JPS63264411A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To obtain a carcinostatic agent exhibiting high sensitivity to a cell cancerized by SV40 oncogenic virus, by using a specific aromatic aldehyde derivative as an active component. CONSTITUTION:The objective carcinostatic agent contains sodium salt of a benzaldehyde-sulfurous acid addition compound, monobenzalpentaerythritol, N-benzylideneethylamine, N-benzylidenemethylamine, N-benzylideneisobutylamine, 1-benzylideneamino-2-ethoxyethane, 1-benzylideneamino-3-dimethylaminopropane, 1-benzylideneamino-2-propanol, 2-benzylideneamino-2-methylpropanol, 3- benzylideneamino-1-propene, 2-benzaldoxime, 3-ethyl-2-phenyloxazolidine, 3-(2'- hydroxyethyl)-2-phenyloxazoline and/or mandelonitrile as active components. The agent has low toxicity and exhibits characteristic carcinostatic action different from the mechanism of conventional chemotherapeutic agent for cancer.

Description

[Detailed description of the invention] The present invention relates to a novel anticancer agent.

Conventionally, cancer chemotherapy agents include alkylating agents (nitrozene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), and plant pre-fission agents (colcemid). , vinblastine, etc.), antibiotics (sarcomycin, carcinophilin, mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, C0PP), etc. The nucleic acid inhibitory effect of substances acts not only on cancer cells but also on normal cells, making them highly toxic and exhibiting serious side effects. The current situation is that it is difficult to give anything.

The present inventor has previously developed a new and useful anticancer agent containing benzaldehyde as an active ingredient (see Japanese Patent No. 1157737). We have newly discovered that it has a specific anticancer effect that is thought to produce therapeutic effects through a mechanism of action that is different from that of other chemotherapy agents. The present inventors discovered substances having anticancer activity among aromatic aldehyde derivatives, obtained new knowledge that these substances can exhibit remarkable effects in cancer treatment, and completed the anticancer agent of the present invention.

Most conventional cancer chemotherapeutic agents are so-called cytotoxic substances that are more sensitive to transplanted cancers such as Ehrlich tumor than to cancerous cells caused by the SV 40 oncogenic virus, whereas the anticancer agent of the present invention The active ingredient is S V
4゜In that it acts on cancerous cells caused by oncogenic viruses and exhibits high sensitivity, it is thought to be based on a specific anticancer effect that differs from that of conventional cancer chemotherapeutic agents, making it an excellent anticancer agent. It has special characteristics.

Aromatic aldehyde derivatives having anticancer activity used in the present invention are shown in Table 1.

Table 1 References, physical properties, and values for the above compounds 1 to 14 (hereinafter, the above compounds are indicated by compound numbers) are listed in the second table.
Shown in the table.

(Production Example 1) 10.6 g of benzaldehyde and 7.0 g of isobutylamine.
3 g was dissolved in a mixed solvent of benzene-toluene, refluxed for 3 hours while removing water azeotropically with toluene, concentrated, and distilled to obtain N-benzylideneisobutylamine (5) (yield: 80%). ). (b, p, 7
8-80℃/2mmHg) (Production Example 2) 10.6 g of benzaldehyde and 9.0 g of 2-nitoxyethylamine were dissolved in benzene, and concentrated by refluxing for 3 hours while removing water that azeotroped with benzene. Then, by distillation, 1-benzylideneamino-2-ethoxyethane (
6) (Yield: 80%) is obtained.

(b, p, 90-91°C/1mm) Ig) (Production Example 3
) 10.6 g of benzaldehyde and 3-dimethylamino-
When 10.5 g of n-propylamine was dissolved in a mixed solvent of benzene-toluene and refluxed for 3 hours while removing azeotropic water, concentrated and distilled, 1-benzylideneamino-3-dimethylamino Propane (7) (Yield: 9
1%). (b, p, 120-123℃/3m+
nHg) Note that the above compounds 5.6 and 7 are compounds that have not been published in any literature.

The anticancer agent of the present invention can be administered either orally or parenterally; when administered orally, it is administered as a soft/hard capsule, tablet, granule, fine granule, or powder; when administered parenterally, can be administered in the form of an injection, an infusion, or a solid or suspended viscous liquid, such as a herbal medicine, so that sustained mucosal absorption can be maintained.

The proportion of the active ingredient in the anticancer drug composition of the present invention may vary depending on the dosage form, but usually, when administered as a suspension or for mucosal absorption, approximately 0.3 to 15.0% by weight is appropriate. When administered parenterally, approximately 0.01 to 10% by weight is appropriate.

In addition, in formulating the active ingredient of the present invention, in the case of the above aromatic aldehyde derivatives 1 to 13, according to a conventional method,
It can be made into a water-soluble suspension, oil-based preparation, etc. for subcutaneous or intravenous injection, and it can also be formulated into capsules, tablets, fine granules, etc. for oral administration. Regarding No. 14, since it is an unstable stimulant, it is required to prevent its deterioration and eliminate its irritating properties in the formulation of the composition. Therefore, when formulating Compound 14, it is necessary to prevent its autoxidation and improve its stimulatory properties.

For this method, for example, it is appropriate to use an inclusion compound that utilizes the inclusion ability of choleic acid or cyclodextrin (Cyclodextrin), and formulation using microcapsules is also effective.

In addition, in order to impart stability and acid resistance that can withstand long-term storage to the above-mentioned clathrate compounds and to fully maintain their medicinal efficacy, it is possible to formulate a formulation with a pharmaceutically acceptable coating. A stable anticancer drug composition can be obtained.

The surfactants, excipients, lubricants, adjuvants, pharmaceutically acceptable film-forming substances, etc. used in formulating the active ingredient of the present invention are as follows.

In order to improve the disintegration and elution of the composition of the present invention,
One or more surfactants such as alcoholobe esters, polyethylene glycol derivatives, fatty acid esters of sorbitan, sulfated fatty alcohols, etc. can be added.

In addition, as excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, soft silicic anhydride, magnesium aluminate, magnesium aluminosilicate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, Carboxymethyl cellulose calcium and the like can be added alone or in combination of two or more.

As the lubricant, for example, one or more types of magnesium stearate, dalc, hydrogenated oil, etc. can be added, and as the flavoring and flavoring agent, salt, saccharin, sugar, mannitol, orange oil, licorice, etc. can be added. Sweeteners such as extracts, citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, coloring agents, preservatives, and the like may be included.

Adjuvants such as suspending agents and wetting agents include, for example, coconut oil, olive oil, sesame oil, peanut oil, calcium lactate,
Safflower oil, soybean phospholipids, etc. can be contained.

In addition, as a film-forming substance, cellulose acetate phthalate (CAP) is used as a carbohydrate derivative such as cellulose and sugars.
In addition, examples of polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters include methyl acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In addition, when coating the above-mentioned film-forming substances, in addition to commonly used coating aids such as plasticizers, the properties of the film-forming agent can be improved by adding various additives to prevent the chemicals from adhering to each other during coating operations. This can improve the coating process and make coating operations easier.

The dosage depends on the desired therapeutic effect and duration of treatment, but for adults it is usually 0.5 to 5.000 mg of the above compound per day, and for children it is usually 0.5 to 3.0 mg per day.
．． 000mg.

Next, the anticancer activity testing method for confirming the anticancer activity of the above compound will be described.

C3H mouse kidney cells were cultured with SV4. Cells W2K-11 that had been made cancerous with an oncogenic virus were used as test cells, and were cultured according to the following method.

(1) Preparation of growth culture solution 9.4 g of Eagle MEM medium and 900 m of distilled water I! After cooling, add 100 mA of revitalized serum and 3 to 5 mj2 of 10% sodium bicarbonate solution, which was separately autoclaved at 115°C for 15 minutes.
Adjust to H7.1-7.2.

10 mf of monoglutamine (2,92 g/l 00 mf) solution filtered through a Millipore filter immediately before grounding use.
Add.

In addition, to preserve the test cells, dimethyl sulfoxide is further added to a final concentration of 10%.

(2) Preparation of transplanted cells Test cells stored in a Jeep freezer (-80°C) were lysed at room temperature, centrifuged at 670 x g for 5 minutes, the supernatant was discarded, and the precipitated cells were suspended in 50 mβ of growth medium. After turbidity, the cells are transferred to a Lou flask and cultured at 37°C. The cells begin to proliferate while adhering to the bottom of the flask, and fully proliferate in 3 to 4 days. Decant the culture solution, and then add 0.2% trypsin solution [Eagle MEM medium (manufactured by Tamizu Seiyaku ■)]
4.7 g z A solution of 0.6 g of sodium bicarbonate and 1 g of trypsin dissolved in 500 m of distilled water and filtered through a Millipore filter) is added to trypsinize for 2-3 minutes at room temperature, and then the trypsin solution is decanted. Further, 50 ml of fresh growth medium is added, and adherent cells are washed off using a Komagome pipette to obtain a cell suspension. Some of them are Lou
Subculture using a flask.

(3) Cell culture and administration of test compound 1.3 mf of the cell suspension was dispensed into a disposable Petri dish (diameter 35 mm) and placed in a carbon dioxide gas incubator (5% CO2, 95% air) at 37°C for 24 hours. Cultivate.

At this point, 0.2 mA of the test compound solution is administered to continue the culture.

The state of cell proliferation is observed every day using an inverted microscope, and the number of surviving cells is counted 48 hours after administration. The test compound is dissolved in distilled water or ethanol (final concentration 2%) and then filtered with a Millipore filter.

(4) How to count the number of cells After administering the test compound, decant the Petri dish for 48 hours, discard the supernatant (culture solution), and remove the 0.2% trypsin solution.
When cells that have adhered to the bottom of a Petri dish (0 ml) are treated, they become single cells. Decant this to remove the trypsin solution, make a cell suspension with physiological saline containing 10 mmol of phosphate buffer (pH 7.0), place 1 to 2 drops of this on a hemocytometer, and place it on a cover glass. Count the number of cells under a microscope.

The inhibition rate of test cell proliferation was determined by the following formula.

×100 Next, regarding the toxicity of the active ingredient compounds of the present invention, all compounds are quickly excreted outside the body due to their low molecular structure, so they do not cause any side effects. LDs in administration.

Both values are low toxicity compared to other anticancer substances.

The present invention will be specifically explained below using formulation examples and test examples.

Formulation Example 1 (Injection/Drop) Add 5 g of powdered glucose to contain 1.500 mg of the above compound, dispense aseptically into vials, seal them, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. Save to. Before use, 500 ml of 0.85% saline
Add this to make an intravenous injection, and administer 10 to 500 mf per day.
Administer by intravenous injection or drip depending on the symptoms.

Formulation Example 2 (Injection/Drop) An intravenous injection for mild symptoms was prepared in the same manner as Formulation Example 1, except that 3.5 flng of the above compound was used.
Administer 500ml by intravenous injection or infusion depending on the symptoms.

Formulation Example 3 (Injection, Capsule) 14.30 mg of the above compound was dissolved in 1 g of purified sesame oil and 1 g of aluminum stearate gel 1001T, sealed, sealed with an inert gas such as nitrogen or helium, and stored in a cool, dark place. The formulation is for subcutaneous injection.

Administer 1 to 10 mj2 by subcutaneous injection once a day depending on the symptoms.

In addition, the above preparation is dispensed into capsules of 0.5 mf each to form oral capsules, and 1 to 10 capsules are orally administered per day depending on the symptoms.

Formulation Example 4 (Enteric-coated tablet) Add 14.15 g of the above compound to 3,000 ml of a saturated aqueous solution of β-cyclodextrin (manufactured by Nippon Shokuhin Kako), mix it, and stir for 5 hours. When the precipitate is dried under reduced pressure, 100 g of the above-mentioned clathrate compound of Compound 14 is obtained.

Enteric-coated tablets for adults (A) and children (A) with the following ingredient composition:
b) 1,000 pieces of each were manufactured.

[A]

(a) (b) Base agent (above compound 14 100 (g) 50 (g)
clathrate compound) Lactose 99.4 49.7
Hydroxypropyl 0.6 0.3 Cellulose magnesium stearate 2.0 1.0 [B] Cellulose acetate phthalate 6.0 (g > 4.0
(g > Hydroxypropyl methyl 6.0 4.0 Take each component of cellulose phthalate [A], mix well, pressurize this directly, or after kneading well, screen the extrusion type granulator. The pellets were formed into granules through a sieve, and the pellets were sufficiently dried and then pressed to produce tablets.

Next, the molded tablet is coated with the well-dissolved base material [B] to form an enteric-coated tablet.

Regarding this tablet, please refer to the Japanese Pharmacopoeia (hereinafter referred to as "diurnal").

) Disintegration test method When an enteric-coated preparation was tested in artificial gastric fluid (pH 1, 2), it did not disintegrate even after shaking for 1 hour, and in the artificial intestinal fluid (pH 7, 5) test, it disintegrated in 5 to 6 minutes.

Formulation example 5 (enteric coated granules) Enteric-coated granules (1.000 g) were manufactured using the following ingredients.

[A]

Main ingredient (clathrate compound of the above compound 11 100 (g)) Lactose 737 Hydroxybropyl 3 Cellulose [B] Cellulose acetate phthalate 80 (g) Hydroxypropyl methyl 80 Cellulose phthalate, each of the ingredients in [A] and well After mixing, the mixture was formed into granules according to a conventional method, thoroughly dried and sieved to produce granules suitable for bottles, heat-seal packaging, etc. Next, the granules are coated with the dissolved base material (B) while floating and flowing to form enteric-coated granules. When this granule was subjected to a disintegration test using a diurnal disintegration tester, it was found that p
It does not disintegrate even when shaken in H1,2 artificial gastric juice for 1 hour. p
In the artificial intestinal fluid of H7.5, it disintegrated in 5 minutes.

Formulation Example 6 (Enteric Coated Capsules) Enteric coated capsules i, o o o were manufactured using the following ingredients.

(A'1 (A) (B) Base agent (above compound 6 100 (g) 50 (g> clathrate) Lactose 24.6 74.4
Hydroxypropyl 0.4 0.4 Cellulose [B] Cellulose acetate phthalate 10 (g) 10 (g) Hydroxypropyl methyl 10 10 Cellulose phthalate Suitable for capsule use with the above ingredients in the same manner as described in Formulation Example 5 Enteric-coated granules were prepared, and the composition was filled into capsules to obtain enteric-coated capsules.

When this capsule was subjected to a disintegration test using a diurnal disintegration tester, no disintegration or dissolution was observed even after shaking for 1 hour in artificial gastric fluid at pH 1.2, and no disintegration or dissolution was observed in artificial intestinal fluid at pH 1.5.
It disintegrated or the entire amount eluted within minutes.

Test Example 1 The above compound 1.2.3.4.5.6.7.8.9.10.11.1 as a test compound of aromatic aldehyde derivative
2. Cancer cells W2 of C3H mice that were made cancerous by the SV4° oncogenic virus according to the above test method using 13 and 14.
The proliferation inhibition rate (%) of K.11 was calculated, and the growth inhibition rate relative activity value of the test compound was calculated when the growth inhibition rate (%) of benzaldehyde (control) was set as 100. Third
The results shown in the table were obtained.

Table 3 [Discussion] As is clear from the results of Test Example 1, all of the above test compounds have an activity equal to or greater than that of benzaldehyde, when the growth inhibition rate of benzaldehyde, which has excellent anticancer activity, is set to 100. It was demonstrated that it has excellent anticancer activity.

Note that among the above test compounds, the activity value of Compound 2 is lower than that of the other compounds. However, it is extremely unique that many of the conventional cancer chemotherapeutic agents have low activity against cancerous cells caused by the SV40 oncogenic virus compared to transplanted animal cancers, whereas the above compounds act on this and show a suppressive effect. It is noteworthy that the

In other words, although many of the conventional cytotoxic anticancer drugs show remarkable activity against experimental tumors in animals, that is, transplanted cancers, they still have many problems clinically, and there are very few effective examples. Ino is the actual situation, and this is thought to be based on the fundamental difference between transplanted cancer and primary cancer, and SV can be said to be an artificial primary cancer. The above-mentioned compounds having activity against I. virus-induced cancer are recognized to have very characteristic anticancer activity.

Claims (1)

[Claims] Benzaldehyde sulfite adduct sodium salt, monobenzalpentaerythritol, N-benzylideneethylamine, N-benzylidenemethylamine, N-benzylideneisobutylamine, 1-benzylideneamino-2-ethoxyethane, 1-benzylideneamino-3-dimethylaminopropane , 1-benzylideneamino-2-propanol, 2-benzylideneamino-2-methylpropanol, 3-benzylideneamino-1-propene, α-benzaldoxime, 3-ethyl-2-phenyloxazolidine, 3-(2'- An anticancer agent containing as an active ingredient at least one compound selected from the group consisting of (hydroxyethyl)-2-phenyloxazoline and mandelonitrile.