WO2011029359A1

WO2011029359A1 - MEDICAMENTS CONTAINING 11β HYDROXYSTEROID DEHYDROGENASE 1 DOUBLE REGULATORS AND USES THEREOF

Info

Publication number: WO2011029359A1
Application number: PCT/CN2010/075829
Authority: WO
Inventors: 李校堃; 梁广; 葛仁山; 王怡; 胡国新
Original assignee: 温州医学院
Priority date: 2009-09-12
Filing date: 2010-08-10
Publication date: 2011-03-17

Abstract

The present invention discloses compounds which can lower the activity of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) reductase and/or improve the activity of 11β-HSD1 oxidase, as well as the uses of said compounds in manufacturing the medicaments for preventing or treating glucose metabolism, tumor, sexual gland degenerative disorder and/or infertility. The pharmaceutical compositions and kits containing said compounds, and the method for screening said compounds are also disclosed.

Description

Medicine containing lip-hydroxyl retention dehydrogenase 1 dual regulator and application thereof

Technical field

The invention belongs to the technical field of medicine, in particular, the invention relates to the pharmaceutical application of a dual modulator of 11β-hydroxyl dehydrogenase 1, in particular for preventing and/or treating diseases of glycolipid metabolism, tumor and hypogonadism. Obstacles and/or infertility, etc. Further, the present invention relates to a pharmaceutical composition comprising a dual modulator of ιΐβ-hydroxydehydrogenase 1, a kit, and the like.

Background technique

Glucocorticoid (herein referred to as GC) is one of the most important hormones that induce insulin resistance. GC mainly reduces insulin-mediated glucose uptake, promotes the breakdown of fat and protein, increases hepatic gluconeogenesis, and inhibits insulin secretion by pancreatic beta cells. GC can directly activate gene expression related to hepatic gluconeogenesis, and promote glucagon release and indirectly increase hepatic glucose output; GC can synergize with CRE binding protein to induce PPAR _Y coactivator and reduce insulin secretion; GC can also interfere with insulin signaling, reducing GLUT4 translocation to the membrane, leading to insulin resistance. Therefore, GC is functionally antagonistic to insulin, and many diseases in clinic also show a relationship between GC and insulin.

GC metabolic abnormalities lead to a wide range of metabolic diseases, including pathological conditions caused by abnormalities in various metabolic components, such as abdominal obesity or overweight, atherosclerotic dyslipidemia (high triglycerides (abbreviated as TG) Hypertension and high-density lipoprotein cholesterol (herein referred to as HDL-C) are low), hypertension, insulin resistance, and impaired glucose tolerance. Common metabolic diseases include diabetes, obesity, atherosclerosis and hypertension, among which diabetes is one of the most important, widespread and serious metabolic diseases. Now, there are about 120 million people with diabetes in the world. According to WHO estimates, by 2020, the global number of diabetic patients will be close to 300 million, which has become the third most serious disease that endangers human health after cardiovascular and cerebrovascular diseases. Epidemiological and clinical studies have confirmed that type 2 diabetes is one of the metabolic diseases caused by insulin resistance.

In addition, there are approximately 500 million people with significant sexual decline in the world. According to a survey conducted by the Chinese Society of Sexual Medicine Professionals on the status of male sex in China, 9.7% of men over the age of 40 have experienced a significant decline in sexual function. The current high-efficiency, fast-paced work style and severe competition have put tremendous psychological and physical pressure on many people. According to a recent large-scale sample survey conducted by the Beijing Communist Youth League, about 60% of people are under stress; these all affect their normal sexual function. Stress stress induces an increase in glucocorticoid levels (Monder C 1994 Endocrinology 134: 1193-1198). Stress stress can induce a decrease in serum testosterone levels, leading to degeneration of testicular function, decreased sexual function, and reduced fertility (Hardy MP et al 2005. Cell Tissue Res. 322: 147-53). Reduced testosterone secretion not only causes sex Dysfunction and causes a wide range of related systemic disorders such as psychological disorders, muscle wasting, obesity and hypertension. In humans, severe psychological stress, death of relatives or spouses, etc., can result in a decrease in sperm count (Fenster L 1997, Journal of Andrology, 18: 194). Stress stress has been clinically considered to be one of the factors contributing to infertility, and reducing stress stress has become a preparation for child development (Hjolhmd NH 2004 Epidemiology 15:21). Physiologically, the stress response maintains the balance of the internal environment through the body's compensatory regulation of stressors. Excessive glucocorticoid activity is a hallmark of stress. In patients with Cushing's syndrome, glucocorticoids increase and plasma testosterone levels decrease. In males, luteinizing hormone is a major driver of testosterone production by testicular stromal cells, whereas glucocorticoids produced by adrenocortical cells are inhibitors. Glucocorticoids also inhibit the secretion of luteinizing hormone. Glucocorticoids act by binding to the glucocorticoid receptor. Leydig cells have a glucocorticoid receptor, suggesting that the glucocorticoid secreted by the adrenal gland can directly affect these cells. However, there is no direct evidence that reducing the amount of active glucocorticoids in the body can treat diseases such as sexual dysfunction and/or infertility.

11β-hydroxysteroid dehydrogenase (abbreviated as Ιΐβ-HSD or 11PHSD) is a key enzyme that catalyzes the synthesis and transformation of glucocorticoids, directly controlling the endocrine hormone-glucocorticoid, which closely affects blood glucose levels and insulin function. Level and activity. Ι ΐβ-HSD includes two enzymes: ΙΙβ-HSD1 and llp-HSD2. In contrast, the main role of ΙΙβ-HSD1 is to promote the conversion of inactive glucocorticoids (herein referred to as GC) to active GC, while 11P-HSD2 only promotes the conversion of active GC to inactive GC. Among them, ΙΙβ-HSD1 is an oxidoreductase, which has two enzyme forms, reductase and oxidase, wherein reductase promotes the conversion of inactive GC to active GC, while oxidase acts in the opposite direction, promoting active GC to inactive GC. Conversion. Since Ιβ-HSD1 reductase is dominant, and its oxidase is in a secondary position, it is apparent that Ιβ-HSD1 usually exhibits reductase activity. According to the type and function of Ιΐβ-HSD, many large pharmaceutical companies in the world have started research on Ιβ-HSD1 inhibitors, such as BVT7702, 138768 and NCB13793. Among them, Incyte's 1 Ιβ-HSD1 selective inhibitor NCB 13793 is undergoing Phase II clinical research.

However, unfortunately, the currently reported ΙΙβ-HSD1 inhibitors simultaneously (mixed) inhibit ΙΙβ-HSD1 oxidase and reductase, some of which do not affect 11P-HSD2, but at the same time The activity of Ιβ-HSD1 oxidase is inhibited. ΙΙβ-HSD1 oxidase has the same effect as l ip-HSD2, which can reduce glucocorticoid concentration, increase insulin expression, reduce glycogen output and blood glucose levels, enhance insulin sensitivity, and resist the development of diabetes.

According to the present inventors, although in the mixed inhibition of ΙΙβ-HSD1 by the ΙΙβ-HSD1 inhibitor, the effect of inhibiting oxidase is less than the effect of inhibiting reductase, apparently ΙΙβ-HSDl The inhibition of reductase thus acts to reduce the activity of GC, but this will inevitably offset the partial efficacy of the ΙΙβ-HSD1 reductase inhibitor. Similarly, if the activity of 11P-HSD2 is inhibited, the ΙΙβ-HSD1 reductase inhibitor will also be offset. Partial efficacy; more serious Inhibition of ΙΙβ-HSD1 reductase can only prevent and prevent the formation of hydrocortisone, prevent blood glucose levels from rising, but does not reduce the already elevated glucocorticoid concentration. Elevated glucocorticoid levels in local tissues will still maintain stimuli glycogen output and insulin resistance.

For this reason, the inventors have worked hard, from the curcumin analog (see Chinese Patent Application CN101003470A), and accidentally discovered that certain compounds have a dual regulatory effect on Ιβ-HSD1 (i.e., inhibit ΙΙβ-HSD1 reductase and activate Ι Ιβ-HSD1 oxidase), this dual regulation will be more important than the current selective inhibitors in the treatment of metabolic diseases such as type 2 diabetes, so the choice of Ιβ-HSDl dual modulators for drug applications, A specific ΙΙβ-HSD1 dual modulator which does not inhibit the activity of llp-HSD2 in the Ιβ-HSD1 dual modulator is particularly preferred. This compound with dual regulation of Ιβ-HSD1, especially a compound with specific regulation of ΙΙβ-HSD1, will form an ideal anti-diabetic drug through the “ΙΙβ-HSD1-glucocorticoid→insulin→blood glucose” channel. . At present, there is no technology to directly predict the ΙΙβ-HSD1 dual regulator through the structure of the compound, and it is even more difficult to predict the specific ΙΙβ-HSD1 dual modulator, which can only rely on excessive experimental screening.

Moreover, it has been found by the inventors that, particularly surprisingly, in contrast to conventionally accepted dose-dependent therapeutic/prophylactic effects, the particularly preferred compound LG13 (BP, B6) is administered at low doses for metabolic diseases such as diabetes. / The effect of prevention is best, and the poor effect at medium and high doses is not directly caused by drug toxicity, which not only reduces the cost of medication, but also reduces the risk of side effects that may be caused by larger doses. In addition, the inventors have also discovered that these ΙΙβ-HSD1 dual modulators, especially the preferred dual 调节β-HSD1 modulators, will form an ideal pathway through the "ΙΙβ-HSD1-glucocorticoid→testosterone" channel. The drug, therefore, will have the effect of preventing and/or treating diseases such as hypogonadal dysfunction and/or infertility, and can be safely administered, meeting the increasing demands of current public and government regulatory authorities for drug safety.

Also, tumors are one of the leading causes of human death. Statistics show that malignant tumors have become the leading cause of death in urban and rural residents. It can be seen that the prevention and treatment of tumors is very urgent. Drug therapy is one of the main treatments for cancer. At present, although dozens of anti-tumor drugs have been developed, which effectively prolong the life of patients or improve the quality of life of patients, some anti-tumor drugs are very effective, such as drugs for the treatment of acute leukemia in children. However, drugs that still require new tumors face enormous challenges in their research and development. For example, anti-tumor drugs are mostly cytotoxic drugs, and their side effects are obvious, which limits the efficacy of these drugs. Although the prior art cannot directly predict the antitumor activity by the compound structure, the inventors have worked hard to select leukemia, oral epithelial cancer, non-small cell non-cancer and brain glue from the above-mentioned ΙΙβ-HSD1 dual modulator. A curative antitumor agent such as a blastoma, which inhibits tumor cell proliferation and is safe and reliable. Brief description of the invention The object of the present invention is to supplement the vacancies of the prior art, and to provide a dual modulator of Ιβ-HSD1, especially a specific ΙΙβ-HSD1 dual modulator, which can be used for pharmaceuticals, safely, effectively and even low-cost for prevention and/or prevention. Therapeutic effects of various diseases. Further, the present invention provides a pharmaceutical composition and a kit comprising the ΙΙβ-HSD1 dual modulator, and a method of screening the ΙΙβ-HSD1 dual modulator, and the like.

Specifically, in a first aspect, the present invention provides a method of using a compound of Formula I in the manufacture of a medicament for preventing and/or treating a disease, wherein the disease is capable of reducing 11β-hydroxydehydrogenase 1 Reductase activity and / or increase 11β-hydroxyl dehydrogenase 1 / or prevent disease,

among them,

Ru and R ₂₁ are each deuterium, halogen, optionally halogen-substituted lower alkyl, or lower alkoxy;

R ₁₂ and R ₂₂ are not alkenyloxy or halogen-substituted lower alkyl, preferably R ₁₂ and R ₂₂ are each H, halogen, or lower alkoxy;

R ₁₃ and R ₂₃ are not alkenyloxy, preferably R ₁₃ and R ₂₃ are each H, halogen, lower alkyl, hydroxy, or optionally hydrazine, fluorenyl-dimethylamino substituted lower alkoxy;

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} synthesized, wherein n is 1, 2, 3. Corresponding to pharmaceutical applications, the first aspect of the invention also provides prevention and/or Or a method of treating a disease, wherein the disease is a disease which can be treated and/or prevented by reducing the activity of 11β-hydroxyl dehydrogenase 1 reductase and/or increasing the activity of 11β-hydroxyl dehydrogenase 1 oxidase. The method comprises administering to a patient a drug comprising an effective amount of a compound of formula I

Formula I

among them,

Ru and R ₂₁ are each H, halogen, optionally halogen-substituted lower alkyl, or lower alkoxy;

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} synthesized, wherein n is 1, 2, 3, or 4.

Preferably, in the first aspect of the invention, the compound of formula I is a dual modulator of 11β-hydroxydehydrogenase 1, preferably a specific 11β-hydroxydehydrogenase 1 dual modulator.

Also preferably in the first aspect of the invention, the compound of formula I is

Formula A

among them,

Ri is oxime, halogen, or lower alkoxy;

R ₂ is an anthracene, a halogen, or a lower alkoxy group;

R ₃ is hydrazine, halogen, or hydrazine, Ν-dimethylamino substituted lower alkoxy;

R ₄ is Η,

Or,

Style Is 11, halogen, or lower alkoxy;

R ₂ is H or lower alkoxy;

R ₃ is H or a hydroxyl group;

R ₄ is H,

Or,

Formula C

among them,

Each is H, halogen, halogen-substituted lower alkyl, or lower alkoxy; 11 or lower alkoxy;

R ₃ is H or a hydroxyl group;

R ₄ is H,

Preferably, the compound of formula I is selected from any of the following compounds

More preferably, the compound of formula I is LG13

Β6 (or LG13) is preferably the first aspect of the invention, wherein the disease is due to an increase in activity of 11β-hydroxydehydrogenase 1 reductase and/or activity of 11 β-hydroxydehydrogenase 1 oxidase A disease caused by a decrease; it is also preferred that the disease is a glycolipid metabolic disease, hypogonadal dysfunction, and/or infertility, preferably selected from the group consisting of diabetes, obesity, atherosclerosis, cirrhosis, fatty liver, high Blood glucose, hyperlipidemia, hypertension, hypogonadal dysfunction and/or infertility, most preferably diabetes, fatty liver, hypogonadal dysfunction caused by excessive glucocorticoids and its induced infertility, and / or stress testosterone reduction, such as sputum type diabetes, stress, disease or aging-induced gonadotropin dysfunction caused by excessive glucocorticoids and its resulting infertility, and so on.

In the first aspect of the invention, it is particularly preferred that the compound of formula I is LG13 in an effective dose of from 1 to 10 mg/kg of the rat, such as 1-10 mg/kg of Wistar rat. This low dose is surprisingly superior to higher doses of diabetes treatment (hypoglycemic), with advantages in both efficacy and safety applications. Therefore, the first aspect of the invention is particularly preferably provided The use of LG13 for the preparation of a medicament for treating diabetes or lowering blood glucose, wherein the medicament comprises LG13 at an effective dose of 1-10 mg/kg rat; accordingly, the first aspect of the invention also provides for treating diabetes or lowering blood glucose Methods, the method comprising administering to a patient an effective amount of a compound of formula I, wherein said effective amount is from 1 to 10 mg/kg of rat.

Further, in a second aspect, the present invention also provides an application method or method similar to the first aspect of the present invention, wherein the only difference is that the disease is capable of apoptotic cells by activating CHOP and/or by activating caspase -3 and caspase-9 and apoptotic cells to treat or prevent diseases. It should be noted that the second aspect of the present invention is not treated and/or prevented by reducing the activity of the 11β-hydroxyl dehydrogenase 1 reductase and/or increasing the activity of the 11β-hydroxyl dehydrogenase 1 oxidase. The disease is contradictory.

Not all tumors and cancers can be treated by the above routes, for example, for human prostate cancer, the compound of the present invention has no inhibitory ability, and therefore, in the second aspect of the invention, the disease is preferably gastric cancer, myeloid leukemia, oral cavity. Epithelial cancer, lung cancer or sarcoma, more preferably non-small cell lung cancer or glioblastoma.

In the second aspect of the invention, it is particularly preferred that the compound of the formula I is B19 or Β63. These compounds have been shown to be safe and effective in treating tumors. Therefore, the second aspect of the present invention particularly preferably provides the use of B19 or Β63 in the preparation of a medicament for treating a tumor, preferably the tumor is non-small cell lung cancer or glioblastoma; accordingly, the second aspect of the invention is also A method of treating a tumor is provided, the method comprising administering to a patient an agent comprising an effective amount of B19 or Β63, preferably the tumor is non-small cell lung cancer or glioblastoma.

Additionally, the ruthenium 63 compound is preferred as a separate aspect.

In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of LG13 and a pharmaceutically acceptable carrier, wherein an effective dose is from 1 to 10 mg/kg of a rat, preferably from 1 to 10 mg/kg of a Wistar rat, such as 5 mg/kg Wistar rats.

Preferably, in the second aspect of the invention, the pharmaceutical composition is a pharmaceutical composition for treating diabetes or preventing stress-induced testosterone reduction.

In a fourth aspect, the present invention provides a kit for treating diabetes comprising LG13, and indicating a rat at 1 - 1 Omg/kg (preferably 1 - 1 Omg/kg Wistar rat, such as 5 mg/kg Wistar rat) The label for dosing. This low dose is surprisingly superior to higher doses of diabetes treatment (hypoglycemic), has advantages in both efficacy and safe use, and can be conveniently used to guide medication.

In a fifth aspect, the invention provides a method of screening a dual modulator of 11β-hydroxydehydrogenase 1 in vitro, comprising

(1) The half-inhibitory concentration (IC50) of the reductase activity of the test compound on the mesenchymal cells, the CHOP cells transfected with human 11PHSD1 and/or the 11β-hydroxydehydrogenase 1 of the microsomal protein was determined in vitro;

(2) The half effective concentration (EC50) of the oxidase activity of the test compound on stromal cells, CHOP cells transfected with human 11PHSD1 and/or 11β-hydroxydehydrogenase 1 of microsomal protein was determined in vitro;

(3) Select a compound with an IC50 of less than 30 μΜ and an EC50 of less than 30 μΜ as a 1β-hydroxydehydrogenase 1 double Heavy regulator.

Preferably, in the fifth aspect of the invention, the method further comprises measuring the half inhibitory concentration of the oxidase activity of the 11β-hydroxydehydrogenase 2 of the microsomal protein in vitro by the dual modulator of iotaβ-hydroxyl dehydrogenase 1 (IC50), then the compound having an IC50 greater than ΙΟΟμΜ was selected as a dual modulator of specific 11β-hydroxyl dehydrogenase 1.

Preferably, in the fifth aspect of the invention, the stromal cells are rat interstitial cells; and/or the microsomal proteins are rat testicular microsomes, human liver microsomes, rat kidney microsomes, and/or human kidneys Microsomes.

Preferably, in the fifth aspect of the invention, the compound to be tested is a compound represented by the formula

Formula II

among them,

Rii, R2i, Ri2, R22 R ₁₄ and R ₂₄ are each H, halogen, hydroxy, amino, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted alkenyloxy, wherein the substituent Including 3⁄4, hydroxy, amino, aryl and/or heterocyclic;

In a particular embodiment of the invention, the invention exemplifies the compounds listed in Example 1. DRAWINGS

Figure 1 is a schematic representation of the chemical synthesis process of the compounds of the invention.

Figure 2 Schematic diagram of the specific dual regulation of the Ιβ-HSD1 mechanism.

As shown in the upper panel of Figure A, the currently reported specific inhibitors of ΙΙβ-HSD1 only refer to the primary selectivity between ΙΙβ-HSD1 and 11P-HSD2, and have not been involved in ΙΙβ-HSD1 reductase activity and oxidation. Enzyme activity is the selectivity between the two catalytic directions. These inhibitors simultaneously inhibit ΙΙβ-HSD1 reductase and oxidase, but because reductase is dominant, it can also be expressed as inhibition of reductase, reducing the body and local cortisol concentration. There have been no reports of screening tests for Ιβ-HSD1 reductase and oxidase, and ΙΙβ-HSD1 inhibitors with secondary selectivity. Although the inhibitory effects of current inhibitors of oxidase are less than the effects of inhibition of reductase, the defects are: (1) will certainly offset some of the efficacy of the inhibitor; (2) two-way inhibition Ι Ιβ-HSDl can only Prevent the conversion of cortisone to cortisol, However, it is not possible to reduce the elevated cortisol concentration. The elevated cortisol levels in the tissue will still maintain stimulating glycogen output and insulin resistance; the existing cortisol concentration needs to be stimulated by Ιβ-HSD1 oxidase. It is converted to inactive cortisone.

The lower panel of Figure A shows the ideal Ιβ-HSD1 dual modulator (the compound of the present invention is a Ιβ-HSD1 dual modulator), which inhibits Ιβ-HSD1 reductase and simultaneously activates Ιβ-HSD1 oxidase. Significantly reduce the activity of glucocorticoids, and finally achieve better anti-diabetes effect, and form an ideal anti-diabetic effect through the "Ιβ-HSD1-glucocorticoid→insulin→blood sugar" channel.

As shown in the upper panel of Figure B, the currently reported selective inhibitors of Ιβ-HSD1 only refer to the first-order selectivity between Ιβ-HSD1 and 11P-HSD2, and have not yet been related to Ιβ-HSD1 reductase. The selectivity between the two catalytic directions of activity and oxidase activity. This type of inhibitor inhibits both Ιβ-HSD1 reductase and oxidase, but since reductase is dominant, it is apparently inhibited by reductase and reduces local cortisol concentration, but may not affect stress or ke Syndrome causes systemic cortisol levels. There have been no reports of l lb-HSDl reductase and oxidase screening tests and secondary-selective Ιβ-HSD1 inhibitors. Although the inhibitory effect of current inhibitors of oxidase is less than the effect of inhibiting reductase, the defects are: (1) will certainly offset some of the efficacy of the inhibitor; (2) two-way inhibition Ι -β-HSDl can only Prevents the conversion of cortisone to cortisol, but does not reduce the already elevated systemic cortisol concentration. The elevated cortisol levels in the tissue will still maintain the inhibition of testosterone synthesis; the existing cortisol concentration needs to be stimulated Ι -β-HSD1 oxidase converts it to inactive cortisone, especially due to excessive circulating glucocorticoid levels, such as stress and Cushing's syndrome.

B The figure below shows an ideal dual regulator of Ιβ-HSD1 (the compound of the present invention is a l lb-HSD1 dual modulator) which inhibits Ιβ-HSD1 reductase and simultaneously activates Ιβ-HSD1 oxidase. Significantly reduce glucocorticoid activity, and ultimately play a better role in preventing or treating hypogonadal dysfunction and/or infertility, forming an ideal anti-drug through the "Ιβ-HSD1-glucocorticoid→testosterone" channel A drug for hypogonadal dysfunction and/or infertility.

Figure 3 LG13 dual regulation of Ιβ-HSD1 reductase and oxidase in murine microsomes. Panel A shows the effect of LG13 on the inhibition of Ιβ-HSD1 oxidase, and Panel B shows the effect of LG13 on the activation of Ιβ-HSD1 reductase (n = 4). * and *** indicate significant differences, where * is P < 0.05 and *** is P < 0.001.

Figure 4 Effect of LG13 and curcumin on blood glucose levels and ester metabolism. Among them, various indicators are: A picture is blood sugar, B picture is triacylglycerol, C picture is total cholesterol, D picture is low density lipoprotein, E picture is apolipoprotein-al, F picture is apolipoprotein- b. * and *** indicate statistical differences, * is P < 0.05, and *** is P < 0.001.

Figure 5 LG13 inhibits the formation of fatty liver in rats. Group A was a normal diet rat; Group B was a high-fat diet rat; Group C was a high-fat diet rat with LG13 at a dose of 1 mg/kg; Group D was administered with a dose of 5 mg/kg for LG13. High fat diet Rat.

Figure 6 Effect of curcumin and LG13 on stress-induced prophylactic low testosterone levels. Mean ± SEM (n = 10). * indicates statistical difference, * is P < 0.05.

Figure 7 Effect of in vitro administration of B19 and B63 on the survival rate of human non-small cell lung cancer H460 cells.

Figure 8 Flow cytometry of B19 and B63-induced apoptosis in human non-small cell lung cancer H460 cells

Figure 9. Effect of B19 and B63 on CHOP protein in the endoplasmic reticulum stress pathway.

Figure 10 B19 and B63 activation of Caspase-3 and Caspase-9.

Figure 11 Effect of toxicity of B19 and B63 on animal body weight and visceral weight. Above: The ordinate indicates the weight difference; the horizontal coordinate indicates the number of days of continuous gavage administration. Bottom: The ordinate indicates the weight (g); the abscissa indicates the animal group; the heart is the heart; the Lung is the lung; the Liver is the liver; and the Kidney is the kidney.

Figure 12 Effect of toxicity of B19 and B63 on blood parameters. Upper right: Red blood cell count detection, the ordinate represents the number of blood cells per liter, and the abscissa represents the animal group. Upper left: The number of white blood cells is detected, the ordinate represents the number of blood cells per liter, and the horizontal coordinate represents the animal group. Right middle: Hemoglobin concentration detection, the ordinate represents the hemoglobin concentration (g/L), and the abscissa represents the animal group. Left middle: White blood cell ratio detection, the ordinate represents the percentage, the abscissa represents the animal group, LYM is the lymphocyte, GRAN is the neutrophil, and MID is the intermediate cell group, including the fin acid, the fin granulocyte and the monocyte. Bottom right: Hematocrit test, the ordinate represents the percentage, and the abscissa represents the animal group. Bottom left: Detection of alanine aminotransferase and aspartate aminotransferase content, the ordinate represents the enzyme content (U/L), and the abscissa represents the animal group. Detailed description of the invention

The object of the present invention is to provide a novel application method or method in which a compound capable of simultaneously reducing the activity of 11β-hydroxysteroid dehydrogenase reductase and increasing the activity of 11β-hydroxysteroid dehydrogenase oxidase is applied. That is, the ΙΙβ-HSD1 dual regulator, especially the specific ΙΙβ-HSD1 dual regulator, can be applied in the fields of pharmaceutical, therapeutic and preventive. Further, another object of the present invention is to provide a method for treating or preventing a disease in which a ΙΙβ-HSD1 dual modulator is apoptotic cells by activating CHAP and/or apoptotic cells by activating caspase-3 and caspase-9 or Pharmaceutical applications. Further, the present invention provides a pharmaceutical composition and kit comprising the ΙΙβ-HSD1 dual modulator, and a method for screening the Ιβ-HSD1 dual modulator. . Additionally, it is an object of the present invention to provide new low dose pharmaceutical compositions, kits and corresponding applications.

Specifically, in a first aspect, the present invention provides a method of using a compound of Formula I in the manufacture of a medicament for preventing and/or treating a disease, wherein the disease is capable of reducing 11β-hydroxydehydrogenase 1 a disease which is treated and/or prevented by the activity of a reductase and/or an activity of increasing 11β-hydroxyl dehydrogenase 1 oxidase,

Formula I

among them,

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} synthesized, wherein n is 1, 2, 3, or 4. Among them, preferred compounds include A2, A6, A10, A12, A19, B6 (i.e., LG13), B12, B19, B63, C2, C6, C13, C19, and C66, and particularly preferably B6.

Corresponding to pharmaceutical applications, the first aspect of the invention also provides a method of preventing and/or treating a disease, wherein the disease is capable of reducing the activity of 11β-hydroxyl dehydrogenase 1 reductase and/or increasing 11β a disease which is treated and/or prevented by the activity of a hydroxydehydrogenase 1 oxidase, the method comprising administering to a patient a drug comprising an effective amount of a compound of formula I

Rl4 R 24

Formula I

among them,

R ₁₃ and R ₂₃ are not alkenyloxy, preferably R ₁₃ and R ₂₃ are each H, halogen, lower alkyl, hydroxy, or optionally Ν, Ν-dimethylamino substituted lower alkoxy;

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} bonded to ^{e3⁄4 1T} wherein n is 1, 2, 3, or 4. Among them, preferred compounds include A2, A6, A10, A12, A19, B6 (i.e., LG13), B12, B19, B63, C2, C6, C13, C19, and C66, and particularly preferably B6.

As used herein, the term "drug" has the meaning well known to those skilled in the art, which comprises an active active ingredient which is effective for the treatment and/or prevention of a disease, and optionally comprises a pharmaceutically acceptable carrier. Although not preferred, an entity which does not comprise a pharmaceutically acceptable carrier and which consists solely of the active active ingredient is also a drug.

As used herein, the terms "treating" and "preventing" have the meanings well known to those skilled in the art, and administering to a patient after the onset of a symptom of the disease an action for eliminating or alleviating the symptoms of the disease or preventing, slowing or delaying the progression of the disease. It is called treatment, and the administration of an action for preventing or preventing the occurrence of a disease or slowing or delaying the deterioration after the occurrence of the disease before the onset of the disease symptoms is called prevention. In a specific embodiment of the present invention, the compound of the present invention can treat diabetes, prevent fatty liver formation, prevent stress testosterone reduction, and treat non-small cell lung cancer and glioblastoma.

In this document, chemical formulas, chemical names, and other chemical terms are defined in standard textbooks unless otherwise stated. Wherein "halogen" means F, Cl, Br and I. "Lower" when used in connection with an alkyl or alkoxy group, means that the corresponding group has 1 to 8 carbon atoms, preferably 1 to 5 carbon atoms, such as 1, 2 or 3, and the like.

Preferably, in the first aspect of the invention, the compound of formula I is a dual modulator of 11β-hydroxydehydrogenase 1, preferably a specific 11β-hydroxydehydrogenase 1 dual modulator. According to the present inventors' research method, it is possible to simultaneously reduce 11β-hydroxysteroid dehydrogenase 1 reductase activity and increase 11β-hydroxydehydrogenase 1 oxidase activity, and it is possible to treat or prevent the corresponding disease of the present invention very effectively.

As used herein, the term "11β-hydroxysteroid dehydrogenase 1 dual modulator" or its abbreviation "dual regulator" refers to the ability to simultaneously reduce 11β-hydroxydehydrogenase 1 reductase activity and increase 11β-hydroxyl Dehydrogenase 1 oxidase activity compound. For example, the semi-inhibitory concentration (EC50) of the 11β-hydroxydehydrogenase 1 reductase activity of the compound is less than 30 μΜ and the semi-effective concentration (EC50) of the 11β-hydroxydehydrogenase 1 oxidase activity is less than 30 μΜ, preferably It has an IC50 of less than 20 μΜ and an EC50 of less than 20 μΜ, more preferably an IC50 of less than ΙΟμΜ and an EC50 of less than 10 μΜ, more preferably an IC50 of less than 5 μΜ and an EC50 of less than 5 μΜ, particularly preferably an IC50 of less than ΙμΜ and an EC50 of less than 1 μΜ.

As used herein, the term "specific ΐβ-hydroxydehydrogenase 1 dual modulator" or its abbreviation "specific dual modulator" refers to a dual regulation that does not substantially affect the activity of 11β-hydroxydehydrogenase 2 oxidase. For example, the half inhibitory concentration (IC50) of the oxidase activity of the dual modulator to 11β-hydroxysteroid dehydrogenase 2 is greater than 100 μΜ. This concentration is much greater than the inhibition of 11β-hydroxydehydrogenase 1 reductase activity and activation of 11β-hydroxyl dehydrogenase 1 oxygen as defined above. The concentration of the enzyme activity, therefore, the effective concentration of the dual modulator does not substantially affect the oxidase activity of 11β-hydroxyl dehydrogenase 2.

As used herein, the term "patient" refers to an animal having, or potentially suffering from, a disease, preferably a mammal, such as a mouse, a rat, a rabbit, a dog, a monkey, a human, etc., and most preferably a human.

In a first aspect of the invention, the patient may have an increased or decreased 11β-hydroxydehydrogenase 1 reductase activity and/or 11β-hydroxyl depoting relative to a healthy or normal individual, organ, tissue or cell Symptoms of decreased activity of hydrogenase 1 oxidase, or symptoms that may or may occur by reducing the activity of 11β-hydroxydehydrogenase 1 reductase and/or increasing the activity of ιΐβ-hydroxydehydrogenase 1 oxidase And treatment and / or prevention. The present inventors have found that the dual modulator of the present invention can simultaneously reduce the activity of ιΐβ-hydroxydehydrogenase reductase and increase the activity of 11β-hydroxysteroid dehydrogenase oxidase, thereby treating or preventing ιββ- in vivo. Increased activity of hydroxydehydrogenase reductase and

A patient with reduced activity of 11β-hydroxydehydrogenase oxidase. Preferably, in the first aspect of the invention, the disease is a glycolipid metabolic disease, hypogonadal dysfunction, and/or infertility, preferably selected from the group consisting of diabetes, obesity, atherosclerosis, cirrhosis, fatty liver, Hyperglycemia, hyperlipidemia, hypertension, hypogonadal dysfunction and/or infertility, most preferably hypoglycemia dysfunction caused by diabetes, fatty liver, glucocorticoid excess and its induced infertility, And/or stress testosterone reduction, such as type 2 diabetes, stress, disease or aging-induced gonadotropin dysfunction caused by excessive glucocorticoids and its resulting infertility, etc.

In the first aspect of the invention, it is particularly preferred that the compound of formula I is LG13, and particularly preferably an effective dose of from 1 to 10 mg/kg of a rat, such as from 1 to 10 mg/kg of Wistar rat. As used herein, "effective dose" refers to the amount required for a single administration in the course of being effective to treat or prevent a disease, which may be in the form of a unit dosage (eg, a tablet, a needle, a pill or a The amount of the drug in the agent may also be the unit dose (e.g., unit weight dose) of the patient in need of treatment/prevention. The drug manufacturer can easily convert the unit weight dose of the patient to be treated/prevented into the amount of the drug in a unit dosage form by the average body weight of the patient population to be treated/prevented, for example, the average of the adult patient. The body weight can be 60 kg, so by multiplying the average body weight by the unit weight dose of the adult, the content in the drug for the unit dosage form for the adult can be obtained. Typically, the patient is a human, but the patient used for the experiment is usually a non-human mammal such as a monkey, rabbit, dog or mouse. According to the equivalent dose conversion relationship between experimental animals and humans known to those skilled in the art (see generally the guidance of FDA, SFDA and other drug regulatory agencies, see also "Huang Jihan et al. Pharmacological tests in animals and animals and humans" Equivalent dose conversion between. Chinese Clinical Pharmacology and Therapeutics, 2004 Sep; 9(9): 1069 - 1072") The human body weight dose can be derived from the dose of experimental animals. For example, for commonly used experimental animal mice, according to the above literature, the conversion relationship with adults is about 12: 1; for commonly used experimental animal rats, according to the above literature, its conversion relationship with adults is about 6: 1. Since various forms of "dose" can be conveniently converted according to the above conversion relationship, the dose is usually expressed herein in terms of rat unit body weight. For example, l-10mg/kg Wistar rats The effective dose refers to an effective dose of 0.167-1.67 mg/kg adult, that is, an effective dose of 10-100 mg in a human drug in a unit dosage form.

Surprisingly, the inventors' research method now has the opposite effect of conventionally approved dose-dependent treatment/prevention, LG13 is best for low-dose treatment/prevention of diabetes, and at medium and high doses. The poor effect is not due to drug toxicity. Therefore, without being limited to theory, it is preferred that in the first aspect, the medicament contains an effective dose of LG13, more preferably a dose of LG13 containing 1-10 mg/kg of Wistar rat, more preferably a dose of 3-8 mg/kg of Wistar rat. LG13, such as LG13 at a dose of 5 mg/kg Wistar rats.

In a second aspect, the present invention provides a method of using a compound of Formula I in the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease is capable of apoptotic cells by activating CHOP and/or by activating caspase- 3 and caspase-9 and apoptotic cells to treat or prevent diseases,

Rl4 R 24

Formula I

among them,

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} synthesized, wherein n is 1, 2, 3, or 4. Among them, preferred compounds include A2, A6, A10, A12, A19, B6 (i.e., LG13), B12, B19, B63, C2, C6, C13, C19, and C66, and particularly preferably B19 and B63.

Corresponding to pharmaceutical applications, the first aspect of the invention also provides a method of preventing and/or treating a disease, wherein the disease is capable of apoptotic cells by activating CHOP and/or by activating caspase-3 and caspase- 9. A disease in which apoptotic cells are treated or prevented, the method comprising administering to the patient a drug comprising an effective amount of a compound of formula I

among them,

R ₁₄ and R ₂₄ are each deuterium or lower alkyl;

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R _{25 are} bonded to ^{e3⁄4 1T} wherein n is 1, 2, 3, or 4. Among them, preferred compounds include A2, A6, A10, A12, A19, B6 (i.e., LG13), B12, B19, B63, C2, C6, C13, C19, and C66, and particularly preferably B19 and B63.

In a second aspect of the invention, the patient may have symptoms that are or will occur relative to healthy or normal individuals, organs, tissues or cells capable of activating cells by activating CHOP and/or by activating caspase-3 and caspase- 9 and apoptotic cells to treat or prevent. It has been found by the present inventors that the compounds of the present invention can also be treated or prevented by activating apoptotic cells by activating CHOP and/or apoptotic cells by activating caspase-3 and caspase-9, and thus can usually treat certain tumors and cancers. . Preferably, in the second aspect of the invention, the disease is gastric cancer, myeloid leukemia, oral epithelial cancer, lung cancer or sarcoma, and particularly preferably non-small cell lung cancer or glioblastoma.

In a separate aspect, the invention provides a compound of the formula

The above compounds are preferably used for the treatment of tumors such as gastric cancer, myeloid leukemia, oral epithelial cancer, lung cancer or sarcoma, particularly preferably non-small cell lung cancer or glioblastoma

In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of LG13 and a pharmaceutically acceptable carrier, preferably a dose of from 1 to 10 mg/kg of a rat (e.g., l-10 mg/kg Wistar rat) LG13. Preferred of the invention Low dose pharmaceutical compositions are preferably used to prevent or treat diabetes or stress testosterone reduction.

As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, adjuvant, encapsulating material or other formulation excipient. The pharmaceutical compositions may be formulated into various dosage forms, such as tablets, films, pills, capsules (including sustained release), depending on the purpose of the treatment, the route of administration, in accordance with the teachings in the art. Or a delayed release form), a powder, a granule, an elixir, a syrup and an emulsion, a sterile solution or suspension, an aerosol or liquid spray, a drop, an injection, an automatic injection device or a suppository. For example, in the form of a tablet or capsule, the dual modulator may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, isotonic glucose solution, glycerol, physiological saline or combination.

The pharmaceutical compositions of this invention may be administered by methods of administration well known to those skilled in the art, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoretic, vaginal and intranasal administration. The pharmaceutical composition of the present invention is preferably administered parenterally, such as subcutaneously, intramuscularly or intravenously. The dose to be administered varies depending on the form of the preparation and the desired time of action and the condition of the subject to be treated, and the amount required for the actual treatment can be conveniently determined by the physician based on the actual conditions (e.g., the condition of the patient, the body weight, etc.).

In a fourth aspect, the invention provides a kit for treating diabetes comprising LG13, and a label indicating administration at a dose of l-10 mg/kg rat. Herein, the low dose refers to a dose of l-10 mg/kg Wistar rat, preferably a dose of 3-8 mg/kg Wistar rat, such as a dose of 5 mg/kg Wistar rat, if not stated to the contrary. The kit is a common product for the general public and can be easily found in pharmacies. In general, the kit comprises a container containing LG13 or a pharmaceutical composition of the third aspect of the invention, in other words, LG13 or the pharmaceutical composition of the third aspect of the invention is contained in a container of the kit of the present invention. The container may be a usual container such as a bottle, a box, a syringe or the like which can accommodate LG13 and the pharmaceutical composition of the second aspect of the invention. The medicine may include only one container, and may also include a plurality of containers. The label may be affixed to the container or printed directly onto the container, or may be present in a separate form, such as a cover for a cartridge that can hold the container or directly provided instructions. The label indicates administration with a low dose of LG13, wherein the indication of the label may be expressed in units of body weight, or may be expressed in absolute doses of a specific population, such as "adult dosage" or "child dosage". It is necessary to perform a simple conversion based on the weight. If the container contains a composition such as a drug, a preparation, etc., the low dose can be converted into a content according to the content of the LG13 in the unit dosage form (e.g., one tablet, one needle), which is indicated by a label, which is for people. Easy. It is also within the scope of the invention to package the kit further into larger packages as needed for convenient transportation and storage.

In a fifth aspect, the present invention provides a method of screening a dual modulator of 11β-hydroxydehydrogenase 1 in vitro, which comprises

(1) The half-inhibitory concentration (IC50) of the reductase activity of the test compound on the mesenchymal cells, the 11OP-hydroxyl dehydrogenase 1 of CHOP cells and/or microsomal proteins transfected with human 11PHSD1 in vitro; (2) In vitro determination of the half-effective concentration (EC50) of the oxidase activity of the test compound on stromal cells, 11β-hydroxyl dehydrogenase 1 of CHOP cells and/or microsomal proteins transfected with human 11PHSD1;

(3) A compound having an IC50 of less than 30 μΜ and an EC50 of less than 30 μΜ is selected as a double regulator of 11β-hydroxydehydrogenase 1. Among them, a compound having an IC50 of less than 20 μΜ and an EC50 of less than 20 μΜ is preferably selected, and a compound having an IC50 of less than ΙΟμΜ and an EC50 of less than ΙΟμΜ is more preferably selected, and a compound having an IC50 of less than 5 μΜ and an EC50 of less than 5 μΜ is more preferably selected, and it is particularly preferable to select an IC50 of less than ΙμΜ and an EC50 of less than ΙμΜ. a compound that acts as a dual regulator of 11β-hydroxysteroid dehydrogenase 1.

Preferably, in the fifth aspect of the invention, the method further comprises measuring in vitro the half-inhibitory concentration of the 11β-hydroxydehydrogenase 1 dual modulator on the oxidase activity of the 11β-hydroxydehydrogenase 2 of the microsomal protein ( IC50), then the compound having an IC50 greater than ΙΟΟμΜ was selected as a dual modulator of specific 11β-hydroxyl dehydrogenase 1.

Preferred in the first aspect of the invention

Formula π

among them,

R _u , R ₂₁ , R ₁₂ , R ₂₂ , R ₁₃ , R ₂₃ , R _{14 and} BR ₂₄ are each H, halogen, hydroxy, amino, optionally substituted alkyl, optionally substituted alkoxy, or any a substituted alkenyloxy group, wherein the substituent includes a 3⁄4 element, a hydroxyl group, an amino group, an aromatic group and/or a heterocyclic ring;

In a particular embodiment of the invention, the invention exemplarily screens the compounds listed in Example 1. In order to facilitate the understanding, the present invention is hereby incorporated by reference in its entirety in its entirety in its entirety herein in its entirety herein in The invention will be described in detail below by means of specific embodiments and the accompanying drawings. It is to be understood that the description is not intended to be limiting of the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the <RTIgt; detailed description

The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Among them, the operations and procedures without specific instructions can be carried out according to the teachings of the "Organic Synthesis Handbook", "Molecular Cloning Experimental Guide", "Cellular Guide" and other experimental manuals or experimental manuals well known in the art; The reagents are all commercially available conventional reagents. Example 1 Compound and preparation example thereof

Referring to the preparation method of the compound described in Chinese Patent Application No. CN101003470A, the compounds of the following formula A, formula B and formula C can be chemically synthesized according to the synthetic route shown in Fig. 1:

An enumerated preparation example is as follows:

(1) Preparation of LG13

10 mmol of 2-bromobenzaldehyde was dissolved in 10 mL of absolute ethanol, stirred at room temperature for 5 min, and then the corresponding cyclopentanone, acetone, and cyclohexanone were added, and stirring was continued for 10 min, and the solution was unchanged. The sodium metal was dissolved in methanol and placed in a 18% (w/v) sodium methoxide/methanol solution. 1.5 mL of sodium methoxide solution (containing 5 mmol of sodium methoxide) was slowly added dropwise to the reaction solution. After stirring for 2 h, a large amount of insoluble yellow matter appeared. The reaction solution was detected by TLC. The raw material was no longer present under the 320 nm UV lamp. - Black spots of bromobenzaldehyde, the product spots are clearly yellow. The reaction was stopped, the reaction solution was filtered, and the product was washed with water, then twice with iced ice and iced acetone, and dried under vacuum at 30 ° C overnight to give the product as a yellow powder of 1,5-bis(2-bromophenyl)-1. , 4-pentadien-3-one (abbreviated as B6 or LG13), yield 81.1%. Melting point 97. C; 1H-NMR (CDC1 ₃ ) δ: 7.03 (2Η, d, J=16 Hz, C=CH-COx2), 7.22-7.63 (8H, m, Ar-H), 8.09 (2H, d, J= ESI-MS m/z: 393.10 (M+l) ⁺ , calcd for C ₁₇ H ₁₂ Br ₂ 0: 392.08.

(2) Preparation of B19

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(1) Experimental method

Isolation method of hepatocytes and interstitial cells: After the rats were sacrificed by C0 ₂ asphyxiation, the testis was removed to extract and purify the interstitial cells, and the method was referred to [Υ^Δ, et al. J Androl. 2001, 22, 665-671]. The purity of mesenchymal cells was determined by 3β-hydroxyindole dehydrogenase activity histochemical staining (with 0.4 mM of this cholesteryl ketone as a substrate), which can stain more than 95% of mature stromal cells; The separation was performed by the method described by Pertoft and Smedsrad [Cell Separation: Methods and Selected Applications. pp: l-23, Academic Press, Orlando, F, 1987], in which the calcium free buffer was infused into the rat liver, and dispersed in 0.05%. The hepatocytes were purified by density gradient centrifugation in the collagenase solution.

Preparation of microsomal proteins: Murine interstitial cells, murine hepatocytes, and human hepatocyte microsomes were prepared by the method reported by [fea, et ^. Endocrinology, 1997, 138: 435-42].

Transfection method of human lipHSD1 gene and culture method of CHOP cell strain: According to the report of Ge et al (Ge et al 2000. J Androl. 21 (;2): 303-10.), the transformation of human Ιβ-HSD1 gene was carried out. The transfected CHOP cell line was stained and cultured.

For 11PHSD oxidase and reductase activity detection method: using detecting ^[3 H] - cortisone or ^[3 H] -11- concentration of hydrocortisone characterized Ι Ιβ-HSDl oxidase or reductase activity, respectively. 25 nM [ ³ H]-cortisone was added to each Ιβ-HSD1 activity assay tube (this concentration was within the physiological concentration range of cortisone). Between primary hepatocytes or interstitial cells containing 25nM ^[3 H] - hydrocortisone in the culture liquid after 30min, ^[3 H] - cortisone or ^[3 H] -11- concentration of hydrocortisone characterized Ι Ιβ-HSD1 oxidase or reductase activity. For the detection of Ιβ-HSD1 activity in the microsomes, the microsomes were incubated with the Ιβ-HSD1 substrates NADPH, G6P, and the reaction was terminated with 2 ml of ice diethyl ether after a certain period of time. Then, the body in the incubation solution is extracted with an organic solvent, dried in a nitrogen gas stream, and then separated by chloroform/methanol (9:1) as a mobile phase, and the steroid is separated by thin layer chromatography, [ ³ H]-cortisone or The radioactivity of [ ³ H]-11-hydrocortisone was detected by scanning radiography (System AR2000, Bioscan Inc., Washington, DC, USA), and the conversion between cortisone and hydrocortisone was determined by themselves. The radioactive standard number is calculated and determined. Oxidase activity can be measured by adding CORT (2 x 10-9 -10-5 M) and 0.2 mM NADP+ for 30 minutes in a 0.15 μ _§ mouse liver microsome system. Ι -β-HSD1 reductase can be measured by adding 11DHC (2 X 10-9 -10-5 M), 0.2 mM NADPH and 0.2 mM G6P in a 1.5 g rat liver microsome system for 30 minutes. l The detection method of lp-HSD2 is as follows: Ιβ-HSD1 oxidase detection method.

Compound treatment and detection: The isolated cells were cultured in a 12-well plate at a density of ⁴ ×10 ⁵ /well. The microsome system directly treated the extract and was incubated with a certain amount of exogenous glucose-6-phosphate (G6P). After 3-6 h, after treatment with different concentrations of compound, the reaction was terminated with 2 ml of ice diethyl ether after a certain period of time. The activity was determined according to the above-mentioned 11PHSD oxidase and reductase activity assay. All tests were performed at least twice independently. The average value is converted into IC50 (semi-inhibitory concentration) and EC50 (semi-effective concentration) based on the concentration at which inhibition or activation occurs. (2) Experimental results

The test results are shown in Table 1 below:

Table 1 Activity of LG13 against ΙΙβ-HSD1 reductase, ΙΙβ-HSD1 oxidase and llp-HSD2

Note: A: rat interstitial cells; B: rat testicular microsomes; C: CHOP cells transfected with human 11PHSD1; D: human liver microsomes; E: rat kidney microsomes; F: human kidney microsomes; -: No detection; Cur: Curcumin. The compounds inhibited the inhibitory or activating activity of ΙΙβ-HSD1 reductase, ΙΙβ-HSD1 oxidase and 11P-HSD2 in six cell or microsomal systems. As can be seen from Table 1, (I) most of the tested curcumin analogs (including curcumin) are not compounds that have dual regulation of ΙΙβ-HSD1, or have little or no inhibition of ΙΙβ-HSD1 reductase activity, or almost no The ability to activate ΙΙβ-HSD1 oxidase activity, especially for alkenyloxy substituted compounds, is not only weakly activated by ΙΙβ-HSD1 oxidase activity, but also weakly inhibits ΙΙβ-HSD1 reductase activity; Most of the tested curcumin analogues have a weak inhibitory effect on 11P-HSD2. In the presence of compounds not greater than ΙΟμΜ, the enzyme activity of 11P-HSD2 is hardly affected, so for ΙΙβ at a level not greater than ΙΟμΜ -HSDl has a specific needle for ΙΙβ-HSD1; (III) comprehensively considers the weight-regulating activity, Α2, Α6, Α10, Α12, Α19, Β6, Β13, Β19, Β63, C6, C13, C19 and C66 are the preferred dual inhibitors of ΙΙβ-HSD1, which can selectively inhibit ΙΙβ-HSD1 reductase without affecting 11P-HSD2. A significantly activate ΙΙβ-HSDl oxidase, couverture play a dual regulatory role ΙΙβ-HSDl can be adjusted more effective in vivo The hormonal activity, some of which inhibit the 50β-HSD1 reductase IC50 values even reach the nM level, and the EC50 value of the activated Ιβ-HSD1 oxidase even reaches the tiM level. Further, referring to Fig. 3 of the present invention, the particularly preferred compound B6 (i.e., LG13) of the present invention has a dose-dependent activity for the inhibitory activity against ΙΙβ-HSD1 reductase and the activation activity for ΙΙβ-HSD1 oxidase. Example 3 B6 (LG13) Decreases blood glucose and lipid levels in animals fed with high-fat foods in vivo

Animal experiments were carried out according to the method recommended by the drug regulatory department: SD rats (purchased from the Shanghai Animal Center of the Chinese Academy of Sciences) were fed with normal diet (CON, control group) and high-fat diet (HFD, high-fat model group), in high fat In the fed rats, LG13 (2 mg/kg, day 13-1) and (4 mg/kg/day, 13-2 group) were administered by gavage, and 10 rats in each group were intragastrically administered daily. Once, continuous administration for 2 months. Then, various indicators were tested by biochemical analyzer. As a result, as shown in Figure 4 of the present invention, LG13 has a good hypoglycemic and lipid-lowering effect at doses of 2 mg/kg and 4 mg/kg. Example 4 B6 (LG13) relieves fatty liver formation in vivo

Obesity and fatty liver caused by high-fat feeding are important stimuli and complications of diabetes. They play an important role in the development of diabetes. Therefore, animal experiments are carried out according to the method recommended by the drug regulatory department: SD rats were purchased from Shanghai, Chinese Academy of Sciences. The animal center was divided into 4 groups, 6 in each group, among which group A was a normal diet rat; group B was a high-fat diet (ie, fed with high-fat words); group C was 1 mg/kg. A high-fat diet rat of LG13 was intragastrically administered at a dose of day/day; a high-fat diet rat of LG13 was intragastrically administered at a dose of 5 mg/kg/day in group D. In addition, 10 high-fat rats were administered with LG13 and C65 at a dose of 10 mg/kg/day, respectively, as B6 and C65, respectively, for the comparison of specific and non-specific ΙΙβ-HSD1 dual regulation. The effect of the agent. After feeding and administering for 2 months on a high-fat diet, the rats were sacrificed and the liver was taken. After observation, the formation of fatty liver was observed by light microscopy. The weight gain of group A was significantly lower than that of other groups. There was no significant difference between group B, group C, group D, group C65 and group B6.

After directly observing the liver, the livers of group A and group B6 were dark red, soft in texture and good in elasticity. The left lobe was significantly larger than the right lobe. The interlobular fissure was clear, the edges were sharp, and the cut surface was smooth. The relevant organs and spleen were also dark. Red slender strips, full kidney, thinner perirenal fat sac. However, in group B and C65, the liver was evenly swollen, the liver was full, the right lobe was enlarged, the ratio of left and right lobe was decreased, the interlobular fissure was small, the liver margin became dull, and the color increased with darkness from dark red to pale white. Or milky white, occasionally the liver is obviously yellowed or fatty granuloma due to cholestatic.

After observation by light microscopy, the hepatic lobules of group A and group B6 were intact, the hepatocyte cords were arranged neatly, and lipid droplets were occasionally seen in hepatocytes. Group B and C65 group formed vesicle-based steatosis, which occurred in different degrees of hepatic lobular inflammation, hepatocyte turbidity, balloon-like changes, point or focal necrosis, and neutrophil or lymphocyte infiltration, and more With varying degrees of fibrosis. In particular, as shown in the sliced photograph of Figure 5 of the present invention, even at an oral dose of 5 mg/kg/day, LG13 has been significantly inhibited. The formation of fatty liver.

The results showed that LG13 intragastric administration can effectively prevent hepatic steatosis caused by high-fat diet in rats, and the titer is high; C65 does not have this effect. Example 5 B6 (LG13) Animal Experiment for Diabetes

1. Establishment of a diabetes model: Male Wistar rats qualified for quarantine (SPF grade rats, purchased from Shandong Lukang Pharmaceutical Co., Ltd., license number: SCXK (Lu) 2008 0002), weight 190~220g modeling . The rats were fasted for 12 h before the model was established. The first dose of 50 mg/kg fasting body weight was injected into the tail vein (iv) with streptozotocin (STZ) (injected in 30 minutes), and the STZ was given for the first time. On the 5th day, STZ was administered once at a dose of 60 mg/kg fasting body weight to obtain experimental diabetic rats with blood glucose ≥11.lmmol/L.

2, Experimental methods: 32 rats identified as experimental diabetes model were randomly divided into 4 groups, namely LG13 high (100mg/kg), medium (25mg/kg), low (5mg/kg) dose group and model control Group, 8 animals per group. Each group of model animals were given the corresponding dose of drugs by oral gavage once a day for 7 days. Blood was collected from the orbital veins of the rats on the 1st, 3rd, and 5th day after the last administration. Serum was prepared and blood glucose was measured. Table 2. Table 2. Effect of LG13 on blood glucose in rats at different time points after administration (mmol/L)

After giving the medicine

Before giving the medicine to the group -

1 day 3 days 5 days Low dose group 28.36±3.17 4.59±1.65 3.36±2.67 27.61±5.12

Medium dose group 28.53±13.66 6.54±2.11 16.97±19.39 24.28±10.1

High dose group 29.10±6.64 6.01±1.59 19.45±12.92 24.25±1.49

Model control group 29.12±1.65 31.63±0.06 36.74±0.06 25.79±1.83

3, conclusion

LG13 has a significant effect on lowering blood glucose at high, medium and low doses, and it is particularly surprising that hypoglycemic effects last longer at low doses (5 mg/kg). Example 6 Safety test of the compound of the present invention

LG13, B19, B63, A6 and B50 were suspended in 1% sodium carboxymethylcellulose solution, taking 14-18g Balb/C mice (male, normal diet), divided into 4 groups, respectively, negative group (normal word, no gavage), solvent group (administer the same amount of sodium hydroxymethylcellulose solution), 400mg /kg dose of LG13 group, 800mg/kg dose of LG 13 group, 400mg/kg dose of B 19 group, 800mg/kg dose of B 19 group, 400mg/kg dose of B63 group, 800mg/kg dose of B63 group, 400 mg/kg dose of A6 group, 800 mg/kg dose of A6 group, 400 mg/kg dose of B50 group, and 800 mg/kg dose of B59 group, 8 rats in each group, intragastric administration, once daily, continuous intragastric administration 14 day. No mice died of toxicity within 14 days, and no abnormal behavior and condition of the administered mice were observed.

After 14 days, all mice in the B19 and B63 groups were killed, the body weight and visceral weight were weighed, and the change values were recorded. The results are shown in Fig. 11; blood was taken from the eyelids, and half of them were directly subjected to whole blood analysis (blood cell analyzer). The amount of red blood cells, white blood cells, and hemoglobin was measured; the other half was centrifuged to separate the serum, and the contents of alanine aminotransferase and aspartate aminotransferase were measured. The results are shown in FIG. From the toxicity test results shown in Figure 11 and Figure 12, 400 mg/kg and 800 mg/kg of gavage for 14 days after continuous gavage, B19 and B63 on animal body weight, visceral weight, plasma red blood cells, white blood cells, hemoglobin, white blood cells There was no significant change in the ratio of pro-basin, alanine aminotransferase and aspartate aminotransferase, indicating that B19 and B63 were almost non-toxic. The safety of B19 and B63 provides the necessary protection for their pharmaceutical applications.

In addition, LG13 was suspended in 1% sodium carboxymethylcellulose solution, and 14-18 g of Balb/C mice (male, normal diet) were taken at a higher dose of 3 g/kg. The dose of LG13 was intragastrically administered once a day for 3 days and then for another 3 days. No mice died of toxicity within 6 days, and no abnormal behavior and condition of the administered mice were observed.

The above results indicate that the compounds of the present invention are safe. Example 7 Pharmacokinetic Experiment of B6 (LG13)

The pharmacokinetics of LG13 mice were measured according to the method recommended by the drug regulatory department and the method described by Liang G, et al. Bioorg. Med. Chem., 2009, 17: 2623-2631. The main pharmacokinetics were determined. The data is shown in Table 3. As can be seen from Table 3, the peak dose of LG13 plasma reached 4.1 g/ml, also at a dose of 500 mg/kg orally. The corresponding LG13 has a lower in vivo clearance (CL) (125.4), indicating a significant decrease in the body's ability to remove compounds. The amount of LG13 entering the plasma was large, and the AUCO-t and AUCO-oo values reached 12.053 and 14.907 mg_h/L, respectively. It can be seen that LG13 has considerable advantages in terms of pharmacokinetic parameters.

Table 3 Pharmacokinetic data of LG13

LG13

AUCo-t (mg h/L) 12.053±4.34

AUCo (mg h/L) 14.907±7.31

Ti/2 (h) 2.92±1.3

MRT _0-t (h) 2.486±0.53

CL (L/kg/h) 38.98±17.16

C g/ml) 4.1±0.19

T (h) 0.25

Note: AUC: area under the curve; t _m : half-life; MRT: mean residence time; CL: clearance rate; C _max: highest blood concentration; T _max: peak time Example 8 Induction of stress by curcumin and LG13 Effect of low testosterone levels on preventive treatment

The experiment was carried out according to the report of Dong Q. et al (Dong Q. et al 2004, J Androl 25: 973-981). SD rats (purchased from the Shanghai Animal Center of the Chinese Academy of Sciences) were fed on a normal diet. The mice in different doses were stressed (stress group) and no stress (no stress group), 10 in each group. Curcumin and LG13 were orally administered at different doses, once daily for 2 days. SD rats were fixed for 3 hours. Take blood and measure changes in serum testosterone. As a result, as shown in Fig. 6 of the present invention, 100 and 200 mg/kg of curcumin can be substantially restored to serum testosterone levels without stress, and only 1 and 5 mg/kg of LG13 are required to substantially return to no stress. Serum testosterone levels. It shows that curcumin and LG13 can prevent the reduction of stress testosterone, and LG13 is much better. Example 9 In vitro antitumor activity of B19 and B63:

The cell strains used were: human gastric cancer cell line BGC 823, human myeloid leukemia cell line HL-60, human oral epithelial cancer cell line KB, human colon adenocarcinoma cell line LS 174T, human prostate cancer cell line PC-3, Human cervical cancer cell line Hela cells were purchased from the Cell Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

The cells were inoculated into 96-well culture plates, and the cell suspension was adjusted to contain 5% heat-inactivated newborn bovine serum, penicillin 100 U/mL, streptomycin 10 (g/mL of 1640 medium, 100 μί per well, so that the cells were The density was 5000/well. It was cultured in an incubator containing 5% C0 ₂ saturated humidity. After 24 h, various compounds dissolved in DMSO were added to the plates to give final concentrations of 100, 33.3, 11.1 and 3.7 g. /mL, after incubation for 72 h, 3 mg/ml MTT 20 μί per well was added 3 h before the incubation. After the incubation, the liquid in the well was carefully aspirated, 100 μL of DMSO was added to each well, and shaken on a shaking bed for 10 min to make crystals. Fully dissolved, the absorbance of each well was measured at 570 nm by enzyme-linked immunosorbent assay (A). The positive control was cisplatin (DDP). The cell growth inhibition rate was calculated according to the absorbance. Cell growth inhibition rate = [OD control - OD experiment] /[OD control-OD blank] xl 00%

The dose response curve was obtained by plotting the different concentrations of the same drug on the growth inhibition rate of the tumor cells. The concentration of the drug against the cell growth inhibition rate of 50% according to the linear regression equation was the half-inhibitory concentration IC _5Q . The results are shown in Table 4. B19 and B63 can make a considerable difference in the ability to inhibit the proliferation of various human tumor cells. As can be seen from the table, the above two The compounds were significantly higher than the curcumin and the positive drug control cisplatin (DDP) in several cell lines such as HL-60 in this experiment, and the inhibitory ability against other cancer cell lines was varied by orders of magnitude. The mechanism is anti-tumor, and the mechanism is not resistant to all tumors.

Table 4 Inhibition of tumor cell proliferation by test compound

Example 10 Effect of B63 and B19 on the survival rate of human non-small cell lung cancer H460 cells

Human non-small cell lung cancer cell line ((NCI-H460), available from US Cell, Strain Bank (ATCC). Human non-small cell lung cancer H460 cells were seeded in 96-well plates at a cell density of 5000 cells/well, The cells were cultured for 24 hours in a CO ₂ incubator, and cells were treated with different concentrations of compounds for 24 hours. The number of cells was measured using a Cell Titer Cell Counting Kit (Promega Co., USA), and the method was performed according to the kit manual. Compared with the blank control group, the number of cells in the blank control group was calculated as 100%.

The cell counting experiment was further shown (see Figure 7 for the results), and the survival rate of H460 was 16.3% after 24 hours of treatment with B19 at 20 uM; the survival rate was only 12.2% at 30 uM. After 24 hours of treatment with BZ at 20 uM, the survival rate of H460 was 14.5%; at 30 uM, the survival rate was only 7.8%. Curcumin did not show good cytotoxicity at the same concentration. Even if the concentration was increased to 50 uM, the apoptotic rate was still about 40%. Example 11 Flow cytometry of apoptosis induced by B19 and B63 in human non-small cell lung cancer H460 cells

Human non-small cell lung cancer cell line (NCI-H460 and H358), derived from American cell, strain cell (ATCC). H460 was inoculated into 6mm well culture plates, and B63 and B19 dissolved in DMSO were added to culture after 24 h. The plates were incubated for a final concentration of 5, 10 and 20 μΜ for 24 h. Then rinsed three times with PBS buffer and added with 0.25% tryptan-EDTA. Centrifuge and then suspend the cells at 0.5 1 ^ 88. Then at 37 , lOOmg /mL RNAse and 0.1% Triton X-100 were stained with Annexin V and propidium iodide (PI) for 30 min and analyzed by flow cytometry (FC500, Beckman, USA). The results are shown in Figure 8, which shows B63 after 24 h. And B19 showed dose-dependent induction of apoptosis, 20 μΜ of Β63 significantly induced apoptosis (16.31%), and B19 induced apoptosis rate at the same concentration (13.88%). Example 12 B19 and Β63 should be on endoplasmic reticulum The effect of CHOP protein in the pathway

Endoplasmic reticulum stress (ER stress) involves and mediates apoptosis induced by multiple drugs. According to Haidara K et al (Haidara K, et al. Toxicol Appl Pharmacol 2008, doi: 10.1016/i.taap.2008.01.010) The test method described is a test for the development of ER stress toward apoptosis in H460 cells using B 19 and B63. Specifically, 1.2χ 10 ⁶ cells were cultured at 37 ° C with culture medium, and after 24 hours, the culture solution was renewed and different concentrations of the compound were added (the control group was added with 3 uL of DMSO), and after the corresponding period of time was continued, the cells were collected. Total protein, Western blot was used to detect CHOP content, and Actin was used as a calibration protein. The result is shown in Figure 9.

Our study found that the three signaling pathways of ER stress are induced to activate in the early stages of B19 and B63. The stimulation of B 19 and B63 causes the ER stress of H460 cells to develop in the direction of apoptosis, so CHOP ( C/EBP- homologous protein Activation is an insurmountable part of it. The data in Figure 9 shows very well that B19 and B63 activate CHOP to the highest value at 12 hours. After 3h, 6h and 12h of stimulating cells of B19 and B63, the CHOP protein in the cells increased significantly. CHOP is an important signaling molecule that is transformed from anti-apoptosis to pro-apoptosis. It can be seen that B19 and B63 activate the apoptotic signaling molecule CHOP through ER stress and exert its anticancer effect. Example 13 Activation of Caspase-3 and Caspase-9 by B19 and B63

The caspase channel is another important pro-apoptotic signaling protein in addition to CHOP downstream of ER stress. We examined the effects of B19 and B63 on caspase-3 and caspase-9. Specifically, 1.2 < 10 ⁶ H460 cells were cultured at 16 ° C in 1640 culture medium, and the culture solution was updated 24 hours later. After treatment with different concentrations of B 19 , B63 and curcumin for 24 hours, the cells were harvested and total protein was collected. Western blot was used to detect caspase-3 p30 and pl7, caspase-9 p46, p35 and B p22, procaspase-3 and B procaspase. -9 content, Actin as a calibration protein.

The result is shown in Fig. 10. The active forms of caspase-3 and caspase-9 were significantly activated after 24 hours of B19 treatment at 20 μΜ, indicating that caspase-3 and caspase-9 are also involved in the signal transduction pathway of B19-induced apoptosis. In the detection of B63, the inactive forms of caspase-3 and caspase-9, procaspase-3 and procaspase-9, were significantly reduced after treatment with 20 μΜ of Β63, indicating that the caspase gradually changed from inactive form to active form. Therefore, the compound of the present invention induces apoptosis by activation of caspase-3 and caspase-9. Example 14 Inhibition rate of mouse S180 sarcoma

Kunming mice, female, 18-22 grams, first grade, purchased from the Animal Center of the Academy of Military Medical Sciences; S 180 sarcoma cells, purchased from the Laboratory of Hematology, Institute of Radiation Medicine, Academy of Military Medical Sciences.

Aseptically inoculated S180 cells in the peritoneal cavity for 7-10 days, the ascites of the mice was diluted with physiological saline to a concentration of S180 cells to 2. 5xl0 ⁶ /ml, and the body weight of 18-22 g of Kunming female mice was taken. Inoculate 0.2 ml containing ⁵ x ^{10 5} cells. The next day, randomized, B63 and B19 were formulated into different concentrations, each mouse was intraperitoneally injected with 0.2 ml; negative control group per mouse peritoneal cavity 2 ml of physiological saline was administered continuously for 7 days; the positive control was administered with cyclophosphamide, administered once in group, and subcutaneous injection of 0.2 ml equivalent to a dose of 50 mg/kg. After the last administration for 24 hours, the mice were sacrificed by cervical dislocation and weighed, and the tumor mass was removed. The tumor weight was called.

Kunming female mice were injected intraperitoneally with B63 and B19 physiological saline solution (750 mg/kg dose) for 7 consecutive days. During the administration period, the animals gained weight and there were no other obvious adverse reactions. Based on this, the doses of B63 and B19 were studied to inhibit the S180 transplanted tumor in mice. The results are shown in Table 5 and Table 6.

Table 5 Inhibition of mouse S180 glioblastoma by B63 Negative control 2.080±0.866 10 - -

5 mg / kg / day 1.824 ± 0.755 10 12.3 P < 0.2

10 mg / kg / day 1.354 ± 0.576 10 34.9 P <0.025

25 mg / kg / 12h 1.127 ± 1.133 10 45.8 P <0.025

50 mg / kg / day 0.801 ± 0.170 10 61.5 P < 0.05

100 mg / kg / day 0.422 ± 0.069 10 79.7 P < 0.05

50 mg / kg cyclophosphamide 0.278 ± 0.116 10 86.6 P <0.01

Table 6 Inhibition of mouse S180 glioblastoma by B19

Dosage Tumor weight (g X±SD) Case number Inhibition rate (%) Significance Negative control 1.680±0.866 10 - -

5 mg / kg / day 1.341 ± 0.355 10 20.2 P < 0.2

10 mg / kg / day 1.113 ± 0.276 10 33.8 P <0.025

25 mg / kg / 12 h 0.787 ± 0.133 10 53.2 P <0.025

50 mg / kg / day 0.329 ± 0.100 10 80.4 P < 0.05

100 mg / kg / day 0.265 ± 0.054 10 84.2 P < 0.05

50 mg / kg cyclophosphamide 0.278 ± 0.116 10 83.5 PO.01 It can be seen from the above experimental results that B63 and B19 have a certain inhibitory effect on mouse S180 sarcoma, 10 mg / kg / day, 25 mg / kg / day 50 mg / kg / day, 100 mg / kg / day, the four dose inhibition rates were more than 30%, and statistically significant; with the increase of the dose, the inhibition has an increasing tendency; the effect of the divided dose is given in a single dose medicine. Example 15 Inhibition of Mouse LLC Non-small Cell Lung Cancer Kunming B6 mice, female, 18-22 grams, first grade, purchased from the Animal Center of the Academy of Military Medical Sciences; LLC non-small cell lung cancer cells, purchased from the Beijing Institute of Neurosurgery.

The tumors of the second-generation LLC non-small cell lung cancer cell tumor mice were inoculated subcutaneously under sterile peeling. The well-growth tissues and normal saline were picked and homogenized at a ratio of about 3× ^{10 6} /ml. The body weight of 18-22 g Kunming female mice, each mouse left sputum subcutaneously inoculated with 0. 2 ml containing lxlO ⁶ cells. The grouping and administration were the same as above, and continuous administration was carried out for 13 days.

Kunming female mice were intraperitoneally injected with B63 and B19 physiological saline solution (750 mg/kg) for 7 consecutive days, and the animals gained weight during the administration period without any other significant adverse reactions. Based on this, the doses of B63 and B19 were tested for the inhibition of mouse LLC non-small cell lung cancer tumors. The results are shown in Tables 7 and 8.

Table 7 Inhibition of B63 on mouse LLC non-small cell lung cancer cells

Dosage Tumor weight (g X±SD) Number of cases Inhibition rate (%) Significance Negative control 2.461±1.600 22 - -

5 mg / kg / day 1.366 ± 0.587 22 44.5 P < 0.1

25 mg / kg / day 1.007 ± 0.249 21 59.1 P <0.025

100 mg / kg / day 0.807 ± 0.087 20 67.2 P < 0.05

50 mg / kg cyclophosphamide 0.199 ± 0.216 22 91.9 PO.01

Table 8 Inhibition of B19 on mouse LLC non-small cell lung cancer cells

5 mg / kg / day 1.443 ± 0.587 22 41.4 P < 0.1

25 mg / kg / day 0.671 ± 0.149 21 72.7 P <0.025

100 mg / kg / day 0.404 ± 0.107 20 83.6 P < 0.05

50 mg / kg cyclophosphamide 0.199 ± 0.216 22 91.9 P <0.01 From the above experimental results, it can be seen that B63 and B19 have a certain inhibitory effect on mouse G422 glioblastoma, 5 mg / kg / day, 25 The three dose inhibition rates of mg/kg/day and 100 mg/kg/day were all over 30%, and were statistically significant. As the dose increased, the inhibition increased.

Claims

Rights request

1. Application method of the compound represented by Formula I in the preparation of drugs for preventing and/or treating diseases, wherein the disease can be caused by reducing the activity of 11β-hydroxybody dehydrogenase 1 reductase and/or increasing 11β-hydroxyl Diseases treated and/or prevented by the activity of body dehydrogenase 1 oxidase,

Formula I

in,

R ₁₃ and R ₂₃ are not alkenyloxy groups, preferably R ₁₃ and R ₂₃ are each H, halogen, lower alkyl, hydroxyl, or optionally N, N-dimethylamino-substituted lower alkoxy;

R ₁₄ and R ₂₄ are each H or lower alkyl; and

R ₁₅ and R ₂₅ are each H or lower alkyl, or R ₁₅ and R ₂₅ are bonded together, where n is 1, 2, 3, or 4.

2. Methods for preventing and/or treating diseases, wherein the diseases can be treated by reducing the activity of 11β-hydroxybody dehydrogenase 1 reductase and/or increasing the activity of 11β-hydroxybody dehydrogenase 1 oxidase. /or prevent diseases, the method includes administering to the patient a medicament containing an effective dose of a compound represented by formula I

Ιίιι

Formula I

in, Ru and R ₂₁ are each H, halogen, optionally halogen-substituted lower alkyl, or lower alkoxy; R ₁₂ and R ₂₂ are not alkenyloxy or halogen-substituted lower alkyl, preferably R ₁₂ and R ₂₂ are each is H, halogen, or lower alkoxy;

R ₁₄ and R ₂₄ are each H or lower alkyl; and

3. The method of claim 1 or 2, wherein the compound represented by formula I is a dual regulator of 11 β-hydroxysome dehydrogenase 1, preferably a specific dual regulator of 11 β-hydroxysome dehydrogenase 1.

4. The method of claim 1 or 2, wherein the compound represented by formula I is

Formula A

in,

Ri is H, halogen, or lower alkoxy;

R ₂ is H, halogen, or lower alkoxy;

R ₃ is H, halogen, or N, N-dimethylamino-substituted lower alkoxy; and

R ₄ is H,

or,

Formula B in,

is 11, halogen, or lower alkoxy;

R ₂ is H or lower alkoxy;

R ₃ is H or hydroxyl; and

R ₄ is H,

or,

Formula C

in,

Each is H, halogen, halogen-substituted lower alkyl, or lower alkoxy; R ₂ is H or lower alkoxy;

R ₃ is H or hydroxyl; and

R ₄ is H,

Optimal formula

More preferably the compound represented by formula I is B6

5. The method of any one of claims 1 to 4, wherein the disease is a glycolipid metabolism disease, hypogonadism, sexual dysfunction and/or infertility, preferably selected from the group consisting of diabetes, obesity, and atherosclerosis. , cirrhosis, fatty liver, hyperglycemia, hyperlipidemia, hypertension, hypogonadism, sexual dysfunction and/or infertility, most preferably diabetes, fatty liver and/or stress-induced testosterone reduction, such as type II diabetes.

6. The method of claim 5, wherein the drug contains an effective dose of B6, and the effective dose is 1-10 mg/kg rat.

7. The method of claim 1 or 2, wherein the disease is a disease that can be treated or prevented by activating CHOP and apoptotic cells and/or by activating caspase-3 and caspase-9 and apoptotic cells, such as tumors. and cancer, preferably gastric cancer, myeloid leukemia, oral epithelial cancer, lung cancer or sarcoma.

8. The method of claim 7, wherein the drug contains an effective dose of B19 or B63.

9. Pharmaceutical composition, which contains an effective dose of B6 and a pharmaceutically acceptable carrier, wherein the effective dose is 1-10 mg/kg rat.

10. The pharmaceutical composition of claim 9, which is used to treat diabetes or prevent stress-induced testosterone reduction.

11. A kit for treating diabetes, comprising B6, and a label indicating administration at a dose of 1-10 mg/kg rat.

12. A method for in vitro screening of 11β-hydroxysome dehydrogenase 1 dual regulators, which includes,

(1 M was used to measure the half inhibitory concentration (IC50) of the test compound on the reductase activity of 11 β-hydroxysome dehydrogenase 1 in Leydig cells, CHOP cells transfected with human 11 β HSD1 and/or microsomal protein. ;

(2) In vitro determination of the effect of the test compound on Leydig cells, CHOP cells transfected with human 11βHSD1 and/or microsomal proteins 11 The half effective concentration (EC50) of the oxidase activity of β-hydroxysteroid dehydrogenase 1;

(3) Select compounds with IC50 less than 30 μM and EC50 less than 30 μM as 11 β-hydroxysteroid dehydrogenase 1 dual regulators.

13. The method of claim 12, further comprising determining in vitro the half inhibitory concentration of the 11 β-hydroxybody dehydrogenase 1 dual modulator on the oxidase activity of the 11 β-hydroxybody dehydrogenase 2 of the microsomal protein ( IC50), and then select compounds with an IC50 greater than 100 μM as specific 11 β-hydroxysteroid dehydrogenase 1 dual modulators.

14. Compound of claim 12 or 13

Formula Π

in,

R _u , R ₂₁ , R ₁₂ , R ₂₂ , R ₁₃ , R ₂₃ , R ₁₄ and BR ₂₄ are each H, halogen, hydroxyl, amino, optionally substituted alkyl, optionally substituted alkoxy, or any optionally substituted alkenyloxy, wherein the substituents include alkyl, hydroxyl, amino, aryl and/or heterocycle; and

15. Compounds represented by the following formula