JP4545463B2 - Production method and use of new transcription factor - Google Patents

Description

      The present invention relates to a method for producing a novel transcription factor that modifies the process by which genetic information is transcribed into messenger RNA and its use.

      The novel transcription factor of the present invention has pharmacological properties essential for the treatment of lifestyle-related diseases such as ischemic heart disease, type II diabetes, hypertension (cerebrovascular disorder), obesity and cancer. The most common cause of death in developed countries except Japan is ischemic heart disease. In Japan, cancer is the leading cause of death. In the United States, ischemic heart disease has long accounted for 60 to 70% of causes of death, but as a result of extensive enlightenment campaigns by the government and medical professionals, deaths have fallen to around 40%. However, even today, it is the top cause of death, and in Scandinavian countries such as Sweden and Norway, it still accounts for 70% of all causes of death, far behind the second place. In developing countries such as China, ischemic heart disease is the leading cause of death. Regardless of industrialized or developing countries, ischemic heart disease treatment and prevention methods are developed for malignant tumors, type II There is no doubt that it is steep as well as diabetes, cerebrovascular disorder, obesity and so on.

      Ischemic heart disease, which is a typical lifestyle-related disease, is fearful for people in industrialized countries because sudden death frequently occurs. The three major risk factors for developing it are (1) genetic predisposition, (2) smoking, abnormal lipid metabolism, environmental factors such as diet, and (3) aging. Of these, genetic predisposition and aging are factors that are difficult to control. On the other hand, the majority of industrialized countries continue to strive to reduce environmental risk factors socially. For example, smoking is considered a bad practice, and there are frequent cases of banning tobacco from the environment or lawsuits against tobacco companies.

      As for the treatment / prevention of ischemic heart disease, only environmental factors can be controlled. Therefore, for the purpose of removing risk factors from the environment, many methods such as (1) diet therapy, (2) exercise therapy, and (3) drug therapy have been proposed. In terms of diet, people in developed countries have come to avoid eating foods high in cholesterol. The representatives are beef and chicken eggs, and their consumption has been declining for a long time. Also, excessive intake of energy by overeating induces obesity. Obesity alone cannot be a risk factor, but when combined with insulin resistance (type II diabetes), hyperlipidemia, and hypertension, it can also be called syndrome X (also known as silent death, multiple risk factor syndrome, visceral fat syndrome) ) Was found to be a risk factor called (Reaven GM Diabetes 37: 155-1607 1988). In order to avoid obesity in the United States, exercise therapy such as jogging and fitness that promotes consumption of ingested energy has become popular.

      Among environmental factors that cause ischemic heart disease, the highest correlation is serum cholesterol concentration. Serum cholesterol as a risk factor is expressed on several scales. That is, when the cholesterol contained in serum cholesterol or serum low density lipoprotein (LDL) is higher than the normal value, or the cholesterol contained in serum high density lipoprotein (HDL) is lower than the normal value. Both have been shown to increase the risk of ischemic heart disease in a concentration-dependent manner. Therefore, pharmacotherapy for reducing hypercholesterolemia with a serum cholesterol lowering agent has also been adopted as a standard method for preventing ischemic heart disease. Serum cholesterol lowering agents have become the most successful pharmacological agents. In particular, HMG CoA reductase inhibitors, which are called statin drugs and reduce hydroxymethylglutaryl CoA to mevalonyl CoA, lower serum cholesterol by inhibiting cholesterol biosynthesis, leading to the top global pharmaceutical sales. Is one of the most successful new drugs in the world. The annual sales of statin drugs reached more than 2.5 trillion yen in FY2002.

      On the other hand, the mechanism by which coronary arteries that develop sclerotic lesions are narrowed and occluded to cause angina and myocardial infarction has been elucidated. Until the 1980s, the mainstream idea was that atheromas (atheromas) formed by the accumulation of cholesterol in the arterial wall swell and narrow the vascular lumen. However, since the 1970s, it has been a thrombus that constricts and occludes the coronary artery, and several clinical trials have been conducted based on the hypothesis that prevention of thrombus formation can prevent or treat ischemic heart disease. For example, a clinical trial using aspirin, which is an anti-inflammatory agent and has a strong platelet aggregation inhibitory action. Since aspirin induces peptic ulcers, it is extremely difficult to set a dosage when long-term administration is required. Long-term clinical trials involving a large number of volunteers are effective in some cases, but ineffective in other trials, and do not achieve a certain level of effectiveness. After all, it has been concluded that aspirin requires a dose that frequently causes peptic ulcer in order to suppress chronic inflammation of the arteries, and that it is ineffective at such a small amount that peptic ulcer does not occur.

      In the 1990s, the idea that the atheroma swelled and occludes the artery was rejected, and the atheroma cause theory and the thrombus cause theory were integrated. In other words, it is probable that the blood vessels that have formed in thickened atheroma are broken due to chronic inflammation, causing bleeding in the coronary arteries, and the resulting thrombus constricts or occludes the blood vessels. (Ross R: New England J Med 340: 115-126, 1999). In other words, the two major risk factors behind ischemic heart disease are hypercholesterolemia and chronic inflammation of the arteries. When both serum total cholesterol / HDL cholesterol level and serum CRP, which is a marker of inflammation, are abnormally high, the risk of death from myocardial infarction and cerebrovascular disorder is more than 8 times higher than that of normal humans. Is clear from epidemiological studies.

      The novel transcription factor of the present invention is expected to show therapeutic effects on hypercholesterolemia, type II diabetes, hypertension and obesity. There are treatments that improve the four abnormalities listed above. However, as is evident from the combination of cerivastatin and gemfibrozil that induces rhabdomyolysis and multiple deaths, the combination of multiple drugs is a complex interaction between drugs. There is a risk of causing effects. Therefore, it is desirable to reduce the number of drugs to be administered as much as possible. However, there is still no drug that can simultaneously treat the above four diseases with one agent.

      There have also been many changes in the treatment of type II diabetes that do not require daily insulin injections. It is known that type II diabetic patients are often obese at a certain time until onset, and the onset is highly correlated with obesity. In the early stage of onset, the serum insulin level is normal or higher than normal, and it develops because of insufficient insulin action, not the amount of insulin as in type I diabetes. In other words, the resistance to insulin in peripheral tissues is on the ground and it develops. The first choice for treatment is diet. Exercise increases the effect of insulin and is used in conjunction with diet.

      If the cause of developing and exacerbating type II diabetes is insulin resistance in peripheral tissues, releasing that insulin resistance should improve type II diabetes. In fact, one of the inventors of the present invention found that one of the ascochlorin derivatives 4-0-carboxymethylascochlorin (AS-6) is a model animal for type II diabetes, an obese diabetic mouse, C57BL ksj (db / db) It has been proved that the improvement of glucose metabolism is caused by the reduced insulin resistance of white adipose tissue (Hosokawa, Ando and Tamura; Diabetes, 34: 267-274, 1985). Year). Furthermore, the AS-6 administration group, the non-administration group, and the individual blood glucose level of normal littermates are inversely correlated with a high correlation coefficient (r = −0.899) of glucose metabolism in white adipose tissue, ie, db / Db mice were found to have insulin resistance due to abnormal energy metabolism of white adipose tissue, and that AS-6 released insulin resistance by improving energy metabolism of white adipose tissue. . Today, when neutral fat is highly accumulated in white adipose tissue and becomes obese, tumor necrosis factor (TNF-α) produced by adipocytes, insulin resistance-inducing factor (Hotamisligil GS) such as interleukin-1β. , Sargarill NS, Spiegelman B.M., et al: Science 259: 87-91, 1993) have been shown to develop type II diabetes. That is, it has been found that white adipose tissue is a target tissue for treatment of type II diabetes along with β cells of pancreatic islets of Langerhans. However, in a society around 1985, white adipose tissue was considered as a simple reservoir of neutral fat and was not considered to play a dynamic role in metabolism.

      On the other hand, when looking at cancer, it is clear that “surgery”, “anticancer agent”, and “radiotherapy”, which are said to be the three major treatments, are not always satisfactory. In particular, cancers that can be completely cured despite the long history of anticancer drugs are limited. Acute myeloid leukemia, malignant lymphoma, Wilms tumor in children, etc. are representative of tumors that can be treated with anticancer drugs. Prostate cancer, lung cancer, breast cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, brain tumor, etc., which account for the majority, are cancers that cannot be expected to have an anticancer drug effect. In addition, most anti-cancer drugs are intense cytotoxins, and the more sensitive the cells are, the more sensitive they are. Therefore, there is a side effect of inhibiting the proliferation of epithelial cells of the small intestine and reducing white blood cells to reduce immunity.

      A further problem is that cancer cells readily acquire resistance to anticancer drugs. Current anticancer drug treatment is administered in combination of multiple anticancer drugs. However, no matter how they are combined, cancer usually gains resistance to anticancer drugs in a short period of time, and the anticancer effect is lost. Although the mechanism by which cancer cells acquire resistance to anti-cancer drugs has been studied, no method for clinically avoiding resistance has been found.

      Ascochlorin compounds have been shown to exhibit time-dependent efficacy. It is an absolute condition for exerting a medicinal effect that the effective blood concentration for showing the medicinal effect lasts for a certain period of time. When AS-6 is orally administered to animals, its blood concentration exceeds the effective concentration and rises to the toxic expression concentration, excretion is rapid and the duration of the effective concentration is short, and the aldehyde group is rapidly oxidized to carboxylic acid. There were drawbacks such as The carboxylic acid in which the aldehyde of AS-6 is oxidized has a significantly reduced medicinal effect as compared with AS-6. In addition, since the blood concentration of AS-6 rapidly increases to a concentration that exhibits toxicity, it is a major drawback that toxicity to the liver is likely to occur.

      4-0-methylascochlorin (MAC), an ascochlorin derivative that preceded AS-6, has a solubility in water of 0.7 μg / ml and is extremely poorly soluble in water. In addition, the dissolution rate is slow. Therefore, the slow absorption from the gastrointestinal tract was a difficulty in developing a medicinal effect. Therefore, most of the orally administered MAC was actually excreted in the feces through the digestive tract. Since the duration of the effective blood concentration is short and the peak of the blood concentration is low, it has been a drawback that the effectiveness of medicinal effects, for example, the rate of decrease in serum total cholesterol, blood glucose, blood pressure, etc. is low.

      In other words, in order for an ascochlorin compound to exhibit a medicinal effect, a derivative is required in which the effective blood concentration lasts for a certain period of time and the blood concentration does not increase to the level of toxicity.

      Research and develop therapeutics to improve multiple risk factor syndrome with a single drug, individual abnormalities in hypercholesterolemia, hypertension, type II diabetes and obesity, and patients with multiple of the four abnormalities This is a problem. Another challenge is to develop an anti-cancer agent that does not weaken the immunity of cancer patients and does not acquire resistance from cancer cells. Both 4-0-methylascochlorin and 4-0-carboxymethylascochlorin, which were previously patented by the present inventors, were derivatives obtained by modifying the 4-position hydroxyl group of orsenylaldehyde of ascochlorin. These derivatives have been in use for over 20 years. A large number of derivatives in which the hydroxyl group at the 4-position of orcenyl aldehyde is modified with an alkyl group or an allyl group have already been synthesized, and it is difficult to synthesize a novel derivative that can be patented only by modifying the hydroxyl group at the 4-position of the aromatic ring.

      Ascochlorin compounds have the property of exerting medicinal effects in a time-dependent manner. That is, the blood concentration of the active substance must be lower than the toxic expression level, and must be maintained for a certain period of time between the thresholds for developing the drug effect. On the other hand, 4-0-alkyl ascochlorin obtained by alkylating the hydroxyl group at the 4-position, which has been studied so far, is highly fat-soluble and therefore hardly soluble in water. In addition, the rate at which a single molecule dissolves in water from a crystal is extremely slow, and when administered orally to small animals such as rats and mice, most of them are excreted in the stool through the digestive tract when fasting. In addition to low bioavailability, gastrointestinal absorption varies depending on whether or not food is consumed (Agr. Biol. Chem. 46: 775-781, 1982). This is because bile secreted by feed intake increases the rate of dissolution in water. The lack of reproducibility in animal experiments was a major obstacle to practical use.

The rate at which a single molecule is dissolved in water from the crystal lattice can be increased by introducing a polar group into the molecule. In fact, 4-0-carboxymethyl ascochlorin in which the hydrogen of the 4-position hydroxyl group of ascochlorin is substituted with —CH ₂ COOH is soluble in water at pH 7.2 to 7.4 of the small intestine. Absorbed. For this reason, the blood concentration exceeds the toxicity expression concentration, and it is a drawback that toxicity is easily developed in humans and animals.

Means to solve the problem

      In order to develop a therapeutic agent for lifestyle-related diseases, multiple risk factor syndrome and cancer using ascochlorin and its related substances as mother compounds, the following conditions are necessary. (1) Low cost synthesis, (2) Slow but reliable absorption from the gastrointestinal tract, (3) Serum cholesterol lowering action in animal experiments, hereditary obesity diabetes model animals, hypertension animal models, etc. 4) it is effective for animal models bearing cancer, 5) it is a transcription factor, 6) it has anti-inflammatory activity that can treat / prevent chronic inflammation of blood vessels, etc. This is a condition to be provided.

      For these purposes, the present inventors focused on the synthesis of the ascochlorin compound and the acetal derivative of the aromatic ring aldehyde group of its derivative. However, surprisingly, a general acid-catalyzed alcohol exchange reaction did not produce any acetal. However, it was discovered that the acetalization of the aldehyde group occurs only when the phenolic hydroxyl group adjacent to the aromatic ring aldehyde group is acylated in the presence of a basic catalyst by completely changing the conditions. No acetals have been reported for ascochlorin and related compounds, and all synthesized acetals are novel compounds.

The invention's effect

      The new acetal compound of the ascochlorin derivative has physical properties as the drug substance between MAC and AS-6. Therefore, when administered orally, it is not as difficult to absorb as MAC, but it is not as rapidly absorbed as AS-6. Moreover, as a result of animal experiments and molecular biological studies, the new acetal compound is an inactive prodrug. However, they regenerate aldehyde groups in vivo to form serum protein and Schiff base, and when that Schiff base reaches the target organ or tissue, it is taken up into the cell by exocytosis, When incorporated, serum albumin is digested, and it has been clarified that ascochlorin or its derivative having an aldehyde group is regenerated and exhibits a medicinal effect as a transcription factor. Medicinal effects refer to the effect of lowering serum cholesterol, improving metabolism in hereditary obese diabetes model animals, lowering blood pressure in hypertensive animal models, and inhibiting fat accumulation in healthy animals. Furthermore, the compound according to the present invention has a preventive / therapeutic effect on chronic inflammation of the arteries, an improvement effect on myxedema that develops due to insufficient action of thyroid hormone, an anticancer effect, a balloon It became clear that prevention / treatment of restenosis of an arterial cavity dilated by a catheter or a stent, engraftment of transplanted cells induced to differentiate outside the body in regenerative medicine, and the present invention was completed.

      When administering a compound of the present invention, it can be formulated and administered in pure form or in the form of a suitable pharmaceutical composition by any route of administration that allows drugs for similar uses. Therefore, administration can be solid, semi-solid, such as tablets, suppositories, pills, capsules, powders, solutions, suspensions, emulsions, creams, lotions, aerosols, ointments, gelling agents, etc. It can be carried out in a lyophilized powder or liquid dosage form, for example orally, intranasally, parenterally or topically, preferably in a suitable unit dosage form which can be administered in a precise volume at once. This composition is composed of a normal pharmaceutical alone or excipient and the compound of the present invention, and may further contain other ethical drugs, carriers, absorption aids and the like. In general, a pharmaceutically acceptable composition contains about 1-99% (by weight) of a compound of the present invention and about 99-1% of a suitable pharmaceutical additive, depending on the dosage form to be administered. Yes. This composition contains about 5 to 75% of a compound of the invention as an ethical drug, the remainder containing suitable pharmaceutical excipients. The effective daily dose for the compound of the present invention to improve the pathological condition is 0.01-100 mg / kg, preferably 0.1-10 mg / kg, per kg body weight of an adult.

A preferable dosage form for the disease described in detail above is to formulate a dosage that can be adjusted according to the degree of the disease. The most important factor in formulation is the restriction derived from the fact that the compound of the present invention is fat-soluble. The ligands of the nuclear receptor superfamily are fat-soluble hormones or vitamins, and it is therefore natural that the compounds of the present invention are also fat-soluble. Pharmaceutically acceptable additives for oral administration include any commonly available additive such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc. It is prepared by adding the above excipients. Such compositions take the form of solutions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition is preferably in the form of tablets or pills, but such compositions are disintegrated with diluents such as lactose, sucrose, 2-calcium phosphate, starch and derivatives thereof together with the compounds of the present invention. Agents, lubricants such as magnesium stearate and the like, and binders such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose and their derivatives, and the compound particles of the present invention that are highly lipid soluble and hydrophobic Contains surfactants that act to wet the surface with water, fat-soluble additives, bile acids, phospholipids, and the like. It is particularly preferable to contain an aliphatic synthetic surfactant or an organic solvent-soluble polymer auxiliary. Examples of these include gum arabic, sodium alginate, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, bentonite, sodium lauryl sulfate, polysorbate 80, sorbitan monofatty acid ester, polyoxyl 40 stearate, and the like.
Examples of the present invention will be given below, but it goes without saying that the present invention is not limited to these examples.

Synthesis method of diacyl derivatives of ascochlorin, silindrochlorin, ascofuranone, chloronectin, LLZ-1272-α, LLZ-1272-δ ascochlorin and related compounds 4-0-alkylascochlorin, 4-0-carboxy Alkyl ascochlorin, ascofuranone, silindrochlorin, chloronectin, LLZ-1272-α, LLZ-1272-δ, etc. were added to the pyridine / acetic anhydride mixture and left at room temperature overnight. The amount of acetic anhydride added in the pyridine / acetic anhydride mixed solution for acylating ascochlorin and its related compounds is such that acetic anhydride is added in a slight excess relative to the former hydroxyl group 1 on a molar ratio basis. After standing overnight, the reaction solution was poured into water, about 3 parts of ethyl acetate was added to 1 part of the mixed solution, and the mixture was vigorously stirred in a separatory funnel to separate the upper ethyl acetate phase. The lower layer was extracted again with ethyl acetate and the ethyl acetate phases were combined. The ethyl acetate phase was washed successively with 1N dilute hydrochloric acid and saturated aqueous sodium hydrogen carbonate, and the washed solution was dried by adding anhydrous sodium sulfate. Anhydrous sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to dryness to obtain a crude acylated product. These acylated products obtained by this operation are almost quantitative in yield and very high in purity, so that they can be used in the next step without further purification.

Alternative Method for Preparing 2,4-Di-0-Acetyl Ascochlorin An alternative method using acetyl chloride in place of acetic anhydride is as follows. Ascochlorin (0.300 g, 0.741 mmol) was dissolved in anhydrous pyridine (1.3 ml), and acetyl chloride (0.158 ml, 2.22 mmol) was added dropwise while cooling in a water bath. The reaction mixture was stirred at room temperature for 4 hr, saturated aqueous NaHCO ₃ solution (2 ml) was added, and the mixture was further stirred for 20 min. The reaction mixture was diluted with water and extracted with ether. The ether layer was washed successively with saturated aqueous CuSO ₄ solution, water and saturated brine, and dried over anhydrous sodium sulfate. The desiccant was filtered off and concentrated under reduced pressure to give 0.320 g (88%) of 2 (colorless viscous oil, as a colorless gum).
NMR (CDCl ₃ , 500 MHz): 0.71 (3H, s), 0.81 (3H, d, J = 6.7 Hz), 0.84 (3H, d, J = 6.7 Hz), 1.63 (1H, qd, J = 13.0, 5.5 Hz), 1.86 (3H, s), 1.90-1.97 (2H, m), 2.34 (3H, s), 2.35 (3H, s), 2.36-2.43 (3H, m), 3.35 (2H, d, J = 7.0 Hz), 5.25 (1H, t, J = 7.0 Hz), 5 .41 (1H, d, J = 16.0 Hz), 5.87 (1 H, d, J = 16.0 Hz), 10.27 (1 H, s).

In previous studies, it has been found that crystalline 4-0-acyl derivatives can be obtained in studies of acylating ascochlorin and related substances. This is because even if a 2,4-di0-acyl compound is formed in the reaction solution, the 2-position acyl group is easily hydrolyzed to return to the hydroxyl group during the purification process. Therefore, the 2,4-di0-acyl derivative which has not been obtained in a crystalline form until now has hardly been used as a starting material for the novel derivative. In this example, in order to confirm the formation of a 2,4-di0-acyl compound in the reaction solution, a methanol / triethylamine mixed solution, which is a solvent / catalyst for reacting an aldehyde with a primary amine for aminocarbonylation, was added. 4-di0-acetylascochlorin (0.1 mmol) was dissolved and allowed to stand overnight at room temperature, and the yield of 4-0-methylascochlorin was obtained by partial decomposition of the acyl group. However, it was not the 4-0-acetyl derivative that was unexpectedly produced, but dimethylacetal of 4-0-acetylascochlorin in which two molecules of methanol were added to the aldehyde group was quantitatively produced (0.095 mmol). . In order to confirm the formation of acetal, 2,4-di0-acetylascochlorin (0.1 mmol) was dissolved in a triethylamine / ethanol solution and treated in the same manner as the methanol / triethylamine mixed solution. Was confirmed to be quantitatively (0.098 mmol). Further, when dimethylacetal of 4-0-acetylascochlorin was dissolved in dilute hydrochloric acid / methanol and hydrolyzed, 4-0-acetylascochlorin was produced. Therefore, the compound produced by the reaction of ascochlorin and its related compound having a hydroxyl group at the 2-position and 4-position with an excess of acetic anhydride in a pyridine solvent is a 2,4-diacetyl compound and is mild. It was confirmed that acetal was formed under the conditions. As a matter of course, when the aldehyde group was protected by an acetal, no Schiff base was produced even when reacted with a primary amine in the presence of a basic catalyst.

1

On the other hand, the 4-0-mono acetyl derivative does not form an acetal even under conditions for generating an acetal using a 2,4-di0-acetyl derivative as a starting material (dissolved in a triethylamine / methanol mixed solvent and left). However, the 2-0-acetyl compound obtained by acetylating 4-0-methylascochlorin or 4-0-carboxymethylascochlorin with acetic anhydride in a pyridine solvent is dissolved in triethylamine / alcohol and left at room temperature overnight. Form. Therefore, it is necessary for the hydroxyl group adjacent to the aldehyde to be acylated in order to produce an acetal. Ascochlorin acylated at the 2-position hydroxyl group and its related compounds and alcohols are condensed using a basic catalyst in the presence or absence of a reaction solvent, and the acetal is quantified as shown in Tables 1-3. Generate automatically.

Table 1

Table 2

Table 3

Generally, the acetal formation reaction is an alcohol exchange reaction in the presence of an acid catalyst. Since direct acetal formation with alcohol by the base catalyst of the present invention can be considered as an unprecedented reaction, the reaction mechanism in the case of 2-0-acetyl form of 4-0-carboxymethyl ascochlorin is estimated. did.

2

That is, after the addition of methanol to the aldehyde, a nucleophilic reaction to the adjacent acetyl group, which is also a mixed acid anhydride, followed by the second addition of methanol with the transfer of the acetyl group and the elimination of the acetoxy group occurs to form an acetal Estimated.

Serum cholesterol lowering effect of mice Thirty ICR male mice having a 5-week-old were reared by optionally ingesting standard mouse feed and water. The mice are randomly divided into 3 groups, one group is a non-drug control group, the other two groups are 4-0-methylascochlorin diethylacetal (MAC-DEA) in the diet, 0.1% and 0%, respectively. Give 05% mixed. Body weight, feed intake and water consumption are measured every other day. Blood is collected one week after feeding and serum total cholesterol and neutral fat are measured.

Table 4

Eighteen 7-week-old male db / db mice were bred for 1 week with optional intake of standard mouse feed and water. The mice were randomly divided into 3 groups of 6 animals, and each group was housed in a rat urine collection cage, and food intake, water consumption, urine output, and urinary glucose excretion were measured daily. The first group is a control group giving CE-2, the second group and the third group are CE-2 mixed with 0.05% or 0.1% of diethyl acetal (MAC-DEA) of 4-0-methylascochlorin. Was fed. Feed and drinking water were freely consumed, blood was collected on the seventh day, and blood glucose, serum neutral fat, and serum insulin were measured.

Table 5

Twenty-four Wistar Imamiti male rats with an average body weight of about 250 g were bred for 2 weeks by removing the right kidney under Nembutal anesthesia, suturing the wound, and optionally ingesting food and drinking water. Twenty-eight animals with well-sealed wounds that were well-healed were selected for the experiment. Rats were randomly divided into 3 groups of 6 animals, one group containing 10 mg / kg acetal (AC-PG) in which propylene glycol was bonded to the aldehyde group of 4-0-acetylascochlorin, and the second group was AC-PG. Was orally administered at 5 mg / kg, and the third group was orally administered with a 0.2% Tween-80 aqueous solution in which AC-PG was suspended as a control group. At the start of the experiment, rats were given CE-2 and 1% saline at random, and deoxycorticosterone acetate 5 mg / kg was subcutaneously administered once a week. 1% saline consumption and urine volume were measured daily, and blood pressure and body weight were measured once a week. The table shows weight gain 42 days after the start of the experiment, daily average water consumption, blood pressure, and serum total cholesterol.

Table 6

Forty Wistar Imamiti male rats having an average weight of about 250 g were bred for 2 weeks by removing the right kidney under Nembutal anesthesia, suturing the wound, and optionally taking food and drinking water. Thirty-six animals in which the stitched wounds were completely fused and in good health were selected for the experiment. At the start of the experiment, rats were given CE-2 and 1% saline at random, and deoxycorticosterone acetate 5 mg / kg was subcutaneously administered once a week. Rats are randomly divided into 6 groups of 6 animals, the first group is 10 mg / kg of acetal (AC-PG) in which propylene glycol is bonded to the aldehyde group of 4-0-acetylascochlorin, and the second group is AC. -PG was orally administered at 5 mg / kg, and the third to sixth groups were orally administered with 0.2% Tween-80 aqueous solution alone. On day 42 from the start, administration of deoxycorticosterone acetate of the fourth group to the sixth group was stopped, and at the same time, the drinking water was switched from 1% saline to tap water. At the same time, oral administration of 5 mg / kg of AG-PG to the fourth group and 10 mg / kg to the fifth group was started, and the experiment was terminated after 56 days. Groups 3 and 6 were administered an aqueous solution containing 0.2% Tween-80 throughout the entire experiment. Blood pressure and body weight were measured once a week. On day 56, the mesenteric artery was removed, and the aneurysm stained with fat and stained orange was measured. The table shows the number of aneurysms per mesenteric artery.

Table 7

Eighteen Wistar-Immati male rats with an average weight of about 250 g were abraded with the carotid artery under anesthesia with diethyl ether to create a carotid-abraded rat (Fraser-Smith EB; J Pharmacol). Exp Ther.1995 Dec; 275 (3): 1204-8). Rats were randomly divided into 3 groups of 6 animals, one group containing 50 mg / kg acetal (AS-6-DM) in which methanol was bonded to the aldehyde group of 4-0-carboxymethylascochlorin, and the second group was AS. -6-DM was orally administered at 25 mg / kg, and the third group was orally administered with 0.2% Tween-80 aqueous solution alone as a control group for 2 weeks. Two weeks later, the carotid artery was removed, fixed with formalin, stained with HE, and the thickness of the most thickened part of the artery under the microscope was measured. (Thickness-healthy carotid artery thickness) was defined as 100%, and suppression of thickening was compared.

Table 8

Thirty 5-week-old ICR male mice were randomly divided into 6 groups of 5 mice each, and were allowed to freely feed the mouse standard feed CE-2 (Clea Japan Co., Ltd.) and water for 1 week. Next, streptozotocin 50 mg. 1 kg of mice were intravenously injected into the first group to the third group, blood was collected from the orbit after one week, blood glucose and serum insulin were measured, and the onset of insulin-deficient diabetes was confirmed. Pancreatic islets of Langerhans were taken from healthy mice of groups 4 to 6 that had been housed at the same time 10 days after the injection of streptozotocin, and 20 mice were transplanted subcutaneously on the back of diabetic mice. Immediately after transplantation, the first group was fed with CE-2 containing 0.1% MAC-DE, the second group was also fed with CE-2 containing 0.05%, and the third group was fed with CE-2. Diabetic mice were bred for an additional 60 days, and blood was collected from the orbit every 15 days to measure blood glucose and serum insulin.

Table 9

Thirty 5-week-old ICR male mice were randomly divided into 3 groups of 10 mice, the first group was given a standard diet for mice CE-2 (Clea Japan Co., Ltd.), and the second group was CE-2 Were fed with 0.1% 4-O-methylascochlorin (MAC-DE) diethyl acetal, and the third group was fed with 0.05% MAC-DE. The final weight and weight gain rate tended to be suppressed in the two MAC-DE groups compared to the control group, but there was no statistically significant difference. After 13 weeks, the liver and the epididymis adipose tissue were excised from the mice, lipids were extracted by the method of Folch, and the triglyceride content was measured. As a result, the content of neutral fat decreased by 27% in the 0.05% MAC-DE group, and decreased by 35% in the 0.1% MAC-DE group. This shows that MAC-DE suppresses fat accumulation in the body.

Thyroid hormone activity enhancement in trans-activation assay In the thyroid hormone transgene activity assay system, the compound of the present invention does not exhibit thyroid hormone activity. However, mixing with a small amount of thyroid hormone increased reporter gene expression. That is, when a reporter gene plasmid whose expression is controlled by a thyroid hormone response element and a thyroid hormone expression plasmid are introduced into COS-1 cells, and the introduced cells are treated with thyroid hormone and the compound of the present invention, the expression level of the reporter gene Will increase. This result shows that the compounds of the present invention are not agonists of thyroid hormone nuclear receptors, but these compounds enhance gene expression through cofactors of the thyroid hormone activity transcription process.

  Thirty 5-week-old male ddY male mice were randomly divided into 3 groups of 10 mice, and were allowed to freely feed and feed for 13 weeks. During this time, the first group was given a mouse standard feed CE-2 (Clea Japan Co., Ltd.), and the second group was CE-2 with 0-methylacetochlorin (MAC-DE) diethyl acetal. The feed containing 1% and the third group were reared with feed containing 0.05% MAC-DE. The final weight and weight gain rate tended to be suppressed in the two MAC-DE groups compared to the control group, but there was no statistically significant difference. After 13 weeks, the liver and the epididymis adipose tissue were excised from the mice, lipids were extracted by the method of Folch, and the triglyceride content was measured. As a result, the content of neutral fat decreased by 27% in the 0.05% MAC-DE group, and decreased by 35% in the 0.1% MAC-DE group. This shows that MAC-DE suppresses fat accumulation in the body.

5 week old ICR male mice 18 animals abdominal subcutaneous inoculated with 10 ⁶ Ehrlich ascites carcinoma cells were divided into 3 groups each 6 animals after 24 hours, 4-O-Mechiruasuko the first group and the second group Chlorine (MAC-DE) diethyl acetal was suspended twice daily at 9:00 and 20:00 in 0.2% Tween-80, and orally administered at 1 mg / kg and 4 mg / kg, respectively. The third group was given a Tween-80 aqueous solution containing no MAC-DE as a control group. The administration was continued for 7 days, and the animals were raised for 2 weeks after the administration. On day 21, nodular tumors were removed from the mice and weighed to examine the effect of MAC-DE on tumor growth inhibition. As is apparent from Table 10, MAC-DE significantly inhibits Erich cancer growth.

Table 10

The invention's effect

In addition to providing new therapeutic agents for arteriosclerosis, hypercholesterolemia, hypertension, non-insulin dependent diabetes (also referred to as type II diabetes), chronic inflammation, myxedema, malignant tumors, etc., It is also useful for multiple risk factor syndrome (also referred to as Syndrome X) for which there is no appropriate treatment, prevention of restenosis of arteries expanded by stents and balloon catheters, and ensuring graft survival in regenerative medicine.

None.

Claims (10)

The following general formula

Or

(Where
R ₁ represents acetyl, methyl or carboxymethyl;
R ₂ and R ₃ represent alkyl having 1 to 4 carbon atoms, or R ₂ and R ₃ together form propylene)
An acetal compound represented by   4-O-acetyl ascochlorin dimethyl acetal, 4-O-acetyl ascochlorin diethyl acetal, 4-O-methyl-acetyl ascochlorin diethyl acetal, 4-O-acetyl ascofuranone diethyl acetal, 4-O-methyl ascochlorin dibutyl The acetal compound according to claim 1, wherein the acetal compound is selected from the group consisting of acetal, 4-O-methyl ascochlorin propylene glycol acetal, and 4-O-carboxymethyl ascochlorin dimethyl acetal. The following general formula

(Where
R is

Or

Represents
R ₀ represents a hydrogen atom, methyl or carboxymethyl)
In the presence of a base after acylating the two phenolic hydroxyl groups (when R ₀ represents a hydrogen atom) or the 2-position hydrogen atom (when R ₀ represents methyl or carboxymethyl) of the compound represented by The following formula, characterized by reacting with alcohol

Or

(Where
R ₁ represents acetyl, methyl or carboxymethyl;
R ₂ and R ₃ represent alkyl having 1 to 4 carbon atoms, or R ₂ and R ₃ together form propylene)
The synthesis method of the acetal compound shown by these. The following general formula

Or

(Where
R ₁ represents acetyl, methyl or carboxymethyl;
R ₂ and R ₃ represent alkyl having 1 to 4 carbon atoms, or R ₂ and R ₃ together form propylene)
The pharmaceutical which uses the acetal compound shown by this as an active ingredient.   The medicament according to claim 4, which is used as a therapeutic drug for diabetes.   The medicament according to claim 4, which is used as a therapeutic drug for arteriosclerosis.   The medicament according to claim 4, which is used as a serum cholesterol lowering agent.   The medicament according to claim 4, which is used as a therapeutic drug for hypertension.   The medicament according to claim 4, which is used as an anticancer agent.   The medicament according to claim 4, which is used as a prophylactic / therapeutic agent for restenosis of the arterial cavity.