JP2011084632A - Derivative of polyethylene glycol and antitumor drug including the same as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide compounds useful as active ingredients of an antitumor drug. <P>SOLUTION: A compound 9bw, which is prepared by producing nonaethylene glycol mono(p-toluenesulfonyl) ether from nonaethylene glycol and deriving nonaethylene glycol mono(4'-iodo-4-biphenyl) ether from the resultant ether, and a compound 8bw, which is prepared by producing dodecaethylene glycol mono(p-toluenesulfonyl) ether from dodecaethylene glycol and deriving dodecaethylene glycol mono(4'-iodo-4-biphenyl) ether from the resultant ether, are used as active ingredients of the antitumor drug. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a derivative of polyethylene glycol and an antitumor agent comprising the same as an active ingredient.

  Various compounds are known as compounds that induce cell death or compounds that enhance the action of compounds that induce cell death, and studies on their use as active ingredients in drugs for the prevention or treatment of various diseases It is actively performed.

  For a polyethylene glycol derivative (for example, polyoxyethylene (2,4-diisooctylphenyl) ether) that can be used as an active ingredient of a cell death inducer and an anticancer agent, and a method for producing the same, WO 2006/043338 (A1) pamphlet, It is described in WO2006 / 043353 (A1) pamphlet and WO2006 / 134648 (A1) pamphlet. Furthermore, the synthesis of such polyethylene glycol derivatives is described in Org. Lett., 2002, 4, 2329-2332.

WO2006 / 043338 pamphlet, WO2006 / 043353 pamphlet WO2006 / 134648 pamphlet

Org. Lett., 2002, 4, 2329-2332

  An object of the present invention is to provide a compound that is useful as an active ingredient of an antitumor agent and can be produced by a simpler method, and an antitumor agent containing the compound as an active ingredient.

  The antitumor agent according to the present invention was selected from the group consisting of a polyethylene glycol monoether compound represented by the following formula (1) and a salt thereof, and a polyethylene glycol monoether compound represented by the following formula (2) and a salt thereof. Including at least one compound.

  According to the present invention, a compound useful as an active ingredient such as an antitumor agent can be provided. In addition, the above efficient production method of an intermediate compound enables efficient production of such a useful compound.

It is a figure which shows the result of the safety test of compound 9bw. It is a figure which shows the result of the safety test of compound 9bw. It is a figure which shows the result of the safety test of compound 8bw. It is a figure which shows the result of the safety test of compound 8bw. It is a figure which shows the DNA synthesis inhibitory effect with respect to Meth-A tumor cell of the compound 9bw. It is a figure which shows the body weight change of the mouse | mouth intraperitoneally administered of compound 9bw. It is a figure which shows the change of Meth-A tumor volume by intraperitoneal administration of compound 9bw. It is a figure which shows the tumor reduction ratio after compound 9bw administration. It is a figure which shows the Meth-A tumor weight after compound 9bw administration. It is a figure which shows the body weight change by continuous administration for 10 days of compound 9bw. It is a figure which shows the change of the tumor volume by continuous administration for 10 days of compound 9bw. It is a figure which shows the change of the tumor volume ratio by continuous administration for 10 days of compound 9bw. It is a figure which shows the change of the body weight of the mouse | mouth in which the compound 9bw is intratumorally administered. It is a figure which shows the change of the tumor volume in the intratumoral administration of compound 9bw. It is a figure which shows the change of the tumor volume in the intratumoral administration of compound 9bw. It is a figure which shows the antitumor effect of compound 9bw. It is a figure which shows the DNA synthesis inhibitory effect with respect to Meth-A tumor cell of the compound 9bw. It is a figure which shows the effect of the compound 9bw with respect to the mitogenic reaction of a mouse | mouth spleen lymphocyte. It is a figure which shows the change of the mouse body weight (at the time of dissection) in Example 5. FIG. It is a figure which shows the change of the tumor weight in Example 5. FIG. It is a figure which shows the change of the red blood cell count in Example 5. FIG. It is a figure which shows the change of the white blood cell count in Example 5. FIG. It is a figure which shows the change of the platelet count in Example 5. FIG. It is a figure which shows the change of the amount of hemoglobin in Example 5. FIG. It is a figure which shows the change of the hematocrit value in Example 5. FIG. It is a figure which shows the change of the body weight in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the tumor weight in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the red blood cell count in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the white blood cell count in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the platelet count in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the amount of hemoglobin in the reference test (1) of Example 5. FIG. It is a figure which shows the change of the hematocrit value in the reference test (1) of Example 5. FIG. It is a figure which shows the effect of 9bw with respect to MIA-PaCa-2 in the reference test (2) of Example 5. FIG. It is a figure which shows the selectivity with respect to the normal cell and tumor cell of compound 9bw and 8bw.

  Polyethylene glycol monoether compounds 8bw and 9bw according to the present invention (hereinafter simply referred to as compounds 8bw and 9bw, 8bw and 9bw, or PEG (8bw) and PEG (9bw)). The above compounds 8bw and 9bw can be synthesized by the following reaction in which the paratoluenesulfonyl group of the intermediate compound corresponding to each of them is substituted with R.

  The reaction can be carried out in an air atmosphere in the presence of water, a solvent immiscible with water in any proportion, and a basic metal salt that is water-soluble. As the solvent, in addition to the above, dichloromethane can be used. Examples of the basic metal salt include sodium carbonate, sodium hydrogen carbonate, lithium hydride, potassium hydroxide and the like in addition to those used in the examples. Examples of the catalyst include quaternary ammonium salts such as tetrabutylammonium bromide, tetrabutylammonium iodide, cetyltrimethylammonium bromide in addition to those used in the examples. The reaction temperature can be appropriately set from a range including room temperature. After completion of the reaction, target compounds 8bw and 9bw are recovered from the reaction solution. For this recovery, conventional methods such as filtration and separation methods using various chromatographies can be used.

  In addition, these intermediate compounds react the corresponding polyethylene glycol and para-toluenesulfonyl chloride in an inert solvent such as toluene in the presence of an aqueous alkali solution such as sodium hydroxide and a phase transfer catalyst such as benzyltriethylammonium chloride. Can be obtained. The reaction temperature can be appropriately set from a range including room temperature. After completion of the reaction, the target intermediate compound is recovered from the reaction solution. For this recovery, conventional methods such as filtration and separation methods using various chromatographies can be used.

  The route in the above synthesis is shown as the following reaction scheme.

  Compounds 8bw and 9bw according to the present invention, and salts thereof, in particular, at least one selected from pharmacologically acceptable salts can be used as an antitumor agent as an active ingredient. Examples of the antitumor agent include solid preparations such as infusions (intravenous injection), tablets, pills, powders, granules, capsules, suppositories, and liquids such as injections, suspensions, syrups, and emulsions. Examples thereof include semi-solid preparations such as preparations and patches. Formulation can be carried out by appropriately selecting carriers, diluents, excipients and various additives according to these dosage forms.

  The pharmacologically acceptable salts of compounds 8bw and 9bw can be obtained by conventional methods. For example, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, or formic acid, acetic acid, fumaric acid, citric acid, maleic acid, oxalic acid, malic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid, p -It can be used as a salt with an organic acid such as toluenesulfonic acid.

  The anticancer agent of the present invention is particularly effective for pancreatic cancer and the like.

  Hereinafter, the present invention will be described in detail by way of examples.

Example 1 Synthesis of Compound 9bw (1) Synthesis of Compound 5
Nonaethylene glycol mono (p-toluenesulfonyl) ester (5)

Nonaethylene glycol 2 (1.00 g, 2.41 mmol) was dissolved in toluene (10 mL). To this, benzyltriethylammonium chloride (0.05 g, 0.24 mmol) and 20% aqueous sodium hydroxide solution (10 mL) were added and stirred. To this was added a solution of p-toluenesulfonyl chloride (0.53 g, 2.75 mmol) in toluene (5.0 mL), and stirring was continued at room temperature for 18 hours. Thereafter, 10% hydrochloric acid (25 mL) was added, and the toluene layer and the aqueous layer were separated. The aqueous layer was extracted with ethyl acetate (25 mL × 3). The toluene layer and the ethyl acetate layer were combined and washed successively with water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL), and saturated brine (20 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 20 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 200 to 1 → 16 to 1), and colorless liquid 5 (0.88 g, 1.55 mmol) , Purity 100%, yield 48%). IR (ATR) v max cm ^-1 : 3433. ¹ H-NMR (CDCl ₃ ) δ: 2.27 (3H, s), 2.93 (1H, br), 3.56-3.64 (34H, br), 4,14 (2H, t, J = 5.0 Hz), 7.34 (2H , d, J = 8.4 Hz), 7.79 (2H, d, J = 8.4 Hz).

(2) Synthesis of compound 9bw
Nonaethylene glycol mono (4'-iodo-4-biphenyl) ether (9bw)

7bw (296 mg, 1.00 mmol) was dissolved in THF (5.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 46 mg, 1.15 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (176 mg, 0.31 mmol) in THF (3.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 8 to 1 → 7 to 2), and 9 bw (119 mg, 0.17 mmol, purity 100%, yield 92%, molecular weight: 675). ¹ H-NMR (CDCl ₃ ) δ: 2.64 (1H, br), 3.56-3.64 (32H, br), 3.88 (2H, t, J = 4.9 Hz), 4.17 (2H, t, J = 4.9 Hz), 6.98 (2H, d, J = 8.6 Hz), 7.29 (2H, d, J = 8.6 Hz), 7.47 (2H, d, J = 8.6 Hz), 7.73 (2H, d, J = 8.6 Hz).

Example 2 Synthesis of Compound 8bw (1) Synthesis of Compound (3)
Dodecaethylene glycol mono (p-toluenesulfonyl) ester (3)

Dodecaethylene glycol 1 (1.00 g, 1.83 mmol) was dissolved in toluene (10 mL). To this, benzyltriethylammonium chloride (0.04 g, 0.19 mmol) and 20% aqueous sodium hydroxide solution (10 mL) were added and stirred. To this was added a solution of p-toluenesulfonyl chloride (0.36 g, 1.90 mmol) in toluene (5.0 mL), and stirring was continued at room temperature for 18 hours. Thereafter, 10% hydrochloric acid (25 mL) was added, and the toluene layer and the aqueous layer were separated. The aqueous layer was extracted with ethyl acetate (25 mL × 3). The toluene layer and the ethyl acetate layer were combined, and washed successively with water (20 mL), saturated aqueous sodium bicarbonate (20 mL), and saturated brine (20 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 16 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 60 to 1 → 8 to 1), and colorless liquid 3 (0.76 g, 1.08 mmol) , Purity 99%, yield 49%). IR (ATR) v max cm ^-1 : 3462. ¹ H-NMR (CDCl ₃ ) δ: 2.03 (1H, br), 2.27 (3H, s), 3.56-3.64 (46H, br), 4.14 (2H, t, J = 5.0 Hz), 7.34 (2H, d , J = 8.4 Hz), 7.79 (2H, d, J = 8.4 Hz).
(2) Synthesis of compound 8bw
Dodecaethylene glycol mono (4'-iodo-4-biphenyl) ether (8bw)

7bw (310 mg, 1.05 mmol) was dissolved in THF (5.0 mL). Sodium hydride (mineral oil suspension, purity 60%, 51 mg, 1.28 mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour. To this was added a solution of 3 (200 mg, 0.29 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated by silica gel column chromatography (silica gel: 5 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 45 to 1 → 45 to 2 → 8 to 1 → 7 to 2) and 8 bw ( 52 mg, 0.06 mmol, purity 100%, yield 75%, molecular weight: 807). ¹ H-NMR (CDCl ₃ ) δ: 2.75 (1H, br), 3.56-3.64 (44H, br), 3.88 (2H, t, J = 4.9 Hz), 4.17 (2H, t, J = 4.9 Hz), 6.98 (2H, d, J = 8.6 Hz), 7.29 (2H, d, J = 8.6 Hz), 7.47 (2H, d, J = 8.6 Hz), 7.73 (2H, d, J = 8.6 Hz).

(Reference synthesis example)
Nonaethylene glycol mono (4-iodophenyl) ether (9ah)

7ah (230 mg, 1.05 mmol) was dissolved in THF (2.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 49 mg, 1.23 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (210 mg, 0.37 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 90 to 1 → 12 to 1 → 4 to 1), and 9ah (154 mg, 0.25 mmol, yield 68%). ¹ H-NMR (CDCl ₃ ) δ: 2.61 (1H, br), 3.57-3.73 (32H, br), 3.84 (2H, t, J = 4.8 Hz), 4.09 (2H, t, J = 4.8 Hz), 6.69 (2H, d, J = 9.2 Hz), 7.54 (2H, d, J = 9.2 Hz).

  Nonaethylene glycol mono (4-bromophenyl) ether (9j)

7j (126 mg, 0.73 mmol) was dissolved in THF (1.0 mL). To this was added sodium hydride (mineral oil suspension, purity 60%, 36 mg, 0.90 mmol), and the mixture was stirred at room temperature for 1 hour. To this was added a solution of 5 (192 mg, 0.34 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 10 to 1), 9j (130 mg, 0.23 mmol, yield 68%) Got. ¹ H-NMR (CDCl ₃ ) δ: 2.78 (1H, br), 3.59-3.74 (32H, br), 3.83 (2H, t, J = 4.8 Hz), 4.09 (2H, t, J = 4.6 Hz), 6.79 (2H, d, J = 9.2 Hz), 7.35 (2H, d, J = 9.2 Hz).

  Nonaethylene glycol mono (4-trifluoromethylphenyl) ether (9o)

7o (170 mg, 1.05 mmol) was dissolved in THF (1.5 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 50 mg, 1.25 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (200 mg, 0.35 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 8 to 1), 9o (99 mg, 0.18 mmol, yield 49%) Got. ¹ H-NMR (CDCl ₃ ) δ: 2.73 (1H, br), 3.57-3.73 (32H, br), 3.86 (2H, t, J = 4.8 Hz), 4.16 (2H, t, J = 4.8 Hz), 6.97 (2H, d, J = 8.7 Hz), 7.52 (2H, d, J = 8.7 Hz).

  Nonaethylene glycol mono (4-cyanophenyl) ether (9r)

7r (200 mg, 1.68 mmol) was dissolved in THF (3.0 mL). Sodium hydride (mineral oil suspension, purity 60%, 82 mg, 2.05 mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour. To this was added a solution of 5 (240 mg, 0.42 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 5 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 100 to 1 → 8 to 1), and 9r (82 mg, 0.16 mmol, collected) Rate 38%). ¹ H-NMR (CDCl ₃ ) δ: 2.64 (1H, br), 3.59-3.74 (32H, br), 3.86 (2H, t, J = 4.8 Hz), 4.16 (2H, t, J = 4.8 Hz), 6.96 (2H, t, J = 8.9 Hz), 7.57 (2H, d, J = 8.9 Hz).

  Nonaethylene glycol mono (6-quinolyl) ether (9s)

7s (200 mg, 1.38 mmol) and benzyltriethylammonium chloride (30 mg, 0.13 mmol) were dissolved in THF (2.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 60 mg, 1.50 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (180 mg, 0.32 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with water (10 mL) and extracted with chloroform (10 mL × 4). The chloroform layer was washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 16 to 1 → 8 to 1 → 7 to 2), and 9s (103 mg, 0.19 mmol, 60% yield). ¹ H-NMR (CDCl ₃ ) δ: 2.89 (1H, br), 3.58-3.79 (32H, br), 3.94 (2H, t, J = 4.9 Hz), 4.26 (2H, t, J = 4.9 Hz), 7.09 (1H, d, J = 2.7 Hz), 7.36 (1H, dd, J = 8.2 Hz, 4.1 Hz), 7.41 (1H, dd, J = 9.4 Hz, 2.7 Hz), 8.00 (1H, d, J = 9.4 Hz), 8.04 (1H, dd, J = 8.2 Hz, 1.7 Hz), 8.77 (1H, dd, J = 4.1 Hz, 1.7 Hz).

  Nonaethylene glycol mono (2,4-bis (1,1-dimethylpropyl) phenyl) ether (9z)

7z (222 mg, 0.95 mmol) was dissolved in THF (2.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 50 mg, 1.25 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (220 mg, 0.39 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 50 to 1 → 8 to 1 → 4 to 1), and 9z (134 mg, 0.21 mmol, yield 55%). ¹ H-NMR (CDCl ₃ ) δ: 0.60 (3H, t, J = 7.3 Hz), 0.66 (3H, t, J = 7.3 Hz), 1.25 (6H, s), 1.34 (6H, s), 1.59 ( 2H, q, J = 7.3 Hz), 1.83 (2H, q, J = 7.3 Hz), 2.843 (1H, br), 3.58-3.74 (32H, br), 3.87 (2H, t, J = 5.2 Hz), 4.10 (2H, t, J = 4.8 Hz), 6.75 (1H, d, J = 8.2 Hz), 7.07 (1H, dd, J = 8.2 Hz, 2.3 Hz), 7.16 (1H, d, J = 2.3 Hz) .

  Dodecaethylene glycol mono (4-iodophenyl) ether (8ah)

7ah (216 mg, 0.98 mmol) was dissolved in THF (2.0 mL). Sodium hydride (mineral oil suspension, purity 60%, 51 mg, 1.28 mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour. To this was added a solution of 3 (172 mg, 0.25 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The resulting residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 45 to 1 → 45 to 2 → 8 to 1), and 8ah (74 mg, 0.10 mmol, 40% yield). ¹ H-NMR (CDCl ₃ ) δ: 2.70 (1H, br), 3.59-3.73 (44H, br), 3.84 (2H, t, J = 4.8 Hz), 4.09 (2H, t, J = 4.8 Hz), 6.70 (2H, d, J = 8.9 Hz), 7.54 (2H, d, J = 8.9 Hz).

  Dodecaethylene glycol mono (p-biphenyl) ether (8aj)

7aj (173 mg, 1.02 mmol) was dissolved in THF (1.5 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 45 mg, 1.13 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 3 (216 mg, 0.31 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 45 to 1 → 30 to 1 → 45 to 2 → 8 to 1 → 7 to 2) Purification was performed to obtain 8aj (105 mg, 0.15 mmol, yield 49%). ¹ H-NMR (CDCl ₃ ) δ: 3.57 (1H, t, J = 6.0 Hz), 3.59-3.75 (44H, br), 3.88 (2H, t, J = 4.9 Hz), 4.18 (2H, t, J = 4.9 Hz), 6.99 (2H, d, J = 8.7 Hz), 7.30-7.57 (7H, m).

  Dodecaethylene glycol mono (4-bromophenyl) ether (8j)

7j (169 mg, 0.98 mmol) was dissolved in THF (1.5 mL). Sodium hydride (mineral oil suspension, purity 60%, 47 mg, 1.18 mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour. A solution of 3 (215 mg, 0.31 mmol) in THF (2.0 mL) was added to this, and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 30 to 1 → 8 to 1 → 7 to 2), and 8j (44 mg, 0.06 mmol, yield 20%). ¹ H-NMR (CDCl ₃ ) δ: 2.78 (1H, br), 3.58-3.65 (44H, br), 3.83 (2H, t, J = 4.6 Hz), 4.08 (2H, t, J = 4.6 Hz), 6.79 (2H, d, J = 9.2 Hz), 7.35 (2H, d, J = 9.2 Hz).

  Dodecaethylene glycol mono (4-trifluoromethylphenyl) ether (8o)

7o (162 mg, 1.00 mmol) was dissolved in THF (2.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 65 mg, 1.63 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 3 (250 mg, 0.36 mmol) in THF (1.5 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 5 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 45 to 1 → 8 to 1), and 8o (54 mg, 0.08 mmol, collected) Rate 22%). ¹ H-NMR (CDCl ₃ ) δ: 2.75 (1H, br), 3.57-3.73 (44H, br), 3.87 (2H, t, J = 4.8 Hz), 4.17 (2H, t, J = 4.8 Hz), 6.98 (2H, d, J = 8.5 Hz), 7.53 (2H, d, J = 8.5 Hz).

  Dodecaethylene glycol mono (2,4-bis (1,1-dimethylpropyl) phenyl) ether (8z)

7z (240 mg, 1.02 mmol) was dissolved in THF (2.0 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 53 mg, 1.33 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 3 (225 mg, 0.32 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 60 to 1 → 30 to 1 → 4 to 1), and 8z (192 mg, 0.25 mmol, yield 79%). ¹ H-NMR (CDCl ₃ ) δ: 0.61 (3H, t, J = 7.5 Hz), 0.66 (3H, t, J = 7.5 Hz), 1.25 (6H, s), 1.34 (6H, s), 1.59 ( 2H, q, J = 7.5 Hz), 1.83 (2H, q, J = 7.5 Hz), 2.73 (1H, br), 3.59-3.74 (44H, br), 3.86 (2H, t, J = 5.2 Hz), 4.10 (2H, t, J = 4.8 Hz), 6.75 (1H, d, J = 8.4 Hz), 7.07 (1H, dd, J = 8.4 Hz, 2.4 Hz), 7.16 (1H, d, J = 2.4 Hz) .

  Nonaethylene glycol mono (p-biphenyl) ether (9aj)

7aj (170 mg, 1.00 mmol) was dissolved in THF (1.5 mL). To this, sodium hydride (mineral oil suspension, purity 60%, 44 mg, 1.10 mmol) was added and stirred at room temperature for 1 hour. To this was added a solution of 5 (205 mg, 0.36 mmol) in THF (2.0 mL) and stirring was continued at room temperature for 24 hours. The reaction solution was diluted with ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate, this was filtered and concentrated with an evaporator. The obtained residue was separated and purified by silica gel column chromatography (silica gel: 4 g, developing solvent: ethyl acetate-methanol mixed solvent 1 to 0 → 90 to 1 → 12 to 1 → 4 to 1), and 9aj (172 mg, 0.30 mmol, yield 84%). ¹ H-NMR (CDCl ₃ ) δ: 2.77 (1H, t, J = 6.0 Hz), 3.59-3.75 (32H, br), 3.88 (2H, t, J = 5.4 Hz), 4.18 (2H, t, J = 5.4 Hz), 6.98 (2H, d, J = 8.7 Hz), 7.30-7.56 (7H, m).

(Example 3) Test on safety of intraperitoneal and intravenous administration of mice (BALB / c, male) (A) Test on safety of compound 9bw [Materials and Methods]
(1) Examination of 50% lethal dose for mouse intraperitoneal / intravenous administration Experimental animals:
80 nude mice (BALB / c Slc-nu / nu male, SLC Co., Ltd.) were purchased at 7 weeks of age and pre-bred for 1 week before use at 8 weeks of age.
2. Sample preparation:
A sample of compound 9bw was prepared by diluting with sterile water and PBS. Sterilization was performed using a 0.45 μm filtration filter.
3. Administration method and dosage:
The administration was a single intraperitoneal and intravenous administration, and both administration volumes were 0.2 ml / mouse. Dose volume is 400mg / kg (9.3mg / 0.2ml / mouse / 23g body weight) as basic concentration, 200mg / kg, 150mg / kg, 100mg / kg, 75mg / kg, 50mg / kg, 40mg / kg, 30mg 8 concentrations of / kg and 20 mg / kg were adjusted with sterile PBS (−).
4). Observation period:
After administration: Observation was performed for 3 weeks. In addition, the body weight of each administration group was measured over time from the start of administration to 3 weeks after administration.
5). The administration groups are shown in Table 1 below.

6). Average body weight before starting the experiment (n = 13):
The average body weight before administration was 23.36 ± 0.89 (SE).
7). Calculation of lethal dose
LD50 was calculated by the probit method.
8). Results Table 2 shows the survival results after intraperitoneal (ip) and intravenous (iv) administration of compound 9bw to nude mice. In addition, changes in body weight of nude mice upon administration of compound 9bw are shown in FIGS.

(B) Test on safety of compound 8bw [Materials and Methods]
(1) Examination of 50% lethal dose for mouse intraperitoneal / intravenous administration Experimental animals:
100 nude mice (BALB / c Slc-nu / nu male, SLC Co., Ltd.) were purchased at 7 weeks of age and pre-bred for 1 week before use at 8 weeks of age.
2. Sample preparation:
A sample of compound 8bw (denoted as PEG (9bw) in the tables and figures) was prepared by diluting with sterile water and PBS. Sterilization was performed using a 0.45 μm filtration filter.
3. Administration method and dosage:
The administration was a single intraperitoneal and intravenous administration, and both administration volumes were 0.2 ml / mouse. Dose volume is 400mg / kg (9.3mg / 0.2ml / mouse / 23g body weight) as the basic concentration, 150mg / kg, 100mg / kg, 75mg / kg, 60mg / kg, 50mg / kg, 40mg / kg, 30mg 8 concentrations of / kg and 20 mg / kg were adjusted with sterile PBS (−).
4). Observation period:
After administration: Observation was performed for 3 weeks. In addition, the body weight of each administration group was measured over time from the start of administration to 3 weeks after administration.
5). The administration groups are shown in the table below.

6). Average body weight before starting the experiment (n = 13):
The average body weight before the start of administration was 23.21 ± 0.25 (SE).
7). Calculation of lethal dose
LD50 was calculated by the probit method.
8). Results Table 4 shows the survival results after intraperitoneal (ip) and intravenous (iv) administration of compound 8bw to nude mice. In addition, changes in body weight of nude mice upon administration of Compound 8bw are shown in FIGS.

(C) Examination of DNA synthesis inhibitory effect on Meth-A tumor cells (in vitro)
1. Tumor cells:
Meth-A tumor cells (mouse sarcoma)
2. Culture medium:
Fetal bovine serum 10%, L-glutamine, gentamycin and streptomycin were added to RPMI-l640 medium (Sigma Chemicals, MO, USA).
3. Sample preparation:
Compound 9bw was adjusted to a concentration of 2 mg / ml with sterilized distilled water, gently stirred, and then stored in a refrigerator overnight to dissolve in a clear state. However, slight cloudiness was confirmed when the dissolved compound was placed at room temperature (at 21-25 ° C.). In this state, the culture solution was diluted 2n in 96-well plates (F96 microwell plate NUNC), and Meth-A cells were added to each well, followed by culture for 24 hours (37 ° C., 5% CO 2).
4). Tritium thymidine incorporation:
After 17 hours of culture, 37 kBq / well of 3H-thymidine (methyl-3H, MP Biomedicals, CA, USA) was added, and after further incubation for 7 hours, a scintillation counter (1450 MicroBeta TriLux. Ma11ac, Finland) was used. The radioactivity was measured.

[Test results]
1. Inhibitory effect of compound 9bw on DNA synthesis on Meth-A tumor cells Radioactive activity of 3H-thymidine incorporated into cells by adding 9 bw of compound diluted in culture medium to Meth-A cells showing active growth at a concentration of 128 μg / ml to 4 μg / ml. Was measured. When the radioactivity (CPM) taken up by compound 9bw-free cells (control) was taken as 100%, the growth and division of the cells were suppressed to about 11% at a concentration of 128 μg / ml, and about 71 at 8 μg / m1. % Inhibitory effect was observed.

(Example 4) Test on antitumor effect of compound on Meth-A tumor transplanted to mouse (BALB / c, male) [Materials and Methods]
(1) Animal test: Determination of antitumor effect of compound 9bw on tumor-transplanted mice Experimental animals:
Eighty normal mice (BALB / c, male, SLC) were purchased at 6 weeks of age and preliminarily raised for 1 week before being used at 7 weeks of age.
2. Transplanted tumor:
Tumors with mouse sarcoma (Meth-A), transplanted about 1 × 10 ⁷ / 60μl of cells present tumors grown (15 animals used) in mice intraperitoneally into mice right thigh subcutaneously at a microsyringe did. One week after transplantation, mice with a tumor diameter of around 7 mm were selected to prepare administration groups and cancer-bearing control groups. In addition, individuals with relatively large tumor growth were selected for the intraperitoneal 60 mg / kg continuous 10 administration group and the intratumoral direct administration group. (Individuals with poor tumor growth and extremely hyperproliferative individuals were excluded.)
3. Sample preparation: A compound 9bw sample was diluted with sterile water and PBS.
4). Administration method and dosage:
The administration route was intraperitoneal and intratumoral direct administration, and the administration volume was intraperitoneal 0.2 ml / mouse and intratumoral 50 μl / mouse. The doses were set to 30 mg / kg and 60 mg / kg for intraperitoneal administration, and 7.5 mg / kg and 15 mg / kg for intratumoral administration.
5). Number and duration of administration:
a) Intraperitoneal administration: Continuous 30 mg / kg × 10 times (10 days), every other day 60 mg / kg × 5 times (10 days) and continuous 60 mg / kg × 10 times (10 days)
b) Intratumoral administration: Five groups of 7.5 mg / kg continuous administration (10 days) and 15 mg / kg continuous administration (10 days) were set.
6). Tumor volume:
The tumor volume transplanted subcutaneously into the right thigh of a mouse was calculated by the following calculation formula by measuring the tumor diameter using a caliper.

Tumor volume (mm ³ ) = major axis (mm) x minor axis (mm) x minor axis (mm) / 2
Tumor volume to be administered = 100 mm ^{3 to} 200 mm ³
7). The administration groups are shown in Table 5 below.

(2) Examination of DNA synthesis inhibitory effect on Meth-A tumor cells (in vitro)
1. Tumor cells:
Meth -A tumor cells (mouse sarcoma)
2. Culture medium:
Fetal bovine serum 10%, L-gluta mne, gentamycin and streptomycin were added to RPMI-1640 medium (Sigma Chemical, MO, USA).
3. Sample preparation:
Compound 9bw was adjusted to a concentration of 2 mg / ml with sterile distilled water and gently stirred. In this state, the culture broth was diluted 2n in 96-well plates (F96 microwell plate NUNC), and Meth-A cells were added to each well and cultured for 24 hours (37 ° C, 5% CO ₂ ). .
4). Tritium-thymidine incorporation:
Before completion of the culture, 37 kBq / well of 3H-thymidine (3H-TdR, methyl-3H, MP Biomedicals, CA, USA) was added, and further cultured for 7 hours. Finland) was used to measure radioactivity.
5). Preparation of splenocytes:
The spleen of normal mice was aseptically collected, and erythrocytes contained in spleen cells were lysed with ammonium chloride / Tris buffer. Spleen cells were washed twice with PBS and prepared with a culture solution so as to be 5 million cells / ml. The prepared splenic lymphocytes were dispensed in a 96-well flat-bottom plate at 500,000 cells / well and cultured with mitogen added.
6). Mitogen reaction:
Concanavalin A (Con A. Pharmacia Fine chemicals) was used as a mitogen for mouse spleen lymphocytes. Con A was added to the culture solution to a concentration of 5 μ / ml.
7). Irradiation of Meth-A cells and splenic lymphocyte cells:
Using an X-ray generator (Hitachi, MBR-1520R-3), irradiation was performed with 3.0 Gy (tube voltage 150 kv / tube current 20 mA, filter A1 1.0 mm, dose rate 2.48 Gy).
8). Incubation time:
Meth-A cells were cultured for 24 hours after the addition of Compound 9bw, and 3H-TdR was added 6 to 7 hours before completion. Normal mouse spleen lymphocyte cells were cultured for 48 hours after the addition of mitogen (Con A) and compound 9bw, and 3H-TdR was added 6 to 7 hours before the end in the same manner as Meth-A cells.
[Test results]
1. Antitumor effect of compound 9bw administration on tumor bearing (Meth-A tumor) mice
A. Judgment of the effect of intraperitoneal administration of Compound 9bw (30mg / kg daily administration and 60mg / kg every other day administration)
The obtained results are shown in FIGS. Regarding the body weight in FIG. 6, no decrease in body weight was observed from the start of administration to the 10th administration after administration of Compound 9bw at 30 mg / kg and 60 mg / kg. In the results of FIG. 7, the tumor volume tended to decrease from the tumor volume at the start of administration for both 30 mg / kg and 60 mg / kg administration of Compound 9bw. In the results of FIG. 8, when the tumor volume ratio is expressed as an index with the tumor volume before administration as 100, a transient increase in tumor is observed after the start of administration, but an apparent reduction effect after 6 days after administration. (Tumor reduction ratio: reduced to about 50% in both 30 mg / kg and 60 mg / kg administration groups). In the results of FIG. 9, regarding the tumor weight at the time of dissection, the tumor weight 24 hours after the final administration was 1459 ± 620 mg in the tumor-bearing control group, 42 ± 22 mg in the 30 mg / kg administration group, and 66 ± 18 mg at 60 mg / kg. there were.
B. Effect of compound 9bw intraperitoneal administration (60mg / kg administered 10 times daily)
The obtained results are shown in FIGS. In the results of FIG. 10, no decrease in body weight and no death were observed from the start to the end of administration after continuous administration of Compound 9bw for 10 times with respect to body weight. In the results shown in FIG. 11, the effect of reducing the tumor volume was observed from 6 days after the administration by continuous administration of 60 mg / kg with respect to the tumor volume. In the results of FIG. 12, a tumor that grew relatively large was selected with respect to the tumor volume ratio, and 60 mg / kg was administered 10 times daily. The index when the pre-dose volume is 1.00 is 4 out of 7 individuals (57%) who have reduced to about 0.5 or 0.5 or less after 10 doses, and 2 individuals have reduced to 0.5 to 1.0 or less. One animal (28%) and one animal (14%) had no effect.

  The obtained results are shown in Tables 6-1 and 6-2 below.

C. Compound 9bw Intratumoral effect assessment (7.5 mg / kg and 15 mg / kg, administered 5 times every other day)
The obtained results are shown in FIGS. In the results shown in FIG. 13, with respect to body weight, no decrease in body weight was observed from the start of administration to the end of administration in both 7.5 mg / kg and 15 mg / kg intratumoral administration of Compound 9bw. In the results shown in FIG. 14, with respect to the tumor volume (C-1), an increase in tumor was observed in one of four animals (No. 3), but an obvious tumor shrinking effect was observed in the remaining three individuals. It was. In addition, intratumoral administration seems to be related to the tumor volume (tumor size) before administration as in No.3. In the results of FIG. 15, regarding the tumor volume (C-2), an increase in tumor was observed in 2 out of 5 (No. 4, No. 5) individuals, but a marked tumor reduction effect was observed in 3 individuals. Was recognized. FIG. 16 summarizes the tumor weight at the time of dissection of cancer-bearing mice by administration of Compound 9bw with respect to the tumor weight. The tumor (A) whose tumor major axis was around 7 mm at the start of administration showed a great antitumor effect.

(2) DNA synthesis inhibitory effect on Meth-A tumor cells (in vitro)
The obtained results are shown in FIGS. In the results of FIG. 17, the non-irradiated 3H-TdR uptake in Meth-A cells without addition of compound 9bw was 24,407 ± 988 cpm, and that with 3.0 Gy irradiation was 15,104 ± 517 cpm. Therefore, it is calculated that 3.0 Gy irradiation has an effect of suppressing DNA synthesis of Meth-A cells by about 38% (3.0 Gy irradiation cpm / non-irradiation−1.0 × 100 == 38.1%). The irradiation effect of 3 Gy is almost equal to 3H-TdR incorporation (15,676 cpm) when 64 μl of compound 9bw is added. In addition, it was suggested that the sensitivity to compound 9bw may be improved by the combined use of irradiation and compound 9bw.

(Example 5) Test on antitumor effect of compounds 9bw and 8bw on human pancreatic cancer tumor transplanted to nude mice (BALB / c-nu / nu, male) [Materials and Methods]
1. Experimental animals:
Nude mice (BALB / c-nu / nu, male, SLC) were purchased at 4 weeks of age and pre-bred for 1 week before being used at 5 weeks of age.
2. Transplanted tumor:
Tumors were purchased from human pancreatic cancer cells (MIA-PaCa-2) and cultured in a culture dish with a diameter of 10 cm using a dedicated culture solution. MIA-PaCa-2 cells grown were transplanted cells about 2.5 × 10 ⁷ / 60pl the right mouse thigh subcutaneously at microsyringe. Mice with tumor diameters of around 7 mm in 3 to 4 weeks after transplantation were selected to create administration groups and tumor-bearing control groups (excluding individuals with poor tumor growth and individuals with excessive growth).
3. Sample preparation: Compound 8bw and Compound 9bw were used. These samples were prepared by diluting with PBS (−).
4). Administration method and dosage:
The administration route was intraperitoneal administration, and the administration volume was 45 ml / kg at 0.2 ml and 60 mg / kg as a reference test. The administration frequency was once a day and 20 daily administrations were set. Similarly, as a reference test (preliminary test prior to this test), 30 mg / kg intraperitoneal administration every other day and the same amount of intratumoral administration were performed, and intra-tumor administration was carried out every other day as 100 μl / tumor / mouse.
5). The test / administration groups are shown in Table 7 below.

[Test results]
1. Main test: Effect of continuous administration of 45 mg / kg of compounds 9bw and 8bw on nude mice bearing tumors 20 times The results obtained are shown in FIGS.

  In the results of FIG. 19, with respect to body weight, no decrease in body weight was observed from the start of administration to the end of administration when Compound 9bw and 8bw were administered at 45 mg / kg (20 times). In the results of FIG. 20, regarding the tumor weight, the results of suppression of tumor growth were obtained by administration of 45 mg / kg of Compound 9bw and 8bw (20 times). However, there was no tendency to decrease from the tumor volume at the start of administration.

  The effective suppression rate of the MIA-PaCa-2 transplanted tumor by compound 9bw or 8bw administration was computed. The results are shown in Table 8 below.

  Effective individual: When an individual having a weight of 1/3 or less (801 mg or less) of the average tumor weight of 2404 mg in the cancer-bearing control group is set as effective. 1): After 20 doses, 45 mg / kg was additionally administered for 2 days, and the dose was further increased to 60 mg / kg. The effective inhibition rate was calculated by adding the tumor weights of each of these 3 continuously administered animals.

  In the results of FIG. 21, the number of red blood cells at the time of dissection showed a tendency to decrease in the cancer-bearing control group compared to the normal control group, but a tendency to slightly increase in the compound 9bw administration group and the compound 8bw administration group. In the results shown in FIG. 22, the leukocyte count was significantly increased in the cancer-bearing control group compared to the normal control group. There was a tendency to increase slightly in the compound 9bw administration group and the compound 8bw administration group, but the values were not significantly different. In the results shown in FIG. 23, the platelet count was significantly increased in the compound 9bw administration group and the compound 8bw administration group with respect to the normal control group. This tendency is also observed in the safety test for normal nude mice (Example 1), which seems to be a feature of this drug.

  In the results of FIG. 24, the amount of hemoglobin was an approximate measured value in the normal control group, the cancer-bearing control group, the compound 9bw administration group and the compound 8bw administration group. The compound 8bw administration group tended to increase slightly, but it was not an increase with a significant difference. In the results of FIG. 25, the hematocrit value tended to increase in the compound 9bw and compound 8bw administration groups, but it was not a change with a significant difference. In addition, in the 8 bw administration group, a slightly high tendency was observed in the measured values of red blood cell count, hemoglobin, hematocrit, and the like.

2. Reference test (1): Antitumor effect of continuous administration of 60 mg / kg of compound 9bw on tumor-bearing nude mice Compound 9wb was administered 20 times daily at 60 mg / kg to relatively small tumors. The obtained results are shown in FIGS.

  In the results of FIG. 26, no decrease in body weight was observed in the administration group even when Compound 9bw was dosed 20 times daily at 60 mg / kg. In the result of FIG. 27, when 60 mg / kg of Compound 9bw was intraperitoneally administered 20 times daily, tumor growth was suppressed and the weight was 24.2% of the control group. In the results of FIG. 28, the number of red blood cells in the compound 9bw administration group showed a tendency of a decrease of 10% on average with respect to the normal control group. In the results of FIG. 29, the white blood cell count showed a 16.2% increasing tendency in the compound 9bw administration group, but there was an individual difference (variation), which was not a change with a significant difference. In the results of FIG. 30, the platelet count tended to increase in the compound 9bw administration group. In the results of FIG. 31, hemoglobin decreased about 10.8% in the compound 9bw administration group and the compound 8bw administration group with respect to the normal control group, but was not significantly different. In the results of FIG. 32, the hematocrit value was significantly decreased as compared with the normal control group. In 20 doses of 60 mg / kg of Compound 9bw, the red blood cell count, hemoglobin and hematocrit values decreased, and suppression of the erythroid system was observed.

3. Reference test (2): Tumor reduction effect of intraperitoneal and intratumoral administration of compound 9bw on tumor-bearing nude mice Prior to this test, the number of transplanted MIA-PaCa-2 tumor cells and their proliferation ability were examined. At that time, the compound 9bw was administered to mice engrafted with tumor cells, and the antitumor effect was examined.
1) Dosage:
Compound 9bw 30 mg / kg-Intraperitoneal administration-Tumor 1 to Tumor 3 (n = 3)
Compound 9bw 30mg / kg-Intratumoral administration-Scars AB (n = 2)
2) Number of doses:
Both intraperitoneal administration and intratumoral administration were administered five times every other day. The results obtained are shown in FIG. In the result of FIG. 33, the tumor volume grew about 4.5 times the pre-dose volume in 1 out of 3 mice administered intraperitoneally, and about 2.4 times the other 2 mice. Intratumoral administration reduced one of two animals to about 50% and the other one an increase of about 1.5.

[Summary]
1. MIA-PaCa-2, which is a human pancreatic cancer cell, is a tumor that shrinks as if it disappeared from the transplant site 10 to 14 days after transplantation, and then gradually grows. In addition, when the tumor starts to proliferate, it shows a certain growth at the transplant site.
2. No decrease in body weight was observed after 20 consecutive doses of Compound 9bw or 8bw (45 mg / kg and 60 mg / kg), and no anatomical findings after the end of administration showed any organ adhesion or tumor metastasis. Blood tests also showed an increase in platelet count.
3. At the time of administration of Compound 9bw or 8bw (45 mg / kg and 60 mg / kg), the locomotor activity of the mice decreased, and the state of stagnation within the gauge continued for about 40-60 minutes. At this time, the body temperature tended to decrease. This condition was prominent at 60 mg / kg.
4). Inhibition of tumor growth was observed when Compound 9bw or 8bw of 45 mg / kg intraperitoneally administered 20 times daily to mice transplanted with MIA-PaCa-2 tumors. However, there was no reduction effect on the tumor at the start of administration.
5). Further inhibition of tumor growth was observed when Compound 9bw was intraperitoneally administered 20 times daily (reference test) to Compound 9bw mice.
6). In the tumor transplanted mice or when the compound 9bw was administered locally at 30 mg / kg every other day (10 days) in the tumor (reference / preliminary test), the tumor volume before the start of administration was reduced to about 50%.
7). From these results, it is presumed that compound 9bw and compound 8bw are small molecules and are absorbed and drained quickly after intraperitoneal administration. This absorbed and drained time appears to be a period of reduced locomotor activity 40-60 minutes after administration. Therefore, it is presumed that the amount of the administered compound 9bw or compound 8 reaching the wound, or the reaching / circulation time is very small and sufficient effects cannot be exerted. If compound 9bw and compound 8bw reach the tumor sufficiently, it may be possible to show a tumor shrinking effect as shown in the results of intratumoral administration shown in the preliminary experiment.

Example 6 Bioassay at cell level
The cytotoxicity against tumor cells was measured using the MTT method. The MTT method is a kind of specific touch method. MTT reagent [3- (4,5-Dimethylthioazol-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide] is converted into insoluble MTT formazan (dark blue) by mitochondrial dehydrogenase in living cells Is done. The amount of MTT formazn produced is measured and expressed as relative cell viability.

Procedure 1. Pre-culture of cells: Hela cell (uterine cancer-derived) suspension adjusted to have an initial cell density of 2 × 10 ⁴ cells / cm ² was dispensed into each well of a 96-well culture plate at 50 μl. The cells were cultured for 24 hours using a CO ₂ incubator (37 ° C., CO ₂ concentration 5%).
2. Dissolution of test compound: Dissolved in ultrapure water to 333 μg / ml. For those which were hardly soluble and clouded, it was considered that the melting operation was completed by continuing stirring with a magnetic stirrer for 2 hours or more. After mixing with an equal volume of double concentration cell culture, it was dispensed into a 48-well culture plate simultaneously with filter sterilization in a clean bench.
3. Preparation of dilution series of test compound: A series of concentrations was prepared by diluting 5 times aseptically using a 48-well culture plate in a clean bench. The following two concentration ranges were prepared according to the strength of cytotoxic activity.
1) 100, 20, 4, 0.8 and O (ppm) (final concentration during main culture)
2) 4, 0.8, 0.16, 0.032 and O (ppm) (final concentration during main culture)
4). Main culture: 75 μl of a culture solution containing a predetermined concentration of test compound was added to each well to initiate main culture (the total volume of the culture solution is 125 μl, so the concentration of the test compound is diluted to 60%. 333 μg / ml (when assay compound is dissolved) → 166.5 μg / ml (when mixed with double concentration culture medium) → 99.9 μg / ml (when main culture is started)). The culture period was 3 days.
5). MTT reaction: a 0.5% MTT reagent into each well of the culture plate after completion of the culture was added 12.5 [mu] l, and allowed to stand for 4 hours in a C0 ₂ incubator base Ta. After stopping the reaction by adding 137.5 μl of 0.04N HCl dissolved in isopropanol to each well, pipetting was sufficiently performed until the blue particles were dissolved.
6). Absorbance measurement: The difference in absorbance at 570 nm (peak) and 630 nm (bottom) was measured with a plate reader.
7). Calculation of cell viability: A relative value with respect to the value in the 0 ppm action group (survival rate 100%) was calculated and used as the cell viability (%) in the concentration action group. In addition, when this value became negative, it was considered that the uniform cell viability was 0%.

  Assays are performed in triplicate and numerical calculations are performed using a simple average of the three values obtained.

(Test results)
The results obtained by the bioassay at the cellular level are shown in Table 9 below. The compound numbers tested are listed on the table.

Example 7 Compound 9bw and 8bw selectivity for normal and tumor cells
The tumor selectivity of compounds 9bw and 8bw was examined by MTT method.

  Human epidermal keratinocytes (normal cells, Kojin Bio Inc.) were used as cells, and MTT method was performed using Cell Counting Kit-8 (Dojindo) to test the tumor selectivity of compounds 9bw and 8bw. As a medium, a commercially available serum-free medium for normal human epidermis keratinocytes (basic medium + bovine pituitary extract) was used by preparing a medium having a double concentration as a standard by concentration by freeze-drying.

  The MTT method was carried out according to the manual attached to the kit (action period: 3 days; action concentration: 0.032, 0.16, 0.8, 4 (ppm)).

  The obtained results are shown in FIG.

Example 8 Effects of Compounds 9bw and 8bw on Various Tumor Cells Test Compounds and Control Compounds Compounds 9bw and 8bw were used as test compounds and CDDP (cisplatin, Sigma, France) was used as a control compound.

A test compound (powder, purity 100%) was dissolved in PBS and diluted to a target concentration with a serum-free medium to prepare a sample. The control compound was used after diluting to a predetermined concentration with PBS.
-Used tumor cells The used tumor cells are shown in Table 10 below.

-Culture conditions Tumor cells were cultured in an attached layer or in a suspended state at 37 ° C in an atmosphere kept at humidity (5% CO2, 95% air). As the culture solution, RPMI1640 containing 2 mM L-glutamine and supplemented with 10% fetal bovine serum (Lonza) was used. In conducting the test, adherent cells are detached from the culture flask by treatment with trypsin-versene (Lonza) diluted with Hank's medium without calcium or magnesium and neutralized with complete medium for 5 minutes. It was. The number of cells was measured with a hemocytometer, and the cell viability was calculated based on the excretion of 0.25% trypan blue. The survival rate of each cell (cell line) when used in each test was at least 84%.
Test Method (A) Mycoplasma detection was performed using a MycoAlert (registered trademark) Maukoplasma detection kit (Lonza) based on the description in its usage manual. The MycoAlert ™ assay is a selective biochemical test that utilizes the activity of mycoplasma enzymes. The enzyme is reacted with MycoAlert (registered trademark) substrate, which is a catalyst that dissolves live mycoplasma and converts ADP to ATP. By measuring the amount (level) of ATP in the sample before and after the addition of the MycoAlert ™ substrate, a ratio indicating the presence or absence of mycoplasma is obtained. Mycoplasma assays were performed in duplicate from cell culture supernatants and compared to positive and negative controls (MycoAlert ™ Assay Control Set, Lonza).
-Results Negative results were obtained for all the cells used.
(B) IC ₅₀ measurement / cell amplification and culture Tumor cells were seeded in a 96-well flat-bottom microtiter plate and cultured in a medium containing 190 μl of a test compound (supplemented with 10% FBS) at 37 ° C. for 24 hours. HeLa cells were used as a control. The planting density of each cell is shown in Table 11 below.

IC ₅₀ measurement Cells were incubated for 72 hours at each of the 10 concentration concentrations obtained at serial dilutions of 1/4 for each test compound. Test compounds (9 bw and 8 bw) were used in the range of 100 μM to 0.28 nM and CDDP was used in the range of 33 μM to 0.126 nM. Cells (190 μl) were incubated at 37 ° C. in an atmosphere of 5% CO 2 in a culture medium (supplemented with 10% FBS) containing a final volume of 200 μl of test compound or control compound. Three independent tests were performed for each compound, and each concentration was obtained from four experiments. About each cell of A-673, KB, MIA-PaCa-2, NIH: OVCAR-3, the 4th test was performed and the result by the 3rd test was confirmed. Control cells (control) were tested under the same conditions except that they did not contain the test compound. At the end of the treatment, it was tested for cytotoxicity by MTS assay.

MTS assay Tetrazolium compounds (MTS, 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethy phenyl) -2- (4-sulfophenyl) -2H-tetrazolium; Promega, France) and PMS ( The in vitro cytotoxicity test of the test compound and the control compound was performed by MTS assay using an electron coupling agent: pheazine methosulfate. This tetrazolium compound is reduced by the cells to produce a formazan compound that dissolves directly in the culture without the need for separate processing operations. At the end of cell treatment, a mixture of MTS (20 ml (2 mg / ml)) and PMS (Sigma, 1 ml (0.92 mg / ml)) in Dulbecco's Phosphate Buffered Saline (Lonza) (filtered: 0.22 μM) 40 μl was added to each well. The culture plate was incubated at 37 ° C. for 24 hours. The 490 nm absorbance (OD) in each well was measured with a Victor ³ ™ 1420 multilabel counter (Perkin Elmer, France).

-Results Tables 12 and 13 show the relative intensities of the compounds 9bw and 8bw in each tumor cell relative to the control compound (relative values when the CDDP IC ₅₀ is taken as 100).

Example 9 Antitumor Effect of 8 bw in MIA-Pa-Ca-2 (Pancreatic Cancer) Tumor-Transplanted Mice A healthy SWISS nude mouse (female) was transplanted with the MIA-Pa-Ca-2 tumor fragment. After 19 days from the transplantation, the mice were randomly divided into 4 groups consisting of 10 mice. Tumor volume in mice was 100 mm ³ to 200 mm ^3. The dose per administration and the number of administrations were set as shown in Table 14, and antitumor effects were observed for each of Compound 8bw and CDDP.

  As a result of each administration under the above-mentioned conditions, group 1 administered with 10 mg / kg showed tolerance in the first two treatments, and no weight loss or death was observed. In this group, there was eventually one death and no further weight loss. From these results, a marked antitumor effect is observed at 8 mg of 10 mg / kg administration in mice having human pancreatic Mia Paca-2 tumor.

Claims (3)

A polyethylene glycol monoether compound represented by the following formula (1) or a salt thereof.

A polyethylene glycol monoether compound represented by the following formula (2) or a salt thereof.

A polyethylene glycol monoether compound represented by the following formula (1) and a salt thereof;

And a polyethylene glycol monoether compound represented by the following formula (2) and a salt thereof:

An antitumor agent comprising one compound selected from the group consisting of as an active ingredient.

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