JPH0314807B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the following general formula (In the formula, R ₁ and R ₂ are the same or different and represent a hydrogen atom, a lower alkoxy group, a lower alkyl group, or a halogen atom, and R ₃ is a hydrogen atom, a lower alkyl group, or a formula R ₄ CO−, R ₄ NHCO− (Here, R ₄ is a lower alkyl group or a phenyl group which may be substituted with a lower alkyl group.) (However, R ₁ = R ₂ = R ₃ =
This is an invention of an anticancer agent containing a benzophenone derivative represented by (excluding the case where it is H) as an active ingredient. Conventionally, cancer chemotherapy agents include alkylating agents such as nitrogen mustards and ethyleneimines, antimetabolites such as folate antagonists, purine antagonists, and pyrimidine antagonists, actinomycin C, D, mitomycin C, etc. Antibiotics, pituitary-adrenal hormones, alkaloids, etc. are used. These compounds suppress the proliferation of cancer cells by inhibiting any step from DNA to protein synthesis, and are used with the aim of directly killing cancer cells. However, since these actions are non-selective, they not only attack cancer cells but also act on normal cells, and are therefore far more toxic than the expected anticancer effects. In view of these circumstances, the present inventors conducted research in search of a drug that specifically acts on cancer cells, and found that
We have discovered the surprising fact that the benzophenone derivative represented by the above general formula [] acts only on cancer cells, causing them to lose their proliferative ability or disappear. The present invention was completed based on these findings. Although the action of this benzophenone derivative is not necessarily clear, it is presumed to be a redifferentiation action against cancer cells generated by abnormal differentiation of normal cells, a so-called anticancer action. The compounds represented by the above formula (1) include known compounds, but the prior literature in which they are described does not describe any cancer-reducing activity or pharmacological activity suggesting it. Further, when a preliminary acute toxicity test was conducted on the compound represented by the above formula (1) using 6-week-old Slc:ddY male mice, the LD ₅₀ was 1 g/Kg or more. The compound of the present invention represented by the above formula (1) (wherein R ₃ =H or an alkyl group) is described by JCESimpson et al. [J.
Chem.Soc.646 (1945)]. On the other hand, in the above formula (1), R ₃ = R ₄ CO−,
The compound represented by R ₄ NHCO- (wherein R ₄ is a lower alkyl group or a phenyl group which may be substituted with a lower alkyl group) can be prepared by a conventional method in formula (1).
It can be easily obtained by acylating a compound where R ₃ =H. Reference example 1 11.3g of 0-fluorobenzoic acid chloride
℃, and gradually added 4.0 g of P-chloroaniline with stirring. Next, the external temperature was raised to 190℃, and zinc chloride was added.
Add 4.0g. Gradually raise the temperature and heat at 210℃ for 3 hours. Next, lower the temperature to 110°C, add 300 ml of 15% hydrochloric acid, reflux for 1 hour, and remove the warm hydrochloric acid by decantation. After performing this operation three times, the residue was dissolved in 200 ml of 75% sulfuric acid, refluxed for 1 hour, and the reaction mixture was poured into ice and the precipitated oil was extracted with chloroform. The chloroform layer was washed with dilute hydrochloric acid, dilute sodium hydroxide solution and water, dried over anhydrous sodium sulfate, chloroform was distilled off under reduced pressure, and the residue was recrystallized from methanol to give 2-amino-5-chloro as yellow needles. −2′−
4.8 g of fluorobenzophenone are obtained. Yield 62% Melting point 93-95℃ Elemental analysis Molecular formula C ₁₃ H ₉ ClFNO C H N Theoretical value (%) 62.54 3.63 5.61 Actual value (%) 62.49 3.66 5.58 Reference example 2 2-Amino-5-chlorobenzophene Non-2.3g,
1.6 g of 0-toluoyl chloride is gradually added to a mixture of 1.1 g of triethylamine and 30 ml of benzene under ice-cooling and stirring. After stirring at room temperature for 1 hour, the reaction solution was washed with dilute hydrochloric acid, dilute sodium hydroxide solution and water, dried with anhydrous sodium sulfate, benzene was distilled off under reduced pressure, and the residue was recrystallized from ethanol to give pale yellow needles. 2.7 g of crystalline 5-chloro-2-(0-toluoylamino)benzophenone was obtained. Yield 77.3% Melting point 135-136℃ Elemental analysis Molecular formula C ₂₁ H ₁₆ ClNO ₂ C H N Theoretical value (%) 72.10 4.61 4.00 Actual value (%) 72.14 4.73 4.11 Reference example 3 2-Amino-5-chlorobenzo Phenone 2.3g,
Add 0.2 ml of triethylamine to a mixture of 1.3 g of cyclohexyl isocyanate and 30 ml of benzene at room temperature while stirring. After stirring at the same temperature for 6 hours, the precipitated crystals were collected, washed with water, and recrystallized from ethanol to obtain 2.1 g of a 5-chloro-2-cyclohexylureidobenzophenone derivative in the form of colorless needles. Yield 59% Melting point 199-200℃ Elemental analysis Molecular formula C ₂₀ H ₂₁ ClN ₂ O ₂ C H N Theoretical value (%) 67.32 5.93 7.85 Actual value (%) 67.18 6.01 7.79 Reference examples 4-28 The above reference examples and The compounds shown in Table 1 were obtained in the same manner.

【table】

[Table] Example 1 (1) Contains 15% horse serum and 2.5% fetal bovine serum
Ham, Mouse Cloudman in sF-12 culture medium
Cultured cell line M-3 derived from S91 melanoma was suspended, placed in a plastic culture bottle, and placed in a carbon dioxide incubator (5% carbon dioxide, 95% air) at 37°C.
The cells were cultured for 10 days. Since the proliferated cells are attached to the bottom of the bottle, remove the supernatant culture solution and
% trypsin solution is added, and after treatment for 2 to 3 minutes, the trypsin solution is removed, and a culture solution is added to wash away the attached cells to obtain a cell suspension. (2) 2 ml of the cell suspension containing 10 ⁵ M-3 cells per ml prepared in (1) was implanted into a plastic jar with a diameter of 3.5 cm, and cultured at 37°C in a carbon dioxide incubator for 48 hours. Remove the supernatant culture solution and add the test compound in advance to culture solution 2.
ml and continue culturing. Similar medium exchange was performed every 48 hours. In addition, the state of cell proliferation was observed every day using an inverted microscope. (3) Measurement of cell proliferation Measurements were performed every other day. The plastic jar was removed from the culture vessel, and the supernatant culture solution was removed by decantation. Next, 1 ml of 0.05% trypsin solution was added and the solution was stirred using a Komagome pipette to sufficiently break up the cells, and then the number of cells was measured using a hemocytometer. The efficacy of the test compound was expressed in the following four stages. There was almost no increase in cells from the day after sample addition (),
There is cell proliferation from the day after the addition of the sample, but the growth rate is clearly suppressed (), and there is no difference between the cells and the control for several days after the addition of the sample, but after that, the proliferation rate becomes obvious compared to the control group. Decreased (+),
No suppression of proliferation was observed at any time point compared to the control group (-). (4) Morphological changes Cells were observed under a microscope on the fourth day after addition of the test compound. The unique morphology with protrusions and the large ratio of nucleus to cell size are differentiation characteristics of normal melanocytes, and the compound of the present invention was found to induce malignant melanoma toward normal melanocytes. The number of protrusions from the cell and the ratio of the size of the nucleus to the cell are used as indicators to express the degree of morphological change, with a ratio of 2 to 3 times the control (+) and a ratio of 4 or more times the control. is displayed as (). (5) Melanin production Cells were taken out on the 10th day of culture, and melanin was measured by Whittaker's method. Melanin production is a differentiation trait of normal melanocytes, and the compound of the present invention was found to induce malignant melanoma toward normal melanocytes. The melanin content was determined as weight per protein (mg), and the rate of increase in melanin production was calculated using the following formula. Melanin production increase rate (%) = (melanin content of test compound added group/melanin content of control - 1) x 100 The protein was determined according to the method of Lowry et al. The above measurement results are illustrated in Table 2. Note that the compound numbers in the table correspond to the numbers in Table 1.

【table】

[Table] Example 2 In the groin area of 5-week-old CDF ₁ male mice
10 ⁶ Cloudman S-91 melanoma cells per animal
The cells were subcutaneously transplanted and the following experiments were conducted. A. Starting 24 hours after transplantation, once a day for 19 days.
A sample dissolved or suspended in a 0.2% carboxymethyl cellulose aqueous solution was orally administered at a dose of 200 mg/Kg. 24 hours after the final administration, tumor tissues were excised and weighed. The measurement results are shown in Table 3 when expressed as the average value of 10 animals in each group.

【table】

[Table] B: The size of the tumor was measured by palpation on the 13th day after transplantation, and after dividing the tumor into groups fairly so that the size of the tumor in each group was equal, samples were collected once a day for 7 days A)
The tumor tissue was orally administered in the same manner as above, and 24 hours after the final administration, the tumor tissue was excised and its weight was measured. The measurement results are shown in Table 4, expressed as the average value of 10 animals in each group.

[Table] Example 3 Human-derived neuroblastoma IMR-32 cell line was transferred to Example 1, (1) Preparation of transplanted cells, (2) Cell culture and addition of test compound, (3) Measurement of cell proliferation. The experiment was conducted according to the section. However, the medium is 90% Ham, sF.
-12-10% fetal bovine serum (contains penicillin G potassium)
100 units/ml, streptomycin 100μg titer/
ml) was used. The effect of the test compound was determined 10 days after implantation when the control cell density reached saturation.
A decrease of 80 to 50% was rated as +, and when observed under a microscope, almost no weight between cells was observed, showing an image similar to contact inhibition of proliferation. The results are shown in Table 5.

[Table] Example 4 Table 6 shows the results of an experiment conducted in the same manner as in Example 3 using human gastric cancer cell line HG-. However, the judgment was made on the 14th day after implantation.

[Table] Example 5 2-Amino-5-chlorobenzophenone 100 parts Potassium hydrogen phosphate 58.5 parts Crystalline cellulose 50 parts Corn starch 40 parts Calcium stearate 1.5 parts These were mixed well and tableted into 250 mg tablets by a conventional method. (contains 100mg of active ingredient) and is used as an anticancer tablet. In carrying out the present invention, in addition to the above formulation examples, ordinary formulation bases listed in the Japanese Pharmacopoeia may be used to prepare the desired capsules, liquids, or suspensions orally or parenterally. Usually 1 day depending on the patient's symptoms
It is administered in a range of 10 mg to 3 g.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ and R ₂ are the same or different and represent a hydrogen atom, a lower alkoxy group, a lower alkyl group, or a halogen atom, and R ₃ is a hydrogen atom, a lower alkyl group, or a formula R ₄ CO−, R ₄ NHCO− (Here, R ₄ is a lower alkyl group or a phenyl group which may be substituted with a lower alkyl group.) (However, R ₁ = R ₂ = R ₃ =
An anticancer agent containing a compound represented by (excluding the case where H is used) as an active ingredient.