JPH08113589A - New taxol derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound derivative holding characteristic of excellent antitumor activity, having an improved water-solubility and a reduced toxicity by introducing a high polar amino sugar into the hydroxyl group at 7-position of taxol by a method of organic synthetic chemistry and then forming salt.

SOLUTION: A new taxol derivative expressed by formula I (R is H or methyl; Ac is acetyl; Pb is phenyl) having an excellent antitumor activity, having an improved water-solubility and reduced toxicity when compared with taxol, and exhibiting excellent antitumor activities on ovarian cancer, breast cancer and the like. This taxol derivative is obtained by reacting taxol of formula II with an amino acid of formula III (Troc is 2,2,2-trichloroethoxycarbonyl) in the presence of an acid catalyst to allow a high polar amino sugar to bind to the hydroxyl group at the 7-position of taxol, then treating the reaction product with zinc powder in an organic solvent to remove a Troc group, and subsequently optionally treating the reaction product with formalin in the presence of a reducing agent to methylate the amino group.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel taxol derivative having antitumor activity.

[0002]

Taxol is an antitumor diterpene compound isolated from the bark of Pacific yew. Taxol inhibits depolymerization of microtubules by binding to tubulin, promoting its polymerization, and stabilizing the formed microtubules, and as a result, the spindle during cell division does not function normally, It has a novel mechanism of action that inhibits cell division. In addition, it has been reported clinically that it has an excellent anticancer effect on ovarian cancer, breast cancer and the like.

Since taxol is extremely difficult to dissolve in water, polyoxyethylene castor oil (trade name: Cremophor EL), which is a solubilizer, and 50/50 (v) of ethanol.
The current situation is to administer an injection prepared by dissolving taxol in a mixed solution of / v) and diluting it 10 times with physiological saline and instilling it.

The side effects of taxol are myelosuppression,
Hypersensitivity reaction, cardiotoxicity, neurotoxicity, digestive tract disorders, hair loss,
Myalgia, joint pain, etc. have been reported. Among these, hypersensitivity reactions (dyspnea due to bronchospasm, skin rash, hypotension, anaphylaxis, etc.) are considered to be derived from polyoxyethylene castor oil used as a solubilizer. In order to suppress this, pretreatment with corticosteroids, histamine H1 antagonists and histamine H2 antagonists has been performed.

Further, in order to avoid side effects derived from polyoxyethylene castor oil, many studies on water-soluble derivatives of taxol and water-soluble preparations have been reported.

For example, WO 90/10443 (V.
J. Stella et al.), United States Patent No. 5,15.
7,049 (RD Haugwitz et al.), European Patent 0537905 (DGI Kingston).
Et al.), US Pat. No. 5,411,984 (DGI Kingston et al.), US Pat. No. 5,422,364 (KC Nicolao).
u, etc.), WO95 / 18804 (K.C. Nicola
eu, et al., European Patent No. 0558959 (Bristol-
Myers Squibb) and European Patent No. 06395
No. 77 (Bristol-Myers Squibb Company) and the like.

These reports indicate that a substituent having a water-soluble functional group such as a carboxylate, a quaternary ammonium salt, a quaternary pyridinium salt, a sulfonate and a phosphate on the hydroxyl group at the 2'-position or 7-position of taxol. The present invention relates to a taxol derivative having a water-solubility improved by an ester bond or an acetal bond.

These water-soluble taxol derivatives are aimed at water-soluble prodrugs of taxol, in which the water-soluble substituent introduced at the hydroxyl group at the 2'-position or the 7-position of taxol is removed by hydrolysis in vivo to produce taxol. It was done.

US Pat. No. 5,424,424
No. 073 (Georgetown University) discloses studies on taxol liposome formulations.

As a glycoside of taxol, a compound represented by the formula (I) in which xylose is bonded to the hydroxyl group at the 7-position is known [J. Nat. Prod. , Vol. 47, No. 13
P. 1, 1984]. formula

[0011]

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This 7-xylosyl taxol has a microtubule depolymerization inhibitory activity equivalent to that of taxol [Proc. Nat
l. Acad. Sci. USA, Volume 81, 4090
P., 1984] and in vitro antitumor activity [Pharm. Res. , Vol. 10, p. 521, 19
1993] has been reported. In addition, the glycosides of taxol and its analogs are obtained only from nature, and there is no report that a sugar was introduced into the 7-position hydroxyl group of taxol through synthetic organic chemistry.

[0013]

The object of the present invention is to synthesize a novel water-soluble taxol derivative, which is different from the conventional prodrug-type water-soluble derivative, that is, a highly polar amino sugar at the 7-position hydroxyl group of taxol, by organic synthetic chemistry. Introduced by the method,
Then, by forming a salt, the excellent antitumor activity of taxol is retained, while avoiding the use of polyoxyethylene castor oil, which is the cause of hypersensitivity reaction due to improvement of water solubility, and other toxicity derived from taxol. The present invention is to provide a novel taxol derivative in which

[0014]

Means for Solving the Problems As a result of earnest studies on introduction of an amino sugar derivative into the 7-position hydroxyl group of taxol, the present inventors have succeeded in synthesizing a 7-position amino sugar glycoside of taxol. It was found that the salt has improved water solubility as compared with taxol, exhibits an antitumor activity equivalent to that of taxol, and has reduced toxicity as compared with taxol, and completed the present invention.

The present invention will be described below.

The compounds of the present invention have the formula

[0017]

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A taxol derivative represented by the formula (wherein R represents a hydrogen atom or a methyl group) and a pharmaceutically acceptable salt thereof.

In the present invention, the pharmaceutically acceptable acid addition salt includes, for example, acetate, propionate, butyrate, lactate, maleate, fumarate, tartrate, citrate, stearate, Succinate, ethyl succinate,
Benzoate, salicylate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,
Benzene sulfonate, paratoluene sulfonate, camphor sulfonate, lauryl sulfate, malate,
Aspartate, glutamate, adipate, cysteine, lactobionate, glucuronate, hydrochloride, hydrobromide, phosphate, sulfate, hydroiodide, oxalate, picric acid Mention may be made of salt.

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).

Production scheme (I)

[0022]

[Chemical 4]

(In the scheme, Z represents a benzyloxycarbonyl group and Troc represents a 2,2,2-trichloroethoxycarbonyl group.) That is, the literature (Bull. Chem. Soc. Jp.
n. , 60, 2205, 1987), the compound (1) is reacted with paraaldehyde in the presence of an acid catalyst at room temperature for about 2 to 48 hours to give the compound (2).
Get.

Examples of the acid of 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. To be

Then, the compound (2) and benzyloxycarbonyl chloride are treated with 1,4 in the presence of a base such as sodium hydroxide, potassium hydroxide or barium hydroxide.
-In a mixed solvent of an organic solvent such as dioxane and water, -10
Compound (3) (α-anomer) and compound (4) (β
A mixture of (anomers). Both compounds can be separated by silica gel column chromatography.

Compound (5) is obtained by reacting compound (3) with 2,2,2-trichloroethoxycarbonyl chloride in an organic solvent in the presence of a base at 0 ° C. to room temperature for about 1 to 24 hours. . Similarly, the compound (9) can be obtained from the 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, diisopropylethylamine and 4-dimethylaminopyridine. Examples of the organic solvent include halogen-based solvents such as chloroform, methylene chloride and 1,2-dichloroethane, or pyridine.

Compound (5) was added with palladium-
Compound (6) is obtained by hydrogenolysis in the presence of a catalyst such as carbon at room temperature. Similarly, a mixture of the compound (6) and its β-anomer can be obtained from the compound (9). Similarly, a mixture of compound (6) and its β-anomer can be obtained from a mixture of compound (5) and compound (9).

The organic solvent used in the above reaction is
Examples thereof include ethyl acetate, tetrahydrofuran, 1,4-dioxane, alcohol solvents such as methanol and ethanol, and mixed solvents thereof. Examples of the catalyst include palladium-carbon, palladium black, platinum oxide, Raney nickel, palladium-barium sulfate, and the like.

Then, the compound (6) and trichloroacetonitrile are added to an organic solvent in the presence of a base at room temperature for 1 to 24 hours.
By reacting for about time, a mixture of compound (7) (α-anomer) and compound (8) (β-anomer) is obtained. Similarly, a mixture of compound (7) and compound (8) can be obtained from a mixture of compound (6) and its β-anomer. Compound (7) and compound (8) can be separated by silica gel column chromatography.

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

Production scheme (II)

[0033]

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(In the scheme, Troc has the same meaning as described above.) Compound (10) [Reference (J. Med. Chem., No. 3)
Volume 5, p. 145, 1992). ] And the 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 -20 ° C to room temperature for 30 minutes to 24 hours to give a compound ( 11) is obtained. Similarly, compound (8) or compound (7) and compound (8)
Compound (11) can also be obtained by using a mixture of

The organic solvent used in the above reaction is
Examples thereof include halogen-based solvents such as methylene chloride, 1,2-dichloroethane and chloroform, and single or mixed solvents such as toluene, benzene, diethyl ether, acetonitrile and nitromethane. Examples of the acid catalyst include trifluoromethanesulfonic acid trimethylsilyl ester, boron trifluoride diethyl ether complex, silver trifluoromethanesulfonic acid, silver perchlorate, silver carbonate, mercury cyanide, mercury bromide, paratoluenesulfonic acid, camphorsulfonic acid. And so on. Examples of the dehydrating agent include powder molecular sieves 4A and anhydrous calcium sulfate.

Zinc powder was added to the compound (11) in an organic solvent in the presence or absence of an acid, and the mixture was stirred at 0 ° C to room temperature for 1 hour.
By reacting for about 24 hours, a compound (12) in which R is a hydrogen atom is obtained among the compounds of the present invention represented by the formula (II).

The organic solvent used in the above reaction is
Examples thereof include alcohol solvents such as methanol, ethanol and propanol, tetrahydrofuran, 1,4-dioxane and ethyl acetate. As the acid, hydrochloric acid,
Examples thereof include sulfuric acid and acetic acid.

Further, the compound (12) is reacted with a reducing agent such as formalin and sodium cyanoborohydride or sodium borohydride in an alcohol solvent such as methanol at 0 ° C. to room temperature for about 1 to 24 hours. Thus, among the compounds of the present invention represented by the formula (II), a compound (13) in which R is a methyl group is obtained.

Each target substance in the above reaction can be obtained by general isolation and purification methods such as extraction, chromatography and recrystallization.

The pharmaceutically acceptable salt of the compound of the present invention can be obtained by subjecting compound (12) or compound (13) to methylene chloride, chloroform, ethyl acetate, tetrahydrofuran, methanol, ethanol, tert according to a conventional method.
-In an organic solvent such as butanol, or in a mixed solvent of these and water, an acid is added, chromatography, recrystallization,
It can be obtained by an isolation or purification method such as reprecipitation or lyophilization.

The effective dose of the compound of the present invention is 1 adult / day.
It is 1 mg to 500 mg, preferably 1 mg to 300 mg per 1 m ^{2 of} body surface area. This dose can be appropriately increased or decreased depending on the age, weight and condition of the patient.

The compound of the present invention thus obtained is
As injections, known additives such as excipients, disintegrating agents, binders, lubricants, antioxidants, coating agents, coloring agents, cross-linking agents, surfactants, plasticizers, etc. It is also possible to mix them by a conventional method to prepare oral preparations such as granules, powders, capsules, tablets, dry syrups and liquids.

As these additives, those generally used in pharmaceuticals can be used.

[0044]

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

[0045]

EXAMPLES The present invention will be specifically described below by showing Examples and Test Examples.

Example 1 Production of Compound (2 ) 79.33 g of Compound (1) was added to 360 ml of paraaldehyde.
And 1.5 ml of concentrated sulfuric acid were added, and the mixture was stirred overnight at room temperature. After the reaction, the precipitated solid was collected by filtration and washed with diethyl ether to give colorless amorphous compound (2) 74.68.
g (yield 87.7%) was obtained.

¹ H-NMR (200 MHz) (DMSO
−d ₆ ) δ: 1.23 (3H, d, J = 5 Hz), 3.
19 (1H, d, J = 8 Hz), 3.37 to 3.53
(3H, m, 1H, exchangeable), 3.
57-3.74 (2H, m), 3.86-3.98 (1
H, m), 4.74 (1H, d, J = 12 Hz), 4.
76 (1H, q, J = 5Hz), 4.83 (1H, d,
J = 12 Hz), 4.99 (1H, br s), 6.7
3 (1H, brs, exchangeable),
7.61 (1H, d, J = 8 Hz, exchangea
ble).

SIMS (+ KI) m / z: 418 (M
K ⁺ ), 420 [(M + 2) K ⁺ ], 422 [(M + 4)
K ⁺ ].

Example 2 Compound (3) and compound
Preparation of (4) 42.39 g of the compound (2) was dissolved in 600 ml of 1,4-dioxane-water (2: 1), and the mixture was stirred at 0 to 0.
23.43 ml of 1N sodium hydroxide was added at 5 ° C. Then, benzyloxycarbonyl chloride 20.
A solution of 07 ml of 80 ml of 1,4-dioxane was added at 0-5 ° C.
Then, add 1N sodium hydroxide 140.5 at the end.
After adding 5 ml and stirring at 0 to 5 ° C for 2 hours, 1 at room temperature
I left it at night.

The reaction solution was diluted with 500 ml of water and extracted with chloroform. 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 column chromatography [chloroform: methanol = 400: 3].
Elution at (v / v)], and 21.50 g of colorless amorphous compound (3) (yield 3
(7.5%) and a fraction eluted later to obtain 10.01 g (yield 17.5%) of colorless amorphous compound (4).

Compound (3); ¹ H-NMR (200 MHz) (CDCl ₃ ) δ: 1.3
7 (3H, d, J = 5Hz), 2.59 (1H, br
s, exchangeable), 3.39 (1H,
t, J = 10 Hz), 3.52 (1H, t, J = 10H
z), 3.70 to 3.82 (1H, m), 3.89 (1
H, t, J = 10 Hz), 3.97-4.17 (2H,
m), 4.60 to 4.83 (2H, m), 4.72 (1
H, q, J = 5 Hz), 5.21 (2H, s), 5.2
3 (1H, d, J = 10Hz), 6.07 (1H, d,
J = 4 Hz), 7.40 (5H, s).

FABMS m / z: 514 (MH ⁺ ).

Compound (4); ¹ H-NMR (200 MHz) (CDCl ₃ ) δ: 1.3
7 (3H, d, J = 5Hz), 2.84 (1H, br
s, exchangeable), 3.30 to 3.65.
(4H, m), 4.07 (1H, t, J = 10Hz),
4.14-4.25 (1H, m), 4.64-4.81
(3H, m), 5.17 (2H, s), 5.31 (1
H, d, J = 9Hz), 5.73 (1H, d, J = 8H
z), 7.36 (5H, s).

FABMS m / z: 514 (MH ⁺ ).

Example 3 Production of compound (5) 2.33 g of compound (3) was dissolved in 23 ml of anhydrous methylene chloride, 23 ml of pyridine was added, and the mixture was cooled in an ice-water bath (0 to 0).
5 ° C.) and 1.25 ml of 2,2,2-trichloroethoxycarbonyl chloride were added, and the mixture was stirred at room temperature for 1.5 hours.

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

¹ H-NMR (200 MHz) (CDC
l ₃ ) δ: 1.32 (3H, d, J = 5Hz), 3.5
4 (1H, t, J = 8Hz), 3.61 (1H, t, J
= 8 Hz), 3.78 to 3.93 (1H, m), 4.0
8 to 4.33 (2H, m), 4.63 to 4.90 (5
H, m), 5.12 (1H, t, J = 8 Hz), 5.2
2 (2H, s), 5.28 (1H, t, J = 8Hz),
6.07 (1H, d, J = 4Hz), 7.41 (5H,
s).

FABMS m / z: 688 (MH ⁺ ),
690 [(M + 2) H ⁺ ].

Example 4 Production of compound (9) 2.02 g of compound (4) was dissolved in 20 ml of anhydrous methylene chloride, 20 ml of pyridine was added, and the mixture was cooled in an ice-water bath (0 to 0).
(5 ° C.) and 2,2,2-trichloroethoxycarbonyl chloride (1.08 ml) were added, and the mixture was stirred at room temperature for 1 hour and 20 minutes.

100 ml of ethyl acetate was added to the reaction solution, and
% Aqueous solution of hydrochloric acid and saturated saline, and then dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was subjected to silica gel column chromatography [hexane: ethyl acetate = 7: 1 (v / v) elution] to give 1.70 g of the colorless amorphous compound (9) ( Yield 62.8%) was obtained.

Anal. Calcd for C ₂₂ H ₂₃
Cl ₆ NO ₁₁ : C, 38.29; H, 3.36; N,
2.03; Cl, 30.82 Found: C, 38.
35; H, 3.29; N, 1.92; Cl, 31.1
8.

IR (KBr) νcm ^-1 : 3384,17
70.

¹ H-NMR (300 MHz) (CDC
l ₃ ) δ: 1.33 (3H, d, J = 5.0Hz),
3.50 to 3.63 (3H, m), 3.81 (1H, d
t, J = 10.0 and 8.9 Hz), 4.16-
4.28 (1H, m), 4.60 to 4.88 (5H,
m), 5.17 (2H, s), 5.24-5.35 (2
H, m, 1H exchangeable), 5.80
(1H, d, J = 8.6Hz), 7.36 (5H,
s).

Example 5 Production of compound (6) 1) Production from compound (5) 2.33 g of compound (5) was dissolved in 120 ml of ethyl acetate, 600 mg of 10% palladium-carbon was added, and the mixture was stirred at room temperature. After introducing hydrogen for 1 hour, the reaction solution was filtered through Celite to remove the catalyst, and the filtrate was evaporated under reduced pressure. The obtained residue was subjected to silica gel column chromatography [eluted with ethyl acetate: hexane = 1: 1 (v / v)] to give 1.60 g of colorless amorphous compound (6) (yield: 85.
2%) was obtained.

¹ H-NMR (200 MHz) (CDC
l ₃ ) δ: 1.33 (3H, d, J = 5Hz), 3.0
6 to 3.12 (1H, m, exchangeable
e), 3.49 to 3.65 (2H, m), 3.93 to
4.20 (3H, m), 4.64 to 4.95 (5H,
m), 5.17 (1H, t, J = 8Hz), 5.31
(1H, t, J = 4Hz), 5.50 (1H, d, J =
8 Hz).

SIMS m / z: 554 (MH ⁺ ), 5
56 [(M + 2) H ⁺ ], 558 [(M + 4) H ⁺ ].

2) Production from Compound (9) 8.051 g of Compound (9) was dissolved in 300 ml of ethyl acetate, 1.610 g of 10% palladium-carbon was added, and hydrogen was introduced for 1 hour under stirring at room temperature. The reaction mixture was filtered through Celite to remove the catalyst, and the filtrate was evaporated under reduced pressure. The obtained residue was subjected to silica gel column chromatography [hexane: ethyl acetate = 4: 1 (v / v) elution],
5.633 g of a mixture of the compound (6) and its β-anomer [(6): β-anomer = 5.3: 1 (from the integral ratio of ¹ H-NMR spectrum)] 5.633 g (yield 86.8)
%) Was obtained.

Example 6 Compound (7) and compound
Production of (8) 1) Production of a mixture of compound (7) and compound (8) 1.04 g of compound (6) was dissolved in 17 ml of anhydrous methylene chloride, and 3.64 ml of trichloroacetonitrile and 1.743 g of anhydrous potassium carbonate were added. The mixture was stirred at room temperature for 6 hours.

After the reaction was completed, the reaction solution was mixed with 180 m of ethyl acetate.
It was diluted with 1 and washed with water and saturated saline, and then dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was subjected to silica gel column chromatography [hexane: ethyl acetate = 2: 1 (v / v) elution] to give a mixture of compound (7) and compound (8). 0.98
0 g (yield 74.8%) was obtained.

2) Production of compound (7) and compound (8) 5.53 mixture of compound (6) and its β-anomer
5 g was dissolved in 80 ml of anhydrous methylene chloride, 20.0 ml of trichloroacetonitrile and 9.631 g of anhydrous potassium carbonate were added, and the mixture was stirred at room temperature for 12 hours.

After completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with water and saturated saline, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was subjected to silica gel column chromatography to give a colorless amorphous compound (7) from the low-polarity fraction [eluted with hexane: ethyl acetate = 9: 1 (v / v)]. ) 3.449 g
(Yield 49.5%) was obtained, and 1.501 g (yield 21.5) of the colorless amorphous compound (8) was obtained from the highly polar fraction [eluted with hexane: ethyl acetate = 7: 1 (v / v)]. %) Was obtained.

Compound (7); IR (KBr) νcm ^-1 : 3384,3344,176
4,1724,1282,1252,798.

¹ H-NMR (400 MHz) (CDC
l ₃ ) δ: 1.35 (3H, d, J = 5.3Hz),
3.59 (1H, t, J = 10.5Hz), 3.68
(1H, t, J = 10.2 Hz), 3.88 to 3.92
(1H, m), 4.19 (1H, dd, J = 10.5)
and 5.0 Hz), 4.33 (1H, ddd, J =
10.2, 9.4 and 3.8 Hz), 4.62
(1H, d, J = 12.2 Hz), 4.75 (1H,
q, J = 5.3 Hz), 4.76 (1H, d, J = 1)
2.2Hz), 4.77 (1H, d, J = 11.8H
z), 4.87 (1H, d, J = 11.8 Hz), 5.
21 (1H, t, J = 10.2 Hz), 5.30 (1
H, d, J = 9.4 Hz, exchangeable
e), 6.37 (1H, d, J = 3.8Hz), 8.7
7 (1H, s, exchangeable).

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

Compound (8); IR (KBr) νcm ^-1 : 3342,1766,128
8,1254,1082,798.

¹ H-NMR (400 MHz) (CDC
l ₃ ) δ: 1.34 (3H, d, J = 5.0Hz),
3.56 to 3.68 (3H, m), 4.00 (1H, d
t, J = 9.7 and 8.8 Hz), 4.24-
4.28 (1H, m), 4.62 (1H, d, J = 1
1.8Hz), 4.69 (1H, d, J = 11.8H)
z), 4.70 to 4.78 (1H, m), 4.75 (1
H, d, J = 11.8 Hz), 4.86 (1H, d, J
= 11.8 Hz), 5.30 (1H, t, J = 9.7H)
z), 5.39 (1H, d, J = 8.8 Hz), 6.0
7 (1H, d, J = 8.8Hz), 8.72 (1H,
s, exchangeable).

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 of compound (7)
90 g of anhydrous methylene chloride was added to 9.00 g and powder molecular sieves 4A (9.00 g) under a nitrogen atmosphere. Subsequently, the mixture was cooled in an ice-water bath, 34 μl of trifluoromethanesulfonic acid trimethylsilyl ester was added with stirring,
After stirring at 2 ° C. for 20 minutes, 17 μl of trifluoromethanesulfonic acid trimethylsilyl ester was added, and 2
Stirred at 30 ° C for 30 minutes.

The reaction solution was filtered to remove insoluble materials, the filtrate was added to saturated aqueous sodium hydrogen carbonate solution, extracted with methylene chloride, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was subjected to silica gel column chromatography [hexane: ethyl acetate = 2: 1 (v /
v)), and 1.106 g of the compound (11) from the fraction eluting first (40.5% yield) and 0.731 g of the compound (10) from the fraction eluting later (40% recovery). 7%).

IR (KBr) νcm ^-1 ; 3392,176
6,1730,1246,1089.

¹ H-NMR (500 MHz) (CDC
l ₃ ) δ: 1.15 (3H, s), 1.21 (3H,
s), 1.31 (3H, d, J = 5.5Hz), 1.6
8 (3H, s), 1.95 (3H, d, J = 1.2H
z), 1.96 to 2.03 (1H, m), 2.21 to
2.29 (1H, m), 2.30 (3H, s), 2.3
8 to 2.44 (1H, m), 2.46 (3H, s),
2.67-2.76 (1H, m), 3.36-3.45
(1H, m), 3.51 (1H, t, J = 9.5H
z), 3.56 (1H, t, J = 10.1Hz), 3.
72-3.80 (1H, m), 3.82 (1H, d, J
= 7.0 Hz), 4.06 to 4.15 (1H, m),
4.16 (1H, d, J = 8.2Hz), 4.20 ~
4.26 (1H, m), 4.30 (1H, d, J = 8.
2Hz), 4.66-4.72 (3H, m), 4.76
~ 4.82 (4H, m), 4.82-4.88 (1H,
m), 4.98 to 5.06 (2H, m), 5.52 (1
H, d, J = 2.4 Hz), 5.67 (1H, d, J =
10.4Hz), 5.69 (1H, d, J = 7.0H
z), 6.03 (1H, dd, J = 9.2 and
2.4 Hz), 6.25 (1H, dif dt, J =
8.5 and 1.2 Hz), 6.36 (1H,
s), 6.90 (1H, d, J = 9.2 Hz), 7.3
5 to 7.47 (7H, m), 7.49 to 7.54 (3
H, m), 7.58 to 7.64 (1H, m), 7.74
~ 7.78 (2H, m), 8.10 to 8.15 (2H,
m).

FABMS m / z: 1563 (M
H ⁺ ), 1565 [(M + 2) H ⁺ ], 1567 [(M +
4) H ⁺ ], 1569 [(M + 6) H ⁺ ].

Example 8 Production of compound (12 ) 3.328 g of compound (11) 30 ml of 0.5N hydrochloric acid
5.830 g of zinc powder was added to a mixed solution of and 60 ml of tetrahydrofuran under ice cooling, and the mixture was stirred at the same temperature for 5 hours. Next, 2.776 g of zinc powder and 15 ml of 0.5N hydrochloric acid were added and stirred for 1 hour, and then 2.776 g of zinc powder and 30 ml of 0.5N hydrochloric acid were added to 1
Stirred for hours.

The reaction solution was diluted with methylene chloride, the insoluble material was filtered off, and the organic layer was separated. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine. 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. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography [chloroform:
Elution with methanol = 50: 1 (v / v)] to obtain 1.502 g (yield: 67.9%) of compound (12).

IR (KBr) νcm ^-1 ; 3436,172
4,1245,1092.

¹ H-NMR (500 MHz) (CDC
l ₃ ) δ: 1.19 (3H, s), 1.21 (3H,
s), 1.37 (3H, d, J = 4.9 Hz), 1.7
6 (3H, s), 1.86 (3H, s), 1.93-
2.00 (1H, m), 2.21 (3H, s), 2.2
7 to 2.38 (2H, m), 2.39 (3H, s),
2.41-2.47 (1H, m), 2.66-2.77
(1H, m), 3.27 to 3.35 (2H, m), 3.
45-3.57 (2H, m), 3.84 (1H, d, J
= 6.7 Hz), 4.07 to 4.17 (2H, m),
4.19 (1H, d, J = 8.5Hz), 4.30 (1
H, d, J = 8.5 Hz), 4.37 (1H, d, J =
7.9 Hz), 4.73 (1H, q, J = 4.9H
z), 4.80 (1H, d, J = 2.6 Hz), 4.8
5 (1H, br d, J = 8.6 Hz), 5.71 (1
H, d, J = 6.7 Hz), 5.79 (1H, dd, J
= 8.9 and 2.6 Hz), 6.19 (1H,
t, J = 9.5 Hz), 6.41 (1H, s), 7.0
2 (1H, d, J = 8.9 Hz), 7.33 to 7.39
(1H, m), 7.39 to 7.45 (4H, m), 7.
46 to 7.54 (5H, m), 7.58 to 7.64 (1
H, m), 7.73 to 7.78 (2H, m), 8.10
~ 8.15 (2H, m).

FABMS m / z: 1041 (M
H ⁺ ).

HRFABMS m / z: Calcd f
or C ₅₅ H ₆₅ N ₂ O ₁₈ (MH), 1041.4228
Found, 1041.4222.

Example 9 Production of compound (13 ) 1.458 g of compound (12) was added to 33.5 ml of methanol.
1.26 ml of 37% formalin and 0.364 g of sodium cyanoborohydride were added to the solution dissolved in
The mixture was stirred at room temperature for 1 hour.

The reaction solution was cooled in an ice-water bath, and 20 ml of 0.5N hydrochloric acid was added to make an acidic solution. Subsequently, a saturated aqueous solution of sodium hydrogen carbonate was added to make the mixture alkaline, extracted with chloroform, and dried with anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the obtained residue was subjected to silica gel column chromatography [eluting with chloroform: methanol = 70: 1 (v / v)] to give 1.263 g of compound (13) (yield 84. 4%).

IR (KBr) νcm ^-1 ; 3401,173
8, 1244, 1094.

¹ H-NMR (500 MHz) (CDC
l ₃ ) δ: 1.19 (3H, s), 1.22 (3H,
s), 1.40 (3H, d, J = 5.0Hz), 1.7
0 (2H, brs, exchangeable),
1.77 (3H, s), 1.87 to 1.93 (1H,
m), 1.88 (3H, d, J = 1.2Hz), 2.1
4 (3H, s), 2.21 (1H, dd, J = 9.8)
and 7.9 Hz), 2.30 (1H, dd, J = 1)
5.6 and 8.9 Hz), 2.35 to 2.41
(1H, m), 2.37 (3H, s), 2.38 (6
H, s), 2.51 to 2.59 (1H, m), 3.30.
(1H, t, J = 9.2 Hz), 3.39 (1H, di
fddd, J = 10.0, 9.2 and 4.8H
z), 3.57 (1H, t, J = 10.0Hz), 3.
60 (1H, dif dd, J = 9.8 and 9.2)
Hz), 3.82 (1H, d, J = 6.7 Hz), 3.
85 (1H, brs, exchangeable),
4.14 (1H, dd, J = 10.0 and 4.8
Hz), 4, 17 (1H, d, J = 8.5 Hz), 4.
30 (1H, d, J = 8.5Hz), 4.40 (1H,
dd, J = 9.5 and 7.6 Hz), 4.68
(1H, d, J = 7.9 Hz), 4.74 (1H, q,
J = 5.0 Hz), 4.83 (1H, d, J = 2.2H
z), 4.84 (1H, d, J = 9.8Hz), 5.7.
1 (1H, d, J = 6.7 Hz), 5.81 (1H, d
d, J = 9.0 and 2.2 Hz, 6.18 (1
H, dt, J = 8.9 and 1.2 Hz), 6.63
(1H, s), 7.04 (1H, d, J = 9.0Hz,
exchangeable), 7.33 to 7.37 (1
H, m), 7.38 to 7.43 (4H, m), 7.47.
~ 7.53 (5H, m), 7.59 ~ 7.63 (1H,
m), 7.72 to 7.76 (2H, m), 8.11
8.14 (2H, m).

FABMS m / z: 1069 (M
H ⁺ ).

HRFABMS m / z: Calcd f
or C ₅₇ H ₆₉ N ₂ O ₁₈ (MH), 1069.4543
Found, 1069.4568.

Example 10 Methane sulphate of compound (13)
Production of phonate [compound (14)] 371 mg of compound (13) was added to tert-butanol 25
It was dissolved in a mixed solution of 10 ml of water and 10 ml of water, and a solution of 27 μl of methanesulfonic acid in 17 ml of water was added under ice cooling. After stirring for 2 minutes, remove insoluble matter with a membrane filter (0.22μ
m) was filtered off and the filtrate was lyophilized to give compound (1
4) 387 mg (yield 95.7%) was obtained.

IR (KBr) νcm ⁻¹ : 3431,17
33,1646.

¹ H-NMR (400 MHz) (CDC
l ₃ ) δ: 1.18 (3H, s), 1.20 (3H,
s), 1.41 (3H, d, J = 5.0Hz), 1.7
5 (3H, s), 1.87 to 1.98 (1H, m),
1.90 (3H, s), 2.14 (3H, s), 2.2
1 (1H, m, exchangeable), 2.30
~ 2.59 (4H, m), 2.39 (3H, s), 2.
74 to 3.13 (7H, m, 1 Hexchangeab
le), 2.78 (3H, s), 3.28 (1H, t,
J = 9.5 Hz), 3.32 to 3.41 (1H, m),
3.50 (1H, t, J = 10.3Hz), 3.84
(1H, d, J = 6.9 Hz), 3.89 (1H, br
t, J = 9.5 Hz), 4.19 (1H, d, J =
8.4 Hz), 4.25 (1H, dd, J = 10.3)
and 5.0 Hz), 4.34 (1H, d, J = 8.
4Hz), 4.51 (1H, dd, J = 9.3 and
7.8Hz), 4.72 (1H, q, J = 5.0H
z), 4.73 to 4.78 (1H, m), 4.84 (1
H, br d, J = 9.2 Hz), 5.03 (1H,
d, J = 7.6 Hz), 5.17 to 5.37 (1H, b
rs, exchangeable), 5.75 (1
H, d, J = 6.9 Hz), 5.86 (1H, dd, J
= 9.5 and 1.9 Hz), 6.10 (1H, br
t, J = 8.6 Hz), 6.57 (2H, br s,
1H exchangeable), 7.31 to 7.5
7 (10H, m), 7.61 to 7.67 (1H, m),
7.83-7.88 (2H, m), 8.11-8.16
(2H, m), 9.37 to 6.91 (1H, brs, e
xchangeable).

FABMS m / z: 1069 (M
H ⁺ ).

Example 11 Hydrochloride of Compound (13)
Compound (15)] for the preparation of compound (13) 63 mg was dissolved in ethyl acetate 2.0 ml, under ice-cooling, 0.5 N hydrogen chloride ethyl acetate solution 0.1
77 ml was added. The solvent was distilled off under reduced pressure, and the resulting product was dried under reduced pressure at room temperature to obtain 60 mg of compound (15) (yield 92.2%).

Example 12 Tartrate salt of compound (13)
[Compound (16)] 53 mg of compound (13) was added to tert-butanol 3.9.
It was dissolved in a mixed liquid of 8 ml and 1.59 ml of water, and 0.266 ml of an aqueous L-tartaric acid solution (L-tartaric acid 7.
(Containing 44 mg) was added. After adding 3 ml of water to the obtained solution, it was freeze-dried to obtain 60 mg of compound (16) (yield 1
00%).

Test Example 1 Growth inhibitory effect on KB cells
(In vitro) 1) Test method 1 × 10 ³ KB cells / well in a flat-bottom 96-well plate
0.1 ml cell suspension (MEM containing 10% fetal bovine serum)
(Floating in the medium) and cultured for 24 hours. To this, 100 μl of the compound solution of the present invention dissolved in dimethyl sulfoxide and diluted with a medium (final concentration of dimethyl sulfoxide was 0.5
%) Was added and the cells were further cultured for 72 hours. After culturing 4,
5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide reagent (coloring reagent) was added, and the mixture was further cultured for 4 hours.

As a control, 100 μl of a medium (final concentration of dimethyl sulfoxide 0.5%) was added, and the cells were cultured in the same manner. After culturing, remove the medium and add 150 μl of cells.
Was dissolved in dimethylsulfoxide and the absorbance was measured, the ratio of the absorbance of the compound of the present invention to the absorbance of the control group was determined, and the 50% growth inhibitory concentration (IC ₅₀ ) was calculated.

2) Test Results Table 1 shows the IC ₅₀ of the compound (12), the compound (13) and the compound represented by the formula (I) for KB cells.

[0104]

[Table 1]

Test Example 2 Mouse B16 melanoma (ascites fluid)
(In vivo) 1) Test method A 10% homogenate solution of B16 melanoma tumor mass subcultured in female C57BL / 6 mice was prepared by adding Hanks balanced salt solution, and 0.5 ml of the solution was used for females. The BDF1 strain mouse was intraperitoneally transplanted. 1 day after tumor cell transplant
The compound of the present invention and taxol were intraperitoneally administered once a day for 5 consecutive days.

Compound (12) and taxol are polyoxyethylene castor oil 50% and ethanol 50%.
Was dissolved in a solvent consisting of and was diluted with physiological saline immediately before administration. That is, physiological saline containing 5% polyoxyethylene castor oil and 5% ethanol was used as a solvent for administration. A formation of a precipitate was observed for taxol in a short time, but the compound (12) was clear for a long time (24 hours or more). Before use, the compound (14) was dissolved in distilled water for injection and administered. The control group was given distilled water for injection, and the solvent control group was given only physiological saline containing 5% polyoxyethylene castor oil and 5% ethanol. 12 in the control group
Six animals were used for each of the animals, the solvent control group and the drug administration group.

After observing the animals for 50 days, the survival rate was calculated by the following formula.

Prolongation of life (%) = median survival time of drug administration group / median survival time of control group × 100 Incidentally, 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 rates and changes in body weight of compound (12), compound (14) and taxol.

[0110]

[Table 2]

Compound (12) is equivalent to taxol,
It showed a slightly weakening effect on life extension.

Compound (14) showed a life-prolonging effect equivalent to that of taxol, and there was no toxicity even at the dose of 30 mg / kg in which weight loss and early death due to administration of taxol were observed. It is considered to have been done.

Test Example 3 Measurement of Solubility in Water 1) Method for Measuring Solubility of Taxol (1) 5 ml of water was added to 5 mg of taxol and treated with ultrasonic waves for 5 minutes to disperse the sample, and further at room temperature for 30 minutes. I was shaken. Then, it is filtered through a 0.45 μm membrane filter to remove taxol present in the filtrate by HPLC.
Was quantified at.

(2) HPLC measurement conditions Column: ODS-80TM 5 μm (4.6 mm ID ×)
150 mm) Column temperature: 50 ° C. Eluent: 0.1% trifluoroacetic acid aqueous solution: Acetonitrile = 1: 1 (v / v) Flow rate: 1.0 ml / min Detection wavelength: 230 nm 2) Measurement of solubility of compound (14) Method (1) 2 ml of water was added to 0.1 mg of the compound (14), treated with ultrasonic waves for 1 minute to disperse the sample, and further shaken at room temperature for 30 minutes and left for 24 hours. Then 0.4
After filtration with a 5 μm membrane filter, the test substance present in the filtrate was quantified by HPLC.

(2) HPLC measurement conditions Column: ODS-80Ts 5 μm (4.6 mm ID ×)
150 mm) Column temperature: 50 ° C. Eluent: 0.1 M ammonium acetate aqueous solution: Acetonitrile = 1: 1 (v / v) Flow rate: 1.0 ml / min Detection wavelength: 230 nm 3) Test results Water of compound (14) and taxol Table 3 shows the solubilities of the above.

[0116]

[Table 3]

[0117]

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yoshinori Sekiguchi 3-24-1 Takada, Toshima-ku, Tokyo Within Taisho Pharmaceutical Co., Ltd. (72) Chihiro Yokoo 3-24-1 Takada, Toshima-ku, Tokyo In Taisho Pharmaceutical Co., Ltd. (72) Inventor Katsuo Hatayama 3-24-1 Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd.

Claims (1)

[Claims] (1) Formula (1) (In the formula, R represents a hydrogen atom or a methyl group.) A taxol derivative and a pharmaceutically acceptable salt thereof.