NO329701B1 - Process for the preparation of combretastanines and intermediate for the process - Google Patents

Abstract

The invention relates to novel processes for the preparation of combretastatins by Wittig condensation between nitromethoxybenzaldehyde and a trimethoxybenzylphosphonium salt or vice versa, a nitromethoxybenzylphosphonium salt with trimethoxybenzaldehyde or further with a nitro amine reaction to the same derivatives, to a Wittig reaction between the same derivatives,

Description

The present invention relates to a new method for the production of combretastatins and their derivatives. The invention also relates to an intermediate product for the method.

The term "combretastatins" or "stilbene derivatives" in connection with the present invention shall be understood to mean derivatives with the following general formula:

ser A represents an amino group in serine salt form, which comprises

where A represents an amino group in serine salt form, and its pharmaceutically acceptable salts.

Synthesis of stilbene derivatives or combretastatins, which may be in the form of a pharmaceutically acceptable salt, and pharmaceutical compositions comprising those described in US Patent Numbers 4,996,237; 5,525,632; 5,731,353 and 5,674,906. These patents describe combretastatins and their metabolites and describe their in vitro oncological activity.

According to these patents, combretastatins are produced from

(3,4,5-trimethoxybenzyl)triphenylphosphbnium salts, which are condensed with a 3-nitro- or 3-hydroxy-4-methoxybenzaldehyde (where the hydroxy group is protected) in the presence of sodium hydride or lithium derivatives, and then the obtained derivatives are reduced in the presence of zinc when this is nitrated.

The isomer with the cis configuration is further produced by exposure to light or by chromatographic separation of the mixture.

The present invention relates to new methods for the production of combretastatins or their derivatives and to improvements in existing methods.

The present invention therefore provides a method for the production of combrestatins with the following general formula (I):

where A represents an amino group, in serine salt form comprising coupling the derivatives of formula (Ha) with the cyclically protected serine derivative of formula (Ilb) where PG means a protecting group for the amine functional group, to give a new intermediate of the following general formula :

which is then deprotected,

wherein the PG group of formulas (IIb) or (III) represents a protecting group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl

(FMOC).

A process route VO 1 for the preparation of the derivatives of formula (I), where A represents an amino group, consists of, after Wittig condensation in the presence of (3,4,5-trimethoxybenzyl)triphenylphosphonium bromide or chloride and of 3-nitro-4 -methoxybenzaldehyde, to carry out the reduction in the presence of iron, instead of zinc, as used in the previous publications, which makes it possible to obtain a total reaction yield with respect to aldehyde load of 60% (yield with respect to the aldehyde load in US patent 5 525,632 are between 21% and 33%).

The first process route VO 2 consists in condensation of

3,4,5-Thymethoxybenzaldehyde with (4-methoxy-3-nitrobenzyl)triphenylphosphonium bromide or chloride. For both of these first two routes VO 1 and VO 2 , the reaction is carried out in the presence of a base selected in particular from potassium tert-butoxide, sodium tert-butoxide, sodium hydride, butyllithium, LDA (lithium diisopropylamine), sodium methoxide, potassium carbonate or alkali derivatives of hexamethyldisilanes .

The reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO and the like), alcohols, aromatic solvents or water, at a temperature adjusted by the person skilled in the art in relation to the base and solvent used .

The reaction of the first route VO 2 is particularly described in the publication by

K. G. Piney in Bioorg. With. Chem., 8 (2000), 2417-2425.

2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)-vinyl]nitrophenyl is reduced according to this method by removing iron. It is advantageous to use an amount of iron in excess, if complete conversion of the starting material is desired. This surplus is preferably greater than 2 equivalents per moles relative to the starting point of nitro derivative.

It has been shown that the same step, carried out in the presence of zinc in acetic acid and a conventional solvent for reductions with zinc, does not enable the achievement of a complete reaction (in US patent number 5,525,623 the yield of the reaction carried out with pure Z-isomer varies between 46 and 66%) and further, the amount of zinc used is greater, thus causing significant industrial loss. Furthermore, the method produces a large amount of "azo" compound, which is the result of coupling between the formed amino and the nitroso intermediate in the reduction.

Reduction with hydrogen formed by ammonium formate in the presence of a conventional catalyst, such as palladium or platinum, leads to high isomerization of the double bond to the undesired E-isomer, and to partial saturation of the double bond.

The above-mentioned Piney publication describes the reduction with sodium hydrosulphite of a pure nitro-Z isomer, obtained after chromatography and recrystallization, and leads to an amino-Z isomer in only 37% yield.

Hydrogenation with molecular hydrogen, catalyzed by platinum or palladium, is rarely complete and results in particular in saturation of the ethylene double bond.

A second process route, which avoids the intermediate reduction step necessary when starting from a nitro derivative, also exists. This is because it is much more economical to condense, according to a first method, to carry out this second method, a (3,4,5-1imethoxybenzyl)triphenylphosphonium bromide or chloride with a 3-amino-4-methoxybenzaldehyde or according to a second method for carrying out this second method, 3,4,5-trimethoxybenzaldehyde with a (3-amino-4-methoxybenzyl)triphenylphosphonium salt.

The second method according to these two alternative variants requires a step in which less CMR (carcinogenic, mutagenic or reproductive toxic) products are released, compared to the first methods V01 and V02, which is a significant advantage at the industrial level, both in terms of safety and production costs.

According to the second process route, VO 3 (3,4,5-1imiethoxybenzyl)triphenylphosphonium salt is brought and

3-amino-4-methoxybenzaldehyde together, and the reaction is advantageously carried out in the presence of a base, selected in particular from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LD A, sodium methoxide, potassium carbonate or alkali derivatives of hexamethyldisilanes. Advantageously, sodium methoxide is used.

The reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO and the like), alcohols, aromatic solvents or water, at a temperature that can be adjusted by the person skilled in the art in relation to the base used and solvent.

The reaction temperature will be adjusted by the specialist in the technique in relation to the base used. When methoxide is used, it is preferred that the temperature is between 0 °C and 10 °C. After the reaction, the base used is neutralized with acid in aqueous solution, the organic phase is washed and concentrated, and the expected product is obtained after chromatography of the impure concentrate.

According to the second process route VO 4 where (3-amino-4-methoxybenzyl)triphenylphosphonium salt and 3,4,5-trimethoxybenzaldehyde are brought together, the reaction is preferably carried out in the presence of an organic base, selected in particular from potassium tert-butoxide, sodium tert-pentoxide , sodium hydride, butyllithium, LD A, sodium methoxide, potassium urn carbonate or alkali derivatives of hexamethyldisilanes. Advantageously, sodium methoxide is used.

This reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO and the like), alcohols, aromatic solvents or water, at a temperature adjusted by the person skilled in the art in relation to the base used and solvent.

The reaction temperature will be adjusted by the specialist in the technique in relation to the base used. When methoxide is used, the temperature is preferably between 0 °C and 10 °C. After the reaction, the base used is neutralized with acid in aqueous solution, the organic phase is washed and concentrated, and the expected product is obtained after chromatography of the impure concentrate.

The derivative obtained according to the second process, route VO 3 or VO 4, or during the second step of the first process, route VO 1 or VO 2, has the following formula:

When serine is coupled with the compound of formula (Ila), it is advantageous to use L-serine doubly protected on the nitrogen of the serine, and on the functional hydroxyl group of the general formula (Ilb) where PG represents a protecting group for the functional amine group, known to those skilled in the art in the art, to give a new intermediate with the following formula:

which is then preferably cleaved simultaneously with opening of the ring by acid hydrolysis, according to a deprotection reaction known to those skilled in the art. The PG group in formula (IIb) or (HI) represents a protecting group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC).

The compound with formula (111) above is new and is stated in the claims as such.

The present invention therefore further provides an intermediate for the coupling between aminocombrestatin and the cyclically protected serine derivative, characterized in that it corresponds to the following formula:

wherein PG is a protecting group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl

(FMOC).

The condensation is preferably carried out in the presence of EDC1

(1-(3-dimethylaniinopropyl)- 3-ethylcarbodiimide chloride) or in the presence of DCC (dicyclohexylcarbodiimide) and of HOBT (hydroxybenzotriazole) or in the presence of DCC (dicyclohexylcarbodiimide) and of HOSU (N-hydroxysuccinide) or in the presence of TOTU (0 -[(ethoxycarbonyl)cyanoethyleneamino]- N3N3N',N'-tetramethyluronium tetrafluoroborate) or of HBTU (O-benzotriazol-1 -yl-NjNjN^N^tetramethyluronium hexafluorophosphate) or of N,N-carbonyldiimidazole. The reaction is preferably carried out in a solvent which is inert with respect to the reaction, the solvent being selected in particular from polar aprotic solvents, such as acetonitrile, dimethylformamide, tetrahydrofuran or chlorinated, aliphatic solvents, such as dichloromethane or esters.

The coupling of the derivative of formula (Ila) can also be carried out by the action of a mixed anhydride, synthesized in situ between a chloroformate or a carboxylic acid chloride, for example pivaloyl chloride, and doubly protected L-serine of formula (Ilb), in the presence of a tertiary base of The NMM (N-methynorpholine) type in various solvents which are inert with respect to the reaction, esters, ethers, chlorinated solvents, acetonitrile and the like. The mixed anhydride is preferably prepared at a temperature of between 0 °C and 10 °C, and the reaction is then carried out at room temperature. After the reaction, the reaction mixture is hydrolyzed with an aqueous solution, the mixture is separated by standing and the organic phase obtained is washed with a hydroxylated base.

The double deprotection of the compound of formula (III) is carried out by the action of an organic or inorganic acid. Advantageously, a concentrated, aqueous hydrochloric acid is used in an alcoholic medium. The reaction temperature according to the best embodiment is between 50 °C and 70 °C.

The invention will be described in greater detail by means of the following examples.

The composition of the mixtures, the monitoring and progression of the reactions, and the yield of the unisolated products/intermediates and their analyses, are determined by HPLC (high performance liquid chromatography) analyses.

EXAMPLE 1 - First procedure, route VO 2

(Z)-N-[2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]-phenyl]-L-serinamide hydrochloride General synthesis scheme

The new "reverse Wittig" method, starting from

(4-Methoxy-3-nitrobenzyl)Mphenylphosphonium bromide and 3,4,5-trimethoxybenzaldehyde, makes it possible to obtain the mixture of Z and E isomers of

2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]-nitrophenyl with a Z/E ratio of 75/25.

This ratio is sufficiently high in the Z-nitroisomer to be able to use the ZÆ mixture directly in the reduction, and to obtain the Z-aminoisomer with an HPLC analysis of 97% IS (international standardization) upon crystallization of the hydrochloride.

(4-methoxy-3-mtrobenzyl) (4) is prepared according to the following example:

3-nitro-4-methoxybenzyl alcohol (2):

To a 2-liter, three-necked flask, equipped with a mechanical stirrer, thermometer, et

T-piece, a solids addition funnel and a reflux condenser with bubble counter, 90.5 g of 3-nitro-4-methoxybenzaldehyde (1) is added, followed by 450 ml of THF and 90 ml of ethanol. The pale yellow solution obtained is cooled to 10 °C and then 10 g of sodium borohydride are added over 40 minutes at 10/15 °C (the reaction is highly exothermic and the temperature must be maintained in an ice/acetone bath); at the end of the addition, the brown solution changes color to navy blue. The solution is stirred for 30 minutes at 10 °C, while completion of the reaction is monitored by TLC (thin layer chromatography), the solution is further stirred for one hour at 10 °C, and then the temperature is adjusted to room temperature.

The addition funnel is replaced by a 500 ml tryldoid equilibration dropping funnel, through which 300 ml of distilled water is added dropwise over 30 minutes, while maintaining the mixture at 20 °C. Gas evolution is observed when the water is added.

The mixture is concentrated to 2/3 on a rotary evaporator (50 °C/20 mm Hg), and a white product crystallizes in the aqueous concentrate in the form of lumps.

The cooled, aqueous phase is extracted with 250 ml and then 150 ml of dichloromethane, and the combined organic phases are washed with 250 ml of distilled water and dried over magnesium sulfate.

After filtration, the dichloromethylene solution is used as is in the following bromination reaction.

The yield from this step is considered to be 100%.

N.B.: The alcohol (2) is commercially available, but very difficult to obtain.

3-nitro-4-methoxybromobenzyl (3):

To a 1-liter, three-necked flask, equipped with a mechanical stirrer, thermometer, et

T-piece, a dropping funnel and a reflux condenser with a bubble counter, the dichloromethyl solution of 3-nitro-4-methoxybenzyl alcohol (2) and 100 ml of dichloromethane are added. The stirred solution is cooled to 5 °C and then 135.4 g of phosphorus tribromide is added dropwise, while the temperature is kept at 5 °C.

The solution is stirred at 5 °C for 90 minutes, while completion of the reaction is monitored by TLC, and then 250 ml of saturated sodium bicarbonate solution are added dropwise, while the temperature is kept at 15 °C. Very strong gas evolution takes place with a slight delay when the phosphorus tribromide is added.

The medium which was separated by standing in a separatory funnel is washed successively with 250 ml of distilled water and 200 ml of saturated sodium hydrogen carbonate solution. The organic phase is dried over magnesium sulphate, filtered and concentrated on a rotary evaporator (50°C/20mmHg).

119 g of solid material in the form of greenish-yellow felt-like needles is obtained with a chemical yield over two steps of 97%.

N.B.: This product (3) can also be prepared according to the following scheme, described in the publication K. G. Piney et al., Bioorg. With. Chem., 8 (2000), 2417-2425.

(3-nitro-4-methoxybenzyl)triphenylphosphonium bromide (4):

To a 2-liter, three-necked flask, equipped with a mechanical stirrer, thermometer, et

T-piece, a funnel for adding solids, and a reflux condenser with bubble counter, containing a stirred stock solution of 1000 ml of toluene, 119 g of 3-nitro-4-methoxybromobenzyl (3) are added, the mixture is heated to 25 °C and the solution is added . 126.5 g of triphenylphosphine is then added, and the solution obtained is gradually heated to 60 °C, and a precipitate forms from 30 °C. The mixture is kept at 60/65 °C for 4 hours, and then cooled to 30 °C and filtered through a sintered glass filter. The filter residue is washed on the filter with 2 x 300 ml of toluene, the liquid is squeezed out and the residue is dried in an oven (35 °C/20 mm Hg/20 hours).

217 g of (4-methoxy-3-nirobenzyl)triphenylphosphonium bromide are obtained with a chemical yield of 88%.

The synthesis described in the publication: (Solvent used: dichloromethane)

K.G. Piney et al., Bioorg. With. Chem., 8 (2000), 2417-2425.

Spectrum No. = 4,865-V

<!>H NMR spectrum (300 MHz, d6-(CD3)2SO, 5 in ppm): 3.90 (s, 3H), 5.26 (d, J= 15 Hz, 2H), 7.33 (mt , 2H), 7.41 (mt, 1H), from 7.65 to 8.05 (mt, 15H).

Mass spectrum No. 212217, m = 428

EI m/z = 262 [PPh3]<+> base peak

DCI m/z = 445MNH3<+>

m/z = 428 M+

m/z = 263 [PPh3H]<+> base peak

IR spectrum 426469 KBr

2869, 2843,2776,1619,1527,1438, 1362, 1287,1270,1111,752,692 and 502 cm-<1 >Z and E mixture of 2-methoxy-5-[2-(3,4,5 -trimethoxyphenyl)vinyl]nitrophenyl (6) and (7):

To a 2-liter, three-necked flask, equipped with a mechanical stirrer, thermometer,

T-piece, a dropping funnel and a reflux condenser with bubble counter, add 54.7 g of 3,4,5-trimethoxybromaldehyde (5), 148.6 g of (4-methoxy-3-nitrobenzyl)triphenylphosphonium bromide (4) and 1300 ml toluene at 20 °C under nitrogen. The stirred suspension is cooled to 5°C using an ice bath, and then 63.2 g of a 25% by weight solution of sodium methoxide in methanol is added at 5°C over 40 minutes.

While this addition is in progress, the suspension changes appearance from off-white to yellow, and then to brown.

The mixture is stirred for 1 hour at 5 °C, and the completion of the reaction is monitored by HPLC (complete consumption of the aldehyde). 3 g (0.05 mol) of acetic acid are then added.

The suspension is heated to, and held at, 40 °C for 30 minutes. At this temperature, only the salts remain insoluble. The mixture is filtered at 40 °C through a sintered glass filter No. 3, and the salts are washed on the filter with 3 x 100 ml of toluene.

The filtrate is transferred to a round-bottomed flask with 250 ml of distilled water, and the two-phase mixture is stirred for 20 minutes at 40 °C, and then separated by standing in a separatory funnel. The toluene phase is washed again with 2 x 250 ml of distilled water and then concentrated to dryness on a rotary evaporator.

The residue is taken up in 600 ml of isopropanol and 12 ml of toluene at 40 °C, the expected product begins to crystallize and the temperature returns to room temperature overnight with slow stirring.

The stirred suspension is cooled to, and maintained at, 5 °C for 1 hour, and then filtered through a sintered glass filter, and the filter cake is washed with 2 x 125 ml of isopropanol, the liquid is squeezed out, and the filter cake is dried in an oven under vacuum (35 ° C/30 mm Hg/18 hours).

A mixture of 91.7 g of Z and E isomers (6) and (7) is obtained with a Z/E ratio of 75/25 (IS HPLC), and a yield of 95%.

Synthesis described in publication: (solvent used: dichloromethane: base used: NaH) K. G. Piney et al., Bioorg. With. Chem., 8 (2000), 2417-2425.

N.B.: Experiments were carried out with several experimental treatments, such as:

Solvents: THF, acetonitrile, methanol and other alcohols, dichloromethane, NMP, DMF, DMSO, and the like.

Bases: potassium t-butoxide, sodium t-pentoxide, sodium hydroxide, NaH, BuLi/LDA, potassium carbonate, and the like.

Temperatures: from -10 °C to reflux temperatures of some solvents Z-2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]-phenylamine hydrochloride (8):

To a 2-Uters, three-necked flask, equipped with mechanical stirrer, thermometer, et

T-piece, a solids addition funnel, and a reflux condenser with a bubble counter and a heating bath, 80 g of 75/25 Z and E mixture of 2-methoxy-[2-(3,4,5-Mmethoxyphenyl) was added )vinyl]nitrophenyl (6) and (7), 640 ml of absolute ethanol and 160 ml of distilled water at 20 °C and under nitrogen.

The mixture is stirred rapidly and heated in an oil bath, and 7.8 ml of 6N hydrochloric acid is added to the suspension at 50 °C, and then the temperature of the mixture is increased to 77 °C ± 2 °C. The mixture is practically soluble. 52 g of iron powder is added in portions over the course of 5 minutes. With the first addition, the mixture is passed into solution and then an almost black precipitate forms on the walls of the round-bottomed flask.

The mixture is kept at 77 °C ± 2 °C for 2 hours and the disappearance of the starting nitro compounds (6) and (7) is monitored by HPLC.

The mixture is cooled to 40 °C and filtered through a sintered glass filter, covered with "Clarcel" and the cake is washed with 2 x 160 ml of 80/20 ethanol/water mixture.

The filtrate, the aqueous mother solution and the aqueous washing solution are concentrated on a rotary evaporator. As soon as the azeotrope is driven off, an oil separates in the remaining water phase.

This aqueous phase is extracted in a separatory funnel with 2 x 300 ml of dichloromethane and then the organic phase is washed with 2 x 300 ml of half-saturated aqueous sodium chloride solution and with 300 ml of distilled water.

The organic phase is concentrated to dryness on a rotary evaporator and 76 g of an oil is obtained, the oil exhibiting a Z/E ratio of 80/20 by HPLC. This oil is dissolved in 591 ml of ethanol and transferred with stirring to a 1 liter round bottom flask, 100 ml of 2.32 N methanolic hydrochloric acid is added, then precipitation is initiated and the mixture is allowed to precipitate overnight with stirring.

The amount of methanol plus methanolic hydrochloric acid is such that the final concentration of Z isomers (determined by HPLC) is equal to 8.8% w/v.

The next morning the mixture is filtered through a sintered glass filter, the dry cake weighs 8.2 g, and consists only of the E-isomer (HPLC).

The filtrate (693 g), ratio Z/E = 86/14 (IS HPLC), is concentrated to half on a rotary evaporator, 400 ml of acetonitrile is added to 347 g of concentrate, and the mixture is concentrated again until a concentrate of 347 g is again obtained. 1000 ml of acetonitrile is then added, and the mixture is concentrated until crystallization starts. The concentrate is then transferred to a stirred 4-litre round-bottom flask, containing 1500 ml of acetonitrile at 60 °C. The mixture precipitates heavily.

The mixture is stirred at 60 °C for 2.5 hours and cooled to 30 °C during approx. 1 hour. The curd is filtered through a sintered glass filter (the E-isomer (9) is soluble in the filtrate). The cake is washed with 2 x 200 ml of acetonitrile and dried in an oven (35 °C/30 mm Hg/18 hours).

45.7 g of Z-2-methoxy-5-[2-3,4,5-trimethoxyphenyl)vmyl]phenylamine (8) is obtained with an IS HPLC analysis of 97% and a yield as such of 56%, i.e. .a yield of the Z-isomer with respect to the added Z-isomer of 72%.

EXAMPLE 2 - Synthesis according to the second procedure, route V0 3.

The advantage of the second procedure, route V0 3 compared to the first reverse Wittig procedure route V0 2 is that the Wittig reaction is carried out between a product that has already been reduced, the aminoaldehyde (1a) and phosphonium (2a), thus eliminating a chemical steps emitting CMR products.

(Z)-N-[2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenyl]-L-serinamide hydrochloride

General synthesis scheme 3-amino-4-methoxybenzaldehyde (la):

To a 2-liter, three-necked flask, inert with argon, and equipped with a mechanical stirrer, thermometer, T-piece, solids addition funnel, reflux condenser with bubble counter, and a heating bath, is added 20 g of 3-nitro-4-methoxybenzaldehyde (1) and 350 ml of absolute ethanol, and the mixture is stirred and heated to 60 °C. The mixture is added to the solution.

The dropwise deposition is carried out at 60 °C with 115 ml of distilled water, followed by 14 ml of 2N hydrochloric acid. 24.7 g of iron powder is then added in portions.

The temperature of the mixture is returned to ambient temperature within 2 hours. The reaction is complete (TLC).

The mixture is filtered through celite and concentrated under vacuum, the residue is then taken up in dichloromethane and the organic solution is washed twice with distilled water and then dried over magnesium sulfate, filtered and concentrated to dryness under vacuum. 16 g of raw material (1a) is obtained and chromatographed on a silica column eluted with dichloromethane.

Two fractions comprising the expected pure product are obtained, and after concentration the fractions give 11.5 g pure (Ia), i.e. a yield of 69%.

<*>H NMR spectrum No. 2810-V (300 MHz, d6-(CD3)2SO, 8 in ppm): ;3.88 (s, 3H), 5.11 (unresolved peak, 2H), 7.01 (d, J= 8 Hz, 1H), 7.14 (d, J= 2 Hz, 1H), 7.18 (d, J= 8 Hz, 1H), 9.53 (s, 1H). ;Mass spectrum no. 210112, m = 151 ;EI m/z =151 M<4>" base peak ;m/z=136[M-CH3]<+>;m/z =108 [136-CO]+ m/ z= 80[108-CO]<+>;IR spectrum: 425135 KBr ;3464, 3437, 3367,3349, 1675,1655,1582,1513, 1293, 1241, 1139, 1023, 803 and 640 cm"<1> ;Z- and E-2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)v^^ (8') and (9'): ;N.B.: The phosphonium (2a) is a starting material that is already described in the original patent Ajinomoto Co., Ltd., US 5,525,632 and WO 01/12579 A2. ;To a 250 mL, three-necked flask inert with nitrogen and equipped with a magnetic stirrer and thermometer, a T-piece, a dropping funnel, a reflux condenser with bubble counter, and a cooling bath, is added 8.0 g of phosphonium salt (2a), followed by 2.2 g of aminobenzaldehyde (1a) and 100 ml of toluene. The stirred suspension is cooled to 5°C and 3.51 ml of a 25% w/w methanolic red trinium methoxide solution is added over 15 minutes. After 2.5 hours at 5 DC, the reaction is still incomplete (DC: 45%), but does not change further (HPLC) and the Z/E ratio is 61/39. 0.2 ml of acetic acid diluted in 50 ml of water is then added, the temperature is increased to 13 °C, the mixture is stirred for 30 minutes, and then separated by standing in a separatory funnel. The organic phase is concentrated under vacuum on a rotary evaporator, and 8 g of yellow oil is obtained. ;By HPLC this oil comprises starting aldehyde, phosphine oxide and the expected Z/E mixture with a ratio of 61/39. The oil is chromatographed on a silica column (40 parts w/w) eluted with a cyclohexane/ethyl acetate/xyethylamine (50/50/2) mixture. ;Two series of combined fractions are concentrated to dryness: The first dry extract of 360 mg comprises 93% of the Z-isomer plus unidentified impurities; the other of 2.09 g, comprises starting aldehyde and a Z/E mixture representing 39% and 37.5% respectively by IS HPLC. ;The weight balance of the Z-isomer (8'), determined by IS HPLC, is 1.15 g in relation to 2.20 g of added aldehyde, i.e. a yield of 24%. ;EXAMPLE 3 - Synthesis according to the second method, route VO 3. ;As with route VO 2, the advantage of route VO 3 over the first "reverse Wittig" method, route VO 2, is that the Wittig reaction is carried out between a product which has already been reduced, (3-ammo-4-methoxybenzyl)triphenylphosphonium bromide (1b), and 3,4,5-trimethoxybenzaldehyde (5), thus eliminating a chemical step that emits CMR products. ;(Z)-N-[2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vmyl]phenyl]-I^series General synthesis scheme ;;(4 methoxy-3-aminobenzyl)trife^ (lb ) ;;To a 1-liter, three-necked flask equipped with a mechanical stirrer, a thermometer, a ;T-piece, a solid settling funnel, a reflux condenser with a bubble counter and a heating bath, add 30 g (4), 240 ml of ethanol and 60 ml of distilled water. ;1.76 ml of 6N hydrochloric acid is added to the stirred suspension and heated to 70 °C. ; 9.9 g of iron powder is then added in portions over the course of 15 minutes; the mixture remains insoluble. The mixture is kept at 75 °C for 2 hours; the organic material is slowly passed into the solution, while a brownish precipitate of iron and iron oxide is formed. ;After monitoring with HPLC, 5% of the starting material still remains; 2 eggs are added again and the heating is continued for one hour; DC is complete. ;The mixture is cooled to 40 °C and filtered through "Clarcel", the filter residue is washed with 100 ml of ethanol containing 20% water and the filtrate is concentrated to dryness under vacuum on a rotary evaporator. ;The residue is taken up in 300 ml of isopropanol and crystallized from the mixture, which is stirred and heated to 50 °C and returned to the solution. 14 ml of 5N hydrochloric acid solution in isopropanol is then added and the mixture precipitates while being held at 50°C for one hour, then cooled to ambient temperature. The gruel is filtered through a sintered glass filter, the filter cake is washed with 50 ml of isopropanol, the liquid is squeezed out, and the filter cake is dried in a vacuum oven. ;Yield of 89.9% is obtained from 27.3 g of (lb). ;<*>H NMR spectrum No. 4584-V (300 MHz, dV(CD3)2SO, 8 in ppm):

3.78 (s, 3H), 5.03 (broad d, /= 15 Hz, 2H), 6.43 (unresolved peak, 1H), 6.62 (broad s, 1H), 6.82 (broad d , J= 8 Hz, 1H), from 7.60 to 8.00 (mt, 15H).

Mass spectrum No. 211915, m = 397

EI m/z = 397 M+

rn/z = 382 [M-CH3]+

m/z = 262 CPPh3]+ base peak

DCI m/z = 398 MN!!/

m/z = 263 [PPh3H]<+>base peak

IR spectrum 426386 KBr

3254,2474,1920,1628,1520,1439, 1433, 1279, 1110, 736, 690, 527 and 511 cm"<1 >Z- and E-2-methoxy-5-[2-(3,4,5 -trimethoxyphenyl)vinyl]phenylanm (8') and (9'):

To a 250 mL, three-necked flask, inert with nitrogen and equipped with a magnetic stirrer, thermometer, a T-piece, dropping funnel, a reflux condenser with bubble counter, and a cooling bath, is added 11.02 g of (lb), 4 g of (5 ), and 70 ml of toluene.

The stirred suspension is cooled to 5°C and 4.92 ml of a 25% w/w solution of sodium methoxide in methanol is added over 15 minutes. The suspension is stirred at 5 °C for 2.5 hours, then 0.2 ml of acetic acid diluted in 50 ml of water is added, the temperature is raised to 14 °C, and the mixture becomes very thick, and diluted with 10 ml of toluene and 10 ml of water. A brown insoluble material is formed.

The mixture is filtered through "Clarcel", the cake is washed 3 times with 50 ml of toluene (the washing water practically only includes the starting aldehyde, and the two-phase filtrate is not added), the clear filtrate (pH 12) is separated by standing in a separatory funnel, and the organic phase is concentrated to dryness under vacuum at 40 °C. The Z/E ratio, determined by IS HPLC is 43/57.

The brown oil obtained (4 g) is chromatographed on a silica column (100 parts w/w) eluted with a cyclohexane/ethyl acetate/tirethylamine (50/50/2) mixture.

Two series of combined fractions are concentrated to dryness: the first dry extract of 1.1 g comprises 14% E-isomer and 59% Z-isomer; the other weighs 1.08 g and comprises 86% E-isomer and 7% Z-isomer.

The weight balance of Z-isomer (8'), determined by IS HPLC, is 0.725 g in relation to 4 g of aldehyde added, i.e. a yield of 11.3%.

Z-4-{2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenylcarbamoyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (11):

Release of the base (8') from the hydrogen chloride (H):

To a 1-liter Erlenmeyer flask, add 44 g of (8), 16 g of sodium bicarbonate and then 200 ml of distilled water and 375 ml of dichloromethane. The mixture is stirred for 20 minutes at room temperature and two clear phases are obtained.

The organic phase is separated on standing, dried over sodium sulphate and filtered. About 400 ml of dichloromethane solution comprising (8') is obtained.

Preparation of 2, 2- QMethyloxazolidine- 3, 4- dicarboxylic acid- 3- tert-- butyl ester ( 101 Although this product is commercially available, it is very difficult to obtain. It has therefore been prepared by cracking with lithium hydroxide from its methyl ester according to J. Org. Chem., 63 (12), p. 3983 (1998).

<J>H NMR spectrum (300 MHz, d6-(CD3)2SO, 8 in ppm):

1.38 (s, 3H), 1.45 (s, 9H), 1.55 (s, 3H), 3.95 (mt, 1H), 4.16 (mt, 1H), 4.31 (mt , 1H), from 12.50 to 13.10 (broad unresolved peak, 1H).

Mass spectrum No. 213135, m = 245

DCI rn/z = 263 MNH4+

m/z = 246 MH +

m/z = 207 [MNH4-t-Bu]<+>base peak

m/z=146 [MH-BOC]<+>

IR spectrum: 426759 KBr

1744, 1704, 1638,1407,1368,1164, 1104, 856, 836 and 623 cm'<1>

Connection:

The solution of (8') is added to a 2-liter, three-necked flask equipped with a mechanical stirrer, a thermometer, a T-piece, a solid settling funnel, a reflux condenser with a bubble counter, and an ice bath, 600 mL of dichloromethane is added, and the mixture is cooled at stirring. 42.9 g of 2,2-dimethyloxazolidine-3,4-dicarboxylic acid-3-tert-butyl ester (10) is added at 5 °C and added to the solution, and then 48 g of 1-(3-dimethylaminopropyl)-3- ethylcarbodiirnide hydrochloride (EDC1) between 5 °C and 10 °C.

The mixture is allowed to slowly return to room temperature by melting the ice bath overnight.

The next morning, 330 ml of distilled water is added and the mixture is stirred vigorously. The mixture becomes cloudy within 30 minutes (hydrolysis of EDC1). Stirring is continued for another 30 minutes.

The mixture is separated by standing in a separatory funnel, and the organic phase is washed successively with 2 x 280 ml of 0.55 N sodium hydroxide solution, and then with 300 ml of distilled water.

The organic phase is concentrated to dryness on a rotary evaporator (50 °C/50 mm Hg).

79.4 g of a sticky oil (11) is obtained, the oil solidifies at 20 °C with a yield by weight in relation to added (8) of 117%.

Spectrum No. = 5 578-V

<J>H NMR spectrum (400 MHz, d6-(CD3)2SO, at a temperature of 373 K, 8 in ppm):

1.41 (s, 9H), 1.53 (s, 3H), 1.64 (s, 3H), 3.64 (s, 6H), 3.71 (s, 3H), 3.86 (s , 3H), 3.99 (dd, J = 9 and 3 Hz, 1H), 4.19 (dd, J = 9 and 7 Hz, 1H), 4.52 (dd, J = 7 and 3 Hz, 1H ), 6.48

(d, J = 12.5 Hz, 1H), 6.55 (d, J = 12.5 Hz, 1H), 6.58 (s, 2H), 7.02 (mt, 2H),

8.13 (broad s, 1H), 8.82 (broad s, 1H).

Mass spectrum No. 213565, m = 542

DCI m/z = 560 MNH4+base peak

m/z = 543 MH +

m/z = 504 [MNH4-t-Bu]<+>

m/z = 443 [MH-BOC] +

IR spectrum: 425857 CCL»

3409,2982,2938,2837,1712, 1698, 1534,1363, 1249, 1133,1092 and 851 cm'<1>

Other coupling conditions were used, such as:

- Mixed anhydride (pivaloyl chloride/(10))

- DCC/HOBT, DCC/HOSU, TOTU, N,N-carbonyldiimidazole, and similar

-1 acetonitrile, DMF, THF, dichloromethane, ester and the like.

EDC1»HC1 in dichloromethane gave the best result.

(Z)-N-[2-methoxy<y>-5-[2-(3,4,5-thyrmethoxy<y>phen<y>l)vin<y>I]phen<y>l]-I ^serinamide hydrochloride

To a 1-liter, three-necked flask, equipped with a mechanical stirrer, thermometer, a T-piece, reflux condenser with bubble counter, and a heating bath, is added 61.8 g of (11) dissolved in 54 mL of methanol at 20 °C, and 150 ml of isopropyl acetate, 99 ml of a 2.3 N hydrochloric acid solution in methanol, and 8.2 ml of distilled water. The mixture is stirred and heated to 60 °C for 3 hours. The solution is cooled to 40 °C, clarified by filtration through a sintered glass filter No. 4 and washed with 40 ml of methanol. The filtrate is returned to the three-necked flask with stirring, and 194 ml of isopropyl acetate is added, and the mixture is again heated to 40 °C, to the solution is added 0.2 g of (12), and then 194 ml of isopropyl acetate dropwise over one hour. The mixture crystallizes slowly as isopropyl acetate is added. The mixture is allowed to reach room temperature, then cooled and then kept at 5°C overnight.

The next morning, the slurry is filtered through a sintered glass filter and the cake pressed dry is washed with 4 x 50 ml isopropyl acetate, pressed thoroughly to remove the liquid, and then dried in an oven to constant weight (35 °C/10 mm Hg). 28 g of (12) is obtained with a yield over 2 steps (coupling, and then deprotection operations) of 56% and an IS HPLC analysis > 98%, i.e. a total yield for the synthesis carried out according to the first procedure, route VO 2 of 30% [(12) obtained in relation to (5) added].

Claims (5)

1. Process for the preparation of combretastatins of the following general formula (I): where A represents an amino group, in serine salt form, characterized by that it comprises coupling the derivatives of formula (Ila) with the cyclically protected serine derivative of formula (IIb) where PG means a protecting group for the amine functional group, to give a new intermediate of the following general formula: which is then deprotected, wherein the PG group of formulas (IIb) or (III) represents a protecting group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC). 2. Method according to claim 1, characterized in that the coupling is carried out in the presence of EDC1 (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride); of DCC (dicyclohexylcarbodiimide) and of HOBT (hydroxybenzotriazole); of DCC (dicyldohexylcarbodiimide) and of HOSU (N-hydrolcysuccinimide); of TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate); of HBTU (O-benzotriazol-1-yl-N,N,NSN'-tetramethyluronium hexafluorophosphate) or of N,N-carbonylm'imidazole. 3. Process according to any one of claims 1-2, characterized in that the protection of the intermediate compound of formula (III) is carried out by the action of an organic or inorganic acid. 4. Method according to claim 3, characterized in that the deprotection is carried out by the action of concentrated aqueous hydrochloric acid in an alcoholic medium. 5. Intermediate product for the coupling between aminocombretastatin and the cyclically protected serine derivative, characterized in that it corresponds to the following formula: wherein PG is a protecting group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC).