Medicaments, compositions, and methods related thereto

Abstract

The present application relates to medicaments, compositions, and methods related thereto. The present disclosure provides compounds of Formula (1)-(3) and compositions and methods of use thereof. The present disclosure also provides methods of making the provided compounds and compositions, and methods of characterizing the provided compounds and compositions.

Description

[0001] This application is a divisional application of the invention patent application with the application date of May 1, 2018, application number 201880027339.7, and invention name “Pharmaceutical agents, compositions and related methods thereof”. Technical Field

[0002] The present application relates to pharmaceutical agents, compositions and related methods.

[0003] Priority claim

[0004] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 508,730, filed May 19, 2017. The entire contents of this priority application are incorporated herein by reference. Background Art

[0005] Diabetes is a group of metabolic diseases in which high blood sugar levels are chronic. There are various types of diabetes. Type I diabetes results from the pancreas's inability to produce enough insulin. Type II diabetes begins with insulin resistance, a condition in which cells fail to respond appropriately to insulin and, as the disease progresses, may lead to insulin deficiency. Worldwide, approximately 400 million people suffer from diabetes, with Type II diabetes accounting for approximately 90% of cases. Insulin or insulin analogs are commonly used to treat Type I diabetes. Metformin is generally recommended as a first-line treatment for Type II diabetes. Summary of the Invention

[0006] Previous work (see WO2015 / 137983, US2016 / 0045533 (granted US Patent #9,642,874), and US2016 / 0082033, each of which is incorporated herein by reference) has identified certain selenium-organic compounds that have shown interesting and valuable activity in certain disease model systems. Specifically, this work has identified compounds of Formulas I, II, and III shown below:

[0007]

[0008] The report stated:

[0009] 1. A combination of the following three compounds: 5'-methylselenoadenosine (compound C, which is a compound of formula I), Se-adenosyl-L-homocysteine ​​(compound D, which is a compound of formula II) and γ-glutamyl-methylseleno-cysteine ​​(compound E, which is a compound of formula III),

[0010]

[0011] But not the single compound, can significantly attenuate G6pc expression, thus representing a novel approach to reduce hepatic glucose output (see WO2015 / 137983);

[0012] 2. Compounds C and D were each able to increase mitochondrial (MT) potential in mouse skeletal muscle myoblasts C2C12 cells, suggesting that compounds C and D may be suitable for T2DM research and control (see WO2015 / 137983);

[0013] 3. The combination of compounds C, D, and E can reduce hepatic glucose output and improve glucose tolerance in an insulin-resistant diabetic mouse model. Therefore, the combination of compounds C, D, and E may be suitable for the treatment of obesity, hyperglycemia, and diabetes (see US20160045533);

[0014] 4. Compounds C and D may be useful for treating sarcopenia caused by the gradual loss of MT function in the kidney or skeletal muscle (see WO2015 / 137983);

[0015] 5. Compound C can enhance gluconeogenesis in brain cells, which may be beneficial for the survival of brain cells in AD (see US20160082033); and

[0016] 6. Compounds C and D can inhibit Tau hyperphosphorylation in AD brain (see US20160082033).

[0017] Thus, previous work suggests that compounds C, D, and E may be useful in certain situations; specifically, it is taught that in some situations (e.g., inhibiting Tau hyperphosphorylation in AD brains and enhancing gluconeogenesis in brain cells), compound C or compound D may be useful alone. In other situations (e.g., reducing hepatic glucose output, improving glucose tolerance, and / or otherwise effectively treating obesity, hyperglycemia, and / or diabetes), it is shown that these compounds are not useful alone, but rather are effective in combination.

[0018] The present disclosure provides novel selenium-containing compounds that share certain structural relationships with the compounds of Formulas I and II above (and specifically with Compounds C and D) that unexpectedly exhibit potent activity alone in a variety of situations.

[0019] The present disclosure particularly shows that the compounds provided show comparable or better biological activity than the CDE combination in reducing the liver glucose output of diabetic subjects with insulin resistance and / or improving glucose tolerance. The present disclosure also teaches that the compounds provided may have comparable or better biological activity than compound C and / or D in enhancing gluconeogenesis (e.g., in brain cells) and / or inhibiting Tau hyperphosphorylation (e.g., in AD brain).

[0020] The present disclosure provides compositions containing and / or delivering such compounds (and / or one or more degradation products and / or active metabolites thereof), as well as various methods (e.g., preparation, characterization and / or use) and / or materials (e.g., intermediates, degradation products, metabolites [particularly active metabolites], etc.) related to such provided compounds. In some embodiments, the provided technology relates to and / or is particularly suitable for regulating glucose metabolism; enhancing AS160 phosphorylation to translocate glucose transporters (GLUTs) from cytoplasmic vesicles to the plasma membrane for glucose uptake; and / or enhancing glucose uptake in both the liver and skeletal muscle. In some embodiments, the provided technology relates to and / or is particularly suitable for treating hyperinsulinemia, obesity, diabetes, hyperglycemia, polycystic ovary syndrome (PCOS), Alzheimer's disease (AD) and / or sarcopenia. In some embodiments, the provided technology relates to and / or is particularly suitable for treating type II diabetes-related conditions, such as diabetic retinopathy, nephropathy, neuropathy and vascular conditions.

[0021] In some embodiments, the present disclosure provides compounds of formula (1):

[0022]

[0023] or a pharmaceutically acceptable salt, prodrug or isomer thereof, wherein

[0024] R 2 and R 3 Each of the is independently H or -C(O)-R, wherein each R is independently C 1-6 Alkyl or 3-8 membered carbocyclic or heterocyclic ring, wherein R 2 and R 3 It can’t all be H;

[0025] or R 2 With R 3 Together they form -(CH2) n -C(O)-(CH2) m -, wherein each of n and m is independently 0-3, and n+m≤3;

[0026] R 5 Yes-C 1-6 Alkyl or -C 1-6 Alkyl -CH(NH2)COOH;

[0027] R 8 is H or halogen; and

[0028] X is H or halogen,

[0029] wherein the carbocyclic ring, heterocyclic ring, -(CH2)n - and -(CH2) m Each of the - moieties may independently be optionally replaced by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and

[0030] Each C 1-6 The alkyl moieties may be optionally substituted 1-3 times independently with -OH, halogen, NH2 or CN.

[0031] In some embodiments, the present disclosure provides compounds of formula (2):

[0032]

[0033] or a pharmaceutically acceptable salt, prodrug or isomer thereof,

[0034] where R 8 is H or halogen;

[0035] X is H or halogen;

[0036] Each R5' is independently H or halogen; and

[0037] Each R is independently C 1-6 Alkyl, each of which independently may be optionally substituted 1-3 times with halogen.

[0038] In some embodiments, the present disclosure provides compounds of formula (3):

[0039]

[0040] or a pharmaceutically acceptable salt, prodrug or isomer thereof,

[0041] where R 8 is H or halogen;

[0042] X is H or halogen; and

[0043] Each R' is independently H or halogen.

[0044] In some embodiments, the present disclosure provides compositions comprising or delivering a compound of any one of formula (1)-(3). In some embodiments, the present disclosure provides compositions comprising a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the present disclosure provides compositions delivering an active moiety of a compound of any one of formula (1)-(3).

[0045] In some embodiments, the present disclosure provides methods for treating a disease, disorder, or condition by administering a compound or composition as described herein. In some embodiments, the methods provided enhance AS160 phosphorylation to translocate glucose transporters (GLUTs) from cytoplasmic vesicles to the plasma membrane for glucose uptake. In some embodiments, the methods provided enhance glucose uptake in both the liver and skeletal muscle. In some embodiments, the methods provided alleviate hyperinsulinemia without impairing renal function and / or causing liver damage.

[0046] In some embodiments, the present disclosure provides methods of treating an insulin-related disorder comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0047] In some embodiments, the present disclosure provides a method of treating an insulin resistance disorder comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0048] In some embodiments, the present disclosure provides a method for treating a glucose metabolism disorder comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the glucose metabolism disorder involves a blood glucose level that is not within a normal range. In some embodiments, the glucose metabolism disorder is associated with a glucose uptake and / or transport defect. In some embodiments, the glucose metabolism disorder is diabetes, glyceraldehyde-3-phosphate dehydrogenase deficiency, diabetes, hyperglycemia, hyperinsulinemia, or hypoglycemia.

[0049] In some embodiments, the present disclosure provides a method for treating a disorder of glucose transport comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the disorder of glucose transport is glucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivo disease (GLUT1 deficiency syndrome (GLUT1DS)).

[0050] In some embodiments, the present disclosure provides a method of treating obesity comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0051] In some embodiments, the present disclosure provides a method of treating diabetes comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0052] In some embodiments, the present disclosure provides a method of treating hyperglycemia comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0053] In some embodiments, the present disclosure provides a method of treating polycystic ovary syndrome (PCOS) comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0054] In some embodiments, the present disclosure provides a method of treating Alzheimer's disease (AD) comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0055] In some embodiments, the present disclosure provides a method of treating sarcopenia comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0056] In some embodiments, the present disclosure provides methods of inhibiting glucose production comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0057] In some embodiments, the present disclosure provides a method of improving glucose tolerance comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0058] In some embodiments, the present disclosure provides a method for activating and / or restoring insulin receptor function and its downstream signaling in a subject in an insulin-resistant state, comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0059] In some embodiments, the present disclosure provides a method for treating a mitochondrial-related disease (e.g., caused by mitochondrial dysfunction), comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the mitochondrial-related disease may be a degenerative disease (e.g., cancer, cardiovascular disease and heart failure, type 2 diabetes, Alzheimer's and Parkinson's disease, fatty liver disease, cataracts, osteoporosis, muscle atrophy such as sarcopenia, sleep disorders, and inflammatory diseases such as psoriasis, arthritis, and colitis). In some embodiments, the present disclosure provides a method for enhancing mitochondrial function, comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0060] In some embodiments, the present disclosure provides a method for enhancing gluconeogenesis in the brain, comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the provided methods increase glucose uptake in the brain. In some embodiments, the provided methods are used to maintain or restore brain function (including memory and learning).

[0061] In some embodiments, the present disclosure provides methods of preparing a compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0062] In some embodiments, the present disclosure provides methods of characterizing a compound of any one of Formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0063] In some embodiments, the present disclosure provides methods of making the compositions described herein.

[0064] In some embodiments, the present disclosure provides methods of characterizing a composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Effects of insulin and pure compounds (listed in Table 1) on glucose production in HepG2 cells. Cells were treated with 0.24% DMSO (maximum volume of test compound solvent), insulin, or the listed compounds in serum-free glucose production medium for 48 hours. Data were normalized by cell number as described above and are presented as mean ± SEM of at least 3 samples per group.

[0066] Figure 2 Comparison of the efficacy of Compound #43 and metformin in both HepG2 and H4IIE cells. HepG2 cells were treated with 0.24% DMSO (maximum volume of Compound #43 solvent), insulin, Compound #43, and metformin in serum-free glucose-producing medium for 48 hours, while rat hepatocytes were treated for 24 hours. Glucose levels in the culture medium were normalized by the number of cells in each sample. Data are presented as mean ± SEM for between 3 and 8 samples per group.

[0067] Figure 3 Effects of compounds #C, #50 and #43 on the long-term treatment of Lepr db / db Differential effects on blood glucose levels and serum HbA1c levels in mice. Starting from 38 days of age, Lepr db / dbMice were intraperitoneally injected with saline (containing 0.2% of the compound solvent DMSO), Compound #C, #50, and #43. Blood glucose levels were measured using a glucometer at 81 days of age after overnight fasting. Serum HbA1c levels were measured at 90 days of age. Relative HbA1c levels were calculated by dividing the HbA1c levels in mice treated with Compound #C, #50, and #43 by the mean HbA1c level in saline-treated mice. Data are presented as mean ± SEM of the indicated number of animals. P values ​​were calculated by comparing the treated and control / saline groups.

[0068] Figure 4 Compound #43 and its sulfur analog #68 have an effect on the long-term treatment of Lepr db / db Effects of daily administration of Lepr on blood glucose and HbA1c levels in mice starting from 38 days of age db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO), compound #43 and #68 at a dose of 25 μg of selenium or sulfur per kg body weight (compound #43 (0.136 mg) or compound #68 (0.298 mg), respectively). Blood glucose and HbA1c levels were measured in mice fasting overnight at 128 days of age. The HbA1c levels in mice treated with compound #43 and #68 were divided by the mean HbA1c levels in mice treated with saline to obtain relative HbA1c levels. Data are presented as the mean ± SEM of the indicated number of animals. Fasting blood glucose levels in non-diabetic / obese mice (indicated by the symbol # in the bar graph) were obtained from 4-month-old wild-type C57 male mice in the laboratory. Different letters in the bar graph indicate P values ​​less than 0.05.

[0069] Figure 5 Effects of compounds #43, #69 and #70 on the long-term treatment of Lepr db / db Differential effects of fasting blood glucose and HbA1c levels in mice. db / db Mice were intraperitoneally injected daily with saline (containing 0.2% of the compound solvent, DMSO), compounds #43 (0.136 mg), #69 (0.145 mg), and #70 (0.153 mg) at a dose of 25 μg of selenium per kg of body weight for 43 days (for blood glucose measurements) and 90 days (for HbA1c measurements). They were fasted overnight and then subjected to blood glucose analysis (using a glucometer) or blood HbA1c measurements. Data are presented as mean ± SEM of the indicated number of animals. P values ​​were derived by comparing the treated and control / saline groups.

[0070] Figure 6 Acute treatment with compound #43 resulted in the db / db Blood glucose levels were reduced in male mice.db / db Male mice were fasted overnight and then injected intraperitoneally with saline (containing 0.2% DMSO, the maximum injection volume of compound #43 stock solvent); 0.0054, 0.054, 0.54 and 5.4 mg compound #43 / kg body weight. db / db Blood glucose levels of mice immediately before injection and 1, 2, 3, 5 and 8 hours after injection (under fasting conditions, but with free access to drinking water). The glucose levels reduced in individual mice were obtained by subtracting the glucose levels immediately before injection from the blood glucose levels of each time period after injection. Data are given as the mean ± SEM of the indicated number of animals. When compared to the corresponding time points of DMSO / saline injection, all other reductions were significant (P < 0.05) except for the P values ​​associated with the early time points treated with 0.0054 mg / kg BW as shown.

[0071] Figure 7 Acute treatment with compound #43 reduces Lepr in ad libitum fed conditions db / db Blood glucose levels in male mice were measured before and 24 hours after intraperitoneal injection of saline (containing 0.2% of the compound solvent DMSO) or Compound #43 at a dose of 5.4 mg / kg body weight in 6-week-old Lepr mice with free access to food and water. db / db Blood glucose levels in male mice. The relative blood glucose levels before ip injection were normalized by the mean glucose levels of all five mice in the group and referred to as 100%. 24 hours after injection, the relative blood glucose levels in each mouse were normalized by their pre-injection glucose levels. Different letters represent statistically significant differences between groups (P < 0.05).

[0072] Figure 8 . Long-term treatment with compound #43 increases Lepr db / db Glucose tolerance in mice. AB. Daily administration of 38-day-old Lepr db / db Mice were intraperitoneally injected with (A) saline (containing 0.2% compound solvent DMSO), compound #43, compound #C, and compound #50 for 43 days, or (B) compound #68 or compound #43 for 60 days. db / dbMice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO), compound #43, compound #69, and compound #70 for 43 days. The daily injection dose for all listed compounds was 25 μg of selenium or sulfur per kg body weight of each test compound. At the end of treatment, the mice were fasted overnight, injected with glucose (2 g / kg body weight), and blood glucose levels were measured immediately before glucose injection (referred to as time zero) and 0.25 hours, 0.5 hours, 1 hour, and 2 hours after glucose injection using a glucometer with a maximum reading of 600 mg / dL. Glucose levels exceeding this limit were recorded as 600 mg / dL. Data are presented as mean ± SEM of the indicated number of animals. *P < 0.05, **P < 0.01, ***P < 0.001 when compared to mice treated with saline (A, C) or compound #68 (B) at the same time points.

[0073] Figure 9 . After long-term treatment with compound #43, Lepr db / db The G6pc mRNA was attenuated in the liver of mice. The dose of 25 μg of selenium per kg body weight per day for each compound was used to treat Lepr on postnatal day 38. db / db Mice were intraperitoneally injected with saline (containing 0.2% compound solvent DMSO), compound #50, and #43 for 52 days. Liver RNA isolated from these compound-treated mice was subjected to QRT-PCR. G6pc mRNA levels in each sample were normalized by Actb mRNA levels, and data are presented as mean ± SEM of five mice per group. P values ​​are relative to the saline group.

[0074] Figure 10Compound #43 inhibits basal G6pc / G6pc expression in AML-12 and human HepG2 cells, and the synergistic effect of Compound #43 and insulin in inhibiting G6pc expression in AML-12 cells. (A) Effect of selenium compounds on G6pc expression in AML-12 cells under serum-free conditions. AML-12 cells were treated with either no (control) or a combination of Compounds C and D, Compound #C, Compound #D, Compound #50, and Compound #43 at a dose of 300 parts per billion (ppb) of selenium (equivalent to 3.8 uM of each compound) for 24 hours in serum-free medium supplemented with insulin-transferrin-sodium selenite (ITS) and without dexamethasone (Dex). (B) Compound #43 inhibits G6pc expression in human HepG2 cells. HepG2 cells were incubated with 100 nM insulin or 600 ppb of Compound #43 in serum-free medium for 40 hours. (C) Compound #43 inhibits G6pc expression, and the synergistic effect of compound #43 and insulin in inhibiting G6pc expression in AML-12 cells pretreated with compound #43. AML-12 cells were pretreated with compound #43 for 24 hours in a culture medium containing FBS but without ITS / Dex, followed by a further treatment of compound #43 for 6 hours in a culture medium without serum / ITS / Dex in the presence or absence of 10 nM insulin. The G6pc mRNA levels in each sample were standardized by Actb mRNA levels, and data were given as the mean ± SEM of the indicated sample numbers in each group. The P values ​​in Figures AB were compared with the control group. The different letters in Figure C represent the statistical significance (P < 0.05) between the two groups.

[0075] Figure 11 Compound #43 inhibits G6pc expression, and the synergistic effect of compound #43 and insulin in inhibiting G6pc expression in AML-12 cells stimulated with diabetes. AML-12 cells were pretreated with compound #43 for 24 hours in a medium containing 10% FBS but without ITS / Dex, followed by treatment of compound #43 for another 6 hours in a serum-free and ITS-free medium in the presence or absence of insulin together with 8-CPT / Dex (diabetes stimulant). The G6pc mRNA level in each sample was normalized by Actb mRNA level, and the data were given as the mean ± SEM of the number of samples indicated in each group. The number at the top of each column is the mean of the G6pc mRNA level normalized by the level in the non-8-CPT / Dex-treated group (column #1). The different letters in the bars represent the statistical significance (P < 0.05) between the two groups.

[0076] Figure 12Long-term treatment with compound #43 enhanced insulin resistance in Lepr cells by Western blot analysis. db / db Phosphorylation of Pdk1 / Akt / Foxo1 signaling in the liver of mice. db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO) or compound #43 for 52 days. Western blot analysis was performed on liver tissue from mice treated with saline or compound #43 (100 μg protein per lane). (A) Western blot images. Protein levels in each sample were normalized by Gapdh levels, and data are presented as mean ± SEM of five mice per group in (B). P values ​​are compared with the saline group.

[0077] Figure 13 Compound #43 transiently activates PDK1 and AKT and subsequently inactivates FOXO1 in human liver HepG2 cells. HepG2 cells were serum-starved overnight and incubated without or with compound #43 (600 ppb) for the indicated times, followed by Western blot analysis. (A) Representative Western blots. (BF) Quantitative data for protein expression of (B) pPDK1, (C) pAKT, (D) total AKT, (E) pFOXO1T24, and (F) total FOXO1 in Western blots are presented as mean ± SEM of three samples. *P < 0.05 when compared to protein levels at 0 minutes (immediately prior to compound treatment) or the control group at each time point.

[0078] Figure 14 Compound #43 transiently activates Pdk1 and Akt and enhances Foxo1 phosphorylation in mouse liver AML-12 cells cultured under simulated diabetic conditions. AML-12 cells were cultured in DMEM / F12 medium with 10% FBS but without ITS / Dex for 24 hours and then serum-starved overnight in standard DMEM / F12 medium. These serum-starved AML12 cells were treated with the diabetic stimulants, 8-CPT (0.1 mM) and Dex (0.5 μM), in standard DMEM / F12 medium without (control) or in combination with 10 nM insulin or compound #43 (300 ppb) at the indicated time points, followed by Western blot analysis. (A) Representative Western blots. (B-E) Quantitative data for protein expression in Western blots are presented as mean ± SEM of three samples. *P < 0.05 when compared to the control (no insulin / compound #43 treatment) at each time point. Different letters in (E) represent statistical significance between the two groups (P < 0.05).

[0079] Figure 15Compound #43 and compound #50 have an effect on Lepr db / db Effects of 25 μg of selenium per kg of body weight per day on the expression of Glut4 gene in the liver of mice on postnatal day 38 Lepr db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO), compound #50, or compound #43 for 52 days. db / db Liver RNA samples isolated from mice were subjected to QRT-PCR analysis. Glut4 mRNA levels in each sample were normalized to Actb mRNA levels, and data are presented as mean ± SEM of four to five mice per group. P values ​​were calculated for treatment groups compared to the saline control group.

[0080] Figure 16 Compound #43 enhances Glut4 mRNA expression in mouse liver AML-12 cells. (A) QRT-PCR of basal Glut4 expression in AML-12 cells. AML-12 cells were expanded, plated on 24-well plates, and cultured overnight in DMEM / F12 medium with 10% FBS and no ITS / Dex. The cells were then incubated with vehicle (0.024% DMSO) or compound #43 (300 ppb) in serum-free DMEM / F12 medium for 24 hours (hr). (B) QRT-PCR of Glut4 expression in AML-12 cells cultured under simulated diabetic conditions. Expanded AML-12 cells were cultured on 24-well plates in DMEM / F12 medium with 10% FBS but no ITS / Dex for 24 hours and then serum-starved overnight in plain DMEM / F12 medium. Serum-starved AML-12 cells were then incubated with vehicle (0.024% DMSO) or with compound #43 (300 ppb) in the presence of diabetic stimulants, 0.1 mM 8-CPT and 0.5 μM Dex in serum-free plain DMEM / F12 medium for 6 and 24 hours. Glut4 mRNA levels in each sample were normalized by Actb mRNA levels, and data are presented as the mean ± SEM of three samples.

[0081] Figure 17 Enhanced glucose uptake in AML-12 cells after 1.5 hours of insulin and compound #43 treatment. Data are presented as mean ± SEM of the indicated number of samples per group. *P value less than 0.05 compared to the basal group.

[0082] Figure 18 In response to treatment with compound #43, in insulin-resistant Lepr db / dbEnhanced phosphorylation of downstream signaling molecules - Pdk1, Akt and Foxo1 - in the skeletal muscle of mice. db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO) or compound #43 for 52 days. db / db Western blot analysis of skeletal muscle protein extracts isolated from mice (100 μg protein per lane). (A) Images of Western blots. (B) Quantified protein levels (normalized by β-tubulin levels in each sample). Data are presented as mean ± SEM of five mice. P values ​​are calculated by comparison with the control (saline) group.

[0083] Figure 19 Effects of insulin and compound #43 on glucose uptake in differentiated mouse C2C12 (skeletal muscle) cells. Equal numbers of C2C12 cells were seeded in 96-well plates (5000 cells / well), cultured in 10% FBSDMEM medium for 5 days, and differentiated in DMEM medium containing 0.5% horse serum for 7 days. Fully differentiated C2C12 cells were pretreated overnight in serum / glucose-free DMEM medium without or with compound #43 (300 or 600 ppb), and then incubated in glucose-free DMEM medium at 37°C for 1.5 hours without (basal) or with insulin, compound #43, or both. After treatment, cells were incubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30 minutes, and then luminescence analysis was performed using Promega's GlucoseUptake-Glo assay kit. Data are given as the mean ± SEM of the indicated number of samples per group. P values ​​were obtained by comparison with the basal group.

[0084] Figure 20 Long-term treatment with compound #43 in insulin-resistant Lepr db / db Restoration of insulin receptor function in skeletal muscle of mice (indicated by increased phosphorylation of Insrβ at tyrosine 1146). db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO) or compound #43 for 52 days. db / dbWestern blot analysis was performed on skeletal muscle protein extracts isolated from mice (100 μg protein per lane). (A) Images of Western blots. (B-C) Quantification of protein levels (normalized by β-tubulin levels in each sample). Data are presented as mean ± SEM of five mice. P values ​​were calculated by comparing treatment values ​​with those in the control / saline group.

[0085] Figure 21 Compound #43 activates Insr and stimulates phosphorylation of Pdk1 / Akt / AS160 in differentiated mouse C2C12 (skeletal muscle) cells. Equal numbers of C2C12 cells were seeded in 12-well plates (60,000 cells / well), cultured in 10% FBS-DMEM medium for 5 days, and differentiated in DMEM medium containing 0.5% horse serum for 7 days. Fully differentiated C2C12 cells were serum-starved overnight and then treated with compound #43 (600 ppb) or insulin (200 nM) in serum-free DMEM medium at 37°C for (A-B) 5 minutes or (B-C) 30 minutes, followed by Western blot analysis. (A, C) Images of Western blots. (B, D) Quantified protein levels (normalized by β-tubulin levels in each sample). Data are presented as mean ± SEM of three samples per group. When compared to the control group (not treated with compound #43), *P<0.05, **P<0.01, ***P<0.001.

[0086] Figure 22 Long-term treatment with compound #43 in insulin-resistant Lepr db / db Restoration of insulin receptor function in the liver of mice (indicated by increased tyrosine phosphorylation of Insrβ). db / db Mice were injected intraperitoneally with saline (containing 0.2% compound solvent DMSO) or compound #43 for 52 days. db / db Liver protein extracts isolated from mice were subjected to ELISA assays for (A) phospho-Insrβ at Y1146 and (B) phospho-Insrβ at Y1150 / 1151, as well as Western blot analysis for β-tubulin. (A) Protein levels of phospho-Insrβ at Y1146 (normalized by β-tubulin levels in each sample). (B) Protein levels of phospho-Insrβ at Y1150 / 1151 (normalized by β-tubulin levels in each sample). Data are presented as mean ± SEM of the indicated number of mice. P values ​​were calculated by comparing treatment values ​​with those in the saline group.

[0087] Figure 23 Compound #43 activates INSR and stimulates AS160 phosphorylation in human liver HepG2 cells. HepG2 cells were seeded in 6-well plates (7×10 5 Cells were plated on 400 μg / mL plates (100 cells / well) and cultured in 10% FBS EMEM medium for 30 hours, followed by serum starvation overnight. These serum-starved HepG2 cells were then treated with compound #43 (600 ppb) at 37°C for 30 and 60 minutes (min) and then subjected to Western blot analysis. (A) Images of Western blots. (B) Quantified protein levels (normalized by ACTB protein levels in each sample). Data are presented as mean ± SEM of three samples per group. **P < 0.01 when compared to the control group (0 minute group, before compound #43 treatment).

[0088] Figure 24 . Long-term treatment with compound #43 leads to the db / db Reduction of serum insulin and alanine aminotransferase (ALT) but not creatinine levels in mice. 38-day-old male Leprazole (0.136 mg / kg body weight diluted in sterile saline) was administered. db / db Mice were injected intraperitoneally (ip) with normal saline (0.09% NaCl) containing 0.2% DMSO or compound #43 every day for 52 days. Serum from 3-month-old wild-type (non-diabetic) C57 mice was also collected. These serum samples were collected and assayed for (A) insulin, (B) ALT, and (C) creatinine. The numbers at the top of each column in (A) are the mean values ​​of insulin levels. The different letters in the bars represent the statistical significance between the two groups.

[0089] Figure 25 .Compound #43 targets the mode of action of type I and II diabetes.

[0090] Figure 26 Compound #43 and insulin directly activate the insulin receptor in a cell-free system. Equal amounts of native insulin receptor protein containing both α and β subunits were incubated with 0.003% DMSO (compound #43 solvent), compound #43, or insulin (Ins, 0.5 μM) in the presence of ATP, followed by Western blot analysis to detect phosphorylated Insrβ (activated Insr) at Y1146, 1150, and 1151. Different letters in the bars indicate statistically significant changes between the groups.

[0091] Figure 27Compound #68 was less effective than Compound #43 in activating the insulin receptor in a cell-free system. Equal amounts of native insulin receptor were incubated with 0.003% DMSO (solvent for Compound #43), Compound #43, or Compound #68 in the presence of ATP. Activated Insr protein was detected by Western blot analysis of phosphorylated Insrβ at Y1146, 1150, and 1151. Different letters in the bar graph indicate statistically significant changes between the groups.

[0092] Figure 28 Compound #43 reduces blood glucose levels in STZ-induced T1D mice following acute treatment. STZ-induced T1D mice with non-fasting blood glucose levels between 500-550 mg / dL were fasted overnight and injected intraperitoneally with either 5.4 mg / kg body weight of Compound #43 or saline containing 2% DMSO (the stock solvent for Compound #43, referred to as the control group) for 1, 2, and 3 hours. Blood glucose was then measured in the mice. P values ​​were calculated by comparing Compound #43 treatment with the control / saline group at each time point. DETAILED DESCRIPTION

[0093] definition

[0094] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover), and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

[0095] As used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds.

[0096] About: When the term "about" is used herein with respect to a value, it refers to a value that is similar to the value being referenced in the context. Generally, one skilled in the art who is familiar with the background will understand the associated degree of variance encompassed by "about" in this context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the value being referenced.

[0097] Administration: As used herein, the term "administering" generally refers to administering a composition to a subject or system. One of ordinary skill in the art will appreciate that a variety of routes can be used for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, and the like. In some specific embodiments, administration can be bronchial (e.g., by bronchial instillation), oral, cutaneous (which can be or include one or more of, for example, topical, intradermal, intradermal, transdermal, etc., of the dermis), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., within the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous administration, and the like. In some embodiments, administration can involve intermittent dosing (e.g., multiple doses separated in time) and / or regular dosing (e.g., single doses separated by a common time period). In some embodiments, administration can involve continuous dosing (eg, infusion) for a selected period of time.

[0098] Alzheimer's disease: As used herein, the term "Alzheimer's disease" or "AD" refers to a progressive disease of the human central nervous system. Brain insulin signaling is important for learning and memory, and insulin resistance in the brain is a major risk factor for AD. Restoration of insulin signaling has become a potential treatment for AD (White MF, Science 2003; 302: 1710–1; De Felice DG et al., Alzheimer's & Dementia 2014; 10: S26–S32). In certain embodiments, it manifests as dementia typical of the elderly; disorientation; memory loss; difficulty with language, calculation or visual-spatial skills; and psychiatric manifestations. In certain embodiments, it is associated with degenerating neurons in several areas of the brain. As used herein, the term "dementia" includes, but is not limited to, Alzheimer's dementia with or without psychotic symptoms. In a certain embodiment, the treatment methods provided herein are effective in treating mild, moderate and severe Alzheimer's disease in a subject. The stages of Alzheimer's disease further include: "moderate to severe cognitive impairment," also known as "moderate or intermediate Alzheimer's disease"; "severe cognitive impairment," also known as "moderate to severe or intermediate Alzheimer's disease"; and "profound cognitive impairment," also known as "severe or late Alzheimer's disease." Moderate to severe cognitive impairment is characterized by major gaps in memory and deficits in cognitive function. At this stage, help with some daily activities becomes essential. In severe cognitive impairment, memory difficulties continue to worsen, significant personality changes may occur, and affected individuals require a great deal of help with daily routines. Late-stage Alzheimer's disease, or profound cognitive impairment, is the final stage of the disease, when individuals lose the ability to respond to their environment, the ability to speak, and eventually the ability to control movement.

[0099] Biological activity: As used herein, the term "biological activity" refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or extent of a biological activity is assessed by detecting a direct or indirect product produced by a biological pathway or event of interest.

[0100] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any doses of a second regimen are administered); in some embodiments, such agents are administered on an overlapping dosing schedule. In some embodiments, "administering" a combination therapy may involve administering one or more agents or tropisms to a subject receiving the other agents or tropisms in the combination. For clarity, combination therapy does not require that the separate agents be administered together in a single composition (or even necessarily be administered simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0101] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another but are sufficiently similar to permit comparisons to be made between them so that one skilled in the art would understand that conclusions can reasonably be drawn based on the observed differences or similarities. In some embodiments, the conditions, circumstances, individuals, or sets of populations are characterized by a plurality of substantially identical features and one or a few different features. One of ordinary skill in the art will understand in context what degree of consistency is required in any given case to consider two or more such agents, entities, situations, sets of conditions, etc. to be comparable. For example, one of ordinary skill in the art will recognize that situations, individuals, or sets of populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant the reasonable conclusion that differences in the results or observed phenomena obtained in or with different situations, individuals, or sets of populations are caused by or indicative of changes in those features that have changed.

[0102] Diabetes: The main characteristic of diabetes is impaired beta cell function. One abnormality that occurs early in the disease progression of both type I and type II diabetes is the loss of the rapid insulin response induced by food. As a result, the liver continues to produce glucose, which increases the glucose intake and absorption from the basic components of the meal.

[0103] Type 2 Diabetes: A hallmark of type 2 diabetes is impaired insulin action, known as insulin resistance. Insulin resistance manifests as both a decrease in the maximum glucose elimination rate (GERmax) and an increase in the insulin concentration required to achieve GERmax. Consequently, to process a given glucose load, more insulin is required, and elevated insulin concentrations must be maintained for longer periods of time. Consequently, diabetics are exposed to elevated glucose concentrations for prolonged periods, further exacerbating insulin resistance. Furthermore, prolonged elevated blood glucose levels are themselves toxic to beta cells. Another characteristic of type 2 diabetes is a delayed response to increased blood glucose levels. While normal individuals begin releasing insulin within 2-3 minutes of food consumption, patients with type 2 diabetes may not secrete endogenous insulin until blood glucose begins to rise, followed by a phase II kinetic in which concentrations slowly increase to a prolonged plateau. Consequently, endogenous glucose production is not shut off after a meal and continues, causing patients to experience hyperglycemia (elevated blood glucose levels). Type 2 diabetes is caused by a different and less understood process. The early loss of insulin release and the subsequent sustained glucose release contribute to elevated glucose concentrations. High glucose levels promote insulin resistance, and insulin resistance causes a long-term increase in serum glucose concentration. This situation can lead to a self-amplification cycle, in which increasing insulin concentrations are less effective in controlling blood sugar levels. In addition, as mentioned above, elevated glucose levels are toxic to β cells, thereby reducing the number of functional β cells. Genetic defects that damage the growth or maintenance of the microvasculature that nourishes the pancreatic islets can also play a role in its worsening (Glee, SM et al., Nature Genetics 38: 688-693, 2006). Eventually, the pancreas becomes overwhelmed, and the individual progresses to produce insulin deficiency, similar to people suffering from type 1 diabetes.

[0104] Type 1 diabetes: Type 1 diabetes develops when the body's own immune system destroys the insulin-producing cells (beta cells) of the pancreas. This eventually leads to a complete deficiency of the insulin hormone.

[0105] Dosage form or unit dosage form: As used herein, the term "dosage form or unit dosage form" refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. In some embodiments, each such unit contains a predetermined amount of active agent. In some embodiments, such an amount is a unit dose (or a full fraction thereof) suitable for administration according to a dosing regimen that has been determined to be associated with a desired or beneficial outcome when administered to a relevant population (i.e., associated with a therapeutic dosing regimen). It is understood by those of ordinary skill in the art that the total amount of a therapeutic composition or therapeutic agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0106] Dosing regimen: As used herein, the term "dosing regimen" refers to a group of unit doses (usually more than one) that are administered separately to a subject, typically separated by time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each dose separated in time from the other doses. In some embodiments, individual doses are separated from each other by time periods of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen includes a first dose of the amount of the first dose, followed by one or more additional doses of the amount of the second dose that is different from the amount of the first dose. In some embodiments, a dosing regimen includes a first dose of the amount of the first dose, followed by one or more additional doses of the amount of the second dose that is the same as the amount of the first dose. In some embodiments, a dosing regimen is associated with a desired or beneficial outcome when administered in a relevant population (i.e., is a therapeutic dosing regimen).

[0107] Excipients: As used herein, the term "excipient" refers to non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like.

[0108] Honeymoon phase: As used herein, the term "honeymoon phase" of type 1 diabetes refers to the early stage of the disease characterized by early loss of insulin release, with remaining beta-cell function producing some insulin released with phase II kinetics.

[0109] Hyperglycemia: As used herein, the term "hyperglycemia" refers to a disease, disorder, or condition characterized by a fasting blood glucose concentration that is higher than normal. In some embodiments, hyperglycemia is characterized by a blood glucose concentration of 126 mg / dL or higher. In some embodiments, hyperglycemia is characterized by a blood glucose concentration of 280 mg / dL (15.6 mM) or higher.

[0110] Hypoglycemia: As used herein, the term "hypoglycemia" refers to a disease, disorder, or condition characterized by a blood glucose concentration below normal. In some embodiments, hypoglycemia is characterized by a blood glucose concentration of 63 mg / dL (3.5 mM) or less. In some embodiments, hypoglycemia causes symptoms such as cognitive impairment, behavioral changes, pallor, hypotonia, flushing, and weakness, which are recognized symptoms of hypoglycemia and disappear with appropriate caloric intake. In some embodiments, hypoglycemia is severe and requires glucagon injections, glucose infusions, or another aid.

[0111] Improve, increase, inhibit, or decrease: As used herein, the terms "increase," "increase," "inhibit," and "decrease," or their grammatical equivalents, indicate values ​​relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement can be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence of a particular agent or treatment (e.g., before and / or after), or in the presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement can be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of a relevant agent or treatment.

[0112] Insulin-related disorders: As used herein, the term "insulin-related disorders" refers to disorders involving insulin production, regulation, metabolism, and action in mammals. Insulin-related disorders include, but are not limited to, prediabetes, type I diabetes, type II diabetes, hypoglycemia, hyperglycemia, insulin resistance, secretory dysfunction, sarcopenia, loss of pancreatic beta cell function, and pancreatic beta cell loss.

[0113] Non-insulin-dependent patients with insulin-related conditions: As used herein, the term "non-insulin-dependent patients with insulin-related conditions" refers to patients with a diagnosed condition for which therapy with exogenously provided insulin is not the current standard of care. Non-insulin-dependent patients with insulin-related conditions not treated with exogenously administered insulin include early-stage type II diabetes, honeymoon stage type I diabetes, prediabetes, and recipients of insulin-producing cell transplants.

[0114] Insulin resistance: As used herein, the term "insulin resistance" refers to the inability of a patient's cells to respond appropriately or effectively to insulin. The pancreas responds to this problem at the cellular level by producing more insulin. Ultimately, the pancreas cannot keep up with the body's demand for insulin, and excess glucose accumulates in the bloodstream. Patients with insulin resistance typically have both high blood sugar levels and high insulin levels circulating in their blood.

[0115] Insulin resistance disorder: As used herein, the term "insulin resistance disorder" refers to any disease or condition caused or contributed to by insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin resistance syndrome, syndrome X, insulin resistance, high blood pressure, hypertension, high cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease (including stroke, coronary artery disease or myocardial infarction), hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications (including coronary heart disease), angina pectoris, congestive heart failure, stroke, dementia, cognitive function, retinopathy, neuropathy , kidney disease, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, some types of cancer (such as endometrial cancer, breast cancer, prostate cancer and colon cancer), pregnancy complications, poor female reproductive health (such as menstrual disorders, infertility, irregular ovulation, polycystic ovary syndrome (PCOS)), lipodystrophy, cholesterol-related conditions (such as cholelithiasis, cholecystitis and gallstones), gout, obstructive sleep apnea and breathing problems, osteoarthritis, and prevention and treatment of bone loss (such as osteoporosis).

[0116] Isomers: As is known in the art, many chemical entities (particularly many organic molecules and / or many small molecules) can exist in multiple structural (e.g., geometric, conformational, isotopic) and / or optical isomeric forms. For example, any chiral center can exist in R and S configurations, a double bond can exist in Z and E conformational isomers, certain structural elements can adopt two or more tautomeric forms, and certain structures can be substituted by one or more isotopically enriched atoms (e.g., deuterium or tritium for hydrogen, 12 C or 14 C substitution 13 C. 131 I replace 129 I, etc.). In some embodiments, it will be clear to those skilled in the art from the context that a description or reference to a particular compound structure herein may represent all of its structures and / or optical isomers. In some embodiments, it will be clear to those skilled in the art from the context that a description or reference to a particular compound structure herein is intended to cover only the isomeric forms described or mentioned. In some embodiments, a composition comprising a chemical entity that can exist in multiple isomeric forms includes multiple such forms; in some embodiments, such compositions include only a single form. For example, in some embodiments, a composition comprising a chemical entity that can exist as multiple optical isomers (e.g., stereoisomers, diastereomers, etc.) includes a racemic population of such optical isomers; in some embodiments, such compositions include only a single optical isomer and / or include multiple optical isomers that together retain optical activity.

[0117] Parenteral: As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, typically by injection. In some embodiments, parenteral administration can be or include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and / or infusion.

[0118] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0119] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, shows a statistically significant likelihood of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including those suitable for the following: oral administration, such as an infusion (aqueous or non-aqueous solution or suspension), tablets, such as those for oral, sublingual and systemic absorption, boluses, powders, granules, pastes for administration to the tongue; parenteral administration, such as a sterile solution or suspension or a sustained-release formulation, such as by subcutaneous, intramuscular, intravenous or epidural injection; topical application, such as a cream, ointment or controlled-release patch or spray applied to the skin, lungs or mouth; intravaginal or rectal, such as as a vaginal suppository, cream or foam; sublingual; ophthalmic; transdermal; or nasal, pulmonary and to other mucosal surfaces. It will be appreciated by those skilled in the art that, in general, any composition prepared for administering to human or animal subjects can be considered as pharmaceutical compositions in some embodiments, no matter whether it administers and needs medical prescription.Therefore, for example, in some embodiments, food or food supplement compositions (for example, liquid or solid consumable compositions, such as milk shake or sports drink or nutritional supplement powder) can be considered as pharmaceutical compositions. Alternatively or in addition, in some embodiments, pharmaceutical compositions can be specially regulated and approved for the preparation administered to relevant subjects by appropriate government agencies (such as the U.S. Food and Drug Administration). In some embodiments, pharmaceutical compositions are pharmaceutical compositions that can not be legally administered in the absence of a prescription from a licensed physician.

[0120] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as applied to a carrier, diluent or excipient used to formulate a composition as disclosed herein means the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0121] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating substance, that participates in carrying the subject compound or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0122] Prediabetes: As used herein, the term "prediabetes" refers to a disease, disorder, or condition in which a patient has impaired fasting glucose and / or impaired glucose tolerance. In some embodiments, a patient with prediabetes has a fasting blood glucose level between 100 mg / dL (5.5 mmol / L) and 126 mg / dL (7.0 mmol / L). In some embodiments, a patient with prediabetes has a 2-hour postprandial blood glucose level between 140 mg / dL (7.8 mmol / L) and 200 mg / dL (11.1 mmol / L).

[0123] Prodrug: As used herein, the term "prodrug" refers to a pharmacologically active or more typically inactive compound that is converted into a pharmacologically active agent by metabolic conversion. Prodrugs of compounds of any of the chemical formulas described herein are prepared by modifying the functional groups present in the compounds of any of the chemical formulas in such a way that the modified compound can be cleaved in vivo to release the parent compound. In vivo, prodrugs readily undergo chemical changes under physiological conditions (e.g., hydrolyzed or acted upon by naturally occurring enzymes), resulting in the release of the pharmacologically active agent. Prodrugs include compounds of any of the chemical formulas described herein, wherein a hydroxyl, amino, or carboxyl group is bonded to any group that can be cleaved in vivo to regenerate a free hydroxyl, amino, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, formate, and benzoate derivatives) of compounds of any of the chemical formulas described herein or any other derivative that is converted into an active parent drug upon reaching physiological pH or by enzymatic action. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the art (see, for example, Bundgaard. Design of Prodrugs. Elsevier, 1985).

[0124] Proliferative condition: As used herein, the term "proliferative condition" refers to a disease or condition associated with cell proliferation. In some embodiments, the proliferative disease or condition is or comprises cancer. In some embodiments, the proliferative disease or condition is an inflammatory disease or condition. In some embodiments, the proliferative disease or condition is an autoimmune disease or condition. In some embodiments, the proliferative disease or condition is a microbial infection (e.g., a bacterial infection).

[0125] Reference: As used herein, the term "reference" refers to a standard or control relative to which comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the test and / or determination of the reference or control is performed substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, the reference or control is measured or characterized under conditions or circumstances comparable to those under which the assessment is being made. Those skilled in the art will understand when there is sufficient similarity to justify reliance on and / or comparison with a particular possible reference or control.

[0126] Risk: As used herein, the "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop a disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, the risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, a reference sample or reference sample group has a known risk of a disease, disorder, condition, and / or event. In some embodiments, a reference sample or reference sample group is from an individual comparable to a particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater.

[0127] Solid Forms: As is known in the art, many chemical entities (particularly many organic molecules and / or many small molecules) can take on a variety of different solid forms, for example, amorphous forms and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such entities are available in a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such entities are available in a mixture of such forms.

[0128] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal forms of humans in some embodiments). In some embodiments, the subject suffers from a disease, disorder, or condition of interest. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a subject that is susceptible to a disease, disorder, or condition or has one or more characteristics unique to a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is receiving and / or has received diagnosis and / or treatment.

[0129] Susceptible: An individual who is "susceptible" to a disease, disorder, or condition (e.g., the flu) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not yet been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with developing the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and / or condition is based on the risk of a population (e.g., family members of an individual who suffers from the disease, disorder, or condition).

[0130] Therapeutically effective amount: The term "therapeutically effective amount" refers to an amount that produces the desired effect of administration. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and / or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition and / or delays its onset. One of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a specific individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a specific desired pharmacological response in a substantial number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., tissues affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, eg, as part of a dosing regimen.

[0131] Treatment: As used herein, the term "treatment" (treatment / treat / treating) refers to the administration of a therapy that partially or completely alleviates, improves, alleviates, inhibits a particular disease, disorder, and / or condition, delays its onset (e.g., relative to a determined time of onset or period), reduces its severity, and / or reduces the incidence of one or more symptoms, features, and / or causes thereof. In some embodiments, treatment may be directed to a subject who does not exhibit signs or symptoms of the relevant disease, disorder, and / or condition, and / or to a subject who has not been diagnosed with the relevant disease, disorder, and / or condition, and / or to a subject who only exhibits early signs of the disease, disorder, and / or condition. Alternatively or in addition, in some embodiments, treatment may be directed to a subject who exhibits one or more confirmed signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to a subject who has been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to a subject who is known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition. In some embodiments, such treatment refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. In some embodiments, treatment is the administration of a therapy according to a protocol that has been shown to achieve statistically significant results (e.g., partially or completely alleviate, ameliorate, alleviate, inhibit, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition) when applied to a relevant population or system (e.g., a model system). In some embodiments, treatment that is administered after diagnosis and / or the onset of one or more symptoms is considered "therapeutic" treatment, while treatment that is administered before diagnosis and / or the onset of symptoms is considered "prophylactic" treatment.

[0132] Unit dose: As used herein, the term "unit dose" refers to an amount administered as a single dose and / or as a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of an agent. In some embodiments, more than one unit dose is administered to obtain a total single dose. In some embodiments, it is necessary or anticipated that multiple unit doses will be administered to obtain the desired effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of one or more solid forms of therapeutic agents, a sustained-release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be understood that a unit dose can be present in a formulation that includes any of a variety of components in addition to the therapeutic agent. For example, as described below, it can include an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc. Those skilled in the art will appreciate that in many embodiments, the total appropriate daily dose of a particular therapeutic agent can comprise a portion of a unit dose or multiple unit doses, and can be determined, for example, by the attending physician within the scope of reasonable medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism may depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific active compound being used; the specific composition being used; the age, weight, general health, sex, and diet of the subject; the time of administration and rate of excretion of the specific active compound being used; the duration of the treatment; drugs and / or adjunctive therapies used in combination or concomitantly with the specific compound being used, and like factors well known in the medical arts.

[0133] Alkyl: As used herein, the term "alkyl" refers to a straight or branched chain alkyl group. 1-6 Alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl.

[0134] Heteroatom: As used herein, the term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of a basic nitrogen; a substitutable nitrogen of a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl)).

[0135] Carbocycle: As used herein, the term "carbocycle" refers to a monocyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation but is not aromatic, and has a single point of attachment to the rest of the molecule.

[0136] Heterocycle: As used herein, the term "heterocycle" refers to a stable monocyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably one to four, heteroatoms as defined above. The term "nitrogen" when used to refer to a ring atom of a heterocycle includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or + NR (as in N-substituted pyrrolidinyl). The heterocycle can be attached to its side group on any heteroatom or carbon atom, which produces a stable structure, and any ring atom can be optionally substituted. Examples of 3-8 membered heterocycles include tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepine, oxazepine, thiazepine and morpholinyl.

[0137] Halogen: As used herein, the terms "halogen" and "halo" refer to F, Cl, Br, or I.

[0138] Compound C: The term "Compound C" as used herein refers to 5'-methylselenoadenosine; also known as (2R,4S,5S)-2-(6-amino-9H-purin-9-yl)-5-((methylseleno)methyl)tetrahydrofuran-3,4-diol, CAS Registry Number 5135-40-0, and includes any pharmaceutically acceptable salts thereof.

[0139]

[0140] Compound D: The term "Compound D" as used herein refers to 5'-selenoadenosylhomocysteine; also known as (2R)-2-amino-4-((((2S,3S,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)seleno)butanoic acid, CAS Registry Number 4053-91-2, and includes any pharmaceutically acceptable salts thereof.

[0141]

[0142] Compound E: The term "Compound E" as used herein refers to γ-glutamyl-methylseleno-cysteine ​​or γ-L-glutamyl-Se-methyl-L-cysteine; also known as N5-(l-carboxy-2-(methylseleno)ethyl)-L-glutamine, or any pharmaceutically acceptable salt thereof.

[0143]

[0144] Compound CDE: The term "Compound CDE" as used herein refers to a mixture of Compound C, Compound D, and Compound E, or pharmaceutically acceptable salts thereof.

[0145] Compound

[0146] The present disclosure provides many exemplary compounds, which, after treatment with a single compound, can reduce glucose levels, improve glucose tolerance, restore or activate insulin receptor function and its downstream signaling, enhance AS160 phosphorylation in diabetic mice, cultured liver and / or skeletal muscle cells to reduce glucose levels, improve glucose tolerance, restore or activate insulin receptor function and its downstream signaling, enhance AS160 phosphorylation to translocate glucose transporter (GLUT) from cytoplasmic vesicles to plasma membrane for glucose uptake, stimulate glucose uptake and alleviate hyperinsulinemia without causing impaired renal function and / or liver damage. For example, Example 2 shows that each of compounds 43, 50, 53, 69 and 70 reduces the glucose production in HepG2 cells. In particular, compound 43 shows a higher efficacy than compound CDE. When compared with the antidiabetic drug metformin in HepG2 and rat H4IIE cells, compound 43 shows greater efficacy and lower cytotoxicity. Compound #43 was also shown to be more effective than Compound 50 in inhibiting the expression of G6pc (a key enzyme gene for liver glucose production) in the liver of diabetic mice, and more effective than Compounds CDE, Compound C, Compound D, and Compound 50 in mouse liver AML-12 cells (Example 4). Further, the present disclosure shows that Compound 43 is more effective than Compound C, Compound 50, Compound 69, and Compound 70 against hyperglycemia in diabetic mice (Example 3); Compound 43 is more effective in insulin-resistant diabetic Lepr db / db Compound 43 significantly improves glucose tolerance in the liver and skeletal muscle of diabetic mice (Example 3) and enhances / restores insulin receptor function and its downstream signaling (Examples 5 and 7-8); Compound 43 elicits a response to glucose challenge in diabetic mice that is similar to the response in wild-type mice (Example 3). In addition, the present disclosure shows that treatment with Compound #43 can enhance the phosphorylation of AS160 in both liver and skeletal muscle cells (to promote the translocation of GLUT from cell vesicles to the plasma membrane) for glucose uptake (Example 8), stimulate GLUT4 expression in the liver of diabetic mice and cultured mouse hepatocytes (Example 6), enhance and / or potentiate insulin action to stimulate glucose uptake in hepatocytes and skeletal muscle cells (Examples 6-7), and reduce hyperinsulinemia in insulin-resistant diabetic mice without causing renal impairment and / or liver damage (Example 9).

[0147] The present disclosure also provides the characteristics of selenocompounds, which may contribute to their activity. For example, it was shown that although selenocompound #43 was highly effective in inhibiting glucose production, its sulfur analog compound 68 was less effective (Example 2); and compound #43 was more effective than compound #68 in reducing blood glucose and HbA1c levels and improving glucose tolerance in insulin-resistant diabetic mice (Example 3). It was also shown that inhibition of glucose production was observed after treatment with selenocompounds, which contained diacetyl esters (#43), cyclic carbonates (compound #50), morpholinocarboxylates (#53), dipropionyl esters (#69) or dibutyryl esters (#70) at the 2' and 3' positions, with the highest efficacy observed after treatment with compound 43 (Example 2). Further, it was shown that compounds containing 5' methylseleno groups and 5' selenohomocysteine ​​groups had similar efficacy in inhibiting glucose production in HepG2 cells (Compound C vs. Compound D in Example 2). In vivo studies also revealed that compound #43 was more effective than compounds #69 and #70 in lowering blood glucose levels and improving glucose tolerance in insulin-resistant diabetic mice (Example 3).

[0148] In some embodiments, the present disclosure relates to compounds of formula (1):

[0149]

[0150] or a pharmaceutically acceptable salt, prodrug or isomer thereof, wherein

[0151] R 2 and R 3 Each of the is independently H or -C(O)-R, wherein each R is independently C 1-6 Alkyl or 3-8 membered carbocyclic or heterocyclic ring, wherein R 2 and R 3 It can’t all be H;

[0152] or R 2 With R 3 Together they form -(CH2) n -C(O)-(CH2) m -, wherein each of n and m is independently 0-3, and n+m≤3;

[0153] R 5 Yes-C 1-6 Alkyl or -C 1-6 Alkyl -CH(NH2)COOH;

[0154] R 8 is H or halogen; and

[0155] X is H or halogen;

[0156] wherein the carbocyclic ring, heterocyclic ring, -(CH2) n - and -(CH2) m Each of the - moieties may independently be optionally replaced by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and

[0157] Each C 1-6 The alkyl moieties may be optionally substituted 1-3 times independently with -OH, halogen, NH2 or CN.

[0158] In some embodiments of formula (1), R 8 is H. In some embodiments of formula (1), R 8 In some embodiments of formula (1), R 8 It's F.

[0159] In some embodiments of formula (1), X is H. In some embodiments of formula (1), X is halogen. In some embodiments of formula (1), X is F.

[0160] In some embodiments of formula (1), R 5 is -C which may be optionally substituted 1-3 times by -OH, halogen, NH2 or CN 1-6 In some embodiments of formula (1), R 5 is -C which may be optionally substituted 1-3 times by halogen 1-6 In some embodiments of formula (1), R 5 is unsubstituted -C 1-6 In some embodiments of formula (1), R 5 is an unsubstituted straight-chain -C 1-6 In some embodiments of formula (1), R 5 In some embodiments of formula (1), R 5 In some embodiments of formula (1), R 5 It is propyl.

[0161] In some embodiments of formula (1), R 5 Yes-C 1-6 Alkyl -CH(NH2)COOH, where C 1-6 The alkyl group may be optionally substituted 1-3 times with -OH, halogen, NH2 or CN. In some embodiments of formula (1), R 5 Yes-C 1-6 Alkyl -CH(NH2)COOH, where C 1-6 Alkyl may be optionally substituted 1-3 times with halogen. In some embodiments of formula (1), R 5Yes-C 1-6 Alkyl -CH(NH2)COOH, where C 1-6 The alkyl group is unsubstituted. In some embodiments of formula (1), R 5 is -CH2CH2-CH(NH2)COOH. In some embodiments of formula (1), R 5 is -CH2-CH(NH2)COOH. In some embodiments of formula (1), R 5 It is -CH2CH2CH2-CH(NH2)COOH.

[0162] In some embodiments of formula (1), R 2 is H, and R 3 is -C(O)-R, where R is C 1-6 Alkyl or 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic ring parts independently can be optionally substituted by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and each C 1-6 Alkyl groups may be optionally substituted 1-3 times independently with -OH, halogen, NH2 or CN.

[0163] In some embodiments of formula (1), R 3 is H, and R in formula (1) 2 is -C(O)-R, where R is C 1-6 Alkyl or 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic ring parts independently can be optionally substituted by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and each C 1-6 Alkyl groups may be optionally substituted 1-3 times independently with -OH, halogen, NH2 or CN.

[0164] In some embodiments of formula (1), R 2 and R 3 Each of the above is independently C(O)-R, wherein each R is independently C 1-6 Alkyl or 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic ring parts independently can be optionally substituted by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and each C 1-6 Alkyl groups may be optionally substituted 1-3 times independently with -OH, halogen, NH2 or CN.

[0165] In some embodiments of formula (1), each R is independently a 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic ring portions is independently optionally substituted with -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and each C1-6 Alkyl is independently optionally substituted 1-3 times by -OH, halogen, NH2 or CN. In some embodiments of formula (1), each R is independently a 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic moieties is independently optionally substituted 1-3 times by halogen. In some embodiments of formula (1), each R is independently a 3-8 membered carbocyclic or heterocyclic ring, wherein each of the carbocyclic and heterocyclic moieties is independently optionally substituted 1-3 times by halogen. In some embodiments of formula (1), each R is independently a 3-8 membered unsubstituted carbocyclic or unsubstituted heterocyclic ring. In some embodiments of formula (1), each R is independently a 6 membered unsubstituted carbocyclic or unsubstituted heterocyclic ring. In some embodiments of formula (1), each R is independently an unsubstituted heterocyclic ring. In some embodiments of formula (1), R is

[0166] In some embodiments of formula (1), each R is independently C 1-6 Alkyl, and each C 1-6 Alkyl groups are independently optionally substituted 1-3 times with -OH, halogen, NH2 or CN. In some embodiments of formula (1), each R is independently C 1-6 Alkyl, and each C 1-6 Alkyl is independently optionally substituted 1-3 times with halogen. In some embodiments of formula (1), each R is independently unsubstituted C 1-6 In some embodiments of formula (1), each R is independently an unsubstituted linear C 1-6 In some embodiments of formula (1), each R is independently methyl, ethyl, or propyl. In some embodiments of formula (1), R is CH3.

[0167] In some embodiments of formula (1), R 2 With R 3 Together they form -(CH2) n -C(O)-(CH2) m -, wherein each of n and m is independently 0-3, and n+m≤3, wherein said -(CH2) n - and -(CH2) m Each of the - moieties may independently be optionally replaced by -OH, halogen, NH2, CN or C 1-6 Alkyl substituted 1-3 times; and each C 1-6 Alkyl groups may be optionally substituted 1 to 3 times by -OH, halogen, NH2 or CN independently. In some embodiments of formula (1), the -(CH2) n - and -(CH2) mEach of the - moieties independently may be optionally substituted 1-3 times with halogen. In some embodiments of formula (1), the -(CH2) n - and -(CH2) m The - moiety is unsubstituted. In some embodiments, n=m=0.

[0168] In some embodiments, the present disclosure relates to compounds of formula (2):

[0169]

[0170] or a pharmaceutically acceptable salt, prodrug or isomer thereof,

[0171] where R 8 is H or halogen;

[0172] X is H or halogen;

[0173] Each R5' is independently H or halogen; and

[0174] Each R is independently C 1-6 Alkyl, each of which independently may be optionally substituted 1-3 times with halogen.

[0175] In some embodiments of formula (2), C(R5')3 is CF3, CHF2, CH2F, or CH3.

[0176] In some embodiments of formula (2), the compound has formula (2'):

[0177]

[0178] In some embodiments of formula (2) or (2'), R 8 is H. In some embodiments of formula (2) or (2'), R 8 In some embodiments of formula (2) or (2'), R 8 It's F.

[0179] In some embodiments of formula (2) or (2'), X is H. In some embodiments of formula (2) or (2'), X is halogen. In some embodiments of formula (2) or (2'), X is F.

[0180] In some embodiments of formula (2) or (2'), each R is independently an unsubstituted C 1-6 In some embodiments of formula (2) or (2'), each R is independently C 1-3 alkyl, each of which independently may be optionally substituted 1-3 times with halogen. In some embodiments of formula (2) or (2'), each R is independently an unsubstituted C 1-3In some embodiments of formula (2) or (2'), each R is independently -CH3, -CH2CH3, or -CH2CH2CH3.

[0181] In some embodiments, the present disclosure relates to compounds of formula (3):

[0182]

[0183] or a pharmaceutically acceptable salt, prodrug or isomer thereof,

[0184] where R 8 is H or halogen;

[0185] X is H or halogen; and

[0186] Each R' is independently H or halogen.

[0187] In some embodiments of formula (3), -Se-C(R')3 is -Se-CH3, -Se-CHF2, -Se-CH2F, or -Se-CF3.

[0188] In some embodiments of formula (3), the compound has formula (3'):

[0189]

[0190] In some embodiments of formula (3) or (3'), R 8 is H. In some embodiments of formula (3) or (3'), R 8 is halogen. In some embodiments of formula (3) or (3'), R 8 It's F.

[0191] In some embodiments of formula (3) or (3'), X is H. In some embodiments of formula (3) or (3'), X is halogen. In some embodiments of formula (3) or (3'), X is F.

[0192] In some embodiments of formula (3) or (3'), each C(R')3 is independently CF3, CHF2 or CH2F or CH3. In some embodiments of formula (3) or (3'), each C(R')3 is independently CH2F or CH3. In some embodiments of formula (3) or (3'), each C(R')3 is CH3.

[0193] In some embodiments, the present disclosure relates to compounds of the formula:

[0194]

[0195]

[0196] or a pharmaceutically acceptable salt, prodrug or isomer thereof.

[0197] preparation

[0198] In some embodiments, the present disclosure provides compositions that contain and / or deliver (i.e., after administration to a system or subject) a compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the present disclosure provides compositions that contain only a single compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the present disclosure provides compositions that contain one or more compounds of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, and one or more carriers or excipients suitable for administration to a human or animal subject according to the present disclosure.

[0199] In some embodiments, the present disclosure provides compositions that deliver the active moiety of a compound of any one of Formulas (1)-(3). In some embodiments, the composition comprises an active metabolite of a compound of any one of Formulas (1)-(3). In some embodiments, the composition comprises a compound that forms a metabolite of a compound of any one of Formulas (1)-(3) upon administration of the composition, the metabolite retaining the relevant biological activity.

[0200] In some embodiments, the present disclosure provides compositions that are pharmaceutical compositions because they contain an active pharmaceutical ingredient (API) and one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the API is or comprises a compound of any one of formulas (1)-(3). In some embodiments, the API consists of a compound of any one of formulas (1)-(3). In some embodiments, the API consists of a single compound of any one of formulas (1)-(3).

[0201] In some embodiments, the present disclosure provides methods for preparing provided compositions, for example, by combining one or more suitable (i.e., pharmaceutically acceptable) carriers or excipients with a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, one or more pharmaceutically acceptable carriers or excipients are suitable for oral administration, and the mixture is formulated into an oral formulation. In some embodiments, the pharmaceutical composition is a solid dosage form. In some embodiments, the solid dosage form is a tablet, capsule, or lozenge. In some embodiments, the pharmaceutical composition is a liquid dosage form (e.g., a beverage).

[0202] Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for formulating pharmaceutical compositions and known techniques for their preparation. In some embodiments, the present disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable amount of a compound as described herein. In some embodiments, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form can vary depending on the host being treated and / or the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the active ingredient, about 5% to about 70%, or about 10% to about 30%.

[0203] In some embodiments, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0204] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0205] In some embodiments, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. In some embodiments, the formulations are conveniently in unit dosage form and can be prepared by any method well known in the pharmaceutical art. In some embodiments, the formulations as described herein comprise: selected from cyclodextrins, liposomes, micelle formers (e.g., bile acids) and polymer carriers (e.g., polyesters and polyanhydrides); and compounds as described herein. In some embodiments, the formulations as described herein make the compounds as described herein orally bioavailable.

[0206] In some embodiments, the method for preparing such formulations may include the step of combining a compound as described herein with one or more pharmaceutically acceptable carriers or excipients and optionally one or more auxiliary ingredients. In some embodiments, the formulations are prepared by uniformly and intimately combining a compound as described herein with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0207] In some embodiments, formulations suitable for oral administration as described herein may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin or sucrose and acacia) and / or as a mouthwash, beverage, or the like, each containing a predetermined amount of a compound as described herein as the active ingredient. In some embodiments, a compound as described herein may alternatively or additionally be administered in the form of a bolus, electuary, or paste.

[0208] In some embodiments, in solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) as described herein, the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following substances: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; humectants, such as glycerol; disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol, glyceryl monostearate and nonionic surfactants; absorbents, such as kaolin and bentonite; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In some embodiments, in the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffering agent. In some embodiments, solid compositions of a similar type may also be employed as fillers in soft and hard shell gelatin capsules using carriers such as lactose or milk sugar and such as high molecular weight polyethylene glycols.

[0209] In some embodiments, tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. In some embodiments, compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant, or a dispersant. In some embodiments, molded tablets can be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent.

[0210] In some embodiments, tablets and other solid dosage forms of pharmaceutical compositions as described herein, such as dragees, capsules, pills, and granules, may optionally be scored or made with coatings and shells, such as enteric coatings and other coatings known in the art of pharmaceutical formulations. In some embodiments, they may be formulated into sustained or controlled release compositions providing active ingredients therein using, for example, hydroxypropyl methylcellulose (to provide the desired release profile), other polymer matrices, liposomes, and / or microspheres in varying proportions. In some embodiments, they may be formulated into rapid release compositions, such as freeze-dried compositions. In some embodiments, they may be sterilized by, for example, filtering through a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved in sterile water or some other sterile injectable medium prior to use. In some embodiments, these compositions may also optionally contain an emulsifier and may be compositions that release the active ingredient only or preferentially in a certain portion of the gastrointestinal tract in a delayed manner. Examples of usable embedding compositions include polymeric substances and waxes. In some embodiments, the active ingredient may be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0211] In some embodiments, liquid dosage forms for oral administration of the compounds described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In some embodiments, in addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuranol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0212] In some embodiments, in addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0213] In some embodiments, suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0214] In some embodiments, formulations for rectal or vaginal administration as described herein may be provided as suppositories, which may be prepared by mixing one or more compounds as described herein with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes, or salicylates, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.

[0215] In some embodiments, formulations suitable for vaginal administration as described herein include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0216] In some embodiments, dosage forms for topical or transdermal administration of a compound as described herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In some embodiments, the active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.

[0217] In some embodiments, ointments, pastes, creams, and gels may contain, in addition to a compound as described herein, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0218] In some embodiments, powders and sprays may contain, in addition to a compound as described herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. In some embodiments, sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0219] In some embodiments, transdermal patches have the added advantage of providing controlled delivery of a compound as described herein to the body. In some embodiments, such dosage forms can be prepared by dissolving or dispersing the compound in an appropriate medium. In some embodiments, absorption enhancers can be used to increase the flow of the compound through the skin. In some embodiments, providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of this flow.

[0220] In some embodiments, the present disclosure provides ophthalmic formulations, eye ointments, powders, solutions, and the like.

[0221] In some embodiments, pharmaceutical compositions suitable for parenteral administration as described herein comprise one or more compounds as described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0222] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions as described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). In some embodiments, proper fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0223] In some embodiments, the compositions described herein may contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. In some embodiments, various antibacterial and antifungal agents may be included to ensure that microorganisms are prevented from acting on the subject compounds, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some embodiments, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the composition. In some embodiments, extended absorption of injectable pharmaceutical forms may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0224] In some embodiments, for example, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. In some embodiments, this can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. In some embodiments, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. In some embodiments, the delayed absorption of a parenteral drug form is achieved by dissolving or suspending the drug in an oil vehicle.

[0225] In some embodiments, injectable reservoir forms are prepared by forming a microcapsule matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). In some embodiments, the rate of drug release can be controlled according to the ratio of the drug to the polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). In some embodiments, reservoir injectable formulations are prepared by embedding the drug in a liposome or microemulsion compatible with body tissues.

[0226] In some embodiments, drug eluting forms include coated or drug-containing stents and implantable devices. In some embodiments, drug eluting stents and other devices may be coated with a compound or drug formulation and may further comprise a polymer designed for timed release.

[0227] In some embodiments, the compound or pharmaceutical formulation is administered orally. In some embodiments, the compound or pharmaceutical formulation is administered intravenously. In some embodiments, the compound is attached to a solid support via a cleavable linker, and the solid support is administered with a catheter. In some embodiments, routes of administration include sublingual, intramuscular, and transdermal administration.

[0228] In some embodiments, the compounds as described herein are administered to humans and animals as pharmaceuticals, either per se or as pharmaceutical compositions containing, for example, 0.1% to 99.5% or 0.5% to 90% active ingredient in combination with a pharmaceutically acceptable carrier.

[0229] In some embodiments, the compounds described herein can be administered orally, parenterally, topically, or rectally. In some embodiments, they are of course administered in a form suitable for each route of administration. In some embodiments, they are administered in tablet or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc.; by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository.

[0230] In some embodiments, the compounds as described herein can be administered to humans and other animals for treatment by any suitable route of administration, including oral, nasal, such as by, for example, aerosol, spray, rectal, intravaginal, parenteral, intracisternal, and topical, such as by powders, ointments, or drops, including buccal and sublingual administration.

[0231] In some embodiments, the compounds as described herein that can be used in a suitable hydrated form and / or the pharmaceutical compositions as described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0232] In some embodiments, actual dosage levels of the active ingredients in the pharmaceutical compositions as described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0233] In some embodiments, the selected dosage level will depend upon a variety of factors, including the activity of the specific compound as described herein, the route of administration, the time of administration, the rate of excretion or metabolism of the specific compound being used, the duration of the treatment, other drugs, compounds and / or substances used in combination with the specific compound being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0234] A physician or veterinarian having ordinary skill in the art can determine the effective amount of the pharmaceutical composition required and prescribe the drug. In some embodiments, the physician or veterinarian may start the pharmaceutical composition at a dose lower than that required to achieve the desired therapeutic effect and then gradually increase the dose until the desired effect is achieved.

[0235] In some embodiments, the subject is provided with a compound or pharmaceutical composition as described herein for a long period of time. In some embodiments, long-term treatment includes repeated administration over a long period of time in any form, such as for one or more months, between one month and one year, one year or more years or longer. In some embodiments, long-term treatment involves repeated administration of a compound or pharmaceutical composition as described herein to a subject throughout life. In some embodiments, long-term treatment involves regular administration, such as once a day or more, once a week or more, or once a month or more. In some embodiments, the appropriate dose (such as daily dose) of a compound as described herein will be the amount of the compound at the lowest dose that effectively produces a therapeutic effect. This effective dose will generally depend on the factors described herein. In some embodiments, when the indicated effect is concerned, the dosage of a compound as described herein for a patient will be in the range of about 0.0001 to about 100 mg per kg body weight per day. In some embodiments, the daily dose will be in the range of 0.001 to 50 mg of compound per kg body weight. In some embodiments, the daily dose will be in the range of 0.01 to 10 mg of compound per kg body weight. However, lower or higher doses may be used. In some embodiments, the dosage administered to a subject may be altered as the subject's physiological condition changes due to age, disease progression, weight, or other factors.

[0236] In some embodiments, the effective daily dose of the active compound may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form.

[0237] In some embodiments, a compound as described herein is administered alone. In some embodiments, a compound as described herein is administered as a pharmaceutical formulation (composition) as described herein.

[0238] In some embodiments, by analogy with other drugs, the compounds as described herein can be formulated for administration in any convenient manner for use in human or veterinary medicine.

[0239] Preparation of compounds and / or compositions

[0240] In some embodiments, the compound of any one of formula (1)-(3) or its pharmaceutically acceptable salt, prodrug or isomer can be prepared in whole or in part by chemical synthesis; in some embodiments, the compound of any one of formula (1)-(3) or its pharmaceutically acceptable salt, prodrug or isomer prepared in part by chemical synthesis is prepared by semi-synthetic methods. In some embodiments, the compound of any one of formula (1)-(3) or its pharmaceutically acceptable salt, prodrug or isomer can be prepared by isolation.

[0241] In some embodiments, the present disclosure provides methods for preparing compounds and / or compositions as described herein, comprising assaying one or more samples (e.g., to detect biological activity therein. In some embodiments, one or more samples comprise a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the methods provided herein include steps of detecting and / or confirming the presence of detectable biological activity in one or more samples. In some embodiments, the methods provided herein include steps of confirming the absence of detectable biological activity in one or more samples.

[0242] In some embodiments, the biological activity is the inhibition of glucose production. In some embodiments, the biological activity is tested in HepG2 cells. In some embodiments, the biological activity is tested in H4IIE cells.

[0243] In some embodiments, the biological activity is a reduction in serum HbAIc levels. In some embodiments, the biological activity is tested in insulin resistant diabetic mice.

[0244] In some embodiments, the biological activity is enhanced glucose tolerance. In some embodiments, the biological activity is tested in insulin resistant and diabetic mice.

[0245] In some embodiments, the biological activity is inhibition of G6pc expression. In some embodiments, the biological activity is tested in AML-12 cells. In some embodiments, the biological activity is tested in AML-12 cells stimulated with a diabetic stimulant. In some embodiments, the biological activity is tested in human HepG2 cells. In some embodiments, the biological activity is tested in the liver of insulin-resistant and diabetic mice.

[0246] In some embodiments, the biological activity is enhanced phosphorylation of Pdk1, Akt, Foxo1, and AS 160. In some embodiments, the biological activity is tested in liver. In some embodiments, the biological activity is tested in skeletal muscle.

[0247] In some embodiments, the biological activity is enhanced Glut4 expression. In some embodiments, the biological activity is tested in mouse liver AML-12 cells. In some embodiments, the biological activity is tested in the liver of insulin-resistant and diabetic mice.

[0248] In some embodiments, the biological activity is activation and / or restoration of insulin signaling in a subject in an insulin resistant state. In some embodiments, the subject is characterized by significant levels of circulating insulin. In some embodiments, the insulin resistant state is characterized by reduced levels and / or activity of phosphorylated insulin receptors in the subject. In some embodiments, the subject suffers from diabetes and / or diabetes-related diseases, disorders, and / or conditions.

[0249] In some embodiments, the biological activity is increased glucose uptake. In some embodiments, the cells are hepatocytes and skeletal muscle cells. In some embodiments, the biological activity is a reduction in serum insulin levels. In some embodiments, the biological activity is tested in insulin-resistant and diabetic mice.

[0250] Identification and / or characterization of compounds and / or compositions

[0251] In some embodiments, the present disclosure provides methods for confirming and / or characterizing compounds and / or compositions as described herein. In some embodiments, such methods include the step of testing a plurality of samples for biological activity, each of the samples comprising a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof; and determining the presence and / or level of the biological activity in one or more such samples. In some embodiments, the methods provided include detecting the biological activity associated with the presence and / or level of a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the methods provided include the step of confirming and / or characterizing a specific compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, by detecting the biological activity of the compound.

[0252] In some embodiments, the biological activity is the inhibition of glucose production. In some embodiments, the biological activity is tested in HepG2 cells. In some embodiments, the biological activity is tested in H4IIE cells.

[0253] In some embodiments, the biological activity is a reduction in serum HbAIc levels. In some embodiments, the biological activity is tested in insulin resistant diabetic mice.

[0254] In some embodiments, the biological activity is enhanced glucose tolerance. In some embodiments, the biological activity is tested in insulin resistant and diabetic mice.

[0255] In some embodiments, the biological activity is inhibition of G6pc expression. In some embodiments, the biological activity is tested in AML-12 cells. In some embodiments, the biological activity is tested in AML-12 cells stimulated with a diabetic stimulant. In some embodiments, the biological activity is tested in human HepG2 cells. In some embodiments, the biological activity is tested in the liver of insulin-resistant and diabetic mice.

[0256] In some embodiments, the biological activity is enhanced phosphorylation of Pdk1, Akt, Foxo1, and AS 160. In some embodiments, the biological activity is tested in liver. In some embodiments, the biological activity is tested in skeletal muscle.

[0257] In some embodiments, the biological activity is enhanced Glut4 expression. In some embodiments, the biological activity is tested in mouse liver AML-12 cells. In some embodiments, the biological activity is tested in the liver of insulin-resistant and diabetic mice.

[0258] In some embodiments, the biological activity is activation and / or restoration of insulin signaling in a subject in an insulin resistant state. In some embodiments, the subject is characterized by significant levels of circulating insulin. In some embodiments, the insulin resistant state is characterized by decreased levels and / or activity of phosphorylated insulin receptors in the subject. In some embodiments, the subject suffers from diabetes and / or a diabetes-related disease, disorder, or condition.

[0259] In some embodiments, the biological activity is enhanced glucose uptake into cells of a subject. In some embodiments, the biological activity is enhanced glucose uptake into hepatocytes and skeletal muscle cells. In some embodiments, the biological activity is a reduction in serum insulin levels. In some embodiments, the biological activity is tested in insulin-resistant and diabetic mice.

[0260] use

[0261] The present disclosure provides that the compounds described herein (e.g., Compound 43) can mimic insulin to inhibit glucose production (see, e.g., Example 2); reduce blood glucose and HbA1c levels, reduce the development of hyperglycemia and improve glucose tolerance in insulin-resistant diabetic subjects (see, e.g., Example 3); inhibit G6pc expression in the liver of insulin-resistant diabetic subjects, mimic but bypass insulin to inhibit G6pc expression in cultured mouse and human hepatocytes, and enhance insulin action (see, e.g., Example 4); mimic but bypass insulin to activate Pdk1 and Akt in the liver and enhance Foxo1 phosphorylation (see, e.g., Example 5); enhance Glut4 expression in the liver of insulin-resistant diabetic subjects, and mimic but bypass insulin to enhance Glut4 expression in mouse hepatocytes (see, e.g., Example 6), phosphorylation of AS160 in human hepatocytes (GLUT4 from the cell) and Glut4 in the liver of insulin-resistant diabetic subjects. , Glutathione 4 (GLUT4) is a key event in the transport of cytoplasmic vesicles to the plasma membrane to promote glucose uptake) (see, e.g., Example 8) and glucose uptake in mouse hepatocytes (see, e.g., Example 6); enhance and / or restore insulin signaling Pdk1 / Akt / Foxo1 in skeletal muscle of insulin-resistant diabetic subjects (see, e.g., Example 7), activate Akt and enhance phosphorylation of AS160 in skeletal muscle cells by bypassing insulin mimicry (a key event in the transport of GLUT4 from cytoplasmic vesicles to the plasma membrane) (see, e.g., Example 8), and potentiate insulin action to stimulate glucose uptake in skeletal muscle cells (see, e.g., Example 7); activate and / or restore insulin receptor (Insr) function in skeletal muscle and liver of insulin-resistant diabetic subjects and in cultured mouse skeletal muscle and human hepatocytes (see, e.g., Example 8); and reduce hyperinsulinemia without impairing renal function and / or causing liver damage (Example 9). Thus, the present disclosure clearly shows that the compounds as described herein are suitable for regulating glucose metabolism and treating insulin-related disorders as described herein.

[0262] In some embodiments, the present disclosure provides a method for regulating glucose metabolism and / or treating a glucose metabolism disorder, comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the glucose metabolism disorder involves a blood glucose level that is not within a normal range. In some embodiments, the glucose metabolism disorder is associated with a glucose uptake and / or transport defect. In some embodiments, the glucose metabolism disorder is diabetes, glyceraldehyde-3-phosphate dehydrogenase deficiency, diabetes, hyperglycemia, hyperinsulinemia, or hypoglycemia.

[0263] In some embodiments, the present disclosure provides a method for treating a disorder of glucose transport comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the disorder of glucose transport is glucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivo disease (GLUT1 deficiency syndrome (GLUT1DS)).

[0264] In some embodiments, the present disclosure provides a method for enhancing AS160 phosphorylation to translocate glucose transporter (GLUT) from cytoplasmic vesicles to the plasma membrane for glucose uptake, comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0265] In some embodiments, the present disclosure provides methods of enhancing glucose uptake in both the liver and skeletal muscle comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0266] In some embodiments, the present disclosure provides a method for treating an insulin-related disorder comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the insulin-related disorder is selected from prediabetes, type I diabetes, type II diabetes, hypoglycemia, hyperglycemia, insulin resistance, secretory dysfunction, loss of pancreatic beta cell function, and pancreatic beta cell loss. In some embodiments, the patient with the insulin-related disorder is a non-insulin-dependent patient suffering from an insulin-related disorder.

[0267] In some embodiments, the present disclosure provides a method for treating an insulin resistance condition comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. Certain examples of insulin resistance conditions are described above. In some embodiments, the provided methods are used to treat type II diabetes, hyperinsulinemia, hyperproinsulinemia, retinopathy, neuropathy, or nephropathy. In some embodiments, the provided methods reduce hyperinsulinemia without impairing renal function and / or causing liver damage.

[0268] In some embodiments, the present disclosure provides a method of treating obesity comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0269] In some embodiments, the present disclosure provides a method for treating diabetes, comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the diabetes is type I diabetes. In some embodiments, the diabetes is type II diabetes.

[0270] In some embodiments, the present disclosure provides a method of treating hyperglycemia comprising administering a therapeutically effective amount of a compound of any one of Formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0271] In some embodiments, the present disclosure provides methods of inhibiting glucose production comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0272] In some embodiments, the present disclosure provides methods of lowering serum HbA1c levels comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0273] In some embodiments, the present disclosure provides a method of improving glucose tolerance comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0274] In some embodiments, the present disclosure provides a method of inhibiting G6pc expression comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0275] In some embodiments, the present disclosure provides methods of enhancing phosphorylation of Pdk1, Akt, and Foxo1 in liver and / or skeletal muscle comprising administering a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0276] In some embodiments, the present disclosure provides a method of increasing Glut4 expression comprising administering a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0277] In some embodiments, the present disclosure provides methods of activating and / or restoring insulin signaling in a subject in an insulin-resistant state, comprising administering a compound of any one of formula (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the subject is characterized by significant levels of circulating insulin. In some embodiments, the insulin-resistant state is characterized by decreased levels and / or activity of phosphorylated insulin receptors in the subject. In some embodiments, the subject suffers from diabetes and / or a diabetes-related disease, disorder, or condition. In some embodiments, the subject suffers from type II diabetes.

[0278] In some embodiments, the present disclosure provides a method of enhancing glucose uptake into a cell of a subject comprising administering a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the cell is a skeletal muscle cell.

[0279] Compound #43 has been shown to mimic but bypass insulin to activate insulin receptor signaling in the liver and skeletal muscle, and to restore insulin receptor function even in insulin-resistant diabetic subjects. These observations suggest that compound #43 could be used to treat diseases or syndromes characterized by defective insulin signaling, such as polycystic ovary syndrome (PCOS), Alzheimer's disease (AD), and sarcopenia.

[0280] In some embodiments, the present disclosure provides a method for treating polycystic ovary syndrome (PCOS) comprising administering a therapeutically effective amount of a compound of any one of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. PCOS is a hormonal imbalance that can lead to irregular menstruation, unwanted hair growth, and acne. Young women with PCOS often have elevated insulin levels, which can cause the ovaries to produce more androgen hormones, leading to increased body hair, acne, and irregular or infrequent menstruation. Having PCOS can lead to insulin resistance and the development of type 2 diabetes. Metformin is a drug often prescribed to women with PCOS to increase insulin sensitivity and prevent the development of type 2 diabetes. The results indicate that compound #43 is more effective than metformin in inhibiting glucose production in cultured hepatocytes and can restore insulin receptor function in insulin-resistant diabetic mice. Therefore, compound #43 may be suitable for treating PCOS.

[0281] In some embodiments, the present disclosure provides a method for treating Alzheimer's disease (AD), comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug or isomer thereof. Brain insulin signaling is important for learning and memory, and insulin resistance in the brain is a major risk factor for AD. Restoring insulin signaling has become a potential therapy for AD (White MF, Science 2003; 302: 1710–1; De Felice DG et al., Alzheimer's & Dementia 2014; 10: S26–S32). Given that the above results show that compound #43 exhibits insulin-like activity and is able to restore insulin receptor function in subjects with insulin resistance, it is likely suitable for treating AD.

[0282] In some embodiments, the present disclosure provides a method for treating sarcopenia, comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. Sarcopenia is characterized by a gradual loss of skeletal muscle mass with age, resulting in decreased muscle strength, decreased mobility and function, increased fatigue, increased risk of fall-related injuries, and frequent weakness (Candow and Chilibeck, 2005; Sakuma and Yamaguchi, 2012). Recent advances in muscle biology have shown that insulin signaling (Insr / PI3K / Akt) is crucial for the synthesis of muscle protein and the inhibition of muscle protein degradation (through Akt / Foxo1-mediated inhibition of the expression of two atrophy genes, Fbxo32 and Trim63). Optimal insulin signaling attenuates muscle atrophy processes, including sarcopenia (Glass and Roubenoff, 2010; Ryall et al., 2008; Sakuma and Yamaguchi, 2012). Thus, stimulators of insulin signaling, amino acid supplementation (to improve protein synthesis), and inhibitors of proteasomal protein degradation are emerging as novel strategies for treating sarcopenia. As described herein, the insulin-like activity of compound #43 in the activation of Insr / Pdk1 / Akt / Foxo1 signaling in skeletal muscle, even in insulin-resistant diabetic mice, suggests that this compound has potential use in treating wasting conditions in muscle.

[0283] The present disclosure also teaches that the compounds provided may enhance mitochondrial function and are therefore suitable for treating mitochondrial diseases and / or dysfunctions. In some embodiments, the present disclosure provides methods for treating mitochondrial-related diseases (e.g., caused by dysfunctional mitochondria) comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug or isomer thereof. In some embodiments, mitochondrial-related diseases may be degenerative diseases (e.g., cancer, cardiovascular disease and heart failure, type 2 diabetes, Alzheimer's and Parkinson's disease, fatty liver disease, cataracts, osteoporosis, muscle atrophy, sleep disorders and inflammatory diseases such as psoriasis, arthritis and colitis). In some embodiments, the present disclosure provides methods for enhancing mitochondrial function comprising administering a therapeutically effective amount of a compound of any one of formula (1)-(3) or a pharmaceutically acceptable salt, prodrug or isomer thereof.

[0284] In some embodiments, the present disclosure provides a method for enhancing gluconeogenesis in the brain, comprising administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3) or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the provided methods increase glucose uptake in the brain. In some embodiments, the provided methods are used to maintain or restore brain function, including memory and learning.

[0285] Combination therapy

[0286] In some embodiments, the present disclosure provides for the use of compounds and / or compositions as described herein for combination therapy of a disease, disorder, or condition as described herein. In some embodiments, the present disclosure provides for methods of treating a patient who has a disease, disorder, or condition and has received, is receiving, or is about to receive one or more different therapies for the disease, disorder, or condition, wherein the method comprises administering a therapeutically effective amount of a compound of any one of Formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.

[0287] In some embodiments, one or more therapies for patients with insulin-related disorders are selected from insulin therapy, such as for type I diabetes; diet and exercise, such as for early stage type II diabetes; oral antidiabetic drugs, such as for early stage type II diabetes; metformin; insulin secretagogues, such as sulfonylureas; glitazones; long-acting basal insulin; intermediate-acting insulin; and short (fast) acting insulin. In some embodiments, insulin therapy involves subcutaneous (SC), intravenous and / or by inhalation administration. In some embodiments, one or more therapies for patients with insulin-related disorders can be a therapy currently under development, such as insulin-fumaryl diketopiperazine (FDKP).

[0288] Example

[0289] Example 1: Synthesis of Compounds #43, #50, #53, #69, #70, and #68

[0290] 1a. Synthesis of Adenosine, 5'-Se-methyl-5'-seleno-, 2', 3'-diacetate (Compound #43)

[0291] plan:

[0292]

[0293] Synthesis procedure: 5'-Se-methyl-5'-seleno-adenosine (1.0 g, 0.0029 mol, 1.0 mol equivalent) and anhydrous pyridine (10 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and a thermometer. The reaction apparatus was placed in an ice / salt bath and stirring was started. When the temperature of the solution dropped to 0°C, acetic anhydride (10 ml, 0.105 mol, 36.47 mol equivalent) was added dropwise for 15 minutes, and the temperature of the reaction mixture was kept below 5°C during the addition of acetic anhydride. The reaction mixture was stirred at 5-10°C for 6 hours. Excess acetic anhydride was quenched by adding ice-cold water (100 ml), and the pH was then adjusted to 7 by adding 10 wt% NaHCO3 aqueous solution. The aqueous mixture was extracted with ethyl acetate (2 x 100 ml). The combined ethyl acetate extracts were dried over anhydrous Na2SO4 (1 gram) and filtered into a 250 ml round-bottom flask. The filtrate was concentrated to dryness under reduced pressure at 35-40 °C to give the crude product as a light yellow syrupy liquid, which was then passed through a silica gel column using a mixture of ethyl acetate and hexane (1:3 v / v) to give the pure product as off-white crystals (1.12 g, yield: 90.3%, HPLC purity: >99%).

[0294] 1b. Synthesis of adenosine, 5'-S-methyl-5'-thio-, 2', 3'-diacetate (Compound #68):

[0295] plan:

[0296]

[0297] Synthesis Procedure: 5'-S-methyl-5'-thio-adenosine (1.0 g, 0.0033 mol, 1.0 mol equivalent) and anhydrous pyridine (10 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and thermometer. The reaction apparatus was placed in an ice / salt bath and agitation was initiated. When the temperature of the solution dropped to 0°C, acetic anhydride (10 ml, 0.105 mol, 31.8 mol equivalent) was added dropwise over 15 minutes, maintaining the temperature of the reaction mixture below 5°C during the addition of acetic anhydride. The reaction mixture was stirred at 5-10°C for 6 hours. Excess acetic anhydride was quenched by the addition of ice-cold water (100 ml), and the pH was then adjusted to 7 by the addition of 10 wt% aqueous NaHCO₃. The aqueous mixture was extracted with ethyl acetate (2 x 100 ml). The combined ethyl acetate extracts were dried over anhydrous Na₂SO₄ (1 g) and filtered into a 250 ml round-bottom flask. The filtrate was concentrated to dryness under reduced pressure at 35-40 °C to give the crude product as a light yellow syrupy liquid, which was then passed through a silica gel column using a mixture of ethyl acetate and hexane (1:3 v / v) to give the pure product as off-white crystals (1.08 g, yield: 87%, HPLC purity: >99%).

[0298] 1c. Synthesis of adenosine, 5'-Se-methyl-5'-seleno-, 2', 3'-dipropionate (Compound #69):

[0299] plan:

[0300]

[0301] Synthesis procedure: 5'-Se-methyl-5'-seleno-adenosine (1.0 g, 0.0029 mol, 1.0 mol equivalent) and anhydrous pyridine (10 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and a thermometer. The reaction apparatus was placed in an ice / salt bath and agitation was initiated. When the temperature of the solution dropped to 0°C, propionic anhydride (10 ml, 0.078 mol, 27.0 mol equivalent) was added dropwise for 15 minutes, and the temperature of the reaction mixture was kept below 5°C during the addition of propionic anhydride. The reaction mixture was stirred at 5-10°C for 6 hours. Excess propionic anhydride was quenched by adding ice-cold water (100 ml), and the pH was then adjusted to 7 by adding a 10 wt% NaHCO3 aqueous solution. The aqueous mixture was extracted with ethyl acetate (2 x 100 ml). The combined ethyl acetate extracts were dried over anhydrous Na2SO4 (1 gram) and filtered into a 250 ml round-bottom flask. The filtrate was concentrated to dryness under reduced pressure at 35-40°C to give the crude product as a light yellow syrupy liquid, which was then passed through a silica gel column using a mixture of ethyl acetate and hexane (1:3 v / v) to give the pure product as off-white crystals (1.18 g, yield: 89.3%, HPLC purity: >99%).

[0302] 1d. Synthesis of adenosine, 5'-Se-methyl-5'-seleno-, 2', 3'-dibutyrate (Compound #70):

[0303] plan:

[0304]

[0305] Synthesis procedure: 5'-Se-methyl-5'-seleno-adenosine (1.0 g, 0.0029 mol, 1.0 mol equivalent) and anhydrous pyridine (10 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and a thermometer. The reaction apparatus was placed in an ice / salt bath and stirring was started. When the temperature of the solution dropped to 0°C, butyric anhydride (10 ml, 0.078 mol, 27.0 mol equivalent) was added dropwise for 15 minutes, and the temperature of the reaction mixture was kept below 5°C during the addition of butyric anhydride. The reaction mixture was stirred at 5-10°C for 6 hours. Excess butyric anhydride was quenched by adding ice-cold water (100 ml), and the pH was then adjusted to 7 by adding 10 wt% NaHCO3 aqueous solution. The aqueous mixture was extracted with ethyl acetate (2 x 100 ml). The combined ethyl acetate extracts were dried over anhydrous Na2SO4 (1 gram) and filtered into a 250 ml round-bottom flask. The filtrate was concentrated to dryness under reduced pressure at 35-40 °C to give the crude product as a light yellow syrupy liquid, which was then passed through a silica gel column using a mixture of ethyl acetate and hexane (1:3 v / v) to give the pure product as off-white crystals (1.20 g, yield: 85.7%, HPLC purity: >99%).

[0306] 1e. Synthesis of Adenosine, 5'-Se-methyl-5'-seleno-, Cyclic 2',3'-Carbonate (Compound #50):

[0307] plan:

[0308]

[0309] Synthesis procedure: 5'-Se-methyl-5'-seleno-adenosine (1.0 g, 0.0029 mol, 1.0 mol equivalent) and anhydrous dimethylformamide (20 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and a thermometer. The reaction apparatus was placed in an ice / salt bath and agitation was initiated. When the temperature of the solution dropped to 0°C, carbonyldiimidazole (CDI, 0.57 g, 0.0035 mol, 1.21 mol equivalents) was added at below 5°C. The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature under an argon atmosphere for 4 hours. The solvent was removed under reduced pressure to obtain a residue, which was dissolved in a mixture of chloroform (5 ml) and ethanol (a few drops) to obtain a clear solution. The organic layer was washed with 1% aqueous acetic acid (2 x 1 ml), dried over anhydrous Na2SO4 (1 g), and filtered into a 250 ml round-bottom flask. The filtrate was concentrated to dryness under reduced pressure at 25-30° C. to give a crude product as a pale yellow syrupy liquid. The crude product was dissolved in an ethanol / water mixture (1:1 v / v) and then concentrated to dryness under reduced pressure at 45-50° C. to give a residue, to which hexane (25 ml) was added and stirred for 10 minutes, and then concentrated to dryness under reduced pressure at 30-35° C. to give the desired product as off-white crystals (1.02 g, yield: 95.3%, HPLC purity:> 99%).

[0310] 1f. Synthesis of a mixture of regioisomers of adenosine, 5'-Se-methyl-5'-seleno-, 2'-morpholinocarbamate and adenosine, 5'-Se-methyl-5'-seleno-, 3'-morpholinocarbamate (Compound #53):

[0311] plan:

[0312]

[0313] Synthesis Procedure: Adenosine, 5'-Se-methyl-5'-seleno-, cyclic 2',3'-carbonate (1.0 g, 0.0027 mol, 1.0 mol equivalent) and anhydrous dimethylformamide (10 ml) were placed in an oven-dried 50 ml three-necked flask equipped with a dropping funnel, inert gas inlet / outlet, and thermometer. Morpholine (0.26 g, 0.0029 mol, 1.1 mol equivalent) was added at 20-25°C. The reaction mixture was stirred at room temperature for 1 hour and then concentrated to dryness under reduced pressure at 45-50°C to give a residue. Hexane (25 ml) was added and stirred for 10 minutes to precipitate the desired regioisomer mixture product as an off-white solid (1.12 g, yield: 91%, HPLC purity: >99%).

[0314] Example 2.

[0315] In the examples described herein, the synthetic compounds listed in Table 1 were tested in cell culture (in vitro) for their effects on glucose production and cell viability. Specifically, the cells tested were human liver cancer HepG2 and rat liver H4IIE hepatocytes.

[0316] Table 1.

[0317]

[0318]

[0319]

[0320] Materials and methods

[0321] Cell lines and compounds

[0322] Human hepatoma HepG2 and rat hepatoma H4IIE cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia).HepG2 cells and H4IIE cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS.

[0323] Compound #43 and the other compounds listed in Table 1 were either synthesized or obtained from commercial sources (if available). The purity of all tested compounds was confirmed to be ≥99% as determined by HPLC. All of these compounds were dissolved in either DMSO or water to obtain 126.7 or 12.67 mM stock solutions for the experiments. Metformin and insulin were purchased from Sigma.

[0324] Glucose production assay

[0325] Equal numbers of human HepG2 or rat H4IIE cells (1-1.5×10 5 The cells were washed twice with PBS and the mixture was stirred for 24 hours. The cells were washed twice with PBS and the mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours. The mixture was stirred for 24 hours.

[0326] After the above treatment, 50 μl of culture medium was collected and analyzed for glucose using Molecular Probes Amplex Red Glucose Assay Kit (Cat. No. A22189) according to the manufacturer's protocol. AQueous One Solution cell proliferation assay kit measures the number of cells or viability in the culture plates after the above-mentioned compound treatment according to the manufacturer's scheme and instructions. In brief, after taking out 50 μl of culture medium for glucose determination, the cells were incubated at 37 ° C for 1 hour with AQueous One solution (25 μl stock solution and 50 μl pre-warmed PBS / per well), and the OD490 nm absorbance in each sample was determined by Bio-Tek microplate reader. The cell viability in the culture wells was determined by subtracting the OD490 nm in the cultured cells from the OD490 nm in the common culture medium (no cell inoculation). Glucose production in the cultured cells was obtained by standardizing the glucose concentration in the culture medium by the cell viability in each well. At least 3 samples were examined for each treatment of the above analysis. Data are given as the mean ± SEM of these samples. The experiment was repeated at least twice.

[0327] The half-maximal inhibitory concentration (IC50 value) of compound #43 or metformin for inhibiting glucose production was determined using ED50 Plus v1.0 online software.

[0328] Results and discussion

[0329] 1. Effects of Insulin and Compound Solvent DMSO on Glucose Production in HepG2 Cells

[0330] like Figure 1 As shown in the left panel, treatment with 10 nM and 100 nM insulin resulted in a 20% and 30% decrease in glucose production in HepG2 cells, respectively, whereas treatment with the largest volume of compound solvent, DMSO, had no effect on glucose levels in cultured HepG2 cells. These results indicate that HepG2 cells are responsive to insulin in the inhibition of glucose production and that the observed effects of the test compounds on glucose production are not due to potential effects of DMSO. These results establish that HepG2 cells constitute an appropriate cell system for screening compounds that possess insulin-like activity and can inhibit glucose production in hepatocytes.

[0331] 2. Compound #43 can mimic insulin to inhibit glucose production in HepG2 cells

[0332] like Figure 1As shown in, HepG2 cells are incubated together with the compound #43 of test dose under serum-free conditions to cause the glucose level in the culture medium to reduce. The HepG2 cells observed after processing with compound #43 (3.8 μM) make the reduction of glucose production comparable to that achieved when using insulin (100nM), while higher doses of compound #43 (7.6, 15.2 and 30.4 μM) are far more effective than 100nM insulin. In addition, after processing with compound #43 under all test doses, no significant reduction (data not shown) in cell viability is observed in HepG2 cells. These results show that compound #43 is an insulin mimetic that can suppress the glucose production in HepG2 cells.

[0333] 3. Compound #43 alone showed higher endpoint efficacy in inhibiting glucose production in HepG2 cells than the three-compound combination CDE

[0334] Treatment of HepG2 cells with compound CDE at all tested doses (1:1:1 ratio of C / D / E) also inhibited glucose production with efficacy comparable to 100nM insulin. Compound #43 at a 3.8μM dose was as effective as the CDE combination product (which contained 3.8μM of each individual compound) in inhibiting glucose production in HepG2 cells. However, compound #43 at a 7.6μM or higher dose was more effective than CDE in inhibiting glucose production in HepG2 cells. These results indicate that compound #43 itself showed a higher endpoint efficacy than the three-compound combination CDE in inhibiting glucose production in HepG2 cells. It should be noted that the selenium concentration in compound CDE was three times that in compound #43 at each dose point, and these results may indicate that one or more structural features of compound #43 (in addition to its selenium molecule) may contribute to its activity, at least in some cases.

[0335] 4. When sulfur replaces the selenium molecule in compound #43, the inhibition of glucose production in HepG2 is eliminated

[0336] like Figure 1 As shown in the right panel, treatment of cells with the sulfur-containing compounds Compound #68 (the sulfur analog of Compound #43) or Compound #64 (SAM), along with the other listed non-selenium compounds, had minimal effect on reducing glucose production in HepG2 cells. The striking difference in inhibition of glucose production between Compound #43 and #68 suggests that the selenium molecule in Compound #43 contributes to its function in inhibiting glucose production in HepG2 cells.

[0337] 5. Differential effects of selenium compounds on the inhibition of glucose production in HepG2 cells

[0338] As described above, an equimolar mixture of selenium-containing compounds C, D, and E (CDE) inhibited glucose production in human HepG2 cells at the indicated doses. Figure 1 The compound concentrations indicated on the X-axis refer to the Se concentration of each Se-containing compound in the mixture, and the total Se concentration is actually three times that indicated on the X-axis. This was done to facilitate direct comparison of the mixture components with each of the single-molecule candidates tested in these experiments.

[0339] If the inhibition of glucose production was due solely to selenium, one would expect that CDE, which contains three times as much selenium as compound #43, would produce a more robust response than the latter. This was clearly not the case.

[0340] Figure 1 (Left panel) shows that each of compounds C, D, 43, 50, 53, 69 and 70 inhibited glucose production, with compound 43 being the most potent. Further, compound E was found to stimulate glucose production in HepG2 cells.

[0341] Taken together, these results suggest that:

[0342] (i) The selenium molecule in the compound is required to effectively inhibit glucose production (compare Figure 1 Compound #43 in the left panel and its appropriate sulfur analog, compound #68 in the right panel).

[0343] (ii) however, selenium molecules alone may not be sufficient to inhibit glucose production (in some cases, structurally similar compounds containing selenium exhibit lower or opposite effects);

[0344] (iii) increasing the concentration of selenium molecules alone may not improve efficacy (CDE has a threefold higher selenium concentration than compound #43, but is still not as effective);

[0345] (iv) Among the compounds tested, compound #43 showed the highest potency in inhibiting glucose production in hepatocytes.

[0346] 6. Analyze the structural features of the listed selenium compounds to identify chemical groups that may contribute to their activity

[0347] Comparison of compounds C and D showed that the 5' methylseleno group and the 5' selenohomocysteine ​​group provided similar inhibition of glucose production. Comparison of compounds 43, 50, 53, 69, and 70 showed that the diacetyl ester at the 2' and 3' positions (#43) provided greater inhibition of glucose production than the cyclic carbonate (compound #50), morpholinocarboxylate (#53), dipropionyl ester (#69), and dibutyryl ester (#70). Adenine, adenosine, and several chemical variants of adenosine did not inhibit glucose production in HepG2 cells. In addition, two adenylate cyclase inhibitors (#59 and 61) also did not inhibit glucose production, indicating that the effect of compound #43 in inhibiting glucose production is unlikely to be due to a decrease in cellular AMP levels (Hardie DG. Cell Metabolism 2013: 17(3): 313–314). In summary, the results suggest that one or more features other than adenine and the seleno molecule may contribute to activity, at least in some cases.

[0348] 7. Comparative study of compound #43 and metformin (a well-known antidiabetic drug) in inhibiting glucose production in HepG2 and rat H4IIE cells

[0349] As described above, a strong inhibition of glucose production in HepG2 cells was observed after treatment with compound #43. The effect of this compound was compared with the well-known antidiabetic drug metformin. Figure 2 As shown in the figure above, a dose-dependent decrease in glucose levels in HepG2 cells after treatment with compound #43 was observed, with an IC50 (half-maximal inhibitory concentration) value of 15.5 μM. As can be seen, a 500 μM dose of metformin did not inhibit glucose production in HepG2 cells, but instead showed some stimulatory effects. Higher doses of metformin (0.5-4 mM) showed cytotoxicity to these cells (data not shown). These results indicate that, similar to insulin, compound #43 can inhibit glucose production in HepG2 cells.

[0350] Rat H4IIE hepatocytes have been reported to respond to metformin with decreased glucose production. Therefore, this rat hepatocyte cell line was used to further confirm the inhibitory activity of compound #43 on glucose production and to compare the potency of compound #43 with metformin. Figure 2As shown in , treatment with compound # 43 and metformin respectively resulted in a dose-dependent reduction in glucose production in H4IIE cells under serum-free conditions. No toxic effects of these compounds on cell viability were observed at the test dose (data not shown). The IC50 of compound # 43 was 17.8 μM, which was almost identical to its IC50 in HepG2 cells. In contrast, metformin at a dose of 36.25 μM showed almost no or no inhibitory activity, and the IC50 of metformin in this experiment was 275 μM. These results indicate that compound # 43 is more effective (at least 15 times more effective) than metformin in inhibiting glucose production in cultured rat hepatocytes.

[0351] In summary, compounds #43, 50, 53, 69, and 70 all showed activity in inhibiting glucose production in cultured hepatocytes. However, compound #43 was the most potent single compound tested to date for inhibitory activity against glucose production, exceeding the activity of a high insulin dose (100 nM) in HepG2 cells.

[0352] Furthermore, compound #43 proved to be significantly more potent in both liver cell lines tested than the biguanide metformin, currently a first-line treatment for type 2 diabetes.

[0353] Example 3: Compound #43 and closely related selenoorganic compounds (#C, #50, #69 and #70) together with compounds A sulfur analogue of compound #43 (compound #68) is shown to be effective in insulin-resistant and diabetic patients with spontaneous null mutations in the leptin receptor (Lepr). Studies in mice to regulate blood glucose and / or HbA1c levels and improve glucose tolerance

[0354] Materials and methods

[0355] Compound

[0356] Compounds #43, #C, #50, #68, #69, and #70 were synthesized in the chemical laboratory of Alltech, Inc. The purity of all tested compounds was confirmed to be > 99% as determined by HPLC.

[0357] animal

[0358] Spontaneous mutation (leptin receptor mutation) in 5-10 week old male diabetic db / db Mice (C57BL / 6J strain) were purchased from Jackson Laboratory (Bar Harbor, Maine) and maintained in a pathogen-free vivarium with free access to food and water.

[0359] Long-term treatment with Compound #43 and other compounds

[0360] 38-day-old male Lepr db / dbMice were injected intraperitoneally (ip) daily with saline (0.09% NaCl) containing 0.2% DMSO, Compound #C, Compound #50, Compound #68, and / or Compound #43 (25 μg of selenium or sulfur equivalent per kg body weight of each compound, diluted in sterile saline) for a period of 43-90 days. The body weight of the treated mice was recorded daily using a scale, and any visible abnormalities in the animal's gross morphology and walking behavior were monitored daily. After treatment, the animals were fasted overnight before blood glucose or HbA1c determination, glucose tolerance test, or tissue collection.

[0361] To investigate the potential antidiabetic effects of compounds #43, #69, and #70, 41-day-old male Lepr db / db Mice were injected intraperitoneally (ip) daily with 0.2% DMSO in saline (0.09% NaCl), Compound #43, Compound #69, or Compound #70 (25 μg of selenium per kg body weight for each compound, diluted in sterile saline). After 43 days of treatment, the animals were fasted overnight and then subjected to blood glucose and glucose tolerance tests. After 90 days of daily treatment with these compounds, serum was collected from the animals and blood HbA1c was measured.

[0362] Compound #43 acute treatment

[0363] After fasting overnight, 8-10 week old Lepr db / db Mice were injected intraperitoneally with saline (0.09% NaCl) containing 0.2% DMSO or compound #43 (0.0054, 0.054, 0.54 or 5.4 mg of compound #43 (stock compound diluted in sterile saline) per kg body weight. After injection of saline or compound, mice were returned to their cages with free access to water but no food for 1, 2, 3, 5 and 8 hours. At each time point after injection, a small drop of blood was collected from the tail of each mouse for glucose measurement.

[0364] Another 6-week-old Lepr db / db Mice were injected intraperitoneally with saline (0.09% NaCl) containing 0.2% DMSO or a single dose of compound #43 (5.4 mg compound / kg body weight). After injection, the animals were returned to their cages with free access to water and food. After 24 hours, a small drop of blood was collected from the tail of the mice for glucose measurement.

[0365] Blood glucose measurement

[0366] After treatment with saline or compound or after glucose bolus, a small drop of blood was collected from each mouse by snipping the tip of the mouse tail. Blood glucose levels were determined using a glucometer capable of measuring a maximum of 600 mg / dL glucose.

[0367] Glucose tolerance test

[0368] Glucose tolerance test was performed as previously described (Li et al., Int J Biol Sci 2008; 4: 29-36). Briefly, Lepr db / db Mice were intraperitoneally injected with 2 grams / kg body weight of 20% D-glucose. Blood glucose levels were measured at time 0 (immediately before glucose injection) and at 0.25, 0.5, 1, and 2 hours after glucose injection using a glucometer capable of measuring a maximum glucose level of 600 mg / dL. Therefore, blood glucose levels exceeding 600 mg / dL were counted as 600 mg / dL in data analysis.

[0369] Blood and serum HbA1c measurement

[0370] After saline or compound treatment, a small drop of blood from the mouse tail was collected in an EDTA-coated eppendorf tube (to prevent blood clotting) (Fisher Scientific) and then HbA1c assayed using a mouse glycated hemoglobin A1c ELISA kit from Crystal Chem or Kamiya according to the manufacturer's protocol. Also after the final treatment, mouse serum was collected and HbA1c assayed using a mouse HbA1c kit from Kamiya Biomedical Company according to the manufacturer's protocol.

[0371] Statistical analysis

[0372] Where applicable, the Student t-test was used to determine the statistical significance of differences between saline-treated and compound-treated groups, with P values ​​less than 0.05 considered significant. Data are presented as mean ± SEM of the number of mice indicated in the figures.

[0373] Results and discussion

[0374] Lepr db / dbThe mice lack all known isoforms of the leptin receptor gene (Lepr). This homozygous mouse model is an aggressive type II diabetic mouse model with impaired glucose tolerance, reduced insulin sensitivity, hyperglycemia, and hyperinsulinemia. These mice show severe obesity at 3 to 4 weeks of age, plasma insulin begins to rise at 10 to 14 days, and develop hyperglycemia (i.e., high blood sugar levels) at approximately 4-8 weeks of age (Coleman DL. 1978 Diabetologia 14: 141-8).

[0375] In vitro studies have shown that compound #43 can mimic but bypass insulin to inhibit glucose production with a potency far greater than that of closely related compounds such as compound #C or #50. In addition, a sulfur-containing analog of compound #43 (compound #68) had little or no inhibitory effect on glucose production in HepG2 cells ( Figure 1-2 ). Therefore, insulin-resistant Lepr db / db The mouse is an ideal in vivo model system for studying the use of experimental compounds to potentially lower glucose in the bloodstream and improve insulin sensitivity and glucose tolerance in a severe diabetic setting.

[0376] 1. After long-term treatment, among the three tested selenoorganic compounds (#C, #50, #43), compound #43 was the most effective against Lepr db / db Most effective compound for treating hyperglycemia in mice

[0377] As in Lepr db / db As shown in Figure 2, the potential effect of the test compound in the treatment of hyperglycemia is studied using the administration scheme of the compound. Treatment is administered to mice daily by roughly intraperitoneal injection of the compound at the onset of hyperglycemia (developed into about 4-8 weeks after birth). Three compounds (i.e., compound #43, compound #C, and compound #50 delivering the same concentration of selenium) are injected every day to study whether these seleno-organic compounds have a measurable effect on the hyperglycemia of insulin-resistant mice.

[0378] It was found that treatment with all tested compounds did not affect the weight gain of these mutant mice (data not shown), suggesting that the tested compounds may have an effect on the function of Lepr db / db There was little or no suppressive effect on the appetite of mice showing abnormally increased food consumption. db / db There were no observable differences in the animals' gross morphology or walking behavior between the mice (data not shown).These results indicate that the compounds had no toxic effects on animal behavior or activity at the doses tested.

[0379] Among compounds #C, #50 and #43, treatment with compound #43 resulted in the db / db The mice showed the most significant decrease in blood glucose levels, which was approximately 45% lower than controls (see Figure 3 Left panel), although Lepr db / db Blood glucose levels in the mice were still higher than those in normal wild-type mice of the same age (approximately 100 mg / dL, data not shown). Furthermore, these results clearly demonstrate that compound #43 can significantly reduce blood glucose levels in this severe type II diabetic mouse model, suggesting that this compound may be useful for preventing hyperglycemia. Furthermore, these results provide good evidence for the differential effects of selenoorganic compounds against hyperglycemia and show that compound #43 is the most effective compound (among the three tested compounds) for treating hyperglycemia.

[0380] In addition, samples from Lepr treated with compounds #C, #50, and #43 were collected. db / db The serum of the mice was tested for HbA1c. The HbA1c level represents a long-term index of blood glucose concentration over the past 2 to 3 months, and this test is widely used in clinical medicine to monitor the blood glucose level history of diabetic patients. Figure 3 As shown in Figure 2, among compounds #C, #50, and #43, treatment with compound #43 resulted in a significant decrease in serum HbA1c levels (by about 20%) compared to the saline-treated group. These results are consistent with the effects of the three compounds on fasting glucose levels in mice ( Figure 3 left image) and in vitro glucose production (HepG2 cells, Figure 1 ) have the same differential impact.

[0381] Thus, the results provide in vivo evidence that selenoorganic compounds have differential effects on hyperglycemia, and that compound #43 is the most potent of the three selenocompounds (#C, #50, and #43), with significant potential for treating hyperglycemia in insulin-resistant subjects.

[0382] 2. Substituting sulfur molecules for selenium molecules in compound #43 at the tested doses had an effect on the long-term treatment of Lepr db / db Hyperglycemia in mice had no significant effect.

[0383] In vitro studies showed that compound #43, but not compound #68, potently inhibited glucose production in HepG2 cells ( Figure 1 ). The above study showed that compound #43 can reduce Lepr db / db Glucose output and HbA1c levels in mice ( Figure 3 To further confirm that compound #43 is in Lepr db / dbTo investigate the anti-diabetic effect in mice and to investigate whether the selenium molecule in compound #43 is required for this anti-hyperglycemic effect, 38-day-old male Lepramine mice were injected intraperitoneally with equal amounts of selenium or sulfur from compound #43 and its direct sulfur analog compound #68. db / db Mice were treated daily for 3 months.

[0384] Again, after a 3-month treatment period, the Lepr db / db There were no visible morphological, walking behavior abnormalities, or weight changes in the mice, indicating that compound #43 or #68 had no overt toxic effects on these mice at the doses tested. However, injections of compound #43 over a 3-month period resulted in a statistically significant decrease in fasting blood glucose levels, whereas compound #68 did not significantly reduce blood glucose levels ( Figure 4 , left panel). More notably, fasting glucose levels decreased to approximately 135 mg / dL after 3 months of Compound #43 treatment, which is close to the reported blood glucose levels of normal non-diabetic / obese mice (approximately 100 mg / dL). Consistent with these reductions in blood glucose levels, compared to the control (saline-treated) group, the fasting glucose levels from Lepr3 mice decreased after 3 months of Compound #43 treatment. db / db The HbA1c levels in the blood samples of the mice were also significantly reduced (approximately 30%). However, treatment with compound #68 did not affect the db / db Blood HbA1c levels in mice. db / db The extent of HbA1c reduction in mice ( Figure 4 ) appeared to be higher than that of Lepr after 42 days of treatment with compound #43. db / db More obvious in mice ( Figure 3 ).

[0385] Together, these in vivo results further confirm the findings that compound #43 significantly reduced blood glucose and HbA1c levels in a mouse model of aggressive type 2 diabetes, suggesting that compound #43 has potential for treating hyperglycemia in diabetic patients. Furthermore, the results also confirmed that the antidiabetic potential of compound #43 was lost after replacing the selenium atom in compound #43 with a sulfur atom.

[0386] 3. Lepr after long-term treatment with compound #43 db / db Shows higher anti-hyperglycemic potential than compounds #69 and #70 in mice

[0387] In vitro studies showed that replacing the diacetyl groups at the 2' and 3' positions of compound #43 with dipropionyl (compound #69) or butyryl (compound #70) attenuated the activity of compound #43 in inhibiting glucose production in HepG2 cells ( Figure 2). The above study showed that compound #43 can significantly reduce Lepr db / db Glucose output and HbA1c levels in mice ( Figure 3-4 To further confirm that compound #43 is in Lepr db / db To investigate the anti-diabetic effect of compound #43 in mice and to investigate the contribution of the diacetyl group in compound #43 to this anti-hyperglycemic effect, 41-day-old male Leprazole mice were treated with intraperitoneal injections of equal amounts of selenium in compounds #43, #69, and #70 for 43 and 90 days. db / db Mice were treated daily.

[0388] Again, after 90 days of treatment with the three compounds, Lepr db / db There were no visible morphological, walking behavior abnormalities, or weight changes in the mice, indicating that Compound #43, #69, or #70 had no overt toxic effects on these mice at the doses tested. It was found that injections of Compound #43 for 43 days resulted in a statistically significant decrease in fasting blood glucose levels (down approximately 35% from approximately 290 mg / dL in the saline group to 188 mg / dL) ( Figure 5 Replacing the diacetyl group in compound #43 with a dibutyryl group (compound #70) also resulted in Lepr db / db Fasting blood glucose levels in mice were reduced, but not as significantly as compound #43 ( Figure 5 Left image). Replacement of the diacetyl group in compound #43 (compound #69) with a dipropionyl group resulted in Lepr db / db Fasting blood sugar levels in mice increased slightly ( Figure 5 To further confirm the above observations, the Lepr db / db Mice were treated (compounds were administered daily for a total of 90 days), serum was collected and blood HbA1c was determined. Figure 5 As shown in the right panel of FIG, compound #43 treatment resulted in a significant decrease in HbA1c levels ( Figure 5 , right image). Preliminary evaluation indicated that treatment with compound #70 also resulted in a reduction of HbA1c levels by approximately 50% ( Figure 5 , right panel), whereas compound #69 treatment did not result in a significant decrease in HbA1c levels ( Figure 5 , right panel); further review of these data determined that neither compound #69 nor compound #70 had any significant effect ( Figure 5, right image). Overall, these results suggest that compound #43 has great potential for treating hyperglycemia in subjects with insulin resistance. Furthermore, the results suggest that the diacetyl groups at the 2' and 3' positions of compound #43 contribute to its anti-hyperglycemic function in vivo, and that extending the diacetyl group by one or two carbon atoms may negatively affect glucose homeostasis in certain circumstances.

[0389] 4. Acute treatment with compound #43 leads to the db / db Dose-dependent reduction of blood glucose levels in mice

[0390] The above studies showed that long-term treatment with compound #43 significantly reduced blood glucose and HbA1c levels. To determine the effective dose range and determine the duration of response to compound #43, 8-10 week old Lepr db / db Male mice were fasted overnight and then injected intraperitoneally with saline (containing 0.2% DMSO, the maximum injection volume of compound #43 stock solvent), 0.0054, 0.054, 0.54, and 5.4 mg of compound #43 (prepared by diluting the stock compound with saline) per kg body weight. Lepr db / db Blood glucose levels in mice were measured and the resulting blood glucose levels for each individual animal at each time point were plotted.

[0391] A single emergency injection of compound #43 at the above dose did not produce any observable toxic effects on gross morphology and walking behavior. db / db In mice, blood glucose levels decreased only slightly (approximately 50-70 mg / dL) during the 8-hour test period ( Figure 6 ), which is consistent with these Lepr db / db The results are consistent with the fact that mice exhibited impaired glucose clearance. However, treatment with Compound #43 resulted in significant reductions in blood glucose levels at all doses tested (when compared to their saline group at each time period), with the exception of the lowest dose of Compound #43 treatment at 2 or 3 hours post-injection (where the reduction in glucose levels approached statistical significance) ( Figure 6 ). The results showed that compound #43 effectively lowered blood glucose levels 1 hour after a single injection at all doses tested. This suggests that compound #43 can reach relevant target tissues in the body within a short period of time (i.e., 1 hour) to trigger its glucose-lowering effect. The activity of compound #43 in lowering blood glucose levels under fasting conditions peaked 2 to 5 hours after administration, and this effect was maintained for at least another 3 hours. Since the test animals (which had been fasted overnight and continued to be fasted during the 8-hour period tested) could not be fasted further, the maximum effective duration of the effect of compound #43 could not be determined from these experiments.

[0392] The results showed that acute treatment with Compound #43 over a 1000-fold concentration range significantly reduced blood glucose levels in a widely used animal model of insulin resistance and type 2 diabetes. Compound #43 was rapidly acting (≤ 1 hour after treatment) and remained active for at least 8 hours.

[0393] 5. Acute treatment with compound #43 weakens young Lepr db / db Progression of hyperglycemia in mice

[0394] As discussed above, Lepr db / db Mice begin to show elevated plasma insulin at 10 to 14 days of age and hyperglycemia (i.e., high blood sugar levels) at approximately 4-8 weeks of age (Coleman DL. 1978 Diabetologia 14:141-8). To test whether Compound #43 has the potential to attenuate the development of hyperglycemia, a single dose of Compound #43 was administered via emergency injection to younger mice. Briefly, 6-week-old Lepr mice under normal feeding conditions were treated with a single dose of Compound #43. db / db Male mice were injected intraperitoneally once with saline containing 0.2% DMSO or 5.4 mg / kg body weight of Compound #43. 24 hours after treatment, the blood glucose levels of the mice were measured while they had free access to food and water.

[0395] like Figure 7 As shown in the db / db Blood glucose levels in mice 24 hours after saline treatment increased significantly (by approximately 20%), indicating that these mice were still in the process of developing hyperglycemia. In contrast, treatment with compound #43 resulted in a decrease in Lepr db / db The blood glucose level of mice was significantly reduced (about 20%) ( Figure 7 ).

[0396] These results suggest that compound #43 has the potential to attenuate the development of hyperglycemia. Furthermore, these studies also demonstrate that compound #43's effectiveness in reducing glucose output in these diabetic mice is likely to persist for at least 24 hours under fed conditions.

[0397] 6. Diabetes Lepr after administration of compound #43 db / db Improved glucose tolerance in mice

[0398] The glucose tolerance test identifies abnormalities in the way the body handles glucose after a rapid rise in blood sugar, such as typically after a meal. Insulin plays a key role not only in suppressing glucose production in the liver, but also in glucose uptake, storage, and metabolism in muscle, liver, and fat cells, resulting in lower glucose levels in the bloodstream.

[0399] Diabetics have very poor glucose tolerance, either because they are unable to produce insulin or because they are unable to respond effectively to insulin to maintain glucose homeostasis. The in vitro studies described herein demonstrate that compound #43 not only mimics insulin but also bypasses insulin to inhibit glucose production ( Figure 1-2 Considering the fact that these mutant mice showed impaired glucose tolerance and insulin resistance, Lepr db / db Mice are an ideal mouse model of type II diabetes for studying the effects of compound #43 on maintaining glucose homeostasis. Therefore, the effects of compound #43 and other structurally similar selenium and sulfur compounds on Lepr db / db Improved glucose tolerance in mice.

[0400] These male mice were injected intraperitoneally with normal saline (containing 0.2% DMSO), compound #C, #43 or #50 (25 μg of selenium per kg body weight for each compound) at 38 days of age for 43 days. At the end of the treatment, the mice were fasted overnight, injected with glucose (2 g / kg body weight), and blood glucose levels were measured at 0.25 hours (15 minutes), 0.5 hours (30 minutes), 1 hour (60 minutes) and 2 hours (120 minutes) after the glucose injection. Blood glucose levels were also recorded just before the glucose injection (referred to as time zero).

[0401] like Figure 8 As shown in A, in saline-treated Lepr db / db A significant increase in blood glucose levels was observed in mice starting at 0.25 hours after glucose injection and at all subsequent time points tested. As described herein, the glucose measurement limit of the glucometer used for these analyses is 600 mg / dL. Therefore, glucose levels exceeding this limit were recorded as 600 mg / dL. Therefore, particularly for saline-treated animals, some of the measured values ​​at the time points tested after glucose injection likely represent an underestimate of the true blood glucose concentration.

[0402] In mice treated with Compound #C, blood glucose levels remained very high at all tested time points after glucose injection, similar to the saline-treated group ( Figure 8 A) These results indicate that compound #C does not improve glucose tolerance at the doses tested in these insulin-resistant diabetic mice.

[0403] Compound #50 treatment resulted in a significant decrease in glucose levels at the 2-hour time point after glucose injection when compared to the saline-treated group, despite Figure 8There was no significant decrease at 0.25, 0.5, or 1 hour after glucose injection in A. These results suggest that compound #50 may have some effect in improving glucose tolerance in these diabetic mice.

[0404] In contrast, in Lepr treated with compound #43 db / db In mice, blood glucose levels at 0.25, 0.5, and 1 hour after glucose injection were significantly lower than those in mice treated with saline, compound #C, or #50 ( Figure 8 A) Due to the measurement limitations of the blood glucose meter, it is possible that the reduction in glucose levels at these time points after glucose injection was less pronounced in mice treated with Compound #43 than in the other treatments. Figure 8 This is much more pronounced as shown in A. 2 hours after glucose injection, Lepr treated with compound #43 db / db Blood glucose levels in mice were much lower than those in littermates treated with saline, Compound #C, or Compound #50 (see Figure 8 A) and almost completely returned to pre-injection glucose levels. When compared to mice treated with saline or compound #C, Lepr db / db The mice showed significantly different reductions in glucose levels 2 hours after glucose injection (P < 0.001). These results indicate that compound #43, but not compound #C, can almost completely restore insulin action in these insulin-resistant diabetic mice at the doses tested, as assessed by improved glucose tolerance, while compound #50 may also have some beneficial effects in improving glucose clearance.

[0405] To further confirm the effect of compound #43 on the enhancement of Lepr db / db To investigate the potential of the selenium atom in compound #43 to improve glucose clearance in mice, and to investigate whether the selenium atom in compound #43 is required for this effect, 38-day-old male Lepr mice were injected intraperitoneally with equal amounts of selenium or sulfur in compound #43 or #68, respectively. db / db The mice were treated daily for 2 months. At the end of treatment, the mice were fasted overnight and subjected to a glucose tolerance test as described above.

[0406] like Figure 8 As shown in B, in Lepr treated with compound #68 db / db A significant increase in blood glucose levels was observed in mice starting at 0.25 hours after glucose injection and at all subsequent time points tested. There was no significant decrease in blood glucose levels at the 2 hour time point after glucose injection (when compared to the levels at the 0.25, 0.5, and 1 hour time periods), indicating that compound #68 had little or no effect on improving glucose tolerance in these insulin-resistant diabetic mice at the doses tested.

[0407] In Lepr treated with compound #43 db / db In mice, blood glucose levels before glucose challenge injection were significantly lower than those in mice treated with compound #68 (P < 0.05). These results are consistent with the above observations that compound #43 is more effective than compound #68 in lowering fasting blood glucose levels in this diabetic mouse model ( Figure 4 ). Lepr treated with compound #43 db / db The blood glucose levels of mice were lower at 0.25, 0.5 and 1 hour after glucose injection than those of mice treated with compound #68. db / db Blood glucose levels in mice were significantly lower than those in littermates treated with Compound #68 (see Figure 8 B, P < 0.001). In this experiment, Lepr treated with compound #43 db / db Glucose clearance curve of mice ( Figure 8 B) is almost identical to the curve observed in the first glucose tolerance test above ( Figure 8 A). Again, due to the measurement limitations of the glucometer, the reduction in glucose levels at these time points after glucose injection in mice treated with Compound #43 relative to mice treated with Compound #68 is likely to be smaller. Figure 8 In any case, the above results further confirm that compound #43 significantly improves glucose tolerance at the tested doses and that replacing the selenium atom in compound #43 with sulfur almost completely abolishes its effect in these insulin-resistant diabetic Lepramines. db / db Ability to promote glucose clearance in mice.

[0408] Finally, we investigated whether replacing the acetyl groups at the 2' and 3' positions of the ribose moiety of compound #43 with propionyl or butyryl groups could db / db Improved glucose clearance in mice. Male 41-day-old male Lepr db / db The mice were treated daily for 43 days. At the end of treatment, the mice were fasted overnight and subjected to a glucose tolerance test as described above.

[0409] like Figure 8 As shown in C, in saline-treated Lepr db / dbA significant increase in blood glucose levels was observed in mice starting at 0.25 hours after glucose injection and at all subsequent time points tested. Blood glucose levels did not decrease significantly until the 1 hour time point after glucose injection (when compared to glucose levels at 0.25 and 0.5 hour time periods), while glucose levels decreased slightly at 2 hours after glucose injection in these insulin-resistant diabetic mice.

[0410] Compound #69 treatment resulted in a slight but non-significant decrease in glucose levels at the 2-hour time point after glucose injection when compared to the saline-treated group, although Figure 8 There was no significant decrease in blood glucose levels at 0.25, 0.5, or 1 hour after glucose injection in C. These results suggest that compound #69 may have some effect in improving glucose tolerance in these diabetic mice.

[0411] In mice treated with compound #70, fasting blood glucose levels before glucose injection were lower than those in the saline-treated group ( Figure 8 C). Blood glucose levels were slightly, but not significantly, lower in mice treated with Compound #70 after glucose injection, particularly at the 2-hour time point, when compared to saline-treated mice. These results suggest that Compound #70, like Compound #69, may have some effect in improving glucose tolerance in these insulin-resistant diabetic mice at the doses tested.

[0412] In contrast, Lepr treated with compound #43 db / db The blood glucose levels of mice at 0.25 and 0.5 hours after glucose injection were significantly lower than those of mice treated with saline, compound #69 or #70 ( Figure 8 C) Lepr treated with compound #43 1 hour after glucose injection. db / db Blood glucose levels in mice were significantly lower than those in littermates treated with saline, Compound #69, or Compound #70 ( Figure 8 C) Lepr treated with compound #43 2 hours after glucose injection. db / db Blood glucose levels in mice were also much lower than in mice treated with saline, Compound #69, or Compound #70. db / db The reduction in glucose levels at 2 hours after glucose injection was significantly different between the mice (P<0.05). Again, due to the measurement limitations of the glucometer, the reduction in glucose levels at each time point after glucose injection in mice treated with Compound #43 may be smaller than in the other treatments. Figure 8C is much more significant. Regardless, the results further confirm that compound #43 significantly improves glucose tolerance in these insulin-resistant diabetic mice at the dose tested. These results also suggest that compounds #69 and #70 may have some beneficial effects in improving glucose clearance. Comparing the chemical structures of compound #43 with those of compounds #69 and #70, it is clear that the acetyl groups at the 2' and 3' positions of the ribose group of compound #43 are essential for optimal glucose clearance activity, and replacing these diacetyl groups in compound #43 with dipropionyl or dibutyryl significantly attenuates its activity in these insulin-resistant diabetic Leprosy mice. db / db Ability to promote glucose clearance in mice.

[0413] In conclusion, the above studies showed that compound #43 significantly increased Lepr db / db Glucose tolerance in mice. The effect of compound #43 in this process is likely mediated by an increase in insulin sensitivity in the clearance of glucose from skeletal muscle, liver, and adipose tissue. Furthermore, while selenium is necessary for the action of compound #43, its presence alone is not sufficient to confer glucose clearance ability in diabetic subjects. The selenium atom must be present in a very specific chemical form. This is evidenced by the lower activity of compound #50 and the lack of activity of compound C; both of which are structurally very similar to compound #43. Furthermore, the acetyl groups at the 2' and 3' positions of the ribose group of compound #43 are also required for its activity in glucose clearance.

[0414] Example 4: Compound #43 treatment in the liver of diabetic leptin receptor (Lepr) spontaneous null mutation mice and inhibition of the expression of the gluconeogenic enzyme gene G6pc in cultured hepatocytes and inhibition of Potentiation of insulin action in G6pc expression

[0415] The liver is the primary organ for producing glucose to maintain normal glucose levels in the bloodstream. Glucose-6-phosphatase catalytic subunit (G6pc) is an essential enzyme for gluconeogenesis in the liver. The effect of compound #43 on regulating G6pc expression was studied both in vivo and in vitro.

[0416] Materials and methods

[0417] Compound

[0418] Compounds #43, #C, #D, #E, and #50 were synthesized in the chemical laboratory of Alltech, Inc. The purity of all tested compounds was confirmed to be > 99% as determined by HPLC.

[0419] In Lepr db / db In vivo treatment of mice with compounds #43 and #50

[0420] 38-day-old male Lepr db / dbMice (C57BL / 6J strain, purchased from The Jackson Laboratory) were injected intraperitoneally daily for 52 days with saline (0.09% NaCl) containing 0.2% DMSO, compound #50, or compound #43 (25 μg of selenium equivalent per kg body weight of each compound, diluted in sterile saline). Following treatment, livers were harvested and subjected to RNA analysis.

[0421] Cell lines and cell expansion

[0422] Human hepatocarcinoma HepG2 and mouse liver AML-12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were amplified in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS. AML-12 cells were amplified in Dulbecco's Modified Eagle's Medium and Ham's F12 (DMEM / F12) culture medium supplemented with 10% fetal bovine serum (FBS), 40 ng / ml dexamethasone (Dex, Sigma), and 1X ITS (containing 0.01 mg / ml bovine insulin, 0.0055 mg / ml human transferrin, 5 ng / ml sodium selenite) solution (Sigma).

[0423] Cell processing for RNA analysis

[0424] For RNA analysis of basal G6pc expression (in the absence of diabetic stimuli: 8-CPT / Dex), expanded AML-12 and HepG2 cells were plated in 24-well plates (0.5-2×10 5 Cells were cultured overnight on PBS (400 cells / well). These cells were rinsed twice with PBS to remove residual serum. Then, HepG2 cells washed with PBS were treated with or without insulin or compound #43 in serum-free EMEM medium for 40 hours. In some experiments, AML-12 cells washed with PBS were incubated in serum-free DMEM / F12 medium for 24 hours without or with compound #43 or other selenium compounds. In other experiments, the expanded AML-12 cells were pretreated with or without compound #43 (150 or 300 ppb) in DMEM / F12 medium with 10% FBS but without ITS / Dex for 24 hours. After 24 hours of treatment, AML-12 cells were washed twice with PBS (to remove any residual serum in the culture) and then treated with insulin, compound #43, or both for 6 hours in serum-free DMEM / Dex medium.

[0425] For RNA analysis of G6pc expression induced by diabetic stimuli, AML-12 cells were pretreated with or without compound #43 (150 or 300 ppb) for 24 hours in DMEM / F12 medium containing 10% FBS but without ITS / Dex. The cells were then washed twice with PBS to remove any residual serum and incubated with compound #43 (150 or 300 ppb) in serum-free plain DMEM / F12 medium for an additional 6 hours in the presence or absence of insulin (10 or 100 nM) or 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex.

[0426] RNA isolation and real-time PCR analysis

[0427] RNA from saline- or seleno compound-treated LeprA was isolated using the Qiagen RNAeasy RNA isolation kit according to the manufacturer's protocol. db / db Total RNA from mice was isolated using Trizol (Invitrogen) according to the manufacturer's protocol and then incubated with DNase I to remove any potential contaminating genomic DNA. RNA samples were subjected to real-time PCR (QRT-PCR) analysis using an Applied-Bioscience RT kit and pre-designed Taqman probes (Invitrogen) as previously described (Lan et al., EMBO J 2003). Data were normalized by the level of actin B (Actb) mRNA in each sample and presented as the mean ± SEM of 3-5 samples.

[0428] Statistical analysis

[0429] Where applicable, the Student t test was used to determine the statistical significance of differences between treatment groups, and P values ​​less than 0.05 were considered statistically significant.

[0430] result:

[0431] 1.Lepr db / db Analysis of G6pc mRNA expression in mouse liver

[0432] Previous experiments have shown that compounds #43 and #50 inhibit Lepr db / db Different effects on blood glucose levels and HbA1c levels in mice ( Figure 3 Without wishing to be bound by any particular hypothesis, this differential effect may be at least in part due to potential differential effects of these compounds on the expression of the gluconeogenic G6pc gene in vivo. Therefore, the expression of Lepr was measured after long-term treatment with these two compounds. db / dbG6pc mRNA expression in mice.

[0433] like Figure 9 As shown in the Figure 2, compound #50 treatment resulted in the expression of db / db G6pc mRNA levels in mouse livers were slightly but not significantly reduced. However, compound #43 treatment resulted in a decrease in Lepr mRNA levels when compared to saline-treated controls. db / db The G6pc mRNA level in the mouse liver was significantly reduced (decreased by about 56%) ( Figure 9 ).

[0434] In summary, the results provide in vivo evidence that these seleno compounds may have differential effects in inhibiting G6pc expression and that compound #43 is a potent inhibitor of G6pc expression in the livers of these severe type II diabetic mice. db / db Blood glucose and HbA1c levels decreased in mice ( Figure 3 ) is at least in part due to decreased G6pc mRNA expression. In addition, because G6pc expression is regulated in response to insulin signaling, and given that Lepr db / db The mice are insulin resistant, so the results suggest that compound #43 may bypass insulin or restore insulin action to regulate G6pc expression in these diabetic mice.

[0435] 2. Inhibition of G6pc mRNA expression and potentiation of insulin action in G6pc expression in mouse and human hepatocytes after treatment with compound #43

[0436] The above studies revealed that compound #43 significantly inhibited G6pc expression in diabetic mice with insulin resistance. Cultured hepatocytes were used to investigate (a) whether seleno compounds differentially affect G6pc expression, (b) whether compound #43 has a direct effect on G6pc expression in the liver, and (c) whether compound #43 can enhance the role of insulin in regulating G6pc expression. Two treatment regimens (direct compound treatment of hepatocytes in serum-free conditions and pretreatment of hepatocytes with compound in serum-containing medium followed by compound re-treatment in serum-free conditions) were implemented to examine the effect of compound #43 on G6pc expression in these hepatocytes.

[0437] First, mouse liver AML-12 cells were left untreated (control) or treated with a combination of Compounds CDE, Compound #C, Compound #D, Compound #50, and Compound #43 at a dose of 300 parts per billion (ppb) selenium (equivalent to 3.8 μM of each compound) in serum-free medium supplemented with insulin-transferrin-sodium selenite (ITS) and without dexamethasone (Dex) for 24 hours to investigate whether these selenium compounds have differential effects on G6pc expression. Figure 10 As shown in A, compounds CDE (300 ppb each) resulted in a significant decrease in G6pc mRNA expression. However, compounds #C, #D, or #50 did not significantly inhibit G6pc expression in AML-12 cells at the doses tested. In contrast, treatment with compound #43 at the same selenium dose resulted in a strong decrease in G6pc expression in AML-12 cells (by approximately 60% when compared to the control group) ( Figure 10 A). The extent of reduction in G6pc expression after treatment with compound #43 (60%) was more pronounced than that after treatment with the combination of compounds CDE (approximately 40% reduction). These results suggest that these selenoorganic compounds may have differential effects on the inhibition of G6pc expression in vitro, and that compound #43 was the most potent compound among all tested compounds in this process. This is consistent with the in vivo mouse studies described above ( Figure 9 Since this experiment was performed in AML-12 cells under completely serum-free conditions (i.e., in the absence of insulin or any other growth factors), the results indicate that compound #43 can mimic but bypass insulin to directly inhibit G6pc expression in AML-12 cells with a higher potency than the combination of compounds CDE.

[0438] Next, another liver cell line, human HepG2 cells, were incubated with 100 nM insulin or 600 ppb compound #43 in serum-free medium for 40 hours to further verify the direct inhibitory effect of compound #43 on G6PC expression. Figure 10 As shown in Figure B, insulin treatment resulted in a significant decrease in G6PC expression, indicating that insulin signaling plays a role in HepG2 cells. Furthermore, when compared to the control group, compound #43 significantly attenuated the G6PC mRNA level in HepG2 cells after treatment under completely serum-free conditions ( Figure 10 B) G6PC expression in HepG2 cells decreased after treatment with compound #43. Figure 1 Therefore, the results further indicate that compound #43 can mimic but bypass insulin to directly downregulate G6PC expression, thereby inhibiting glucose production in HepG2 cells.

[0439] Finally, AML-12 cells were pretreated with compound #43 in serum-containing medium without ITS / Dex for 24 hours, and then treated with this compound in medium without FBS / ITS / Dex for an additional 6 hours in the presence or absence of insulin to further investigate whether compound #43 could inhibit G6pc expression and whether there was an additive or synergistic effect between insulin and compound #43 in downregulating G6pc expression. Figure 10 As shown in C, 10 nM insulin treatment resulted in a significant decrease (about 65%) in G6pc mRNA levels when compared to the control group ( Figure 10 C, bar 1). Like insulin, treatment with compound #43 (at 150 and 300 ppb) also resulted in a significant decrease in G6pc expression, comparable to that of 10 nM insulin. Furthermore, the reduction in G6pc mRNA levels in AML-12 cells was more pronounced after pretreatment with compound #43 followed by co-treatment with both compound #43 and insulin, compared to treatment with compound #43 or insulin alone. These results further support the observation that compound #43 can mimic but bypass insulin to inhibit G6pc expression in AML-12 cells. The results also suggest that compound #43 can potentiate the effects of insulin in downregulating G6pc expression in AML-12 cells.

[0440] 3. Compound #43 inhibits G6pc expression and enhances insulin action in the regulation of G6pc expression in AML-12 cells cultured under simulated diabetic conditions (stimulated by both 8-CPT and Dex)

[0441] Cyclic AMP (8-CPT) and Dex are well-known stimulators of G6pc expression and glucose production in the liver, which mimic diabetic conditions in vivo. To further investigate the effect of compound #43 on G6pc expression, G6pc mRNA expression was examined in AML-12 cells co-treated with cell-permeable 8-(4-chlorophenylthio) cAMP (8-CPT) and dexamethasone (Dex). Briefly, AML-12 hepatocytes were pretreated for 24 hours without or with 150 ppb or 300 ppb of compound #43 in DMEM / F12 medium containing 10% FBS but without ITS / Dex. After washing twice with PBS, the cells were treated with these seleno compounds in serum-free medium for an additional 6 hours in the presence or absence of 10 nM or 100 nM insulin, 0.1 mM 8-CPT, and 0.5 μM Dex. Following these treatments, cells were harvested and subjected to QRT-PCR analysis.

[0442] like Figure 11As shown in Figure 2, treatment of AML-12 hepatocytes with 8-CPT / Dex resulted in a 41.5-fold increase in the expression of G6pc mRNA (bars #1 vs. #2). Treatment with both doses of insulin significantly reduced 8-CPT / Dex-induced G6pc expression in AML-12 cells when compared to the 8-CPT / Dex group ( Figure 11 Furthermore, compound #43 at 150 and 300 ppb doses also significantly attenuated 8-CPT / Dex-induced G6pc expression (from 41.5 in column #2 to 13 in column #5 and 13.5 in column #8, Figure 11 ), potency with 10 nM insulin (column #3, Figure 11 These studies showed that, like insulin, compound #43 alone could inhibit 8-CPT / Dex-induced G6pc expression at the doses tested (a decrease of approximately 68% when compared to the 8-CPT / Dex group, Figure 11 Columns #5 and #8 vs. #2).

[0443] In addition, when treated with no insulin / Compound #43 (column #2), insulin alone (columns #3-4), or Compound #43 alone ( Figure 11 Compound #43 combined with insulin treatment ( Figure 11 Columns #6-7 and #9-10 in the Figure 3 further inhibited 8-CPT / Dex-induced G6pc expression in AML-12 cells. More significantly, in the treatment with 150 ppb of compound #43 and 100 nM insulin together with 8-CPT / Dex (column #7) and in the treatment with 300 ppb of compound #43 and 100 nM insulin together with 8-CPT / Dex (column #10), G6pc mRNA levels dropped strongly from the level of 8-CPT / Dex treatment alone (column #2) to nearly that of the control group without 8-CPT / Dex ( Figure 11 In other words, compound #43 (150 or 300 ppb) co-treatment with 100 nM insulin almost completely abolished 8-CPT / Dex-induced G6pc expression in AML-12 cells.

[0444] In summary, these results indicate that, like insulin, compound #43 alone can inhibit 8-CPT / Dex-induced G6pc expression at the tested doses, and the combination of insulin and compound #43 is even more effective than insulin alone or compound #43 alone in inhibiting the increase in G6pc expression caused by 8-CPT / Dex treatment in AML-12 cells.

[0445] The aforementioned effect of reducing G6pc expression in response to selenocompounds was not due to potential toxic effects of these selenocompounds on cell survival, as these compounds did not affect the viability of AML-12 or HepG2 cells under the same experimental conditions at the doses tested (data not shown).

[0446] In summary, these results indicate that compounds #43 and #50 have differential effects on the inhibition of G6pc expression in the liver. At the very least, the data indicate that compound #43 is an effective compound for inhibiting G6pc expression in the liver both in vivo and in vitro. The study further revealed that compound #43 can mimic but bypass insulin to directly inhibit G6pc expression in mouse and human hepatocytes cultured under normal conditions and conditions that simulate diabetes (i.e., cells treated with 8-CPT / Dex). In addition, compound #43 can enhance insulin action to inhibit G6pc expression in AML-12 cells cultured under normal and simulated diabetes conditions. In summary, these results provide molecular evidence that compound #43 can inhibit G6pc expression in the liver both in vivo and in vitro, and therefore may be valuable for treating patients with type I and type II diabetes.

[0447] Example 5: Compound #43 mimics but bypasses insulin to activate phosphoinositide-dependent proteins in vivo and in vitro kinase 1 (PDK1) and protein kinase B (AKT) signaling to enhance the phosphorylation of Forkhead box protein O1 (FOXO1) in the liver acidification

[0448] The forkhead transcription factor FOXO1 plays a key role in metabolism, gluconeogenesis, and insulin sensitivity in the liver. The intracellular activity of FOXO1 is tightly regulated by post-translational modifications. Specifically, phosphorylation of FOXO1 excludes FOXO1 from the nucleus, preventing it from accessing its target genes, such as G6pc, which is required for glucose production in the liver. In individuals with insulin resistance or diabetes, there is no signal to exclude FOXO1 from the nucleus, so it remains there and stimulates the transcription of G6pc. Increased G6pc expression drives gluconeogenesis, leading to hyperglycemia.

[0449] As described above, the in vivo and in vitro results indicate that compound #43 can mimic but bypass the action of insulin to inhibit G6pc expression and enhance the insulin action in the process. Since FOXO1 is the main signaling molecule for gluconeogenesis and insulin sensitivity in the liver, and PDK1 and AKT are two major intermediate signaling molecules upstream of FOXO1, the research on the effect of compound #43 on G6pc expression in Lepr db / db The question is whether compound #43, like insulin, will target FOXO1 and its upstream signaling molecules PDK1 and AKT in mouse, human liver HepG2, and mouse liver AML-12 cells.

[0450] Materials and methods

[0451] Compound

[0452] Compound #43 was synthesized in the chemical laboratory of Alltech, Inc. The purity of this test compound was confirmed to be > 99% as determined by HPLC.

[0453] Compound #43 in Lepr db / db In vivo processing in mice and liver protein preparations

[0454] On the 38th day after birth, male Lepr db / db Mice (C57BL / 6J strain, purchased from The Jackson Laboratory) were injected intraperitoneally daily for 52 days with saline (0.09% NaCl) containing 0.2% DMSO and compound #43 (25 μg of selenium or sulfur equivalent per kg body weight of each compound, diluted in sterile saline). After treatment, livers were harvested and stored at -80°C.

[0455] Frozen liver tissue is minced in sterile ice-cold PBS containing complete protease and phosphatase inhibitors (Thermo-FisherScientific, Waltham, MA) and homogenized using a tissue homogenizer (Thermo-FisherScientific, Waltham, MA). These tissue homogenates are diluted (1 part homogenate / 2 parts RIPA buffer) in Themo-Fisher pre-made RIPA buffer containing complete protease / phosphatase inhibitors to extract protein. At 4°C, the protein in the homogenate is extracted overnight in RIPA buffer. The protein lysates extracted overnight are centrifuged at 12000x g for 30 minutes at 4°C, and the protein level in the supernatant of these tissue lysates is determined according to the manufacturer's protocol using Pierce Micro-BCA protein assay kit (Thermo Scientific-Piece Biotechnology, Rockford, IL).

[0456] Cell culture

[0457] Human hepatocarcinoma HepG2 and mouse liver AML-12 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS. AML-12 cells were amplified in Dulbecco's Modified Eagle's Medium and Ham's F12 (DMEM / F12) culture medium supplemented with 10% fetal bovine serum (FBS), 40 ng / ml dexamethasone (Dex, Sigma), and 1X ITS (containing 0.01 mg / ml bovine insulin, 0.0055 mg / ml human transferrin, 5 ng / ml sodium selenite) solution (Sigma).

[0458] Cell processing for protein analysis

[0459] HepG2 cells were seeded in 6-well plates (7 × 10 5 Cells were plated on 400 cells / well and cultured in 10% FBS EMEM medium for 30 hours. The cells were then washed twice with PBS to remove residual serum and serum-starved overnight in normal EMEM medium. Serum-starved HepG2 cells were left untreated or treated with compound #43 (600 ppb) for 0 minutes (immediately before treatment), 30 minutes, 60 minutes, 90 minutes, 24 hours, 30 hours, and 40 hours.

[0460] AML-12 cells were used to investigate whether compound #43 could modulate Pdk1 / Akt / Foxo1 signaling molecules in hepatocytes after induction with diabetic stimuli. The expanded AML-12 cells were seeded in 6-well plates (1×10 6 Cells were plated on 400 cells / well plates and cultured in DMEM / F12 medium with 10% FBS but without ITS / Dex for 24 hours. The cells were then washed twice with PBS to remove residual serum and serum-starved overnight in plain DMEM / F12 medium. These serum-starved AML12 cells were treated with the diabetes stimulants 8-CPT (0.1 mM) and Dex (0.5 μM) without (control group) or in combination with 10 nM insulin or compound #43 (300 ppb) in serum-free plain DMEM / F12 medium for 60 minutes, 90 minutes, and 6 hours, respectively.

[0461] After the above treatment, cultured HepG2 and AML-12 cells were rinsed twice with ice-cold PBS on ice and lysed for 30 minutes in ice-cold RIPA buffer containing complete protease and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA). Cell lysates were collected using a cell scraper and transfer pipette, and then centrifuged at 12000 x g for 30 minutes at 4 ° C to remove DNA precipitation and obtain protein extracts. Protein levels in the supernatants of these cell lysates were determined using Pierce Micro-BCA protein assay kit (Thermo Scientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.

[0462] Western blot analysis

[0463] As previously described (Reddy et al., 2008 Science), one hundred micrograms of liver tissue protein or five micrograms of total protein from control and compound-treated HepG2 or AML-12 cells were subjected to SDS-PAGE gel separation and then transferred to a PVDF membrane. The membrane was blocked in phosphate-buffered saline (PBS) containing 5% (w / v) bovine serum albumin (Sigma, St. Louis, MO) and incubated with specific primary antibodies, followed by incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000 dilution, Cell Signaling Inc.). All primary antibodies except Gapdh (Li-COR, Lincoln, Nebraska) were purchased from Cell Signaling Inc. Positive signals on membrane blots were detected using Amersham's enhanced chemiluminescent Western blot primary detection reagent (GE Healthcare Lifescience, Pittsburgh, PA). Images of these luminescent signals on membrane blots were captured using the LI-COR Odyssey Fc Image System (Lincoln, Nebraska). The same membrane blot was stripped and reblotted with another antibody as described in the GE WB ECL primary detection protocol (GE healthcare Lifescience, Pittsburgh, PA). Protein band density in the Western blot was determined using NIH ImageJ software and then normalized by Gapdh or Actb / ACTB levels in each sample. Data are presented as mean ± SEM of three samples per group.

[0464] Statistical analysis

[0465] Where applicable, Student's t-test was performed to determine the statistical differences between the two groups. A P value of less than 0.05 was considered significant.

[0466] result:

[0467] 1. Insulin-resistant Lepr after long-term treatment with compound #43 db / db Increased phosphorylation of Pdk1, Akt, and Foxo1 in mouse liver

[0468] Animal studies reveal compound #43 can be used in Lepr db / db Reduced blood glucose and HbA1c levels and inhibited liver G6pc expression in mice ( Figure 3-7 , 9). The reduced fasting glucose levels and blood HbA1c levels are at least in part due to Lepr db / db The expression of the gluconeogenic G6pc gene in the liver of mice was reduced. G6pc expression in the liver is controlled by the insulin signaling Pdk1 / Akt / Foxo1 cascade. Therefore, this application investigated whether long-term treatment with compound #43 could inhibit the expression of the gluconeogenic G6pc gene in these insulin-resistant Lepr db / db Insulin signaling was restored, at least to some extent, in the liver of mice (ie, enhanced phosphorylation of Pdk1 / Akt / Foxo).

[0469] Lepr on the 38th day after birth db / db Mice were intraperitoneally injected with saline or compound #43 (at a dose of 25 μg selenium per kg body weight) daily for 52 days. After the above treatments, liver tissues were collected and subjected to Western blot analysis using specific antibodies against insulin signaling molecules. Figure 12 As shown in A, protein signals for phosphorylated Pdk1 at threonine 308, phosphorylated Akt, and phosphorylated Foxo1 at serine 256 were significantly more abundant in the livers of mice treated with compound #43 than those in mice treated with saline. Quantitative analysis of these Western blots showed that Lepr db / db The protein levels of pPdk1, pAkt, and pFoxo1 were significantly increased in the liver of mice ( Figure 12 B). In contrast, total Akt levels were not significantly altered in mice treated with compound #43. Increased phosphorylation of Pdk1, Akt, and Foxo1 strongly suggests that the insulin downstream signaling cascade is disrupted in Lepr db / db is active in the liver of mice, although Lepr db / dbIn other words, the results suggest that compound #43 can at least partially restore insulin action, bypass insulin, or both to stimulate phosphorylation of Pdk1 / Akt / Foxo1, leading to attenuated G6pc expression and glucose production in the livers of these insulin-resistant diabetic mice.

[0470] 2. In human HepG2 cells cultured in serum-free medium, compound #43 mimics but bypasses insulin to transiently activate PDK1 / AKT and subsequently inactivate FOXO1

[0471] To investigate whether compound #43 has an insulin-dependent but insulin-like effect on regulating the phosphorylation of PDK1, AKT, and FOXO1 in the liver, serum-starved human HepG2 cells were treated with control and 600 ppb compound #43 in serum-free medium for periods ranging from 30 minutes to 48 hours. Western blot analysis was performed on the treated cells.

[0472] like Figure 13 As shown in Figure A, there was a significant increase in the protein signal of phosphorylated PDK1 in HepG2 cells after 30, 60, and 90 minutes of treatment with compound #43, but not at longer treatment time points (after 24 hours of treatment). Quantitative studies showed a significant and transient increase in pPDK1 in HepG2 cells after compound #43 treatment, with the peak increase occurring approximately 60-90 minutes after compound treatment ( Figure 13 Similarly, a significant and transient increase in phosphorylated AKT at T308 was observed in HepG2 cells after treatment with compound #43 for 30, 60, 90 minutes and 24 hours, with peak increases at 60 and 90 minutes ( Figure 13 A, C). In contrast, total AKT protein levels did not change significantly in HepG2 cells at all tested time points after treatment with compound #43 ( Figure 13 A, D).

[0473] After treatment with compound #43 for 90 minutes and longer, the protein level of phosphorylated FOXO1 at T24 in HepG2 cells was significantly increased ( Figure 13 A, E). Increased FOXO1 phosphorylation was observed later than the increase in pPDK1 and pAKT ( Figure 13 A, 13E vs. 13B-C). There was no significant change in total FOXO1 protein levels in HepG2 cells after treatment with compound #43 for less than 24 hours ( Figure 13 However, prolonged treatment (30 or 48 hours) with compound #43 resulted in a slight but statistically significant decrease in total FOXO1 protein in HepG2 cells ( Figure 13A, F), which may be due to the continued increase in phosphorylated FOXO1 in HepG2 cells, leading to a potential increase in proteasomal protein degradation of FOXO1. Phosphorylated FOXO1 is excluded from the nucleus, which means that the direct result is less nuclear FOXO1 and less G6pc expression. In addition, a significant decrease in glucose production was observed in HepG2 cells treated with compound #43 ( Figure 1 ) and G6pc expression ( Figure 10 B), and in Lepr treated with compound #43 db / db Reduced hyperglycemia and attenuated G6pc expression in mice ( Figure 3-7 , 9).

[0474] In summary, the above results indicate that compound #43 can mimic but bypass insulin to transiently activate PDK1 and AKT and subsequently inactivate FOXO1 in human liver HepG2 cells.

[0475] 3. In AML-12 cells cultured under simulated diabetic conditions (stimulated by both 8-CPT and Dex), compound #43 mimics but bypasses insulin to transiently activate Pdk1 / Akt and subsequently inactivate Foxo1

[0476] As described in the previous examples, compound #43 can mimic but bypass insulin to inhibit 8-CPT / Dex-induced G6pc expression in AML-12 cells ( Figure 11 This effect may be due to the potential insulin-like activity of compound #43 inactivating Foxo1 in the liver cells of these mice. Therefore, the present application examined the protein expression of insulin signaling molecules in AML-12 cells cultured under simulated diabetic conditions (stimulated with 8-CPT and Dex).

[0477] like Figure 14 As shown in Figure A, insulin treatment enhanced the phosphorylation of Pdk1, Akt, and Foxo1 at 60 and 90 minutes, indicating that AML-12 cells cultured under mimicking diabetic conditions are responsive to insulin. Like insulin, compound #43 also significantly induced the phosphorylation of Pdk1, Akt, and Foxo1 in these 8-CPT / Dex-treated AML-12 cells after 60 minutes of compound treatment ( Figure 14 AB). A significant increase in pFoxo1 protein levels was observed in these AML-12 cells 90 minutes after compound treatment, whereas the protein levels of all other tested molecules including pPdk1, pAkt, Akt, and Foxo1 did not change significantly after compound #43 treatment ( Figure 14A, C). At 6 hours of compound #43 treatment, pFoxo1 levels were still significantly increased, and total Foxo1 protein levels were slightly but significantly decreased ( Figure 14 A, D). Increased pFoxo1 and slightly decreased total Foxo1 protein levels were also observed in AML-12 cells after treatment with 10 nM insulin at 6 h ( Figure 14 A, E). Again, the slight decrease in total Foxo1 at 6 hours of treatment may be due to the continued phosphorylation of Foxo1 in the cytoplasm of these AML-12 cells that have been stimulated by diabetes, leading to targeted proteasomal protein degradation of Foxo1 protein. The enhanced phosphorylation of Foxo1 after compound #43 treatment may lead to Figure 11 The results indicate that compound #43, like insulin, transiently induces phosphorylation of Pdk1 and Akt, and subsequently induces phosphorylation of Foxo1, in AML-12 cells cultured under diabetic-like conditions.

[0478] In summary, the above in vitro and in vivo studies indicate that compound #43 can mimic but bypass insulin to transiently activate Pdk / Akt in the liver and then inactivate Foxo1.

[0479] Example 6: After treatment with compound #43, db / db Glut4 in mouse liver and cultured hepatocytes Increased SLC2A4 expression and glucose uptake in cultured hepatocytes

[0480] In vivo studies reveal Lepr in insulin resistance db / db Compound #43 lowered blood glucose levels and increased glucose clearance in mice ( Figure 3-8 This is partly due to decreased glucose production in the liver, but may also be due to increased glucose uptake from the bloodstream to peripheral tissues, including the liver. Glut4 is a key glucose transporter in the liver and an indirect Foxo1 target gene for glucose uptake in response to systemic insulin stimulation. Therefore, treatment of Lepr with compound #43 db / db mice and cultured mouse liver AML-12 cells to test its potential effects on Glut4 expression and glucose uptake in the liver.

[0481] Materials and methods

[0482] Compound

[0483] Compound #43 and Compound #50 were synthesized in the chemical laboratory of Alltech, Inc. All of these compounds were confirmed to be ≥99% pure as determined by HPLC.

[0484] In Lepr db / dbIn vivo treatment of mice with compounds #43 and #50

[0485] On the 38th day after birth, male Lepr db / db Mice (C57BL / 6J strain, purchased from The Jackson Laboratory) were injected intraperitoneally daily for 52 days with saline (0.09% NaCl) containing 0.2% DMSO, compound #43, or compound #50 (25 μg of selenium per kg body weight of each compound, diluted in sterile saline). Following treatment, livers were harvested and subjected to RNA analysis.

[0486] Cell culture

[0487] Mouse liver AML-12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). These cells were expanded in Dulbecco's modified Eagle's medium and Ham's F12 (DMEM / F12) medium supplemented with 10% fetal bovine serum (FBS), 40 ng / ml dexamethasone (Dex, Sigma), and 1X ITS (containing 0.01 mg / ml bovine insulin, 0.0055 mg / ml human transferrin, and 5 ng / ml sodium selenite) solution (Sigma).

[0488] Cell processing for RNA analysis

[0489] For RNA analysis of basal Glut4 (Slc2a4) expression (in the absence of the diabetic stimulator 8-CPT / Dex), expanded AML-12 cells were plated in 24-well plates (1×10 5 The cells were cultured overnight on plates (100 cells / well). The cells were washed twice with PBS to remove residual serum and then incubated with vehicle (0.024% DMSO) or compound #43 (300 ppb) in serum-free DMEM / F12 medium for 24 hours.

[0490] To perform RNA analysis of Glut4 expression in AML-12 cells cultured under simulated diabetic conditions, expanded AML-12 cells were plated in 24-well plates (2×10 5Cells were cultured overnight on 500 cells / well plates. The cells were then washed twice with PBS to remove any potential residual serum and then serum-starved overnight in plain DMEM / F12 medium. Serum-starved AML-12 cells were then incubated with vehicle (0.024% DMSO) or compound #43 (300 ppb) in the presence of the diabetes stimulants 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex in serum-free plain DMEM / F12 medium for 6 and 24 hours.

[0491] RNA isolation and real-time PCR analysis

[0492] RNA from saline- or compound #43-treated LeprA cells was isolated using the Qiagen RNAeasy RNA isolation kit according to the manufacturer's protocol. db / db Total liver RNA from mice. Total RNA from cultured cells was isolated using Trizol (Invitrogen) according to the manufacturer's protocol and then incubated with DNase I to remove any potential contaminating genomic DNA. Real-time PCR analysis of RNA samples was performed using an Applied-Bioscience RT kit and pre-designed Taqman probes (Invitrogen) as previously described (Lan et al., EMBO J 2003). Data were normalized by the level of actin B (Actb) mRNA in each sample and presented as the mean ± SEM of 3-5 samples.

[0493] Glucose uptake assay

[0494] Equal numbers of expanded AML-12 cells were seeded in 96-well plates (1.5 × 10 5 44. The cells were then plated on PBS (400 μg / well) and cultured overnight in DMEM / F12 medium containing 10% FBS but without ITS / Dex. The cells were then washed twice with PBS (to remove any potential residual serum) and serum starved overnight in ordinary DMEM / F12 medium. The serum-starved AML-12 cells were not treated (basal), with insulin (10 and 100 nM) or compound #43 (150, 300 and 600 ppb) for 1.5 hours at 37°C in DMEM medium without glucose / phenol red. After treatment, the culture medium was removed, the cells were washed once with PBS, and incubated for 30 minutes at room temperature with 1 mM 2-deoxyglucose (2DG). Glucose uptake was then measured using Promega's Glucose Uptake-Glo assay kit according to the manufacturer's protocol for cells treated with 2DG. Luminescence signals were recorded using a Bio-Tek luminometer.

[0495] Statistical analysis

[0496] Where applicable, the Student t test was used to determine the statistical significance of differences between treatment groups, and a P value of less than 0.05 was used to indicate a significant result.

[0497] result:

[0498] 1. Lepr after long-term treatment with compound #43 and compound #50 db / db Analysis of Glut4 mRNA Expression in Mouse Liver

[0499] On the 38th day after birth, male Lepr db / db Mice were injected intraperitoneally daily with 0.2% DMSO in saline (0.09% NaCl), compound #43, or compound #50 (25 μg of selenium equivalent per kg body weight of each compound, diluted in sterile saline) for 52 days. Following treatment with these compounds, livers were harvested and subjected to RNA analysis for Glut4 and Actb mRNA.

[0500] like Figure 15 As shown in Figure 5, compound #50 treatment numerically increased the expression of Lepr db / db Glut4 mRNA levels in the liver of mice (when compared to saline-treated groups). However, Compound #43 treatment caused db / db A large and significant increase (5-fold; P = 0.048) in Glut4 mRNA expression levels in the liver of mice ( Figure 15 These results indicate that there is a clear difference between the two seleno compounds in stimulating Glut4 expression in the liver and that compound #43 is a potent enhancer of Glut4 expression in this organ. db / db The reduction in blood sugar levels and improvement in glucose tolerance observed in mice ( Figure 3-8 ) may be due in part to enhanced Glut4 expression, leading to increased glucose uptake from the bloodstream to the liver in diabetic subjects.

[0501] 2. Compound #43 treatment enhanced Glut4 mRNA expression in mouse liver AML-12 cells with or without diabetic stimuli (8-CPT / Dex) stimulation

[0502] The above in vivo studies revealed that compound #43 could significantly stimulate the insulin-resistant Lepr db / dbGlut4 expression in the liver of mice. This could be due to a potential systemic effect of compound #43 or a potential direct effect of compound #43 on liver tissue. To test the latter, cultured hepatocytes were used to examine whether compound #43 could directly modulate Glut4 expression in the liver.

[0503] We investigated whether compound #43 could modulate basal Glut4 expression in normal AML-12 cells (without diabetic stimulation). Briefly, AML-12 cells were treated with vehicle (0.024% DMSO) and compound #43 (300 ppb) in serum-free and ITS / Dex-free medium for 24 hours, and RNA analysis of Glut4 and Actb expression was performed. Figure 16 As shown in Figure A, treatment with compound #43 (300 ppb) resulted in a significant increase in Glut4 mRNA expression in AML-12 cells (approximately 2.1-fold increase). Considering that this experiment was performed on AML-12 cells cultured in complete serum-free conditions, the results indicate that the enhanced Glut4 expression in AML-12 cells following compound #43 treatment is independent of insulin, serum, or any growth factors.

[0504] To further test whether compound #43 can modulate Glut4 expression, AML-12 cells that had been stimulated with diabetes were used. Briefly, AML-12 cells were serum starved overnight and then incubated with vehicle (0.024% DMSO) or compound #43 (300 ppb) in the presence of the diabetes stimulators 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex in serum-free plain DMEM / F12 medium for 6 and 24 hours. Figure 16 As shown in Figure B, both 6 and 24 hour treatment with compound #43 resulted in a significant increase in Glut4 mRNA expression in these AML-12 cells co-treated with the diabetic stimulants 8-CPT and Dex. Thus, these results indicate that compound #43 can directly regulate Glut4 expression in AML-12 cells cultured under mimicking diabetic conditions.

[0505] 3. Compound #43 mimics but bypasses insulin to enhance glucose uptake in mouse liver AML-12 cells

[0506] Enhanced Glut4 expression suggests that compound #43 has the potential to mimic but bypass insulin to enhance glucose uptake in the liver. To test this, a glucose uptake experiment was performed on mouse liver AML-12 cells. Briefly, equal numbers of AML-12 cells were seeded on 96-well plates and cultured for 24 hours in DMEM / F12 medium containing 10% FBS but no ITS / Dex, and then serum starved overnight in regular DMEM / F12 medium. These serum-starved AML-12 cells were treated with insulin (10 and 100 nM) or compound #43 (150, 300, and 600 ppb) in DMEM medium without glucose / phenol red / serum at 37°C for 1.5 hours. After treatment, the cells were incubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30 minutes, and then luminescence analysis was performed using Promega's Glucose Uptake-Glo assay kit. The luminescent signal detected represents glucose uptake into the cultured cells.

[0507] like Figure 17 As shown in , glucose uptake is not promoted by 10nM insulin treatment. However, 100nM insulin treatment causes the glucose uptake in AML-12 cells to increase significantly. Further, compound #43 treatment under all three test doses all causes glucose uptake to increase significantly, and the degree of glucose uptake increase in the AML-12 cells treated with 600ppb compound #43 is comparable to 100nM insulin ( Figure 17 Since AML-12 cells were treated with compound #43 under serum-free conditions, the results suggest that, like insulin, compound #43 can act rapidly (less than 1.5 hours) to directly stimulate glucose uptake in hepatocytes. Increased glucose uptake in the liver may be a potential mechanism by which compound #43 can stimulate insulin-resistant Leptin. db / db One of the reasons for the lowered blood glucose levels and improved glucose clearance in mice ( Figure 3-8 ).

[0508] Example 7: Lepr in insulin resistance db / db Key downstream targets of insulin signaling in mouse skeletal muscle The expression of phosphorylated Pdk1, Akt, and Foxo1 was enhanced, and both insulin and compound #43 stimulated differentiation. Synergy in glucose uptake in C2C12 (skeletal muscle) cells

[0509] Materials and methods

[0510] Compound

[0511] Compound #43 was synthesized in the chemical laboratory of Alltech, Inc. The purity of this test compound was confirmed to be > 99% as determined by HPLC.

[0512] Lepr db / db In vivo treatment of compound #43 in mice

[0513] 38-day-old male Lepr db / db Mice (C57BL / 6J strain, purchased from The Jackson Laboratory) were injected intraperitoneally daily for 52 days with 0.2% DMSO in saline (0.09% NaCl) or 0.136 mg of compound #43 diluted in sterile saline per kg body weight. After treatment, gastrocnemius skeletal muscle samples were collected and stored at -80°C.

[0514] Skeletal muscle protein preparation and western blot analysis

[0515] Frozen skeletal muscle was minced in sterile ice-cold PBS containing complete protease and phosphatase inhibitors (Thermo-FisherScientific, Waltham, MA) and homogenized using a tissue homogenizer (Thermo-FisherScientific, Waltham, MA). These tissue homogenates were diluted (1 part homogenate / 2 parts RIPA buffer) in Themo-Fisher's RIPA buffer containing complete protease / phosphatase inhibitors to extract protein. The protein in the homogenate was extracted overnight in RIPA buffer at 4°C. The protein / tissue lysates extracted overnight were centrifuged at 12000x g for 30 minutes at 4°C, and the protein level in the supernatant of these tissue lysates was determined using a Pierce Micro-BCA protein assay kit (ThermoScientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.

[0516] As previously described (Reddy et al., 2008Science), one hundred micrograms of skeletal muscle protein were separated by SDS-PAGE gel and then transferred to a PVDF membrane. The membrane was blocked in phosphate-buffered saline (PBS) containing 5% (w / v) bovine serum albumin (Sigma, St. Louis, MO) and incubated with specific primary antibodies, followed by incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000 dilution, Cell Signaling Inc.). All primary antibodies except the antibody against β-tubulin (LI-COR bioscience) were purchased from Cell Signaling Inc. Positive signals on the membrane blot were detected using Amersham's enhanced chemiluminescent Western blot primary detection reagent (GE Healthcare Lifescience, Pittsburgh, PA). Images of these luminescent signals on the membrane blot were captured using the LI-COR Odyssey Fc Image System (Lincoln, Nebraska). The same membrane blot was stripped and reblotted with another antibody as described in the GE WB ECL primary detection protocol (GE healthcare Lifescience, Pittsburgh, PA). Protein band density in the Western blot was determined using NIH ImageJ software and then normalized by the β-tubulin level in each sample. Data are presented as mean ± SEM for 5 animal samples.

[0517] C2C12 cell culture, C2C12 cell differentiation and glucose uptake analysis

[0518] Mouse myoblast C2C12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). These cells were expanded in DMEM medium supplemented with 10% FBS. Equal numbers of C2C12 cells were then seeded in 96-well plates (5,000 cells / well) and cultured at 37°C in 10% FBS-DMEM medium for 5 days. The cells were replenished with fresh 10% FBS-DMEM medium daily. As previously described (Misu et al., Cell Metabolism 12, 483–495, 2010), on day 5 of culture, C2C12 cells were differentiated using DMEM medium containing 0.5% horse serum (Sigma) (differentiation medium) for 7 days, with fresh differentiation medium replaced daily. On the 7th day after differentiation, the C2C12 cells of differentiation were rinsed twice with PBS and pretreated overnight in serum-free, glucose-free DMEM culture medium without or with 0.006% DMSO (compound #43 solvent) or compound #43 (300 or 600 ppb). These cells were then washed once with PBS and then processed for 1.5 hours at 37°C in the DMEM culture medium without glucose / phenol red without (basal) or with insulin (200 nM), compound #43 (300 and 600 ppb) or both insulin and compound #43. After treatment, the culture medium was removed, the cells were washed once with PBS, and incubated for 30 minutes at room temperature with 1 mM 2-deoxyglucose (2DG). The GlucoseUptake-Glo assay kit was then used to determine that the cells processed with 2DG were subjected to glucose uptake according to the manufacturer's protocol. Bio-Tek photometer was used to record the luminescent signal.

[0519] Statistical analysis

[0520] Where applicable, Student's t test was used to determine the statistical significance of differences between treatment groups, with P values ​​less than 0.05.

[0521] result:

[0522] 1. After long-term treatment with compound #43, insulin-resistant Lepr db / db Increased phosphorylation of insulin downstream signaling molecules Pdk1, Akt, and Foxo1 in mouse skeletal muscle

[0523] Besides the liver, skeletal muscle is another major organ that is crucial for glucose homeostasis in response to systemic insulin. Glucose uptake in skeletal muscle plays a key role in maintaining normal glucose levels in the bloodstream. Animal studies have revealed that in insulin-resistant Lepr db / dbIn mice, compound #43 reduced blood glucose and HbA1c levels and significantly improved glucose tolerance ( Figure 3-8 These effects may be due to the restoration of insulin signaling (i.e., Pdk1 / Akt) in skeletal muscle, stimulating glucose uptake. Therefore, we investigated whether long-term treatment with compound #43 could partially restore the insulin-resistant Lepr db / db Impaired insulin signaling in skeletal muscle of mice.

[0524] Lepr on the 38th day after birth db / db Mice were injected intraperitoneally with saline (containing 0.2% DMSO) or compound #43 (at a dose of 0.136 mg compound #43 per kg body weight) daily for 52 days. Following the above treatments, skeletal muscle was collected and subjected to Western blot analysis using specific antibodies against those insulin signaling molecules.

[0525] like Figure 18 As shown in A, the protein band densities of phosphorylated Pdk1 at threonine 308, phosphorylated Akt, and phosphorylated Foxo1 at serine 256, but not total Akt or Foxo1, were significantly higher in skeletal muscle of mice treated with compound #43 than in skeletal muscle of mice treated with saline. Quantitative analysis of these Western blots showed that Lepr db / db Increased pPdk1 protein levels in skeletal muscle of mice ( Figure 18 B). Increased pPdk1 levels in skeletal muscle of mice treated with Compound #43 approached statistical significance (P=0.11). db / db In the skeletal muscle of mice, the protein levels of two insulin / Pdk1 downstream signaling molecules, pAkt and pFoxo1, were significantly increased ( Figure 18 B) In contrast, there were no significant changes in total Akt and Foxo1 protein levels in skeletal muscle of mice treated with compound #43. The increased phosphorylation of Pdk1 and the significant increase in phosphorylated Akt and phosphorylated Foxo1 suggest that long-term treatment with compound #43 may lead to the activation of Lepr db / db Insulin downstream signaling cascades are active in mouse skeletal muscle, despite the known Lepr db / db The mice were unable to respond to insulin. In other words, the results suggest that compound #43 can restore insulin action, bypass insulin, or both to activate PI3K and induce phosphorylation of Pdk1 / Akt in skeletal muscle, thereby enabling them to play a key role in regulating glucose homeostasis in these severely insulin-resistant type 2 diabetic mice.

[0526] 2. Enhanced glucose uptake in differentiated mouse C2C12 (skeletal muscle) cells following both insulin and compound #43 treatment

[0527] In humans and other mammals, skeletal muscle typically accounts for 75% of systemic insulin-stimulated glucose uptake. Impaired skeletal muscle response to insulin can severely disrupt systemic glucose homeostasis. It is well documented that glucose uptake in skeletal muscle is primarily mediated by the PI3K / Pdk1 / Akt signaling cascade in response to insulin. db / db Activation of PI3K / Pdk1 / Akt signaling molecules in mouse skeletal muscle ( Figure 18 ) suggest that compound #43 has the potential to directly regulate glucose uptake in skeletal muscle and enhance the effects of insulin in this process. To test these possibilities, glucose uptake experiments were performed on differentiated mouse C2C12 (skeletal muscle) cells.

[0528] Briefly, equal numbers of C2C12 cells were seeded in 96-well plates (5000 cells / well) and cultured at 37°C in 10% FBS DMEM medium for 5 days. As previously described (Misu et al., Cell Metabolism 12, 483–495, 2010), these cells were differentiated into skeletal muscle cells for 7 days using DMEM medium supplemented with 0.5% horse serum (Sigma). Fully differentiated C2C12 cells were pretreated overnight in serum-free, glucose-free DMEM medium without or with 0.006% DMSO (compound #43 solvent) or compound #43 (300 or 600 ppb). These cells were then incubated at 37°C in glucose-free / phenol red-free DMEM medium for 1.5 hours without (basal) or with insulin (200 nM), compound #43 (300 and 600 ppb), or both insulin and compound #43. After treatment, cells were incubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30 minutes and then analyzed for luminescence using Promega's Glucose Uptake-Glo assay kit. The detected luminescence signal represents glucose uptake into cultured differentiated C2C12 cells.

[0529] like Figure 19As shown in Figure 2, treatment with 200 nM insulin alone or 600 ppb Compound #43, but not 300 ppb Compound #43, resulted in a 16-19% increase in glucose uptake in these cultured skeletal muscle cells (although not statistically significant when compared to the basal group). Since there was a trend toward increased glucose uptake in these cultured skeletal muscle cells following treatment with the higher dose (600 ppb) of Compound #43 (with potency similar to 200 nM insulin), it is possible that Compound #43 directly and significantly enhances glucose uptake in these differentiated muscle cells at doses above 600 ppb. Regardless, the results indicate that Compound #43 stimulates glucose uptake at the tested dose of 600 ppb with potency comparable to 200 nM insulin.

[0530] Co-treatment of both insulin (200 nM) and compound #43 (300 ppb) resulted in a robust and significant increase (63%) in glucose uptake by differentiated C2C12 cells ( Figure 19 A more pronounced increase in glucose uptake (79%) was observed in these differentiated cells following co-treatment with 200 nM insulin and 600 ppb Compound #43. The increase in glucose uptake in differentiated C2C12 cells following co-treatment with both insulin and Compound #43 was much greater than that observed with either insulin or Compound #43 alone, indicating a synergistic effect between insulin and Compound #43. Therefore, these studies demonstrate that Compound #43 can work in conjunction with insulin to significantly enhance glucose uptake in differentiated skeletal muscle cells.

[0531] In conclusion, the above studies indicate that compound #43 can activate or restore insulin-resistant diabetic Lepr db / db Compound #43 can enhance insulin signaling in skeletal muscle of mice (as indicated by enhanced phosphorylation of Pdk1 / Akt / Foxo1) and can potentiate insulin action to enhance glucose uptake in cultured skeletal muscle (differentiated C2C12) cells. db / db Further molecular evidence for lowering blood sugar levels and improving glucose tolerance in mice ( Figure 3-8 ).

[0532] Example 8: Insulin-resistant Lepr db / db In skeletal muscle and liver of mice Activation of insulin receptor (Insr) signaling in cultured mouse skeletal muscle and human hepatocytes, and the effect of compound #43 on AS160 in differentiated mouse C2C12 (skeletal muscle) cells and human liver HepG2 cells (for GLUT4 translocation from cellular vesicles to cytoplasm) Insulin-like effects of phosphorylation (critical for glucose uptake)

[0533] It is well documented that after insulin binds to the α subunit of Insr, Insrβ undergoes tyrosine autophosphorylation starting at Y1146 and then at Y1150 / 51, which subsequently activates PI3K / Pdk1 to induce Akt phosphorylation in the liver and skeletal muscle. AS160, also known as TBC1 domain family member 4 (TBC1D4), is an Akt substrate that plays a key role in retaining GLUT4 protein in cytoplasmic vesicles. Insr / PI3k / Pdk1 / Akt signaling phosphorylates AS160 in response to insulin activation, promoting the translocation of GLUT4 protein from cytoplasmic vesicles to the plasma membrane, thereby promoting glucose uptake in skeletal muscle cells and glucose uptake in hepatocytes. Therefore, in insulin-resistant Lepr db / db The potential of Compound #43 to induce tyrosine phosphorylation of Insrβ was investigated in mouse skeletal muscle and liver, as well as in differentiated mouse C2C12 (skeletal muscle) cells and human liver HepG2 cells. Furthermore, the potential of Compound #43 to mimic but bypass insulin to activate Akt, a downstream signaling molecule of the insulin receptor, in differentiated mouse C2C12 cells was investigated. Furthermore, the effect of Compound #43 on phosphorylation of the Akt target substrate AS160 was investigated in both differentiated mouse C2C12 and human liver HepG2 cells.

[0534] Materials and methods

[0535] Compound

[0536] Compound #43 was synthesized in the chemical laboratory of Alltech, Inc. The purity of this test compound was confirmed to be > 99% as determined by HPLC.

[0537] Lepr db / db In vivo treatment of compound #43 in mice

[0538] On the 38th day after birth, male Lepr db / db Mice (C57BL / 6J strain, purchased from The Jackson Laboratory) were injected intraperitoneally daily for 52 days with 0.136 mg of compound #43 per kg body weight in saline (0.09% NaCl) containing 0.2% DMSO or diluted in sterile saline. Following treatment, gastrocnemius skeletal muscle and liver samples were collected and stored at -80°C.

[0539] Skeletal muscle and liver protein preparations

[0540] Frozen skeletal muscle and liver tissues were minced in sterile ice-cold PBS containing complete protease and phosphatase inhibitors (Thermo-FisherScientific, Waltham, MA) and homogenized using a tissue homogenizer (Thermo-FisherScientific, Waltham, MA). These tissue homogenates were diluted (1 part homogenate / 2 parts RIPA buffer) in Themo-Fisher pre-made RIPA buffer containing complete protease / phosphatase inhibitors to extract protein. The protein in the homogenate was extracted overnight in RIPA buffer at 4°C. The protein / tissue lysates extracted overnight were centrifuged at 12000x g for 30 minutes at 4°C, and the protein level in the supernatant of these tissue lysates was determined according to the manufacturer's protocol using Pierce Micro-BCA protein assay kit (ThermoScientific-Piece Biotechnology, Rockford, IL).

[0541] Cell culture of HepG2 and C2C12 cells, differentiation of C2C12 cells, cell treatment, and preparation of protein extracts from cultured cells

[0542] Human hepatocellular carcinoma HepG2 cells and mouse myoblast C2C12 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS, while C2C12 cells were expanded in DMEM supplemented with 10% FBS.

[0543] HepG2 cells were seeded in 6-well plates (7 × 10 5 Cells were plated on 400 cells / well and cultured in 10% FBS EMEM medium for 30 hours. The cells were then washed twice with PBS to remove residual serum and serum-starved overnight in regular EMEM medium. These serum-starved HepG2 cells were treated with or without compound #43 (600 ppb) in serum-free medium for 0 minutes (immediately before treatment), 30 minutes, and 60 minutes.

[0544] To differentiate C2C12 cells, equal numbers of these myoblasts were first seeded onto 12-well plates (60,000 cells / well) and cultured at 37°C in 10% FBSDMEM medium for 5 days. Fresh 10% FBSDMEM medium was added to the cells daily. On day 5 of culture, C2C12 cells were differentiated using DMEM medium containing 0.5% horse serum (Sigma) (differentiation medium) for 7 days, with fresh differentiation medium replaced daily, similar to what was previously described (Misu et al., Cell Metabolism 12, 483–495, 2010). On day 7 of differentiation, the differentiated C2C12 cells were rinsed twice with PBS and incubated overnight in serum-free DMEM medium. These serum-starved cells were then treated with either no (basal) or insulin (200 nM), compound #43 (600 ppb), or both insulin and compound #43 in serum-free DMEM medium at 37°C for 5 and 60 minutes.

[0545] After the above treatment, the cultured HepG2 cells or differentiated C2C12 cells were rinsed twice with ice-cold PBS and lysed in ice-cold RIPA buffer containing complete protease and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) on ice for 30 minutes. Cell lysates were collected using a cell scraper and transfer pipette, and then centrifuged at 12000 x g for 30 minutes at 4°C to remove DNA precipitation and obtain protein extracts. Protein levels in the supernatants of these cell lysates were determined using the Pierce Micro-BCA protein assay kit (Thermo Scientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.

[0546] Western blot analysis

[0547] As previously described (Reddy et al., 2008Science), one hundred micrograms of skeletal muscle or liver tissue protein, five micrograms of HepG2 cell protein extracts, or eight micrograms of differentiated C2C12 cell protein extracts were subjected to SDS-PAGE gel separation and then transferred to a PVDF membrane. The membrane was blocked in phosphate-buffered saline (PBS) containing 5% (w / v) bovine serum albumin (Sigma, St. Louis, MO) and incubated with specific primary antibodies, followed by incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000 dilution, Cell Signaling Inc.). All primary antibodies except the antibody against β-tubulin (LI-COR bioscience) were purchased from Cell Signaling Inc. Positive signals on the membrane blot were detected using Amersham's enhanced chemiluminescent Western blot primary detection reagent (GE Healthcare Lifescience, Pittsburgh, PA). Images of these luminescent signals on membrane blots were captured using the LI-COR Odyssey Fc Image System (Lincoln, Nebraska). The same membrane blot was stripped and reblotted with another antibody as described in the GE WB ECL primary detection protocol (GE healthcare Lifescience, Pittsburgh, PA). Protein band density in Western blots was determined using NIH ImageJ software and then normalized by the β-tubulin or ACTB protein levels in each sample. Data are presented as mean ± SEM for 3-5 samples per group.

[0548] Enzyme-linked immunosorbent assay (ELISA) of phospho-Insrβ at Y1146 or Y1150 / 51

[0549] The PathScan Phospho-Insulin Receptor β (Tyr1146 or Tyr1150 / 1151) Sandwish ELISA kit (Cell Signaling Technology, Danvers, MA) was used according to the manufacturer's protocol to detect the expression of insulin in Leprβ cells treated with saline or compound #43. db / dbLiver protein samples from mice were subjected to ELISA assays, except that the protein extracts were incubated with capture pInsrβY1146 or pInsrβY1150 / 1151 antibodies overnight at 4°C (instead of incubation at 37°C for 2 hours as described in the protocol). Four hundred micrograms of liver protein extract were used to detect phospho-Insrβ at Y1146, and six hundred micrograms of liver protein extract were used to detect phospho-Insrβ at Y1150 / 1151. The absorbance at 450 nm (OD450) of the test samples was recorded using a Bio-tek microplate reader. The level of the internal protein control β-tubulin in each sample was determined by Western blot analysis using 100 μg of protein extract and a specific β-tubulin monoclonal antibody, followed by quantitative analysis of the β-tubulin protein band density in the Western blot using NIH Image J software. The OD450 of each test sample was then normalized by its β-tubulin protein level to obtain the level of phospho-Insrβ at Y1146 or Y1150 / 1151.

[0550] Statistical analysis

[0551] Where applicable, the Student t test was used to determine the statistical significance of differences between treatment groups, and P values ​​less than 0.05 were considered significant.

[0552] result:

[0553] 1. Insulin-resistant Lepr after long-term treatment with compound #43 db / db Tyrosine phosphorylation of Insrβ is enhanced in skeletal muscle of mice

[0554] As discussed above, skeletal muscle is absolutely essential for glucose homeostasis in response to systemic insulin. Animal studies have revealed that compound #43 can be used in insulin-resistant Lepr db / db Reduced blood glucose and HbA1c levels and improved glucose tolerance in mice ( Figure 3-8 These effects may be due to a potential restoration of insulin receptor function in skeletal muscle, thereby allowing for the activation of insulin in these insulin-resistant Lepr db / db Glucose uptake in mice. Insulin-resistant Lepr treated with compound #43 db / db Increased phosphorylation of Pdk1 and Akt in mouse skeletal muscle ( Figure 18) suggests that compound #43 can bypass but mimic or restore insulin action to activate insulin signaling cascade molecules upstream of the Pdk1 / Akt cascade in skeletal muscle. Tyrosine phosphorylation of Insrβ at Y1146 reflects the first step of insulin receptor signaling activated after insulin binding to Insrα and is a key event upstream of PI3K / Pdk1 / Akt signaling in skeletal muscle. Therefore, whether long-term treatment with compound #43 can modulate these insulin-resistant Lepr db / db Phosphorylation of Insrβ in mouse skeletal muscle.

[0555] Lepr on the 38th day after birth db / db Mice were intraperitoneally injected daily with saline (containing 0.2% of the compound solvent DMSO) or compound #43 (at a dose of 0.136 mg of compound #43 per kg body weight) for 52 days. Following treatment, skeletal muscle samples were collected and subjected to Western blot analysis using specific antibodies against the aforementioned insulin signaling molecules.

[0556] like Figure 20 As shown in A, in Lepr treated with compound #43 db / db The protein band density of phosphorylated Insrβ at tyrosine 1146, but not total Insrβ, was much higher in skeletal muscle of mice treated with compound #43 than in saline-treated mice. Quantitative analysis of these Western blots showed that Leprβ was upregulated after treatment with compound #43 compared to saline-treated mice. db / db The phosphorylated Insrβ protein level was strongly increased (approximately 2.5-fold increase) in the skeletal muscle of mice ( Figure 20 B). In contrast, there was no significant change in total Insrβ protein levels in skeletal muscle of mice treated with compound #43 ( Figure 20 C) These results are consistent with those in Lepr treated with compound #43. db / db This is consistent with the observation that phosphorylation of Pdk1 and Akt, key insulin signaling molecules downstream of Insr, is increased in mouse skeletal muscle ( Figure 18 In conclusion, the results clearly show that long-term treatment with compound #43 upregulates insulin receptor expression in Lepr db / db is activated in the skeletal muscle of mice, although Lepr db / db The mice were engineered to be unable to respond to insulin. In other words, the results suggest that compound #43 can restore insulin action, bypass insulin, or both to stimulate tyrosine phosphorylation of Insrβ and subsequently activate PI3K / Pdk1 / Akt signaling in skeletal muscle of these severely type II insulin-resistant diabetic mice.

[0557] These findings, along with the observations of improved clearance of glucose from the bloodstream, reduced fasting blood glucose levels, reduced levels of HbA1C, and enhanced glucose uptake in skeletal muscle in concert with insulin, strongly suggest that compound #43 may be effective in treating both type I and type II diabetes.

[0558] 2. In differentiated mouse C2C12 (skeletal muscle) cells, compound #43 mimics but bypasses insulin to stimulate phosphorylation of Insrβ, Pdk1, Akt, and AS160 at Y1146

[0559] To further investigate whether compound #43 can directly activate insulin receptor signaling in skeletal muscle, serum-starved differentiated mouse C2C12 (skeletal muscle) cells were incubated without or with compound #43 (600 ppb), insulin (200 nM, positive control), or both for a very short period of time (i.e., 5 minutes) and 60 minutes in serum-free and glucose-free DMEM medium. Western blot analysis was then performed to examine the protein expression levels of activated Insr (i.e., pInsrβ at Y1146) and its downstream signaling molecules (including phosphorylated Pdk1, Akt, and As160) in these differentiated skeletal muscle cells.

[0560] Protein expression of activated Insr was measured in these cultured skeletal muscle cells after treatment with insulin or compound #43. As expected, treatment with insulin for 5 minutes resulted in a significant increase in pInsrβ, but not total Insrβ, at Y1146 in differentiated C2C12 cells ( Figure 21 AB), indicating that these differentiated C2C12 cells respond rapidly to insulin. Furthermore, treatment with 600 ppb compound #43 also resulted in a significant increase in pInsrβ but not total Insrβ at Y1146 in these cultured skeletal muscle cells ( Figure 21 AB). Co-treatment with insulin and compound #43 for 5 minutes tended to increase the pInsrβ protein level in these differentiated C2C12 cells ( Figure 21 AB), indicating that stimulation of Insrβ following treatment with either compound #43 or insulin is a transient event. Indeed, prolonged treatment (60 min) with insulin, compound #43, or both resulted in decreased pInsrβ levels ( Figure 21 CD), further suggesting that the activation of Insr after insulin or compound #43 treatment is indeed a transient event and that negative feedback exists to regulate Insrβ tyrosine phosphorylation in skeletal muscle cells. Regardless, the enhanced tyrosine phosphorylation of Insrβ at Y1146 observed in these cultured skeletal muscle cells after short-term (5 min) treatment with compound #43 is consistent with the above-mentioned insulin-resistant Lepr db / dbInsrβ activation in mice is consistent ( Figure 20 Since these differentiated C2C12 cells were serum-starved, cultured, and treated with Compound #43 under completely serum-free conditions, the results suggest that Compound #43 can mimic but bypass insulin to directly and rapidly activate Insrβ in these differentiated skeletal muscle cells.

[0561] Since tyrosine phosphorylation of Insrβ activates PI3K / Pdk1 signaling to enhance Akt phosphorylation in response to insulin, we investigated whether compound #43 could mimic insulin to regulate Akt phosphorylation in these differentiated C2C12 skeletal muscle cells. Figure 21 As shown in A, 5 min of insulin treatment resulted in a significant increase in phosphorylated Akt but not total Akt protein levels. However, no increase in Akt phosphorylation was observed in these differentiated C2C12 cells after 60 min of insulin treatment ( Figure 21 CD). In contrast, treatment with compound #43 for 5 minutes did not cause a significant increase in phosphorylated Akt protein levels in these C2C12 cells, whereas a strong increase in phosphorylated Akt protein levels was observed in these cells after treatment with compound #43 for 60 minutes ( Figure 21 CD). Co-treatment of both insulin and compound #43 resulted in a decrease in the expression of IL-12 at both test time points (6 minutes and 60 minutes, Figure 21 ) significantly increased pAkt protein levels in both groups. The levels of increased phosphorylated Akt in the insulin and compound #43 co-treatment group were comparable to those observed with insulin alone at 5 minutes, while the protein levels of phosphorylated Akt at 60 minutes after insulin and compound #43 co-treatment were almost identical to those observed after compound #43 treatment alone. These results suggest that insulin can transiently activate Akt, a signaling molecule downstream of Insr, in these skeletal muscle cells. Furthermore, the results reveal that compound #43 can mimic but bypass insulin to activate Akt in these cells, although not as rapidly as insulin. Furthermore, the results also indicate that there is no synergistic effect between insulin and compound #43 in stimulating Akt phosphorylation in these differentiated skeletal muscle cells. However, the results reveal that co-treatment with compound #43 and insulin can prolong the duration of activated Akt signaling.

[0562] As discussed above, AS160 is an Akt target substrate and is required to retain GLUT4 protein in vesicles within skeletal muscle cells. Phosphorylation of AS160 at S588 in response to insulin / Insrβ / PI3K / Akt signaling leads to translocation of GLUT4 protein from cytoplasmic vesicles to the plasma membrane to facilitate glucose uptake. In vivo studies revealed that compound #43 can inhibit the expression of GLUT4 in insulin-resistant Lepramine. db / dbLowers blood sugar levels and improves glucose tolerance in diabetic mice ( Figure 3-8 Furthermore, in cultured skeletal muscle cells, increased glucose uptake was observed following treatment with Compound #43, particularly following co-treatment with both insulin and Compound #43 ( Figure 19 ). Therefore, it is possible that compound #43 can regulate GLUT4 translocation in skeletal muscle cells for glucose uptake through phosphorylation of AS160. Therefore, the protein level of phosphorylated AS160 at S588 was measured in differentiated C2C12 (skeletal muscle) cells after treatment with insulin, compound #43, or both for 5 and 60 minutes in serum-free medium.

[0563] like Figure 21 As shown in Figures AB, treatment with insulin for 5 minutes did not affect the protein expression of AS160, but resulted in a significant increase (approximately 2-fold) in the phosphorylation of AS160 at S588 in these differentiated C2C12 cells. However, no significant increase in phosphorylated AS160 was observed after 60 minutes of insulin treatment ( Figure 21 CD). These results indicate that insulin transiently enhances the phosphorylation of AS160 for GLUT4 translocation in skeletal muscle cells.

[0564] In contrast, treatment with compound #43 alone for 5 minutes did not affect the protein levels of phosphorylated and total AS160 in these differentiated C2C12 cells ( Figure 21 AB). However, a strong increase (approximately 6-fold) in the levels of phosphorylated AS160 at S588 was observed in skeletal muscle cells after 60 minutes of treatment with compound #43 ( Figure 21 CD). These results indicate that compound #43 can mimic insulin (although not as rapidly as insulin) to induce phosphorylation of AS160 for GLUT4 translocation in these cultured skeletal muscle cells.

[0565] Co-treatment with both insulin and compound #43 resulted in a significant increase in phosphorylated AS160 protein levels at both time points tested (5 and 60 min). Figure 21 The increased phosphorylation level of AS160 in the co-treated group at 5 minutes was comparable to that of insulin alone ( Figure 21 AB), indicating that the AS160 phosphorylation observed in the co-treated group was mainly due to the effect of insulin at this time point. Similarly, at 60 minutes after treatment, the protein level of phosphorylated AS160 in the co-treated group was almost the same as that after treatment with compound #43 alone ( Figure 21CD), indicating that the AS160 phosphorylation observed in the co-treated group at this time point was primarily due to the effect of compound #43 alone. These results suggest that co-treatment with compound #43 and insulin can prolong the duration of AS160 phosphorylation to promote GLUT4 translocation for glucose uptake in these differentiated, insulin-responsive cells.

[0566] In summary, the results indicate that compound #43 can mimic but bypass insulin to directly and rapidly activate Insr (indicated by tyrosine phosphorylation of Insrβ) in differentiated C2C12 cells. db / db This is consistent with the activation of Insrβ observed in skeletal muscle of diabetic mice ( Figure 20 ). Activated Insr can then activate PI3K / Pdk1 / Akt signaling in skeletal muscle (which is supported by Figure 18 ) to subsequently phosphorylate AS160 (a known Akt target substrate), leading to enhanced translocation of GLUT4 from cytoplasmic vesicles to the cell membrane for glucose uptake. Indeed, an increase in glucose uptake was observed in cultured skeletal muscle cells following treatment with compound #43, particularly following co-treatment with insulin and compound #43. Figure 19 Enhanced glucose uptake in skeletal muscle after treatment with compound #43 lowered blood glucose levels and improved glucose tolerance in diabetic mice ( Figure 3-8 In short, these results indicate that compound #43 can mimic but bypass insulin to directly activate Insrβ / PI3K / Pdk1 / Akt signaling, thereby phosphorylating AS160 in skeletal muscle cells, leading to enhanced GLUT4 translocation from cytoplasmic vesicles to the plasma membrane to promote glucose uptake into skeletal muscle, thereby counteracting the key features and pathologies of both type 1 and type 2 diabetes.

[0567] 3. Long-term treatment with compound #43 in insulin-resistant Lepr db / db Tyrosine phosphorylation of Insrβ is enhanced in the liver of mice

[0568] The studies described in Example 6 revealed that compound #43 can enhance the phosphorylation of Pdk1 / Akt / Foxo1 in the liver in vivo and in vitro ( Figure 12-14All of these effects can be attributed to the activation of its upstream signaling molecule Insr in hepatocytes after treatment with compound #43, similar to those in skeletal muscle cells described above. As discussed above, tyrosine phosphorylation of Insrβ at Y1146 and subsequently at Y1150 / 1151 reflects the first few steps of insulin receptor signaling activated after insulin binding to Insrα and is a key event upstream of PI3K / Pdk1 / Akt / Foxo1 signaling in the liver. Therefore, it was investigated whether long-term treatment with compound #43 could modulate these insulin-resistant Lepr db / db Tyrosine phosphorylation of Insrβ in mouse liver.

[0569] Lepr on the 38th day after birth db / db Mice were intraperitoneally injected daily with saline (containing 0.2% compound solvent DMSO) or compound #43 (at a dose of 0.136 mg of compound #43 per kg body weight) for 52 days. Following treatment, liver samples were collected and subjected to ELISA assays for phospho-Insrβ at Y1146 and phospho-Insrβ at Y1150 / 1151 (to obtain OD450) as well as Western blot analysis of the internal control β-tubulin. The OD450 in each sample was normalized by its β-tubulin protein level to obtain the level of phospho-Insrβ at Y1146 or Y1150 / 1151 in each sample.

[0570] like Figure 22 As shown in A, Lepr after treatment with compound #43 was elevated compared to saline-treated mice. db / db Protein levels of phosphorylated Insrβ at tyrosine 1146 were significantly increased in the livers of mice (approximately 2.9-fold increase). Similarly, Leprβ levels were significantly increased in the livers of mice following chronic treatment with Compound #43 when compared to saline-treated mice. db / db In the liver of mice, the protein level of phosphorylated Insrβ at tyrosine 1150 / 1151 was also significantly increased (approximately 2.95-fold increase) ( Figure 22 B) These results are consistent with those in Lepr treated with compound #43. db / db This is consistent with the observation that phosphorylation of key insulin signaling molecules Pdk1 and Akt downstream of Insr in mouse liver is increased ( Figure 12 In conclusion, the results clearly show that after long-term treatment with compound #43, insulin receptors are activated in Lepr db / db is activated in the liver of mice, although Lepr db / dbThe mice were engineered to be unable to respond to insulin. In other words, the results suggest that compound #43 can restore insulin action, bypass insulin, or both to stimulate tyrosine phosphorylation of Insrβ and subsequently activate PI3K / Pdk1 / Akt signaling in the livers of these severely type II insulin-resistant diabetic mice.

[0571] 4. Compound #43 mimics but bypasses insulin to stimulate phosphorylation of Insrβ at Y1146 and AS160 at S588 in human liver HepG2 cells

[0572] To further investigate whether compound #43 can mimic but bypass insulin to directly activate the insulin receptor in hepatocytes, human liver HepG2 cells were serum starved overnight and then incubated with compound #43 (600 ppb) in serum-free and glucose-free DMEM medium for 30 and 60 minutes. Western blot analysis was performed to examine the protein expression levels of activated INSR (i.e., pINSRβ at Y1146) in these human hepatocytes. Figure 23 As shown in Figure 2, treatment with 600 ppb compound #43 for 30 and 60 minutes resulted in a significant increase in phosphorylated INSRβ at Y1146, but not total INSRβ, in these cultured hepatocytes. These results are consistent with the increased phosphorylation of INSR downstream signaling molecules PDK1 and AKT in HepG2 cells after compound #43 treatment for the same time period ( Figure 13 Since these hepatocytes were serum-starved and compound #43 treatment was performed under completely serum-free conditions, the results suggest that compound #43 can mimic but bypass insulin to directly stimulate tyrosine phosphorylation of INSRβ, leading to activation of PI3K / PI3K / AKT to inactivate FOXO1, inhibiting G6PC expression for glucose production, and stimulating GLUT4 expression for glucose uptake in hepatocytes.

[0573] In addition to FOXO1, AS160 is another AKT target substrate that plays a key role in glucose translocation from cell vesicles to the plasma membrane in insulin target tissues such as adipocytes and skeletal muscle. In vivo and in vitro studies have shown that compound #43 can reduce blood glucose levels and improve glucose tolerance in diabetic mice ( Figure 3-8 ) and can enhance glucose uptake in cultured AML-12 hepatocytes ( Figure 17 ), suggesting that enhanced glucose uptake in the liver may be one of the mechanisms by which compound #43 can lower blood glucose levels and improve glucose tolerance against type I and type II diabetes. The enhanced glucose uptake induced by compound #43 treatment is likely to be caused by enhanced GLUT4 expression (by Figure 15-16Indicator), potential enhanced GLUT4 translocation, or both, to stimulate glucose uptake into hepatocytes. To address the latter scenario, phosphorylated AS160 (AKT target substrate) protein levels were measured in HepG2 cells treated with compound #43.

[0574] like Figure 23 As shown in Figure 2, treatment with 600 ppb compound #43 for 30 and 60 minutes resulted in a significant increase in phosphorylated AS160 at S588, but not total AS160 protein levels, in these cultured human hepatocytes. These results are consistent with the increased phosphorylation of two key signaling molecules upstream of AS160, PDK1 and AKT, in HepG2 cells after treatment with compound #43 for the same period of time ( Figure 13 Since phosphorylation of AS160 can enhance GLUT4 translocation from cytoplasmic vesicles to the plasma membrane for glucose uptake, the results suggest that compound #43 can mimic but bypass insulin to stimulate GLUT4 translocation from cytoplasmic vesicles to the plasma membrane for glucose uptake in human hepatocytes. In support of this, it will be reviewed that enhanced glucose uptake was observed in cultured AML-12 hepatocytes after 1.5 hours of compound #43 treatment ( Figure 17 Thus, in hepatocytes, compound #43 will not only stimulate GLUT4 expression mediated by Insr / PI3k / Pdk1 / Akt / Foxo1 signaling (mediated by Figure 12-17 ), but also enhances the translocation of GLUT4 from cytoplasmic vesicles to the plasma membrane mediated by Insr / PI3K / Pdk1 / Akt / AS160 (indicated by Figure 12-17 , 23 instructions), thereby enhancing glucose uptake.

[0575] In conclusion, all the above studies revealed that compound #43 can be used in diabetic Lepr db / db Insulin receptor function was restored in both skeletal muscle and liver of mice (as indicated by enhanced tyrosine phosphorylation of Insrβ). In vitro studies further demonstrated that compound #43 closely mimics insulin to activate the insulin receptor in both skeletal muscle cells and human hepatocytes. In addition, the results indicate that compound #43, like insulin, can activate Pdk1 / Akt signaling to induce phosphorylation of AS160 (an AKT target substrate) in both cultured skeletal muscle cells and human hepatocytes. Enhanced phosphorylation of AS160 promotes GLUT4 translocation from cytoplasmic vesicles to the plasma membrane to enhance glucose uptake in both liver and skeletal muscle cells, leading to lower blood glucose levels and improved glucose tolerance in diabetic patients.

[0576] It should be emphasized that the liver and skeletal muscle are by far the two tissues most critical in the development and pathogenesis of type 2 diabetes. Compound #43 may have great therapeutic value in the treatment of type 2 diabetes by virtue of its ability to restore the insulin signaling cascade in these tissues.

[0577] Furthermore, since compound #43 can act as an insulin mimetic in insulin-responsive cells without the addition of insulin thereto, it is also highly likely to be effective in treating type I diabetes.

[0578] Example 9: Long-term treatment with compound #43 results in serum islet in insulin-resistant diabetic db / db mice Decreased serum creatinine and alanine aminotransferase (ALT) levels but not serum creatinine levels

[0579] Materials and methods

[0580] Compound

[0581] Compound #43 was synthesized in the chemical laboratory of Alltech, Inc. All tested compounds were confirmed to be ≥99% pure as determined by HPLC.

[0582] animal

[0583] Spontaneous mutation (leptin receptor mutation) Lepr in 5-week-old male diabetic db / db Mice (C57BL / 6J strain) were purchased from Jackson Laboratory (Bar Harbor, Maine) and housed in a pathogen-free vivarium with free access to food and water. 3-month-old wild-type (non-diabetic) C57 mice were also purchased from Jackson Laboratory.

[0584] Chronic treatment with compound #43

[0585] 38-day-old male Lepr db / db Mice were injected intraperitoneally (ip) daily with saline (0.09% NaCl) containing 0.2% DMSO and compound #43 (0.136 mg per kg body weight, diluted in sterile saline) for 52 days. Serum was also collected from 3-month-old wild-type (non-diabetic) C57 mice. After treatment, these serum samples were collected and assayed for insulin, ALT, and creatinine.

[0586] Serum insulin, ALT, and creatinine measurements

[0587] Serum levels of insulin, ALT, and creatinine were determined using the Insulin Mouse ELISA Kit from Themo-Fisher Scientific (Cat. No. EMINS), the ALT Activity Assay Kit from Sigma (Cat. No. MAK052), and the Creatinine Assay Kit from Abcam (Cat. No. Ab65340), respectively, according to the manufacturers' protocols.

[0588] Statistical analysis

[0589] Where applicable, the Student t-test was used to determine the statistical significance of differences between saline-treated and compound-treated groups, with P values ​​less than 0.05 considered significant. Data are presented as mean ± SEM of the number of mice indicated in the figures.

[0590] Results and discussion

[0591] like Figure 24 As shown in A, Lepr db / db The mice exhibited hyperinsulinemia, with insulin levels of approximately 2586 μIU / ml (approximately 100-fold that of non-diabetic wild-type mice). However, treatment with compound #43 resulted in a significant decrease in serum insulin levels (by approximately 80%), although levels remained higher than those of non-diabetic wild-type mice ( Figure 24 A). These results suggest that compound #43 has the potential to treat hyperinsulinemia in diabetic patients.

[0592] Alanine aminotransferase (ALT) tests are often used to detect liver damage. Figure 24 As shown in B, saline-treated Lepr db / db Serum ALT levels in mice were significantly higher than in non-diabetic mice. However, treatment with compound #43 resulted in a significant decrease in serum ALT levels. These results suggest that long-term treatment with compound #43 (daily for 52 days) did not show liver toxicity; instead, it may have a protective effect against liver damage.

[0593] Blood creatinine tests are widely used to assess kidney function, and elevated creatinine levels can indicate impaired kidney function or kidney disease. Figure 24 As shown in C, after treatment with compound #43, Lepr db / db There were no significant changes in serum creatinine levels in the mice.These results suggest that long-term treatment with Compound #43 (daily treatment for 52 days) may not have toxic effects on renal function.

[0594] In summary, these results demonstrate the potential utility of Compound #43 in combating hyperinsulinemia in diabetic subjects. Furthermore, these results suggest that Compound #43 has little or no toxic effects on liver and kidney function. Conversely, Compound #43 may have some protective effects against liver damage.

[0595] Conclusion (mode of action; Figure 25 )

[0596] The experimental results in this disclosure are represented by solid red arrows, while the expected results (based on published literature) are represented by dotted arrows. Compound #43 can mimic but bypass insulin to rapidly induce tyrosine phosphorylation of the insulin receptor β subunit on the inner surface of the cell membrane, thereby eliminating the need for insulin to bind to and activate the insulin receptor on the cell surface. This leads to activation of the PI3K / PDK1 / AKT signaling cascade in both the liver and skeletal muscle. In other words, normal insulin signaling can be restored without the presence of insulin or active cell surface insulin receptors. In hepatocytes, activation of AKT leads to a robust increase in FOXO1 phosphorylation, resulting in a significant decrease in the expression of the FOXO1-direct target gene G6PC; this leads to inhibition of glucose production in hepatocytes of diabetic subjects. In addition, in hepatocytes treated with compound #43, the expression of the FOXO1-indirect target gene GLUT4 and the phosphorylation of the AKT target substrate AS160 (TBC1D4), which is key to the translocation of GLUT4 from cytoplasmic vesicles to the plasma membrane, were enhanced, resulting in more GLUT4 transporters in the hepatocyte membrane and increased glucose uptake from the bloodstream. All of this points to improved glucose tolerance in subjects with type I and type II diabetes. In skeletal muscle cells, compound #43 can also mimic but bypass insulin to activate INSRβ / PDK1 / AKT signaling, leading to phosphorylation of AS160 (TBC1D4). Again, this leads to enhanced translocation of GLUT4 from cytoplasmic vesicles to the plasma membrane to promote glucose uptake into skeletal muscle cells, ultimately leading to a significant decrease in blood sugar in subjects with type I and type II diabetes, and a significant improvement in glucose tolerance. As shown, compound #43 can enhance insulin action by inhibiting G6pC expression in hepatocytes. Uncontrolled glucose production by the liver—a process driven by G6pC expression—is a key characteristic and a key problem in type 2 diabetes. Inhibiting glucose production in the diabetic liver is a key mechanism of action for biguanides (e.g., metformin), the most widely used class of antidiabetic drugs. The ability of compound #43 to block this process makes it potentially very valuable in the treatment of type 2 diabetes. In addition, compound #43 can restore insulin receptor function in skeletal muscle of insulin-resistant diabetic mice and can enhance insulin action to stimulate glucose uptake in cultured skeletal muscle cells. Taken together, these results suggest that the use of compound #43 has great potential to combat both type I and type II diabetes in humans.

[0597] Example 10: Compound #43 directly activates insulin receptor protein in a cell-free system

[0598] Materials and methods

[0599] Compound

[0600] Compound #43 and its sulfur analog, Compound #68, were synthesized in the chemical laboratory of Alltech, Inc. The purity of these test compounds was confirmed to be > 99% as determined by HPLC.

[0601] In vitro phosphorylation of insulin receptor (Insr) and detection of activated Insr by Western blot analysis Phosphorylated tyrosine residues 1146, 1150, and 1151 of the Insrβ subunit are indicated)

[0602] In vitro phosphorylation of Insr was performed according to the Sigma protocol with the following modifications. Briefly, 10 μl of native insulin receptor solution containing 0.8 μl of original native INSR stock solution (Sigma, catalog number I9266; diluted in enzyme dilution buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100) was incubated on ice for 30 minutes with an equal volume of a solution containing insulin (Sigma), DMSO (solvent for compound #43), compound #43, or compound #68 (diluted in 50 mM HEPES (pH 7.6) and 100 μg / ml bovine serum albumin). 20 μl of 2X kinase buffer containing 0.2 mM ATP, 50 mM HEPES (pH 7.6), 50 mM MgCl2, and 4 mM MnCl2 was then added to the reaction, mixed, and incubated on ice for 45 minutes.

[0603] Five microliters of the reaction were immediately subjected to Western blot analysis using antibodies specific for phosphorylated tyrosine residues 1146, 1150, and 1151 of the Insrβ protein (Cell Signaling Inc.) Protein band density was determined using NIH Image J software.

[0604] Statistical analysis

[0605] Where applicable, the Student t test was used to determine the statistical significance of differences between treatment groups, and P values ​​less than 0.05 were considered significant.

[0606] result:

[0607] 1. Activation of native insulin receptor protein purified from rat liver tissue by compound #43 and insulin in an in vitro cell-free system

[0608] Studies in cultured liver and differentiated skeletal muscle cells and in T2D diabetic mice revealed that compound #43 can activate the insulin receptor in vitro and in vivo. To investigate whether compound #43 has a direct effect on the activation of Insr, an in vitro cell-free phosphorylation assay of native insulin receptor protein (purified from rat liver tissue) was performed. Activated Insr was detected using specific antibodies against phosphorylated Insrβ at tyrosine residues 1146, 1150, and 1151. As expected, insulin treatment was able to induce phosphorylation of Insrβ at Y1146 / 1150 / 1151 in this cell-free in vitro system. Figure 26 Importantly, compound #43 also significantly enhanced phosphorylation of Insrβ at Y1146 / 1150 / 1151 at doses of 1.9 and 3.8 μM, with the elevated phosphorylation levels directly comparable to those at 0.5 μM insulin ( Figure 26 These results demonstrate that compound #43 can reliably mimic insulin to directly activate the insulin receptor, providing further molecular evidence that compound #43 has the potential to replace insulin in the fight against diabetes, including type 1 diabetes.

[0609] 2. Compound #68, a sulfur analog of compound #43, is far less effective than compound #43 in activating Insr in a cell-free system.

[0610] Cultured hepatocytes and type 2 diabetes (T2D) Lepr db / db Studies in mice have shown that inhibition of glucose production in vitro ( Figure 1 ) and alleviated hyperglycemia in T2D mice ( Figure 4 ), compound #68 (the sulfur analog of compound #43) was less effective than compound #43. To investigate whether there were differential effects between compound #43 and compound #68 in the activation of Insr in a cell-free system, equal amounts of native insulin receptor protein were incubated with the same molar concentration (3.8 μM) of compound #43 or compound #68, and then subjected to in vitro phosphorylation assays. Figure 27 As shown in Figure 3, compound #43 but not compound #68 was able to activate Insr at the doses tested. These studies indicate that compound #68 was less effective than compound #43 at the doses tested in activating Insr, which may explain the inhibition of hepatic glucose production by compound #68 in T2D mice ( Figure 1 ) and combating hyperglycemia ( Figure 4 ) is the reason for the low effectiveness.

[0611] Example 11: After acute treatment with compound #43, streptozotocin (STZ)-induced type 1 diabetes (T1D) Lowered blood sugar levels in mice

[0612] Materials and methods

[0613] Compound

[0614] Streptozotocin (STZ) was purchased from Sigma. Compound #43 was synthesized in the chemical laboratory of Alltech, Inc. The purity of Compound #43 was confirmed to be ≥99% as determined by HPLC.

[0615] Type 1 Diabetes (T1D) Mouse Model and Effects of Compound #43 on Blood Glucose Levels in These T1D Mice

[0616] Five-week-old C57 / BL6 male mice were injected intraperitoneally with streptozotocin (STZ, 55 mg / kg mouse body weight) every day for 5 days and then raised in a feeding box for another 14 days to recover. Blood glucose levels in these mice were measured using a glucometer. Those animals with blood glucose levels higher than 500 mg / dL were considered to have type 1 diabetes (T1D). These T1D mice with non-fasting blood glucose levels between 500-550 mg / dL were fasted overnight and injected intraperitoneally with compound #43 (at a dose of 5.4 mg / kg body weight) or normal saline containing 2% DMSO (compound #43 stock solvent). Blood glucose levels in these mice were measured using a glucometer 1, 2, and 3 hours after injection.

[0617] Statistical analysis

[0618] The Student's t-test was used to determine the statistical significance of the differences between the control (DMSO) group and the compound #43 group at each time point. A P value of less than 0.05 indicated that the difference between the two groups was statistically significant.

[0619] result

[0620] To investigate the potential of compound #43 to combat hyperglycemia in T1D mice, STZ-induced T1D mice with blood glucose levels between 500-550 mg / dL were fasted overnight and injected intraperitoneally with saline containing DMSO or compound #43 (5.4 mg / kg body weight) for 1, 2, and 3 hours, followed by measurement of blood glucose levels. Figure 28 As shown in Figure 2, there was no significant change in blood glucose levels in control mice (injected with saline containing DMSO) over a 3-hour period. However, a trend toward lower blood glucose levels was observed in these T1D mice after 1 hour of treatment with compound #43 ( Figure 28 More importantly, a significant decrease in blood glucose levels was observed in these T1D mice 2 and 3 hours after treatment with compound #43 ( Figure 28 ). Taken together, these results demonstrate that compound #43 alleviates hyperglycemia in T1D mice.

[0621] Although some embodiments have been shown in the examples, it will be apparent that they can be modified to provide other embodiments of the present disclosure. It is therefore to be understood that the scope of the invention is to be limited by the appended claims rather than by the specific embodiments that have been described by way of example.

Claims

1. A compound, wherein the compound has the formula:

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an insulin-related disorder, wherein the treatment comprises administering a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating insulin resistance, wherein the treatment comprises administering a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

5. The use according to claim 3 or 4, wherein the disorder is hyperglycemia, retinopathy, neuropathy, nephropathy, hyperinsulinemia, polycystic ovary syndrome (PCOS) or type II diabetes-related vascular disorders.

6. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diabetes, wherein the treatment comprises administering a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

7. The use according to claim 6, wherein the diabetes is type I diabetes or type II diabetes.

8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating mitochondrial-related diseases, wherein the treatment comprises administering a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

9. The use according to claim 8, wherein the mitochondrial-related disease is a degenerative disease selected from Alzheimer's disease, Parkinson's disease and sarcopenia.

10. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting glucose production, wherein the inhibition comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

11. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for lowering serum HbA1c levels, wherein the lowering comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

12. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving glucose tolerance, wherein the improvement comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

13. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting G6pc expression, wherein the inhibition comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

14. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the phosphorylation of Pdk1, Akt, AS160 and Foxo1 in liver and / or skeletal muscle, wherein the enhancement comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

15. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for increasing Glut4 expression, wherein the increase comprises administering the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

16. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for activating and / or restoring insulin signaling in a subject in an insulin-resistant state, wherein the activation and / or restoration comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

17. The use of claim 16, wherein the subject is characterized by significant levels of circulating insulin.

18. The use of claim 16, wherein the insulin resistance state is characterized by a decrease in appropriately or properly phosphorylated insulin receptors in the subject.

19. The method of claim 16, wherein the subject suffers from diabetes and / or a diabetes-related disease, disorder or condition.

20. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing glucose uptake into cells of a subject, wherein the enhancement comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

21. The use of claim 20, wherein the cells are selected from the group consisting of skeletal muscle cells and liver cells.

22. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the translocation of glucose transporter (GLUT) from cytoplasmic vesicles to the plasma membrane for glucose uptake, wherein the enhancement comprises administering the compound of claim 1 or a pharmaceutically acceptable salt thereof.

23. The use of any one of claims 3 to 22, wherein the administering comprises administering a composition comprising and / or delivering the compound, and wherein the composition comprising and / or delivering the compound is administered according to a regimen that provides a therapeutically effective amount.

24. The use according to claim 23, wherein the composition is a pharmaceutical composition comprising an active pharmaceutical ingredient and one or more carriers or excipients, wherein the active pharmaceutical ingredient comprises or consists of the compound according to claim 1.

25. The use according to claim 23, wherein the active pharmaceutical ingredient consists of the compound according to claim 1.

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