EP2252572A1

EP2252572A1 - Method for preparing combretastatin

Info

Publication number: EP2252572A1
Application number: EP09725421A
Authority: EP
Inventors: Marc Frederic; Sylviane Lutz; Joël MALPART; Philippe Masson; Stéphane Mutti
Original assignee: Sanofi Aventis France
Current assignee: Sanofi SA
Priority date: 2008-02-28
Filing date: 2009-02-27
Publication date: 2010-11-24
Also published as: DOP2010000250A; ZA201006137B; EP2562154A1; WO2009118474A1; IL225776A0; BRPI0907999A2; HN2010001700A; CA2716541A1; CL2012001744A1; AR072772A1; CR11635A; FR2928148B1; JP2011513286A; AU2009229027A1; PE20091559A1; CO6241150A2; EA201300249A1; UY31681A1; CL2009000463A1; TW200948753A

Abstract

The invention relates to a method for preparing a combretastatin (A): formula (I) in the form of a base or of an addition salt with an acid, which comprises coupling, in the presence of a base and of T3P, the salt of the (Z)-amino compound of formula (II) with a doubly protected L-serine derivative of formula (III) in which PG denotes a group protecting the amine function, so as to obtain the compound of formula (Z)-(Ib): formula (IV), then deprotecting and opening the ring of (Z)-(Ib) in the presence of an acid, so as to obtain the combretastatin (A) in the form of a salt; and, optionally, adding a base, so as to obtain the combretastatin (A) in the form of a base, the salt of the (Z)-amino compound having been obtained by enrichment of the salt of the amino compound of formula (V) in (Z) isomer.

Description

The present application relates to a process for preparing a combretastatin (A):

as a base or an addition salt with an acid.

[Prior art]

US 6759555 discloses a process for the preparation of a combretastatin of formula

wherein X is -NH ₂ or one of two groups:

PG designating a protective group of the amine function.

Bioorg. Med. Chem. 2000, δ, 2417-2425 and US 2003/0220404 also describe processes for the preparation of combretastatins.

J. Pept. Res. 1999, 54 (1), 54-65 compares acid activators in the formation of peptide bonds and concludes that the TDBTU is the best.

Bioorg. Med. Chem. 2006, 14, 3231-3244 describes the preparation of combretastatin of formula (A) with a coupling using DCC, HOBt-H ₂ O as the acid activator (step e, compound 10).

Chem. Common. 1999, 1847-1848 describes T3P in the preparation of amide bonds; however, T3P is described as leading to more epimerization and lower yields than HAPyU (see Table 3). In these documents, the method forming the subject of claim 1 is not described or suggested. Similarly, the enrichment of the salt of the amino compound with benzyl alcohol and acetonitrile as well as coupling with T3P is not described.

[The technical problem]

Combretastatins or stilbene derivatives exhibit high cytotoxic activity and are therefore useful as anticancer agents. However, it is the (Z) isomers that have the highest cytotoxic activity. These compounds are in particular described in US 5731353, US 5561122 or US 6759555. The Applicant has improved the process for the preparation of combretastine (A).

[Brief description of the invention]

The invention relates to a process for preparing a combretastatin (A):

in the form of a base or an addition salt with an acid, consisting of coupling in the presence of a base and of T3P of formula (III):

the compound (Z) -amino the salt of the compound (Z) -

, B ^" designating a counter-anion, with a derivative of L-

doubly protected serine of formula (II) in which PG designates a group protecting the amine function so as to obtain the compound of formula (Z) - (Ib):

then to deprotect and open the cycle of (Z) - (Ib) in the presence of an acid so as to obtain combretastatin (A) in the form of salt (-NH ₃ ⁺ ) and optionally to add a base so as to obtain combretastatin (A) in base form (-NH ₂ ),

the salt of the compound (Z) -amino having been obtained by an enrichment of the salt of the amino compound of formula:

in isomer (Z), B ^" denoting a counteranion, consisting of:

• adding benzyl alcohol to a suspension of a mixture of the salts of the compounds (Z) - and (E) -amino in acetonitrile then

• to mechanically separate the salt of the amino compound enriched in isomer (Z).

The invention also relates to the enrichment of the salt of the amino compound of formula

in isomer (Z), B ^' denoting a counteranion, consisting of:

Adding benzyl alcohol to a suspension of a mixture of the salts of the (Z) - and (E) -amino compounds in acetonitrile and mechanically separating the salt of the amino compound enriched in isomer (Z).

The invention also relates to the use of T3P of formula (III)

to perform in the presence of a base the coupling between the compound (Z) -amino or the salt of the compound (Z) -anπino with the derivative of L-serine doubly protected. The proportion acetonitrile / salts of the compounds (Z) - and (E) -amino expressed by weight is between 5 and 17, preferably between 10 and 12. The temperature at which the enrichment is conducted is preferably between 20 and 7O ⁰ C. The proportion benzyl alcohol / salts of the compounds (Z) - and (E) -amino expressed by weight is between 1 and 4, preferably between 2 and 3.

[Detailed description of the invention]

Scheme 1 below describes the reaction steps (i) to (iv) of a process for the preparation of combretastatin (A):

1 step (i): Wittig reaction between nitromethoxybenzaldehyde and trimethoxybenzylphosphonium bromide or chloride in the presence of a base leading to the salt of the nitro compound, which is in the form of a mixture of the two isomers (Z) and (E) ) 2-methoxy-5- [2- (3,4,5-trimethoxyphenyl) vinyl] nitrobenzene ((Z) - and (E) -nitro);

step (ii): reduction of the mixture leading to a mixture of compounds (Z) - and (E) -amino which are converted into salts (B ^' denotes a counteranion, for example CI ^" or SO ₄ ^2" ), followed by a step of separating the compound (Z) -amino;

step (iii): coupling of the salt of the compound (Z) -amino with a doubly protected L-serine derivative on the -OH and amino functions (compound of formula (H)) leading to the compound (Z) - (Ib) . PG denotes a protective group for the amine function: it may be, for example, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC);

Steps (iv) and (v): deprotection and opening of the cycle leading to combretastatin (A) in salified form or not.

Step (i) is described in US 5731353 as well as in Bioorg. Med. Chβm. 2000, 8, 2417-2425 (Scheme 1). The reaction is carried out in an organic solvent such as for example an aromatic solvent (eg toluene) in the presence of a base, preferably strong such as MeONa or NaH. The ratio (Z) / (E) is of the order of 75/25.

The reduction step (ii) described in US 6759555 is conducted in the presence of excess iron (more than 2 equivalents relative to (Ia)). The reduction in the presence of zinc as described in US

5525632 (ex.2, step 2) is also possible but it is not complete (yield = 49.3%) and it also leads to the formation of a significant amount of "azo" by-product. The compound

(Z) -amino of sufficient purity is then obtained by one or more complicated separation (s). The separation following the reduction is carried out by successive crystallizations. A 1 ^st crystallization allows to eliminate the compound (E) -amino, then a 2 ^nd crystallization allows to isolate the (Z) -amino (see US 6,759,555, ex.1).

The coupling of step (iii) is advantageously carried out in the presence of an acid activator such as, for example, EDCI (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide chloride), DCC (dicyclohexylcarbodiimide), TOTU (o- [ethoxycarbonyl] cyanomethyleneamino) -N, N, N ', N'-tetramethyluronium tetrafluoroborate), HBTU (o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate), PivCl (pivaloyl chloride) or N, N-carboπyldiimidazole. The term "coupling agent" means a compound whose function is to activate the -COOH acid function in order to promote the formation of a peptide bond. For more details on the acid activators, reference may be made to the review Ch0mFiles Vol.7, No. 2, page 3 edited by Aldrich Chemical or Tetrahedron report No. 672, 2004, 60, 2447- 2467, "Recent Development of Peptide Coupling Reagents in Organic Synthesis". The review Tetrahedron 2005, 61, 10827-10852 reveals that there are many available acid activators.

Step (iv) is conducted in the presence of an acid to promote ring opening and to obtain combretastatin (A) as a salt (-NH ₃ ⁺ ). This is a deprotectioπ / cycle opening. In the presence of BOC, hydrochloric acid is advantageously used, for example, in the form of methanolic solution, and the hydrochloride is obtained. Combretastatin (A) in the base form is obtained by neutralizing the salt with the aid of a base, for example sodium hydroxide (cf..ex.1-step (Ni)).

Process according to the invention

The process of the present invention follows the same steps of Scheme 1 but steps (ii) and (iii) have been improved. Indeed, in the invention, during step (ii), the (Z) isomer of the salt of the amino compound is obtained by the process of: "adding benzyl alcohol to a suspension of a mixing the salts of the (Z) - and (E) -amino compounds in acetonitrile; and • mechanically separating the salt of the amino compound enriched in the (Z) isomer.

The isomer (Z) is thus obtained by "enrichment", this term meaning that at the end of the two steps of the process described above, the proportion of the isomer (Z) is increased relative to the isomer (E); we can also speak of separation process of the (Z) isomer from a mixture of isomers (Z) and (E)). Compared to (re) crystallization, enrichment has the advantage of being both direct and simple to implement. It makes it possible to obtain the amino compound enriched in isomer (Z) with a low content of residual isomer (E) (which can be up to <1 mol%, cf ex.1, where the purity of the product is 99.93%). % in (Z) and 0.07% in (E)). The mechanical separation may be, for example, filtration or centrifugation. At the end of the mechanical separation, the salt of the isomer (Z) may be optionally washed and dried.

The suspension preferably has a proportion of acetoπitrile / salts of the compounds (Z) - and (E) - amino expressed by weight of between 5 and 17, preferably between 10 and 12 (i.e. acetonitrile in the suspension is between 5 to 17 x weight of the compounds (Z) - and (E) -amino).

The amount of benzyl alcohol which is added is preferably such that the proportion benzyl alcohol / salts of the compounds (Z) - and (E) -amino expressed by weight is between 1 and 4, preferably between 2 and 3 (c). that is, the weight of benzyl alcohol added is from 1 to 4 x weight of the compounds (Z) - and (E) -amino). This proportion makes it possible to maintain a high ratio (Z) / (E) for the final product. The benzyl alcohol has the function of preferentially dissolving the salt of the compound (E) -amino.

Enrichment is preferably carried out at a temperature between 20 and 70 ° C, preferably between 30 and 70 ° C, preferably between 35 and 65 ° C. Beyond 70 ⁰ C, the amino compound begins to degrade slowly. A preferred method of preparing the suspension is described below. Compounds (Z) - and (E) -nitro are reduced with sodium dithionite (Na ₂ S ₂ O ₄ ) in a solvent which may be a mixture of water and alcohol, for example a mixture water / methanol. After the reduction, a strong acid (eg HCl or H ₂ SO ₄ ) is introduced into the reaction medium which reacts with the reaction intermediates as well as with the dithionite and disulfite residues. A mixture of the salts of the compounds (Z) - and (E) -amino (eg hydrochloride or sulfate) is then obtained. A strong base is then added to obtain the free bases (-NH ₃ ⁺ • => -NH ₂ ) which are extracted with an organic solvent, for example a chlorinated solvent such as dichloromethane (DCM). A strong acid in an alcohol is added to the organic phase, and the alcohol is then displaced with acetonitrile so as to obtain a suspension of the salts of the (Z) - and (E) -amino compounds in acetonitrile. The strong acid may be HCl or H ₂ SO ₄ (B ^" = Cl or SO ₄ ) The solvent can be displaced by adding acetonitrile after removing the alcohol in a more or less complete vacuum. The acetonitrile may also be concomitantly with the removal of the alcohol under vacuum The alcohol is preferably a light alcohol such as methanol or ethanol.

For step (iii), the coupling between the salt of the (Z) -amino compound and the doubly protected L-serine derivative of formula (II) is carried out in an organic solvent using anhydride. phosphonic propane acid (T3P) as an acid activator in the presence of a base. The function of the base is to trap the acid species and move the salt to the free base. The amount of base added is between 2 and 3 eq. relative to the salt of the compound (Z) -amino (see ex.1 where 2.7 eq.) were used. It is also possible to directly couple the (Z) -amino compound with the compound of formula (II) in the presence of T3P and a base; in this case, the base proportion added is smaller, between 1 and 2 eq. (about 1 eq less than if you start with salt).

The base may be a tertiary amine, for example triethylamine (TEA), diisopropylethylamine (DIEA), N-methyl morpholine (NMM) or methylpiperidine. The organic solvent may be dichloromethane (DCM), toluene, methyl isobutyl ketone (MIBK), ethyl acetate, acetonitrile, tetrahydrofuran (THF), Me-tetrahydrofuran (Me-THF) or cyclopentylmethyl ether.

The T3P has the formula:

n-Pr n-Pr-l I n-Pr

(III) The advantage of using T3P over other acid activators is that the by-products of T3P are easily removed (all by-products soluble in water) and that it is a good activator. walk. Moreover, the reaction can be carried out in the presence of the compound (Z) -amino (in salt or base form) and of the compound (II) put together according to a "one pot" process: it is therefore possible to react together in the same container the compound (Z) -amino (as salt or base), the compound (II) in the presence of T3P and a base. This is not the case for all the coupling agents, since some, such as PivCl (pivaloyl chloride), require for the same reaction a prior activation of the acid function of the compound (II) before the salt of the compound can be added. compound (Z) -amino.

Finally, we found that T3P does not lead to an epimerization of the asymmetric center, which makes it possible to obtain combretastatin (A) with a good purity and a good yield. It has also been found that T3P makes it possible to obtain a good yield of coupling product (see Table III).

The coupling reaction is generally conducted at a temperature between 5 ° C and 70 ° C, for example at reflux of DCM. The proportion of T3P relative to the (Z) -amino compound is between 1 and 2 eq, preferably between 1.5 and 1.8 eq.

[Examples]

Example 1 (according to the invention) Step (i):

On a mixture at a temperature of 5-10 ^° C., consisting of toluene (91.1 liters) of trimethoxybenzylphosphonium bromide (15.35 kg, 29.33 moles) and 4-methoxy-3-nitrobenzaldehyde (5.06 g). kg, 27.93 moles), a solution of sodium methoxide (5.66 kg, 29.34 moles) is poured. At the end of the reaction, 0.32 liter (5.59 moles) of acetic acid is poured. After maintaining at 20 ^° C., the medium is filtered. The cake is washed with toluene (11.1 liters). The filtrates are washed several times with water (20.2 liters) and then concentrated under vacuum. The isopropyl alcohol (87.6 liters) is then charged and the medium is concentrated and then cooled. The suspension is then filtered at 100 ^° C. The isolated product is dried under vacuum (6.46 kg, 52.2%). The purity of the isolated product is of the order of 78% in (Z) and 22% in (E).

Step (ii):

On a medium consisting of methanol (100 ml), the compounds (Z) and (E) -nitro to be reduced (20 g, 0.058 mol) sodium dithionite (36.8 g, 0.179 mol, 3.1 eq), is poured water (80 ml) at 50 ° C. Once the reaction is complete (1 h allowed to 5O ⁰ C) ₁ Hydrochloric acid (36.3 ml, 0.406 mol) is added. The treatment is carried out by pouring water (70 ml) and DCM (80 ml), then sodium hydroxide (30.5% sodium hydroxide solution, 10N) to pH = 7. The aqueous phase is removed after re-extraction with DCM (20 ml) then the DCM phase is concentrated under vacuum (about 200 mbar at 35 ° C) and replaced with acetonitrile (160 ml); the solvent is changed from DCM to acetonitrile under reduced pressure so as to keep a reaction volume of about 200 ml. The methanolic hydrochloric acid (27 ml, 0.081 mol) is cast and the solvent is then evaporated under reduced pressure (90 mbar) so as to effect the solvent change from methanol-acetonitrile to acetonitrile at a constant volume of about 233 ml. . At the end of the change of solvent, the total volume (solvent + organic compounds) is adjusted to 280 ml.

A suspension is then obtained in the form of a white slurry. The benzyl alcohol (46 ml) is then added to the suspension at 45 ± 3 ° C. After cooling to 25 ° C., the medium is then filtered and washed with acetonitrile (30 ml) and benzyl alcohol (3.3 ml). The isolated product is then dried under vacuum (11.7 g, 74.4%). This product has a purity determined by HPLC of 99.93% in (Z) and 0.07% in (E). At the end of step (ii), the salt of the compound (Z) -amino was thus obtained simply and directly with a good purity.

Step (in):

On a medium consisting of DCM (500 ml), the compound (Z) -amino in hydrochloride form (50 g, 0.142 mol) and the doubly protected L-serine of formula (II) with PG = BOC (L-serine N, BOC-acetonide, 41.8 g, 0.170 mol) is poured from the TEA (53.4 ml, 0.383 mol) at 20 ^° C. and then a solution of T3P in DCM (154 g, 0.242 mol). The mixture is brought to reflux and then water (500 ml) is added. After decantation and concentration, methanolic hydrochloric acid (189.5 ml, 1.136 mol) and methanol (189.5 ml) are added. Water (300 ml) is added to the medium and the phases are decanted. Isopropyl acetate (650 mL) was added to the aqueous phase and then sodium hydroxide (115 ml, 1, 150 moles) is cast at 20 ⁰ C. The organic phase is decanted and washed concentrated in vacuo and then heated at 65 ⁰ C. Methanol (35 ml) and then methanolic hydrochloric acid (47.4 ml, 0.142 mol) are added at this temperature. After cooling and filtration, the product is isolated (49.6 g, 79.5%). The final product has a purity of 99.2%.

Example 2 (according to the invention)

The compound (Z) -amino in the form of 2-aminostil hydrochloride, HCl (50.0 g), N-BOC-acetonide (41.0 g) was charged to a 1.6 liter reactor and equipped with a double jacket. 8 g) and 500 ml of DCM. 53.4 ml of triethylamine (2.7 eq.) Are then added at 22 ± 3 ° C., followed by a solution of T3P in 50% DCM (1.7 eq.). The mixture is stirred under reflux for 1 hour. The mixture is then cooled to 22 ± 3 ° C. and 500 ml of demineralized water are added. It is allowed to settle and the phases are separated. The DCM phase is washed with 500 ml of an aqueous solution of 6% sodium hydrogenphosphate (30 g) and then with 500 ml of demineralized water. The DCM phase is concentrated under reduced pressure from 360 to 150 mbar at 40-50 ^° C. then a HCl solution in methanol at 3 mol / l (379 ml, 8 eq.) and 300 ml of demineralized water. It is allowed to settle and the phases are separated. The DCM / methanol phase was re-extracted with deionized water (200 ml) and the phases were separated. 650 ml isopropyl acetate and then 115 ml of 30% sodium hydroxide solution (8.1 eq) are added. The mixture is left to settle and the organic phase is washed with 400 ml of demineralised water. It is allowed to settle and the phases are separated. The organic phase is then concentrated under reduced pressure from 160 to 60 mbar to 300-350 ml and a change of solvent DCM isopropyl acetate. The mixture is then heated to 62 ° C., 35 ml of methanol are added and a solution of hydrochloric acid in 3 mol / l methanol (47.4 ml, 1 eq) is poured. It is then allowed to cool to ambient temperature and the white mud is filtered. We wash the final solid

In Examples 3-6, step (ii) is repeated with different amounts of (Z) and (E) -nitro compounds initially. The proportion acetonitrile / salts of the compounds (Z) - and (E) -amino is fixed at 10.8 and the proportion benzyl alcohol / salts of the compounds (Z) - and (E) -amino is itself variable.

Example 3 (according to the invention)

On a medium consisting of methanol (50 ml), the compounds (Z) and (E) -nitro to be reduced (15 g, 0.043 mol) of sodium dithionite (27.2 g, 0.133 mol) are poured from the water (60 ml) at 50 ° C. After the reaction is complete, hydrochloric acid (26.2 ml, 0.314 mol) is added. The treatment is performed by pouring water and DCM, then sodium hydroxide to pH = 7. On the DCM phase is added methanolic hydrochloric acid (18.9 ml, 0.0586 mol) and the solvent is replaced by acetonitrile. A slurry is then obtained. The benzyl alcohol (31 ml) is then added to the suspension at 50 ^° C. and maintained at 65 ° C. for 2 hours. After cooling, the medium is then filtered and washed. The isolated product is then dried under vacuum.

Examples 4-6: Same as Example 3 but with a different amount of benzyl alcohol

Table I

proportion acetonitrile / salts Z + E amino = 10.8

In Examples 5 and 7-9, the proportion benzyl alcohol / salts of the compounds (Z) - and (E) -amino is fixed at 2.25 and the proportion acetonitrile / salts of the compounds (Z) - and (E) - amino is variable. Table II

proportion benzyl alcohol / salts Z + E amino = 2,25

Examples 10-14 describe coupling results (step (Ni)) using other coupling agents than T3P.

Example 10 (comparative): use of TOTU

The conditions of step (iii) are repeated but in the presence of TOTU as acid activator (1 eq of TOTU + 5 eq of TEA). The final yield is only 71%.

Example 11 (comparative): use of TOTU

On a medium consisting of DCM (10 ml), the compound (Z) -amino in hydrochloride form (0.50 g, 1.4 mmol) and the doubly protected L-serine of formula (II) with PG = BOC (L-serine-NBOC-acetonide, 0.35 g, 1.4 mmol) was poured from the TEA (0.71 g, 7.0 mmol) at 5 ° C and then TOTU (0.46 g, 4 mmol). The medium is kept at 5 ° C. for 24 hours and then water (5 ml) is added. After decantation, the organic phase is analyzed by HPLC. The metered yield of the coupling product is 50.1% and its purity is 69.3%.

Example 12 (comparative): Use of BOP-Cl (Bis (2-oxo-3-oxazolidinyl) phosphinic chloride) on a medium consisting of DCM (5 ml), compound (Z) -amino (0.50 g, 1 g) , 4 mmol) and doubly protected L-serine of formula (II) with PG = BOC (L-serine-NBOC-acetonide, 0.35 g, 1.4 mmol) is poured from NMM (0.42). g, 4.2 mmol) at 5 ° C and then BOP-CI (0.72 g, 2.8 mmol). The medium is kept at 5 ° C. for 24 hours and then water (5 ml) is added. After decantation, the organic phase is analyzed by HPLC. The measured yield of the coupling product is 29.1% and its purity is 91.6%.

Example 13 (comparative): use of PyCIOP (chlorotripyrrolidinophosphonium hexafluorophosphate)

On a medium consisting of ethyl acetate (10 ml), compound (Z) -amino (0.50 g, 1.4 mmol) and doubly protected L-serine of formula (II) with PG = BOC (L-serine-NBOC-acetonide;

0.70 g, 2.8 mmol) was poured from the NMN (0.42 g, 4.2 mmol) at 5 ° C followed by PyCIOP (1.2 g, 2.8 mmol). The medium is kept at 5 ° C. for 24 hours and then water (5 ml) is added. After decantation, the organic phase is analyzed by HPLC. The metered yield of the coupling product is 53.3% and its purity is 83.9%.

Example 14 (Comparative): Use of Pybrox (bromo-tris-pyrrolidino phosphoniumhexafluorophosphate)

On a medium consisting of Me-CN (5 ml), the compound (Z) -amino (0.50 g, 1.4 mmol) and the doubly protected L-serine of formula (II) with PG = BOC ( L-serine-NBOC-acetonide, 0.35 g, 1.4 mmol) is poured from the NMN (0.42 g, 4.2 mmol) at 25 ° C. and then the PyBROP (0.65 g, 1, 4 mmol). The medium is kept at 25 ° C for 24 hours and then water (5 ml) is added. After decantation, the organic phase is analyzed by HPLC. The metered yield of the coupling product is 24.9% and its purity is 75.4%.

Table III

It can be seen that T3P makes it possible to obtain a better yield of coupling product than TOTU, BOP-CI, PyCIOP or PyBROP.

Claims

claims

1. Process for the preparation of combretastatin (A)

in the form of a base or an addition salt with an acid, consisting of:

• coupling in the presence of a base and T3P of formula (III)

the compound (Z) -amino or the salt of the compound (Z) -amino

, B ^" designating a counter-anion,

with a doubly protected L-serine derivative of formula (II) wherein PG denotes a protecting group of the amine function so as to obtain the compound

of formula (Z) - (Ib):

• then deprotect and open the cycle of (Z) - (Ib) in the presence of an acid so as to obtain combretastatin of formula (A) in the form of salt;

And optionally to add a base so as to obtain combretastatin of formula (A) in base form, the salt of the compound (Z) -amino having been obtained by an enrichment of the salt of the amino compound of formula:

in isomer (Z), B ^" denoting a counteranion, consisting of:

2. Process for enriching the salt of the amino compound of formula

in isomer (Z), B ^" denoting a counteranion, consisting of:

Adding benzyl alcohol to a suspension of a mixture of the salts of the (Z) - and (E) -amino compounds in acetonitrile and

3. The method of claim 1 or 2 wherein the mechanical separation is filtration.

4. Process for the preparation of combretastatin (A)

in the form of a base or an addition salt with an acid, consisting of:

• coupling in the presence of a base and T3P, the compound (Z) -

amino or the salt of the compound (Z) -amino

B-

, B ^' denoting a counteranion, with a doubly protected L-serine derivative of formula (II) wherein PG denotes a protecting group of the amino function so as to obtain the compound of formula (Z) - (Ib):

• then deprotect and open the cycle of (Z) - (Ib) in the presence of an acid so as to obtain combretastatin (A) in the form of salt;

And optionally to add a base so as to obtain combretastatin (A) in base form.

5. Method according to one of claims 1 to 4 wherein PG represents BOC, CBZ or FMOC.

6. Method according to one of claims 1 to 5 wherein B ^" designates CI ^" or SO ₄ ^{2 "} .

7. Method according to one of claims 1 to 6 wherein PG is BOC and B ^" designates CI ^" .

8. Method according to one of claims 1 to 7 wherein the base is a tertiary amine.

The process according to claim 8 wherein the base is triethylamine (TEA), diisopropylethylamine (DIEA), N-methyl morpholine (NMM) or methylpiperidine.

The method according to one of claims 1 to 9 wherein the (Z) -amino compound or the salt of the (Z) -amino compound and the compound of formula (II) react together in the same container in the presence of T3P and from the base.

11. Method according to one of claims 1 to 10 wherein the proportion acetonitrile / salts of compounds (Z) - and (E) -amino expressed by weight is between 5 and 17, preferably between 10 and 12.

12. Method according to one of claims 1 to 11 wherein the temperature at which the enrichment is conducted is between 20 and 70 ° C.

13. Method according to one of claims 1 to 12 wherein the proportion benzyl alcohol / salts of the compounds (Z) - and (E) -amino expressed by weight is between 1 and 4, preferably between 2 and 3.

14. Use of T3P of formula (III)

ppoouurr rreeaalliissuee cooouuppllaaggee eennttrree ccoommppoosséé (Z) -amino of formula

or the salt of the compound (Z) -amino

formula designating a counter-anion

with a doubly protected L-series derivative of formula (II) wherein PG denotes a protecting group of the amine function.

15. Use according to claim 14 wherein PG designates BOC, CBZ or FMOC and / or B ^" refers to Cl ^" or SO ₄ ^{2 "} .

16. Use according to claim 14 or 15 wherein the base is a tertiary amine.

17. Use according to one of claims 14 to 16 wherein the compound (Z) -amino or the salt of the compound (Z) -amino and the compound of formula (II) react together in the same container in the presence of T3P and from the base