WO1996006852A1

WO1996006852A1 - Novel taxol derivative

Info

Publication number: WO1996006852A1
Application number: PCT/JP1995/001684
Authority: WO
Inventors: Morihiro Mitsukuchi; Hisaya Wada; Yoshinori Sekiguchi; Chihiro Yokoo; Katsuo Hatayama
Original assignee: Taisho Pharmaceutical Co., Ltd.; Institute Of Materia Medica Chinese Academy Of Medical Sciences
Priority date: 1994-08-26
Filing date: 1995-08-25
Publication date: 1996-03-07
Also published as: AU3265195A

Abstract

A water-soluble taxol derivative represented by general formula (II) and different from conventional prodrug type of compounds, (wherein R represents hydrogen or methyl). The water solubility has been improved by introducing a highly polar amino sugar unit into the 7-hydroxy group of taxol by an organic synthetic chemical technique, followed by salt formation. As compared with taxol itself, this derivative has an excellent antitumor activity, an improved water solubility and a reduced toxicity.

Description

Description New Taxol Derivative Technical Field

The present invention relates to a novel taxol derivative having antitumor activity. Background art

Yuxol is an antitumor diterbene compound isolated from the bark of Pacific yew. Taxol binds to tubulin, promotes its polymerization, stabilizes the formed microtubules, thereby inhibiting microtubule depolymerization, and as a result, the spindle force during cell division does not function normally It has a novel mechanism of action in which cell division is inhibited. It has also been reported in clinical practice that it has excellent anticancer effects on ovarian cancer and breast cancer.

Since taxol is very insoluble in water, it is dissolved in a mixture of polyoxyethylene castor oil (Cremophor EL), a solubilizing agent, and 50Z50 (v / v) of ethanol. At present, injections prepared by diluting 10-fold are administered intravenously by intravenous drip.

As side effects of taxol, myelosuppression, hypersensitivity reaction, cardiotoxicity, neurotoxicity, gastrointestinal tract disorders, hair loss, myalgia, joint pain and the like have been reported. Of these, hypersensitivity reactions (dyspnea due to bronchospasm, rash, hypotension, anaphylaxis, etc.) are thought to be derived from polyoxyethylene castor oil used as a solubilizing agent. To reduce this, pretreatment with corticosteroids, histamine H I antagonists and histamine H 2 antagonists has been performed.

Also, in order to avoid side effects caused by polyoxyethylene castor oil, many studies on water-soluble derivatives and water-soluble preparations of taxol have been reported. For example, WO9004104 (V.J.Ste.11a etc.), U.S. Pat. No. 5,157,049 (A.R.D. Haugwitz etc.), Europe Patent No. 0 537 905 (D. G. I. Kingston et al.), United States Patent No. 5, 411, 984 (DG I. Kingston et al.), U.S. Pat. No. 5,422,364 (KC Nico 1 aou, etc.), WO 95 Z 18804 (KCNico I aou, etc.), European Patent 0558959 (Bristol-Myers Squibb) and EP 0639577 (Bristol-Myers Squibb). In these reports, the hydroxyl group at the 2'-position or 7-position of taxol is esterified with a substituent having a water-soluble functional group such as carboxylate, quaternary ammonium salt, quaternary pyridinium salt, sulfonate and phosphate. The present invention relates to a taxol derivative having improved water solubility by bonding or acetanol bonding. These water-soluble taxol derivatives are water-soluble taxol prodrugs in which a water-soluble substituent introduced into the hydroxyl group at the 2'-position or 7-position of taxol is hydrolyzed in vivo to produce taxol. It is oriented. Also, US Pat. No. 5,424,073 (Georgetown University) discloses a study on taxol ribosome formulations.

As a taxol glycoside, a compound represented by the formula (I) in which xylose is bonded to the hydroxyl group at position 7 is known [J. Nat. Pr 0 d., Vol. 47, p. 131, 1984].

Expression

This 7-xylosyltaxol has a microtubule depolymerization inhibitory activity equivalent to that of oxol [Proc. Natl. Acad. Sci. USA, 81, 4090, 1984] and It has been reported to have in Vitro antitumor activity [Pharm. Res., 10, 521, 1993]. Also, Taki W

3 Glycosides of sole and its analogs are obtained only from nature, and there is no report that a sugar was introduced into the hydroxyl group at the 7-position of taxol by organic synthetic chemistry.

An object of the present invention is to introduce a novel water-soluble hexol derivative different from the conventional water-soluble prodrug derivative, that is, a highly polar amino sugar into the hydroxyl group at the 7-position of taxol by an organic synthetic chemistry technique, and then add a salt. By forming it, it avoids the use of polyoxyethylene castor oil, which is a cause of hypersensitivity reactions due to improved water solubility, while retaining the excellent antitumor activity of taxol, and other toxicities derived from taxol And to provide a novel taxol derivative which also reduces the above. Disclosure of the invention

As a result of intensive studies on the introduction of an amino sugar derivative into the hydroxyl group at the 7-position of taxol, the present inventors have succeeded in synthesizing the 7-amino sugar glycoside of taxol. The salt was found to have improved water-solubility compared to taxol, and exhibited the same antitumor activity as taxol, and further reduced compared to taxol. 5 completed.

Hereinafter, the present invention will be described.

The compounds of the present invention have the formula

[Wherein, R represents a hydrogen atom or a methyl group. ] The taxol derivative represented by these, and its medical ^ JL acceptable salt.

In the present invention, the medically acceptable acid addition salts include, for example, acetate, propionic acid Salt, butyrate, lactate, maleate, fumarate, tartrate, citrate, stearate, succinate, ethylsuccinate, benzoate, salicylate, methansulfonate, ethanesulfone Acid salt, 2-hydroxyethanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, radium sulfate, malate, aspartate, glutamate, adipate, cysteine salt , Lactobionate, glucuronate, hydrochloride, hydrobromide, phosphate, sulfate, hydroiodide, oxalate, picrate.

The method for producing the compound of the present invention represented by the formula (II) is as shown in the following production schemes (I) and (II).

Manufacturing scheme (I)

(2)

ZCI ₊ o ⁰

TrocHN ^ _z NHTroc

(3) (4)

(3)

H

CCI

I,

TrocCI ο

(4) OZ hydrocracking

TrocO

Yuki TrocHN

(9)

(6) + β-anoma of (6) ~-(7) + (8)

[In the scheme, Z represents a benzyloxycarbonyl group, and Troc represents a 2-hydroxy-2-chloroethoxycarbonyl group. ] In other words, the compound (1) described in the literature (Bull. Chem. Soc. Jpn., Vol. 60, p. 2205, 1987) can be prepared by adding paraaldehyde to the compound (1) in the presence of an acid catalyst. Reaction is carried out at room temperature for about 2 to 48 hours to obtain compound (2).

Examples of the acid for the acid catalyst include inorganic acids such as sulfuric acid, hydrochloric acid, and phosphoric acid, and organic acids such as acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, camphorsulfonic acid, and paratoluenesulfonic acid. .

Then, the compound (2) and benzyloxycarbonyl chloride are mixed with an organic solvent such as 1,4-dioxane and water in the presence of a base such as sodium hydroxide, hydroxylammonium hydroxide or sodium hydroxide. A mixture of compound (3) —anomer) and compound (4) {β-anomer) is obtained by reacting at −10 to −48 hours at room temperature. Both compounds can be separated by silica gel column chromatography.

Compound (5) is obtained by reacting compound (3) with 2,2,2-trichloromouth ethoxycarbonyl chloride in an organic solvent in the presence of a base at 0 to room temperature for about 1 to 24 hours. Similarly, compound (9) can be obtained from compound (4). Similarly, a mixture of compound (5) and compound (9) can be obtained from a mixture of compound (3) and compound (4).

Examples of the base used in the above reaction include organic bases such as pyridine, triethylamine, diisopyrupyramine, and 4-dimethylaminopyridine. Examples of the organic solvent include halogen solvents such as chloroform, methylene chloride and 1,2-dichloroethane, and pyridine.

Hydrogenolysis of compound (5) at room temperature in the presence of a catalyst such as palladium-carbon in an organic solvent gives compound (6). Similarly, a mixture of compound (6) and its yS-anomer can be obtained from compound (9). Similarly, a mixture of compound (6) and its —anomer can be obtained from a mixture of compound (5) and compound (9).

Examples of the organic solvent used in the above reaction include ethyl acetate, tetrahydrofuran, 1,4-dioxane, an alcoholic solvent such as methanol and ethanol, or a mixed solvent thereof. As a catalyst, besides palladium-carbon Examples include palladium black, platinum oxide, Raney nickel, and barium palladium monosulfate.

Then, the compound (6) is reacted with trichloroacetonitrile in an organic solvent in the presence of a base at room temperature for about 1 to 24 hours to give the compound (7) (α-anomer) and the compound (8) (; δ-anomer). Obtain the mixture of 1). Similarly, a mixture of compound (7) and compound (8) can be obtained from a mixture of compound (6) and its 9-anomer. Compound (7) and compound (8) can be separated by silica gel column chromatography.

The most suitable organic solvents used in the above reaction include halogenated solvents such as methylene chloride, 1,2-dichloroethane and chloroform, and the bases include potassium carbonate, sodium hydride, 1,8- Diazabicyclo [5,4,0) -7-decene and cesium carbonate.

Manufacturing scheme (II)

[In the scheme, Troc is as defined above. ] Compound (10) A known compound described in the literature (J. Med. Chem., Vol. 35, p. 145, 19992). With compound (7) in an inert gas atmosphere such as nitrogen or argon, in an anhydrous organic solvent, in the presence of an acid catalyst and a dehydrating agent, at a temperature of from 120 to room temperature for 30 minutes to 24 hours to give the compound (7). 11) is obtained. Similarly, compound (11) can be obtained also using compound (8) or a mixture of compound (7) and compound (8).

Examples of the organic solvent used in the above reaction include a halogen-based solvent such as methylene chloride, 1,2-dichloroethane, and chloroform, or a single or mixed solvent such as toluene, benzene, getyl ether, acetonitrile, and nitromethane. Examples of the acid catalyst include trimethylsilyl trifluoromethanesulfonate, boron trifluoride dimethyl ether complex, silver trifluoromethanesulfonate, silver perchlorate, silver carbonate, mercury cyanide, mercury bromide, Examples include toluenesulfonic acid and camphorsulfonic acid. Examples of the dehydrating agent include powdered molecular sieve 4A and anhydrous calcium sulfate.

The compound (11) is reacted with the zinc powder in an organic solvent in the presence or absence of an acid at 0 to room temperature for about 1 to 24 hours to give the compound of the present invention represented by the formula (II). A compound (12) in which R is a hydrogen atom is obtained.

Examples of the organic solvent used in the above reaction include alcohol solvents such as methanol, ethanol, and propanol, and tetrahydrofuran, 1,4-dioxane, and ethyl acetate. Examples of the acid include hydrochloric acid, sulfuric acid, and acetic acid. Further, by reacting compound (12) with a reducing agent such as formalin and sodium cyanoborohydride or sodium borohydride in an alcoholic solvent such as methanol at room temperature for about 1 to 24 hours after reducing the temperature from zero. The compound (13) of the present invention represented by the formula (II) wherein R is a methyl group is obtained.

Each target compound in the above reaction can be obtained by general isolation and purification methods, for example, extraction, chromatography, and recrystallization.

The pharmaceutically acceptable salts of the compounds of the present invention can be obtained by converting compound (12) or compound (13) into methylene chloride, chloroform, ethyl acetate, tetrahydrofuran, methanol, ethanol, tert -Organic solvents such as butanol It can be obtained by adding an acid in or in a mixed solvent of water and these, and by an isolation or purification method such as chromatography, recrystallization, reprecipitation or lyophilization.

The effective dose of the compound of the present invention is 1 mg 500 mg, preferably 1 mg 30 Omg per lm ^{2 of} adult daily Z body surface area. This dosage may be adjusted as appropriate according to the patient's age, weight and condition.

The compound of the present invention thus obtained can be used as an injection or as a known additive such as an excipient, a disintegrant, a binder, a lubricant, an antioxidant, a coating agent, a coloring agent, Hashimi Bridge Oral preparations such as granules, powders, capsules, tablets, dry syrups, and liquid preparations can be prepared by mixing odorants, surfactants, plasticizers, and the like in a conventional manner. These additives can be used in general. Industrial applicability

The compound of the present invention exhibits excellent antitumor activity, and has been found to have improved water solubility and reduced toxicity compared to taxol, and is useful as an antitumor agent. Example

Hereinafter, the present invention will be specifically described with reference to Examples and Test Examples. Example 1 Production of Compound (2)

To 73.33 g of the compound (1), 36 Om1 of paraaldehyde and 1.5 ml of concentrated sulfuric acid were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the precipitated solid was collected by filtration and washed with getyl ether to obtain colorless amorphous (2) 74.68 ^ (yield 87.7%) o

NMR-NMR (20 OMH z) (DMS 0- d ₆ ) 6: 1.23 (3 H, d, J = 5 Hz), 3.19 (1 Hd, J = 8 Hz), 3.3 7 3.53 (3 H, m, 1 H, exchangeable), 3.5 7 3.74 (2 H, m), 3.86 6 3.98 (1 H, m), 4 74 (1H, dJ = 12Hz), 4.76 (1H, q, J = 5Hz), 4.83 (1Hd, J = 12Hz), 4. 9 9 (1 H, brs), 6.73 (1 H, brs, exchange ab le), 7.61 (1 H, d, J = 8 Hz, exc hange ab le) ₀

SI MS (+ KI) m / z: 418 (MK ⁺ ), 420 [(M + 2) K +], 422 [(M + 4) K ⁺ ]. Example 2 Preparation of compound (3) and compound (4)

To a solution of 42.39 g of the compound (2) dissolved in 600 ml of 1,4-dioxane / aqueous solution (2: 1) was added 23.43 ml of 1N sodium hydroxide at 0 to 5 with stirring. Then, a 80 ml solution of benzyloxycarbonyl chloride 20.071111 in 1,4-dioxane 80 ml was added dropwise at 0 to 5, and 140.55 ml of 1N sodium hydroxide was added. After stirring for 1 hour, it was left at room temperature for 1 晚. The reaction solution was diluted with 50 Om1 of water and extracted with a black hole form. The extract was washed with saturated saline and water, and dried over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel gel chromatography (eluted with gel form: methanol = 400: 3 (v / v)), and the fraction eluted first 21.50 g (yield 37.5%) of color amorphous (3) and 10.01 g (17.5% yield) of colorless amorphous (4) were obtained from the fraction eluted later.

Compound (3);

'Η— NMR (200 MHz) (CDC) ₃ ) (5: 1.37 (3 H, d, J = 5 Hz), 2.59 (1 H, brs, exchange ab le), 3.39 ( 1 H, t, J = 1 OH z), 3.52 (1 H, t, J = 1 OH z), 3.70-3.82 (1 H, m), 3.89 (1 H, t , J = 10 Hz), 3.97 to 4.17 (2H, m), 4.60 to 4.83 (2 H, m), 4.72 (1 H, q, J = 5 Hz), 5.21 (2H, s), 5.23 (1H, d, J = 10Hz), 6.07 (1H, d, J = 4Hz), 7.40 (5H, s).

F ABMS m / z: 514 (MH ⁺ ).

Compound (4);

Ή-NMR (200 MHz) ( CD C 1 3) δ: 1. 37 (3 H, d, J = 5 H z), 2. 84 (1 H, brs, ex chage ab le), 3. 30~ 3. 65 (4 H, m), 4.07 (1 H, t, J = 10 Hz), 4.14 to 4.25 (1 H, m), 4.64 to 4.81 (3H, m) , 5.17 (2 H, s), 5.

31 (1H, d, J = 9Hz), 5.73 (1H, d, J = 8Hz), 7.36 (5H, s).

F ABMS m / z: 514 (MH +) Example 3 Preparation of Compound (5)

Compound (3) 2.33 g was dissolved in anhydrous methylene chloride (23 ml), pyridine (23 ml) was added, and the mixture was cooled with an ice-water bath (0 to 5 "€) to give 2,2,2-trichloromouth ethoxycarbonyl chloride 1. 25 ml was added, and the mixture was stirred at room temperature for 1.5 hours.

The reaction mixture was added with 100 ml of ethyl acetate, washed sequentially with a 5% aqueous hydrochloric acid solution and saturated saline, and dried over anhydrous magnesium sulfate. The residue obtained by distilling off the solvent under reduced pressure was subjected to silica gel column chromatography [eluted with chloroform: methanol = 400: 1 (v / v)] to give a colorless amorphous (5) 2.38 g (Yield 76.2%).

'H-NMR (200 MHz) (CDC 1 3) δ: 1. 32 (3Η, d, J = 5H z), 3. 54 (1 H, t, J = 8Hz), 3. 61 (1 H, t, J = 8Hz), 3.78 to 3.93 (1H, m), 4.08 to 4.33 (2H, m), 4.63 to 4.90 (5H, m), 5.12 (1H, t, J = 8Hz), 5.22 (2H, s), 5.28 (1H, t, J = 8Hz), 6.07 (1H, d, J = 4Hz), 7.41 (5H, s).

F ABMS m / z: 688 (MH ⁺ ), 690 [(M + 2) H ⁺ ]. Example 4 Production of Compound (9)

Compound (4) (2.02 g) was dissolved in anhydrous methylene chloride (20 ml), pyridine (20 ml) was added thereto, and the mixture was cooled in an ice-water bath (0 to 5) to give 2,2,2-trichloroethoxycarbonylcarbonyl chloride. 08 ml was added and the mixture was stirred at room temperature for 1 hour and 20 minutes.

The reaction mixture was added with 100 ml of ethyl acetate, washed sequentially with a 5% aqueous hydrochloric acid solution and saturated saline, and dried over anhydrous magnesium sulfate. The solvent is obtained by evaporation under reduced pressure. The residue was subjected to silica gel column chromatography [eluted with hexane: ethyl acetate = 7: 1 (v / v)] to obtain 1.70 s (62.8% yield) of colorless amorphous (9) Was.

An a l .C a l c dfo r C 22H23 C 1 eNOn: C, 38.29; H, 336; N, 2.03; C 1, 30.82 F o u n d: C, 38.35; H, 3.

29; N, 1.92; C1, 31.18.

IR (KB r) cm- ¹ : 3384, 1770.

Ή-NMR (30 OMH z) (CDC 13) δ: 1.33 (3H, d, J = 5.0 Hz), 3.50 to 3.63 (3H, m), 3.81 (1H , Dt, J = 10.0 and 8.9 Hz), 4.16 to 4.28 (1H, m), 4.60 to 4.88 (5H, m), 5.17 (2H , s), 5.24 to 5.35 (2 H, m, 1 H exchangeable), 5.80 (1 H, d, J = 8.6 Hz), 7.

36 (5 H, s). Example 5 Production of Compound (6)

1) Production from compound (5)

Dissolve 2.33 g of compound (5) in 120 ml of ethyl acetate, add 60 mg of 10% palladium-carbon, and introduce hydrogen for 1 hour with stirring at room temperature. Was removed by filtration, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography [acetate Echiru: hexane = 1: 1 (VZV) elute at] subjected to a colorless amorphous (6) 1.60 ₈ (yield 85.2%) Obtained. Ή-NMR (200 MHz) (CDC 13) δ 1.33 (3 H, d, J = 5 Hz), 3.06 to 3.12 (1 H, m, exchangeable), 3.49 to 3.65 (2 H, m), 3.93 to 4.20 (3 H, m), 4.64 to 4.95 (5 H, m), 5.17 (1 H, t, J = 8 Hz ), 5.31 (1H, t, J = 4Hz), 5.50 (1H, d, J = 8Hz).

S IMS m / z: 554 (MH ⁺ ), 556 [(M + 2) H ⁺ ], 558 [(M

+ 4) H ⁺ ] o

2) Production from compound (9) Dissolve 8.051 g of compound (9) in 300 ml of ethyl acetate, add 1.610 g of 10% paradigmum carbon, introduce hydrogen under stirring at room temperature for 1 hour, and filter the reaction mixture through celite. The catalyst was removed by filtration, and the filtrate was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography [eluted with hexane: ethyl acetate = 4: 1 (vZv)] to obtain a mixture of compound (6) and its 8-anomer [(6): β — Anomer = 5.3: 1 (from the integration ratio of the NMR spectrum)] 5.633 g (86.8% yield). Example 6 Production of Compound (7) and Compound (8)

1) Preparation of a mixture of compound (7) and compound (8)

1.04 g of the compound (6) was dissolved in 17 ml of anhydrous methylene chloride, 3.64 ml of trichloromethylacetonitrile and 1.743 g of anhydrous potassium carbonate were added, and the mixture was stirred at room temperature for 6 hours.

After completion of the reaction, the reaction solution was diluted with 180 ml of ethyl acetate, washed with water and saturated saline, and then dried over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography [elution with hexane: ethyl acetate = 2: 1 (v / v)] to obtain compound (7) and compound (8). Mixture 0.9

80 g (74.8% yield) were obtained.

2) Production of compound (7) and compound (8)

5.535 g of the mixture of compound (6) and its 3-anomer was dissolved in 8 Oml of anhydrous methylene chloride, and 20.Oml of trichloroacetonitrile and 9.631 g of anhydrous potassium carbonate were added. Stirred for hours.

After the completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with water and saturated saline, and then dried over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography, and the low-polar fraction [hexane: ethyl acetate =

9: 1 (v / v)] to obtain 3.449 g (yield 49.5%) of colorless amorphous (7), and a highly polar fraction [hexane: ethyl acetate = 7: 1 (v / v)] to give colorless amorphous (8) 1.50 lg (yield 21.5%).

Compound (7); IR (KB r) cmcm-]: 3384, 3344, 1764, 1724, 1282, 1252, 798 o

- NMR (400 MHz) (CDC 1 3) δ: 1. 35 (3 H, d, J = 5. 3 H z), 3. 59 (1 H, t, J = 10. 5Hz), 3. 68 (1H, t, J = 10.2Hz), 3.88 to 3.92 (1H, m), 4.19 (1H, dd, J = 10.5 and 5.0Hz), 4 33 (1 H, ddd, J = 10.2, 9.4 and 3.8 Hz), 4.62 (1 H, d, J = 12.2 Hz), 4.75 (1 H, q, J = 5.3 Hz), 4.76 (1H, d, J = 12.2Hz), 4.77 (1H, d, J = 11.8Hz), 4.87 (1H, d, J = 11.8 Hz), 5.21 (1H, t, J = 10.2Hz), 5.30 (1H, d, J = 9.4Hz, ex change table), 6.37 (1H, d, J = 3.8Hz), 8.77 (1H, s, exchange table).

FABMS (+ KI) m / z: 735 (MK ⁺ ), 737 [(M + 2) K ⁺ ], 739 [(M + 4) K ⁺ ], 741 [(M + 6) K ⁺ ], 743 [(M + 8) K ⁺ ] compound (8);

I R (KB r) cm— 3342, 1766, 1288, 1254, 1082, 798 o

'H-NMR (400 MHz) (CDC 1 a) δ: 1.34 (3 H, d, J = 5.0 Hz), 3.56 to 3.68 (3H, m), 4.00 (1H, dt, J = 9.7 and 8.8Hz), 4.24 to 4.28 (1H, m), 4.62 (1H, d, J = 11.8Hz), 4.69 (1H, d, J = 11.8 Hz), 4.70 to 4.78 (1 H, m), 4.75 (1 H, d, J = l l.8 Hz), 4.86 (1H, d, J = 11.8Hz), 5.30 (1H, t, J = 9.7Hz), 5.39 (1H, d, J = 8.8Hz), 6.07 (1H, d, J = 8.8 Hz), 8.72 (1 H, s, exchange table).

FABMS (+ KI) m / z: 735 (MK ⁺ ), 737 [(M + 2) K ⁺ ], 739 [(M + 4) K ⁺ ], 741 [(M + 6) K ⁺ ], 743 [( M + 8) K ⁺ ] Example 7 Production of Compound (11)

1.794 g of compound (10), 2.444 g of compound (7) and powdered molecular compound Under a nitrogen atmosphere, 90 ml of anhydrous methylene chloride was added to 9000 g of Lashibus 4A. Subsequently, the mixture was cooled in an ice-water bath, trifluoromethanesulfonic acid trimethylsilyl ester 341 was added thereto with stirring, and the mixture was stirred for 20 minutes at 2, and then trifluoromethanesulfonic acid trimethylsilyl ester 1Ίa1 was further added. , 2 for 30 minutes.

The reaction solution was filtered to remove insolubles, and the filtrate was added to a saturated aqueous solution of sodium hydrogen carbonate, extracted with methylene chloride, and dried over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography [eluted with hexane: ethyl acetate = 2: 1 (v / v)], and the compound eluted from the fraction eluted first

(11) 1.106 g (yield 40.5%) and compound eluted from later eluted fraction

(10) 0.731 g (recovery rate 40.7%) was obtained.

^{IR (KB r) vera "1} ; 3392, 1766, 1730, 1246, 1089. Ή-NMR (50 OMH z) (CDC 1 3) δ 1. 15 (3H, s), 1. 2 1 (3 H, s), 1.31 (3 H, d, J = 5.5 Hz), 1.68 (3 H, s) 1.95 (3 H, d, J = 1.2 Hz), 1.96 to 2. 03 (1H, m), 2.21 to 2.29 (1H, m), 2.30 (3H, s), 2.38 to 2.44 (1H, m), 2.46 (3 H, s), 2.67 to 2.76 (1 H, m), 3.36 to 3.45 (1 H, m), 3.51 (1 H, t, J = 9.5 Hz), 3 56 (1H, t, J = 10.1Hz), 3.72 to 3.80 (1H, m), 3.82 (1H, d, J = 7.OHz), 4. 06 to 4.15 (1H, m), 4.16 (1H, d, J = 8.2Hz), 4.20 to 4.26 (1H, m), 4.30 (1H, d , J = 8.2 Hz), 4.66 to 4.72 (3H, m), 4.76 to 4.82 (4H, m), 4.82 to 4.88 (1H, m), 4. 98 to 5.06 (2 H, m), 5.52 (1 H, d, J = 2.4 Hz), 5.67 (1 H, d, J = 1 0.4 Hz), 5.69 ( 1 H, d, J = 7.0 Hz), 6.03 (1 H, dd, J = 9.2 and 2.4 H z), 6.25 (1 H, difdt, J = 8.5 and 1 2H z), 6.36 (1 H, s), 6.90 (1 H, d, J = 9.2 H z), 7.35 to 7.47 (7H, m), 7.49 to 7.54 (3 H, m), 7.58 to 7.64 ( 1 H, m), 7.74 to 7.78 (2 H, m), 8.10 to 8.15 (2H, m :).

FABMS m / z: 1563 (MH ⁺ ), 1565 [(M + 2) H ⁺ ], 1567 [(M + 4) H ⁺ ], 1569 [(M + 6) H ⁺ ]. Example 8 Production of Compound (12)

Compound (11) 3.328 gO.5 830 g of sub-forced powder was added to a mixed solution of 0.5 N hydrochloric acid 3 Om1 and tetrahydrofuran 6 Om1 under ice-cooling, and the mixture was stirred at the same temperature for 5 hours. . Then, 2.776 g of zinc powder and 15 ml of 0.5 N hydrochloric acid were added and stirred for 1 hour, and then 2.776 g of zinc powder and 30 ml of 0.5 N hydrochloric acid were added and stirred for 1 hour.

The reaction solution was diluted with methylene chloride, the insolubles were separated by filtration, and the organic layer was separated. The organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated saline solution. The separated aqueous layer was neutralized with sodium hydrogen carbonate, extracted with methylene chloride, and the combined organic layers were dried over anhydrous sodium sulfate. Evaporating the solvent under reduced pressure to give residue was purified by silica gel column chromatography and subjected to [Black port Holm: methanol = 50 1 eluted (VZV)], Compound (12) 1.502 ₈ (yield: 67 9%).

IR (KB r) cm- ¹ ; 3436, 1724, 1245, 1092.

Ή-NMR (500 MHz) ( CD C 1 3) <5:. 1. 1 9 (3 H, s), 1. 2 1 (3 H, s), 1. 37 (3 H, d, J = 4.9Hz), 1.76 (3H, s) 1.86 (3H, s), 1.93 to 2.00 (1H, m), 2.21 (3H, s) 2.27 to 2.38 (2H, m), 2.39 (3H, s), 2.41 to 2.47 (1H, m), 2.66 to 2.77 (1H, m), 3.27 ~ 3.35 (2H, m), 3.45 to 3.57 (2 H, m), 3.84 (1 H, d, J = 6.7 Hz) 4.07 to 4.17 (2H , M), 4.19 (1H, d, J = 8.5Hz), 4.30 (1H, d, J = 8.5Hz), 4.37 (1H, d, J = 7.9Hz), 4.73 (1H, q, J = 4.9Hz), 4.80 (1H, d, J = 2.6Hz) 4.85 (1H, brd, J = 8.6 Hz), 5.71 (1 H, d, J = 6.7 Hz), 5.79 (1 H, dd, J = 8.9 and 2.6 Hz), 6.1 9 (1H, t, J = 9.5Hz), 6.41 (1H, s), 7.02 (1H, d, J = 8.9Hz), 7.33 Up to 7.39 (1H, m), 7.39 to 7.45 (4H, m), 7.46 to 7.54 (5H, m), 7.58 to 7. 64 (1H, m), 7.73 to 7.78 (2H, m), 8.10 to 8.15 (2H, m).

FABMS / z: 1041 (MH ⁺ ).

HRF ABMS m / z: C a 1 cdfor C 55 H 65 N 2 0 18 (MH), 1 04 1. 4 2 2 8 F ound, 1 04 1. 4 2 2 2. Example 9 Production of Compound (13)

To a solution of 1.458 g of compound (1 2) in 33.5 ml of methanol was added 1.26 ml of 37% formalin and 0.364 g of sodium cyanoborohydride, and the mixture was added at room temperature. Stir for 1 hour.

The reaction solution was cooled in an ice-water bath, and 0.5N hydrochloric acid (20 ml) was added to make an acidic solution. Subsequently, a saturated aqueous solution of sodium hydrogencarbonate was added to make the solution alkaline, followed by extraction with chloroform and drying over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography [eluted with chloroform: methanol = 70: 1 (v / v)] to obtain compound (13) 1.2 63 g (84.4% yield) were obtained.

1 R (KB r) vein- '; 34 01, 1 738, 1 244, 1 94.

Ή-NMR (500 MHz) (CDC la) δ: 1.19 (3 H, s), 1.2

2 (3 H, s), 1.40 (3 H, d, J = 5.0 Hz), 1.70 (2 H, brs, exchangeable), 1.77 (3 H, s) , 1.87-; I.93 (1H, m), 1.88 (3H, d, J = 1.2Hz), 2.14 (3H, s), 2.2 1 (1 H, dd, J = 9.8 and 7.9 Hz), 2.30

(1 H, dd, J = 15.6 and 8.9 Hz), 2.35 to 2.4 1 (1 H, m), 2.37 (3 H, s), 2.3 8 (6 H, s), 2.5 1 to 2.5 9

(1 H, m), 3.30 (1 H, t, J = 9.2 Hz), 3.39 (1 H, difddd, J = 10.0, 9.2 and 4.8 H z), 3.57 (1 H, t, J = 10 .OH z), 3.60 (1 H, difdd, J = 9.8 and 9.2 Hz), 3.82 (1 H, d, J = 6.7 Hz), 3.85 (1 H, brs, exchangeable), 4.14 (1 H, dd, J = 10. 0 and 4.8 Hz), 4, 1 7 (1 H, d, J = 8.5 Hz), 4.30 (1 H, d, J = 8.5 Hz), 4.40 (1 H, dd, J = 9.5 and 7.6 Hz), 4.68 (1 H, d, J = 7.9 Hz), 4.74 (1 H, q, J = 5.OHz), 4.83 (1H, d, J = 2.2Hz), 4.84 (1H, d, J = 9.8Hz), 5.71 (1H, d, J = 6.7Hz), 5.81 (1H, dd, J = 9.0 and 2.2Hz), 6.18 (1H, dt, J = 8.9 and 1.2Hz), 6.63 (1 H, s), 7.04 (1 H, d, J = 9.0 Hz, exchangeable), 7.33 to 7.37 (1 H, m), 7.38 to 7.43 (4 H, m ), 7.47 to 7.53 (5H, m), 7.59 to 7.63 (1H, m), 7.72 to 7.76 (2H, m), 8.11 to 8 . 1 4 (2

H, m).

F ABMS mZ z: 1069 (MH ⁺ ).

HRFABMS / z: C a 1 cdfor C 57 H 69 N 2 0 18 (MH), 106 9. 4543 F ound, 1 069. 4568. Example 10 Methanesulfonate of Compound (13) "Production of Compound (14) 1 ^ 1 (37) 1 mg was dissolved in a mixed solution of tert-butanol 25 ml and water 10 ml, Under ice-cooling, a solution of methanesulfonic acid 27] in 17 ml of water was added, and after stirring for 2 minutes, insolubles were filtered off using a membrane filter (0.22 ^ m), the filtrate was lyophilized, and the compound ( 14) 387 mg (95.7% yield) IR (KB r) cm ¹ : 343 1,1 733, 1646.

Ή-NMR (400 MHz) (CDC) ₃ ) S: 1.18 (3 H, s), 1.20 (3H, s), 1.41 (3H, d, J = 5.0 H) z), 1.75 (3H, s)

I. 87-: I. 98 (1H, m), 1.90 (3H, s), 2.14 (3H, s) 2.21 (1H, m, exchangeable), 2.30- 2.59 (4 H, m), 2.39 (3H, s), 2.74 to 3.13 (7H, m, 1 H exchangeab 1 e), 2.78 (3H, s), 3. 28 (1 H, t, J = 9.5 Hz), 3.32 to 3.41 (1H, m), 3.50 (1H, t, J = 10.3Hz), 3.84 (1H, d, J = 6.9Hz) ), 3.89 (1H, brt, J = 9.5Hz), 4.19 (1H, d, J = 8.4Hz), 4.25 (1H, dd, J = 10.3) and 5.0 Hz), 4.34 (1 H, d, J = 8.4 Hz), 4.51 (1 H, dd, J = 9.3 and 7.8 Hz), 4.72 (1H, q, J = 5.0Hz), 4.73 to 4.78 (1H, m), 4.84 (1H, brd, J = 9.2Hz), 5.03 (1H, d, J = 7.6Hz), 5.17 to 5.37 (1H, brs, exchangeable), 5.75 (1H, d, J = 6.9Hz), 5.86 (1H, dd) , J = 9.5 and 1.9 Hz), 6.10 (1 H, brt, J = 8.6 Hz), 6.57 (2H, brs, 1 H exchangeable), 7.31 to 7.57 (1 0 H, m), 7.61 to 7.67 (1 H, m), 7.83 to 7.88 (2 H, m), 8.1 1 to 8.16 (2 H, m), 9 .37〜6.91 (1 H, brs, exchangeable) ₀

F ABMS mZz: 1069 (MH <+>). Example 11 1 Hydrochloride of Compound (13) “Production of Compound (15) 1

63 mg of the compound (13) was dissolved in 2. Oml of ethyl acetate, and 0.177 ml of a 0.5N hydrogen chloride / ethyl acetate solution was added under ice-cooling. The product obtained by evaporating the solvent under reduced pressure was dried at room temperature under reduced pressure to obtain 6 Omg of the compound (15) (yield 92.2%). Example 12 Tartrate of Compound (13) “Production of Compound (16) 1

53 mg of the compound (13) is dissolved in a mixture of 3.98 ml of tert-butanol and 1.59 ml of water, and under ice-cooling, 0.266 ml of an aqueous L-tartaric acid solution (containing 7.44 mg of L-tartaric acid) ) Was added. After adding 3 ml of water to the obtained solution, it was freeze-dried to obtain 6 Omg of the compound (16) (100% yield). Test Example 1 Growth inhibitory effect on KB cells (Invitro) 1) Test method

To each well of a flat-bottomed 96-well plate, 1 × 10 ³ KB cells (0.1 ml) of cell suspension (suspended in MEM medium supplemented with 10% fetal bovine serum) was added and cultured for 24 hours. To this, 100 1 of the compound solution of the present invention (final concentration of dimethyl sulfoxide 0.5%) dissolved in dimethyl sulfoxide and diluted with a medium was added, and the cells were further cultured for 72 hours. After the culture, 4,5-dimethylthiazoyl 2-yl 2,5-diphenyltetrazolium promide reagent (coloring reagent) was added, and the cells were further cultured for 4 hours.

As a control, medium 100 1 (dimethylsulfoxide final concentration 0.5%) was added, and the cells were cultured in the same manner. After completion of the culture, the medium was removed, the cells were measured for absorbance was dissolved in 150 1 of dimethyl sulfoxide to obtain the ratio of the absorbance of the present invention compounds to the absorbance of the control group, 50% growth inhibitory concentration (IC _5. ) Was calculated.

2) Test results

IC _{5 of} compound (12), compound (13) and compound represented by formula (I) for KB cells. Are shown in Table 1.

Compound I Cso (g / m 1) Compound represented by the formula (I) 0.027

(12) 0.0241

(13) 0.061 1 Test Example 2 Antitumor effect on mouse B 16 melanoma (ascites type) (In Vivo)

1) Test method

A 10% homogenate solution of B16 melanoma tumor mass passaged in female C57B LZ6 mice was prepared by adding Hank's balanced salt solution, and 0.5 ml of the solution was intraperitoneally injected into female BDF1 mice. Transplanted. The compound of the present invention and taxol were intraperitoneally administered once a day for 5 consecutive days from the day after the tumor cell transplantation.

Compound (12) and taxol were dissolved in a solvent consisting of 50% polyoxyethylene castor oil and 50% ethanol, and diluted with physiological saline immediately before administration. That is, physiological saline containing 5% of polyoxyethylene castor oil and 5% of ethanol was used as a solvent for administration. Taxol formed a precipitate in a short time, whereas compound (12) was clear for a long time (over 24 hours). Compound (14) was administered by dissolving in distilled water for injection before use. The control group received distilled water for injection, and the vehicle control group received only saline containing 5% of polyoxetylene castor oil and 5% of ethanol. The control group used 12 mice, and the vehicle control group and drug administration group used 6 mice per group.

After breeding and observation for 50 days, the survival rate was determined by the following formula. Median days of survival in the drug-administered group

Life extension (%) = X 100

Median survival time of control group The median survival time of the control group was 23.3 days, and the median survival time of the solvent control group was 22.3 days.

2) Test results

Table 2 shows the survival rate and weight change of compound (12), compound (14) and taxol. Table 2

Compound (12) exhibited a life-promoting effect that was equivalent to or slightly weaker than taxol. Compound (14) showed comparable survival benefit and taxol, since toxicity was seen in 3 Omg / k _g dose weight loss and early death was observed by administration in taxol, toxic or Gensa is It is thought that it is. Test example 3 Measure the resolution to 7k

1) How to measure taxol solubility

(1) 5 ml of water was added to 5 mg of taxol, treated with ultrasonic waves for 5 minutes to disperse the sample, and shaken at room temperature for 30 minutes. Then, the mixture was passed through a 0.45 μπι membrane finoletter, and the amount of taxol present in the solution was quantified by HP LC.

(2) HP LC measurement conditions

Column: 〇DS—80TM 5 (4.6 mm ID x 15 Omm) Column temperature: 50

Eluent: 0.1% trifluoroacetic acid aqueous solution: acetonitrile = 1: 1

(v / v)

Flow rate: 1. Oml / min

Detection wavelength: 230 nm 2) Method for measuring the solubility of compound (14)

(1) Compound (14) 0.1 ml of water was added to 2 ml of water, treated with ultrasonic waves for 1 minute to disperse the sample, shaken at room temperature for 30 minutes, and left for 24 hours. Then, the mixture was passed through a 0.45 membrane filter, and the test substance present in the filtrate was quantified by HP LC.

(2) HP LC measurement conditions

Column: ODS—80T s 5 (4.6 mm ID x 150 mm) Column temperature: 50V

Solution: 0.1M aqueous solution of ammonium nitrate: acetonitrile = 1: 1

(v / v)

Flow velocity: 1.0 m 1 / min

Detection wavelength: 230 nm

3) Test results

Table 3 shows the solubility of compound (14) and taxol in water.

Table 3 Compound solubility (/ m 1) Relative ratio

(1 4) 1 6, 8 6 4 6

Taxol 0, 0 2 6 1

Claims

The scope of the claims

1 set

[Wherein, R represents a hydrogen atom or a methyl group. ] The taxol derivative represented by these, and its pharmaceutically acceptable salt.