OA12804A - Processes for the preparation of combrestastatin - Google Patents

Abstract

Preparation of combrestatins (I) by a Wittig condensation of (3,4,5-trimethoxybenzyl) triphenyl phosphonium bromide or chloride with 3-nitro-4-methoxy benzaldehyde, or of 3,4,5-trimethoxy benzaldehyde with a (4-methoxy-3-nitrobenzyl) triphenyl phosphonium salt, followed by reduction of the nitro group on the product in the presence of iron. Preparation of combrestatins (I) by a Wittig condensation of (3,4,5-trimethoxybenzyl) triphenyl phosphonium bromide or chloride with 3-nitro-4-methoxy benzaldehyde, or of 3,4,5-trimethoxy benzaldehyde with a (4-methoxy-3-nitrobenzyl) triphenyl phosphonium salt, followed by reduction of the nitro group on the product in the presence of iron. [Image] A' : an amino group. Independent claims are included for (1) the process as described but in which the nitro group in the starting material is replaced by an amino group; (2) preparation of 2-amino-3-hydroxy-N-{2-methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]-phenyl}-propionamide hydrochloride salt of formula (IV) by reacting (I; A = NH 2) with a protected cyclic derivative of serine of formula (IIb), to give an intermediate (III) followed by removal of the protecting group PG; (3) intermediates of formula (III). [Image] [Image] PG : a protecting group such as tert. butoxycarbonyl, benzyloxy carbonyl, or 9-fluorenylmethyloxy carbonyl. ACTIVITY : Cytostatic. MECHANISM OF ACTION : Not given in source material.

Description

012804 1

The present invention relates to a novel process for the preparation of combretastatins and their derivatives.

Combretastatins or derivatives of stilbene are understood to mean the following derivatives of general formula:

wherein A represents a hydroxyl group or an amino group as well as its pharmaceutically acceptable salts.

Among the salts, mention may be made of hydrochloride, acetate, phosphate and methanesulphonate. When the compound or A is an amino group it can also be coupled to amino acids to yield amides as well as their pharmaceutically acceptable salts. ·

The synthesis of stilbene derivatives or combretastatines which can be in the form of a pharmaceutically acceptable salt as well as the pharmaceutical compositions containing them are described in U.S. Patents 4,996,237; US 5,525,632; US 5,731,353 and US 5,674,906. These patents describe combretastatins as well as their metabolites and describe their oncological activity in vitro.

According to these patents combretastatins are prepared from (3,4,5-trimethoxy-benzyl) -triphenylphosphonium salts which are condensed on a 3-nitro or 3-hydroxy (the hydroxyl group is protected) 4-methoxybenzaldehyde The cis-isomer is then reduced by the action of light or by chromatographic separation of the mixture.

The present invention relates to novel processes for the preparation of combretastatines or derivatives thereof and improvements to existing methods. 012804 2

It was first discovered a first method V0 1 preparation of derivatives of formula (I) for which A represents an amino group, which is an improvement to the process described in the patents cited above which consists, after Wittig condensation in the presence 5 bromide or (3,4,5-trimethoxy-benzyl) -triphenylphosphonium chloride and 3-nitro-4-methoxybenzaldehyde, to achieve reduction in the presence of iron, in place of the zinc used in [prior art], which allows to achieve an overall reaction yield of 60% (relative to the aldehyde involved in US Pat. No. 5,525,632 compared with the aldehyde employed in the range of from 21% to 33%).

The first method VO 2 is to condense 3,4,5-trimethoxy-benzaldehyde with bromide or chloride (4-methoxy-3-nitro-benzyl) -triphenylphosphonium. For all these first two processes VO 1 and VO 2, the reaction is carried out in the presence of a base chosen from potassium tert-butoxide, sodium teramylate, sodium hydride, butyl lithium, LDA (Lithium diisopropylamine), sodium methylate, potassium carbonate or alkaline derivatives of hexamethyldisilanes.

This reaction is carried out in various solvents such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO.), Alcohols, aromatic solvents or water, at a temperature which will be adapted by [male from the art to the base used and to the solvent used.

This reaction with respect to the first VO 2 route method is notably described in K. G. Piney's publication published in Bioorg. Med. Chem. 8 (2000) 2417-2425.

2-Methoxy-5- [2- (3,4,5-trimethoxy-phenyl) -vinyl] -nitrophenyl is reduced according to the improved method of [invention by [action of iron. It is advantageous to use an excess amount of iron if a complete transformation of the starting material is desired. This excess is advantageously greater than 2 equivalents for one mole of nitrided derivative.

It has been proved that the same step carried out in the presence of zinc in [acetic acid, a conventional solvent for zinc reductions, does not allow a complete reaction to be obtained (in US Pat. No. 5,525,632, the 012804 3). the pure Z isomer varies between 46 and 66%), and on the other hand the quantities of zinc used are enormous and consequently result in considerable industrial waste, in addition the process degenerates a large amount of "azo" compound resulting from the coupling between 5 Famino formed and intermediate nitroso reduction.

The reduction to nascent hydrogen generated by the ammonium formiate in the presence of conventional catalysts such as palladium or platinum results in high isomerization of the undesirable E-isomer double bond as well as partial saturation of the double bond. The aforementioned Piney publication describes the reduction by

Sodium hydrosulphite of a pure nitro Z isomer, obtained after chromatography and recrystallization, resulting in an amino Z isomer with a yield of only 37%.

Molecular hydrogen hydrogenations, catalyzed by platinum or palladium, are rarely complete and lead mainly to the saturation of the ethylenic double bond. A second method has also been found which avoids the necessary intermediate reduction step when starting from a nitro derivative. In fact it is much more economical to condense according to a first method of implementation of this second process a (3,4,5-trimethoxy-benzyl) -triphenylphosphonium bromide or chloride with a 3-amino-4-methoxybenzaldehyde or according to a second method of implementing this second method 3,4,5-trimethoxybenzaldehyde with a salt of (3-amino-4-methoxy-benzyl) -triphenylphosphonium. This second method according to its two variants requires a step of less engaging CMR products (Carcinogenic, Mutagenic, Toxic for Reproduction), compared to the first V0 1 and VO 2 pathway processes, which on the industrial level is an advantage from the point of view of safety. and considerable cost of production. According to the second method V 03 of implementation of the invention, the salt of (3,4,5-trimethoxybenzyl) -triphenylphosphonium is brought into contact with 3-amino-4-methoxybenzaldehyde, the reaction is carried out, preferably, in the presence of a base selected, for example, potassium terbutylate, sodium teramylate, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilanes. It is preferred to use sodium methoxide.

This reaction is carried out in various solvents such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, etc.), alcohols, aromatic solvents or water, at a temperature which will be adapted by man. Wax at the base used and the solvent used.

The reaction temperature will be adapted by those of ordinary skill in the art, when using methylate it is preferably from 0 to 10 ° C. After reaction, the base used is neutralized with an acid in aqueous solution, the organic phase is washed, concentrated and the expected product is obtained after chromatography of the crude concentrate.

According to the second method VO 4 of implementation of the invention, in which the salt of (3-amino-4-methoxybenzyl) -triphenylphosphonium is brought into contact with 3,4,5-trimethoxy-benzaIdehyde, the reaction is, preferably, carried out in the presence of an organic base chosen from, in particular, potassium tert-butoxide, sodium tetramylate, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or the alkaline derivatives of hexamethyldisilanes. It is preferred to use sodium methoxide.

This reaction is carried out in various solvents such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, etc.), alcohols, aromatic solvents or water, at a temperature which will be adapted by those skilled in the art. the base used and the solvent used.

The reaction temperature will be adapted by those of ordinary skill in the art, when using the methylate it is preferably from 0 to 10 ° C. After reaction, the base used is neutralized with an acid in aqueous solution, the organic phase is washed, concentrated and the expected product is obtained after chromatography of the crude concentrate.

The derivative obtained according to the second method V0 3 or V0 4 route or during the second step of the first method V0 1 or V0 2 pathway has the following formula:

A 012804

formula (lia)

It is advantageous when coupling serine with the compound of formula (IIa) to introduce doubly protected L-serine on serine nitrogen and on the hydroxyl function of general formula (IIb).

O

wherein PG represents a protecting group of the amine function known to anyone skilled in the art to give a novel intermediate of the following general formula:

Which is then preferably cleaved simultaneously with the opening of the acidic hydrolysis ring according to a deprotection reaction known to any man in the art. Preferably the PG group of the formulas (IIb) or (III) represents a protective group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC). The compound of formula (III) above is new and is claimed as such.

The condensation is advantageously carried out in the presence of EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide chloride) or in the presence of DCC (dicyclohexylcarbodiimide) and HOBT (hydroxybenzotriazole) or

Still in the presence of DCC (dicyclohexylcarbodiimide) and HOSU 012804 6 (N-Hydroxy succinimide) or finally in the presence of TOTU (O - [(Ethoxycarbonyl) cyanomethyleneamino] -NNN'.N'-tetramethyluronium tetrafluoroborate or HBTU (O-Benzotriazol) 1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate) or NN carbonyldiimidazole.The reaction is preferably carried out in a solvent which is inert with respect to the reaction which is chosen in particular from aproticpolar solvents such as acetonitrile, dimethylformamide, tetrahydrofuran or chlorinated aliphatic solvents such as dichloromethane or finally lesesters.

The coupling on the derivative of formula (IIa) 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, and the protected L-serine of formula ( 1b) in the presence of a tertiary base of the NMM (N-methyl morpholine) type in various reaction-inert solvents, esters, ethers, chlorinated solvents, acetonitrile, etc. Anhydridemixte is preferably prepared at a temperature in the range of from 0.degree. between 0 and 10 ° C then the reaction is carried out at room temperature. After reaction, the reaction medium hydrolyses the reaction medium with an aqueous solution and then the medium is corrosive 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, it is preferred to use aqueous concentrated hydrochloric acid in an alcoholic medium. The reaction temperature is included in a better way of carrying out the invention between 50 and 70.degree. . The invention will be more fully described with the aid of the following examples which should not be considered as limiting the invention.

The composition of the mixtures, the monitoring and the evolution of the reactions as well as the yield of the uninsulated products / intermediates and their titers are determined by HPLC analysis (High Performance Liquid Chromatography). 012804 7

Example 1 - first method VO 2 way according to the invention

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

General scheme of the synthesis The new process, called "reverse Witty", from (4-methoxy) bromide

Wittlg reaction OMe

MeO OMe

PPhj.Br 3,4,5-Trimethyloxy-benzaidehyde

Commercial wo4

Phosphonium knows K.G. Piney et al.Bioorg. Med. Chem.8 (2000) 2417-2425

MeONa Z toluene

1) Reduction-FelHCl k 2) HCl purification

MeOH-HCl

Acétonltriie

ο ο I

Boc ~ L -ser-PG EDCl / Dichloromethane

Deprotectlon

MeOH / HCl HCl CrystalllzatlonAcOlPr

MeO OMe

OMe

OHNK, H-3-nitrobenzyl) -triphenylphosphonium and 3,4,5-trimethoxy-benzaldehyde can be used to obtain the mixture of Z and E isomers of 2-methoxy-5- [2- (3,4,5- trimethoxy-phenyl) -vinyl] -nitro-phenyl with a Z / E ratio of 75/25.

This ratio is sufficiently high in the Z nitro isomer to be able to directly engage the reducing Z / E mixture and to obtain, by crystallization of the hydrochloride, [Z amino isomer with an HPLC titer of 97% N1 (internal standardization).

The preparation of (4-methoxy-3-nitro-benzyl) -triphenylphosphonium bromide (4) is carried out according to [Example: 012804 8

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

In a 2-liter 3-neck with mechanical agitation, a thermometer, a Y-tube, an addition funnel for solid and a condenser surmounted by a bubble counter, 90.5 3-nitro-4-methoxy-benzaldehyde (1) is charged, followed by 450 ml THF and 90 ml ethanol. The light yellow solution obtained is cooled to 10 ° C., and then 10 g of sodium borohydride are charged in 40 minutes at 10/15 ° C. (the reaction is very exothermic and the temperature must be maintained with an ice-bath acetone), at the end. addition the brown solution turns to navy blue. The solution is stirred for 30 minutes at 10 ° C., the end of the reaction is checked by TLC (thin layer chromatography) and the mixture is stirred for another hour at 10 ° C. and the temperature is then allowed to return to room temperature. The addition funnel is replaced by a 500 ml isobaric dropping funnel through which 300 ml of distilled water are poured drop by drop in 30 minutes while maintaining the medium at 20 ° C. There is a gas release at the beginning of casting.

The medium is concentrated in a rotary evaporator (50 ° C./20 mmHg) in a white product crystallized in the aqueous concentrate in the form of blocks. The cooled aqueous phase is extracted with 250 ml and then with 150 ml of dichloromethane, the combined organic phases are washed with 250 ml of water-distilled water and then dried over magnesium sulphate.

After filtration, the chloromethylenic solution is used as it is in the next bromination reaction. The yield of this step is considered to be 100%. NB: [alcohol (2) is commercial but very little available 3-nitro-4-methoxy-bromobenzyl (3): 012804 9

In a 1-liter three-neck with mechanical agitation, a thermometer, a Y-tube, a dropping funnel and a condenser surmounted by a bubble counter, the chloromethylenic alcohol solution is charged. 3-nitro-4-methoxy benzyl (2) and 100 ml of dichloromethane are added. The stirred solution is cooled to 5 ° C. and 135.4 g of phosphorus tribromide are then added dropwise while keeping the temperature at 5 ° C.

The solution is stirred for 1 hour 30 minutes at 5 ° C. the end of reaction is checked by TLC and then poured drop by drop while maintaining the temperature at 15 ° C. 250 ml of saturated sodium hydrogencarbonate solution gives rise to a very strong gas evolution with slight delay during casting.

The medium decanted into an ampoule is washed successively with 250 ml of water and 200 ml of saturated solution of sodium hydrogencarbonate. The . The organic phase is dried over magnesium sulphate, filtered and concentrated by rotary evaporation (50 ° C./20 mmHg). 119 g of solid are obtained in the form of yellow-green felted needles with a 97% 2-stage chemical yield. NB: This product (3) can also be prepared according to the following scheme described in K. G. Piney et al. Bioorg. Med. Chem. 8 (2000) 2417-2425

(3-Nitro-4-methoxy-benzyl) -triphenylphosphonium bromide (4): In a 2-liter tricolor equipped with mechanical stirring, a thermometer, a Y tube, a funnel of addition for solid and a condenser surmounted by a bubble counter, 119 g of 3-nitro-4-methoxy-bromobenzyl (3) are charged on a stirred foot of 1000 ml of toluene, warm medium at 25 ° C. solution. 126.5 g of triphenylphosphine are then added, the solution obtained is gradually heated to 60 ° C., from 30 ° C. a precipitate is formed. The medium is maintained for 4 hours at 60 ° / 65 ° C. and then cooled to 30 ° C., filtered on sintered glass, washed on a filter with 2 × 300 ml of toluene, drained and dried in a oven (35 ° C./20 mmHg / 20 ° C.). hours).

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

Synthesis described in the publication: (solvent used: dichloromethane) K. G. Pineyetal. Bloorg. Med. Chem. 8 (2000) 2417-2425

Spectrum no. = 4,865 - V

Spectrum of R.M.N. 1H (300 MHz, (CD3) 2SO d6, δ 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

El m / z = 262 [PPh3f DCI m / z = 445 MNH3 + m / z = 428 M + m / z = 263 [PPh3Hf peak base peak base

IR Spectrum 426469 KBr 2869; 2843; 2776; 1619; 1527 1438; 1362; 1287; 1270; 1111; 752; 692 and 502 cm · 1 Mixture Z and E of 2-methoxy-5- [2- (3,4,5-trimethoxy-phenyl) -vinyl] -nitro-phenyl (6) and (7);

In a 2 liter knit equipped with mechanical stirring, a thermometer, a Y tube, a dropping funnel and a refrigerant surmounted by a bubble counter, it is charged at 20 ° C. And under nitrogen, 54.7 g of 3,4,5-trimethoxybenzaldehyde (5), 148.6 g of (4-methoxy-3-nitro-benzyl) -triphenylphosphonium bromide (4) and 1300 ml of toluene.

The stirred suspension is cooled to 5 ° C. with an ice bath and then poured at 5 ° C. over 40 minutes, 63.2 g of a 25% w / w sodium methoxide solution in methanol. .

As the casting progresses, the suspension changes from off-white to yellow to brown.

Stirred 1 hour at 5 ° C, the end of reaction is monitored by HPLC (total consumption of aldehyde), 3 g (0.05 mole) of acetic acid are then added.

The suspension is heated to 40 ° C. and maintained for 30 minutes at 40 ° C., at this temperature, only the salts remain insoluble, filtered at 40 ° C. on sintered glass No. 3 and the salts are washed on a filter with 3 times 100 ml of toluene. The filtrate is tank-fed with 250 ml of distilled water, the biphasic mixture is stirred for 20 minutes at 40 ° C. and then decanted into an ampoule. The toluenetoluene is washed twice more with 250 ml of distilled water and then concentrated to dryness with a rotary evaporator.

The pellet is taken up in 600 ml of isopropanol and 12 ml of toluene at 40 ° C., the mixture begins to crystallize and the temperature is allowed to stir slowly until overnight.

The stirred suspension is cooled and maintained for 1 hour at 5 ° C. and then filtered on sintered glass, the cake is washed twice with 125 ml of isopropanol, drained and dried under vacuum (35 ° C./30 mmHg / 18 hours). 91.7 g of a mixture of isomers Z and E (6) and (7) are obtained with a Z / E ratio of 75/25 (HPLC NI) and a yield of 95%.

Synthesis described in the publication: (solvent used: dichloromethane: baseutilized: NaH) KG. Pineyetal. Bioorg. Med. Chem. 8 (2000) 2417-2425 NB: Many operating conditions have been experimented such as:

Solvents: THF, acetonitrile, methanol and other alcohols, dichloromethane, NMP, DMF, DMSO, etc. 012804 12

Bases: Potassium t-butoxide, sodium t-amylate, sodium hydroxide, NaH, Buli / LDA, potassium carbonate, etc.

Temperatures: -10 ° C to the reflux of certain solvents

Z-2-methoxy-5- [2- (3,4,5-trimethoxyphenyl) -vinyl] -5-phenylamine (8) chlodbydrate:

In a 2-liter three-neck with mechanical agitation, a thermometer, a Y-tube, an addition funnel for solid, a refrigerant surmounted by a bubble-counter and a heating bath 80 g of 2-methoxy-5- [2- (3,4,5-trimethoxy-phenyl) -vinyl] -Nitro-phenyl (6) are charged at 20 ° C. under nitrogen. ) and (7), 640 ml of absolute ethanol and 160 ml of distilled water.

After stirring rapidly and heating in an oil bath at 50 ° C., 7.8 ml of 6N hydrochloric acid are added to the suspension and the temperature of the medium is then raised to 77 ± 2 ° C., the medium is almost soluble. add in 5 minutes in fractions, 52 g of iron powder. From the first addition the medium goes into solution and a blackish deposit is formed on the walls of the balloon.

The mixture is kept at 77 ± 2 ° C. for 2 hours and the disappearance of the starting nosnitros (6) and (7) is monitored by HPLC. The mixture is allowed to cool to 40 ° C. and the medium is filtered through a sintered glass filled with clarcel, the cake is rinsed with twice 160 ml of 80/20 ethanol / water mixture.

The filtrate, mother liquors and washings, is concentrated in a rotary evaporator, as soon as the azeotrope has been expelled an oil begins to precipitate in the residual aqueous phase. 012804 13

This aqueous phase is extracted into an ampoule with twice 300 ml of dichloromethane and the organic phase is then washed twice with 300 ml of half-saturated aqueous sodium chloride solution and with 300 ml of water-distilled water. The organic phase is concentrated to dryness on a rotary evaporator to give 76 g of an oil which has a Z / E ratio of 80/20 in HPLC. This oil is dissolved in 591 ml of methanol and transferred to a flask of 1 liter, then 100 ml of 2,32N hydrochloric methanol are added, the mixture is initiated and left to precipitate overnight with stirring. The amount of methanol + hydrochloric methanol is such that the final concentration in Z isomer (determined by HPLC) is equal to 8.8% w / v.

The following day the medium is filtered on sintered glass, the dried cake weighs 8.2 g and is composed only of E isomer (HPLC). The filtrate (693 g) Z / E ratio = 86/14 (HPLC NI) is concentrated by half a rotary evaporator, 400 ml of acetonitrile are added to the 347 g of concentrate and the mixture is reconcentrated until a further reaction is obtained. concentrate of 347 g. We add 1000m! The concentrate is then transferred to a stirred 4-liter flask containing 1500 ml of acetonitrile at 60 ° C. and the mixture precipitated abundantly.

The mixture is stirred for 2.5 hours at 60 ° C., allowed to cool to 30 ° C. in approximately 1 hour, the slurry is filtered on sintered glass, the E isomer (9) is solved in the filtrate, the cake is washed twice ml of acetonitrile and dried in a oven (35 ° C, 30 mmHg / 18 hours). 45.7 g of Z-2-methoxy-5- [2- (3,4,5-trimethoxy-phenyl) -vinyl] -phenylamine (8) are obtained with an HPLC titer of 97% and a yield as such. of 56% is a yield of isomer Z obtained on isomer Z engaged by 72%. EXAMPLE 2 - Synthesis according to the second method, route V0 3 according to the invention The interest of the second method V0 3 compared to the first method V0 30 2, referred to as "reverse Wittig" is to carry out the Wittig reaction between an already reduced product, Famino aldehyde (1a) and phosphonium (2a) and thus remove a chemical step involving CMR products. 012804 14

(Z) -N- [2-Methoxy-5- [2- (3,4,5- (t-dimethoxy-phenyl) vinyl] phenyl] -L-serinamide hydrochloride

General outline of the synthesis

Trans isomer

DeproteetionMeOH / HCl HCl CrystallizationAcOiPr

3-Amino-4-methoxybenzaldehyde (1a):

In a 2 liter tricoî inert with argon and provided with a mechanical stirrer, a thermometer, a tube Y, a funnel of addition for solid, a cooler surmounted of a bubble-counter 20 g of 3-nitro-4-methoxy-benzaldehyde (1) and 350 ml of ethanol were charged with a heating bath. absolute, onagite, heated to 60 ° C, the medium goes into solution. At 60 ° C, 115 ml of distilled water and then 14 ml of 2N hydrochloric acid are added dropwise. 24.7 g of iron in powder are then introduced in small portions.

The temperature of the medium is allowed to return to Fambiante in 2 hours. The reaction is complete (CCM).

The medium is filtered on celite and concentrated in vacuo, the pellet is taken up in dichloromethane and the organic solution is washed twice with distilled water and then dried over magnesium sulphate, filtered and concentrated to dryness in vacuo.

16 g of (1a) are obtained, which are chromatographed on a column of silica eluted with dichloromethane.

2 fractions are obtained containing clean expected after which 11.5 g of (1 a) pure, giving a yield of 69%. Spectrum No. 2810-V of R.M.N. 1H (300 MHz, (CD3) 2SO d6, δ in ppm): 3.88 (s: 3H), 5.11 (mf: 2H); 7.01 (d, J = 8Hz: 1H); 7.14 (d, J = 2 Hz: 1H), 7.18 (d, J = 8 Hz: 1H); 9.53 (s: 1H). 20

Mass Spectrum no. 210112, m = 151

El m / z = 151 M + 'm / z = 136 [M-CHsf m / z = 108 [136-CO] + m / z = 80 [108-CO] * base peak

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-trimethoxy-phenyl) -vinyl-phenylamine (8 ') and (9'): 01 2804

NB: Phosphonium (2a) is a raw material already described in the original patent Ajinomoto Co., Ltd. US 5,525,632 and WO 01/12579 A2.

In a 250 ml knit inert with nitrogen and equipped with a magnetic stirrer, a thermometer, a tube Y, a dropping funnel, a refrigerant surmounted by a bubble counter and a 8.2 g of phosphonium (2a) and then 2.20 g of amino benzaldehyde (1a) and 100 ml of toluene were charged to the cooling bath. The stirred suspension is cooled to 5 ° C. and 3.51 ml of 25% w / w methanolic sodium methylate are run in over 15 minutes. After 2.5 hours at 50 ° C the reaction remains incomplete (TT: 45%) but rvvolved more (HPLC) and the Z / E ratio is 61/39. 0.2 ml of acetic acid diluted in 50 ml of water is then poured in. The temperature rises to 13 ° C., the mixture is stirred for 30 minutes and then decanted into an ampoule, the organic phase is concentrated under vacuum and evaporated to give 8 g of yellow oil. . In HPLC this oil contains the starting aldehyde of the 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 mixture of cyclohexane / ethyl acetate / triethylamine (50/50/2). 2 sets of pooled fractions are concentrated to dryness, the first 360 mg dry extract contains 93% isomer Z + unidentified impurities, the second of 2.09 g contains the starting aldehyde and a 7JE mixture representing 39 and 37.5%. in HPLC NI.

The weight balance in Z isomer (8 ') determined by HPLC NI is 1.15 g out of 2.20 g of committed aldehyde, ie a yield of 24%. EXAMPLE 3 - Synthesis according to the second method VO 3 route according to the invention

As for the VO 2 channel, the interest of the VO 3 path compared to the first "reverse Wittig" VO 2 process is to carry out the Wittigentre reaction to an already reduced product, the (3-amino-4-methoxy) -bromide. benzyl) - triphenylphosphonium (1b) and 3,4,5-trimethoxybenzaldehyde (5) and thus remove a chemical step involving CMR products.

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

General outline of the synthesis

Trans isomer

Deprotecûon

MeOH / HCl HCl CrystalliZaiionAcOiPr

(4-Methoxy-3-amino-benzyl) -triphenylphosphonium bromide (1b)

PPh3, Br

Fe! HCl

. EtOH-H2O PPh3, Br (1b)

In a 1-liter three-necked flask equipped with a mechanical stirrer, a thermometer, a tube Y, an addition funnel for solid, a refrigerant surmounted by a bubble-counter and a heating bath, 30 g of (4), 240 ml of ethanol are charged with 60 ml of distilled water. "

To the stirred suspension was added 1.76 ml of 6N hydrochloric acid and heated to 70 ° C.

Then added in 15 minutes in small portions, 9.9 g of iron powder the medium remains insoluble. The medium is maintained for 2 hours at 75 ° C., the organic substances slowly pass into solution while a brownish deposit of iron oxide iron is formed.

After HPLC control there is still 5% starting, 2 g iron is added again and heating is continued for 1 hour, the TT is complete.

It is cooled to 40 ° C. and the medium is filtered on clarel, rinsed with 100 ml of 20% ethanol and the filtrate is concentrated to dryness under vacuum in a rotary evaporator.

The pellet is taken up in 300 ml of isopropanol and crystallized in the medium, the mixture is stirred and heated to 50 ° C. and the medium returns to solution. 14 ml of 5N hydrochloric isopropanol are then poured in, the medium is precipitated, the mixture is kept at 50 ° C. for 1 hour and then allowed to return to room temperature.

The film is filtered on sintered glass, the cake is washed with 50 ml of isopropanol, drained thoroughly and dried under vacuum.

27.3 g of (1b) are obtained with a yield of 89.9%.

Spectrum No. 4584-V of R.M.N. 1h (300 MHz, (CD3) 2SO d6, δ in ppm): 3.78 (s: 3H); 5.03 (broad d, J = 15 Hz: 2H); 6.43 (mf: 1H); 6.62 (bs: 1H), 6.82 (d, J = 8 Hz: 1H); from 7.60 to 8.00 (mt: 15H).

Mass spectrum no. 211915, m = 397 19 01 2804

El m / z = 397 m / z = 382 m / z = 262 M + · [M-CH 3] + [PPh 3] + base peak DCI m / z = 398 min / 4 + m / z = 263 [PPh 3 H] + base peak IR Spectrum 426386 KBr 3254; 2474; 1920; 1628 1520 1439; 1433; 1279; 1110; 736; 690; 527 and 511cm'1 Z and E -2-methoxy-5- [2- (3,4,5-trimethoxy-phenyl) -vinyl] -phenylamine (8 ') and (9')

In a 250 ml tricoi inerfé fazote and equipped with a magnetic stirrer, a thermometer, a tube Y, a dropping funnel, a refrigeranturemounted of a bubble and a bath 11.02 g of (1b), 4 g of (5) and 70 ml of toluene are charged to the refrigerant. .

The stirred suspension is cooled to 5 ° C. and 4.92 ml of sodium methylate solution in 25% w / w methanol are poured in over 15 minutes. The suspension is stirred for 2.5 hours at 5.degree. C., then 0.2 ml of dilute acetic acid is poured into 50 ml of water, the temperature rises to 14.degree. C. and the medium becomes very thick, diluted with 10 ml. of toluene and 10 ml of water, it remains an insoluble brown.

The mixture is filtered on clarcel, the cake is washed with 3 times 50 ml of toluene (the washings contain practically only starting formaldehyde and are not joined to the bi-phasic filtrate), the clear filtrate (pH 12) is decanted into ampoules. and the organic phase is concentrated to dryness under vacuum at 40 ° C. The Z / E ratio determined by HPLC NI is 43/57. The brown oil obtained (4 g) is chromatographed on a silica column (100 parts w / w) eluted with a mixture of cyclohexane / ethyl acetate / triethylamine (50/50/2). 2 sets of pooled fractions are concentrated to dryness, the first dry extract of 1.1 g contains 14% E-isomer and 59% Z, the second weighs 1.08 g and contains 86% E-isomer and 7% Z- .

The weight balance in Z isomer (8 ') determined by HPLC NI is 0.725 g of 4 g of aldehyde employed, ie a yield of 11.3% .Z-4- {2-methoxy-5- [2- ( 3,4,5-trimethoxy-phenyl) -vinyl] -phenylcarbamoyl} -2,2-dimethyl-oxazolidine-3-carboxy acid tert-butyl ester (11):

ου

Release of the base (8 ') from the hydrochloride (8):

In a 1 liter Erlenmayer, 44 g of (8), 16 g of sodium hydrogen carbonate and then 200 ml of distilled water and 375 ml of dichloromethane are charged. It is stirred for 20 minutes at room temperature, two impregnated phases are obtained.

The organic phase is separated by decantation, dried over sodium sulfate and filtered.

About 400 ml of chloromethylene solution containing (8 ') is obtained. Obtaining 2,2-dimethyl-oxazolidine-3,4-dicarboxylic acid-3-tert-butyl ester (10)

This product is well commercial that is very little available, so it wasprepared by saponification lithia of its methyl ester according to: J.ORG. CHEM. 63 (12) P. 3983 (1998).

I 21 01 280 4

Spectrum of R.M.N. 1H (300 MHz, (CD3) 2SO d6, δ 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 (mf spread: 1H).

Mass Spectrom: 213135, m = 2455 DCI m / z = 263 MNH4 + m / z = 246 MH + m / z = 207 [MNH4-tBu] + base peak m / z = 146 [MH-BOC] +

IR spectrum: 426759 KBr 101744; 1704; 1638; 1407; 1368; 1164; 1104; 856; 836 and 623 cm'1 t *

Coupling:

In a two-liter three-necked machine equipped with a mechanical stirrer, a thermometer, a Y tube, an addition funnel for solid, a refrigerant surmounted by a bubble-counter and a bath of The solution is cooled (8 '), 600 ml of dichloromethane are added and the mixture is cooled with stirring. At 42 ° C., 42.9 g of 2,2-dimethyloxazolidine-3,4-dicarboxylic acid-3-tert-butyl ester (10) are added, which solution is then added in portions of 5 ° to 10 ° C. 48 g of 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide, hydrochloride (EDCI). The medium is slowly allowed to return to room temperature, allowing the bath water to melt overnight.

The next day, 330 ml of distilled water are added and shaken vigorously. In 30 minutes the medium is cloudy (hydrolysis FEDCI) stirring is maintained for another 30 minutes. The medium is decanted into an ampoule and the organic phase is washed successively with water. 2 times 280 ml of 0.55 N sodium hydroxide then 300 ml of water.

The organic phase is concentrated to dryness on a rotary evaporator (50 ° C./50 mmHg). 79.4 g of a sticky oil (11) which hardens at 20 ° C. with a weight yield of (8) of 117% is obtained. .

Spectrum no. 5 578 - V 012804 22

Spectrum of R.M.N. 1H (400 MHz, (CD3) 2SO d6, at a temperature of 373 K, δ 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 3Hz: 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 (brs: 1H); 8.82 (s wide: 1H). 10

Mass Spectrum no. 213565, m = 542DCI m / z = 560 MNH4 + m / z = 543 MH + m / z = 504 [MNH4-tBu] + m / z = 443 · [MH-BOC] + base peak

IR Spectrum 425857 CCI4,3409; 2982; 2938; 2837; 1712; 1698 1534; 1363; 1249; 1133; 1092 and 851 cm -1 Other coupling conditions were implemented such as: - mixed anhydride (pivaloyl chloride / (10)) - DCC / HOBT - COD / HOSU - TOTU-NN carbonyldiimidazole - etc. - In acetonitrile, DMF, THF, Dichloromethane, ester, etc. EDCI, HCl in dichloromethane gave the best result. (Z) -N- [2-Methoxy-5- [2- (3,4,5- (trimethoxy-phenyl) vinyl] phenyl] -L-serinamide hydrochloride

(11) (12)

In a 1 liter tricolor equipped with a mechanical stirrer, a thermometer, a Y tube, a refrigerant surmounted by a bubble counter and a heating bath, the mixture is charged at 20 ° C. 8 g of (11) in solution in 54 ml of methanol are added 150 ml of isopropyl acetate, 99 ml of 2.3N methanolchlorhydrique and 8.2 ml of distilled water. Stir and heat at 60 ° C for 3 hours. The solution cooled to 40 ° C. is clarified by filtration on a sintered glass No. 4 rinsed with 40 ml of methanol. The filtrate was refluxed with stirring and 194 ml of isopropyl acetate were added, heated to 40 ° C., the solution was initiated with 0.2 g of (12) and then poured dropwise over 1 hour. isopropyl acetate, the medium crystallizes slowly during casting.

The medium is allowed to come to room temperature then cooled and kept overnight at 5 ° C.

The next day the slurry is filtered on sintered glass, the cake drained is washed with 4 times 50 ml of isopropyl acetate, wrung thoroughly and then dried in a constant weight oven (35 ° C / 10 mmHg).

28 g of (12) are obtained with a yield on 2 stages (coupling then deprotections) of: 56%, and a HPLC titre of> 98%.

That is, an overall yield as it stands, for the synthesis carried out according to the first method V0 2 of: 30%. [(12) obtained on (5) engaged].

Claims (11)

012804 24 Claims 1 - Process for the preparation of combretastatines of general formula (I) below: Wherein A represents an amino group characterized in that after Wittig condensation in the presence of bromide or chloride (3,4,5-trimethoxy-benzyl) -triphenylphosphonium and 3-nitro-4-methoxybenzaldehyde or 3, 4,5-trimethoxybenzaldehyde with a salt of (4-methoxy-3-nitro-benzyl) -triphenylphosphonium the reduction of the nitro group in the presence of iron is carried out. 2 - Process according to claim 1, characterized in that the amount of iron used is in excess. 3 - Process according to claim 2, characterized in thatexis excess is greater than 2 equivalents for one mole of starting nitro derivative. 4 - Process for the preparation of combretastatines of general formula (I) below: wherein A represents an amino group characterized in that oncondense according to the second method of implementation a salt of (3,4,5-trimethoxy-benzyl) -triphenylphosphonium with 3-amino-4-methoxy-benzaldehyde or (3-Amino-4-methoxy-benzyl) -triphenylphosphonium salt with 3,4,5-trimethoxybenzaldehyde. 012804 25 5 - Process according to claim 4 characterized in that Toncondense a (3,4,5-trimethoxy-benzyl) -triphenylphosphonium salt with 3-amino-4-methoxy-benzaldehyde. 6-Process according to claims 4 or 5, characterized in that the reaction is preferably carried out in the presence of a base selected from potassium terbutylate, sodium teramylate, desodium hydride, butyliithium, LDA (Lithium Diisopropyl Amine), sodium methylate, potassium carbonate or alkaline derivatives of hexamethyldisilanes. 7 - Process according to claims 4 or 5, characterized in that the sodium methyiate is used. 8 - Process according to claims 4 or 5, characterized in that the reaction inert solvent of the reaction is selected from ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO ..) alcohols, aromatic solvents or water 9 - Process according to claim 7, characterized in that the reaction temperature is preferably between 0 and 10 ° C 10 - Process for preparing compounds of formula (I) in the form of serine salt by coupling derivatives of formula (IIa) with the cyclic derivative of the protected serine of formula (IIb) O wherein PG represents a protecting group of the amine function to give a new intermediate of the following general formula: ## STR2 ## (111) which is then deprotected. 11 - Intermediate coupling between aminoconcetastatin and the cyclic vector of the protected serine, characterized in that it corresponds to the following formula: wherein PG is a protecting group. 12 - Intermediate according to claim 11 characterized in that PGrepresente a protecting group selected from the following groups: terbutoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyl-oxycarbonyl (FMOC).