KR20020084110A - Novel diphenylethylene compounds - Google Patents

Abstract

A novel diphenylethylene compound which is orally administered to reduce the circulating concentration of glucoses is provided. It also shows the effect on insulin resistant rats. The effect on lipid and leptin concentrations is also shown. The compound is an orally active antidiabetic agent capable of normalizing glucose and lipid metabolism in diabetic patients.

Description

Novel diphenylethylene compounds {NOVEL DIPHENYLETHYLENE COMPOUNDS}

The leaves, flowers and gum extracts of Pterocarpus marsupium Roxb. { Leguminosae } wood, also known as Indian Kino Tree, are traditionally found in diarrhea, toothache, fever and urinary and skin Have been used to treat infections. Extracts of bark have long been thought to be useful for treating diabetes. Naturally occurring pterostilbene, trans-1- (3, 5-dihydroxyphenyl) propionate, which is isolated from the core of pterocarpus maspium, 5-dimethoxyphenyl) -2- (4-hydroxyphenyl) -ethylene have been reported to some extent. However, these sterols were insoluble in water and did not appear to be effective in the treatment of diabetes.

The causes of Type I and Type II diabetes have not yet been elucidated, both genetic and environmental factors appear to be involved. Type I diabetes (or insulin-dependent diabetes) is an autoimmune disease in which the causative autoantigen has not yet been identified. Patients with type I diabetes need to administer parenteral insulin for survival. Type II diabetes {also referred to as non-insulin dependent diabetes mellitus (NIDDM)) is a metabolic disorder caused by a disorder in the body that does not produce sufficient insulin or does not adequately use the produced insulin. Insulin secretion and insulin resistance are thought to be major metabolic defects, but the exact genetic factors involved are not known.

Diabetic patients generally have one or more of the following defects:

Lack of production of insulin by the pancreas

Hypercholesterolemia

Defects in Glucose Carrier

Desensitization of insulin receptor

Defects in metabolism of polysaccharides

In addition to the parenterally administered insulin, currently available drugs for diabetes include the four types of oral hypoglycemic agents shown in the following table.

Kinds Trading agent Mechanism of action limit Sulfonylurea First generation: 2 Second generation: 3 Signaling β-cells to release more insulin Generation of resistance Hypoglycemia Biguanide Metformin Reduce the production of liver glucoose Disadvantages of improving sensitivity to insulin Lactic acid Non-boosting gastrointestinal action Glucosidase inhibitor Acarbose Reduce Glucose Absorption in the Bowl GI side effects only after meals Thiazolidinedione Troglitazone (canceled) Rosiglitazone (Pioglitazone) Reduces insulin resistance Ineffective in 25% of patients. Requires frequent liver function tests. It takes a long time to initiate. It causes weight gain.

As is apparent from the above table, currently available antidiabetics have drawbacks. Therefore, there is a continuing interest in the identification and development of new drugs that can be used in the treatment of diabetes, particularly water-soluble compounds that are orally administered.

(-) - epicatechin in addition to the above-described pterostilbene was isolated from phthalocarpus maspium (Sheehan et al., J. Nat. Prod. 1983; 46: 232) (See also Chakravarthy et al. Life Sciences 1981; 29: 2043-2047). Other phenolic compounds have also been isolated from pterocarpus maspium (Maurya et al. J. Nat. Prod. 1984; 47: 179-181, Jahromi et al., J. Nat. Prod. 1993; 56: 989-994 And Maurya et al. Heterocycles 1982; 19: 2103-2107).

The present invention relates to novel diphenylethylene compounds and their use for use in the treatment of disorders associated with diabetes.

Figure 1 is a graph showing that Compound Ia reduces blood glucose levels in rats subjected to streptozotocin-induced diabetes.

Figure 2 is a graph showing that Compound Ia in ob / ob mice decreases blood glucose levels.

Figures 3a, 3b, 3c are graphs showing that Compound Ia in ob / ob mice decreases insulin, triglyceride, and free fatty acid concentrations.

4 is a graph showing that Compound Ia decreases blood glucose concentration in db / db mice.

Figures 5a, 5b and 5c are graphs showing that compound Ia in db / db mice decreases triglyceride and free fatty acid concentrations.

Figure 6 is a graph showing that orally administered Compound Ia is more effective at maintaining reduced blood glucose levels than IP administration.

Figures 7a and 7b are graphs showing that compound Ia reduces blood glucose levels in female obesity (fa / fa) jurker rats without affecting body weight.

Figures 8a, 8b, 8c, 8d are graphs showing that compound Ia increases glucose tolerance in female obese Zucker rats.

Figures 9a and 9b are graphs showing that compound Ia decreases serum insulin and increases leptin concentration in female obese zooker fa / fa rats.

10 is a graph showing that compound Ia decreases cholesterol, triglyceride, and free fatty acid concentrations in female zooker fa / fa rats.

11a, 11b, 11c, and 11d are graphs showing that Compound Ia (20 mg / kg daily) decreases insulin, triglyceride, free fatty acid and cholesterol concentrations in male obese zooker fa / fa rats.

Figures 12a and 12b are graphs showing that compound Ia does not decrease glucose concentration in normal animals.

Figures 13a and 13b are graphs showing that compound Ia stimulates glucose uptake in adipocytes.

Figures 14a, 14b, 14c and 14d are graphs showing that compound Ia increases GLUT-1 and GLUT-4 carriers in 3T3-L1 cells.

Figures 15a, 15b and 15c show lethal effect studies on Swiss Webster mice by administering compound Ia at doses of 16.7, 167 and 333 mg / kg / BW at day 0, respectively.

16 is a graph showing that Wortmannin (a known PI-3 kinase inhibitor) blocks compound Ia mediated glucoside absorption in adipocytes.

Figure 17 is a graph showing that compound Ia stimulates insulin receptor beta subunit and phosphorylation of insulin receptor substrate 1 in CHO.IR cells.

Figure 18 is a graph showing that compound Ia does not stimulate phosphorylation of the IGF-1 receptor in CHO.IGF-1R cells.

19 is a graph showing that compound Ia stimulates phosphorylation of Akt (protein kinase B) in CH). IR cells.

Figure 20 is a description of western blot showing that wortmannin inhibits Compound Ia stimulated Akt phosphorylation.

Figure 21 is a graph showing that compound Ia did not upregulate the expression of PPAR-y in 3T3-L1 adipocytes.

Figure 22 summarizes the results of a binding study showing that compound Ia is not an agonist of nuclear PPARs.

23 is a graph showing that Compound Ia inhibits the binding of insulin to the insulin receptor.

Figures 24a and 24b are graphs showing that the two isoforms Ia and Ib (E and Z) stimulate rapid glucose uptake in rat adipocytes.

25a and 25b are graphs showing the results of drug bioassay studies of Compound Ia in Sprague-Dole rats.

26 is a chart summarizing the results of toxicity studies using Compound Ia under Good Laboratory Practice regulation.

The kinds of compounds having the general formulas (I) and (II) have a hypoglycemic activity.

In formula (I), the bond indicated by the dashed line may be a selective double bond, and the geometry for the bond is E or Z.

In Formulas I and II, A is -COOR, -CONR'R ", -CN or -COR ₇ , and R, R ', R "and R ₇ are defined as follows:

X is H, OH or a C ₁ -C ₁₀ linear or branched alkyl or alkenyl group which may be substituted by COOR, carbonyl or halo;

R is H, linear or branched C ₁ -C ₂₀ alkyl or aryl or aralkyl, Na, K, or other pharmaceutically acceptable counterion such as calcium, magnesium, ammonium, tromethamine and the like;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are independently H; A C ₁ -C ₂₀ straight chain or branched chain alkyl or alkenyl group which may be optionally substituted, COOR; NR'R "or CONR'R" (R 'and R "are independently H or C ₁ -C ₂₀ straight or branched chain alkyl, or aryl); OH; C ₁ -C ₂₀ alkoxy; C ₁ -C ₂₀ acyl Amino, C ₁ -C ₂₀ acyloxy, C ₁ -C ₂₀ alkanoyl, C ₁ -C ₂₀ alkoxycarbonyl, halo, NO ₂ , SO ₂ R '"; CZ _{3 wherein} each Z is independently a halo atom, H, alkyl, chloro, or fluoro substituted alkyl; Or SR '" wherein R''is H or linear or branched C ₁ -C ₂₀ alkyl; Or R ₂ and R ₃ may be combined together, or R ₅ and R ₆ may be bonded together to form a methylenedioxy or ethylenedioxy group.

In the compounds of formula II, the bond indicated by the dashed line may be a selective double bond, the geometry for the bond is E or Z; The naphthyl group may be bonded to the? Or? Position.

The pharmaceutical compositions of the compounds of Formulas I and / or II are provided for the treatment of diabetes comprising a therapeutically effective amount of a compound in a pharmaceutically acceptable carrier.

The method of treating diabetes is also provided comprising orally administering a therapeutically effective amount of a compound of Formulas I and / or II to a diabetic patient.

The compounds of formulas I and II are provided by synthetic methods generally known in the art. (See Pettit et al., J. Nat. Prod., 1988, 51 (3), pp 517-527 for methods of preparing E-isomers analogous to Ia and Kessar et al al., Indlan J. of Chem., 1981, 20B, pp 1-3).

A is -COOR; Wherein R ₁ , R ₄ and R ₆ are H, R ₂ and R ₃ are methoxy (OCH ₃ ), R is H, R ₅ is OH and the intermittent line is a carbon-carbon double bond ≪ / RTI > are preferred. And R _1, R _4, R ₆ is H, R ₂ is OCH ₃ in 3-position, R ₃ is OCH ₃ in the 5-position, R ₅ is OH in the 4-position, intermittent lines E or Z And X is H and R is a pharmaceutically acceptable cation such as H or lithium, sodium, potassium, calcium, magnesium, ammonium, tromethamine, and the like, Particularly preferred are compounds of formula I which can be introduced parenterally.

A is -COOR; Wherein R ₁ , R ₄ and R ₆ are H, R ₂ and R ₃ are methoxy (OCH ₃ ), R is H, R ₅ is OH and the intermittent line is a carbon-carbon double bond Lt; RTI ID = 0.0 > (II) < / RTI > R ₁ , R ₄ , R ₆ are H, R ₂ is OCH ₃ at the 3-position and the intermittent line is a carbon-carbon double bond in E or Z form; X is H and R is H or a pharmaceutically acceptable cation such as lithium, sodium, potassium, calcium, magnesium, ammonium, tromethamine, and the like, and can be either orally or parenterally Compounds are particularly preferred.

In general, the compounds of formula I A) a suitably substituted _{(R 1, R 2, R} 3) benzaldehyde or phenyl the ketone with a suitably substituted _{(R 4, R 5, R} 6) phenylacetic acid or petil ethyl ester ; B) (R ₄ , R ₅ , R ₆ ) phenylacetamide suitably substituted with a suitably substituted (R ₁ , R ₂ , R ₃ ) benzaldehyde or phenyl ketone; C) condensation of appropriately substituted (R ₁ , R ₂ , R ₃ ) benzaldehyde or phenyl ketone with appropriately substituted (R ₄ , R ₅ , R ₆ ) phenylacetonitrile.

In general, the compounds of formula (II) can be prepared by reaction of a) (R ₄ , R ₅ , R ₆ ) naphthylacetic acid or naphthyl, suitably substituted with a suitably substituted (R ₁ , R ₂ , R ₃ ) benzaldehyde or phenylketone Acetic acid esters; B) (R ₄ , R ₅ , R ₆ ) naphthylacetamides suitably substituted with suitably substituted (R ₁ , R ₂ , R ₃ ) benzaldehyde or phenyl ketone; C) condensation of appropriately substituted (R ₁ , R ₂ , R ₃ ) benzaldehyde or phenyl ketone with appropriately substituted (R ₄ , R ₅ , R ₆ ) naphthyl acetonitrile.

In Scheme I, the synthesis of Formula Ia is illustrated by an exemplary synthetic method. An exemplary synthetic method for converting Ia to the Z-isomer is shown in Scheme II.

In the compounds of formulas I and II, the alkyl group may be straight or branched including but not limited to methyl, ethyl, propyl, isopropyl, sec-butyl, n-butyl, pentyl, isopentyl and the like. The alkenyl group having 1 to 20 carbon atoms includes, but is not limited to, ethylene, propylene, butylene, isobutylene, and the like. The aryl group includes phenyl, and other multi-ring aromatic structures. Alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, methylenedioxy, ethylenedioxy, and the like. Halo includes bromo, chloro, fluoro, and iodo.

Acylamino has the following structure:

(Wherein R is hydrogen, alkyl or aryl)

.

Acyloxy has the following structure:

(Wherein R is hydrogen, alkyl or aryl)

.

The alkanoyl is the next group:

(Wherein R may be hydrogen, alkyl or aryl)

.

The alkoxycarbonyl may be substituted with one or more of the following groups:

(Wherein R may be hydrogen, alkyl, aryl or aralkyl)

.

The compounds according to the present invention may be combined with various compositions suitable for oral or parenteral delivery with pharmaceutically acceptable carriers and vehicles. A particularly preferred form of the composition is an orally administered capsule or solution in which the compound is delivered in water, saline or phosphate buffer; Or freeze-dried powder in tablet or capsule form containing various fillers and binding agents. Effective doses of the compounds in the compositions will be selected by those skilled in the art and can be determined empirically.

The compounds of the present invention are useful for the treatment of diseases characterized by the presence of elevated blood glucose levels, such as hyperglycemia, such as diabetes, including both type I and type II diabetes, and hyperglycemia, such as obesity, increased cholesterol concentration, Are useful in the treatment of other related diseases.

&Quot; Therapeutic " is administered such that the compound at least reduces the blood glucose concentration in a patient with hyperglycemia; Such a compound also means that it can reduce insulin or lipid concentration, or both. The compound is administered in an amount sufficient to reduce the blood glucose concentration to an acceptable extent and the acceptable range means within about ± 10% of the normal average blood glucose concentration for the patient of that species. A variety of patients, such as livestock, expensive or rare animals, and pets, as well as humans can be treated with these compounds to reduce blood glucose levels. The compounds may be administered to patients with hyperglycemia using any administration technique including intravenous, endothelial, intramuscular, subcutaneous, or oral. However, oral administration routes are particularly preferred. The dose delivered to the patient will depend on the route through which the compound is delivered, but is generally in the range of 5 to 500 mg per 70 kg person, and typically in the range of 50 to 200 mg per 70 kg person.

Administering the compound to a person afflicted with hyperglycemia to reduce the patient ' s blood glucose concentration to at least about a normal blood glucose range for a person; Also of particular interest are methods of treating hyperglycemia in humans, such as diabetes (both type I and type II), wherein the compound is capable of reducing insulin or lipid concentrations, or both.

The following examples are provided in an illustrative manner and should not be construed as limiting the invention in any way.

(Example 1)

Synthesis of E-3- (3,5-dimethoxy-phenyl) -2- (4-hydroxy-phenyl) -acrylic acid

5 ml acetic anhydride and 2.5 ml triethylamine (TEA) were added to a mixture of 3,5-dimethoxybenzaldehyde (30 mmol) and p-hydroxyphenylacetic acid (30 mmol). After stirring at 130-140 ℃ for 24 hours, the mixture was cooled to room temperature and stopped by 25㎖ concentrated HCl and extracted with CH ₂ Cl _2. The organic extract was further extracted with 1N NaOH, then the NaOH extract was washed with CH ₂ Cl ₂ , the aqueous layer was acidified with concentrated HCl and washed with water to give the crude product. The crude product was recrystallized from ethanol / water to give acid Ia.

Four lots of Ia (E-isomer) prepared as described above were separated into 40 시 samples by HPLC on an Intersil ODS-3 (GL Sciences) column, 250 x 4.6 mm and eluted with 62% eluent A and 38% eluent B Respectively. Eluent A is 0.1% formic acid in water; B is 0.1% formic acid in ACN. All samples showed a large amount of E-isomer with a small amount of Ib (Z-isomer) at a relative retention time of 1.073 ± 0.001. By this method, it is estimated that the presence of the Z-isomer is 0.27% to 3.09% in these samples.

Synthesis of Z-3- (3,5-dimethoxy-phenyl) -2- (4-hydroxy-phenyl) -acrylic acid

The Z-acid Ib was synthesized by the method described by Kessar et al. Showing that continuous heating under basic conditions can convert E-α-phenyl cinnamic acid to a similar Z-α-phenyl cinnamic acid. The E-acid Ia (1.2 g, 4.0 mmol) was dissolved in a mixture of triethylamine (5.0 mL) and acetic anhydride (0.5 mL) and heated with reflux for 24 h. The mixture was then cooled, diluted with ethyl acetate, first extracted with 5% HCl (aq) and extracted with 2N NaOH and water. The combined basic aqueous solution was acidified to pH 5 using acetic acid and cooled, and the solid was filtered. The filtrate was further acidified with concentrated HCl. The precipitation occurred as it cooled down. The solid was collected by filtration and washed with fresh water. The solid compound was air dried to obtain Ib.

The isomers were analyzed by NMR, pKa, HPLC, and UV spectra.

E-isomer. The E-isomer in the free acid form showed a chemical shift to an olefinic proton (in DMSO-d ₆ ) of 隆 7.59. The free acid has a melting point of 225-227 캜 and a pKa of 6.2.

Z-isomer. ¹ H NMR analysis of the Z-isomer produced as above showed that the chemical evolution of the olefinic proton was δ 6.81 as the free acid in DMSO-d ₆ . The free acid form has a melting point of 135-137 [deg.] C and a pKa of 5.3.

Comparison of the resulting isomers. The chemical shifts of the olefinic proton of the E- and Z-isomers prepared as described above are? 7.59 and? 6.81, respectively. As reported in the literature (Gadre and Marathe, Synth Commun 1988; 18: 1015-1027), the higher chemical shift of the olefinic proton is the E-isomer, and each transition exhibited by the prepared compounds is consistent with that .

Analysis of the Perkin reaction product of phenylacetic acid and benzaldehyde (similar compounds) showed that the pKa of the isomer of a-phenylcinnamic acid was 6.1 for the E-isomer and 4.8 for the Z-isomer (Fieser LF and Williamson KL, Exp. In Org. Chem (3rd ed.), Lexington, Mass .; Heath and Company). Thus, between the two isomers, it is the E-isomer that has a higher pKa.

HPLC and UV spectrum analysis

G.L. Reverse-phase HPLC analysis of the E- and Z-isomers was performed by a linear gradient on a Scientific Intersil ODS-3 column (250 x 4.6 mm, 5 μm) using a 0.1% formic acid / water / acetonitrile system and irradiated at 280 nm . In this system, the E- and Z-isomers eluted at 17.4 min and 17.9 min, respectively.

Each isomer has a completely different UV spectrum. The? Max values for the E-isomer are 227 nm and 284 nm, and the values for the Z-isomer are 221 nm and 303 nm.

Synthesis of E-4- [2- (3,5-dimethoxy-phenyl) -vinyl] -phenol

To decarboxylation to Ia, with the addition of quinoline in the 3g Cu powder and the 30㎖ Ia of 1g under N ₂ and stirred for 4 hours to reflux (still with N _2). The reaction mixture was filtered, acidified with concentrated HCl and extracted with CH ₂ Cl ₂ . The organic layer was washed with water saturated NaCl, dried and concentrated. The decarboxylated product was purified by flash chromatography on silica gel.

Synthesis of E-3- (3,5-dimethoxy-phenyl) -2- (4-hydroxy-phenyl) -acrylic acid sodium salt

To convert the acid Ia to a sodium salt, 1 g of NaOH solution is added to 1 g at room temperature; The mixture was shaken and freeze-dried to obtain sodium salt of Ia.

(Example 2)

Synthesis of 3- (3,4-dimethoxy-phenyl) -2- (4-hydroxy-phenyl) -acrylic acid

Acetic anhydride (12 ml) and triethylamine (8.0 ml, 58 mmol) were added to a mixture of 3,4-dimethoxybenzaldehyde (9.97 g, 60 mmol) and p- hydroxyphenylacetic acid (10.0 g, Was added. The mixture was stirred at 140 < 0 > C for 18 hours. The reaction mixture was cooled to 5 [deg.] C and dichloromethane (100 ml) was added. To this yellow suspension, concentrated HCl (20 mL) was added and the suspension was stirred for 20 minutes. The separated solid was filtered off, dissolved in aqueous sodium hydroxide (2M, 225 mL) and re-precipitated with concentrated HCl (40 mL). The yellow solid was filtered off, washed with water (2 x 30 mL), and the wet solid recrystallized from a water-ethanol mixture.

_{^{1 H NMR (DMSO-d 6}} ): δ12.38 (. Br 1H), 9.47 (br, 1H), 7.61 (s, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.81-6.77 ( overlapping , 4H), 6.54 (br, IH), 3.40 (s, 3H) and 3.37 (s, 3H).

Synthesis of 3- (3,4-dimethoxy-phenyl) -2- (4-fluoro-p-phenyl) -acrylic acid

Acetic anhydride (5 ml) and triethylamine (5.0 ml, 36 mmol) were added to a mixture of 3,5-dimethoxybenzaldehyde (4.98 g, 30 mmol) and p- fluorophenylacetic acid (4.62 g, 30 mmol) Was added. The mixture was stirred at 140 < 0 > C for 18 hours. The reaction mixture was cooled to room temperature and diethyl ether (100 ml) was added. The ether solution was further cooled to 10 < 0 > C and acidified with concentrated HCl (35 ml). The aqueous layer was discarded and the organic layer was extracted with aqueous sodium hydroxide solution (2M, 3 x 75 mL). The aqueous layer was pooled and acidified with concentrated HCl (40 mL). The resulting precipitate was filtered off, washed with water (2 x 30 mL) and recrystallized from a water-ethanol mixture.

_{^{1 H NMR (DMSO-d 6}} ): δ12.73 (. Br 1H), 7.69 (s, 1H), 7.22 (d, J = 7.2 Hz, 4H), 6.38 (t, J = 2.5 Hz, 1H), 6.23 (d, 2.5 Hz, 2H), and 3.33 (s, 6H).

Synthesis of 2- (4-acetylamino-phenyl) -3- (3,5-dimethoxy-phenyl) -acrylic acid

Acetic anhydride (5 mL) and triethylamine (3.4 mL, 24 mmol) were added to a mixture of 3,5-dimethoxybenzaldehyde (2.5 g, 15 mmol) and p- aminophenylacetic acid (2.28 g, . The mixture was stirred at 140 < 0 > C for 2 hours. The reaction mixture was cooled to room temperature and chloroform (50 mL) was added. The chloroform solution was further cooled to 10 < 0 > C and acidified with concentrated HCl (10 ml). The aqueous layer was discarded and the organic layer was extracted with aqueous sodium hydroxide (2M, 3 x 50 mL). The aqueous layer was pooled and acidified to pH 1 with concentrated HCl (40 mL). The resulting precipitate was filtered off, washed with water (2 x 30 mL) and recrystallized from a water-ethanol mixture.

_{^{1 H NMR (DMSO-d 6}} ): δ12.70 (. Br 1H), 10.04 (s, 1H), 7.63 (s, 1H), 7.54 (d, J = 7.5 Hz, 2H), 7.08 (d, J = 7.5 Hz, 2H), 6.36 (t, J = 2.4 Hz, IH), 6.25 (d, J = 2.4 Hz, 2H), 3.56 (s, 6H) and 2.04 (s, 3H).

Synthesis of 3- (3,4-dimethoxy-phenyl) -2- (4-hydroxy-phenyl) -propionic acid

Acrylic acid was dissolved in ethanol (100 ml) and palladium-charcoal (10%, 50% wet, 0.3 g) was added to a solution of 3- (3,4- dimethoxy- phenyl) -2- . The mixture was stirred at room temperature under hydrogen overnight. The reaction mixture was filtered through Celite Filtered through a layer of diatomaceous earth and the solvent was evaporated.

_{^{1 H NMR (DMSO-d 6}} ): δ12.15 (. Br 1H), 9.22 (br, 1H), 7.10 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), (Dd, J = 13.3 and 8.6 Hz, 2H), and 2.80 (dd, J = 2.2 Hz, 2H) J = 13.3 and 8.6 Hz, 2H).

(Example 3)

Diabetes was induced in Sprague-Doll (SD) male rats (average body weight, 180 g) by IV injection of 60 mg of streptozotocin. After 5 days, the average glucose concentration was about 350-400 mg / dl. The rats were then divided into 5 groups (n = 7) and given orally phosphate buffered saline (PBS) (vehicle) or Compound Ia (10, 20, 40 or 80 mg / kg body weight) daily for 8 days. Treatment with Ia at doses of 20, 40, or 80 mg / kg resulted in a reduction in blood glucose levels in these rats from day 2 to day 6 (as compared to vehicle-treated control). This decrease was statistically significant (p <0.05) from day 4 to day 6 in rats given 80 mg / kg. The results are shown in Fig.

(Example 4)

Obesity (ob / ob) mice developed spontaneously in diabetes, and glucose concentrations ranged from 200 to 300 mg / dl. In this experiment, Ia was daily administered to ob / ob mice at doses of 0 (vehicle only), 10, 20, 40, or 80 mg / kg body weight for 4 days. On day 4, blood glucose levels in the animals that gave Ia were lower than in animals that gave only vehicle; The difference was statistically significant for the groups given 20 and 40 mg / kg. The results are shown in Fig.

(Example 5)

Serum concentrations of insulin, triglyceride and free fatty acid (FFA) in ob / ob mice were measured at day 8 after oral treatment with daily doses of 20 mg / kg body weight for 7 days. Serum insulin concentrations were 42% lower in Ia treated animals than in vehicle-treated animals (A). Serum glyceride levels were 24% lower in Ia-treated mice than in vehicle-treated mice (B). Serum FFA concentrations were not significantly reduced (C). The results are shown in Figs. 3A, 3B and 3C.

(Example 6)

The ability of Ia to decrease glucose concentration was further investigated in db / db mice. 8-week old db / db mice with an average blood glucose concentration of 280-300 mg / dl were treated with vehicle or Ia (20 mg / kg single dose daily for 20 days; 20 mg / kg twice daily for 8 days; 50 mg / kg twice). Blood glucose levels in mice that received Ia were 20% lower than in mice that received vehicle at the end of the first 20 days of treatment. Treatment with higher doses of Ia did not improve the glucoside reducing effect. The results are shown in Fig.

(Example 7)

Figures 5a, 5b and 5c show serum insulin, triglyceride and free fatty acid (FFA) concentrations found in db / db mice treated with Ia in the experiment shown in Figure 4 (analysis was done at the end of the experiment). (A), the concentrations of triglyceride (B) and FFA (C) were significantly lower in mice treated with Ia than in mice treated with vehicle, and the concentrations of triglyceride Was 32% lower than in vehicle-treated mice, and the free fatty acid concentration was 28% lower than vehicle-treated mice.

(Example 8)

Compound Ia was orally or intraperitoneally (IP) administered at a dose of 20 mg / kg to db / db mice for 22 days. After 9 days, the glucose-reducing effect of IP-administered Ia disappeared, but the glucosecure-reducing effect of orally administered Ia was maintained. The difference between the mice receiving oral Ia and those receiving IP Ia was statistically significant at 13 and 15 days (p <0.05). The results are shown in Fig.

(Example 9)

The effect of Ia was studied in female faux (fa / fa) rats (considered as a superior natural developmental genetic model for insulin-resistant diabetes mellitus). Female fa / fa rats were given vehicle or Ia (20 mg / kg) daily for 58 days. (A) From 10th to the end of the experiment, the blood glucose level of the rats submitting Ia was lower than that of the vehicle-fed rats, and the difference was statistically significant from 9th to 34th day. (B) In all experiments, the body weights of rats in both treatment groups were substantially the same. The results are shown in Figs. 7A and 7B.

(Example 10)

Zucker fa / fa rats were given vehicle or Ia (20 mg / kg) daily for 58 days; 3 days, 14 days, 30 days and 44 days, glucose tolerance test (glucoside, 2 mg / kg in water) was administered. As a result of this test, the difference between the treatment groups was statistically significant (C) at 30 and 180 minutes (B) at 30 days and at 30 and 60 minutes after 30 days of immunosurvement at 14 days (p < 0.05). On day 44, the effect of Ia on glucose tolerance disappeared. The results are shown in Figs. 8A to 8D.

(Example 11)

(A) The zuga fa / fa rats treated with Ia (20 mg / kg) for 58 days (see FIG. 7) showed a 70-78% decrease in serum insulin concentration compared to vehicle-fed rats (P <0.05). This suggests that the mechanism by which Ia affects diabetes includes insulin-sensitization.

(B) Furthermore, serum leptin levels in Ia-treated rats were 45% higher than in vehicle-treated rats. The results are shown in Figs. 9A and 9B.

(Example 12)

Serum concentrations of triglyceride, free fatty acids, and cholesterol were measured in jugular fa / fa rats that gave vehicle or Ia (20 mg / kg) daily for 58 days (see Figures 7 and 9). At the end of the study, triglyceride, free fatty acid, and cholesterol concentrations found in rats subjected to Ia were reduced by 70%, 89%, and 68%, respectively, compared to rats that received vehicle. The results are shown in Fig.

(Example 13)

When the tests described in connection with Figures 7-10 were performed using male obesity zooker fa / fa rats treated with vehicle or Ia (20 mg / kg) daily for 65 days, the Ia-treated animal's glucose concentration, Tolerance and leptin levels were not different from vehicle treated animals. However, the insulin, triglyceride, free fatty acid, and cholesterol concentrations identified in Ia-treated rats at the end of the experiment were all lower than those found in vehicle-treated animals. The results are shown in Figs. 11A to 11D.

(Example 14)

Compound Ia does not reduce blood glucose levels in normal animals. This was confirmed in two studies using rats and dogs. The daily dose of Ia for 28 days, or even at very high doses (1000 mg / kg), did not result in the hypoglycemic activity of this compound. The results are shown in Figs. 12A and 12B.

(Example 15)

Glucose uptake in normal adipose cells freshly prepared from epidermal fat pads of (A) in the presence of indicated concentrations of Ia, and of epidermal fat pads of (B) Sprague Dawley (SD) rats (170 g) in differentiated 3T3-L1 adipocytes Respectively. Insulin was used as a positive control in both experiments. In both cases, Ia stimulated glucose uptake in a manner similar to insulin. The results are shown in Figs. 13A and 13B.

(Example 16)

Differentiated 3T3-L1 adipocytes were treated with Ia, insulin or vehicle alone to determine if Ia treatment affected the expression of GLUT-1 and GLUT-4 in the glucocorticoids. Cells were lysed, 4-20% gradient SDS-PAGE, electroblotted and probed with monoclonal antibodies to anti-GLUT-1 or anti-GLUT-4. As shown in the figure, GLUT-1 (A) and GLUT-4 (B) were up-regulated in 3T3-L1 cells after exposure of cells to Ia. The results are shown in 14a to 14d.

(Example 17)

Differentiated 3T3-L1 adipocytes were treated with either 10 μM vehicle (medium alone) or Ia for 30 min at 37 ° C after stopping the serum supply for 3 h. Cells were washed with phosphate buffered saline (PBS), fixed with methanol for 20 minutes at -20 째 C, rinsed three times with PBS, and incubated with PBS containing 10% bovine serum at 37 째 C for 30 minutes. Slides were incubated with anti-GLUT-4 polyclonal antibody (1:50 dilution) in 10% bovine serum for 2 hours at 37 ° C. After this incubation, the slides were rinsed three times with PBS and incubated with secondary antibody conjugated to Alexa-Fluor (EX _max 495 nm; Em _max 519 nm) for 30 minutes. Finally, the slides were rinsed with PBS and increased with continuous anti-fade growth media. The photographs taken using a Nikon confocal PCM 2000 microscope coupled to an image analyzer showed high fluorescence in Ia-treated cells. Fluorescence staining appears in the cell membrane, indicating that treatment with Ia promotes transposition of the GLUT-4 glucocorticoid carrier on the cell surface.

(Example 18)

Nine healthy male Swiss Webster mice were divided into three study groups of three mice. The first study group (Figure 15a) received 167 mg / kg / BW of compound Ia, the second study group 15b received 167 mg / kg / BW and the third study group 15c 333 mg / kg / BW. During the entire study period, mice were given certain nutrients and water. During the study, mice were closely monitored and their characteristics, total physiological function and mortality / survival were investigated. Figures 15a, 15b and 15c show that the survival rate of these mice is 100% in the course of the study.

(Example 19)

Wortmannin is a known inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme required for the insulin-signaling pathway. In this experiment, the ability of wortmannin to inhibit Ia-stimulated glucose uptake was measured. Freshly prepared adipocytes were cultured in the presence or absence of 4 [mu] M wortmannin at various concentrations of insulin or Ia. The ability of adipocytes to absorb glucose was then examined using a ¹⁴ C-deoxyglucose tracer. As shown here, treatment of adipocytes with wortmannin strongly inhibits insulin or Ia-dependent glucose uptake. This result suggests that Ia affects the PI 3-kinase pathway. The results are shown in Fig.

(Example 20)

Chinese hamster ovary cells (CHO.IR cells) overexpressing human insulin receptor were grown in F12 ham medium with 10% fetal bovine serum (FBS) at 37 ° C in 5% CO ₂ . Cells were sacrificed for 6 hours and then incubated with vehicle, insulin (10 nM) or Ia (12.5, 25 or 50 μM) for 30 minutes at 37 ° C. The cells were then washed with cold phosphate buffered saline (PBS) and electrophoresis (4-20% SDS-PAGE) to separate 13 μg total cell lysate, blunt over the nitrocellulose membrane, Phosphorylation was detected with a monoclonal antibody against tyrosine (Transduction Laboratories, clone PY20). Western blotting was carried out using an enhanced chemical luminescence detection system, the results were scanned and quantified and expressed in units of rams. As a result, it can be seen that Ia phosphorylates the insulin receptor or insulin receptor substrate 1 (like insulin) in a dose-dependent manner. The results are shown in Fig.

(Example 21)

Chinese hamster ovary cells (CHO.IGF-1R cells) overexpressing human insulin-like growth factor 1 receptor were grown in F12 ham medium with 10% FBS at 37 ° C in 5% CO ₂ . Cells were terminated in serum for 6 hours and then incubated with vehicle, IGF-1 (100 nM) or Ia (12.5, 25 or 50 μM) for 30 minutes at 37 ° C. The cells were then washed with cold phosphate buffered saline (PBS) and electrophoresed (4-20% SDS-PAGE) to isolate 21 μg total cell lysate and blotted onto the nitrocellulose membrane; Phosphorylation was detected with a monoclonal antibody to phosphotyrosine (Transduction Laboratories, clone PY20). As a result, it can be seen that Ia does not phosphorylate insulin-like growth factor 1 receptor or insulin receptor substrate 1 in CHO.IGF-1R cells. The results are shown in Fig.

(Example 22)

Chinese hamster ovary cells (CHO.IR cells) overexpressing human insulin receptor were grown in F12 ham medium with 10% fetal bovine serum (FBS) at 37 ° C in 5% CO ₂ . Cells were sacrificed for 6 hours and incubated at 37 ° C for 30 minutes with vehicle, insulin (10 nM), tolbutamide (50 μM), or with either Ia (12.5, 25 or 50 μM) in three different doses Respectively. The cells were then washed with cold phosphate buffered saline (PBS) and electrophoresed (4-20% SDS-PAGE) to separate 25 μg total cell lysate, blotted onto the membrane, Was detected with phospho-Akt (Ser 473) (New England Biolabs). Western blotting was carried out using an enhanced chemical luminescence detection system, the results were scanned and quantified and expressed in units of rams. As a result, it can be seen that the phosphorylation of Akt increased in a dose-dependent manner in the presence of Ia. The results are shown in Fig.

(Example 23)

(10 nM), tolbutamide (50 μM) or one of three different doses of Ia (12.5 μM) for 30 min at 37 ° C after stopping the serum supply to Chinese hamster ovary (CHO) 25 or 50 [mu] M). Both groups were preincubated with 100 nM wortmannin (lanes 7 and 8), at which time insulin (10 nM) and Ia (50 μM) were added. The cells were then washed with cold phosphate buffered saline (PBS) and 20 μg total cell lysate was separated by electrophoresis (4-20% SDS-PAGE), blotted onto the membrane, and Akt phosphorylation was detected by antibody [ Anti-phospho-Akt (Ser 473), New England Biolabs]. As a result, it can be seen that wortmannin inhibited Akt-phosphorylation stimulated by insulin or by Ia. The results are shown in Fig.

(Example 24)

All thiazolidinedione compounds are known to stimulate glucose uptake through a mechanism involving increased expression by binding to a nuclear receptor transcription factor known as PPAR-y (peroxisome proliferator activated receptor-gamma). 3T3-L1 adipocytes were incubated with vehicle, Ia (5 [mu] M), or troglitazone (5 [mu] M) for 40 hours to confirm if Ia upregulated glucose uptake by a mechanism involving PPAR-y. PPAR-y expression was evaluated by immunoblotting. Western blotting was carried out using an enhanced chemical luminescence detection system, the results were scanned and quantified and expressed in units of rams. The results shown in the figure show that troglitazone induces an increase in PPAR-y while Ia did not induce an increase in PPAR-y beyond the basal level of this transcription factor. The results are shown in Fig.

(Example 25)

Differentiation of fibroblasts into adipocytes involves the expression of PPAR-y. All members of the thiazolidinediones of antidiabetic compounds stimulate PPAR-y expression and promote the differentiation of fibroblasts into adipocytes. Similarly, insulin stimulates the differentiation of fibroblasts into adipocytes. To investigate the effect of Ia on this differentiation process, 3T3-L1 fibroblasts were incubated with either Ia (1 μM), insulin (0.17 mM), or a combination of both. After incubation, the cells were lysed and the amount of expressed PPAR-y was analyzed by ECL blot analysis using anti-PPAR-y antibody. Treatment of fibroblasts with Ia did not enhance the differentiation process. In the positive control, treatment of fibroblasts with insulin stimulated the differentiation of these cells into adipocytes with increasing levels of PPAR-γ.

(Example 26)

Figure 22 shows the results of three tests performed to determine whether Ia is an agonist of nuclear PPAR receptors. The ability of Ia to bind to human recombinant PPAR-alpha, PPAR-gamma or PPAR-delta was demonstrated using radioligand-binding assays measuring the substitution of set radioconjugated ligands. In this assay, IC values for all three nuclear receptors were> 50 μM. Ligand-induced structural changes in PPARs are known to promote binding of the coactivator molecules. Cofactor binding was measured by time-resolved fluorescence (HTRF) using energy transfer between two adjacent molecules to determine the ability of Ia to promote binding of PPARs to the cofactor protein. Ia did not induce any binding of the cofactor. Finally, we examined the effect of Ia on PPARs in biological systems by performing cell-cycle transcriptional activation assay. In this experiment, COS cells with chimeric receptors were treated with Ia and transcription activity was measured by increased luciferase activity. Activation of PPARs by Ia was not observed. All these results confirm that Ia is not an agent for these PPARs.

(Example 27)

The differentiated 3T3-L1 cells (in the triple well) were treated with either Ia or cold insulin for 1 hour at 37 [deg.] C at the indicated concentrations. After incubation, excess compounds were washed out and the cells were incubated with a fixed amount of ¹²⁵ I-insulin (10 pM; 2000 Ci / mmol) at 4 ° C for 12 hours. The cells were washed and then lysed with 0.1% SDS and counted on a scintillation counter. As expected, increasing the dose of cold insulin inhibited the binding of radioactive insulin, whereas pre-incubation with Ia resulted in 45% inhibition. The results are shown in Fig.

(Example 28)

Real-time direct binding of Ia to the insulin receptor was confirmed using Biocore 3000 (measuring surface plasmon resonance). The intensity and wavelength of the light reflected from the metal surface with the thin film solution thereon is influenced by the mass concentration of the components at the liquid-surface interface. The interaction of molecules in the liquid changes the intensity of the reflected light at a certain angle. In this experiment, a purified insulin receptor containing both alpha and beta subunits was immobilized on the sensor surface of a flow cell 2 of a Biocore 3000 with a gold membrane; Flow cell 1 was used as a control for background. When Ia was injected at a concentration of 200 μM, 100 μM and 10 μM, the response label of the binding to the insulin receptor appeared within a few seconds, similar to the binding curve obtained using insulin.

(Example 29)

Glucose uptake was measured in normal adipose cells freshly prepared from epidermal fat pad of SD rats in the presence of the E or Z-isomer of Ia or Ib. The cells were preincubated with the indicated concentrations of isomer for 30 minutes, then ¹⁴ C-deoxyglucose was added and the sample was further incubated for 5 minutes. Figure 24a shows that the extent of glucose uptake stimulated by both isomers is similar. Figure 24b shows that the stimulation effect of Z-form (Ib) is greater than that of insulin, as shown for Form E of (Ia) (see Figure 16) . &Lt; / RTI &gt;

(Example 30)

We have developed a sensitive method for detecting Ia in Sprague Dawley (SD) rat serum, where Ia can be detected at levels of 10-25 ng. The dynamics of drug absorption and elimination from circulation were studied in a rat model. SD rats were given an oral dose of Ia (20 mg / kg). At different time intervals, blood was collected and serum was assayed for Ia. As shown in the figure, Ia was maximally absorbed at 1 hour after oral delivery of the drug and was removed from the circulation within 24 hours. The results are shown in Figs. 25A and 25B.

(Example 31)

A variety of toxicity studies were performed, and their status and results are summarized in Fig. No serious toxicological problems were found when administered at high doses of 1000 mg / kg.

Claims (23)

The compounds of formula (I) (I) (Wherein, The dashed bond may be a selective double bond, the geometry for the bond may be E or Z, A is -COOR, -CONR'R &quot;, -CN or -COR ₇ , R, R ', R "and R ₇ are defined as follows; X is H, OH, or a C ₁ -C ₁₀ straight chain or branched chain alkyl or alkenyl group which may be optionally substituted with COOR, carbonyl or halo; R is H or C ₁ -C ₂₀ linear or branched alkyl or aryl or aralkyl, or a pharmaceutically acceptable counterion; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are independently H; A C ₁ -C ₂₀ straight chain or branched chain alkyl or alkenyl group which may be optionally substituted; COOR (R is as defined above); NR'R "or CONR'R" (R 'and R "are independently H or C ₁ -C ₂₀ straight or branched chain alkyl, or aryl); OH; C ₁ -C ₂₀ alkoxy; C ₁ -C ₂₀ acyl Amino, C ₁ -C ₂₀ acyloxy, C ₁ -C ₂₀ alkanoyl, C ₁ -C ₂₀ alkoxycarbonyl, halo, NO _2, SO ₂ R '"; CZ _{3 wherein} each Z is independently a halo atom, H, alkyl, chloro, or fluoro substituted alkyl; Or SR '" wherein R''is H or linear or branched C ₁ -C ₂₀ alkyl; Or R ₂ and R ₃ together, or R ₅ and R ₆ may combine together to form a methylenedioxy or ethylenedioxy group; X, R ₃ , R ₅ and R ₆ are H; R &lt; ₄ &gt; is p-hydroxy; R ₁ and R ₂ is a 3,5-dimethoxy-together, and the dotted line is not a double bond of E- form) The method according to claim 1, A is -COOR. Compounds of formula II: &Lt; RTI ID = 0.0 & (Wherein, The bond indicated by the dashed line may be a selective double bond, the geometry for the bond may be E or Z, the naphthyl group may be bonded at the? Or? A is -COOR, -CONR'R &quot;, -CN or -COR ₇ , R, R ', R "and R ₇ are defined as follows; X is H, OH, or a C ₁ -C ₁₀ straight chain or branched chain alkyl or alkenyl group which may be substituted by COOR, carbonyl or halo; R is H or C ₁ -C ₂₀ linear or branched alkyl or aryl or aralkyl, or a pharmaceutically acceptable counterion; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are independently H; A C ₁ -C ₂₀ straight chain or branched chain alkyl or alkenyl group which may be optionally substituted; COOR (R is as defined above); R; NR'R "or CONR'R" (R 'and R "are independently H or C ₁ -C ₂₀ straight or branched chain alkyl, or aryl); OH; C ₁ -C ₂₀ alkoxy; C ₁ -C ₂₀ acyl Amino, C ₁ -C ₂₀ acyloxy, C ₁ -C ₂₀ alkanoyl, C ₁ -C ₂₀ alkoxycarbonyl, halo, NO ₂ , SO ₂ R '"; CZ _{3 wherein} each Z is independently a halo atom, H, alkyl, chloro, or fluoro substituted alkyl; Or SR ''', wherein R''is H or a linear or branched C ₁ -C ₂₀ alkyl, or R ₂ and R ₃ together or R ₅ and R ₆ together form a methylenedioxy or ethylenedioxy group Lt; / RTI &gt; The method according to claim 1, A is -COOR; X, R ₃ , R ₅ and R ₆ are H; R ₄ is p-hydroxy, R ₁ , R ₂ together are 3,5-dimethoxy; And the dotted line is a Z-type double bond. 5. The method of claim 4, Lt; RTI ID = 0.0 &gt; R &lt; / RTI &gt; 5. The method of claim 4, And R is Na +. 3. The method of claim 2, R &lt; ₄ &gt; is p-hydroxy; R ₁ and R ₂ together are 3,5-dimethoxy; And the dotted line represents a double bond. The method of claim 3, R ₁ and R ₂ together are 3,5-dimethoxy and the dotted line represents a double bond. A pharmaceutical composition for the treatment of diabetes comprising a therapeutically effective amount of a compound according to any one of claims 1 to 8 or a mixture thereof in a pharmaceutically acceptable carrier. 10. The method of claim 9, Wherein said composition is suitable for oral administration. 8. A method for treating diabetes comprising administering to a diabetic patient a therapeutically effective amount of a compound according to any one of claims 1 to 8 or a mixture thereof in a pharmaceutically acceptable carrier. 12. The method of claim 11, Wherein said compound is administered orally to said patient. Comprising a therapeutically effective amount of a compound according to any one of claims 1 to 8 in a physiologically acceptable carrier, The dashed bond may be a selective double bond, the geometry for the bond may be E or Z; R is H, linear or branched C ₁ -C ₂₀ alkyl, aryl or aralkyl, or a pharmaceutically acceptable counterion. 14. The method of claim 13, Wherein R is H or Na &lt; + &gt;, wherein said double bond is E-form. 14. The method of claim 13, R is H or Na &lt; + &gt;, and the double bond is in the Z-form. 16. The method of claim 15, And R is Na +. 15. The method of claim 14, And R is Na +. 14. The method of claim 13, Wherein said composition is suitable for oral administration. Comprising administering to a diabetic patient a therapeutically effective amount of a compound according to any one of claims 1 to 8 in a physiologically acceptable carrier, The dashed bond may be a selective double bond, the geometry for the bond may be E or Z; R is H, linear or branched C ₁ -C ₂₀ alkyl or aryl, or a pharmaceutically acceptable counterion. 20. The method of claim 19, R is H or Na &lt; + &gt;, and the double bond is E-form. 20. The method of claim 19, R is H or Na &lt; + &gt;, and the double bond is in the Z-form. 21. The method of claim 20, And R is Na +. 22. The method of claim 21, And R is Na +.