JPS63192793A - Novel ester of 4'-demethyl-epipodophyllotoxin derivative - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R1-R3 are OH, sulfate group or phosphoric acid group, provided that at least one of them presents the other than OH) or salt thereof. EXAMPLE:4'-o-Phosphono-4'-demethyl-epipodophyllotoxin-beta-D-4,6-,o-et hylidenegly coxide. USE:An antitumor agent having high water-solubility. PREPARATION:For example, a protecting group X1 and/or X2 of 4'-o- protected-4'-demethyl-epipodophyllotoin-beta-D-2,3-o-diprotected-4,6-0- ethylideneglycoxide expressed by formula II (X1 and X2 are protecting groups) is removed and the resultant product is reacted with a phosphorus oxidizing agent such as phosphorus oxychloride, in the presence of an aprotic solvent such as THF, at -10 deg.C- boiling point of the solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The compounds of the present invention are expected to be used as antitumor agents.

[Conventional technology]

4′-demethyl-epipodophyllotoxin-β-D −
4,6-o-ethylidene glycoside is known as a compound having antitumor activity, such as in Japanese Patent Publication No. 38258/1983.

[Problem that the invention seeks to solve]

4'-demethyl-epipodophyllotoxin-β-D-4,6-〇-ethylidene glycoside, which has been conventionally used as an antitumor agent, is extremely poorly soluble in water, so it cannot be used as a regular injection. It is difficult to prepare and has been used as a polyethylene glycol solution.

However, it has drawbacks such as pain during injection, and an aqueous solution formulation is desired.

Therefore, the present inventors conducted various studies on 4'-demethyl-epipodophyllotoxin derivatives with increased water solubility.

As a result, a 4'- The present invention was completed based on the discovery that a novel ester of a demethyl-epipodophyllotoxin derivative or a pharmacologically acceptable salt thereof has high water solubility.

Examples of the compound of general formula (T) of the present invention include the compounds shown in Table 1 below.

Table 1 Compounds Represented by General Formula (T) The compounds of the present invention may be produced, for example, as follows.

4-〇-protected-4'-demethyl-ehyphodophyllotoxin-β-D-2,3 represented by the general formula (wherein, Xl and X2 each represent a selectively removable protecting group) -0-protection-4,6
A phosphorylating agent or a sulfating agent is applied to a compound obtained by removing either or both of the protecting group X of -0-ethylidene glycoside in an aprotic solvent. If either the protecting group of Xl or n remains, the remaining protecting group of the obtained compound may be removed.

The amount of the phosphorylating agent or sulfating agent is generally equal to or more than the equivalent mole based on the compound represented by the general formula, and depending on the number of groups to be substituted, it may be used in an amount equal to or more than the theoretical number of moles.

In the case of a compound in which only one of R1 and R2 is substituted, 1 mol of 4'-o-protected-4'-demethyl-epipodophyllotoxin-β-D-4,6-0-ethylidene glycoside A phosphorylating agent or sulfating agent in a range of 1^2 mole is added to the reaction mixture under mild conditions, and R1 and R2 are unsubstituted, mono-substituted, and di-substituted. A mixture may be obtained, and the desired monosubstituted product may be separated from the mixture by a conventional method such as chromatography.

In the case of disubstituted R1 and R2, 4-o-protected-4
'-demethylepipodophyllotoxin-β-4,6-o
- It can be obtained by adding 2 or more moles of a phosphorylating agent or sulfating agent to 1 mole of ethylidene glycoside, sufficiently carrying out the reaction, and then isolating the target compound.

When R1, R2 and R3 are all substituted, a phosphorylating agent or sulfuric acid is added per mole of 4'-demethyl-epipodophyllotoxin-β-4,6-o-ethylidene glycoside. It can be obtained by adding 3 moles or more of a curing agent and sufficiently carrying out the reaction. The reaction temperature may be -10°C to the boiling point of the solvent.

The protecting group for hydroxyl group in the present invention is not particularly limited as long as it is a commonly used group, but R+, R of the general formula (?)
When either 2 or R3 is a hydroxyl group, Xl
and must be able to be removed by different removal methods. For example, when Xl is eliminated by reduction using zinc-acetic acid, such as 2.2.2-trichloroethoxycarbonyl group, Xl is eliminated by catalytic reduction using a palladium catalyst, etc. For example, substituted or unsubstituted benzyloxy Examples of the substituted benzyloxycarbonyl group include a p-methoxybenzyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group, and a p-nitrobenzyloxycarbonyl group.

Examples include Lopenzyloxycarbonyl group.

Examples of the phosphorylating agent include phosphorus oxychloride and birophosphoric acid. Examples of the sulfating agent include sulfuric anhydride/pyridine complex salt, chlorosulfonic acid, and the like.

There are no particular restrictions on the solvent for phosphorylation, but ethyl ether, tetrahydrofuran, benzene, toluene, ■, 2
- Aprotic solvents that do not adversely affect the reaction, such as dichloroethane, are preferred. Also, as a solvent for sulfation, N
, N-dimethylformamide, pyridine and the like are preferred.

As a neutralizing agent added during the reaction, organic bases such as pyridine and triethylamine, and inorganic bases such as calcium oxide, sodium bicarbonate, and potassium carbonate can be used.

The reaction temperature is usually below the boiling point of the solvent used, but
Preferably, it is carried out at about 0°C to room temperature.

After the phosphorylated or sulfated compound is extracted using water and ethyl acetate or dichloromethane, the layer containing the target compound is concentrated.

After the obtained concentrated residue is purified directly or by column chromatography using a non-polar adsorption resin, the protecting group is removed, and if necessary, after purification by column chromatography using a non-polar adsorption resin, etc., it is purified by a conventional method. The compound of the present invention can be obtained by isolation.

Phosphoric acid ester or sulfuric acid ester of the present invention [Effect] Table 2 shows the solubility in water of the sodium salt of a representative compound of the general formula fT) obtained by the present invention.

In addition, as a control compound, R1= R2= R3= OH
The following compounds are listed.

Table 20 Water Solubility of Compounds of the Present Invention As shown in Table 2, the obtained compounds represented by general formula The process has become extremely advantageous.

[For production]

Next, the antitumor activity of the sodium salt of the representative compound of the present invention represented by the general formula (n) will be shown by experimental examples.

(1) Experimental method L1210 leukemia cells I x 10510.2 ml
Han K'5balanced 5ol
tion K was suspended and intraperitoneally transplanted into CDF mice (6 weeks old).

The drug was intraperitoneally administered every day for 5 days starting from the next day.

The drug was dissolved and diluted with physiological saline, and the volume was 0.1 ml/1.
It was administered at a rate of 0 g body weight. The observation date was 60 days.

For evaluation, the average survival days of each group was determined from the survival days of each mouse, and the ratio to the average survival days of the control group was determined as T/C (%).

(2) Table 3 shows the antitumor activity of representative examples of the compounds of the present invention.

As a control, a compound represented by the general formula (■) where R+ = R2 = R3 = OH was used.

The present invention will be specifically explained below using Examples.

The Rf value of thin layer chromatography was measured using a Merck thin layer plate, Arti'b 5715.

Note that detection was performed using ultraviolet irradiation and dilute sulfuric acid.

Example 1 Preparation of the sodium salt of 4'-o-phosphono-47-dimethyl-epipodophyllotoxin-β-D-4,6-o-ethylidene glucoside (compound %1)tl) 4'-o-
Phosphono-4'-demethyl-epipodophyllotoxin-
β-D-2,3-G0-β, β.

β-Trichloroethoxycarbonyl-4,6-.

-Sodium salt of ethylidene glucoside 4'-demethyl-epipodophyllotoxin-β-D-2,3-di0-
β, β, β-trichloroethoxycarbonyl-4,6-
o-ethylidene glucoside 4.70 g (5,00m
mol) to 1,2-dikuo.

Add 30 ml of ethane and 5 ml of dry pyridine to dissolve and cool to -60°C. Then, 1.2-shikuo
o, x Phosphorous oxychloride O1 dissolved in 20 ml of tan
After dropping 92mB (10mmol) for 5 minutes, the internal temperature was about -
Stir at 15°C for 1 hour, at 0°C for 1 hour, then at room temperature overnight.

After the reaction, the reaction solution was concentrated under reduced pressure, and ethyl acetate and ethyl acetate were added to the concentrated residue.
After extraction with ice and brine followed by IN hydrochloric acid, the ethyl acetate layer is washed twice with brine and dehydrated using anhydrous magnesium sulfate.

After removing the magnesium sulfate from the furnace, the ethyl acetate solution was concentrated under reduced pressure. 50 ml of ethyl acetate was added to the residue, and 0.9 g of sodium 2-ethylhexanoate dissolved in 50 ml of isopropanol was added to the solution. ~20m
When concentrated to 1, add 100 ml of isopropanol
was added, the crystals were filtered, and dried under reduced pressure to obtain 4.8 g of crude crystals.
(Gross yield: 92%) is obtained.

ru = o, s (developing solvent n-butanol ° acetic acid: water =
4:1:1) (2) 4'-phosphono-47-temethyl-epipodophyllotoxin-β-D-4,6-o-
Sodium salt of ethylidene glucoside (compound ventilation 1) 400 g of the crude crystals obtained in (1) above are dissolved in 120 ml of acetic acid, and then 6.0 g of zinc powder is added and stirred vigorously at room temperature for 2 hours. After the reaction is complete, the zinc is filtered using 10,100 O of water, and the cloudy filtrate is filtered again using cellulose filter aid and 800 ml of water.

Next, this filtrate (approximately 2000 ml) was injected into Hypo-5.
x Ho IJ z - (Mitsubishi Chemical Industries KK, HP-2
0') Adsorbed on a 200ml column and washed with water, 3
Elution is performed using 0% acetone water, and a fraction containing the target product is collected.

The fractionated eluate was concentrated under reduced pressure, the acetone was distilled off, and then lyophilized to produce 4'-phosphono-4'-temethyl-epipodophyllotoxin-β-D-4,6-〇-ethylidene glucoside. 1.50 g of sodium salt (2,17
mmol).

Rf=02 (developing solvent n-butanol; acetic acid; water=4
:1:1) Nuclear magnetic resonance spectrum (dimethylsulfoxide-da solution, internal standard tetramethylsilane) δ=1.24 (3B, d) δ=2.89 (I H, m) δ=3.0 ~3.4 (5H, m) δ=3.50 (IH, t) δ=3.57 (6H, s) δ=4.08 (1)1. aa) δ=4.28
(2H, m) δ=4.54 (IH, d) δ=4.58 (1) 1. a) δ=4.73 (IH, m) δ=4.94 (IH, d) δ=5.24 (2H, broad) δ=6.03
(21-1,s) δ=6.20 (2H,s) δ=6.54 (LH,s) δ=7.01 (IH,s) Example 2゜4'-demethyl-epipod Phyllotoxin-β-D-2
．． Agricultural method of disodium salt of 3-di-0-phosphono-4,5-Q-ethylidene glucoside (compound Nn2) (1) 4'-o-benzyloxycar,bonyl-4'-
Demethyl-epipodophyllotoxin-β-D-2゜3-
Disodium salt of di-0-phosphono-4,6-o-ethylidene glycoside 4'-〇-benzyloxycarbonyl-4'-temethyl-evivodophyllotoxin-β-D-4゜6-o-ethylidene glycoside 1. 45 g (2,00 mmol
), 1.08 ml of dry pyridine and 7710 ml of tetrahydro 7
ml (4.4 mmol of ethyl ether/L'10 m
1 into the solution under cooling at -60°C.

After the dropwise addition, the mixture was stirred at the same temperature for 2 hours, in a water bath for 1 hour, and then at room temperature overnight.

After the reaction, ethyl acetate and ice water were added to the reaction solution, and while stirring, sodium hydrogen carbonate was added, and after stirring at room temperature for 3 hours,
- Set up at night and separate the aqueous layer.

Dissolved ethyl acetate in the aqueous layer was distilled off by concentration under reduced pressure, and water was added to the remaining solution to make 150 ml. Next, high porous polymer (Mitsubishi Chemical Industries KK, HP-20■) 50m10
It is adsorbed onto a column, washed with water, and then eluted with 50% acetone water to collect a fraction containing the target product.

After concentrating the fractionated eluate under reduced pressure, Improper Tool is added to crystallize it. The precipitated crystals were filtered and dried under reduced pressure to yield 4'-0-benzyloxycarbonyl-4'-demethyl-epipodophyllotoxin-β-D-2,3-di-0-phosphono-4°6-o-ethylidene. Glycoside 2
0.44 g (0.45 mmol) of the sodium salt is obtained.

Rf=0.5 (exhibited solvent chloroform:methanol:acetic acid=75:25:3) Nuclear magnetic resonance spectrum (dimethyl sulfoxide-d6,
Internal standard substance, tetramethylsilane) δ = 1.23 (3)], aa δ = 2.95 (IH, m) δ = 3.20 < I H, m) δ = 3.3 to 3.7 ( 5H,m)δ=3.62
(6H,s) δ=3.94(IH,t) δ=4.03 (IH,dd) δ=4.26 C2H,d) δ=4.63CxH,d) δ=4.79 (IH , m) δ=4.97 (IH,d) δ=5.23 (2H,s) δ=6.03. 6.05 (2B, s, s
) δ=6.31 (2H, s) δ26.55 (IH, S) δ=7.10 (18, s) δ=7.40 (5B, m) (2) 4'-demethyl-epi Podophyllotoxin
β-D-2,3-di-0-phosphono-4,6-o-
: Disodium salt of f-tylidene glycoside (compound Nn2) 4'-〇-benzyloxycarbonyl-4'-temethyl-evibodophyllotoxin- obtained in (1) above, #
-D-2,3-di-0-phosphono-4,6-o-ethylidene glycoside disodium salt 300■ (0,30
(9 mmol) were added with IQ ml of water, 1 Q ml of acetone, and 0.1 g of 5% palladium on carbon, and catalytic reduction was carried out at normal pressure and room temperature for 2 hours.

After the reduction, the radium on carbon was filtered, the filtrate was concentrated under reduced pressure, 20 to 3 Q ml of water was added to the resulting residue, and the water-insoluble material was filtered into Millipore filtrate (Mi1lex181-G).
S.

0.22 μm).

The filtrate was then freeze-dried to yield 250% of the disodium salt of 4'-demethyl-epipodophyllotoxin-β-D-2,3-di-0-phosphono-4,6-o-ethylidene glycoside. [1 g (0,315 mmoJ) is prayed.

Rf = 0.4 (Developing solvent chloroform: methanol: acetic acid = 75:25:3) Nuclear magnetic resonance spectrum (heavy water, external standard, water (4,80
ppm) ) δ=1.36 (3H, d) δ=3.07 (I H, m) δ=3.73 (6B, s) δ=3.96 (LH, m) δ=5.97. 5.99(2H,s,s)δ=6
．． 37 (2H, s) δ=6.58 (1B, s) δ=7.02 (1B, s) Example 3 4'-Hydroxysulfonyl-47-temethylepipodophyllotoxin-β-D4, Method for producing sodium salt of 6-o-ethylidene glucoside (compound % 3) (1) 4'-Hydroxysulfonyl-4'-demethylepipodophyllotoxin-β-D-2,3-cy.

-β,β,β-trichloroethoxycarbonyl-4゜6
- Sodium salt of o-ethylidene glucoside 4'-demethylepipodophyllotoxin-β-D-2,3-di. -β,β,β-trichloroethoxycarbonyl-4,
6-o-ethylidene glucoside (0.47g) with N,N
-dimethylformamide (1om+) and viridi7 (
After dissolving sulfuric anhydride/pyridine complex salt (2.5 g) in N,N-dimethylformamide (tom
l) Add gi, stir at room temperature for 7 days, and concentrate the reaction solution under reduced pressure. Methanol and sodium 2-ethylhexanoate are added to the concentrated residue, and after concentration under reduced pressure, ethyl acetate and water are added to the residue for extraction.

The ethyl acetate layer was separated and concentrated under reduced pressure. Ether was added to the resulting residue, and the crystals were filtered and dried under reduced pressure to obtain the target product C.
0.21 g) is obtained.

Rf=0.46 (Developing solvent n-butanol, acetic acid:
Water = 4:1:1) (2) 4'-Hydroxysulfonyl-4'-temethylehifodophyllotoxin-β-D
= 4.6- Add the sodium salt of o-ethylidene glucoside (0.4 ml) and stir at room temperature for 1 hour.

Water (100ml) was added to the reaction solution, zinc was filtered, and the filtrate was treated in the same manner as in Example 1-(2) to obtain the desired product
66■) is obtained.

Rf=0.2 (Developing solvent n-butanol; acetic acid; water = 4+141) Reference example (1) 4'-benzyloxycarbonyl-4'-demethylepipodophyllotoxin-β-D-2.3-di 0-β, β, β-trichloroethoxycarbonyl-4,
Preparation of 5-Q-ethylidene glucoside 4'-benzyloxycarbonyl-4'-temethylevipodophyllotoxin (72.5 g) and 4.6-o-ethylidene-
After dissolving 2,3-di0-β,β,β-trichloroethoxycarbonyl-β-D-glucobylanose (83 g, 1.1 equivalents) in 1,2-dicoooz tough (800n++), - Boron trifluoride etherate (29 g) dissolved in dichloroethane (300 ml), of which m-10 to -
Keep at 15°C and add dropwise over 3 hours. -10~15 after dropping
Stir at ℃ for 2 hours.

Then, a solution of pyridine (21 ml) and 1,2-dichloroethane (80 ml) was added to the reaction mixture. After slow dropwise addition at -10°C, water (sooml) is added and the organic layer is separated. This organic layer is washed successively with dilute hydrochloric acid and then brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting concentrated residue is purified by silica gel chromatography to obtain the desired product (121 g).

Rf=0.8 (Developing solvent chloroform: methanol
/l/=10:1)t2) 4'-f methylepipodophyllotoxin-β-D-2,3-di0-β,
β, β-trichloroethoxycarbonyl-4,6-0-
4'-benzyloxycarbonyl-4'-temethylepipodophyllotoxin-β-D obtained in reference example (1) for the production of ethylidene glucoside
-2,3-di-O-β,β,β-trichloroethoxycarbonyl-4,6-0-ethylidene glucoside (20,
0 g) in aceto7 (300 m+) in the presence of 5% palladium on carbon (1 g) in an autoclave under hydrogen pressure of 30
Catalytic reduction is carried out at room temperature for 2 hours at kg/cm2.

Then, after removing the catalyst, the solution was concentrated under reduced pressure, and ethyl acetate was added to the resulting residue to dissolve it. The ethyl acetate solution is washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Improper tool is added to the resulting residue for crystallization.

The precipitated crystals are filtered, washed and dried under reduced pressure to obtain the desired product (1
6.2 g) is obtained.

Claims (1)

[Claims] (1) General formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I) A novel ester of a 4'-demethyl-epipodophyllotoxin derivative represented by (representing a group other than a hydroxyl group) or a pharmacologically acceptable salt thereof.