WO2013048204A2

WO2013048204A2 - Process for preparing paclitaxel from 10-deacetylpaclitaxel

Info

Publication number: WO2013048204A2
Application number: PCT/KR2012/007951
Authority: WO
Inventors: Na-Young Lee; Jin Suk Cho
Original assignee: Samyang Genexbio Corporation
Priority date: 2011-09-30
Filing date: 2012-09-28
Publication date: 2013-04-04
Also published as: KR101378537B1; KR20130035731A; WO2013048204A3

Abstract

This disclosure relates to a process of preparing paclitaxel with high yield by semi-synthesis of paclitaxel from paclitaxel intermediate in one pot. According to the preparation process of the present invention, total steps may be progressed in one-pot reaction without separating intermediate by using 10-deacetylpaclitaxel as starting material, the process is very economical because less amount of reagents may be used, paclitaxel may be obtained with high yield amounting to 80~95% even when the process is applied for mass production by including crystallization and purification processes, thus obtaining paclitaxel more easily and efficiently, and the process is very advantageous when applied for industrial mass production of paclitaxel.

Description

[Title of the Invention]

PROCESS FOR PREPARING PACLITAXEL FROM 10- DEACETYLPACLITAXEL

[Field of the Invention]

The present invention relates to a process for preparing paclitaxel with high yield by semi-synthesis of paclitaxel from paclitaxel intermediate in one pot.

[Background of the Invention]

In the paper relating to preparation of paclitaxel [Rao, K. V.; Bhakuni, R. S.; Johnson, J.; Oruganti, R. S. J. Med. Chem. 1995, 38, 3411-3414], a technology of using hexamethyldisilazane (HMDS) to obtain paclitaxel from 10-deacetylpaclitaxel is described. However, it has disadvantage of very low paclitaxel yield because undesired by-product paclitaxel 2' -acetate is produced in large quantity due to deprotection of the unstable protection group of a trimethylsilyl group during the reaction, and thus, it is difficult to be applied for large scale industrial synthesis system.

And, US Patent No. 7,563,914 describes a method of preparing protected baccatin derivatives from baccatin in one pot with high yield (80%), however, to prepare paclitaxel according to this method, the obtained baccatin derivatives should be subjected to additional process. Specifically, according to the method described in US Patent No. 7,563,914, after obtaining protected baccatin derivatives in one pot, paclitaxel is prepared through continuous condensation reaction using the obtained intermediate, wherein total yield of paclitaxel from the raw material baccatin is reported as 65%, and thus the paclitaxel yield is very low.

As described, a process of progressing total steps of obtaining paclitaxel from 10-deacetylpaclitaxel in one pot has not be reported yet. Despite increasing demand of paclitaxel in medicinal industries, and the like, the existing technology is inefficient for producing a large quantity of paclitaxel and has limitation of low productivity. Therefore, there is urgent need for development of technology for efficiently preparing paclitaxel with high yield.

[Detailed Description of the Invention]

[Technical Problem] It is an object of the present invention to provide a process for preparing paclitaxel more easily with high yield in one pot without separation of intermediate using 10-deacetylpaclitaxel as starting material.

More specifically, the present invention provides a process for preparing paclitaxel comprising the steps of

1) reacting 10-deacetylpaclitaxel with a silylating agent to protect 2'-, 7- hydroxyl groups of the 10-deacetylpaclitaxel with silylated radicals;

2) acetylating a 10-hydroxyl group; and

3) deprotecting 2'-, 7-hydroxyl groups from the silylated radicals,

wherein the steps 1) to 3) are conducted in one-pot reaction.

[Technical Solution]

Despite increasing demand of paclitaxel in medical industries, and the like, so far developed preparation methods of paclitaxel are very inefficient when applied for mass production, and had limitations due to low productivity. Therefore, as results of studies for developing a process of preparing paclitaxel with high yield within a short time in a more efficient way, the inventors confirmed that by using 10- deacetylpaclitaxel as starting material, undesired side reactions and the resulting byproduct generation may be inhibited, total steps for obtaining paclitaxel may be progressed in one-pot reaction without separation of intermediate, paclitaxel may be prepared within a short time using less amount of reagents, thus making the process very economical, and by including crystallization and purification processes, paclitaxel may be obtained with high yield of 80-95% even when applied for mass production, thus obtaining paclitaxel in a more easy and efficient way, and completed the invention.

Therefore, the present invention provides a process for preparing paclitaxel more easily with high yield in one-pot without separation of intermediate using 10- deacetylpaclitaxel as starting material.

Hereinafter, the present invention will be explained in detail.

The present invention provides a process for preparing paclitaxel comprising the steps of

1) reacting 10-deacetylpaclitaxel (10-DAP) with a silylating agent to protect 2'-, 7-hydroxyl groups of the 10-deacetylpaclitaxel with silylated radicals;

2) acetylating a 10-hydroxyl group; and

3) deprotecting 2'-, 7-hydroxyl groups from the silylated radicals,

wherein the steps 1) to 3) are conducted in one-pot reaction.

As used herein, the term "one-pot reaction" means that the processes of 1) to 3) are continuously conducted without separating, isolating or separately obtaining intermediate formed in all steps until obtaining final product paclitaxel from starting material 10-deacetylpaclitaxel.

As such, according to the present invention, a preparation process of paclitaxel may be conducted in one-pot reaction, thus remarkably simplifying the whole preparation process, and thereby, paclitaxel may be efficiently prepared within a short time. Furthermore, since the purification process that may be optionally conducted after the step of 3) may also be conducted in one-pot like the step 3), loss of materials that may be generated at work up after the reaction or during recovery of intermediate or transfer of container may be prevented, and thus, yield and purity of paclitaxel may be remarkably improved.

Meanwhile, with regard to description of a carbon position, a single quotation mark (') after the number "2" indicating a carbon position such as "2'-" refers to the carbon position of side chain precursor, more specifically side chain precursor condensed by esterification at 13-hydroxyl group of 10-DAP. For example, if the side chain precursor is a compound of the following Chemical Formula 5, mark of each carbon position is as described in the following Chemical Formula 5.

[Chemical Formula 5]

The process for preparing paclitaxel according to one embodiment of the invention may be preferably represented by the following Reaction Formula 1.

[Reaction Formula 1]

2 ( 1 O-deacetylpaclitaxel) m

1 tpaelitaxel

In the above Reaction Formula 1 , "M" of the Chemical compound (Chemical Formula 3) prepared after the step 1), is an silylated protective radical to protect hydroxyl group.

The silylated radical may be preferably a trialkylsilyl, a dialkylarylsilyl, an alkyldiarylsilyl, or a triarylsilyl group (wherein the alkyl radical may be a linear or branched Cl-4 alkyl group, and the aryl may be a C6-10 aryl, preferably phenyl).

For example, the silylated radical may be a triethylsilyl or a trimethylsilyl group, however, in case a trimethylsilyl group is used as a protection group, deprotection of the unstable trimethylsilyl group may occur during the acetylation process and undesired by-products may be generated, and thus, a triethylsiyl group is more preferable.

To protect hydroxyl group with the silylated radical, preferably triethylsilyl group, TESC1 (chlorotriethylsilane), TBSC1 (tert-Butyldimethylsilyl chloride), or TBDPSC1 (tert-Butylchlorodiphenylsilane) may be preferably used as a silylating agent to react with 10-deacetylpaclitaxel.

And, the silylating agent may be preferably reacted in an amount of 3 to 10 moles, more preferably 4 to 6 moles per 1 mol of 10-deacetylpaclitaxel (Chemical Formula 2). If the amount of the silylating agent is under the above range, reaction rate of the silylating agent to hydroxyl group may be lowered to decrease silylation reaction efficiency, and it the amount of the silylating agent exceeds the above range, preparation cost may be increased, and thus, the process may become economically infeasible compared to effect increase.

The step 1), namely the step of reacting 10-deacetylpaclitaxel with a silylating agent to optionally silylate 2'-, 7-hydroxyl groups of the 10-deacetylpaclitaxel thus obtaining a compound (Chemical Formula 3) having 2'-, 7-hydroxyl groups protected with silylated radicals, may be preferably conducted at a temperature of 0 to 40 ^°C, more preferably 15 to 25 ^°C, or room temperature, and thus, the process is very convenient.

The step 1) may be preferably performed in a basic organic solvent or a mixed reaction solution of an inert organic solvent and base.

The basic organic solvent may be pyridine or 2,6-lutidine, preferably pyridine. And, the mixed reaction solution of an inert organic solvent and base may include an inert organic solvent selected from the group consisting of toluene, tetrahydrofuran, dichloromethane, 1 ,2-dichloroethane, ethylacetate, and chloroform, and at least one base selected from the group consisting of pyridine, 2,6-lutidine, dimethylaminopyridine, piperidine, and triethylamine.

For example, the step 1) may be performed by the action of triethylsilyl chloride, in the presence of pyridine as described in EP336840, or in the presence of basic material such as 4-dimethylamino pyridine in an inert organic solvent such as dichloromethane as described in WO 94/14787.

The mixed reaction solution of an inert organic solvent and base may be preferably used in an amount of 0.01 M to 0.8 M, more preferably 0.1 M to 0.5 M based on 10-deacetylpaclitaxel. If the concentration of the mixed reaction solution is under the above range and it is used in a diluted concentration, it takes a long time to -react, thereby delaying reaction. If the concentration exceeds the above range and it is used under concentrated condition, fluidity of the reaction solution may become lowered, and the reaction may not be uniformly progressed.

And, total volume of the basic organic solvent or the mixed reaction solution of an inert organic solvent and base may be preferably 0.5 to 10 ml per lg of 10- deacetylpaclitaxel, more preferably 1.0 to 3 ml per lg of 10-deacetylpaclitaxel.

As explained, according to the process for preparing paclitaxel of the invention, the amount of the solvent used in the step 1) may be minimized, thereby increasing the concentration of the reactants in the step 1), and thus, reaction may efficiently occur within a short time to decrease reaction time and the amount of a silylating agent, and the like used in the reaction, thereby reducing preparation cost.

Meanwhile, the base of the basic organic solvent may be preferably used in 5 to 15 equivalents based on 10-DAP, and more preferably 8 to 13 equivalents of base may be used. If the amount of the base is less than the above range, reaction rate may become lowered and thus the amount of impurities may be increased, and if it is greater than the above range, it may be difficult to remove remaining base after completion of the reaction.

Furthermore, in the mixed reaction solution of an inert organic solvent and base, the inert organic solvent may be used at the ratio of 1 : l(w/w) to 1 :20(w/w) based on the amount of the base (base : inert organic solvent). If the ratio of the inert organic solvent is greater than the above range, namely, the amount of the solvent is large compared to the amount of the base, reaction time may be lengthened, and if the ratio of the inert organic solvent is less than the above range, fluidity of the reaction solution may become lowered, and reaction may not be uniformly progressed.

The step 2), namely, the step of acetylating a 10-hydroxyl group of the compound of the Chemical Formula 3 to obtain a compound of the Chemical Formula 4 may be conducted using an acetylating agent, and the acetylating agent may be preferably at least one selected from the group consisting of acetic anhydride and acetyl halide (for example, acetyl chloride), and more preferably, acetic acid anhydride.

The acetylation process may be performed under the conditions described in EP336840, preferably by adding acetic anhydride at a temperature of 0 to 25 ^°C , in the presence of dimethylamino-4-pyridine. And preferably, the acetylating agent may be used in an amount of 1.2 to 5 moles, preferably 1.5 to 2.5 moles per 1 mol of 10— deacetylpaclitaxel .

The step 3), namely, the step of removing silylated radicals introduced at 2'-, 7- positions to obtain the compound of the Chemical Formula 1 (paclitaxel), may be preferably performed at a temperature of -10 to 30 ^°C, more preferably 0 to 10 °C, and the deprotection may be achieved by adding dropwise at least one acid selected from the group consisting of inorganic acid and organic acid, or adding dropwise the mixed solvent of the acid and organic solvent.

The inorganic acid may be for example hydrohalic acid, sulfuric acid, nitric acid, phosphoric acid, sulphamic acid, perchloric acid, chromic acid, sulfurous acid, or nitrous acid, and the like, and the organic acid may be for example halogenated acid, formic acid, acetic acid, oxalacetic acid, propionic acid, oxalic acid, glycolic acid, tartaric acid, citric acid, fumaric acid, malic acid, succinic acid, butyric acid, citric acid, or trifluoroacetic acid, and the like. And, the organic solvent may be preferably CI -4 linear or branched alcohol.

When the deprotection is achieved by adding dropwise the mixed solvent of acid and an organic solvent, the volume ratio of the acid and organic solvent included in the mixed solvent of acid and organic solvent may be preferably 1 : 0.5 to 1 : 10, more preferably 1 : 2 to 1 : 4, but is not limited thereto. And, the mixed solvent may preferably include halogenated acid (for example, hydrochloric acid) and CI -4 linear or branched alcohol (for example, ethanol or methanol).

Preferably, the process for preparing paclitaxel may further include the step of 4) separating and purifying the paclitaxel prepared through the deprotection step, after the step 3).

Preferably, the process may further include the step of 3-2) liquid-liquid extracting paclitaxel obtained through the step 3), before the step 4). The liquid-liquid extracting step may be conducted by a method commonly used in the art, and for example, it may be achieved using a common organic solvent such as dichloromethane, ethylacetate, chloroform, ether, and the like together with water.

The step 4), namely, the step of separating and purifying obtained paclitaxel may be preferably achieved by crystallization and chromatography, and specifically, paclitaxel obtained through the step 3) may be subjected to a crystallization process and separated as paclitaxel containing solid, and then, the paclitaxel containing solid may be purified by chromatography to obtain paclitaxel with high yield.

As such, according to preferred embodiment of the invention, by further including the step 4), paclitaxel may be recovered with high yield amounting to about 80-95% within a short time even if reduced amount of reagents are used in one pot. Moreover, since the step 4) may be composed in one pot like the previous steps, loss of materials, which may be generated at work up after the reaction or during recovery of intermediate or transfer of container, may be prevented, thus remarkably improving yield and purity of obtained paclitaxel.

Preferably, the step of 4) separating may be achieved by adding water or an aqueous solution of inorganic salt, and CI -4 linear or branched alcohol, or adding two or more kinds of organic solvents to precipitate and crystallize paclitaxel.

If the precipitation and crystallization step is achieved by adding water or an aqueous solution of inorganic salt, and CI -4 linear or branched alcohol, the water or an aqueous solution of inorganic salt, and CI -4 linear or branched alcohol may be preferably used in the volume ratio of 20 : 1 to 4 : 1, preferably 10 : 1 to 7 :1 to precipitate and crystallize paclitaxel. If the ratio of alcohol to water or an aqueous solution of inorganic salt is less than the above range, solubility of material in the solvent may be lowered and selective precipitation of material to be obtained may not be smoothly achieved, and thus, purity of the final product may be lowered, and if the ratio of alcohol is greater than the above range, the amount of water may become smaller to render selective precipitation and crystallization difficult, and thus, yield of the final product may become lowered.

The water or water used in the aqueous solution of inorganic salt may be tap water, distilled water, deionized water, and the like, preferably distilled water. And, the aqueous solution of inorganic salt may be aqueous solution of inorganic salt such as sodium chloride (NaCl), or ammonium chloride (NH₄C1) in the water, more preferably an aqueous solution of sodium chloride (NaCl). Meanwhile, the concentration of the inorganic salt is not specifically limited, but preferably, a saturated solution of inorganic salt may be used.

If the precipitation and crystallization step is achieved by adding two kinds of organic solvents, a polar solvent and a non-polar solvent that are miscible with each other may be preferably used as the two kinds of organic solvents, and specifically, a polar solvent may be added to dissolve paclitaxel, and then, a non-polar solvent may be slowly added to precipitate and crystallize paclitaxel. And preferably, the precipitation and crystallization step may be conducted at a temperature of 0 to 30 ^°C for 5 to 60 minutes, more preferably 10 to 30 minutes, and then, maintained at about 0 to 10 ^°C for 5 minutes to 24 hours, preferably 10 minutes to 15 hours, more preferably 30 to 60 minutes.

The polar solvent may be C2-4 linear or branched alcohol, dichloromethane, etylacetate or acetone, and the non-polar solvent may be hexane, heptane or pentane. Preferably, the polar solvent may be C2-4 linear or branched alcohol, and the non-polar solvent may be hexane.

Preferably, the polar solvent and the non-polar solvent may be used in the volume ratio of 1 :3 to 15, more preferably 1 :5 to 10.

If the ratio of the polar solvent is less than the above range, solubility of materials in the solvent may be lowered and selective precipitation of material to be obtained may not be smoothly achieved, and thus, purity of the final product may be lowered, and if the ratio of the polar solvent is greater than the above range, selective precipitation and crystallization may be difficult to lower yield of the final product.

The step of 4) separating may preferably include removing excessive amount of remaining solvents in paclitaxel to obtain solid paclitaxel, using immiscible two kinds of organic solvent.

As the two kinds of organic solvents, a polar solvent and a non-polar solvent that are immiscible with each other may be used, preferably, a polar organic solvent selected from the group consisting of methanol and acetonitrile; and a non-polar organic solvent selected from hydrocarbons having 6 or more carbon number such as hexane, heptanes, and pentane, and the like may be used, and more preferably, methanol as the polar solvent and hexane as the non-polar solvent may be used.

More specifically, the step of removing remaining solvents to obtain solid paclitaxel may include dissolving the synthesized product through the step 3) in an organic solvent, adding a non-polar organic solvent immiscible with the polar organic solvent, and vigorously stirring the reaction solution, and then, discarding the non-polar organic solvent layer and concentrating the polar organic solvent layer to obtain solidified paclitaxel. Through the process, non-polar remaining reagents may be removed as well as the non-polar organic solvent.

The crystallization of paclitaxel through removal of remaining solvents may be achieved using the polar organic solvent and non-polar organic solvent in the volume ratio of 1 :0.1 to 10, more preferably 1: 0.5 to 1 (polar organic solvent: non-polar organic solvent).

If the ratio of the polar organic solvent to the non-polar organic solvent does not fall within the above range, layer separation may become difficult, and there is a . concern for loss of synthesized paclitaxel.

The solid containing separated paclitaxel may be purified by chromatography or recrystallization, preferably chromatography.

As the solvent used in the chromatography, solvents commonly used for chromatography purification such as hexane: ethylacetate, dichlocomethane:methanol, hexane: ether, and the like may be used, and preferably, it may be purified by silica gel chromatography using hexane: ethylacetate (1 :1 volume ratio), but is not limited thereto.

The amount of paclitaxel finally obtained by the preparation process according to one embodiment of the invention corresponds to about 80-95% of 10- deacetylpaclitaxel used as starting material, thus confirming that paclitaxel may be obtained with very high yield.

[Advantageous Effects]

According to the preparation process of the present invention, all steps for obtaining paclitaxel may be progressed in one-pot reaction without separating intermediate by using 10-deacetylpaclitaxel as starting material, less amount of reagents may be used thus rendering the process very economical, paclitaxel may be obtained with high yield of 80~95% even when the process is applied for mass production by including crystallization and purification processes, thus obtaining paclitaxel more easily and efficiently, and the process is very advantageous when applied for industrial mass production of paclitaxel.

[Brief Description of Drawings]

Fig. 1 schematically shows the process of preparing paclitaxel according to one example of the invention.

[Examples]

Hereinafter, the present invention will be explained in detail with reference to the following Examples. However, these examples are only to illustrate the invention, and the scope of the invention is not limited thereto.

TESC1 : Triethylsilyl chloride

DCM : Dichloromethane

IPA : Iso-propyl alcohol

EA : Ethyl acetate

sat. NaCl : Saturated sodium chloride

cone. HC1 : Concentrated hydrochloric acid DMAP : 4-Dimethylaminopyridine

HMDS : hexamethyldisilazane

TLC : Thin layer chromatography (TLC developer: hexane/ethylacetate = 1/2) HPLC : High Performance Liquid Chromatography

column: eclipse plus CI 8, 4.6 x 150 mm, 3.5 um

temperature: 35 ^°C

flow rate: lml/min

measurement wavelength: 227 nm

concentration gradient condition: as described in the following Table 1.

[Table 1 ]

Example 1.

7.35 g of 10-deacetylpaclitaxel(9.95 mmol) was dissolved in a mixed solution of 10 ml of pyridine and 10 ml of DCM. 10 ml of TESCl (59.6 mmol) was added thereto, and then, the reaction solution was stirred at room temperature for 20 hours, and after 20 hours, completion of the reaction was confirmed by TLC. A small amount of the sample of the reaction solution was gathered, and as a result of structure determination, it was confirmed that 2,7-bis(triethylsilyl)-10-deacetyl-paclitaxel (intermediate I ) was produced.

And then, 2.2 ml of Ac₂0 (23.3 mmol) was added, the reaction solution was stirred for 2 hours, and then, the completion of the reaction was confirmed by HPLC, 10 ml of DCM was added, and the reaction solution was cooled to 0 ^°C . The sample of the solution of the completed reaction was gathered, and as a result of structure determination, it was confirmed that 2,7-bis(triethylsilyl)-paclitaxel (intermediate Π ) was produced.

20 ml of cone. HC1 and 40 ml of methanol were mixed in a separate container, and the hydrochloric acid mixture was slowly added dropwise to the reaction solution. The temperature of the reaction solution was maintained at 0 ^°C . After the addition of the hydrochloric acid mixture was completed, the reaction solution was stirred for 1 hour while maintaining the temperature of the reaction solution at 5 ^°C . After 1 hour, the completion of the reaction was confirmed by TLC, and after the completion of the reaction, 50ml of DCM and 50 ml of distilled water were added to the reaction solution to cause layer separation, and then, the DCM layer was taken. 50 ml of DCM was added again to the aqueous layer, and then, the reaction solution was extracted once again. The obtained DCM layers were combined and washed with sat. NaCl(50ml) twice, and then, dried with MgS0₄ (6g), filtered and concentrated.

30 ml of IP A was added to the concentrate to dissolve it, and 300 ml of hexane was slowly added thereto to form precipitation of paclitaxel. After the reaction solution was stirred at room temperature for 1 hour, it was filtered and dried to obtain white paclitaxel containing solid, and purified by silica gel chromatography using hexane: ethylacetate (1 :1 volume ratio) to obtain paclitaxel (6.5g) with 84% (96.8% HPLC area) yield. The properties of the each step intermediate I, II, and the final product paclitaxel were confirmed by 1H NMR and ¹³C NMR. Specifically, after completion of the reaction of each step was confirmed, a small amount of the sample of the reaction solution was gathered, separated and purified using prep-TLC (condition: hex/EA=6/l), and then, structure-determined, and the results are described as follows. intermediate I : 2,7-bis(triethylsilyl)-10-diacetyl-paclitaxel> Ή NMR (400 MHz, CDC1₃) 8.12 (d, J = 6.8 Hz, 1H), 7.76 (d, J = 6.8 Hz, 1H), 7.59 (m, 1H), 7.53-7.48 (m, 3H), 7.41-7.26 (m, 7H), 7.14 (d, J= 8.8 Hz, 1H), 6.31 (t, J= 8.8 Hz, 1H), 5.69 (dd, 7 =8.8, 2.0 Hz, 1H), 5.66 (d, J = 7.2 Hz, 1H), 5.11 (d, J = 1.6 Hz, 1H), 4.95 (dd, J = 7.6, 1.6 Hz, 1H), 4.70 (dd, J= 6.0, 1.6 Hz, 1H), 4.39 (dd, J = 10.8, 6.8 Hz, 1H), 4.32 (d, J = 8.4 Hz, 1H), 4.29 (d, J= 1.6 Hz, 1H), 4.22 (d, J = 8.4 Hz, 1H), 3.89 (d, J = 7.2 Hz, 1H), 2.56 (s, 3H), 2.47 (m, 1H), 2.37 (m, 1H), 1.96 (s, 3H), 1.95 (m, 1H), 1.21 (s, 3H), 1.09 (s, 3H), 0.94 (m, 9H), 0.82 (m, 9H), 0.55 (m, 6H), 0.48 (m, 6H); ¹³C NMR (100 MHz, CDC1₃) 209.99, 171.64, 170.37, 167.16, 167.07, 138.64, 138.31, 136.18, 134.18, 133.78, 131.91, 130.37, 129.35, 128.90, 128.83, 128.80, 128.09, 127.21, 126.62, 84.43, 81.16, 79.10, 76.75, 75.14, 75.01, 74.17, 72.96, 71.65, 57.87, 55.88, 46.57, 43.36, 37.37, 36.05, 26.79, 23.21, 21.14, 14.31, 10.30, 6.93, 6.69, 5.31, 4.49; LRMS (ESI) m/z 1062.6 (M+Na)⁺. intermediate Π : 2,7-bis(triethylsilyl)-paclitaxel>

1H NMR (400 MHz, CDC1₃) 8.12 (d, J = 6.8 Hz, 1H), 7.74 (d, J = 6.8 Hz,

1H), 7.59 (m, 1H), 7.52-7.46 (m, 3H), 7.41-7.27 (m, 3H), 7.12 (d, J= 8.8 Hz, 1H), 6.45 (s, 1H), 6.25 (t, J= 8.8 Hz, 1H), 5.71 (dd, J = 8.8, 2.0 Hz, 1H), 5.70 (d, J= 7.2 Hz, 1H), 4.95 (dd, J= 7.6 Hz, 1.6 Hz, 1H), 4.70 (dd, J = 6.0, 1.6 Hz, 1H), 4.48 (dd, J = 10.8, 6.8 Hz, 1H), 4.31 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H), 3.83 (d, J = 7.2 Hz, 1H), 2.55 (s, 3H), 2.52 (m, 1H), 2.40 (m, 1H), 2.12 (m, 1H), 2.17 (s, 3H), 2.12 (m, 1H), 2.02 (s, 3H), 1.90 (m, 1H), 1.22 (s, 3H), 1.18 (s, 3H), 0.93 (m, 9H), 0.81 (m, 9H), 0.61-0.57 (m, 6H), 0.49-0.42 (m, 6H); ¹³C NMR (100 MHz, CDC1₃) 201.87, 171.73, 170.24, 169.47, 167.18, 167.07, 140.33, 138.56, 134.19, 133.80, 133.79, 131.90, 130.37, 129.33, 128.87, 128.82, 128.80, 128.10, 127.19, 126.61, 84.38, 81.27, 78.96, 76.69, 75.12, 75.05, 74.96, 72.36, 71.57, 58.52, 55.85, 46.80, 43.45, 37.35, 35.68, 26.69, 23.51, 21.61, 21.04, 14.33, 10.26, 6.91, 6.68, 5.42, 4.49; LRMS (ESI) 1104.4 (M+Na)⁺.

1H NMR (400 MHz, CDC1₃) 8.11 (m, 2H), 7.73 (m, 2H), 7.43 (m, 5H), 7.36 (m, 5H), 7.08 (d, J= 8.8 Hz, 1H), 6.27 (s, 1H), 6.21 (t, J= 8.8 Hz, 1H), 5.77 (dd, J= 8.8 Hz, 2.5 Hz, 1H), 5.66 (d, J= 6.96 Hz, 1H), 4.93 (dd, J= 9.84, 2.16 Hz, 1H), 4.78 (d, J= 2.92 Hz, 1H), 4.38 (dd, J= 10.62 Hz, 6.6 Hz, 1H), 4.28 (d, J= 8.4 Hz, 1H), 4.18 (d, J = 8.44 Hz, 1H), 3.78 (dd, J= 6.96, 1.0 Hz, 1H), 2.54 (m, 1H), 2.37 (s, 3H), 2.29 (m, 2H), 2.24 (s, 3H), 1.88 (m, 1H), 1.77 (s, 3H), 1.66 (s, 3H), 1.21 (s, 3H), 1.12 (s, 3H); ¹³C NMR (100 MHz, CDC1₃) 203.7, 172.8, 171.3, 170.5, 167.3, 167.0, 142.0, 138.1, 133.7, 133.6, 133.2, 132.0, 130.3, 129.3, 129.0, 128.8, 128.7, 128,4, 127.2, 127.1, 84.5, 81.2, 79.0, 76.6, 75.7, 75.1, 73.3, 72.3, 72.2, 58.6, 55.2, 45.8, 43.3, 35.8, 35.7, 26.9, 22.7, 21.9, 20.9, 14.9, 9.7; LRMS (ESI) 876.32 (M+Na)⁺.

Example 2.

7.35 g of 10-DAP (9.95 mmol) was dissolved in a mixed solution of 10 ml of pyridine and 10 ml of DCM. 10 ml of TESC1 (59.6 mmol) was added thereto, and then, the reaction solution was stirred at room temperature for 20 hours. After 20 hours, the completion of the reaction was confirmed by TLC, and 2 2ml of Ac₂0 (23.3 mmol) was added, and the reaction solution was stirred for 2 hours. After stirring for 2 hours, the completion of the reaction was confirmed by HPLC, 10 ml of DCM was added, and the reaction solution was cooled to 0 ^°C . In a separate container, 20 ml of cone. HC1 and 40 ml of methanol were mixed, and then, the hydrochloric acid mixture was slowly added dropwise to the reaction solution. The temperature of the reaction solution was maintained at 0 ^°C . After the addition was completed, the reaction solution was stirred for 1 hour while maintaining the temperature of the reaction solution at 5 ^°C . After 1 hour, the completion of the reaction was confirmed by TLC, and after the reaction was completed, 50 ml of DCM and 50 ml of distilled water were added to the reaction solution, and then, the DCM layer was obtained after liquid/liquid extraction. 50 ml of DCM was added again to the aqueous layer, and then, the reaction solution was extracted again. The obtained DCM layers were combined and washed with sat. NaCl (50ml) twice, and then, dried with MgS0₄ (6g), filtered and concentrated.

30 ml of methanol was added to the concentrate to dissolve it, and 300 ml of a saturated aqueous solution of sodium chloride was slowly added to form precipitation of paclitaxel. The reaction solution was stirred at room temperature for 1 hour and filtered, the obtained precipitate was washed with 100 ml of distilled water twice and dried to obtain white paclitaxel containing solid. It was purified by silica gel chromatography using hexane:ethylacetate (1:1) to obtain paclitaxel (6.5g) with yield of 84% (97.2% HPLC area). Homology of each reaction step product was confirmed by NMR result as described in Example 1.

Example 3

7.35 g of 10-DAP (9.95 mmol) was dissolved in a mixed solution of 10 ml of pyridine and 10 ml of EA. 10 ml of TESC1 (59.6 mmol) was added thereto, and then, the reaction solution was stirred at room temperature for 20 hours. After 20 hours, the completion of the reaction was confirmed by TLC, and 2.2ml of Ac₂0 (23.3 mmol) was added, and the reaction solution was stirred for 2 hours. After stirring for 2 hours, the completion of the reaction was confirmed by HPLC, 10 ml of EA was added, and the reaction solution was cooled to 0 ^°C . In a separate container, 20 ml of cone. HC1 and 40 ml of ethanol were mixed, and then, the hydrochloric acid mixture was slowly added dropwise to the reaction solution. The temperature of the reaction solution was maintained at 0^°C . After the addition of the hydrochloric acid mixed solution was completed, the reaction solution was agitated for 1 hour while maintaining the temperature of the reaction solution at 5 ^°C . After 1 hour, the completion of the reaction was confirmed by TLC, and after the reaction was completed, 50 ml of EA and 50 ml of distilled water were added to the reaction solution, and then, the EA layer was obtained after liquid/liquid extraction. 50 ml of EA was added again to the aqueous layer, and then, the reaction solution was extracted again. The obtained EA layers were combined and washed with sat. NaCl (50ml) twice, and then, dried with MgS0₄ (6g), filtered and concentrated.

The concentrate was dissolved in 200 ml of methanol, and then, 100 ml of hexane was added to extract. After layer separation, the hexane layer was discarded, 100 ml of hexane was added again to the methanol to extract, the methanol layer was taken and concentrated, thereby removing remaining reagents. And then, the methanol layer was taken and concentrated. After further drying, white paclitaxel containing solid was obtained. The solid was purified by silica gel chromatography using hexane:ethylacetate (1 :1) to obtain paclitaxel (6.3g) with 82% yield (97.8% HPLC area). Homology of each reaction step product was confirmed by NMR result as described in Example 1. Example 4

73.5 g of 10-DAP (99.5 mmol) was dissolved in 500 ml of DCM. 100 g of DMAP (818.5 mmol) and 100 ml of TESC1 (596 mmol) were added thereto, and then, the reaction solution was stirred at room temperature for 1 hour. After 1 hour, the completion of the reaction was confirmed by TLC, 13 ml of Ac₂0 (137 mmol) was added, and the reaction solution was stirred for 1 hour. After stirring for 1 hour, the completion of the reaction was confirmed by HPLC, and the reaction solution was concentrated under reduced pressure. 400 ml of methanol was added to the concentrate, and the reaction solution was cooled to 0 ^°C . In a separate container, 200 ml of cone. HC1 and 400 ml of methanol were mixed, and then, the hydrochloric acid mixture was slowly added dropwise to the reaction solution. The temperature of the reaction solution was maintained at 0^°C . After the addition was completed, the reaction solution was stirred for 1 hour while maintaining the temperature of the reaction solution at 5 ^°C . After 1 hour, the completion of the reaction was confirmed by TLC, and after the reaction was completed, 500 ml of DCM and 500 ml of distilled water were added to the reaction solution, and then, the DCM layer was obtained after liquid/liquid extraction. 500 ml of DCM was added again to the aqueous layer, and then, the reaction solution was extracted again. The obtained DCM layers were combined and washed with 5% (w/v) NaCl aqueous solution. And, the DCM layer was dried with MgS0 (60g), filtered and concentrated.

500 ml of DCM was added to the concentrate to dissolve it. The dissolved solution was added to 2.5 L of hexane that was cooled to 4 ^°C over 10 minutes to form precipitation of paclitaxel. The precipitated mixed solution was additionally stirred at 10 ^°C for 30 minutes, and then, the precipitate was filtered and dried in a 40 ^°C dry oven to obtain white paclitaxel containing solid. The solid was purified by silica gel chromatography using hexane:ethylacetate (1 :1) to obtain paclitaxel (7.4 g) with 95% yield (97.5% HPLC area).

Comparative Example 1.

874 g of 10-DAP (1.077 mol) and 331 g of DMAP were dissolved in 5.5 L of acetonitrile, and HMDS(1121 ml) was added thereto. The reaction solution was stirred at room temperature for 2 hours, and then, concentrated. The concentrated reactants were dissolved in 5.5 L of THF, 115 ml of acetic anhydride was added, and then, the reaction solution was reacted for 6 hours. After the completion of the reaction was confirmed, reaction mixture was dissolved in 4L of methanol, and the reaction solution was cooled to 0^°C . After cooling the reaction mixture, 4N HC1 (3.3L) was slowly added dropwise over about 1 hour. After 1 hour, the completion of the reaction was confirmed, the reaction solution was extracted using saturated NaCl (3.3L) and DCM (12L), and the obtained DCM layer was washed with saturated NaHC0₃ aqueous solution (4L). Subsequently, the DCM layer was dried with MgS0₄, filtered and concentrated to obtain crude paclitaxel semi-synthetic material. The obtained crude paclitaxel semi-synthetic material was purified by silica gel chromatography (DCM→DCM:MeOH=10) to obtain paclitaxel 400g (yield 43%, 93.2% HPLC area), 10-deacetylpaclitaxel 150 g (yield 17%, 80.5% HPLC. area), and paclitaxel 2'-acetate 100 g (yield 10%, 84.5% HPLC area) [see Rao, K. V.; Bhakuni, R. S.; Johnson, J.; Oruganti, R. S. J. Med. Chem. 1995, 38, 3411-3414].

Experimental Example 1. Comparison of yield and purity of paclitaxel prepared according to Example 4 and Comparative Example 1

Yield, purity, and by-product production and the amount of by-product of paclitaxel respectively prepared according to Example 4 and Comparative Example 1 were confirmed and described in the following Table 2 [see Rao, K. V.; Bhakuni, R. S.; Johnson, J.; Oruganti, R. S. J. Med. Chem. 1995, 38, 3411-3414].

[Table 2]

Yield % [purity%] paclitaxel 10-deacetylpaclitaxel paclitaxel 2'-acetate

(recovered)

Comparative 43% [93.2%] 17% [80.5%] 10% [84.5%] Example 1

Example 4 95% [97.5%] - -

As shown in Table 2, when paclitaxel was prepared according to Comparative Example 1, paclitaxel yield was only 43%, which was less than half, undesired byproduct paclitaxel 2'-acetate was produced with 10% yield due to deprotection of the protecting group of a tnmethylsilyl group during the reaction process, and raw material 10-deacetylpaclitaxel was also recovered with yield amounting to 17%. It appears that the recovered raw material 10-deacetylpaclitaxel is not unreacted material, but is produced by deprotection of the unstable trimethylsilyl (TMS) group during the acetylation process, and in practice, it was observed that when synthetic intermediate 2', 7-bis(trimethylsilyl)-l 0-deacetylpaclitaxel was allowed to stand in the air at room temperature, deprotection of TMS was detected.

On the other hand, when paclitaxel was prepared according to Example 4, paclitaxel was obtained with remarkably high yield amounting to 2 times or more of the yield of paclitaxel according to Comparative Example 1, and by-product paclitaxel 2'- acetate or raw material 10-deacetylpaclitaxel was not produced, and thus, paclitaxel may be very efficiently prepared. Moreover, the purity of the prepared paclitaxel is 97.5%, thus confirming that paclitaxle haying remarkably higher purity than the purity of 93.2% of the paclitaxel obtained according to Comparative Example 1 may be prepared.

As explained, since the preparation process of the present invention may prepare high purity paclitaxel with high yield, it may be very usefully applied for industrial large scale synthesis system.

Claims

[Claims]

1. A process for preparing paclitaxel comprising the steps of

1) reacting 10-deacetylpaclitaxel with a silylating agent to protect 2'-, hydroxyl groups of the 10-deacetylpaclitaxel with silylated radicals;

2) acetylating a 10-hydroxyl group; and

3) deprotecting 2'-, 7-hydroxyl groups from the silylated radicals,

wherein the steps 1) to 3) are conducted in one-pot reaction.

2. The process according to claim 1, wherein the silylated radical is at least one selected from the group consisting of trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl.

3. The process according to claim 1 or 2, wherein in the step 1), 3 to 10 moles of the silylating agent are reacted per 1 mole of the 10-deacetylpaclitaxel.

4. The process according to claim 1, wherein the step 1) is performed in a basic organic solvent or a mixed reaction solution of an inert organic solvent and base.

5. The process according to claim 4, wherein the inert organic solvent is toluene, tetrahydrofuran, dichloromethane, 1 ,2-dichloroethane, ethylacetate, or chloroform, and

the base is at least one selected from the group consisting of pyridine, dimethylaminopyridine, piperidine, and triethylamine.

6. The process according to claim 1, wherein the step 2) is performed using at least one acetylating agent selected from the group consisting of acetic anhydride and acetyl halide.

7. The process according to claim 1 or claim 6, wherein the acetylating agent is used in an amount of 1.2 to 5 moles per 1 mole of 10-deacetylpaclitaxel.

8. The process according to claim 1, wherein the step 3) is achieved by adding dropwise at least one acid selected from the group consisting of inorganic acid and organic acid, or a mixed solvent of the acid and an organic solvent.

9. The process according to claim 8, wherein the organic solvent is CI -4 linear or branched alcohol.

TO. The process according to claim 1, further comprising the step of 4) separating and purifying paclitaxel prepared after the deprotection.

11. The process according to claim 10, wherein the step of 4) separating comprises adding water or an aqueous solution of inorganic salt, and CI -4 linear or branched alcohol to precipitate and crystallize paclitaxel.

12. The process according to claim 11, wherein the water or an aqueous solution of inorganic salt, and CI -4 linear or branched alcohol are added in the volume ratio of 20 : 1 to 4 : 1.

13. The process according to claim 10, wherein the step of 4) separating comprises adding two kinds of organic solvents to precipitate and crystallize paclitaxel.

14. The process according to claim 13, wherein the two kinds of organic solvents includes a polar organic solvent and a non-polar organic solvent.

15. The process according to claim 14, wherein the polar solvent is C2-4 linear or branched alcohol, and the non-polar solvent is hexane.

16. The process according to claim 14, wherein the polar organic solvent and the non-polar organic solvent are added in the volume ratio of 1 : 3 to 15.

17. The process according to claim 10, wherein the step of 4) separating comprises removing residual solvents in paclitaxel using two kinds of immiscible organic solvents to obtain solid paclitaxel.

18. The process according to claim 17, wherein the two kinds of organic solvents include

a polar organic solvent selected from methanol and acetonitrile; and

a non-polar organic solvent selected from hydrocarbon having carbon number of 6 or more.

19. The process according to claim 18, wherein the polar organic solvent and the non-polar organic solvent are used in the volume ratio of 1 : 0.1 to 10.