JPS6352608B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer agent, which has the general formula: (However, R represents a dibutylamino group, a chloro group, or a pyrrolidine group.) An anticancer agent containing a compound represented by the following as an active ingredient is provided. Conventionally, cancer chemotherapy agents include alkylating agents (nitrozene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), and plant fission toxins (colcemid). , vinblastine, etc.),
Antibiotics (sarcomycin, carcinophilin,
mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and volufiline complex salts (maafilin, COPP), etc.; however, in general, the nucleic acid inhibiting effect of anticancer substances affects not only cancer cells but also normal cells. Because of their strong toxicity and serious side effects, it is currently difficult to achieve sufficient effects by using large amounts of drugs such as chemotherapeutic agents for infectious diseases. The present inventor previously developed a new and useful anticancer agent containing benzaldehyde as an active ingredient (Japanese Patent Publication No. 54
(Refer to Publication No. 962), the action of this active ingredient does not directly attack cancer cells, but is thought to produce therapeutic effects through a mechanism of action that is different from that of conventionally thought chemotherapeutic agents. Furthermore, as a result of intensive research to search for anticancer active substances, it was discovered that each of the compounds represented by the above formula has anticancer activity, and these substances have been found to have anticancer activity. We have now completed the anticancer agent of the present invention by obtaining new knowledge that it can exhibit remarkable therapeutic effects. Most conventional cancer chemotherapeutic agents are so-called cytotoxic substances, which are more sensitive to transplanted cancers such as Ehrlichi tumors than to cancerous cells caused by the SV ₄₀ oncogenic virus. The organic component is highly sensitive to cancerous cells caused by the SV ₄₀ oncogenic virus.
It is thought to be based on a specific anticancer effect that is different from the mechanism of action of conventional cancer chemotherapeutic agents, and is effective against humans, livestock,
It can be an excellent cancer chemotherapy agent for warm-blooded animals such as dogs and cats. Examples of the compounds represented by the above formula having anticancer activity used in the present invention are shown in Table 1.

[Table] Table 2 shows the physical properties and references (or NMR values) of the above compounds (1) to (3) (hereinafter, the above compounds are indicated by compound numbers).

[Table] Production method example 1 Dissolve di-n-butylamine in an amount equal to the mole of parachloroformylbenzaldehyde in anhydrous benzene, boil and reflux for 3 to 5 hours in the presence of twice the mole of triethylamine or pyridine, and then process in a conventional manner. Purify with silica gel column chromatography to obtain the desired product in high yield. Production method example 2 Pyrrolidine in an amount equal to that of parachloroformylbenzaldehyde is dissolved in anhydrous benzene, and in the presence of 2 times the mole of triethylamine or pyridine,
After boiling and refluxing for 5 hours, the mixture is treated in a conventional manner and purified by silica gel column chromatography to obtain the desired product in high yield. The anticancer agent of the present invention can be administered either orally or parenterally.
Administered as hard capsules, tablets, granules, fine granules, or powders; when administered parenterally, injections,
It can be administered in a dosage form such as a suppository so as to maintain sustained mucosal absorption in the form of a drop, a solid or a viscous liquid suspension. The proportion of the active ingredient in the anticancer drug composition of the present invention may vary depending on the dosage form, but usually approximately 0.3 to 15.0% by weight is appropriate when administered orally or by mucosal absorption, and when administered parenterally, When the value is approximately 0.01~
10% by weight is suitable. Furthermore, when formulating the active ingredient of the present invention, the compound represented by the above formula can be made into an aqueous suspension, oily preparation, etc., into a preparation for subcutaneous or intravenous injection according to a conventional method. It can be formulated into dosage forms such as capsules, tablets, and fine granules for oral use. In addition, it can be made into an inclusion compound that utilizes the inclusion ability of choleic acid or 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. Generally, the active ingredients of the present invention can be formulated using surfactants, excipients, lubricants, leftover agents, pharmaceutically acceptable film-forming substances, etc.
Specific examples are as follows. In order to improve disintegration and elution of the composition of the present invention, one or more surfactants such as alcohols, esters, polyethylene glycol derivatives, sorbitan fatty acid esters, sulfated fatty alcohols, etc. are added. Can be added. In addition, excipients such as sucrose, lactose, starch, crystalline cellulose, mannite, light silicic anhydride, magnesium aluminate, magnesium aluminate metasilicate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, carboxylic Methylcellulose calcium and the like can be added alone or in combination of two or more. As a lubricant, for example, one or more types of magnesium stearate, talc, hydrogenated oil, etc. can be added, and as a flavoring agent and a flavoring agent,
Sweeteners such as salt, saccharin, sugar, mannitrite, orange oil, licorice extract, citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, colorants, preservatives, and the like may be included. Adjuvants such as suspending agents and wetting agents may include, for example, coconut oil, olive oil, sesame oil, fall seed oil, calcium lactate, safflower oil, soybean phospholipid, and the like. Film-forming substances include cellulose, cellulose acetate phthalate (CAP) as a carbohydrate derivative such as sugars, methyl acrylate and methacrylate as polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters. Examples include copolymers and methyl methacrylate/methacrylic acid copolymers. In addition, when coating the above-mentioned film-forming substance, in addition to commonly used coating aids such as plasticizers, various additives to prevent chemicals from adhering to each other during coating operations can be added to the film-forming agent. properties and make coating operations easier. A typical dosage form of the anticancer agent of the present invention is a dosage form for oral administration, particularly an enteric-coated dosage form. Dosage forms in which the active ingredient is coated with a film-forming substance are particularly preferred because of the relative instability of the active ingredient and because it is enteric-coated. When the active ingredient is in liquid form, it is preferable to form it into a clathrate compound and then mix it with an excipient or the like, or to impregnate it in a porous excipient and then coat it with a film-forming substance. When the active ingredient is in solid form, it is preferable to thoroughly mix it with excipients and the like before coating it with a film-forming substance. Note that a dosage form may be prepared in which the active ingredient is microencapsulated using a film-forming substance and then mixed with excipients and the like. In particular, the blending ratio in typical dosage forms is as follows.

Table: Substances Particularly preferred excipients are lactose, crystalline cellulose, calcium carboxymethyl cellulose. In addition, the dosage is sufficient to effectively treat the target tumor, and depends on the symptoms of the tumor, route of administration, dosage form, etc., but in general, in the case of oral administration, the amount per day for adults is The range is about 0.5 to 5000 mg/Kg body weight (0.5 to 3000 mg/Kg body weight for dwarfs), and the upper limit is preferably about 200 mg/Kg body weight, more preferably about 50 mg/Kg body weight, and in the case of injections. , its upper limit is about 100 mg/Kg body weight, preferably 50 mg/Kg body weight, more preferably 20 mg/Kg
Appropriate weight. As a result of various tests, it is understood that the anticancer agent of the present invention is effective against human cancer. Next, the anticancer activity testing method for confirming the anticancer activity of the above compound will be described. The test cells were W2K-11 cells obtained by turning C3H mouse kidney cells into cancer with the SV ₄₀ oncogenic virus, and were cultured by the following method. (1) Preparation of growth culture solution Dissolve 9.4 g of Eagle's MEM medium in 900 ml of distilled water, autoclave at 120°C for 15 minutes, cool, add 100 ml of calf serum and 10% carbon dioxide that was separately autoclaved at 115°C for 15 minutes. Add 3-5ml of sodium hydrogen solution
Correct the pH to 7.1-7.2. Immediately before using the medium, add 10 ml of Millipore filtered L-glutamine (2.92 g/100 ml) solution. In addition, to preserve the test cells, the final concentration is 10.
% dimethyl sulfoxide. (2) Preparation of transplanted cells The test cells stored in a Jeep freezer (-80℃) 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 ml of growth medium. After it becomes cloudy, transfer it to a roux flask,
When 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, then add 0.2% trypsin solution [Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.)
Dissolve 4.7 g of the product, 0.6 g of sodium bicarbonate, and 1 g of trypsin in 500 ml of distilled water and filter it with a Millipore filter. Add 10 ml of the solution, treat with trypsin for 2 to 3 minutes at room temperature, and then decant the trypsin solution. Furthermore, add 50 ml of fresh growth medium and wash off adhering cells using a Komagome pipette to obtain a cell suspension. A portion is subcultured using Roux flasks. (3) Cell culture and administration of test compound 1.8 ml of the above cell suspension was dispensed into a disposal chamber (diameter 35 mm) and incubated at 37°C in a carbon dioxide incubator (5% CO ₂ , 95% air).
Incubate for 24 hours. At this point, 0.2 ml 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 cells After administering the test compound, decant the petri dish for 48 hours, discard the supernatant (culture medium), and treat the cells attached to the bottom of the petri dish with 1.0 ml of the above 0.2% trypsin solution to become single cells. . Decant this to remove the trypsin solution, make a cell suspension with physiological saline containing 10 mmol of phosphate buffer (PH7.0), place 1 to 2 drops of this on a hemocytometer, and put 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. Inhibition rate (%) = 1 - (number of cells in the test compound administered shear) / (cell number in the test compound not administered shear)
×100 Next, regarding the toxicity of the active ingredient compounds of the present invention, all compounds are rapidly excreted outside the body due to their low molecular structure, so they do not cause side effects. in administration
Both _LD50 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 500 mg of the above compound (3) and aseptically dispense into vials.
Seal tightly, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. Before use, add 500 ml of 0.85% physiological saline to make an intravenous injection,
Administer 10 to 500 ml per day by intravenous injection or drip depending on symptoms. Formulation Example 2 (Injection/Drop) An intravenous injection for mild symptoms was prepared in the same manner as Formulation Example 1, except that 50 mg of the above compound (1) was used.
Administer 10 to 500 ml per day by intravenous injection or drip depending on symptoms. Formulation Example 3 (Injection/Capsule) Dissolve 30 mg of the above compound (1) in 1 g of refined sesame oil and 100 mg of aluminum stearate gel, seal the solution, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. , as a preparation for subcutaneous injection. Administer 1 to 10 ml subcutaneously once a day depending on the symptoms. In addition, the above preparation is dispensed into capsules in 0.5 ml portions to prepare oral capsules, and 1 to 10 capsules are orally administered per day depending on the symptoms. Formulation Example 4 (Enteric-coated tablets) 1000 enteric-coated tablets each for adults (a) and children (b) were manufactured with the following ingredient composition.

[A]

Main ingredient (β-cyclodextrin clathrate compound of compound (2) above) 100 (g) Lactose 737 Hydroxypropyl cellulose 3 [B] Cellulose acetate phthalate 80 (g) Hydroxypropyl methyl cellulose phthalate 80 Each component of [A] After mixing well, 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 Japanese Pharmacopoeia disintegration tester, it did not disintegrate even if it was shaken in artificial gastric juice of pH 1.2 for one hour. In artificial intestinal fluid with a pH of 7.5, it disintegrated in 5 minutes. Formulation Example 6 (Enteric-coated capsules) 1000 enteric-coated capsules were manufactured using the following ingredients.

[Table] Cellulose phthalate
Enteric-coated granules suitable for capsules were prepared using the above ingredients in the same manner as described in Formulation Example 5, and the composition was filled into capsules to obtain enteric-coated capsules. When this capsule was subjected to a disintegration test using a Japanese Pharmacopoeia disintegration tester, no disintegration or dissolution was observed even after shaking in artificial gastric fluid at pH 1.2 for 1 hour, and no disintegration or dissolution was observed in artificial intestinal fluid at pH 7.5 in 5 minutes. Collapsed or the entire amount eluted. Test Example Using the above compounds (1) to (4), according to the test method,
When the inhibition rate (%) of the proliferation of cancer cells W2K-11 in C3H mice that had become cancerous due to the SV ₄₀ oncogenic virus was calculated, the results shown in Table 3 were obtained.

[Table] [Discussion] As is clear from the results of the above test example, the above test compound was proven to have excellent anticancer activity. In addition, the above test compound has an extremely low activity against cancerous cells caused by the SV ₄₀ oncogenic virus, whereas most of the conventional cancer chemotherapeutic agents have an extremely low activity against cancerous cells caused by the SV 40 oncogenic virus. It should be noted that it is extremely characteristic that both compounds exhibit extremely low toxicity. In other words, many of the conventional cytotoxic anticancer drugs show remarkable activity against experimental tumors in animals, that is, metastatic cancers, but many clinical problems remain, and there are very few effective examples. The reality is that the number of cases is small, and this is thought to be due to the fundamental difference between metastatic cancer and primary cancer, and it can be said that this is an artificial primary cancer.
The above-mentioned compound, which is active against SV ₄₀ virus-induced cancer and has low toxicity, is recognized to have very specific anticancer activity.

Claims (1)

[Claims] 1. General formula: (However, R represents a dibutylamino group, a chloro group, or a pyrrolidino group.) An anticancer agent containing a compound represented by the following as an active ingredient. 2. The anticancer agent according to claim 1 in a parenteral administration form. 3. The anticancer agent according to claim 1 in an oral administration form.