A process for preparing intermediates useful for the synthesis of colchicine

Abstract

A process is described for preparing the compound of formula (S)-3 for optical enzymatic or chiral amine resolution of compounds of formula 4 wherein R is a cyano, carboxy or C1-C6 alkoxy carbonyl group and P is a protecting group of the hydroxy group.

Description

[0001] " A PROCESS FOR PREPARING INTERMEDIATES USEFUL FOR THE SYNTHESIS OF COLCHICINE"

[0002] * * *

[0003] Field of the invention

[0004] The present invention relates to an efficient, economical and industrially scalable process that allows the preparation of a chiral intermediate of colchicine.

[0005] Background of the invention

[0006] Colchicine 1 (N-[(7S)-5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]eptalen-7-yl]-acetamide)

[0007]

[0008] a tricyclic alkaloid present in the bulbs and seeds of plants of the colchicaceae family, in particular Gloriosa superba and Colchicum autumnalis, is useful as a drug for the treatment of various diseases such as gout, Mediterranean fever, Behcet's syndrome and autoimmune or cardiovascular diseases. Colchicine is mainly prepared by extraction from plant seeds. A semisynthetic process for preparing colchicine from colchicoside is described in WO 2021255618. The structural complexity and the presence of asymmetry centres have hindered the industrial development of total colchicine synthesis processes.

[0009] A review of the synthetic methodologies of colchicine is reported in Graening, T.; Schmalz, H. G. Angew. Chem., Int. Ed. 2004, 43, 3230-3256. Recently, a seven-step synthetic pathway has been described (Xiao Liang et al., Org. Lett. 2021, 23, 2731-2735) which uses an iridium-based chiral catalyst for preparing intermediate 2:

[0010]

[0011] by amination of the corresponding racemic allyl alcohol.

[0012] Intermediate 2, after conversion to alkyl borane with 9-borabicyclo(3.3.1)nonane (9-BBN), is then reacted with 3,4,5-trimethoxybenzene bromide in the presence of triphenylphosphine and a palladium-based catalyst according to Suzuki reaction conditions to give the chiral intermediate of formula GS')-3

[0013] NHAc

[0014]

[0015] OMe

[0016] (S)-3

[0017] which is then transformed into colchicine by means of successive steps of oxidative intramolecular condensation, oxidation, cyclopropanation and ring expansion.

[0018] There remains a need for more efficient and economical processes, which do not require the use of expensive reagents such as the chiral iridium-based catalysts used in the process described above.

[0019] Description of the invention

[0020] An efficient, economical and industrially scalable process has now been found that allows the preparation of the chiral intermediate of formula GS')-3

[0021] NHAc

[0022]

[0023] OMe

[0024] (S)-3

[0025] which can then be conveniently converted to colchicine in high yields and high purity. According to the invention, intermediate (> S’)-3 is prepared by resolution of compounds of formula 4

[0026]

[0027] 4

[0028] wherein R is a cyano (compounds 4A), carboxy (compounds 4B) or C1-C6 alkoxycarbonyl (compounds 4C) group and P is a protecting group of the hydroxy group, to give a compound of formula (A’)-4B

[0029] COOH

[0030]

[0031] OMe

[0032] ()-4B

[0033] wherein P is as defined above.

[0034] The compound (A’)-4B is then transformed into compound 3 by conversion of the carboxy group to the amino group, acetylation and removal of the protecting group P.

[0035] In a first aspect thereof, the invention therefore relates to a process for preparing a compound of formula (. S’)-3

[0036] NHAc

[0037]

[0038] OMe

[0039] (5)-3

[0040] which comprises:

[0041] a) separation of enantiomers by optical resolution of a compound of formula 4

[0042]

[0043] 4

[0044] wherein R is a cyano (compounds 4A), carboxy (compounds 4B) or C1-C6 alkoxycarbonyl (compounds 4C) group and P is a protecting group of the hydroxy group to give a compound of formula (S)-4B

[0045] COOH

[0046]

[0047] OMe

[0048] (5)4B

[0049] b) conversion of the carboxy group to the amino group, acetylation and removal of the protecting group P.

[0050] The compound of formula (A’)-4B is novel and is a further object of the invention. The invention further relates to the use of the compound of formula fS')-4B in a colchicine synthesis process.

[0051] Detailed description of the invention

[0052] Resolution of the compounds of formula 4 can be carried out enzymatically when R is a cyano (compounds 4A) or C1-C6 alkoxy carbonyl (compounds 4C) group, using a nitrilase and an esterase respectively. Nitrilases are known enzymes isolatable from microorganisms of the genus Pseudomonas, Nocardia and Fusarium.

[0053] A review of carboxyl esterases and their use in the optical resolution of compounds containing a chiral centre bearing a carboxyl group is reported in Uwe T. Bornscheuer, FEMS Microbiology Reviews, Volume 26, Issue 1, March 2002, Pages 73-81.

[0054] When R is a carboxy group (compounds 4B), resolution is conveniently carried out by salification with a chiral amine, preferably with an amine selected from (R)-(+)-l-phenylethylamine, (R)-(+)-l-(2-naphthyl)ethylamine, (S)-(+)-leucinol, (S)-(-)-4-methoxy- alpha-methylbenzylamine, cinchonidine, (R)-(+)-N, N-dimethyl-1-phenylethylamine, (R)-(+)-N-benzyl-1 -phenylethylamine. Resolution with chiral amines is typically carried out in a solvent selected from isopropanol, methyl ethyl ketone, tetrahydrofuran, acetonitrile and mixtures thereof. Preferred is (S)-(-)-4-methoxy-alpha-methylbenzylamine in acetonitrile or isopropanol.

[0055] The undesired enantiomer (R) can be racemized, for example by acid treatment, to then be subjected to resolution again, so as to increase the overall yields of the process.

[0056] The conversion of the carboxy group to the amino group can be carried out by means of a Curtius, Hoffman, Lessen or Schmidt reaction.

[0057] The Curtius reaction is preferably carried out by treating the compound of formula (A)-4B with diphenylphosphorylazide in toluene in the presence of a base such as triethylamine.

[0058] As an alternative to the Curtius reaction, other known reactions can be used for the conversion of a carboxy group to an amino group. For example, the Hofmann reaction comprises treatment with bromine and an alkali hydroxide. The bromine may be replaced by sodium hypochlorite, lead tetraacetate, N-bromosuccinimide or bis(trifluoroacetoxy)iodobenzene.

[0059] According to another method, the compound (A)-4B can be converted to the corresponding isocyanate generating amine by treatment with water under Lessen rearrangement conditions. Yet another alternative is treatment with hydrazoic acid in the presence of acids according to the Schmidt reaction.

[0060] Acetylation of the amino group can be carried out by conventional methods, for example by reaction with acetic anhydride in the presence of a base such as triethylamine in an aprotic solvent.

[0061] The protecting group P is preferably the benzyl group, removable by catalytic hydrogenation.

[0062] The compound 4A wherein P is benzyl(Bn) can be obtained by reaction of (3,4,5-trimethoxyphenylj-ethanol (possibly activating the -OH group) with 2-(3-(benzyloxy)-4-methoxyphenyl)acetonitrile. The compound 4B is obtained by hydrolysis of the cyano group. Esterification of the carboxyl group with a C1-C6 alcohol leads to the compounds 4C, usable for enzymatic resolution.

[0063] The compound 3 is useful for the synthesis of colchicine 1 by sequential steps of oxidative intramolecular condensation, oxidation, cyclopropanation and ring expansion. The following diagram summarizes the overall process for preparing colchicine 1 from the compound of formula 3.

[0064]

[0065] OMe More in detail, the intramolecular oxidative condensation to give the compound of formula (5)-5

[0066]

[0067] can be carried out by treating the compound GS')-3 with iodobenzene diacetate and boron trifluoride-ethyl ether in a polar solvent such as methanol or ethanol.

[0068] The compound thus obtained is then oxidized to give the compound of formula

[0069]

[0070] (5)-6

[0071] Oxidation can also be carried out by treatment with iodobenzene diacetate in methanol. Cyclopropanation of the oxidized compound by treatment with trimethyl sulphoxonium bromide in polar aprotic solvents and in the presence of bases provides the compound of formula

[0072]

[0073] (5)-7

[0074] which is converted to colchicine 1 by treatment with trifluoroacetic acid.

[0075] The invention is illustrated in more detail in the following examples, in which:

[0076] the NMR spectra were run on Varian MERCURY 300 MHz (300 and 75 MHz for ¹H and13C, respectively), Bruker Avance I 400 MHz (400 and 101 MHz for ¹H and13C, respectively) or Agilent MR400 DD2 VNMRJ™ Software Rev. 4.2 (400 and 101 MHz for ¹H and13C, respectively) spectrometers. The chemical shifts (δ) are indicated in ppm relative to the internal standard and coupling constants J in Hertz (Hz).

[0077] The mass spectra were recorded on a Thermo Scientific Fleet LCQ spectrometer and on a triple quadrupole 3500 mass spectrometer (AB SCIEX) equipped with ESI interface. The optical rotations were measured on a PerkinElmer 343 polarimeter (concentration in g / 100 mL).

[0078] The HPLC analyses were performed with HPLC equipment equipped with quaternary pump, automatic sampler with thermostat, column and UV detector. The stationary phase was Daicel Chiralpak AD, used to measure the enantiomeric excess (abbreviated to e.e.) of the following compounds:

[0079] carboxylic acid 4B-a: isocratic elution with heptane / iPrOH (7:3) + 1% AcOH; flow 0.7 mL / min; T = 25 °C; two λ set at 210 and 254 nm; t₁ = 18.67 min; t₂ = 21.58 min.

[0080] Methyl ester 4C-a: isocratic elution with heptane / iPrOH (7:3) + 1% AcOH; flow 0.7 mL / min; T = 25 °C; two λ set at 210 and 254 nm; t₁ = 17.08 min; t₂ = 20.90 min.

[0081] Acetamide (S)-3: isocratic elution with hexane / iPrOH (3:2); flow 0.8 mL / min; T = 25 °C; two λ set at 210 and 254 nm; t₁ = 9.00 min; t₂ = 17.34 min.

[0082] Colchicine 1: isocratic elution with hexane / iPrOH (4:1); flow 1.0 mL / min; T = 25 °C; λ = 210 nm; t₁ = 8.23 min; t₂ = 13.49 min (higher).

[0083] With regard to enzymatic reactions, the value E is calculated as follows: E = ln[1-substrate conversion *(1 +ee of the product)] / ln[1-conversion *(1-ee of the product)]. The melting points were recorded with Stuart Scientific Melting Point Apparatus SMP3 version 5.0.

[0084] Example 1 - Synthesis of 2-(3-(benzyloxy)-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)butanenitrile (compound 4A-a)

[0085] BnO MeO. MeO MeO'

[0086] 8

[0087]

[0088] By operating under nitrogen and stirring, nitrile 8 (5 g, 19.82 mmol) and mesylate 9 (5.75 g, 19.82 mmol) were added to a mixture of anhydrous toluene (25 mL) and DMSO (6.2 mL) at 25±5 °C. The mixture was heated to 70±3 °C and a solution of tBuOK (2.69 g, 23.78 mmol) in a mixture of anhydrous toluene (16 mL) and DMSO (4 mL) was added dropwise into the reaction mixture over 1 h, keeping the temperature below 75 °C. The suspension was kept under stirring at 70±3 °C and monitored by TLC [a sample was dissolved in AcOEt (1 mL) and a saturated solution of NH4 Cl (1 mL); hexane / AcOEt, 7:3; R 8, 0.52; 9, 0.15; 4A-a, 0.30;

[0089] 1019. 0.70] until complete conversion of mesylate 9. After 18 hours, the solution (which turned from light orange to black) was cooled to 25±3 °C and added dropwise over 15 minutes to a saturated aqueous solution of NH4CI (50 mL) cooled to 5±3 °C to pH = 7. The layers were separated and the aqueous one was extracted with AcOEt (2^25 mL). The combined organic layers were washed with H2O (4x20 mL) to remove traces of DMSO and the solvent was evaporated under reduced pressure. The raw matter was purified by column chromatography [185 g silica gel; gradient from hexane / AcOEt 9:1 to 7:3], Four fractions were obtained: a) styrenic by-product 10 (1 g, 26%) as colourless oil. ¹H NMR (DMSO-d₆, 400 MHz): δ 6.75 (2H, s; Ar), 6.67-6.56 (1H, m; CH=CH2), 5.76 (1H, d, J = 17.8 Hz; CH=CH2), 5.18 (1H, d, J = 10.9 Hz; CH=CH2), 3.77 (6H, s; OMe), 3.63 (3H, s; OMe); ¹³C NMR (DMSO-d6, 101 MHz): δ 153.4 (Ar), 138.0 (Ar), 137.1 (Ar-C=CH2), 133.3 (Ar), 114.0 (Ar-CH=CH2), 104.0 (Ar), 60.5 (OMe), 56.3 (2C; OMe); MS (ESI): m / z calculated for C11H14O3+H+: 195.10; it was found: 195.05 [M+H]+.

[0090] b) unreacted nitrile 8 (1.21 g, 24%; NMR and MS analyses agree with the data reported in example 6);

[0091] c) the product 4A-a (4.34 g, 49%), which was used in the next step without further purification. The product 4A-a can be crystallized as follows: by operating under stirring, the compound 4A-a (0.63 g) was dissolved in AcOEt (1.8 mL) at 25±5 °C. Hexane (4.2 mL) was added dropwise over 5 minutes until precipitation was observed. After 30 minutes, the crystallized solid was filtered under vacuum, washed twice with hexane (2 mL) and dried under vacuum at 45±3 °C until constant weight, obtaining the product 4A-a (0.39 g, crystallization yield 62%) as a white solid. 'H NMR (DMSO-d6, 400 MHz): δ 7.44-7.26 (5H, m; Bn), 7.08-6.90 (3H, m; Ar), 6.45 (2H, s; Ar), 5.05 (2H, s; Bn), 4.06-4.00 (1H, m; CH-2), 3.74 (3H, s; OMe), 3.72 (6H, s; OMe), 3.58 (3H, s; OMe), 2.64-2.50 (2H, m; CH2-4), 2.22-2.03 (2H, m; CH2-3); ¹³C NMR (DMSO-d6, 101 MHz): δ 153.2 (2C; Ar), 149.3 (Ar), 148.4 (Ar), 137.3 (Bn), 136.4 (Ar), 136.3 (Ar), 128.8 (2C; Bn), 128.6 (Ar), 128.4 (2C; Bn), 128.3 (Bn), 121.9 (CN), 120.5 (Ar), 113.6 (Ar), 112.8 (Ar), 106.0 (2C; Ar), 70.6 (Bn), 60.4 (OMe), 56.2 (2C, OMe), 56.1 (OMe), 36.2 (CH2-3), 35.6 (CH-2), 33.4 (CH2-4); MS (ESI): m / z calculated for C27H29NO5+Na+: 470.19; it was found: 470.23 [M+Na],

[0092] Example 2 - Synthesis of 2-(3-(benzyloxy)-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)butanoic acid (compound 4B-a)

[0093] CN CO2H MeO...,, OBn KOH (14 eq) MSQ OBn MeO' O Y O 'OMe a EtOOHH / HH? OOt (1l / 1), MeO' I J (I 'OMe OMereflux 36hOMe

[0094] 4A-a77%4B-a

[0095] By operating under stirring, the compound 4A-a (32.9 g, 73.51 mmol) was suspended in EtOH (95%; 100 mL) at 25±3 °C. A solution of KOH (85% pellet; 65.2 g, 987.7 mmol) in H2O (100 mL) was added to the reaction, keeping the temperature below 40 °C. The suspension was refluxed under stirring and monitored by TLC [a sample was dissolved in CH2C12(1 mL) and treated with HC1 (IM; 1 mL) until pH= 1; hexane / AcOEt, 7:3; R / : 4A-a, 0.50; 4B-a, 0.14; heptane / iPrOH (7:3) + 1% AcOH; Rf: 4A-a, 0.95; 4B-a, 0.45], After 36 hours, the reaction mixture was cooled to 30±5 °C and EtOH was evaporated in vacuo. tBuOMe (100 mL) was added to the aqueous mixture and the biphasic mixture was stirred for 15 minutes at 25±5 °C. The organic layer was removed to remove any impurities. Extraction with tBuOMe was repeated twice as described above. AcOH (42 mL) was added dropwise over 20 min into the aqueous layer until pH = 6, keeping the temperature below 30 °C. AcOEt (15 mL) was added and the mixture was allowed to stir at 25±5 °C for 15 min, then cooled to 0±5 °C. After 15 minutes, the solid was vacuum filtered, washed with H2O (3^100 mL) to remove salts, then with AcOEt (30 mL) at 0±5 °C. The crystallized solid was dried under vacuum at 50±3 °C until constant weight, obtaining racemic carboxylic acid 4B-a (26.4 g, 77%) as a white solid. Mp = 149-150 °C; 'H NMR (CDCh, 400 MHz): δ 7.50 (br s; CO2H), 7.44-7.21 (5H, m; Bn), 6.92-6.78 (3H, m; Ar), 6.31 (2H, s; Ar), 5.09 (2H, s; Bn), 3.83 (3H, s; OMe), 3.81 (3H, s; OMe), 3.80 (6H, s; OMe), 3.43 (1H, t, J = 7.6 Hz; CH-2), 2.45 (2H, t, J = 7.7 Hz; CH2-4), 2.36-2.24 (1H, m; CH2-3), 2.03-1.93 (1H, m; CH2-3); ¹³C NMR (CDCl3, 101 MHz): δ 179.0 (CO2H), 153.1 (2C; Ar), 149.1 (Ar), 148.2 (Ar), 137.2 (Bn), 137.0 (Ar), 136.2 (Ar), 131.6 (Ar), 128.5 (2C; Bn), 127.9 (Bn), 127.6 (2C; Bn), 121.1 (Ar), 114.3 (Ar), 111.9 (Ar), 105.5 (2C; Ar), 71.2 (Bn), 60.8 (OMe), 56.1 (2C, OMe), 56.0 (OMe), 51.1 (CH2-3), 34.6 (CH-2), 33.9 (CH2-4); MS (ESI): m / z calculated for C27H30O7+H+: 467.21; it was found: 467.12 [M+H]+

[0096] Example 3 - Optical resolution of carboxylic acid 4B-a by (S)-(-)-4-methoxy-a-methylbenzylamine

[0097] By operating under stirring, the racemic carboxylic acid 4B-a (6.23 g, 13.36 mmol) was suspended in iPrOH (31 mL) at 25±3 °C, and (5)-(-)-4-methoxy-a-methylbenzylamine (2.0 mL, 13.36 mmol) was added dropwise over 5 min at 25±3 °C, fluidizing the suspension. The suspension was heated under reflux and iPrOH (37 mL) was added portionwise until dissolved. After 15 min, the solution was cooled to 61 °C (internal T) in 3 h and a few mg of enantiomeric salt were added, observing that they remained in suspension. After 30 min, the suspension was cooled to 40±3 °C in 3:30 h, then to 25 °C (internal T) in 1:20 h. An additional amount of iPrOH (12 mL) was added at 25±3 °C to fluidize the suspension. After 15 minutes, the crystallized salt was filtered under vacuum, washed twice with iPrOH (12 mL) and dried under vacuum at 25±3 °C until constant weight, obtaining 4B-a ammonium salt (3.17 g) as a white solid. Then, it was dissolved in CH2Q2 (15 mL), washed with an aqueous solution of HC1 (0.5 M; 13 mL; until pH = 3) and H2O (3x15 mL; until pH = 6), evaporated and dried under vacuum at 25±3 °C until constant weight, obtaining the enantiomeric fS')-carboxylic acid (A’)-4B-a (2.55 g, 41%) as a white solid with e.e. of 88% (see General information for HPLC method). The enantiomeric excess of (5)-carboxylic acid (A’)-4B-a can be increased as follows: by operating under stirring, the enantiomeric salt of carboxylic acid (A’)-4B-a (200 mg; obtained as above) was suspended in iPrOH (2.6 mL). The suspension was heated under reflux and iPrOH (1.2 mL) was added in portions until dissolved. After 15 minutes, the solution was cooled to 40±5 °C for 3 hours, then to 25±5 °C for 1 hour. After 15 min, the crystallized salt was filtered in vacuo, washed with iPrOH (1 mL) and dried in vacuo at 25±3 °C until constant weight, obtaining the ammonium salt (135 mg) as a white solid. Then, it was dissolved in CH2Cl2 (5 mL), washed with an aqueous solution of HCl (0.5 M; 5 mL; until pH = 3) and H2O (3×5 mL; until pH = 6), evaporated and dried under vacuum at 25±3 °C until constant weight, obtaining enantiopure (5)-carboxylic acid (A’)-4B-a (100 mg, 67%) as a white solid with an e.e. of 98% (see General information for HPLC method). Mp = 137-138 °C; [OC]D20= +32.0 (c 1.1, CH2 Cl2).

[0098] The mother liquor from the first crystallization was evaporated, then dissolved in CH2Cl2 (25 mL), washed with an aqueous solution of HCl (0.5 M; 13 mL; until pH = 3) and H2O (3×15 mL; until pH = 6), evaporated and dried under vacuum at 25+3 °C until constant weight obtaining (R) -carboxylic acid (R)-4B-a (2.61 g, 42%), which can be used for racemization without further purification. It is possible to crystallize (R)-carboxylic acid (R)-4B-a as follows: by operating under stirring, the compound 3 (2.61 g) was dissolved in AcOEt under reflux (13 mL). The solution was then cooled to 25+5 °C for 1.30 h and stirred at this temperature for 18 h. The crystallized solid was filtered under vacuum, washed with AcOEt (1.5 mL), and dried under vacuum at 25+3 °C to constant weight, obtaining the (R -carboxylic acid (R)-4B-a (1.96 g, 75% crystallization yield) as a white solid with an e.e. of 80% (see General information for HPLC method). Mp = 137-138 °C; [OC]D20= -30.7 (c 1.0, CH2 Ch).

[0099] Example 4 - Racemization of the (R -carboxylic acid (R)-4 -n

[0100] By operating under nitrogen and under stirring, (R -carboxylic acid R -4B-a (e.e. = 80% - see General information for HPLC method; 150 mg, 0.321 mmol) was dissolved in dimethylacetamide (1.13 mL) at 25+5 °C. The solution was cooled to 5+5 °C and AC2 O (60 pL, 0.611 mmol) was added. The reaction mixture was stirred at 25+5 °C for 3 days. Then, a solution of AcONa (155 mg, 1,890 mmol) in H2O (3 mL) stirred at 5+5 °C (pH = 10) was added dropwise over 5 minutes. Then, aqueous AcOH (80% v / v, 0.10 mL, 0.848 mmol) was added dropwise for 5 min to the mixture at 5+5 °C until pH = 5-6. The resulting biphasic mixture was heated and allowed under stirring at 25+5 °C for Ih, then diluted with H2O (4 mL) and extracted with CH2CI2 (2x10 mL). The organic layer was washed with H2O (3x10 mL; until pH = 7), dried with Na2SO4 and evaporated to obtain racemic acid 4B-a (145 mg, 97%). The NMR and MS analyses were in agreement with the data reported in Example 2.

[0101] Example 5 - Synthesis of methyl 2-(3-(benzyloxy)-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl) butanoate ) (4C-a)

[0102] CO2Me OBn SOCI2(1.5 eq)Me0OMe MeOH, 0-25 °C MeO OMe 86%

[0103]

[0104] 4C-a By operating under nitrogen and stirring, the racemic carboxylic acid 4B-a (250 mg, 0.536 mmol) was suspended in anhydrous MeOH (6 µL) at 20±5 °C under nitrogen. The solution was cooled to 5±5 °C and SOCl2 (60 mL, 0.804 mmol) was added dropwise, keeping the temperature below 20 °C. The solution was heated to 40±5 °C and monitored by TLC [hexane / AcOEt, 3:7; R / : 4B-a, 0.22; 4C-a, 0.77], After 5h, the reaction mixture was diluted with toluene (5 mL) and cooled to 5±5 °C. A saturated aqueous solution of Na2CO3 (4 mL) was added per drop over 5 min, until pH = 10. The aqueous layer was separated and extracted with toluene (2x10 mL). The combined organic layers were washed with H2O (20 mL) and evaporated. The raw matter was purified by column chromatography [10 g silica gel; gradient hexane / AcOEt from 9:1 to 3:1] to obtain racemic methyl ester 4C-a (221 mg, 86%) as white wax.1H NMR (CDCl3, 400 MHz): d 7.43-7.25 (5H, m; Bn), 6.98-6.88 (2H, m; Ar), 6.82-6.77 (1H, m; Ar), 6.38 (2H, s; Ar), 5.02 (2H, system AB, J = 11.9 Hz; Bn), 3.71 (3H, s; ArOAfe), 3.70 (6H, s; ArOAfe), 3.58 (3H, s; ArOAfe), 3.52 (3H, s; CCWe), 3.49 (1H, t, J= 7.6 Hz; CH-2), 2.42-2.32 (2H, m; CH2-4), 2.24-2.13 (1H, m; CH2-3), 1.98-1.87 (1H, m; CH2-3);13C NMR (CDCl3, 101 MHz): d 174.2 (CO2Me), 153.2 (2C; Ar), 148.9 (Ar), 148.1 (Ar), 137.5 (Ar), 137.3 (Ar), 136.1 (Ar), 131.6 (Bn), 128.8 (2C; Bn), 128.3 (2C; Bn), 128.2 (Bn), 120.9 (Ar), 114.0 (Ar), 112.7 (Ar), 105.9 (2C; Ar), 70.5 (Bn), 60.4 (ArOAfe), 56.2 (2C, ArOAfe), 56.1 (ArOAfe), 52.1 (CCWe), 50.0 (CH2-3), 34.8 (CH2-2), 33.7 (CH2-4); MS (ESI): m / z calculated for C28H32O7+H+: 467.21; it was found: 467.12 [M+H]+ Mass 1344 / 46 / D - BA 240036 LRS -16 / 1 / 24

[0105] Example 6 - Enzymatic resolution of 4C-a methyl ester with porcine liver esterase The methyl ester 4C-a (100 mg) was dissolved in DMSO (1 mL) at 30±5 °C. 10 µL of this solution was added to each freeze-dried porcine liver esterase of the kit (purchased from Syncozymes - SynKit PLE), previously suspended for 5 min with milli-Q H2O (90 pL). The multiple well was incubated at 37 °C and stirred constantly at 400 rpm. After 20 hours, 50 µL of mixture was taken from each enzymatic reaction and extracted separately with AcOEt (100 µL). The mixtures were stirred and allowed to stand for 15 minutes. Each organic phase was analysed by HPLC. After the above analyses, each sample was incubated for another 48 hours, then processed as above and analysed by HPLC obtaining the following data:

[0106] Conversion of ester 4C-a Value Conversion of ester 4C-a and Value Enzyme

[0107] and e.e. after 20h E e.e. after 72h

[0108] 26% conversion to (S)- 45% conversion to (S)- carboxylic acid (A)-4B-a with 280 carboxylic acid (A')-4B-a with e.e. > 99% e.e. = 94%

[0109] 50% conversion to (S)- 57% conversion to (S)- carboxylic acid (A)-4B-a with carboxylic acid (A)-4B-a with e.e. = 80% e.e. = 66%

[0110] 23% conversion to (S)- 39% conversion to (S)- carboxylic acid (A)-4B-a with carboxylic acid (A)-4B-a with e.e. = 92% e.e. = 90%

[0111] 0% Conversion | 10% conversion to (R)- 0 | carboxylic acid (S)-4B-a with 0.2

[0112]

[0113] e.e. > 99%.

[0114] Example 7 - Synthesis of (S)-benzyl-(l-(3-(benzyloxy)-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propyl)carbamate (S)-3a)

[0115] CO2H 1) TEA (2.5 eq),

[0116] DPPA (1.2 eq), NHCBz

[0117] 2) BnOH (3 eq) Toluene, 50-55 °C OMe 82% OMe

[0118]

[0119] (S)-4B-a

[0120] CBz = C(O)OBn

[0121] (S)-23b: By operating under gentle nitrogen flow and stirring, the carboxylic acid (S)-4B-a (1.00 g, 2.14 mmol) was suspended in anhydrous toluene (20 mL). Triethylamine (0.74 mL, 5.36 mmol) was added, then the suspension was heated to 53±3 °C until dissolution (pH = 8). A solution of diphenylphosphorylazide (0.56 mL, 2.57 mmol) in anhydrous toluene (2 mL) was added dropwise at 53±3 °C over 40 min. The reaction solution was stirred at 53±3 °C and monitored according to the development of gas and TLC until complete conversion of the acid (S)-4B-a [hexane / iPrOH (7:3) + 1% AcOH; Rf: (S)-4B-a, 0.33], After Ih, a solution of benzyl alcohol (0.67 mL, 6.43 mmol) in anhydrous toluene (2 mL) was added dropwise into the reaction mixture for 10 min at 53±3 °C (pH = 7). The reaction solution was then stirred at 53±3 °C and monitored by TLC [hexane / AcOEt (7:3); Rf: (S)-4B-a, 0.05; (S)-11a, 0.14], After 4h, the reaction mixture was cooled for 30 min at 25±5 °C (pH = 6) and washed with a saturated solution of NaHCO3 (10 mL; until pH = 9). The aqueous solution was extracted with toluene (2x15 mL). The combined organic layers were washed with HC1 solution (IM, 10 mL; until pH = 1), then with H2O (3x10 mL; until pH = 7). The combined organic layers were evaporated in vacuo. The raw matter was purified by column chromatography [50 g silica gel; gradient hexane / AcOEt 7:3 to 3:2] to obtain the raw matter benzyl carbamate (S)-11a (1.003 g, 82%). The raw compound (1.00 g) was suspended twice in AcOEt (9 mL) and evaporated. By operating under stirring, the compound was dissolved in AcOEt (3 mL) at 25±5 °C. Hexane (12 mL) was added dropwise over 5 minutes at 25±5 °C, then cooled to 5±5 °C. After 30 minutes, the crystallized solid was vacuum filtered, washed with hexane (0.5 mL) at 5±5 °C and dried under vacuum at 25±3 °C until constant weight, benzyl carbamate (S)-311a (0.856 g, 70% after crystallization) as a white solid. Mp = 94-95 °C; [α]D20= -32.0 (c 1.0, CH2Cl2). δ 7.51-7.21 (10H, m; Bn, Ar-Cbz overlapped), 6.95-6.80 (3H, m; Ar), 6.35 (2H, s; Ar), 5.13 (2H, s; Bn overlapped), 5.10 (2H, system AB, J = 12.2 Hz; CH2-Cbz overlapped), 4.99 (1H, br d, J = 6.2 Hz; NH), 4.65 (1H, br d, J = 6.2 Hz; CH-1), 3.90 (3H, s; OMe), 3.84 (9H, s; OMe), 2.61-2.44 (2H, m; CH2-3), 2.19-2.05 (1H, m; CH2-2), 2.05-1.93 (1H, m; CH2-2);13C NMR (CDCl3, 101 MHz): δ 155.7 (CO-Cbz), 153.2 (2C; Ar), 149.2 (Ar), 148.3 (Ar), 137.01 (Ar), 136.98 (Ar), 136.4 (Ar-Cbz), 136.2 (Ar), 134.6 (Bn), 128.5 (4C; Bn, Ar-Cbz overlapped), 128.2 (Ar-Cbz), 127.9 (Bn), 127.5 (4C; Bn, Ar-Cbz overlapped), 119.3 (Ar), 113.0 (Ar), 111.9 (Ar), 105.3 (2C; Ar), 71.3 (Bn), 66.8 (CH2-Cbz), 60.9 (OMe), 56.1 (3C, OMe), 54.8 (CH-1), 38.0 (CH2-2), 32.9 (CH2-3); MS (ESI): m / z calculated for C34H37NO7+Na+: 594.25; it was found: 594.81 [M+Na]+ Example 8 - Synthesis of (S)-5-(l-amino-3-(3,4,5-trimethoxyphenyl)propyl)-2-methoxyphenol ((S)-12)

[0122] NHCbz NH2

[0123] H2(6 atm), Pd-C (0.05 eq) MeO^z^^X / vOH MeO'^'y THF, 25 C MeO^^p ^x??^x0Me OMe 99% OMe

[0124]

[0125] (S)-11a (SJ-12 Benzyl carbamate ($-lla (0.750 g, 1.312 mmol) was dissolved in THF (8 mL) at 25±5 °C. Pd on carbon (10 wt %, 55% wet; 125 mg, 0.062 mmol) was then added, then the reaction mixture was stirred under hydrogen atmosphere (6±1 atm) and monitored by TLC [hexane / AcOEt (3:2); R / : ($-lla, 0.37; ( -12, 0.05; CH2CI2 / MeOH (9.1); R / ($-lla, 0.90;

[0126] ($-12, 0.6], After 24 hours, the catalyst was filtered over celite, then washed with THF (3x4 mL). The filtered solution was evaporated to obtain amine ($-12 (0.45 g, 99%), which can be used in subsequent steps without further purification. The amine ($-12 (0.45 g) was suspended twice in Et2O (5 mL) and evaporated. Then, by operating under stirring, the compound was suspended in Et2O (2.5 mL) at 25±5 °C for 10 min, then cooled to 5±5 °C. After 1 h, the crystallized solid was vacuum filtered and vacuum dried at 25±3 °C until constant weight, obtaining amine (A)-12 (0.396 g, 87% after crystallization) as a white solid.

[0127] Mp = 155-156 °C; [α]D20= -14.2 (c 1.0, THF). Mp = 163-164 °C;1H NMR (MeOD, 300 MHz): δ 6.91 (1H, d, J = 8.2 Hz; Ar), 6.82 (1H, d, J = 2.0 Hz; Ar), 6.78 (1H, dd, J = 8.2, 2.0 Hz; Ar), 6.42 (2H, s; Ar), 3.84 (3H, s; OMe), 3.79 (6H, s; OMe), 3.78-3.72 (1H, m; CH-1), 3.71 (3H, s; OMe), 2.45 (2H, t, J = 7.8 Hz; CH2-3), 2.13-1.90 (2H, m; CH2-2);13C NMR (MeOD, 75 MHz):

[0128] 3 154.4 (2C; Ar) 148.5 (Ar), 137.9 (Ar), 139.3 (2C; Ar), 137.8 (Ar), 119.1 (Ar), 114.9 (Ar), 112.8 (Ar), 106.6 (2C; Ar), 61.1 (OMe), 56.5 (2C, OMe), 56.4 (OMe), 56.3 (CH-1), 40.7 (CH2-2), 34.0 (CH2-3); MS (ESI): m / z calculated for C19H25NO5: 347.17; it was found: 347.35 [M]

[0129] Example 9 - Synthesis of (S)-N-(l-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propyl)acetamide ((S)-3; from(S)-12 at 7 °C)

[0130] NHAc py (1.05 eq), AC2O (1.05 eq) CH2CI2, 0*7 °C y = 80%

[0131]

[0132] (S)-12 (SJ-3 (S)-3. By operating under nitrogen and stirring, (S)-amine (S)-12 (396 mg, 1.140 mmol) was suspended in anhydrous CH2Cl2 (9.6 mL) at 25±5 °C. The mixture was cooled to -1±3 °C, then pyridine (100 pL, 1.140 mmol) (pH = 9) was added. After 5 min, a solution of Ac2O (110 pL, 1.140 mmol) in anhydrous CH2C12(2.4 mL) was added dropwise over 5 min at -2±2 °C, dissolving the reaction mixture (pH = 6). The reaction was heated to 7±2 °C in 30 min, stirred at the same temperature and monitored by TLC [CH2Cl2 / MeOH (9:1); Rf: (S)-12, 0.5; (S)-3, 0.66], After 1 h, MeOH (0.24 mL) was added dropwise to the reaction solution, which was stirred at 9±3 °C for 5 min; then HC1 (IM; 5 mL) was added dropwise until pH = 1 while keeping the temperature below 15 °C. The layers were separated and the aqueous layer was extracted with CH2Cl2 (2×40 mL). The combined organic layers were washed with saturated H2O (2×40 mL; up to pH = 6), dried over Na2SO4 and evaporated. The raw matter was purified by column chromatography [120 g silica gel; petroleum ether / AcOEt (1:3)] to give (S)-acetamide (. S’)-3 (355 mg, 80%) as a white foam. It is possible to crystallize the 6S -acetamide (. S’)-3 as follows: by operating under stirring, the 6S -acetamide (. S’)-3 (350 mg) was suspended in iPr2O (6.8 mL) at 25±5 °C for 10 min, then cooled to 5±5 °C. After 30 minutes, iPr2O was poured off and the residue was dried under vacuum at 25±3 °C until constant weight, obtaining (S)-acetamide (S)-3 (345 mg, crystallization yield 98%) as a white solid. Mp = 58-59 °C; [α]D20= -46.3 (c 0.6, CHCl3).1H NMR (CDCl3, 300 MHz): δ 6.86 (1H, d, J= 1.7 Hz; Ar), 6.83-6.75 (2H, m; Ar), 6.35 (2H, s; Ar), 5.66 (1H, br s; OH), 5.59 (1H, d, J= 8.2 Hz; NH), 4.91 (1H, q, J = 8.2 Hz; CH-1), 3.87 (3H, s; OMe), 3.82 (6H, s; OMe), 3.80 (3H, s; OMe), 2.60-2.41 (2H, m; CH2-3), 2.22-2.08 (1H, m; CH2-2), 2.07- 1.97 (1H, m; CH2-2,), 1.95 (3H, s; CH3-Ac); MS (ESI): m / z calculated for C21H27NO6+H+: 390.19; it was found: 390.29 [M+H]+.

[0133] Example 10 - Synthesis of (S)-N-(l-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propyl)acetamide ((S)-3; from by-product (S)-3a)

[0134]

[0135] By operating under stirring, the by-product (S)-O-acetyl-acetamide (S)-3a (100 mg, 0.232 mmol) was dissolved in CH2Cl2 (0.8 mL) and MeOH (3.1 mL) at 25±5 °C. Then H2O (0.15 mL) and K2CO3(128 mg, 0.926 mmol) were added at 25±5 °C. The resulting suspension was stirred at 25±5 °C and monitored by TLC [CH2Cl2 / MeOH (95:5); Rf: (S)-3a, 0.44; (S)-3, 0.26], After 1 h, the suspended solid was filtered and washed with MeOH (3×2 mL) until no product was present in the wash solvent (TLC control). The filtered solution was concentrated to 0.2 mL, then the residue was partitioned between CH2C12(10 mL) and H2O (10 mL). The aqueous layer was extracted with CH2C12(2x10 mL). The combined organic layers were dried over Na2SO4 and evaporated obtaining the (S)-acetamide (S)-3 (72 mg, 80%; for analysis see example 9). NMR spectra, MS (ESI) and the [α]D analyses were in agreement with the data reported in Example 22.

[0136] Example 11 - Synthesis of (S)-acetamide ((S)-5)

[0137]

[0138] By operating under nitrogen and stirring, the (S)-acetamide (S)-3 (130 mg, 0.341 mmol) was dissolved in anhydrous MeOH (0.70 mL) at 25±5 °C. PhI(OAc)2 (113 mg, 0.341 mmol) was then added at 25±5 °C. After 5 minutes, the methanol solution was added by dripping over 30 minutes to a stirred solution of BF3OEt2 (0.14 mL, 1.03 mmol) in anhydrous CH2Cl2 (34 mL) using a syringe pump. The solution obtained (pH = 3; the mixture turned from colourless to orange) was stirred at 25±5 °C and monitored by TLC [AcOEt; R / : (S)-3, 0.55; (S)-5, 0.43], After 20 minutes, a saturated aqueous solution of NaHCOs (20 mL; up to pH = 8) was added dropwise over 3 minutes. The layers were separated and the aqueous one was extracted with CH2 Ch (3x10 mL). The combined organic layers were washed with H2O / brine (1:1; 10 mL), dried over Na2SO4 and the solvent was evaporated to obtain the intermediate raw matter (S)-5, which was used without further purification.1H NMR (CD3OD, 300 MHz): δ 7.00 (1H, s; Ar), 6.80 (1H, s; Ar), 6.72 (1H, s; Ar), 4.58 (1H, dd, J= 12.4, 6.0 Hz; CH-7), 3.88 (3H, s; OMe), 3.87 (3H, s; OMe), 3.85 (3H, s; OMe), 3.53 (3H, s; OMe), 2.56-2.44 (1H, m; CH2-5), 2.31-2.21 (2H, m; CH2-5, CH2-6, overlapped), 2.00 (3H, s; CH3-Ac), 1.94-1.86 (1H, m; CH2-6).

[0139] Example 12 - Synthesis of (S)-acetamide ((S)-6)

[0140]

[0141] (S)-5 (S)-6

[0142] By operating under stirring, the 6S -intermediate raw matter (5)-5 (obtained from example 11) was dissolved in anhydrous MeOH (1.80 mL) at 25±5 °C. Subsequently, NaHCO3 powder (58 mg, 0.682 mmol; solution turned from dark brown to light brown) and Phi (OAc)2 (113 mg, 0.341 mmol; solution turned red) were added at 25±5 °C. The resulting solution was stirred at 25±5 °C and monitored by TLC [AcOEt; (S)-5, R 0.43; (S)-6, 0.44], After 40 min, the reaction mixture was diluted with a mixture of Et2O / AcOEt (1:1, 10 mL) and dropped into a saturated aqueous solution of Na2SO3 (10 mL). The layers were separated and the aqueous was extracted with Et2O / AcOEt (1:1, 4x10 mL). The combined organic layers were washed with H2O / brine (1:1; 10 mL), dried over Na2SO4 and the solvent was evaporated to obtain intermediate (S)-6. which was used without further purification.1H NMR (CD3Cl, 300 MHz): δ 6.49 (1H, s; Ar), 6.43 (1H, s; Ar), 6.11 (1H, d, J= 7.8 Hz; NH), 6.01 (1H, s; Ar), 4.57-4.44 (1H, m; CH-7), 3.89 (3H, s; OMe), 3.87 (3H, s; OMe), 3.76 (3H, s; OMe), 3.43 (3H, s; OMe), 3.41 (3H, s; OMe), 2.72 (1H, td, J= 13.5, 6.9 Hz; CH2-5), 2.59 (1H, dd, J= 13.8, 6.2 Hz; CH2-5), 2.18 (1H, tt, J= 13.2, 6.9 Hz; CH2-6), 1.98 (3H, s; CH3-Ac), 1.73-1.62 (1H, m; CH2-6).

[0143] Example 13 - Synthesis of (S)-acetamide ((S)-7)

[0144]

[0145] By operating under nitrogen and stirring, NaH (60% dispersed in mineral oil; 60 mg, 1.50 mmol) was slowly added to a solution of trimethyl sulphoxonium iodide (300 mg, 1.36 mmol) in anhydrous DMSO (2.8 mL), monitoring effervescence. After 10 minutes (evolution of H2 ceased), an aliquot of this solution (0.70 mL, 1 eq) was added dropwise over 5 minutes to a stirred solution of (S)-intermediate raw matter (S)-6 (obtained from Example 26) in anhydrous DMSO (0.85 mL) at 25±5 °C (turned from brown to very dark brown). The resulting solution was stirred at 25±5 °C and monitored by TLC [AcOEt; R / : (S)-6, 0.44; (S)-7, 0.32], After 1 h, the reaction mixture was diluted with a mixture of Et2O / AcOEt (1:1, 10 mL) and dripped into H2O (10 mL). The layers were separated and the aqueous was extracted with Et2O / AcOEt (1:1, 4×10 mL). The combined organic layers were washed with H2O / brine (1:1; 10 mL), dried over Na2SO4 and the solvent was evaporated to obtain raw intermediate (S)- (S)-7, which was used without further purification.

[0146] 1H NMR (CD3OD, 300 MHz): δ 6.76 (1H, s; Ar), 5.68 (1H, d, J= 1.1 Hz; Ar), 4.25 (1H, dd, J = 11.6, 6.7 Hz; CH-7), 3.86 (3H, s; OMe), 3.82 (3H, s; OMe), 3.73 (3H, s; OMe), 3.49 (3H, s; OMe), 3.26-3.20 (1H, m; CH2-5), 3.23 (3H, s; OMe), 2.79 (1H, dd, J= 13.9, 8.2 Hz; CH2-5), 2.10-2.00 (1H, m; CH2-6), 1.94 (3H, s; CH3-Ac), 1.86 (1H, td, J= 12.2, 8.6 Hz; CH2-6), 1.36 (1H, dd, J= 9.0, 4.0 Hz; cyclopropane CH2), 1.14 (1H, dd, J= 7.3, 4.0 Hz; cyclopropane CH2).

[0147] Example 14 - Synthesis of colchicine 1

[0148]

[0149]

[0150] By operating under nitrogen and stirring, the raw intermediate (S)-7 (obtained from Example 13) was dissolved in anhydrous CH2Cl2 (6.80 mL) at 25±5 °C. Then 4A molecular sieves (410 mg) were added and the resulting suspension was stirred at 25±5 °C for 15 minutes. TFA (0.26 mL, 3.41 mmol) was then added batchwise over 3 minutes at 25±5 °C. The reaction mixture was stirred at 43±3 °C (external temperature) and monitored by TLC [AcOEt; R / : (S)-7, 0.44; 1, 0.0], After 2 hours, the molecular sieves were filtered and washed with CHCl3 (2×5 mL). Then, the resulting solution was poured into a saturated aqueous solution of NaHCO3 (20 mL; until pH = 8). The layers were separated and the aqueous one was extracted with CHCl3 (4×10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and the solvent was evaporated. The raw matter was purified by column chromatography [7 g silica gel; gradient from petroleum ether / acetone (1:4) to acetone] to obtain 6S -colchicine 1 (60 mg, 52% overall yield in 4 steps from (S)-3) as a dark yellow solid.1H NMR (CD3Cl, 300 MHz): δ 8.53 (1H, d, J= 5.9 Hz; NH), 7.64 (1H; Ar), 7.33 (1H, d, J= 10.7 Hz, 1H; Ar), 6.88 (1H, d, J= 10.9 Hz; Ar), 6.51 (1H, s; Ar), 4.63 (1H, dt, J= 11.7, 5.6 Hz, CH-7), 3.99 (3H, s; OMe), 3.91 (3H, s; OMe), 3.88 (3H, s; OMe), 3.63 (3H, s; OMe), 2.57-2.42 (1H, m; CH2-5), 2.41-2.26 (2H, m; CH2-5, CH2-6, overlapped), 2.02-1.90 (1H, m; CH2-6), 1.92 (3H, s; CH3-Ac). NMR analysis was in agreement with the literature.

Claims

CLAIMS1. A process for preparing a compound of formula GS')-3NHAcOMe(S)-3which comprises:a) the separation of enantiomers by resolution with chiral amines of a compound of formula 44wherein R is a carboxy group and P is a protecting group of the hydroxy group to give a compound of formula (A)-4BCOOHOMe()-4Bb) conversion of the carboxy group to the amino group, acetylation and removal of the protecting group P.

2. Process according to claim 1 wherein the chiral amines are selected from (R)-(+)-l-phenylethylamine, (R)-(+)-l-(2-naphthyl)ethylamine, (S)-(+)-leucinol, (S)-(-)-4-methoxy-alpha-methylbenzylamine, cinchonidine, (R)-(+)-N, N-dimethyl-1-phenylethylamine, (R)-(+)-N-benzyl-1-phenylethylamine.

3. Process according to claim 2 wherein the chiral amine is S)-(-)-4-methoxy-alpha-methylbenzylamine.

4. Process according to any one of the preceding claims, wherein the resolution is carried out in a solvent selected from isopropanol, methyl ethyl ketone, tetrahydrofuran, acetonitrile and mixtures thereof.

5. Process according to any one of the preceding claims, wherein the conversion of the carboxy group to the amino group in step b) is carried out by means of a Curtius, Hoffman, Lessen or Schmidt reaction.

6. Process according to claim 5 wherein step b) is carried out by Curtius reaction.

7. Compound of formulaOMe()-4B8. Use of the compound of claim 7 for preparing colchicine.

9. Use according to claim 8 comprising sequential steps of oxidative intramolecular condensation, oxidation, cyclopropanation and ring expansion.

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