JPH0770130A - New cyclic enediyne antitumor agent - Google Patents

Abstract

PURPOSE:To obtain a new cyclic enediyne compound having really new structure designed by using dynemicin A as the mother compound and exhibiting extremely excellent antitumor activity. CONSTITUTION:A cyclic enediyne compound of formula I[R1 is H or a R5-OCO; R5 is a (substituted)phenyl, a (substituted)benzyl, a (substituted)1-10C alkyl, a (substituted)naphthyl, 8-quinolyl, 9-fluorenylmethyl, 2-morpholinoethyl, 4- methoxy-9-acridinyl, etc.; R2 is H, OH, a lower alkoxy or a lower acyloxy; R3 is H, OH, a lower alkoxy, a lower alkylsilyloxy, etc.; R4 is a lower alkyl], e.g. (2R*, 5Z, 9S*, 10R*, 16R*)-3,4,7, 8-tetradehydro-2,9-dihydro-9-acetoxy-9- methyl-1H-10,2,10-(ethoxymetheno)-1-benzazacyclododecyne-1-carboxylic acid ethyl ester. The compound of formula Ia which is one of the compounds of formula I can be produced from the compound of formula I via 6 steps.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclic enediyne compound and an antitumor agent containing them as an active ingredient.

[0002]

2. Description of the Related Art In recent years, antitumor antibiotics having a characteristic enyne structure in the molecule and exhibiting extremely strong anticancer activity have been successively discovered. Calichemicin [J. Am. Chem.So
c., Vol. 109, 3466-3468 (1987)], Esperamicin
[J. Am. Chem. Soc., Vol. 109, 3462-3464 (1987)], Dynemicin A [J. Antibiot., Vol. 42, 1449-1452 (199)
0)], and neocarzinostatin chromophore [Tetrahe
dron Lett., Vol. 26, 331-334 (1985)]. The former three have a common 10-membered cyclic enediyne structure, while the latter have a 9-membered cyclic enediyne structure. In all of these compounds, the internal structure of the molecule undergoes a Bergman-type reaction on the internal part of the molecule to cause aromatic cyclization, and the resulting benzenoid diradical cleaves DNA, resulting in the development of anticancer activity. Proven.

[0003] Synthetic studies of these compounds have been actively conducted in the past several years on the basis of the expression mechanism of this completely new anticancer activity and the characteristic structure. SL Schreiber et al. Reported the synthesis of pentacyclic analogs of dynemicin A [J. Am. Chem.
Soc., Vol. 112, 7410-7411 (1990)]. KC Nicolaou
Et al. Synthesized a tetracyclic analog of dynemicin A and reported its antitumor activity [International Patent WO 92/02522]. or,
The tricyclic analog of dynemicin A is described by PA Wender et al. [J.
Am. Chem. Soc., Vol. 113, 2311-2313 (1991)], and
P. Magnus et al. [J. Chem. Soc., Chem Commun., 544-546.
(1991)], and the present inventors have also reported the synthesis of a tricyclic analog of dynemicin A [Chem. L.
ett., 1271-1274 (1991)].

[0004]

DISCLOSURE OF THE INVENTION The present invention has a completely new cyclic enediyne structure and has a novel DN.
It is an object of the present invention to provide a compound having an antitumor activity superior to that of conventional antitumor agents by an A-cleavage mechanism and a clinically useful antitumor agent containing the compound as an active ingredient.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a cyclic enediyne compound having a completely novel structure designed using dynemicin A as a mother compound is extremely effective. They have found that they exhibit excellent antitumor activity, and completed the present invention.

That is, the present invention has the general formula (I)

[0007]

[Chemical 2]

(In the formula, R ₁ is hydrogen, or R ₅ --OCO--
Means a group, R ₅ is phenyl, substituted phenyl, benzyl, substituted benzyl, C ₁ -C ₁₀ alkyl, substituted C ₁ -C
₁₀ alkyl, naphthyl, substituted naphthyl, 8-quinolyl, 9-
Means fluorenylmethyl, 2-morpholinoethyl, 4-methoxy-9-acridinyl, 2-anthraquinolylmethyl, 6-hydroxy-2-anthraquinolyl group, R ₂ is hydrogen, hydroxy, a lower alkoxy group, It means a lower acyloxy group, R ₃ means hydrogen, hydroxy, lower alkoxy, lower alkylsilyloxy or lower acyloxy group, and R ₄ means a lower alkyl group. ), A novel cyclic enediyne compound and an antitumor agent containing them as an active ingredient.

The compound represented by the general formula (I) can be produced by the processes and methods shown in the following reaction steps 1, 2, 3 and 4. These manufacturing methods are merely examples and are not limited to these.

Reaction step 1 is a process for producing a compound (Ia) in which R ₁ is an R ₅ —OCO— group and R ₃ is a hydroxy group among the compounds represented by the general formula (I). is there. The starting material, quinoline derivative 1, is Chem. Let.
T., 1271-1274 (1991). To this compound was added Grignard reagent in an ethereal solvent such as tetrahydrofuran and then R
By reacting with _5- OCOCl, 1 (2H) -quinolinecarboxylic acid derivative 2 is obtained. Compound 2 in the presence of an acid catalyst such as p- toluenesulfonic acid in an alcohol solvent to give a compound 3 is tert- butyldimethylsilyl group protecting group is removed.

The olefin of compound 3 is stereoselectively epoxidized with a peroxide, preferably m-chloroperbenzoic acid, in a suitable solvent such as dichloromethane to give epoxy alcohol 4 . Epoxy ketone 5 is obtained by oxidizing the hydroxyl group of compound 4 with an oxidizing agent, preferably DMSO.

Compound 5 is treated with Pt complex-Cu (I) as a catalyst in the presence of a base such as n-propylamine or n-butylamine in a solvent such as benzene or ether and Tetr.
ahedron Lett., Vol. 29, 4217-4220 (1988), Compound (Z) -1-chloro-4-trimethylsilyl-1-
Coupling with buten-3-yne gives enediyne 6 . As a Pd complex, bis (triphenylphosphine)
Palladium (II) acetate complex, tetrakis (triphenylphosphine) palladium (0) complex, or tris (dibenzylideneacetone) dipalladium (0) -chloroform complex is preferable.

Further, copper (I) iodide is preferable as the Cu (I) catalyst. Then, Compound 6 is cyclized with cesium fluoride in the presence of crown ether in a solvent such as tetrahydrofuran or acetonitrile to give a compound of the general formula (I-
The cyclic enediyne compound represented by a) can be obtained.

[0014]

[Chemical 3]

The reaction step 2 shows another method for synthesizing the compound (Ia). That is, in the compound represented by the general formula (I), R ₁ is a PhOCO- group and R ₃ is a hydroxy group (Ib), and the compound (Ia) is converted to a compound (Ia) by a nucleophilic substitution reaction with R ₅ -ONa. It can be manufactured.
As the reaction solvent, tetrahydrofuran, ether solvents such as 1,4-dioxane, acetonitol, N, N-dimethylformamide and the like solvents are preferable, the reaction temperature is 0 ~
50 ° C is preferred.

[0016]

[Chemical 4]

Reaction step 3 shows a method for producing a compound (Ic) from the compound (Ia) in which R ₁ is an R ₅ —OCO— group among the compounds represented by the general formula (I). Alkylation, acylation, silylation, and dehydroxylation of the hydroxy group can be easily performed by known general methods.

[0018]

[Chemical 5]

The reaction step 4 is a process for producing a compound (Id) from the compound (Ic) in which R ₁ is hydrogen among the compounds represented by the general formula (I). It can be produced by reducing only carbamate with a reducing agent in an ether solvent such as tetrahydrofuran, preferably lithium aluminum hydride.

[0020]

[Chemical 6]

Next, typical examples of the cyclic enediyne compound represented by the general formula (I) obtained by the above production method are shown in Table 1.
Shown in.

[0022]

[Table 1]

[0023]

INDUSTRIAL APPLICABILITY The novel cyclic enediyne compound according to the present invention has a completely new structure and has a novel structure.
It has excellent antitumor activity due to the mechanism of DNA cleavage action, and an antitumor agent containing the compound as an active ingredient is extremely clinically useful.

[0024]

[Dosage Form and Dosage when Used as a Pharmaceutical] There is no limitation on the dosage form when the cyclic enediyne compound represented by the general formula (I) according to the present invention is formulated as an active ingredient, and therefore, tablets,
Solid preparations such as capsules, powders, and granules, and liquid preparations such as solutions, suspensions, and emulsions can be prepared by conventional methods.

In the case of solid preparations, excipients such as starch, lactose, glucose, calcium phosphate, magnesium stearate and gum arabic can be used, and if necessary, lubricants, disintegrants, coating agents. , Colorants and the like can also be used. Liquid preparations may contain stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives and the like.

The dose of the compound according to the present invention depends on the type,
Although it depends on factors such as the dosage form, the degree of disease, and the age of the patient, an amount of 0.1 to 100 mg / day is usually suitable for an adult.

[0027]

[Examples] Next, the present invention will be specifically described with reference to production examples, drug efficacy test examples, and formulation examples.

Example 1 4- [1-[[(1,1-dimethylethyl) dimethylsi
Ryl] oxy] ethyl] -2-ethynyl-1 (2H)-
Phenyl quinolinecarboxylate 4- [1-[[(1,1-dimethylethyl) dimethylsilyl] oxy] ethyl] quinoline 1.00 g (3.48 mmol) was dissolved in anhydrous tetrahydrofuran 15 ml, and 0.5 M was stirred at -60 ° C with stirring. 8.35 ml (4.18 mmol) of an ethynylmagnesium bromide-tetrahydrofuran solution was added dropwise. Reaction mixture at 0 ° C
After the temperature was raised to -60 ° C. and cooled again to −60 ° C., 654 mg (4.18 mmol) of phenyl chlorocarbonate was added dropwise. Reaction mixture at 25 ° C
After the temperature was raised to 1, the mixture was stirred for 1 hour, a saturated aqueous ammonium chloride solution was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. This is subjected to silica gel column chromatography (ether-n-hexane).
= 1: 1) to obtain 1.50 g (quantitative) of the title compound.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.11 (3H,
s), 0.13 (3H, s), 1.49 (3H, d, J = 6.4Hz), 2.21 (1H,
d, J = 2.0Hz), 4.73 (1H, q, J = 6.4Hz), 5.96 (1H, dd, J
= 6.8, 2.4Hz), 6.08 (1H, d, J = 6.8Hz), 7.12 ~ 7.41 (8
H, m), 7.80 (1H, dd, J = 7.8, 2Hz). EI-MS m / z: 433 (M +).

Example 2 4- (1-hydroxyethyl) -2-ethynyl-1 (2
H) -Phenylquinolinecarboxylate 4- [1-[[(1,1-dimethylethyl) dimethylsilyl] oxy] ethyl] -2-ethynyl-1 (2H)-
Dissolve 1.50 g (3.48 mmol) of phenyl quinolinecarboxylate in 20 ml of methanol to obtain p-toluenesulfonic acid monohydrate.
330 mg (1.74 mmol) was added and the mixture was stirred at 25 ° C for 3 hours. After adding 138 mg (1.74 mmol) of pyridine to the reaction mixture, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. This was purified by silica gel column chromatography (ether-n-hexane = 1: 1) to obtain 1.00 g (yield 91%) of the title compound.

¹ H-NMR (270 MHz, CDCl ₃ ): δ1.56 (3H,
d, J = 6Hz), 1.87 (1H, d, J = 4Hz), 2.23 (1H, d, J = 3H
z), 4.91 (1H, m), 6.00 (1H, dd, J = 6, 3Hz), 6.18 (1
H, dd, J = 6, 1Hz), 7.15 ~ 7.42 (7H, m), 7.60 (1H, d
d, J = 8, 2Hz), 7.76 (1H, d, J = 8Hz). EI-MS m / z: 319 (M +).

Example 3 7b- (1-hydroxyethyl) -2-ethynyl-1
a, 7b-Dihydrooxyreno [c] quinoline-3 (2
H) -phenyl phenyl 4- (1-hydroxyethyl) -2-ethynyl-1 (2
H) -phenyl quinolinecarboxylate 550 mg (1.72 mmol)
Was dissolved in 10 ml of dichloromethane, 740 mg (5.20 mmol) of sodium hydrogen phosphate was added, the mixture was cooled to 5 ° C, and 450 mg (2.60 mmol) of m-chloroperbenzoic acid was added to this for about 5 minutes.
The reaction mixture was stirred at 5 ° C for 2 hours, an aqueous sodium thiosulfate solution was added to reduce excess m-chloroperbenzoic acid, and the mixture was extracted twice with dichloromethane. Wash the organic layer with water,
The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (550 mg, yield 95%).

¹ H-NMR (270 MHz, CDCl ₃ ): δ1.44 (3H,
d, J = 6Hz), 1.58 (1H, s), 2.23 (1H, d, J = 2Hz), 4.09
(1H, d, J = 3Hz), 4.84 (1H, q, J = 6Hz), 5.94 (1H, t, J
= 2Hz), 7.13 (2H, d, J = 7Hz), 7.18 ~ 7.30 (2H, m),
7.32 ~ 7.43 (3H, m), 7.54 (1H, dd, J = 8, 1Hz), 7.60
(1H, d, J = 8Hz) .EI-MS m / z: 335 (M +).

Example 4 7b-Acetyl-2-ethynyl-1a, 7b-dihydro
Oxyleno [c] quinoline-3 (2H) -carboxylic acid
Eniru 7b-(1-hydroxyethyl) -2-ethynyl -1
a, 7b-Dihydrooxyreno [c] quinoline-3 (2
520 mg (1.55 mmol) of H) -carboxylate was dissolved in 4 ml of dichloromethane and 8 ml of anhydrous dimethyl sulfoxide, and 3.20 ml (23.0 mmol) of triethylamine was added. The solution was cooled on an ice bath and the sulfur trioxide-pyridine complex 2.4
7 g (15.5 mmol) was added over about 5 minutes, and the mixture was stirred at 25 ° C for 1.5 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. This was purified by silica gel column chromatography (dichloromethane) to obtain 450 mg (yield 87%) of the title compound.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 2.27 (1H,
d, J = 2Hz), 2.37 (3H, s), 4.04 (1H, d, J = 3Hz), 5.99
(1H, d, J = 3Hz), 7.13 (2H, d, J = 8Hz), 7.26 (2H, br-
t, J = 8Hz), 7.32 to 7.45 (3H, m), 7.58 (1H, br-d, J
= 8Hz), 7.69 (1H, dd, J = 8, 1Hz). EI-MS m / z: 333 (M +).

Example 5 7b-acetyl-1a, 7b-dihydro-2-[(Z)
-6- (Trimethylsilyl) -3-hexene-1,5-
Diynyl] oxyreno [c] quinoline-3 (2H) -carb
Phenyl rubonate 7b-acetyl-2-ethynyl-1a, 7b-dihydrooxyreno [c] quinoline-3 (2H) -phenyl phenylate 1.23 g (3.70 mmol), palladium (II) acetate 42 mg
(0.185mmol), and triphenylphosphine 97mg (0.3
(7 mmol) was dissolved in 30 ml of degassed anhydrous benzene, and (Z) -1-chloro-4-trimethylsilyl-1-
5.86 g (37.0 mmol) of butene-3-yne and 0.76 ml (9.25 mmol) of n-propylamine were added, and the mixture was stirred at 25 ° C for 1.5 hours. Saturated ammonium chloride aqueous solution was added to the reaction mixture,
The mixture was extracted 3 times with ether, the organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. This was purified by silica gel column chromatography (ether-n-hexane = 1: 1) to obtain 1.17 g (yield 78%) of the title compound.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.23 (9H,
s), 2.37 (3H, s), 4.07 (1H, d, J = 2.5Hz), 5.69 (1
H, dd, J = 11, 2.0Hz), 5.84 (1H, d, J = 11Hz), 6.24 (1
H, d, J = 2.5Hz), 7.14 (2H, d, J = 8.0Hz), 7.19 ~ 7.29
(2H, m), 7.33 ~ 7.44 (3H, m), 7.59 (1H, br-d, J = 8.0H
z), 7.71 (1H, dd, J = 8.0, 1.0Hz). EI-MS m / z 455 (M +).

Example 6 (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
2,10- (Epoxy metheno) -1-benzazacyclo
Dodecyn-1-carboxylic acid phenyl cesium fluoride 43.8mg of (0.288 mmol) was suspended in anhydrous tetrahydrofuran 20 ml, to 7b- acetyl -1
Phenyl a, 7b-dihydro-2-[(Z) -6- (trimethylsilyl) -3-hexene-1,5-diynyl] oxyreno [c] quinoline-3 (2H) -carboxylate 7
8 mg (0.19 mmol) was dissolved in 2.5 ml of anhydrous tetrahydrofuran and added, and further 76 mg of 18-crown-6 ether (0.
288 mmol) was dissolved in 2 ml of anhydrous tetrahydrofuran and added, and the mixture was stirred at 25 ° C. for 3 hours. A saturated ammonium chloride solution was added to the reaction mixture, followed by extraction with ethyl acetate three times. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. This was purified by silica gel thin layer chromatography (ether-n-hexane = 1: 1) to give 11 mg of the title compound (yield 16%).
Obtained.

¹ H-NMR (270 MHz, CDCl ₃ ): δ1.73 (3H,
s), 4.07 (1H, d, J = 3.0Hz), 5.67 (1H, dd, J = 10, 2.0
Hz), 5.82 (1H, d, J = 10Hz), 5.89 (1H, dd, J = 3.0,2.0
Hz), 7.14 (2H, br-d, J = 8.0Hz), 7.20 ~ 7.28 (2H, m),
7.31 ~ 7.41 (3H, m), 7.51 (1H, br-d, J = 8.0Hz), 8.80
(1H, br-d, J = 8.0Hz). EI-MS m / z: 383 (M +).

Example 7 (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Acetoxy-9-methyl-1H-10,2,10- (d
Poximetheno) -1-benzazacyclododecin-1-
Ethyl carboxylate (2R ^* , 5Z, 9S ^* , 10
R ^* , 16R ^* )-3,4,7,8-tetradehydro-
2,9-dihydro-9-trimethylsilyloxy-9-
Methyl-1H-10,2,10- (epoxy metheno)-
Ethyl 1-benzazacyclododecine-1-carboxylate
17 mg (0.051 mmol) was dissolved in 0.8 ml of pyridine, 0.8 ml of acetic anhydride was added thereto, and the mixture was stirred at room temperature for 34 hours. Toluene was added to the reaction mixture and concentrated under reduced pressure to obtain a crude product. This was purified by silica gel thin layer chromatography (dichloromethane) to give the title compound (8.2 mg, yield 43
% )Obtained.

¹ H-NMR (270 MHz, CDCl ₃ ): δ1.29 (3H,
t, J = 7.0Hz), 1.80 (3H, s), 2.21 (3H, s), 4.09 (1H,
d, J = 3.0Hz), 4.23 (2H, m), 5.66 (1H, dd, J = 9.5,
1.5 Hz), 5.83 (1H, d, J = 9.5Hz), 5.84 (1H, m), 7.17
(1H, m), 7.32 (1H, dt, J = 8.0, 1.5Hz), 7.39 (1H, b
rd, J = 8.0Hz), 8.00 (1H, br-d, J = 8.0Hz) .EI-MS m /
z: 377 (M +).

Example 8 (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
2,10- (Epoxy metheno) -1-benzazacyclo
Phenyl dodecine-1-carboxylate (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
Phenyl 2,10- (epoxymetheno) -1-benzazacyclododecine-1-carboxylate 2.0 mg (5.4 μmol)
Was dissolved in 0.1 ml of anhydrous pyridine, 0.1 ml (0.4 mmol) of bis (trimethylsilyl) trifluoroacetamide was added, and the mixture was stirred at 25 ° C. for 24 hours. The reaction mixture was concentrated under reduced pressure to obtain 2.4 mg (quantitative) of the title compound.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.29 (9H,
s), 1.71 (3H, s), 4.06 (1H, d, J = 2.9Hz), 5.64 (1H,
dd, J = 10, 2.0Hz), 5.81 (1H, d, J = 10Hz), 5.89 (1H,
m), 7.14 (2H, br-d, J = 8.0Hz), 7.20 ~ 7.28 (2H, m),
7.31 ~ 7.41 (3H, m), 7.51 (1H, br-d, J = 8.0Hz), 8.69
(1H, dd, J = 8.3, 1.5Hz). EI-MS m / z: 455 (M +).

Example 9 (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
2,10- (Epoxy metheno) -1-benzazacyclo
Dodecine (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
2.4 mg (5.4 μmol) of phenyl 2,10- (epoxymetheno) -1-benzazacyclododecine-1-carboxylate was dissolved in 1 ml of anhydrous tetrahydrofuran and cooled to 5 ° C.
14 μl (14 μmol) of 1.0 M lithium aluminum hydride-tetrahydrofuran solution was added dropwise to this solution, and the mixture was added at 30 ° C at 30 ° C.
Stir for minutes. After adding 50 μl of saturated sodium hydrogen carbonate solution to the reaction mixture, about 5 ml of ether was added, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product of the title compound. The title compound is extremely unstable at room temperature. FAB-MS m / z: 336 [(M + H) +].

Drug efficacy test example 1 (DNA cleaving activity) A DNA solution was prepared at 32.5 µg / ml with a sterilized 50 mM phosphate buffer solution (pH 7.4) of bacteriophage φX174 containing 90% of Supercoiled DNA. The cyclic enediyne compound according to the present invention was dissolved in dimethyl sulfoxide to prepare a 10 mM solution. The reaction solution was prepared by mixing 2 μl of the DNA solution, 1 μl of the cyclic enediyne compound solution, and 10 μl of the 50 mM phosphate buffer solution. The above reaction solution was incubated at 37 ° C. for 18 hours. After the reaction was completed, 2 μl of a 0.25% bromophenol blue-containing 30% glycerol solution was added and mixed, and 1% agarose gel electrophoresis (50 V, 40 minutes) was performed. After the electrophoresis, the gel was treated with 0.5 µg / ml ethidium bromide solution for 15 minutes, and irradiated with ultraviolet rays (312 nm) to detect DNA. The determination of the DNA-cleaving activity was carried out by visual inspection using the increase of Linear DNA and Nicked DNA as an index. The results of electrophoresis are shown in FIG. Linear DNA and Nicked DNA for all tested drugs
Was detected, which revealed that it showed DNA-cleaving activity.

Drug efficacy test example 2 (proliferation inhibitory activity against KB cells
Sex) 5 × 10 ⁴ KB cells / ml in a 12-well culture plate at 0.7 ml / we
ll, the cells were cultured at 37 ° C. for 24 hours, replaced with a medium containing a drug, and further cultured for 48 hours. The test drug was dissolved in dimethyl sulfoxide to prepare a 1 mg / ml solution, and EMEM 1
Drug concentrations of 0.1, 1, and 10 μg / ml were prepared by diluting with 0% fetal bovine serum medium. At the time of drug addition and 48 hours after drug addition,
Cell number (nuclear number) after detaching cells with trypsin-EDTA
Was measured with a Coulter counter. The growth inhibition rate was calculated by the following formula, and the 50% growth inhibition concentration (IC ₅₀ ) was calculated from the graph. The results are shown in Table 2.

Proliferation inhibition rate (%) = (A−B) / (A−C) × 100 A: Number of cells 48 hours after drug-free group B: Number of cells 48 hours after drug-added group C: Number of cells when drug is added

Drug efficacy test example 3 (Proliferation inhibition against L1210 cells
Harmful activity) L1210 cells in a 12-well culture plate 2.5 × 10 ⁴ cells / ml / we
ll, the drug was added at the same time, and the cells were cultured at 37 ° C for 48 hours. The test drug was prepared in the same manner as in the drug efficacy test example 2.
However, the medium used was RPMI 1640 10% fetal bovine serum medium.
48 hours after the addition of the drug, the number of cells (nuclear number) was counted with a Coulter counter. The growth inhibition rate is calculated by the following formula,
The results of the 50% growth inhibitory concentration (IC ₅₀ ) determined from the graph are shown in Table 2.

Proliferation inhibition rate (%) = (A−B) / (A−C) × 100 A: Number of cells 48 hours after drug-free group B: Number of cells 48 hours after drug-added group C: Number of cells when drug is added

Drug efficacy test example 4 (proliferation inhibition against MOLT-4 cells
Harmful activity) MOLT-4 cells in a 12-well culture plate at 1 × 10 ⁵ cells / ml / well
, The drug was added at the same time, and the mixture was cultured at 37 ° C. for 48 hours. The test drug was prepared in the same manner as in the drug efficacy test example 3. 48 hours after the addition of the drug, the number of cells (nuclear number) was counted with a Coulter counter. The growth inhibition rate was calculated by the following formula, and the 50% growth inhibition concentration IC ₅₀ ) was calculated from the graph. The results are shown in Table 2.

Growth inhibition rate (%) = (A−B) / (A−C) × 100 A: Number of cells 48 hours after drug-free group B: Number of cells 48 hours after drug-added group C: Number of cells when drug is added

[0052]

[Table 2]

Formulation Example 1 (Tablet) The following ingredients were blended to produce a tablet by a conventional method.

Formulation Example 2 (Injection) An injection was prepared by a conventional method by mixing the following components, and then it was aseptically charged into a vial.

[Brief description of drawings]

FIG. 1 shows the results of electrophoresis in drug efficacy test example 1 (DNA cleavage activity).

[Explanation of symbols]

 1-Linear DNA 2-Control 3-Compound of Example 7 4-Compound of Example 6 5-Compound of Example 9

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryoichi Umino 35 Higashi Sotobori-cho, Higashi-ku, Nagoya City Sanwa Chemical Research Institute Co., Ltd. In-house (72) Takahito Shiromori, 35, Higashi Sotobori-cho, Higashi-ku, Nagoya City, Sanwa Chemical Research Institute Co., Ltd.

Claims (3)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R ₁ represents hydrogen or an R ₅ —OCO— group,
R ₅ is phenyl, substituted phenyl, benzyl, substituted benzyl, C ₁ -C ₁₀ alkyl, substituted C ₁ -C ₁₀ alkyl, naphthyl, substituted naphthyl, 8-quinolyl, 9-fluorenylmethyl, 2-morpholinoethyl, 4-methoxy-9-acridinyl, 2-anthraquinolylmethyl, 6-hydroxy-2-anthraquinolyl group is meant, R ₂ is hydrogen, hydroxy, lower alkoxy group, lower acyloxy group,
R ₃ represents hydrogen, hydroxy, lower alkoxy, lower alkylsilyloxy, lower acyloxy group, R ₄
Means a lower alkyl group. ) A novel cyclic enediyne compound represented by 2. The compound according to claim 1 is (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Acetoxy-9-methyl-1H-10,2,10- (epoxymetheno) -1-benzazacyclododesin-1-
Ethyl carboxylate (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Trimethylsilyloxy-9-methyl-1H-10,
Phenyl 2,10- (epoxymetheno) -1-benzazacyclododecine-1-carboxylate (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
Hydroxy-9-methyl-1H-10,2,10- (epoxymetheno) -1-benzazacyclododesin-1-
Phenyl carboxylate (2R ^* , 5Z, 9S ^* , 10R ^* , 16R ^* )-3,
4,7,8-tetradehydro-2,9-dihydro-9-
A novel cyclic enediyne compound, which is hydroxy-9-methyl-1H-10,2,10- (epoxymetheno) -1-benzazacyclododecine. 3. An antitumor agent containing the compound according to claim 1 or 2 as an active ingredient.