparation of compound 1.

[0033] According to a preferred embodiment, the reaction is carried out using anhydrous dichloromethane as solvent, molecular sieves as dehydrating agent and TMSOTs as catalyst. Preferably, the reaction is carried out at a temperature of -20 °C, with an equivalent ratio of compound 3a to compound 2a comprised between 1.5: 1 and 1: 1.

[0034] The compound 2 is prepared by reaction of a compound 4 and a compound 5

[0035] 2c: R6= R5; 2d: R6=H in the presence of a dehydrating agent and a catalyst wherein:

[0036] Re is selected from H, Rs and X;

[0037] R, Rs and X are defined as above, wherein Rs is allyl.

[0038] Typically, the reaction between compound 4 and compound 5 is carried out using an equivalent ratio of the two compounds comprised between 2: 1 and 1: 1, preferably 1.2: 1.

[0039] Typically, the reaction between compound 4 and compound 5 is carried out in the presence of a dehydrating agent selected from molecular sieves, acid-washed molecular sieves (4 A AW 300 MS), sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride; preferably, acid-washed molecular sieves.

[0040] Typically, the reaction between compound 4 and compound 5 is carried out in the presence of a catalyst selected from BFs-EtiO, TI'iO, In(OTf)3, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably, TMSOTf; typically, the equivalent ratio of compound of formula 4 to catalyst is comprised between 1:0.005 and 1:0.5.

[0041] Typically, the reaction between compound 4 and compound 5 is carried out in several steps, each at a temperature lower than that of the next step; the reaction is carried out in at least two steps at different temperatures, preferably at least three, more preferably at least four. The first step is carried out at a temperature comprised between -40 °C and -15°C, the second at a temperature comprised between -30 °C and 0 °C, the third at a temperature comprised between -10 °C and 4°C and the fourth at room temperature.

[0042] Typically, the protecting group Rs is removed using a catalyst containing a Group 9 or 10 transition metal; preferably, a catalyst containing iridium (Ir) or palladium (Pd).

[0043] Preferably, compound 2a is prepared by reaction of a compound 4a and a compound 5a in the presence of a dehydrating agent and a catalyst to give a compound 2c’ under the conditions described above for the preparation of compound 2; followed by conversion of compound 2c 'first to compound 2d’, then to compound 2a

[0044] The compound 3 is prepared by reaction of a compound of formula 6 with a compound of formula 7 in the presence of a dehydrating agent and a catalyst, followed by removal of the R? group wherein R7 is selected from benzoyl, acetyl, levulin, fluorenylmethyloxycarbonyl and pivaloyl; preferably, acetyl;

[0045] X, Ri, R2, R3, R4, and R5 are defined as above.

[0046] Typically, the reaction between compound 6 and compound 7 is carried out using an equivalent ratio of the two compounds comprised between 2: 1 and 1: 1, preferably 1.2: 1.

[0047] Typically, the reaction between compound 6 and compound 7 is carried out in the presence of a dehydrating agent selected from molecular sieves, acid washed molecular sieves, sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride.

[0048] Typically, the reaction between compound 6 and compound 7 is carried out in the presence of a catalyst selected from BFs-EtiO, TI2O, In(OTf)3, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably TMSOTf; typically, the equivalent ratio of the compound of formula 6 to the catalyst is comprised between 1:0.005 and 1:0.5.

[0049] Preferably, compound 3 a is obtained by reaction of compound 6a and compound 7a to give compound 3a' in the presence of a dehydrating agent and a catalyst, under the conditions described above for the preparation of compound 3, followed by removal of the acyl group.

[0050] 6a 7a 3a' 3a

[0051] As can be seen from preparation examples 2a and 2b, by applying the conditions of the process of the invention, in particular using molecular sieves as dehydrating agent, TMSOTf as catalyst, ethyl ether as solvent and operating at temperatures between 0 °C and -15°C, it is possible to obtain, with respect to the conditions described by Wang, an increase of at leastlO- 25 percentage yield points for the preparation of compound 3 a’.

[0052] In a second preferred aspect thereof, the invention relates to a process for preparing the compound of formula 8 by reaction of a compound of formula 9 and a compound of formula 3 in the presence of a dehydrating agent and a catalyst wherein Rs is selected from hydrogen, benzoyl, acetyl, levulin, fluorenylmethyloxycarbonyl and pivaloyl; preferably, acetyl;

[0053] X, R, Ri, R2, R3, R4, and R5 are defined as above.

[0054] Typically, the reaction between compound 9 and compound 3 is carried out using an equivalent ratio between the two compounds comprised between 1: 1 and 1:2, preferably, 1: 1.5.

[0055] Typically, compounds 9 and 3, before being used in the process of the invention, are anhydrified by dissolution in a first solvent capable of forming an azeotropic mixture with water; preferably the solvent is selected from toluene, benzene, chloroform, ethyl acetate, pentane, cyclohexane, cyclohexene, 1,2-dimethoxyethane and hexane. Upon completion of the azeotropic distillation the solvent is removed and compounds 9 and 3 are kept under vacuum until use.

[0056] Typically, the reaction between compound 9 and compound 3 is carried out using an anhydrous solvent selected from tetrahydrofuran, 2-methyl-tetrahydrofuran, dichloromethane and chloroform; preferably anhydrous dichloromethane.

[0057] Typically, the reaction of compound 9 and compound 3 is carried out in the presence of a dehydrating agent selected from acid washed molecular sieves (4 A AW 300 MS), molecular sieves, sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride; preferably acid washed molecular sieves.

[0058] Typically, the reaction of compound 9 and compound 3 is carried out in the presence of a catalyst selected from BFs EtiO, TI2O, In(OTf)3, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably TMSOTf; typically, the equivalent ratio of the compound of formula 9 to the catalyst is comprised between 1:0.005 and 1:0.25.

[0059] Preferably, the invention relates to a process for preparing the compound of formula 8a

[0060] 8a by reaction of a compound of formula 9a and a compound of formula 3a in the presence of a dehydrating agent and a catalyst, under the conditions described for the preparation of compound 8,

[0061] 9a 3a

[0062] Compound 9 is prepared by reaction of a compound 10 and a compound 5 in the presence of a dehydrating agent and a catalyst wherein X, R, Rs and Rs are defined as above, wherein Rs is allyl. Typically, the reaction between compound 10 and compound 5 is carried out using an equivalent ratio of the two compounds comprised between 2: 1 and 1: 1, preferably 1.2: 1.

[0063] Typically, the reaction between compound 10 and compound 5 is carried out in the presence of a dehydrating agent selected from acid- washed molecular sieves, molecular sieves, sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride. Typically, the reaction between compound 10 and compound 5 is carried out in the presence of a catalyst selected from BFs-EtiO, TI'iO, In(OTf)s, Yb(OTf)3, Bi(OTf)3 and TMSOTf; preferably TMSOTf, typically, the equivalent ratio of the compound of formula 10 to the catalyst is comprised between 1:0.005 and 1:0.5.

[0064] Typically, the reaction between compound 10 and compound 9 is carried out in several steps, each at a temperature lower than that of the next step; the reaction is carried out in at least two steps at different temperatures, preferably at least three, more preferably at least 4. The first step is carried out at a temperature comprised between -40 °C and -15°C, the second at a temperature comprised between -30 °C and 0 °C, the third at a temperature comprised between -10 °C and 4°C and the fourth at room temperature.

[0065] Preferably, compound 9a is prepared by reaction of a compound 10a and a compound 5a in the presence of a dehydrating agent and a catalyst under the conditions used for the preparation of compound 9.

[0066] Compounds 3 and 3 a are prepared according to the methods described above.

[0067] EXPERIMENTAL SECTION

[0068] 1H-NMR analyses were carried out using a frequency of 400 MHz and dissolving the samples in a deuterated solvent, such as CDCh.

[0069] Example 1 - Preparation of compound 2c’

[0070] Compounds 4a (120 mg, 1.2 eq.) and 5a (65 mg, 1 eq.) were dissolved in anhydrous dichloromethane (1.2 mL) in the presence of 4 A AW 300 MS washed molecular sieves; to the obtained solution, cooled to -20 °C, TMSOTf was added (4.7 pL;

[0071] 0.036 mmol; 0.2 eq.), then this solution was kept under stirring at -20 °C for 30 minutes, then at -10 °C for 30 minutes, then at 0 °C for 30 minutes, and finally at room temperature for one hour. The reaction was quenched by addition to the reaction mixture of triethylamine (100 pL). The solvent was distilled off under reduced pressure and the residue obtained was purified by chromatography on silica gel, using an eluent mixture of hexane / ethyl acetate 8 / 2. After purification, compound 2c’ status is obtained as a white solid (79 mg, yield: 59%).

[0072] ' H-NMR 2c’ (400 MHz, CDCl3) 5 (ppm) 5.85 (m, 1H), 5.28 (d, 1H), 5.20 (d, 1H), 5.15 (t, 1H), 5.08 (d, 1H), 4.94-4.73 (m, 4H), 4.58 (dd, 1H), 4.33 (t, 1H), 4.14 (dd, 1H), 4.08 (dd, 1H), 3.89 (dd, 1H), 3.76 (dd, 1H), 3.48 (t, 1H), 3.24 (t, 1H), 1.28 (s, 9H), 1.20 (s, 9H), 1.15 (s, 9H), 1.11 (s, 9H), 1.10 (s, 9H).

[0073] Example 2a - Preparation of compound 3 a'

[0074] Compounds 6a (140 mg, 1 eq) and 7a (132 mg, 1.5 eq) were dissolved in anhydrous ethyl ether (1.2 mL) in the presence of 4 A AW 300 MS molecular sieves; to the obtained solution, kept under stirring and cooled to 0 °C for 90 minutes, TMSOTf was added (10 pL; 0.054 mmol; 0.15eq). The obtained solution was maintained at 0 °C for further 30 min, then allowed to rise to room temperature and finally quenched by addition of triethylamine (0.3 mL). The solvent was distilled off under reduced pressure and the residue obtained was purified by silica gel chromatography, using a 6 / 4 hexane / ethyl acetate eluent mixture. After purification, compound 3a' was obtained as a white solid (54 mg, yield: 32%).

[0075] JH NMR 3a’ (400 MHz, CDCl3) 5 (ppm) 5.92 (m, 1H), 5.34 (d, 2H), 5.22 (d, 1H), 4.86 (dt, 1H), 4.83 (d, 1H), 4.29 (q, 2H), 4.21-4.15 (m, 2H), 4.12 (m, 1H), 4.06 (dd, 1H), 4.00 (dd, 1H), 3.81 (dd, 1H), 3.75 (dd, 1H), 2.09 (s, 1H), 1.56 (s, 3H), 1.54 (s, 3H), 1.37-1.32 (m, 9H), 1.12 (d, 3H).

[0076] The table below shows the result of two other experiments carried out according to example 2a with some modifications (concentration of compound 6a, temperature and equivalents of compound 7a)

[0077] These further experiments demonstrate that as the temperature decreases and the concentration of compound 6a increases, the yield increases significantly.

[0078] Example 2b - Preparation of compound 3a' (comparison example - conditions according to Wang et al)

[0079] 6b7a 3a'

[0080] A solution of Ir-(COD)(PMePh 2)2]PFe -base (11 mg, 0.013 mmol) in THF was degassed and added to compound 6b (150 mg, 0.52 mmol) dissolved in THF. After Ih at room temperature, the solvent was distilled off under reduced pressure and the residue was combined with compound 7a (220 mg, 0.34 mmol) and dried by azeotropic distillation with toluene. The residue was then dissolved in 20 mF of acetonitrile. N-iodosuccinimide (NIS) (118 mg, 0.52 mmol) and TfOH (0.4 pF, 0.020 mmol) were added at room temperature. After 5 minutes 0.1 mF of triethylamine was added and the reaction mixture was concentrated under reduced pressure. The residue obtained was purified by silica gel chromatography (eluent mixture of hexane / ethyl acetate 6 / 4). After purification, compound 3 a' was obtained as a white solid (11- 34 mg, yield: 7-22%).

[0081] Example 3 - Preparation of compound 3 a

[0082] A solution of compound 3a' (85 mg, leq.) in 0.1M sodium methoxide (1.4 mF) was stirred under nitrogen at room temperature for 2.5 hours; to the reaction mixture was added 120 H+amberlite resin until pH 7 was reached. The solvent was then evaporated and compound 3 a was obtained as a white solid (75 mg, yield; 97%).

[0083] 1H NMR 3a (400 MHz, CDCl3) 5 (ppm) 5.91 (m, 1H), 5.32 (d, 1H), 5.27 (s, 1H), 5.22 (d, 1H), 4.85 (d, 1H), 4.32-4.25 (m, 2H), 4.18 (dd, 1H), 4.14-4.08 (m, 2H), 4.05 (dd, 1H), 4.00 (dd, 1H), 3.82 (dd, 1H), 3.71 (dd, 1H), 3.41 (t, 1H), 1.54 (s, 3H), 1.52 (s, 3H), 1.38-1.32 (m,

[0084] 9H), 1.26 (d, 3H).

[0085] Example 4 - Preparation of compound 2d’

[0086] With Ir catalyst

[0087] A first solution of [Ir-(COD)(PMePh2)2]PF6 (1.1 mg; 0.025 eq.) in THF (0.5 mL) was degassed and kept under stirring under hydrogen atmosphere for 15 minutes at room temperature. This first solution was injected into a second solution of compound 2c’ (40 mg; leq.) in THF (0.6 mL), then the reaction mixture was stirred for 60 minutes, and finally the solvent was distilled off in vacuo. The residue was dissolved in a mixture of acetonitrile (5.3 mL) and water (5.8 pL; 6 eq) and to the obtained solution, kept under stirring at room temperature, N-iodo- succinimide (12.2 mg; 1 eq) and triflic acid (0.1 pL; 0.02 eq) were added sequentially. At 5 minutes after the addition, the reaction was quenched by the addition of triethylamine (0.20 pL). After removal of the solvent and chromatographic purification on silica gel (eluent mixture hexane / ethyl acetate 7:3), compound 2d was obtained as a white solid (30 mg, yield: 80%).

[0088] ' H NMR 2d’ (400 MHz, CDCl3) 6 (ppm) 5.38 (t, 1H), 5.17 (t, 1H), 4.96-4.84 (m, 2H), 4.82-4.74 (m, 2H), 4.61 (dd, 1H), 4.35 (t, 1H), 4.09 (dd, 1H), 3.81 (dd, 1H), 3.74 (t, 1H), 3.26 (t, 1H), 1.30 (s, 9H), 1.21 (s, 9H), 1.16 (s, 9H), 1.13-1.11 (m, 18H).

[0089] With Pd catalyst

[0090] A solution of compound 2c’ (1 eq.) in anhydrous dichloromethane (1 mL) was added to a second solution of Pd(OAc)2 (22 mg; 0.3 eq.), PPI13 (110 mg; 1.2 eq.) and DEA (360 pL, 10 eq.) dissolved in anhydrous dichloromethane (3 mL) and methanol (0.4 ml). The reaction mixture was heated to 40 degrees and stirred for 6 hours, at the end of which the solvent was distilled off in vacuo and the residue obtained purified on silica gel (hexane / ethyl acetate 1: 1); after purification, compound 2d’ (85 mg; yield: 98%).

[0091] Example 5 - Preparation of compound 2a

[0092] Compound 2d’ (40 mg, 1 eq.) was dissolved in anhydrous dichloromethane (0.6 mL), and to the obtained solution, maintained under stirring and at room temperature were added CChCN (57 pL; lOeq.) and DBU (4.2 pL, 0.5 eq.). The reaction mixture was stirred at ambient temperature for 2.5 hours, then brought to dryness by vacuum distillation, taken up with ethyl acetate (35 mL) and filtered over silica gel. After removal of the solvent from the filtered solution, compound 2a was obtained as white-yellow solid (45 mg, yield: 93%).

[0093] JH NMR 2a (400 MHz, CDCl3) 6 (ppm) 6.49 (d, 1H), 5.16 (t, 1H), 4.96-4.83 (m, 4H), 4.81 (d, 1H), 4.41 (t, 1H), 4.17-4.08 (m, 2H), 3.96 (dd, 1H), 3.67 (t, 1H), 3.27 (t, 1H), 1.25 (s, 9H), 1.22 (s, 9H), 1.15 (s, 9H), 1.14-1.11 (m, 18H).

[0094] Example 6 - Preparation of compound la

[0095] A solution of compounds 2a (45 mg, 1 eq.) and 3a (34 mg, 1.5 eq.) in toluene was subjected to azeotropic distillation, at the end of which anhydrous dichloromethane (0.9 mL) and molecular sieves were added. After keeping the solution under stirring for 1.5 hours, this solution is cooled to -20 °C and added with TMSOTs (1.5 pL; 0.008 mmol; 0.15 eq.). After 20 min, the reaction is warmed to room temperature and quenched by the addition of triethylamine (100 pL). After removal of the solvent by vacuum distillation and purification on silica gel (eluent mixture hexane / ethyl acetate 8:2), compound la was obtained as a white solid (27 mg, yield: 46%).

[0096] JH NMR la (400 MHz, CDCl3) 5 (ppm) 5.88 (m, 1H), 5.31 (d, 1H), 5.27 (s, 1H), 5.22 (d, 1H), 5.16 (t, 1H), 4.92 (d, 1H), 4.90-4.77 (m, 5H), 4.74 (d, 1H), 4.27 (dd, 1H), 4.21 (d, 1H), 4.18-4.02 (m, 7H), 3.99 (dd, 1H), 3.79 (dd, 1H), 3.60 (m, 1H), 3.52 (dd, 1H), 3.24 (m, 2H), 1.52 (s, 3H), 1.48 (s, 3H), 1.34 (m, 6H), 1.30 (s, 3H), 1.26 (s, 9H), 1.15 (s, 9H), 1.14 (s, 9H), 1.11 (s, 9H).

[0097] The table below shows the result of two further experiments carried out according to example 6 with some modifications (concentration of compound 2a and equivalents of compound 3 a).

[0098] These results demonstrate that by decreasing the ratio of equivalents of compound 3a to compound 2a, the yield increases markedly.

[0099] Example 7 - Preparation of compound 11

[0100] D-xylose (1 g, 1 eq.) was added to a solution of allyl alcohol (4.1 mL, 9 eq.) and acetyl chloride (0.95 mL, 2 eq.), previously cooled in acetone / ice. After removing the bath, the reaction mixture was stirred for 25 hours, allowing the temperature to rise to room temperature. The reaction was then quenched by addition of solid sodium bicarbonate (1 g). The reaction mixture was diluted with MeOH and filtered over celite. The filtrate was evaporated in vacuo and the obtained residue was purified on automatic chromatography column [gradient eluent mixture ethyl acetate (A) / methanol (B): A / B 1% for one column volume, A / B 1 to 10% for 10 column volumes, A / B 10% for 2 column volumes.] From the combined and dried fractions, compound 11 was obtained as white precipitate (1.05 g, yield: 82%).

[0101] JH NMR 11 (400 MHz, D2O) 5 (ppm) 5.99 (m, 1H), 5.39 (d, 1H), 5.29 (d, 1H), 4.95 (d, 1H), 4.24 (dd, 1H), 4.08 (dd, 1H), 3.70 (m, 1H), 3.63 (m, 2H), 3.56 (m, 2H).

[0102] Example 8 - Preparation of compounds 5a and 12

[0103] - 5a synthesis

[0104] In a solution of compound 11 (300 mg, 1 eq.) and pyridine (1.75 mL, C = 0.9 M), under an inert atmosphere, was dripped pivaloyl chloride (426pL; 3.48 mmol; 2.2eq.); the reaction mixture obtained was kept under stirring for 4.5 hours, at the end of which the reaction was quenched by the addition of water (5 mL) and then the product was extracted four times with ethyl acetate, before being concentrated in vacuo. The residue was purified by silica gel chromatography (hexane / ethyl acetate 8:2) to give compounds 5a (323 mg, yield: 57%) and 12 (140 mg, yield: 20%).

[0105] -synthesis of 12

[0106] Into a solution of compound 11 (200 mg, 1 eq.) and pyridine (0.95 mL, C= 1.1 M), under an inert atmosphere, was dripped pivaloyl chloride (540 pL; 4.41 mmol; 4.2 eq.); the reaction mixture obtained was kept under stirring for 16 hours at 80 °C. At the end, the reaction was quenched by addition of MeOH (0.12 mL), the crude diluted with water (10 mL) and then the product was extracted four times with ethyl acetate. The combined organic phases were washed with water, then brine and evaporated. The residue was purified by silica gel chromatography (hexane / ethyl acetate 8:2) to give compounds 12 (220 mg, yield: 40%) and 5a (14 mg, yield: 5%).

[0107] JH NMR 5a (400 MHz, CDCl3) 5 (ppm) 5.85 (m, 1H), 5.60 (t, 1H), 5.31 (d, 1H), 5.20 (d, 1H) 5.05 (d, 1H), 4.98 (m, 1H), 4.79 (dd, 1H), 4.19 (dd, 1H), 3.94 (dd, 1H), 3.76 (dd, 1H), 3.60 (t, 1H), 1.17 (s, 9H), 1.15 (s, 9H), 1.13 (s, 9H).

[0108] JH NMR 12 (400 MHz, CDCh)'. 6 (ppm) 5.85 (m, 1H), 5.31 (d, 1H), 5.20 (d, 1H) 5.02 (d, 1H), 4.85 (m, 1H), 4.68 (dd, 1H), 4.15 (m, 2H), 3.95 (dd, 1H), 3.73 (dd, 1H), 3.57 (t, 1H), 1.23 (s, 9H), 1.21 (s, 9H).

[0109] Example 9 - Preparation of compound 13

[0110] Ir catalyst

[0111] A solution of [Ir(COD)(PPh2Me)2]PF6 in 0.6 mL of THF was degassed and kept under stirring under hydrogen atmosphere for 15 minutes. The pale yellow solution of the catalyst was injected into a solution of compound 12 in 0.6 mL of THF at room temperature. The reaction mixture is allowed to stir for 1.5 h and then concentrated under vacuum. The obtained residue is dissolved in a mixture of acetonitrile (1 mL) and water (15 pL; 3 eq.), and to the obtained solution are sequentially added N-iodo- succinimide (55 mg, 1 eq.) and triflic acid (11 pL, in 7 mL of acetonitrile) at room temperature. After 10 min, the reaction was quenched by addition of triethylamine (0.2 mL) and concentrated in vacuo. The residue was taken up in ethyl acetate, filtered over silica, and dried by vacuum distillation to afford compound 13 as a yellowwhite solid (104 mg, >95% yield).

[0112] Pd catalyst

[0113] A solution of Pd(OAc)2 (500 mg, 0.3 eq), PhsP (2.41 g, 1.2 eq) and Et2NH 7.2 mL, 10 eq) in anhydrous dichloromethane (32 mL) was added to a solution of 12 (3.29 g, 1 eq) in anhydrous dichloromethane (32 mL). After 5 min, add 8.6 mL of MeOH and stir for 6 h at 40 °C. At the end of the reaction, the solvent is evaporated, the residue is taken up in dichloromethane and filtered over celite. The crude is then purified by silica chromatography (8:2 hexane: acetate) to afford compound 13 in 98% yield.

[0114] JH NMR 13 (400 MHz, CDCl3) 6 (ppm) 5.63 (t, 1H), 5.41 (d, 1H), 4.98 (m, 1H), 4.81 (dd, 1H), 3.81 (m, 2H), 1.18 (s, 9H), 1.15 (s, 9H), 1.14 (s, 9H).

[0115] Example 10 - Preparation of compound 4a

[0116] Compound 13 (29mg; leq.) was dissolved in anhydrous dichloromethane (1 mL) and to the solution obtained, maintained under stirring and at room temperature, CCI3CN (43 pL; 6eq.) and DBU (4.2 pL, 0.5 eq.) were added. The reaction mixture is kept under stirring at room temperature for 2.5 hours, then brought to dryness by vacuum distillation, taken up with ethyl acetate (10 mL) and filtered over silica gel. After removal of the solvent from the filtered solution, compound 4a is obtained as a yellow syrup (46 mg, yield> 95%).

[0117] JH NMR 4a (400 MHz, CDCI3) 6 (ppm) 6.50 (d, 1H), 5.67 (t, 1H), 5.11 (m, 2H), 3.96 (dd, 1H), 3.77 (t, 1H), 1.16 (s, 9H), 1.14 (s, 9H), 1.13 (s, 9H).

[0118] Example 11 - Preparation of compound 14

[0119] D-fucose (7 g, leq.) was added to a solution of allyl alcohol (26.2 mL, 9 eq.) and acetyl chloride (6.1 mL; 2eq.), previously cooled in acetone / ice. After removing the bath, the reaction mixture is kept under stirring, allowing the temperature to rise to room temperature, for 3 hours. The reaction mixture was then concentrated in vacuo, washed with 2-Me-THF (70 mL), cooled to 0 °C, and allowed to stir for 1 h until crystallized, then isolated by vacuum filtration. The obtained crystallized product was washed with 2-Me-THF (14 mL), then dried at 30 °C under vacuum; compound 14 is obtained as white powder (6.5 g, yield: 74%).

[0120] ' H NMR 14 (400 MHz, D2O) 6 (ppm) 6.00 (m, 1H), 5.38 (d, 1H), 5.28 (d, 1H) 4.95 (d, 1H), 4.22 (dd, 1H), 4.10 (m, 2H), 3.88 (dd, 1H), 3.80 (m, 2H), 1.23 (d, 3H). Example 12 - Preparation of compound 7a

[0121] Compound 14 (6.3 g; 30.8 mmol; 1 eq.) was added at room temperature to a solution of 2,2-dimethoxypropane (37 mL, 10 eq.); TsOH (530 mg, 0.09 eq.) was then added to the reaction mixture, then stirred for 1.5 h, at which point the reaction was quenched by addition of dichloromethane (37.7 mL) and saturated aqueous bicarbonate solution (38 mL). After separating the phai L-Rham noseISP^ase waswashed with dichloromethane (37.7 mL) and the combined organic pnuscs wcic concentrated in vacuo to give compound 7a as a colourless oil (7.4 g. yield> 95%).

[0122] JH NMR 7a (400 MHz, CDCl3) 5 (ppm) 5.91 (m, 1H), 5.29 (d, 1H), 5.21 (d, 1H) 4.87 (d, 1H), 4.24 (m, 2H), 4.13 (m, 1H), 4.06 (m, 2H), 3.78 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H). 1.31 (d, 3H).

[0123] Example 13 - Preparation of compound 15

[0124] L-rhamnose monohydrate (10 g, 1 eq.) was dissolved in 1,2-dimethoxy ethane (200 mL) and dehydrated by azeotropic distillation (atmospheric pressure, T: 83-85 °C). The solvent was distilled to half the initial volume, then returned to the initial volume by addition of additional 1,2-dimethoxyethane; the distillation / solvent addition cycle was repeated three times. Separately, acetyl chloride (3.9 mL; 1 eq.) was added dropwise, at 0 °C, to allyl alcohol; after 30 minutes, the allyl alcohol-containing solution was added dropwise to the rhamnose- containing solution, and the obtained mixture was first kept under stirring for 3.5 hours, then concentrated in vacuo. The reaction was quenched by addition of sodium bicarbonate (27 mL) and extracted 10 times with 2-Me-THF (45 mL per single extraction). The combined organic phases were concentrated in vacuo to give compound 15 (8.6 g; yield: 77%), in oil form.

[0125] JH NMR 15 (400 MHz, D2O) 5 (ppm) 5.99 (m, 1H), 5.38 (d, 1H), 5.30 (d, 1H) 4.86 (d, 1H), 4.23 (dd, 1H), 4.09 (dd, 1H), 3.96 (m, 1H), 3.75 (m, 2H), 3.46 (t, 1H), 1.31 (d, 3H). Example 14 - Preparation of compound 16

[0126] P-Toluenesulfonic acid monohydrate (0.7 g; 0.09 eq.) was added to a solution of compound 15 (8.2 g, 1 eq.) in 2,2-dimethoxyethane (49 mL; 10 eq.); the reaction mixture was kept under stirring for 2 h, at the end of which this mixture was first diluted with dichloromethane, then slowly added to a saturated aqueous solution of sodium bicarbonate. After separating the two phases formed, the aqueous one was washed with dichloromethane (49 mL x 3 times), then the combined organic phases were dried by vacuum distillation, to give compound 16 (8.5 g, yield:87%) in the form of yellow syrup.

[0127] JH NMR 16 (400 MHz, CDCh)'. 6 (ppm) 5.91 (m, 1H), 5.31 (d, 1H), 5.22 (d, 1H) 5.00 (s, 1H), 4.19 (m, 2H), 4.10 (t, 1H), 4.00 (dd, 1H), 3.69 (m, 1H), 3.40 (t, 1H), 1.53 (s, 3H), 1.36 (s, 3H), 1.30 (d, 3H).

[0128] Example 15 - Preparation of compound 17

[0129] To a solution of compound 16 (8.5g, leq.) in dichloromethane (42.5 mL) it was added at room temperature, in sequence, triethylamine (5.3 mL; 1.1 eq.), DMAP (130 mg; 0.03 eq.) and, finally, acetic anhydride (8.2 mL; 2.5 eq.). The reaction mixture was stirred for 4 hours, then concentrated in vacuo. The residue obtained was taken up with ethyl acetate (42 mL) and water (21 mL) and the two phases were separated. The aqueous phase was washed with ethyl acetate (42 mL x 3 times). From the combined and vacuum-dried organic phases a crude (9.6 g) was obtained, then purified on silica gel (20 parts of silica 20 micron, fractional volume; 170 mL, eluent mixture: hexane / ethyl acetate 95:5, flow 1 cm / min). After purification, compound 17 was obtained (7.3 g, yield: 73%).

[0130] JH NMR 17 (400 MHz, CDCl3) 5 (ppm) 5.91 (m, 1H), 5.30 (d, 1H), 5.22 (d, 1H) 5.05 (s, 1H), 4.86 (dd, 1H), 4.18 (m, 3H), 4.01 (dd, 1H), 3.75 (m, 1H), 2.10 (s, 3H), 1.57 (s, 3H), 1.34 (s, 3H), 1.16 (d, 3H). Example 16 - Preparation of compound 18

[0131] A solution of compound 17 (100 mg, 1 eq.) in anhydrous dichloromethane (1 mL) was added to a second solution of Pd(OAc)2 (22 mg; 0.3 eq.), PPI13 (110 mg; 1.2 eq.) and DEA (360 pL, 10 eq.) dissolved in anhydrous dichloromethane (3 mL) and methanol (0.4 ml). The reaction mixture was heated to 30 degrees and stirred for 4.5 hours, at the end of which the solvent was distilled off in vacuo and the residue obtained purified on silica gel (hexane / ethyl acetate 1: 1); after purification, compound 18 (85 mg; yield: 98%).

[0132] JH NMR 18 (400 MHz, CDCI3) 6 (ppm) 5.43 (d, 1H), 4.87 (dd, 1H), 4.21 (m, 2H), 3.96 (m, 1H), 2.10 (s, 3H), 1.57 (s, 3H), 1.36 (s, 3H), 1.16 (d, 3H).

[0133] Example 17 - Preparation of compound 6a

[0134] CCECN (210 pL, 6 eq.) and DBU (25 pL, 0.5 eq.) are added sequentially to a solution of compound 19 (85 mg; 1 eq.) in anhydrous dichloromethane cooled to 0 °C. The reaction mixture is then kept under stirring at 0 °C for 30 minutes, then brought to room temperature and stirred for a further 2 hours. Then, the solvent is distilled off under vacuum, and the obtained residue is diluted with ethyl acetate (15 mL), filtered on silica, washed with ethyl acetate (50 mL). From solvent removal, compound 6a (140 mg, >95% yield) was obtained.

[0135] JH NMR 6a (400 MHz, CDCl3) 6 (ppm) 6.45 (s, 1H), 4.95 (m, 1H), 4.29 (m, 2H), 3.94 (m, 1H), 2.12 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H), 1.20 (d, 3H).

Claims

CLAIMS1. A process for preparing a compound of formula 1 :wherein:R is selected from hydrogen, benzoyl, acetyl, levulin, fluorenylmethyloxycarbonyl and pivaloyl;Ri, R2, R3, and R4 are hydrogen or Ri and R2 and / or R3 and R4 form together with the OHs that they protect a 5-membered ring by reaction with a ketone selected from acetone, cyclopentanone and cyclohexanone;R5 is selected from: methyl or ethyl; by reaction of a compound of formula 2:wherein:R is as defined above andX = -C(NH)CC13or -C(NPh)CF3with a compound of formula 3:wherein Ri, R2, R3, R4, and R5 are as previously defined, with the provision that, in the compound of formula 3, R5 is allyl; or for preparing a compound of formula 8:by reaction of a compound of formula 9:wherein the compound of formula 3 and R, Ri, R2, R3 and R5 and X are as previously defined and Rs is selected from benzoyl, acetyl, levunyl, fluoreny Ime thy loxy carbonyl and pivaloyl.

2. The process according to claim 1 wherein R is pivaloyl and Ri and R2 and / or R3 and R4form together with the OHs that they protect a 5-membered ring by reaction with acetone.

3. The process according to claim 1 or 2, where the equivalent ratio between compound 2 and compound 3 and between compound 9 and compound 3 is comprised between 1: 1 and 1:2.

4. The process according to any one of claims 1 to 3, wherein the dehydrating agent is selected from molecular sieves, acid-washed molecular sieves sodium sulfate, calcium sulfate, magnesium sulfate and calcium chloride.

5. The process according to any one of Claims 1 to 4, wherein the catalyst is selected from BF3Et2O, Tf2O, In(OTf)3, Yb(OTf)3, Bi(OTf)3, and TMSOTf.

6. The process according to any of claims 1 to 5, wherein the equivalent ratio between the compound of formula 2 or the compound of formula 9 and the catalyst is comprised between 1:0.005 and 1:0.25.

7. The process according to any one of claims 1 to 6, wherein the addition of the catalyst is preceded by cooling the reaction mixture to a temperature comprised between -78°C and 0 °C.

8. The process according to any one of claims 1 to 7, wherein compound 1 is a compound of formula la, compound 2 is a compound of formula 2a, compound 3 is a compound of formula 3’, compound 8 is a compound of formula 8a, and compound 9 is a compound of formula 9a.

9. The process according to claim 8 for the preparation of compound la, wherein the reaction between compound 2a and compound 3a is carried out using dichloromethane asa solvent, molecular sieves as a dehydrating agent, and TMSOTf as a catalyst.

10. The process according to claim 9, wherein the reaction is carried out at a temperature of -20 °C with an equivalent ratio between 3a and 2a comprised between 1.5 and 1:1.

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