JP2005029554A - Chemically synthesized, conjugated highly unsaturated fatty acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cancer cytocidal agent capable of killing cancer cells specifically and effectively and a composition useful for the prevention and treatment of cancer diseases. <P>SOLUTION: This specific cancer cytocidal agent consists of a highly unsaturated fatty acid having a structure having ≥3 conjugated double bonds and obtained by a chemical synthesis, and its derivative as an active ingredient. Also a medicinal or food composition blended with the cancer cytocidal agent is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Detailed Description of the Invention

Technical field to which the invention belongs

The present invention relates to a chemically synthesized polyunsaturated fatty acid having various physiological activities, a chemically synthesized polyunsaturated fatty acid having three or more conjugated double bond structures, a derivative thereof, and a cancer killing agent containing the same as an active ingredient. It is related with the composition formed by mix | blending. More specifically, a cancer cell killing agent that efficiently kills human-derived cancer cells containing conjugated trienoic acid produced by a novel synthesis method as an active ingredient, and a pharmaceutical composition or an edible composition comprising the same It is about things.

In recent years, research on the physiological activity and pharmacological action of lipids has progressed, and conversion to various chemical substances and the function thereof have been elucidated in the metabolism of unsaturated fatty acids. In particular, the nutritional ratio of saturated fatty acids, monoenoic acids and unsaturated fatty acids, and the ratio of omega-3 fatty acids derived from fish carp such as icosapentaenoic acid and docosahexaenoic acid, and omega-6 fatty acids derived from plants, mainly linoleic acid, It is regarded as important in relation to illness.

ω6-based linoleic acid is an essential fatty acid for skin formation and undergoes chain extension and desaturation in the human body to become arachidonic acid and ω6 docosapentaenoic acid. Arachidonic acid is an indispensable fatty acid for growth in early childhood, and it becomes a prostaglandin and leukotriene by the arachidonic acid cascade and plays a physiological function in each part of human leave. There is also a series of ω3 fatty acids such as icosapentaenoic acid (IPA) and docosahexaenoic acid (DHA) by chain extension and desaturation using α-linolenic acid (ALN) as a starting material. IPA and DHA are ω3 polyunsaturated fatty acids that are abundant in fish oil and the like, and are not converted from other series of fatty acids such as ω6 in human leave. Therefore, it is said that the deficiency of these fatty acids causes various diseases.

Physiological functions of these polyunsaturated fatty acids include ALN's ability to improve memory learning ability (Japanese Patent Laid-Open No. 1-153629), antiallergic action, serum lipid improving action, etc., and AA has an effect on platelet aggregation and growth in infancy In EPA, hyperlipidemia improvement effect has been recognized for pharmaceutical use, cholesterol reduction effect and erythrocyte deformability improvement effect (Yasu Tamura et al. “Food Science” Vol. 161, 33-39 1991) Blood lipid lowering action (Katsuaki Imaizumi, “Clinical Nutrition”, 83: 4, 440, 1993, etc.) Inhibiting platelet aggregation (Yuzo Nagakawa et al., “Blood and Vascular”, 15: 2, 138-141, 1984) ) improvement of learning and memory (A.Hucas, et al., the Lancet, 339, 261,1992) anti-dementia (M.Soderberg et al., J.Immun logy, 150, 3525,1993), the anti-tumor effect (Tomio Narusawa et al., "Medical History" Section 145, Volume 911, pp. 1988) anti-allergic effect (M.Shikano et al., J.Immunology, 150,3525,1933 ) And the like have been found, and further functional studies on DPA and mead acid are underway.

On the other hand, in the chemical structure of polyunsaturated fatty acids, it has been clarified that conjugated linoleic acid is contained in bovine lipid, which prevents fat accumulation, and alkali-conjugated conjugated linoleic acid is effective in reducing body fat (Lipids, 31 , 853 (1997)) Functions such as reduction of blood cholesterol and antitumor action (MA Belury, Nutr. ReS., 53, 83, (1995)) have been found. In addition, it has been announced that eleostearic acid and the like contained in tung oil and bittern seed oil are more effective.

The inventors have already filed Japanese Patent Application Laid-Open Nos. 2000-281572 and 2001-288079 for cancer cell killing agents containing polyunsaturated fatty acids conjugated with alkali as active ingredients. Since these contain a lot of conjugated trienes, they are more effective than conjugated dienes, and it has become clear that long-chain fatty acids having 20 or more carbon atoms are less toxic to normal cells than conjugated trienes. .

Problems to be solved by the invention

As described above, IPA and DHA having a conjugated triene structure were found to have an excellent cancer killing effect and not to affect normal cells. However, in studying the mechanism of action in actual treatment, The fact that the conjugated triene has a very large number of positions and geometric isomers makes it difficult to confirm in terms of its structure. Further, in medicine, it is desirable that it is a single component as much as possible in terms of purity, and when many isomers are contained, each structure is isolated, and evaluation results regarding each function and toxicity are required. It is difficult to isolate dozens of these isomers and to evaluate the toxicity and cancer killing action. Accordingly, it was an object of the present invention to establish a method for producing a chemically or as few isomers as possible, to evaluate the action of cancer killing cells and to provide a composition for food and food.

Means for solving the problem

In order to solve the above-mentioned problems, the present inventors have intensively studied a method for synthesizing polyunsaturated fatty acids having a conjugated triene structure and a method having few isomers. The inventors have established a method for synthesizing conjugated triene-type polyunsaturated fatty acids having the same chemical structural formula as the obtained conjugated triene type, and found that the method has a remarkable cancer-killing cell effect, thereby completing the present invention.

The object of the present invention is to obtain a composition having a conjugated triene having a single composition or two to three chemical structures. Therefore, a method of conjugating naturally-occurring polyunsaturated fatty acids so as to selectively form a single structure or a method such as column separation of polyunsaturated fatty acids containing many isomers of ordinary alkali conjugates Although some studies have been made on the use of several isomers, no good results have been obtained.

As described below, an object of the present invention is to form a target compound by constituting a conjugated triene structure by chemical synthesis and extending the chain by a chemical reaction. The goal is to make a compound consisting of only a single or a few isomers by chemical synthesis. This facilitates identification of the compound in the body metabolism of a compound effective for physiological activity.

In the present invention, the method of producing a chemically unsaturated polyunsaturated fatty acid having three or more conjugated double bond structures and derivatives thereof is a single or a few isomers represented by chemical formulas in chemical synthesis. The goal is to make fewer compounds. These compounds may be in the form of fatty acids but can also be used in the form of various derivatives. Triglycerides, diglycerides, monoglycerides and phosphorus, including normal esters, metal salts such as alkali metals and alkali metals, amides with ammonium, ethanolamine, etc. It can be used in derivatives such as lipids, sulphagolipids containing ceramide and glycosides thereof, glycoglycerides such as sulfoquinovosyl diglycerides and monoglucosyl di. Pharmaceutical compositions and edible compositions can be produced using these chemically synthesized polyunsaturated fatty acids having three or more conjugated double bond structures and derivatives thereof as active ingredients of cancer killing agents.

The polyunsaturated fatty acid having 3 or more chemically conjugated double bond structures and derivatives thereof as referred to in the present invention are not particularly limited, but 5 (Z), 7 (E), 9 (E ), 14 (Z), 17 (Z) icosapentaenoic acid and 5 (E), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid, 5 (Z), 7 (E) , 9 (E), 14 (Z), 17 (Z) docosapentaenoic acid and 5 (E), 7 (E), 9 (E), 14 (Z), 17 (Z) docosapentaenoic acid, and 5 (Z), 7 (E), 9 (E), 14 (Z), 17 (Z), 20 (Z) docosahexaenoic acid and 5 (E), 7 (E), 9 (E), 14 (Z ), 17 (Z), 20 (Z) docosahexaenoic acid and the like. The basic chemical reaction of the present invention is to form a conjugated triene structure and does not necessarily limit the compounds. Extending the chain or further increasing the number of double bonds can be produced by changing the raw materials used.

In the present invention, it is possible to use a concomitant substance in the same manner as other cancer cell killing agents. Moreover, the usage form does not limit the form used as a normal pharmaceutical composition or an edible composition. Moreover, if the object as a cancer cell killing agent said by this invention is a thing derived from a human tissue, a site | part will not specifically limit. It exerts a remarkable killing effect on cancer cells such as large intestine, hepatocytes, lungs, stomach and breast cancer.

Examples of production methods of polyunsaturated fatty acids having 3 or more chemically conjugated double bond structures and derivatives thereof as described in the present invention are shown below.

Example 1 Synthesis Method of Compound 2 Concentrated sulfuric acid (0.1 ml) was added to a solution of δ-valerolactone (5 g) dissolved in methanol (104 ml) and refluxed for 6 hours. After allowing to cool, sodium hydrogen carbonate (0.6 g) was added at 0 ° C. and stirred for 10 minutes. After filtration through celite, concentration under reduced pressure gave ester (HO (CH ₂ ) ₄ COOMe).
This ester was Swern oxidized. Under a nitrogen atmosphere, a three-necked flask equipped with a dropping funnel was charged with Oxary Chloride (5 ml) and anhydrous methylene chloride (75 ml), cooled to −78 ° C., and DMSO (9 ml) was dissolved in methylene chloride (15 ml) with stirring. The solution was slowly added dropwise. After stirring for 30 minutes, an anhydrous methylene chloride (6.4 g) solution of ester (HO (CH ₂ ) ₄ COOMe) (50 mmol) was added dropwise thereto. After stirring at −78 ° C. for 30 minutes, triethylamine was added dropwise. Cooling was stopped and the reaction solution was returned to room temperature. Thereafter, water was added to the reaction solution and stirred. The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate (anhydrous), and concentrated under reduced pressure. The obtained crude product was purified by distillation under reduced pressure (bp: 72-75 ° C./5 mmHg) to obtain a colorless and transparent liquid resting compound 2 (3.1 g).

Example 2 Compound 3 Synthesis dropping funnel and cooling tube Wittig reagent a three-necked flask equipped with _{(Br (C 6 H 5)} 3 PCH 2) 2 CH) (9g, 12.3mmol) -THF a (120 ml) solution Cooled to -78 ° C. A KHMDS (KN (SiMe ₃ ) ₂ ) toluene solution (20% toluene solution, 25 g) was added dropwise to the Wittig reagent-THF suspension at −78 ° C. in a nitrogen atmosphere. The cooling bath was removed and the reaction solution was returned to room temperature and stirred for 30 minutes. HMPA (6 ml) was added and cooled again to -78 ° C. Propionaldehyde (CH3CH2CHO) (1 ml, 14 mmol) was slowly added dropwise to the diylide reaction solution. After the addition, the reaction solution was brought to 0 ° C. over 2 hours and stirred for 30 minutes. After cooling to −78 ° C. again, a solution of compound 2 (1 g, 7.7 mmol) in anhydrous THF (15 ml) was added dropwise. The reaction solution was returned to room temperature and stirred for 30 minutes.
Saturated aqueous ammonium chloride solution (30 ml) was added and stirred, and the organic layer was separated. The aqueous layer was further extracted with ether, and these organic layers were washed with saturated brine and dried over magnesium sulfate (anhydrous). After drying, the filtrate was concentrated under reduced pressure and purified by silica gel chromatography (hexane / ethyl acetate-90 / 1) to obtain Compound 3 as a transparent liquid (30%).

Example 3 Synthesis Method of Compound 4 A compound 3 (200 mg) -anhydrous methyl chloride (8 ml) solution was cooled to −78 ° C. in a three-necked flask equipped with a condenser tube in a nitrogen atmosphere. A hexane solution (1.1 ml) of DIBAL (diisobutyryl aluminum hydride) was slowly added dropwise along the wall of the three-necked flask. After confirming that Compound 3 was reduced to aldehyde by TLC analysis, the mixture was further stirred for 30 minutes. A saturated aqueous ammonium chloride solution (3 ml) was added dropwise, and the reaction solution was returned to room temperature. The mixture was diluted with ether, celite was added, and the mixture was stirred for 15 min. Celite was filtered, and the organic layer was dried over magnesium sulfate (anhydrous) and concentrated under reduced pressure. This composition was purified by silica gel chromatography (hexane / ethyl acetate-100 / 1) to obtain an oily aldehyde compound 7. Ph3 = CHCHO (242 mg) was added to a toluene (10 ml) solution of this substance (120 mg) and refluxed for 2 hours. Thereafter, Ph3 = CHCHO (212 mg) and 5 ml of toluene were further added and refluxed overnight. After concentration under reduced pressure, hexane extraction was performed, and this was concentrated to obtain a crude reaction product. This was purified by silica gel chromatography (hexane / chloroform = 1/1) to obtain Compound 4 (10%).

Example 4 Synthesis Method of Compounds 5 and 6 LiHMDS (0.5 ml) was added dropwise to a Wittig reagent (Ph3P (CH2) COOH, 100 mg) -anhydrous THF (1 ml) suspension in a nitrogen atmosphere. After stirring at room temperature for 30 minutes, Compound 4 (34 mg) -anhydrous THF was added dropwise and stirred at room temperature at 25 ° C. for 30 minutes. A saturated aqueous oxalic acid solution and ether were added and stirred to separate the organic layer. The aqueous layer was further extracted with ether and combined with the organic layer. The organic layer was concentrated under reduced pressure to 1 ml, transferred to an Erlenmeyer flask, and a diazomethane / ether solution was added dropwise. The ether layer was concentrated under reduced pressure to obtain a crude reaction product. This was purified by silica gel column chromatography (hexane / ethyl acetate = 30/1) to obtain 22 mg of a mixture of compounds 5 and 6. (47%)
Compound 5 and 6 mixed samples were subjected to HPLC fractionation (hexane / acetonitrile = 100 / 0.3 (v / v), CHROMPAC 250 × 4.6 mm, CHROMSPHER 5 LIPIDS), and the structure was confirmed by NMR. Compound 5 was preferentially synthesized.

Example 5 Compound 7 (1 g, 6 mmol), which is a synthetic aldehyde of compound 8, was added to Me- (4) -4- (dimethylphosphophoryl) but-2-enoate (1.6 g, 6.7 mmol), powdery molecular sieves 4Å. (6.5 g), lithium hydroxide / THF (275 mg Li · H 2 O, 50 ml THF) was added, and the mixture was refluxed for 14 hours. After refluxing, the mixture was returned to room temperature and filtered through celite with ether. After concentration, the residue was purified by silica gel chromatography (hexane / ethyl acetate = 40 · 1) to obtain 684 mg (46%) of Compound 8 as a colorless and transparent liquid rest, ester.

Example 6 Synthesis Method of Compound 9 A DIBAL-xan solution (0.96 mol / L, 6.5 ml) was slowly added dropwise at −78 ° C. to an anhydrous methylene chloride solution (674 mg, 2.7 mmol ester, 15 ml CH2C12) of ester compound 8. did. After stirring for 1 hour, a saturated aqueous ammonium chloride solution was added, diluted with ether, celite was added, and the mixture was stirred for 15 minutes. The reaction solution was filtered through Celite and concentrated under reduced pressure to obtain crude reaction product compound 9 (allyl alcohol, 594 mg).

Example 7 Synthesis Method of Compound 4 Active manganese dioxide (MnO 2, 8 g) was added to a solution of methylene chloride (250 ml) containing allyl alcohol compound 9 (584 mg), and the mixture was stirred with ultrasound for 4 hours. The reaction solution was filtered through Celite and concentrated under reduced pressure to obtain 456 mg of Compound 4 as a crude reaction product.

Test example 1
The cancer-killing cell action of the above-mentioned highly unsaturated fatty acids having three or more conjugated double bond structures and their derivatives was evaluated by the method described below. That is, colon cancer cells (cell number (hereinafter the same): TKG0379, hereinafter referred to as DLD-1), liver cancer, which are the five types of human-derived cancer cells that were obtained and obtained from the Cancer Cell Facility attached to Tohoku University Institute of Aging Medicine Using cells (TKG0205, hereinafter referred to as HepG2), lung cancer cells (TKG0184, hereinafter referred to as A549), breast cancer cells (TKG0479, hereinafter referred to as MCF7) and gastric cancer cells (TKG0228, hereinafter referred to as MKKN-7), 96 cells each When seeding on a microplate and reaching 80% confluence (cell fullness), 15 μM of 5 (Z), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid and 5 0.5% serum albumin containing a mixture of (E), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid In addition, after 24 hours of incubation at 37 ° C. in a 5% carbon dioxide atmosphere, the number of viable cells was examined by the MTT (3- (4,5-dimethylazol-2-yl) -2,5-2H-tetrazolium bromide) method. It was. The MTT method can be applied to almost all cells because it uses the enzyme activity in living cells as an index, and since this result is also relatively stable, it was adopted as a method for evaluating cell killing action. The results are shown in Table 1. Moreover, the result tested similarly about the fish oil (comparative sample 2) which uses nonconjugated EPA and nonconjugated DHA as a constituent fatty acid was written together.

In Table 1, the numerical values represent the MTT activity (cell viability,%) when no test sample is added as 100, and the relative value at the test sample addition concentration (15 μM) is expressed as an average value ± standard deviation (n = 6). (The same applies hereinafter). The numerical value of each test sample was superior to the values of comparative samples 1 and 2 (P <0.05). From this data, the chemically synthesized polyunsaturated fatty acids having three or more conjugated double bond structures according to the present invention are similar in value to alkali-conjugated EPA and DHA, and are more resistant to colon cancer than fish oil. A cell killing effect was observed on the cells. Similar cell-killing effects were observed for liver cancer cells, lung cancer cells, and gastric cancer cells.

Test example 2
The effect of the various conjugated polyunsaturated fatty acids described above on normal cells was evaluated in the same manner as described in Test Example 1. Normal human-derived cells include lung fibroblasts (hereinafter referred to as MRC-5), skin fibroblasts (hereinafter referred to as TIG-103), and lung fibroblasts (hereinafter referred to as KMS-6) obtained from Human Science Research Resource Bank (Osaka). Used). The results are shown in Table 2 together with the results for conjugated linoleic acid ethyl ester and fish oil (unmodified). The meanings of numerical values and symbols in the table are the same as those in Table 1. From these data, conjugated polyunsaturated fatty acids such as α-linolenic acid, EPA, DHA, which are one of n-3 fatty acids, have little effect on the growth (division, proliferation, etc.) of normal human cells, In particular, those containing a large amount of a conjugated triene have been found to have a strong effect whether it is an alkali conjugate or a chemically synthesized product.

Example 8
The chemical structural formula obtained by Example 4 is 5 (Z), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid and 5 (E), 7 (E), 9 ( E), 14 (Z), 17 (Z) 250 mg of a mixture of icosapentaenoic acid, 30 mg of refined soybean oil, 10 mg of beeswax, and 10 mg of vitamin E are heated to about 40 ° C. in a nitrogen gas atmosphere and mixed thoroughly to form a homogeneous liquid It was a thing. This was supplied to a capsule filling machine, and a gelatin-coated capsule preparation having an inner volume of 300 mg was made as a prototype. These preparations can be used as pharmaceutical compositions or edible compositions.

The invention's effect

According to the present invention, a cancer cell killing agent comprising polyunsaturated fatty acids having three or more conjugated double bond structures and derivatives thereof as active ingredients is provided. This cancer cell killing agent has an effect of killing cancer cells of human-derived colorectal cancer, liver cancer, lung cancer, breast cancer, or stomach cancer, and does not adversely affect the maintenance of normal cells. Such an effect is not observed with conjugated dienes such as conjugated linoleic acid. According to the present invention, highly unsaturated fatty acids having three or more conjugated double bond structures limited to a single or a few isomers and their derivatives have the same effects as alkali-conjugated EPA and DHA. To study the metabolism etc. easily by using polyunsaturated fatty acids and their derivatives having three or more conjugated double bond structures that contain a single or a few isomers It is easy to use for medicinal purposes. According to the present invention, a pharmaceutical composition or an edible composition comprising a cancer cell killing agent is provided. The composition can be used for prevention or treatment of cancer diseases.

Claims (8)

Highly unsaturated fatty acids and derivatives thereof having three or more conjugated double bond structures synthesized chemically Highly unsaturated fatty acids and their derivatives with chemically synthesized conjugated triene structures Conjugated icosapentaenoic acid with chemically synthesized conjugated triene structure The chemical structural formula of claim 3 is 5 (Z), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid and / or 5 (E), 7 (E), 9 ( E), 14 (Z), 17 (Z) chemically synthesized conjugated icosapentaenoic acid which is icosapentaenoic acid The chemical structural formula of claim 3 is 5 (Z), 7 (E), 9 (E), 14 (Z), 17 (Z) icosapentaenoic acid and 5 (E), 7 (E), 9 (E) , 14 (Z), 17 (Z) chemically synthesized conjugated icosapentaenoic acid which is a mixture of icosapentaenoic acid A process for producing a highly unsaturated fatty acid having three or more conjugated double bonds and a derivative thereof chemically synthesized according to claim 1. The biocidal agent against the cancer cell derived from a human whose bioactivity of the compound according to claim 1 is 5. A pharmaceutical composition or an edible composition comprising the cancer cell killing agent against human-derived cancer cells according to claim 7.