HRP20040935A2 - Method for preparing combretastatins - Google Patents

Description

The presented invention relates to a new process for the preparation of combretastatins and their derivatives.

The name "combretastatins" or "stilbene derivatives" means derivatives of the following general formula:

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in which A represents a hydroxyl group or an amino group and their pharmaceutically acceptable salts.

Of the salts, it is necessary to mention hydrochloride, acetate, phosphate or methanesulfonate. When compound A represents an amino group, it can also be attached to amino acids forming amides and their pharmaceutically acceptable salts.

The synthesis of stilbene or combretastatin derivatives, which can be in the form of a pharmaceutically acceptable salt and pharmaceutical preparations containing them, is shown in Patents US 4,996,237, US 5,525,632, US 5,731,353 and US 5,674,906. Said patents show combretastatins and their metabolites, and also show their oncogenic activity.

According to the mentioned patents, combretastatins are prepared from (3,4,5-trimethoxybenzyl)triphenyl-phosphonium salts, and condensed with 3-nitro or 3-hydroxy-4-methoxybenzaldehyde (which has a protected hydroxyl group), in the presence of sodium hydride or lithium derivative, then the obtained derivative, in case it is nitrated, is reduced in the presence of zinc.

The isomer of the cis configuration was then prepared under the action of light or by chromatographic separation of the mixture.

The presented invention relates to new processes for the preparation of combretastatin or their derivatives and to the improvement of existing processes.

First, the first process of route VO 1 was disclosed, for the preparation of derivatives of formula (I) in which A represents an amino group, and which was improved to the process shown in the aforementioned patents, which comprises, after Wittig condensation in the presence of (3,4,5 -trimethoxybenzyl)triphenylphosphonium bromide or chloride and 3-nitro-4-methoxybenzaldehyde, reduction in the presence of iron, instead of zinc used in previous publications, which enables obtaining a total yield of the reaction, considering the aldehyde used, of 60% (yield considering the aldehyde used in Patent US 5,525,632 amounts from 21% to 33%).

The first process of the VO 2 pathway includes the condensation of 3,4,5-trimethoxybenzaldehyde with (4-methoxy-3-nitrobenzyl)triphenyl-phosphonium bromide or chloride. For both of the first two procedures of the V0 1 and V0 2 pathways, the reaction is carried out in the presence of a base particularly selected from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA (lithium cliisopropylamine), sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilane.

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

The mentioned reaction, regarding the first procedure of the V0 2 pathway, is described in particular in the publication of K.G. Piney in Bioorg. Honey. Chem., 8 (2000), 2417-2425.

2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)-vinyl]nitrophenyl was reduced in accordance with the improved process of the invention, and by the action of iron. It is appropriate to use iron in excess, if complete conversion of the starting material is to be achieved. The excess is advantageously more than 2 equivalents per mole of the starting nitro derivative.

It was observed that the same phase, carried out in the presence of zinc in acetic acid, a common solvent for zinc reduction, does not allow a complete reaction to be achieved (in US Patent 5,525,632, the yield of the reduction reaction carried out on the pure Z isomer varies between 46 and 66%), and moreover, the quantities of zinc used are large and thus lead to the creation of a considerable amount of industrial waste. Furthermore, the process yields a large amount of an "azo" compound formed by joining the created amino group and the nitroso intermediate during the reduction.

Reduction of the generated hydrogen, derived from ammonium formate in the presence of a common 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 aforementioned Piney publication describes sodium hydrosulfite reduction of the pure nitro Z isomer, obtained by chromatography and recrystallization, to the formation of the amino Z isomer in a yield of 37%.

Hydrogenations with molecular hydrogen, catalyzed by platinum or palladium, are rarely complete, and in particular lead to saturation of the ethylene double bond.

Another process has been discovered in which the reduction of the intermediate product, which is inevitable when starting the reaction with a nitro derivative, does not occur. This means that it is significantly more economical to condense (3,4,5-trimethoxybenzyl)triphenyl-phosphonium bromide or chloride with 3-amino-4-methoxybenzaldehyde or, in accordance with the second method of carrying out the second procedure, 3,4,5-trimethoxybenzaldehyde with (3-amino-4-methoxybenzyl)-trimethylphosphonium salt.

The second procedure, in accordance with the two alternative forms, requires the existence of a phase in which CMR (Carcinogenic, Mutagenic or Reproductively Toxic) products are obtained in smaller quantities compared to the first procedures of the V0 1 and V0 2 pathways, which is of particular advantage at the industrial level in terms of safety and production costs.

According to the second process of route V0 3 of the invention, (3,4,5-trimethoxybenzyl)triphenylphosphonium salt and 3-amino-4-methoxybenzaldehyde are left together and the reaction is carried out, conveniently, in the presence of a base selected from potassium tert-butoxide , sodium tert-pentoxide, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilane. Sodium methoxide is suitably used.

The above 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 determined by a person skilled in the art depending on the used base and solvent.

The reaction temperature will be determined by a person skilled in the art depending on the base used. When methoxide is used, the temperature ranges conveniently between 0 and 10°C. At the end of the reaction, the used base was neutralized with an aqueous acid solution, the organic phase was washed and concentrated, and the expected product was obtained by chromatography of the unpurified concentrate.

According to the second process of route V0 4 for the application of the invention, in which (3-amino-4-methoxybenzyl)triphenylphosphonium salt and 3,4,5-trimethoxybenzaldehyde are left together, the reaction is conveniently carried out in the presence of an organic base selected in particular from potassium tert -butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilane. Sodium methoxide is particularly suitable for use.

The above reaction was carried out in different solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, and the like), alcohols, aromatic solvents and water, at a temperature determined by a person skilled in the art depending on the used base and solvent.

The reaction temperature will be determined by a person skilled in the art depending on the base used. When methoxide is used, the temperature is conveniently between 0 and 10°C. At the end of the reaction, the used base was neutralized with an aqueous acid solution, the organic phase was washed and concentrated, and the expected product was obtained after chromatography of the unpurified concentrate.

The derivative obtained in accordance with the second procedure of the V0 3 or V0 4 pathways or during the second stage of the first procedure of the V0 1 or V0 2 pathways is represented by the following formula:

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The advantage, when it is necessary to bind serine with the compound of formula (IIa), is to use L-serine doubly protected, on the nitrogen atom of serine and on the hydroxyl functional group, which is represented by the general formula (IIb)

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in which PG represents the protective group of the amine functional group, known to every person skilled in the art, until the formation of a new intermediate product of the following general formula:

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which is then cleaved off, conveniently, simultaneously with ring opening, by acid hydrolysis according to a deprotection reaction known to any person skilled in the art. Suitably 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).

The compound of the aforementioned formula (III) is considered new and is stated as such in the claims.

Condensation is conveniently carried out in the presence of EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride) or in the presence of DCC (dicyclohexylcarbodiimide) and HOBT (hydroxybenzotriazole) or in the presence of DCC (dicyclohexylcarbodiimide) and HOSU (N-hydroxysuccinimide) or, finally , in the presence of TOTU (O-[(ethoxycarbonyl)-cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate) or HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate) or N,N-carbonyldiimidazole. The reaction is conveniently carried out in an inert solvent with regard to the reaction itself, which is particularly selected from polar aprotic solvents, such as acetonitrile, dimethylformamide, tetrahydrofuran or chlorinated aliphatic solvents, such as dichloromethane or, finally, esters.

Binding to the derivative of formula (Ha) can certainly also be carried out by the action of a mixed anhydride, synthesized in situ between chloroformate or carboxylic acid chloride, for example pivaloyl chloride and doubly protected L-serine of formula (IIb), in the presence of the tertiary base NMM (N- methylmorpholine) types in various solvents that are inert regarding the reaction itself, esters, ethers, chlorinated solvents, acetonitrile and the like. The mixed anhydride was suitably prepared at a temperature between 0 and 10°C, and then the reaction was carried out at ambient temperature. After the reaction was completed, the reaction mixture was hydrolyzed with an aqueous solution, then the mixture was separated by phase precipitation, and the obtained organic phase was washed with a hydroxylated base.

The removal of the double protection from the compound of formula (III) is performed by the action of an organic or inorganic acid. It is suitable to use a concentrated aqueous solution of hydrochloric acid in an alcoholic medium. The temperature of the reaction mixture ranges, in accordance with a better way of realizing the invention, between 50 and 70°C.

The invention is described in more detail with the help of the following examples, which should not be understood as limiting the scope of the presented invention.

Preparations of mixtures, monitoring the course and progress of reactions and yield of non-isolated products/intermediates and their tests were determined by means of HPLC (High Performance Liquid Chromatography) analysis.

Example 1 - The first process of the V0 2 pathway in accordance with the invention

(Z)-N-[2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenyl]-1-serinamide hydrochloride

General synthesis scheme

The new "inverse Wittig" procedure, starting from (4-methoxy-3-nitrobenzyl)triphenylphosphonium bromide and 3,4,5-trimethoxy-benzaldehyde, allows obtaining a mixture of Z and E isomers of 2-methoxy-5-[2-(3, 4,5-trimethoxyphenyl)vinyl]-nitrophenyl Z/E ratio 75/25.

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The given ratio is high enough in the amount of Z nitro isomer, that the Z/E mixture can be used directly in the reduction and obtain, by crystallization of the hydrochloride, the Z amino isomer with an HPLC test of 97% IS (internal standardization).

(4-Methoxy-3-nitrobenzyl)triphenylphosphonium bromide (4) was prepared according to the following example:

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

90.5 g of 3-nitro-4-methoxybenzaldehyde (1), then 450 ml of THF and 90 ml of ethanol, was charged into a 2-liter three-necked flask equipped with a mechanical stirrer, a thermometer, a T-piece, a solids addition funnel, and a reflux condenser covered with bladder counter. The resulting light yellow solution was cooled to 10°C, and then the vessel was filled with 10 g of sodium borohydride for 40 minutes at a temperature of 10/15°C (the reaction is extremely exothermic, and the temperature must be maintained with an ice/acetone bath); at the end of the addition, the brown solution turned dark blue. The solution was stirred for 30 minutes at 10°C and the completion of the reaction was monitored by TLC (thin layer chromatography), the solution was stirred for a further hour at 10°C and then the mixture was allowed to warm to room temperature.

The addition funnel was replaced with a 500 ml dropping and pressure equalization funnel, through which 300 ml of distilled water was added drop by drop over 30 minutes, while maintaining the temperature of the mixture at 20°C. The evolution of gas was monitored when the water began to influence.

The mixture was concentrated to 2/3 on a rotary evaporator (50°C/20 mmHg), and the white product was obtained by crystallization in the aqueous concentrate in the form of pieces.

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

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

The yield at this stage is considered 100%.

N.B.: Alcohol (2) is available on the market, but it is very difficult to find. 3-Nitro-4-methoxybromobenzyl (3):

A dichloromethylene solution of 3-nitro-4-methoxybenzyl alcohol (2) was charged to a 1 L three-necked vessel equipped with a mechanical stirrer, thermometer, T-piece, solids addition funnel, and reflux condenser covered with a bubble counter, and 100 mL of dichloromethane was added. The mixed solution was cooled to 5°C, and then 135.4 g of phosphorus tribromide was added drop by drop while maintaining the temperature at 5°C.

The solution was stirred at 5°C for 1 hour and 30 minutes, the end of the reaction was monitored by TLC, and then 250 ml of saturated sodium bicarbonate solution was added drop by drop while maintaining the temperature at 15°C. A very strong evolution of gas occurred with a slight delay after the addition of phosphorus tribromide was started.

The mixture, separated by settling in a separatory funnel, was washed successively with 250 ml of distilled water and 200 ml of saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate, filtered and concentrated on a rotary evaporator (50°C/20 mmHg).

119 g of solid substance was obtained in the form of a green-yellow solid substance with a needle-like surface, chemical yield during two stages was 97%.

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

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

119 g of 3-nitro-4-methoxybromobenzyl (3) was charged into a stirred portion of 1,000 ml of toluene in a 2 L three-necked vessel equipped with a mechanical stirrer, a thermometer, a T-piece, a solids addition funnel, and a reflux condenser covered with a bubble counter, and the mixture, heated to 25°C, was added to the solution. Then 126.5 g of triphenylphosphine was added and the resulting solution was gradually heated to 60°C; a precipitate was formed at 30°C. The mixture was left at 60/65°C for 4 hours, then cooled to 30°C and filtered through a sintered glass filter. The residue obtained by filtration was washed on the filter with 300 ml of toluene twice, extracted to dryness, and dried in an oven (35°C/20 nmHg/20 hours).

217 g of (4-methoxy-3-nitrobenzyl)triphenyl-phosphonium bromide with a chemical yield of 88% were obtained.

Synthesis described in the publication: (solvent used: dichloromethane)

K.G. Piney et al., Bioorg. Honey. chem., 8(2000), 2417-2425. Spectrum no. = 4 865-V

1H N.M.R. spectrum (300 MHz, d6-(CD3)2SO, 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 EI

m/z = 262 [PPh3]* DCI base peak

m/z = 445 MNH3+

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-trimethoxy-phenyl)vinyl]-nitrophenyl (6) and (7):

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54.7 g of 3,4,5-trimethoxybenzaldehyde (5), 148.6 g of (4-methoxy-3-nitrobenzyl)triphenylphosphonium bromide (4) and 1,300 ml of toluene were charged, at a temperature of 20°C under nitrogen, in 2 liter triple-door vessel equipped with mechanical stirrer, thermometer, T-piece, dropping funnel and reflux condenser covered with bubble counter. The stirred suspension was cooled to 5°C using an ice bath, and then 63.2 g of a 25% w/w solution of sodium methoxide in methanol was added at a temperature of 5°C over 40 minutes.

As the addition progressed, the suspension changed color from off-white to yellow and then brown.

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

The suspension was heated to 40°C and left at 40°C for 30 minutes. At this temperature, only salts remain in insoluble form. The mixture was filtered at a temperature of 40°C through a sinter glass filter No. 3, and the salts were washed on the filter with 100 ml of toluene 3 times.

The filtrate was returned to a round-bottomed vessel filled with 250 ml of distilled water, and the mixture containing the two phases was stirred for 20 minutes at a temperature of 40°C and then separated by sedimentation in a separatory funnel. The toluene phase was washed again with 2 times 250 ml of distilled water and then concentrated to dryness on a rotary evaporator.

The residue was treated in 600 ml of isopropanol and 12 ml of toluene at 40°C, the expected product began to crystallize, and the mixture was allowed to warm to ambient temperature overnight with slow stirring.

The mixed suspension was cooled and left at a temperature of 5°C for 1 hour, then it was filtered through a sintered glass filter and the cake was washed twice with 125 ml of isopropanol, drawn dry and dried in an oven under vacuum (35°C/ 30 mmHg/ 18 hours).

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

Synthesis described in the publication: {solvent used: dichloromethane: : base used: NaH)

K.G. Piney et al., Bioorg. Honey. Chem., 8 (2000), 2417-2425. N.B.: A number of operating conditions have been tested, as follows: 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 -10c to the reflux temperature of the same solvents Z-2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]-phenylamine hydrochloride (8):

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80 g of 75/25 Z and E mixture

2-Methoxy-[2-(3,4,5-trimethoxyphenyl)vinyl]nitrophenyl (6} and (7), 640 ml of absolute ethanol and 160 ml of distilled water were charged, at a temperature of 20°C and under nitrogen, in 2 liter triple door vessel equipped with mechanical stirrer, thermometer, T-piece, solids addition funnel, reflux condenser covered with bubble counter and heating bath.

The mixture was quickly mixed and heated in an oil bath, 7.8 ml of 6N hydrochloric acid was added to the suspension at a temperature of 50°C, and then the mixture was heated to 77 ± 2°C. The mixture is virtually soluble.

52 g of iron powder was added in portions over 5 minutes. With the first addition, the mixture turns into a solution, and then a black deposit is formed on the walls of the round-bottomed container.

The mixture was left at a temperature of 77 ± 2°C for 2 hours, and the disappearance of the initial nitro compounds (6) and (7) was monitored by HPLC.

The mixture was allowed to cool to 40°C and was filtered through a sintered glass filter covered with clarcel, and the cake was washed twice with 160 ml of 80/20 ethanol/water mixture.

The filtrate, aqueous mother liquors and aqueous wash liquors, is concentrated on a rotary evaporator. As soon as the azeotrope was removed, the oil began to settle in the remaining aqueous phase.

The aforementioned aqueous phase was extracted on a separatory funnel with 2 times 300 ml of dichloromethane, then the organic phase was washed with 2 times 300 ml of semi-saturated sodium chloride aqueous solution and with 300 ml of distilled water.

The organic phase was concentrated to dryness on a rotary evaporator to give 76 g of an oil showing a Z/E ratio of 80/20 as determined by HPLC. Said oil was dissolved in 591 ml of methanol, mixed and transferred to a 1 liter round-bottom vessel, then 100 ml of 2.32N methanolic hydrochloric acid was added, precipitation was started and the mixture was left to settle overnight with stirring.

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

In the morning, the mixture was filtered through a sintered glass filter. The dried cake weighs 8.2 g and consists only of the E isomer (HPLC).

The filtrate (693 g), ratio Z/E = 86/14 (IS HPLC), was concentrated to half on a rotary evaporator, 400 ml of acetonitrile was added to 347 g of concentrate and the mixture was concentrated again until a concentrate of 347 g was formed again.

Then 1000 ml of acetonitrile was added and the mixture was concentrated until crystallization began. The concentrate was then mixed and transferred to a 4-liter round-bottom vessel containing 1,500 ml of acetonitrile at a temperature of 60°C. The mixture precipitates profusely.

Stirring of the mixture was continued at 60°C for 2.5 hours and then the mixture was allowed to cool to 30°C for approximately 1 hour. The mass was filtered through a sintered glass filter (E isomer (9) is soluble in the filtrate). The cake was washed twice with 200 ml of acetonitrile and dried in an oven (35°C/30 mmHg/18 hours).

45.7 g of Z-2-methoxy-5-[2-(3,4,5-trimethoxy-phenyl)vinyl]phenylamine (8) were obtained with an IS HPLC test of 97% and a yield of 56%, i.e. the yield of the obtained Z of the isomer compared to the charged Z isomer is 72%.

EXAMPLE 2 - Synthesis in accordance with the second method of route V0 3 of the invention

The advantage of the second procedure of the V0 3 pathway compared to the first «reverse Wittig» procedure of the V0 2 pathway is in the performance of the Wittig reaction between the already reduced product, aminoaldehyde (1a) and phosphonium (2a), and thus the chemical stage in which the creation of CMR products.

(Z)-N-[2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenyl]-1-serinamide hydrochloride

General synthesis scheme

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20 g of 3-nitro-4-methoxybenzaldehyde (1) and 350 ml of absolute ethanol filled in a 2-liter three-necked vessel were left in an inert atmosphere of argon and equipped with a mechanical stirrer, thermometer, T-piece, solids addition funnel, reflux condenser covered with a bubble counter, and a heating bath and the mixture was stirred and heated to 60°C. The mixture was turned into a solution.

115 ml of distilled water at 60°C was added drop by drop, then 14 ml of 2N hydrochloric acid was added. Then 24.7 g of iron powder was added in portions.

The mixture was allowed to cool at ambient temperature for 2 hours. The reaction was complete (TLC).

The mixture was filtered through celite and concentrated under vacuum, the residue was treated in dichloromethane, and the organic solution was washed twice with distilled water, then dried over magnesium sulfate, filtered and concentrated to dryness under vacuum.

16 g of unpurified (1a) product was obtained, which was then chromatographed on a silica gel column eluting with dichloromethane.

Two fractions containing the pure expected product were obtained, from which, after concentration, 11.5 g of the pure (1a) product was obtained, i.e. a yield of 69%.

1H N.M.R. spectrum No. 2810-V (300 MHz, d6-(CD3)2SO, δ in ppm): 3.88 (s, 3H), 5.11 (unmelted 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+ DCI 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)vinyl]-phenylamine (8') and (9'):

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N.B.: Phosphonium (2a) represents the starting material already known from the original Ajinomoto Co. patent. Ltd, US 5 525 632 and WO 01/12579 A2.

8.0 g of phosphonium salt (2a), then 2.20 g of aminobenzaldehyde (la) and 100 ml of toluene, were filled into a 250 ml three-necked vessel and left under an inert nitrogen atmosphere, which was equipped with a magnetic stirrer, a thermometer, a T part, a funnel for dropwise, with a reflux condenser covered with a bubble counter and a cooling bath. The stirred suspension was cooled to 5°C and 3.51 ml of a 25% w/w solution of methanolic sodium methoxide was added over 15 minutes. After 2.5 hours at 5°C, the reaction remained incomplete (DC: 45%), but no further change occurred (HPLC), and the Z/E ratio was 61/39. Then 0.2 ml of acetic acid diluted with 50 ml of water was added, the temperature rose to 13°C and the mixture was stirred for 30 minutes and then separated by settling in a separatory funnel. The organic phase was concentrated under vacuum on a rotary evaporator to give 8 g of a yellow oil.

HPLC showed that the mentioned oil contains the starting aldehyde, phosphine oxide and the expected mixture of Z/E ratio 61/39.

The oil was chromatographed on a silica gel column (40 parts w/w) eluting with a mixture of cyclohexane/ethyl acetate/triethylamine (50/50/2).

Two series of combined fractions were concentrated to dryness: the first dry extract of 360 mg contained 93% Z isomer + unidentified impurities; the second, of 2.09 g, contains the starting aldehyde and a Z/E mixture of 39 and 37.5% determined 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 the aldehyde charge, i.e. a yield of 24%.

EXAMPLE 3 - Synthesis according to the second process of the V0 3 pathway according to the invention

Compared to the V0 2 route, the advantage of the V0 3 route with respect to the first "inverse Wittig" procedure of the V0 2 route is that the performance of the Wittig reaction between the previously reduced product, (3-amino-4-methoxybenzyl)triphenylphosphonium bromide (1b) and 3,4,5-trimethoxybenzaldehyde (5), leads to the elimination of the chemical phase in which CMR products are formed. (Z)-N-[2-Methoxy-5-[2-(3-4,5-trimethoxyphenyl)vinyl]-phenyl]-1-serinamide hydrochloride

General synthesis scheme

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30 g (4), 240 ml of ethanol and 60 ml of distilled water were charged into a 1 L three-necked vessel equipped with a mechanical stirrer, a thermometer, a T-piece, a solids addition funnel, a reflux condenser covered with a bubble counter, and a heating bath.

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

Then, 9.9 g of iron powder was added in portions over 15 minutes; the mixture remained insoluble. The mixture was left at a temperature of 75°C for 2 hours; organic materials were slowly added to the solution until a brownish precipitate of iron and iron oxide was formed.

After monitoring by HPLC, 5% of the starting material still remained; 2 g of iron were again added and heating was continued for 1 hour; DC is complete.

The mixture was cooled to 40°C and filtered through clarcel, the residue obtained by filtration was washed with 100 ml of ethanol containing 20% water and the filtrate was concentrated to dryness under vacuum on a rotary evaporator.

The residue was treated with 300 ml of isopropanol and crystallized from the mixture, which was further stirred and heated to 50°C and turned back into a liquid. Then 14 ml of a 5N solution of hydrochloric acid in isopropanol was added, the mixture precipitated, it was left at 50°C for 1 hour, and then it was allowed to return to room temperature.

The mass was filtered through a sintered glass filter, and then the cake was washed with 50 ml of isopropanol, taken out and dried well in an oven under vacuum.

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

1H N.M.R. spectrum No. 4584-V (300 MHz, d6-(CD3)2SO, δ in ppm): 3.78 (s, 3H), 5.03 (broad d, J = 15 Hz, 2H), 6.43 (unmelted 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

[image] 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]-phenylamine (8') and (9'):

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11.02 g (1b), 4 g (5) and 70 ml of toluene were charged into a 250 ml three-necked vessel and left under an inert atmosphere of nitrogen, which was equipped with a magnetic stirrer, a thermometer, a T-piece, a dropping funnel, a reflux condenser covered with a counter bubbles and cooling baths.

The stirred suspension was cooled to 5°C and 4.92 ml of a 25% w/w solution of sodium methoxide in methanol was added over 15 minutes. The suspension was stirred at 5°C for 2.5 hours, then 0.2 ml of acetic acid diluted in 50 ml of water was added and allowed to warm up to 14°C, and the mixture became extremely thick. It was diluted with 10 ml of toluene and 10 ml of water. A brown insoluble material remains.

The mixture was filtered through clarcel, the cake was washed 3 times with 50 ml of toluene (the washings contained virtually only the initial aldehyde and were not added to the biphasic filtrate), the clear filtrate (pH 12) was separated by settling in a separatory funnel, and the organic phase was concentrated to dryness under vacuum at a temperature of 40°C. The Z/E ratio, determined by IS HPLC, is 43/57.

The resulting brown oil (4 g) was chromatographed on a silica gel column (100 parts w/w) eluting with a mixture of cyclohexane/ethyl acetate/triethylamine (50/50/2).

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

The mass balance of the Z isomer (8'), determined by IS HPLC, is 0.725 g in relation to 4 g of charged aldehyde, i.e. the yield as such is 11.3%.

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

[image]

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

44 g (8), 16 g of sodium bicarbonate, then 200 ml of distilled water and 37 5 ml of dichloromethane were filled into a 1 liter Erlenmeyer flask. The mixture was stirred for 20 minutes at ambient temperature and two clear phases were obtained.

The organic phase was separated by precipitation, dried over sodium sulfate, and then filtered.

Approximately 400 ml of dichloromethane solution containing (8') was obtained

Preparation of 2,2-dimethyloxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester (10)

Although available on the market, the product is difficult to find. Therefore, it is prepared by saponification with lithium hydroxide of its methyl ester in accordance with: J. Org. Chem., 63(12), p. 3983 (1998).

lH N.M.R. spectrum (300 MHz, d6-(CD3)2SO, 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 (broad undissolved peak, 1H).

Mass spectrum no. 213135, m = 245

EI m/z = 263 NMH4+

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

Binding:

The solution (8') was charged to a 2 L three-necked vessel equipped with a mechanical stirrer, thermometer, T-piece, solids addition funnel, reflux condenser covered with a bubble counter, and an ice bath, 600 ml of dichloromethane was added and the mixture was cooled with stirring. .

42.9 g of 2,2-dirnethyloxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester (10) was added to the solution at 5°C, and then 48 g of 1-{3-dimethylaminopropyl)-3- of ethylcarbodiimide hydrochloride (EDCl) at a temperature between 5 and 10°C.

The mixture was allowed to slowly return to ambient temperature by melting the ice in an ice bath overnight.

In the morning, 330 ml of distilled water was added and the mixture was stirred vigorously. It became cloudy over 30 minutes (EDCl hydrolysis). Mixing was continued for the next 30 minutes.

The mixture was separated by settling in a separatory funnel, and the organic phase was washed sequentially with 2 times 180 mg of 0.55 N sodium hydroxide solution and then with 300 ml of distilled water.

The organic phase was concentrated to dryness on a rotary evaporator (50°C/50 mmHg).

79.4 g of sticky oil (11) was obtained, which turns into a solid phase at a temperature of 20°C, in a yield of 117% compared to the filled (8).

Spectrum no. = 5 578-V

1H N.M.R. spectrum (400 MHz, d6-(CD3)2SO, 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 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 NMH4* base peak

m/z = 543 MH+

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

m/z = 443 [MH-BOC]+

IR spectrum 425857 CCl4

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

Other join conditions are used, such as:

- Mixed anhydride (pivaloyl chloride/(10)).

- DCC/HOBT, DCC/HOSU, TOTU, N,N-carbonyldiimidazole and the like.

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

EDCl-HCl in dichloromethane gives the best result.

(Z)-N-[2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]-phenyl]-1-serinamide hydrochloride

[image]

61.8 g of (11), in a solution of 54 ml of methanol, was charged at a temperature of 20°C into a 1 L three-necked vessel equipped with a mechanical stirrer, a thermometer, a T-piece, a reflux condenser covered with a bubble counter, and a heating bath, and 150 ml of isopropyl acetate, 99 ml of a 2.3N solution of hydrochloric acid in methanol and 8.2 ml of distilled water. The mixture was stirred and heated to 60°C for 3 hours. The solution, cooled to 40°C, was purified by filtration through sintered glass filter no. 4 washed with 40 ml of methanol. The filtrate was returned to a stirred three-neck vessel and 194 ml of isopropyl acetate was added, the mixture was reheated to 40°C, 0.2 g (12) was added to the solution, and then 194 ml of isopropyl acetate was added dropwise over 1 hour. The mixture slowly crystallized as isopropyl acetate was slowly added.

The mixture was allowed to return to ambient temperature and then cooled and left overnight at 5°C.

In the morning, the mass was filtered through a sinter glass funnel and cake, taken out dry, washed 4 times with 50 ml of isopropyl acetate, dried well, and dried in an oven until reaching a constant weight (35°C/10 mmHg).

28 g (12) was obtained in a yield, during 2 stages (binding and deprotection reactions), of 56%, and determined by the IS HPLC test > 98%, i.e. the total yield, for the synthesis performed in accordance with the first procedure of the V0 2 pathway, of 30% [(12) obtained in relation to the amount of loaded (5)].

Claims (12)

1. Process for the preparation of combretastatins, indicated by the fact that they are selected from the group of compounds represented by the general formula (I): [image] in which A represents an amino group, and is characterized by the fact that after Wittig condensation in the presence of (3,4,5-trimethoxybenzyl)triphenylphosphonium bromide or chloride and 3-nitro-4-methoxybenzaldehyde or 3,4,5-trimethoxybenzaldehyde with (4 -methoxy-3-nitrobenzyl)triphenylphosphonium salt, reduction of the nitro group with iron occurs. 2. The process according to claim 1, characterized in that iron is used in excess. 3. The process according to claim 2, characterized in that the stated excess of iron is greater than 2 equivalents per one mole of the initial nitro derivative. 4. Process for the preparation of combretastatins, indicated by the fact that they are selected from the group of compounds represented by the following general formula (I): [image] in which A represents an amino group, and is characterized by the fact that, in accordance with another applied procedure, the {3,4,5-trimethoxybenzyl)triphenylphosphonium salt is condensed with 3-amino-4-methoxybenzaldehyde or is (3-amino-4- methoxybenzyl)triphenylphosphonium salt condensed with 3,4,5-trimethoxybenzaldehyde. 5. The method according to claim 4, characterized in that the (3,4,5-triaethoxybenzyl)triphenylphosphonium salt is condensed with 3-amino-4-methoxybenzaldehyde. 6. The method according to claim 4 or claim 5, characterized in that the reaction is conveniently carried out in the presence of a base selected from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA (lithium diisopropylamine), sodium ethoxide, potassium carbonate or alkali derivatives or hexamethyldisilane. 7. Process according to claim 4 or claim 5, characterized in that sodium methoxide is used. 8. The method according to claim 4 or claim 5, characterized in that the reaction solvent, inert in relation to the selected reaction, is chosen from ether (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO and the like), alcohol , aromatic solvents or water. 9. The method according to claim 7, characterized in that the temperature of the reaction mixture is suitably between 0 and 10°C. 10. Process for the preparation of compounds of formula (I), characterized in that the serine salt is obtained by the reaction of derivatives of formula (IIa) [image] with a cyclic protected serine derivative of the formula (IIb) [image] in which PG represents the protective group of the amino functional group, until the formation of a new intermediate product of the following general formula: [image] which was then deprotected. 11. An intermediate product of the coupling of aminocombretastatin and a cyclic protected serine derivative, characterized by the fact that it is represented by the following general formula: [image] where PG denotes a protecting group. 12. Intermediate product according to claim 11, characterized in that PG represents a protecting group selected from the following compounds: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC).