CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority from Italian patent application no. 102020000008515 filed on 21/04/2020, the entire disclosure of which is incorporated herein by reference. TECHNICAL FIELD

The present invention concerns a method for preparing a cholane derivative, in particular for preparing 6a-q11ir1-24-hoG-5b- cholan-3 ,7 -23-triol (BAR502), having formula (I).

BACKGROUND ART

BAR502 was described for the first time as a reaction intermediate in the synthesis of derivatives of 6- ethylchenodeoxycholic acid (D'Amore et al. J. Med. Chem. 2014, 57, 937-954).

WO2015181275 describes the use of BAR502 as an FXR/TGR5 modulator for use in the treatment of gastrointestinal disorders, hepatic diseases, cardiovascular diseases, atherosclerosis, metabolic diseases, infectious diseases, cancer, renal disorders, inflammatory and neurological disorders.

WO2015181275 describes the synthesis of BAR502. However, the methodology illustrated is not suited to the production of BAR502 in industrial quantities.

The search for a new method for the preparation of BAR502 which can be scaled-up to industrial level is therefore ongoing.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a new method for the preparation of BAR502. Said object is achieved by means of a method according to claim 1, a method according to claim 2 and reaction intermediates according to claim 14.

BEST MODE FOR CARRYING OUT THE INVENTION

In particular, according to a first aspect of the invention, a method is provided for the preparation of a compound of formula BAR-7: comprising the step of protecting the position 7 of the steroid ring of a compound of formula BAR-6 by means of the formation of an acetal.

According to a further aspect of the invention, a method is provided for the preparation of a compound of formula (I): comprising the step of f) protecting the position 7 of the steroid ring of a compound of formula BAR-6 by means of the formation of an acetal of formula BAR-7.

Preferably, the protection step is carried out in the presence of 1,2-ethanediol in an acidic environment. Advantageously the formation of the acetal BAR-7 avoids reduction of the carbonyl group to an alcohol.

Subsequently the compound of formula BAR-7 is transformed into the compound of formula (I), as illustrated below (step g).

According to an embodiment of the method of the invention, the step g) comprises the step of gl) transforming a compound of formula BAR-7 into a compound of formula BAR-9 by reduction of the ester group on the lateral chain in position 17 of the steroid ring and hydrolysis of the acetal in position 7 of the steroid ring. According to a further embodiment, the step g) further comprises the step of g2) protecting the alcohol groups of the compound of formula BAR-9 to obtain a compound of formula BAR-IO-G: in which P is a protecting group. Said step is preferably carried out by using a reagent included in the following list: chloro- dimethylether (MOMC1), chloromethyl benzylether (BOMC1), chloromethyl p-methoxybenzylether (PMBMC1), chloromethyl 2- methoxyethylether (MEMC1), 3,4-dihydropyran (DHP), a trialkyl silyl derivative, like chlorotriethylsilane (TESC1), chlorotriisopropylsilane (TIPSC1), chlorotert- butyldimethylsilane (TBDMSC1) but preferably chlorotrimethylsilane (TMSC1).

According to a further embodiment step g) further comprises the step g3) of transforming the compound of formula BAR-10-G into a compound of formula BAR-ll-G by the formation of an enolate between positions 6 and 7 of the steroid ring: in which P is a protecting group as described above.

According to a further embodiment, the step g) further comprises the step g4) of transforming the compound of formula BAR-11-G into a compound of formula BAR-12 by means of alkylidenation in position 6 of the steroid ring and deprotection of the alcohol groups in position 3 of the steroid ring and in lateral chain. According to a further embodiment, step g) further comprises the step g5) of transforming the compound of formula BAR-12 into a compound of formula BAR-13 by means of reduction, preferably stereoselective, of the double bond in position 6 of the steroid ring.

According to a further embodiment, step g) further comprises the step g6) of transforming the compound of formula BAR-13 into the compound of formula (I) by reduction, preferably stereoselective, of the carbonyl in position 7 of the steroid ring.

Advantageously, the method of the invention can be carried out starting from known reagents commonly available on the market.

In particular, the method of the invention can comprise the step of a) transforming the 7-ketolithocholic acid (BAR-1) by means of formylation in position 3 of the steroid ring to obtain a compound of formula BAR-2.

Subsequently the compound of formula BAR-2 can be transformed (step b), by shortening of the lateral chain in position 17 of the steroid ring, to obtain a compound of formula BAR-3.

The compound of formula BAR-3 can then be transformed (step c) by deformylation in position 3 of the steroid ring to obtain a compound of formula BAR-4.

The compound of formula BAR-4 can then be transformed (step d) by hydrolysis of the nitrile group on the lateral chain in position 17 of the steroid ring to obtain a compound of formula BAR-5.

BAR-5

The compound of formula BAR-5 can then be transformed (step e) by esterification of the carboxyl on the lateral chain in position 17 of the steroid ring to obtain a compound of formula BAR-6.

According to a further aspect of the invention the following compounds are furthermore provided as reaction intermediates selected from the group consisting of:

in which P is a protecting group; useful as reaction intermediates in the synthesis of BAR502.

Further characteristics of the present invention will become clear from the following description of some purely illustrative and non-limiting examples.

Hereinafter, the following acronyms will be used: para- toluenesulfonic acid monohydrate (p-TSA-H ₂ 0), formic acid (HCOOH), acetic anhydride (AC2O), trifluoroacetic acid (TFA), trifluoroacetic anhydride (TFAA), 7-ketolithocholic acid (7K- LCA), sodium nitrite (NaNCh), potassium hydroxide (KOH), sodium hydroxide (NaOH), hydrochloric acid (HC1), methanol (MeOH), ethanol (EtOH) 1,2-ethanediol (MEG) (EG), sodium methoxide (MeONa), N,N-diisopropylethylamine (DIPEA), chlorotrimethylsilane (TMSC1), diisopropylamine (DIPA), tetrahydrofuran (THF), n-butyl lithium (n-BuLi), dichloromethane (DCM), methylisobutylketone (MIBK), triethylamine (TEA), sodium bicarbonate (NaHCCb), acetaldehyde (CH3CHO), palladium on carbon (Pd/C), sodium borohydride (NaBH ₄ ), boron trifluoride complexed with acetonitrile (BF3-CH3CN).

EXAMPLE 1

Step 1: preparation of BAR-2 - protection of the hydroxyl group of the 7-ketolithocholic acid (7K-LCA, BAR-1) by formylation 7K-LCA (BAR-1) BAR-2

BAR-1 (10 Kg, 0.0256 Kmol) is suspended in HCOOH (20 L). p- TSA-H ₂ 0 (50 g, 0.3 mol) is added to this suspension. The mixture is heated to 50°C and stirred at this temperature for 1 hour 30 minutes. The reaction mixture is then cooled to ambient temperature and AC2O (11 L) is slowly added in approximately 60 minutes maintaining the reaction temperature below 30°C.

The reaction is heated to 40°C and kept at this temperature for 30 minutes. The reaction mixture is cooled to ambient temperature and loaded in water (100 L). The solid is filtered and washed with water up to neutral pH. BAR-2 is obtained (10.5 Kg, 97%).

Step 2: preparation of BAR-3 - Beckmann rearrangement of BAR-2

BAR-2 BAR -3

BAR-2 (10.5 Kg, 0.0251 Kmol) is dissolved in TFA (32 L) and TFAA (11 L) at ambient temperature. NaNCk (2.1 Kg, 0.0301 Kmol) is added in portions. The reaction is heated to 40°C and kept at this temperature for two hours. The reaction mixture is brought to ambient temperature and loaded in water (147 L). The pH of the mixture is neutralized by adding a solution composed of KOH (32 Kg) and water (32 L). The solid is filtered and washed with water up to neutral pH. BAR-3 is obtained (9.5 Kg, 98%). Step 2A: preparation of BAR-4 - deformylation of BAR-3

BAR-3 BAR-4

BAR-3 (9.5 Kg, 0.0246 Kmol) is suspended in water (95 L), then KOH is added (1.4 Kg, 0.0246 Kmol). The suspension is heated to reflux and maintained for 4 hours. The suspension is cooled to ambient temperature. The solid is filtered and washed with water up to neutral pH. BAR-4 is obtained (6.4 Kg, 73%).

Step 3: preparation of BAR-5 - Hydrolysis of the BAR-4

BAR-4 (6.4 Kg, 0.0179 Kmol) is suspended in EtOH (32 L) and water (64 L) and KOH (9.0 Kg) is added to this suspension. The reaction is brought to reflux and left under stirring for 72h. At the end of the conversion, the reaction mixture is cooled to ambient temperature.

The pH of the mixture is corrected to 2.0 by addition of HCI. The solid obtained is filtered and washed with water up to neutral pH. BAR-5 is obtained (5.1 Kg, 75%).

Step 4: preparation of the BAR-6 - Preparation of the methyl ester of the BAR-5

BAR-5 BAR-6

BAR-5 (5.1 Kg; 0.0135 Kmol) is suspended in MeOH (25.5 L). p- TSA-thO (0.28 Kg, 0.0015 Kmol) is added to this suspension. The mixture is brought to reflux and kept at this temperature for 4 5 hours. The reaction mixture is cooled to ambient temperature with precipitation of the product. BAR-6 is obtained (4.9 Kg; 92%).

BAR-6 (4.9 Kg, 0.0125 Kmol) is dissolved in toluene (49 L) at ambient temperature. p-TSAdhO (48 g, 0.2 mol) and MEG (4.9 L) are added to this solution. The two-phase solution is brought to distillation and the MEG and the water are collected in the 15 Dean-Stark trap until a single-phase solution is obtained.

The reaction mixture is cooled to ambient temperature in approximately 1 hour and washed with NaOH 3N (3.2 L).

20 The phases are separated and the organic phase is washed with water (13 L). The organic phase is used in the subsequent step.

For the characterization, BAR-7 is isolated by chromatography (Si0 ₂ /Et ₃ N, DCM (2.5% Et ₃ N)/DCM:MeOH:Et ₃ N (80:20:2.5%)).

¹ H NMR (CDCls) : 3.99 (m, 2H, CH ₂ -CH ₂₎ , 3.89 (m, 2H, CH ₂ -CH ₂₎ , 3.66 (s, 3H, COOCHs), 3.51 (m, 1H, H3), 2.45 (dxd, Ji=12, J ₂ =4 Hz, 1H, CH), 2.20-1.05 (m, 23H, CH/CH ₂₎ , 0.97 (d, J=4 Hz, 3H, H21), 0.94 (s,3H, CH ₃₎ , 0.69 (s, 3H, CH ₃₎ .

¹³ C NMR (CDCls) : 174.2 (Cq, COOCHs), 110.9 (Cq, C7), 72.0 (CH,

C3), 64.1 (CH ₂ -CH ₂ ), 62.3 (CH ₂ -CH ₂ ), 55.2 (CH), 51.5 (CH ₃ ,

COOCHs), 49.7 (CH), 43.6 (Cq), 42.3 (CH), 41.7 (CH ₂ ), 41.7 (CH), 39.7 (CH), 37.8 (CH), 37.2 (CH ₂ ), 35.2 (CH ₂ ), 35.1 (CH ₂ ), 34.7

(Cq), 33.9 (CH ₂ ), 30.8 (CH ₂ ), 28.8 (CH ₂ ), 26.0 (CH ₂ ), 23.1 (CH ₃ ), 21.6 (CH ₂ ), 19.8 (CHs, C21), 12.1 (CH ₃ ). m/z= 435.10 and 891.60 correspond to [M+H] ⁺ and [2M+Na] ⁺ , respectively .

Step 6: preparation of BAR-9 - Reduction of the ester BAR-7 and hydrolysis of the acetal

Sodium dihydro-bis (2-methoxyethoxy) aluminium hydride 70% by weight is loaded in toluene (8.7 L Kg, 0.0312 Kmol) under nitrogen flow. The solution is cooled to 0-10°C and the solution of BAR-7 is added (weight: 5.4 Kg, 0.0125 Kmol), prepared in the previous step, in approximately 2 hours, maintaining the reaction temperature below 10°C.

The reaction is kept at 0-10°C for 1 hour. Subsequently, water (4.9 L) is added to the mixture, maintaining the temperature below 15°C. The reaction mixture is stirred for approximately 60 minutes.

For the BAR-8 characterization, 24-Nor-5 -cholane-7- ethylendioxy-3a,23-diol is isolated.

¾ NMR (CDCls): 4.01 (m, 2H, CH ₂ -CH ₂ ), 3.82 (m, 2H, CH ₂ -CH ₂ ), 3.72 (m, 1H, CHHOH), 3.64 (m, 1H, CHHOH), 3.63 (m, 1H, H3), 2.09 (m,

IH, CH), 2.01 (m, 1H, CHH), 1.84-1.10 (m, 22H, CH/CH ₂ ), 0.95 (s,

3H, CH ₃ ), 0.94 (d, J=4 Hz, 3H, H21), 0.67 (s, 3H, CH ₃ ).

¹³ C NMR (CDCls): 110.9 (Cq, C7), 71.9 (CH, C3), 64.0 (CH ₂ -CH ₂ ),

62.3 (CH ₂ -CH ₂ ), 61.0 (CH ₂ , C23), 55.5 (CH), 49.7 (CH), 43.6 (Cq),

42.3 (CH ₂ ), 41.7 (CH), 39.8 (CH ₂ ), 39.1 (CH), 37.8 (CH ₂ ), 37.1

(CH), 35.2 (Cq), 35.1 (CH ₂ ), 34.6 (CH ₂ ), 33.1 (CH), 30.7 (CH ₂ ),

28.9 (CH ₂ ), 26.1 (CH ₂ ), 23.1 (CH ₃ ), 21.5 (CH ₂ ), 19.1 (CH ₃ , C21),

II.7 (CH ₃ ). m/z= 407.32 and 835.61 correspond to [M+H] ⁺ and [2M+Na] ⁺ respectively .

HC1 6N (29 L) is slowly added to the suspension, in approximately 60 minutes. The mixture is heated to reflux and kept at this temperature for 2 hours. The reaction mixture is cooled to ambient temperature. The product BAR-9 is isolated and washed with water up to neutral pH. BAR-9 is obtained (3.4 Kg, 76% in two passages).

¹ H NMR (d6-DMSO): 4.48 (d, 1H, J=4 Hz, -OH), 4.25 (t, 1H, J=4

Hz, -OH), 3.39 (m, 3H, H3 + CH ₂ OH), 2.90 (dxd, 1H, Ji=12, J ₂ = 8Hz, CH), 2.44 (m, 1H, CH), 2.41 (m, 1H, CHH), 1.97 (m, 1H, CHH), 1.93-0.89 (m, 20H, CH/CH ₂ ), 1.13 (s, 3H, CH ₃ ), 0.88 (d, J=8 Hz, 3H, H21), 0.62 (s, 3H, CH ₃ ).

¹³ C NMR (d6-DMSO): 211.4 (Cq, C7), 69.1 (CH, C3), 58.5 (CH ₂ ,

C23), 54.9 (CH), 48.8 (CH), 48.6 (CH), 45.4 (CH), 45.1 (CH ₂ ),

42.2 (CH), 42.2 (Cq), 38.8 (CH ₂ ), 38.6 (CH ₂ ), 37.4 (CH), 34.8

(CH ₂ ), 33.8 (Cq), 32.3 (CH ₂ ), 29.8 (CH ₂ ), 28.0 (CH ₂ ), 24.4 (CH ₂ ),

22.8 (CH ₃ ), 21.2 (CH ₂ ), 18.8 (CH ₃ , C21), 11.9 (CH ₃ ). m/z= 363.29, 725.57 and 747.55 correspond to [M+H] ⁺ , [2M+H] ⁺ and

[2M+Na] ⁺ respectively.

Step 7: preparation of BAR-10 - Transformation of the BAR-9 into bis (trimethylsilyl) ether

BAR-9 BAR-10

BAR-9 (3.4 Kg, 0.0094 Kmol) is suspended in toluene (34 L) under N ₂ flow. DIPEA (9.6 L, 0.0564 Kmol) is added at ambient temperature to this suspension. TMSC1 (5.9 L, 0.0470 Kmol) is then added dropwise. The reaction mixture is brought to 80°C and kept under stirring at this temperature for 3 hours.

It is then brought back to ambient temperature and water is added (27 L), the phases are separated and the organic phase is concentrated to residue. BAR-10 (4.8 Kg, quantitative yield) is obtained .

¾ NMR (CDCls): 3.56 (m, 1H, H3), 3.64 (m, 1H, CHHOSi), 3.53 (m, 1H, CHHOSi), 2.81 (dxd, Ji=12, J ₂ =4 Hz, 1H, CH), 2.35 (t, J=12 Hz, 1H, CH), 2.17 (m, 1H, CHH), 1.89-0.86 (m, 21H, CH/CH ₂ ), 1.17 (s, 3H, CH ₃ ), 0.92 (d, J=4 Hz, 3H, H21), 0.64 (s, 3H, CH ₃ ), 0.10 (s, 9H, Si(CH ₃ ) ₃ ), 0.08 (s, 9H, Si(CH ₃ ) ₃ ).

¹³ C NMR (CDCls): 211.9 (Cq, Cl), 71.4 (CH, C3), 60.8 (CH ₂ , C23), 55.3 (CH), 49.6 (CH), 49.0 (CH), 46.2 (CH ₂ ), 45.6 (CH), 42.8 (Cq), 42.5 (CH), 39.1 (CH ₂ ), 39.0 (CH ₂ ), 37.8 (CH ₂ ), 35.2 (Cq), 34.5 (CH ₂ ), 32.9 (CH), 30.5 (CH ₂ ), 28.5 (CH ₂ ), 25.1 (CH ₂ ), 23.2 (CH ₃ ), 21.8 (CH ₂ ), 19.1 (CH ₃ , C21), 12.1 (CH ₃ ), 032 (CH ₃ ,

Si (CH ₃ ) ₃ ), -0.24 (CH ₃ , Si(CH ₃ ) ₃ ). m/z= 507.37, 1013.73 and 1035.71, correspond to [M+H] ⁺ , [2M+H] ⁺ and [2M+Na] ⁺ respectively.

Step 8: preparation of the BAR-11 - Transformation of the BAR- 10 into enolate

^BAR ' ¹⁰ BAR-11

Under N ₂ flow, the solution of n-BuLi (9.0 L, 2.5 M in hexane, 0.0226 Kmol) is added dropwise to a solution of DIPA (3.2 L, 0.0226 Kmol) in anhydrous THF (24 L). After 30 minutes, TMSC1 is added (3.0 L, 0.0235 Kmol). After a further 30 minutes, the solution of BAR-10 is added dropwise (4.8 Kg, 0.0094 Kmol) in anhydrous THF (24 L). The reaction is stirred for 60 minutes at -78°C and then TEA is added (6.6 L, 0.0047 Kmol). After 1 hour, the reaction is heated to -20°C and treated with a saturated aqueous solution of NaHC0 ₃ (24 L) allowing the mixture to heat to ambient temperature. The mixture is stirred at ambient temperature for 30 minutes and then the phases are separated. The organic phase is evaporated at reduced pressure until a residue is obtained.

The residue is dissolved in DCM (48 L) and the organic layer is washed twice with a saturated aqueous solution of NaHC0 ₃ (2x14 L) and then with water (14 L). The organic phase is evaporated at reduced pressure until a residue is obtained. For the characterization, BAR-11 is isolated as a white solid (5.4 Kg, quantitative yield).

¹ H NMR (CDCI3) : 4.72 (dxd, Ji=8, J ₂ =4 Hz, 1H, H6), 3.64 (m, 1H, CHHOSi), 3.57 (m, 1H, H3), 3.51 (m, 1H, CHHOSi), 1.90 (m, 1H, CH), 1.81-0.93 (m, 21H, CH/CH ₂ ), 0.93 (d, J=8 Hz, 3H, H21), 0.82 (s, 3H, CH ₃ ), 0.68 (s, 3H, CH ₃ ), 0.16 (s, 9H, C7-OSi(CH ₃ ) ₃ ), 0.11 (s, 9H, Si(CH ₃ ) ₃ ), 0.10 (s, 9H, Si(CH ₃ ) ₃ ).

¹³ C NMR (CDCI3) : 151.9 (Cq, C7), 109.0 (CH, C6), 71.7 (CH, C3), 61.0 (CH ₂ , C23), 55.5 (CH), 54.2 (CH), 44.6 (Cq), 42.8 (CH),

41.1 (CH ₂ ), 41.1 (CH ₂ ), 40.5 (CH ₂ ), 40.3 (CH), 39.1 (CH ₂ ), 34.8

(CH ₂ ), 33.1 (Cq), 33.0 (CH), 30.8 (CH ₂ ), 28.9 (CH ₂ ), 27.3 (CH),

22.7 (CH ₃ ), 21.1 (CH ₂ ), 19.3 (CH ₃ , C21), 12.4 (CH ₃ ), 0.53 (Cq,

C7-OSi (CH ₃ ) ₃ ), 0.40 (CH ₃ , Si(CH ₃ ) ₃ ), -0.21 (CH ₃ , Si(CH ₃ ) ₃ ). m/z= 579.41 corresponds to [M+H] ⁺ .

Step 9: preparation of the BAR-12 - Transformation of the BAR-

Under N ₂ flow, BAR-11 (5.4 Kg, 0.0094 Kmol) is dissolved in DCM (54 L) at ambient temperature. The reaction mixture is cooled to -78°C. At this temperature CH ₃ CHO (1.1 L, 0.0188 Kmol) and BF ₃ -CH ₃ CN (13.7 L, 0.0282 Kmol) are added. The reaction mixture is stirred at -78°C for 2 hours and then heated to 0°C. NaOH 3N at pH = 7.0 is then added. The mixture is left to reach ambient temperature and water is then added (11 L). The phases are separated and the organic phase is evaporated at reduced pressure until dry. BAR-12 is obtained (3.6 Kg, quantitative yield).

¹ H NMR (CDCI3): 6.17 (dxt, Ji=12, J ₂ =8 Hz, 1H, H24), 3.72 (m, 3H, H3 + CH2OH), 2.59 (dxd, Ji=12, J ₂ =4 Hz, 1H, CH), 2.33 (m, 1H, CH), 2.25 (dxd, Ji=12, J ₂ =8 Hz, 1H, CH), 2.02-1.09 (m, 19H,

CH/CH ₂ ), 1.68 (d, J=8 Hz, 3H, H25), 1.00 (s, 3H, CH ₃ ), 0.96 (d,

J=8 Hz, 3H, H21), 0.65 (s, 3H, CH ₃ ).

¹³ C NMR (CDCI3): 205.0 (Cq, C7), 143.5 (Cq, C6), 130.0 (CH, C24), 70.7 (CH, C3), 61.0 (CH ₂ , C23), 55.2 (CH), 50.9 (CH), 48.9 (CH ₂ ), 45.7 (CH), 43.8 (Cq), 39.3 (CH ₂ ), 39.2 (CH ₂ ), 37.7 (CH), 34.7

(Cq), 34.6 (CH ₂ ), 32.9 (CH), 29.9 (CH ₂ ), 29.8 (CH ₂ ), 28.8 (CH ₂ ),

26.1 (CH ₂ ), 23.0 (CH ₃ ), 21.5 (CH ₂ ), 19.1 (CH ₃ , C21), 12.8 (CH ₃ ),

12.2 (CH ₃ , C25). m/z= 389.30 and 799.58 correspond to [M+H] ⁺ and [2M+Na] ⁺ respectively .

Step 10: preparation of BAR-13 - Transformation of the BAR-12 The solution of BAR-12 (3.6 Kg, 0.0094 Kmol) in anhydrous MeOH (14.4 L) and MeONa 25% by weight in MeOH (2.2 L, 0.0094 Kmol) is hydrogenated in the presence of 5% Pd/C (0.18 Kg). The hydrogenation tank is flushed with three empty/refill cycles with N2 at P=2 bar.

The tank is then filled with H ₂ at P=4 bar. The mixture is stirred at ambient temperature for 24 hours. The catalyst is filtered and washed with MeOH (3.6 L). The pH is corrected to neutrality with HC1 6M and the solvent is removed at reduced pressure. MIBK (36 L) and water (7.2 L) are loaded, the layers are separated and the organic layer is concentrated to 1 volume. N-heptane (10 L) is loaded and the suspension obtained is cooled to ambient temperature. BAR-13 (3.1 Kg, 85%) is obtained.

¹ H NMR (CDCls): 3.70 (m, 1H, CHHOH), 3.62 (m, 1H, CHHOH), 3.52

(m, 1H, H3), 2.68 (dxt, Ji=12, J ₂ =4Hz, 1H, CH), 2.34 (t, J=8 Hz, 1H, CH), 2.16 (m, 1H, CHH), 1.98 (dxt, Ji=12, J ₂ =4 Hz, 1H, CHH), 1.92-0.84 (m, 21H, CH/CH ₂ ), 1.20 (s, 3H, CH ₃ ), 0.94 (d, J=8 Hz,

3H, H21), 0.79 (t, J=8Hz, 3H, H25), 0.66 (s, 3H, CH ₃ ).

¹³ C NMR (CDCls): 212.9 (Cq, C7), 71.3 (CH, C3), 61.0 (CH ₂ , C23), 55.5 (CH), 52.1 (CH), 50.8 (CH), 50.1 (CH), 49.2 (CH), 43.8

(CH), 42.8 (Cq), 39.2 (CH ₂ ), 39.1 (CH ₂ ), 35.8 (Cq), 34.4 (CH ₂ ),

32.9 (CH), 31.9 (CH ₂ ), 30.0 (CH ₂ ), 28.7 (CH ₂ ), 24.8 (CH ₂ ), 23.7

(CH ₃ ), 22.0 (CH ₂ ), 19.0 (CH ₂ ), 19.0 (C21), 12.1 (CH ₃ ), 12.2 (CH ₃ ,

C25). m/z= 391.32, 781.63, 798.66 and 803.61 correspond to [M+H] ⁺ ,

[2M+H] ⁺ , [2M+NH ₄ ] ⁺ and [2M+Na] ⁺ respectively.

For the characterization, the intermediate BAR-13/? is purified by chromatography (Si0 ₂ , DCM/DCM:MeOH (80:20)) and characterized.

¾ NMR (CDCls): 3.53 (m, 1H, H3), 3.72 (m, 1H, CHHOH), 3.61 (m,

1H, CHHOH), 2.56 (dxd, Ji=12, J ₂ =12 Hz, 1 H, CH), 2.16 (m, 1H,

CHH), 2.03-0.87 (m, 23H, CH/CH ₂ ), 1.18 (m, 3H, CH ₃ ), 0.96 (d, J= 4Hz, 3H, H21), 0.84 (t, J=8 Hz, 3 H, H25), 0.70 (s, 3H, CH ₃ ).

¹³ C NMR (CDCls): 215.6 (Cq, C7), 70.8 (CH, C3), 62.2 (CH), 61.0

(CH ₂ , C23), 55.5 (CH), 49.8 (CH), 48.9 (CH), 45.7 (CH), 43.1

(CH), 42.7 (Cq), 40.0 (CH ₂ ), 39.1 (CH ₂ ), 38.9 (CH ₂ ), 35.7 (Cq), 35.5 (CH ₂ ), 32.9 (CH ₂ ), 29.8 (CH ₂ ), 28.6 (CH ₂ ), 26.1 (CH), 26.1

(CH ₃ ), 25.1 (CH ₂ ), 21.6 (CH ₂ ), 19.0 (CH ₃ , C21), 13.2 (CH ₃ , C25),

12.2 (CH ₃ ). m/z= 391.32; 413.30; 781.63 and 803.62 correspond to [M+H] ⁺

[M+Na] ⁺ , [2M+H] ⁺ and [2M+Na] ⁺ respectively.

Step 11: Transformation of the BAR-13 into BAR502

The solution of BAR-13 (3.1 Kg, 0.0079 Kmol) in anhydrous MeOH (3.1 L) and toluene (28 L) is cooled to 0°C. NaBH ₄ (0.45 Kg, 0.0118 Kmol) is loaded in portions. The reaction is heated to ambient temperature and stirred for 60 minutes. Water (6 L) is loaded and the organic solvents are evaporated at reduced pressure. MIBK (31 L) and water (6 L) are loaded on the residue. The phases are separated and the organic phase is concentrated to 2 volumes. BAR502 (2.3 Kg, 75%) precipitates.