RU2798603C1 - Agent for increasing the sensitivity of tissues to insulin in type 2 diabetes mellitus - Google Patents

Abstract

FIELD: organic chemistry; pharmacology.

SUBSTANCE: invention is directed to the development of new agents for the control of metabolic disorders. The use of the compound 1a-e of the following structure as an agent to increase the sensitivity of tissues to insulin in type 2 diabetes mellitus is disclosed.

EFFECT: invention provides an effective increase in tissue sensitivity to insulin in type 2 diabetes mellitus.

1 cl, 8 dwg, 2 tbl, 7 ex

Description

Hyperglycemia is the main symptom of a number of metabolic disorders, including type 2 diabetes mellitus, which has recently become epidemic [Roglic, G. WHO Global report on diabetes: A summary. Int. J. Non - Commun. Dis. 2016 , 1 , 3-8.]. Hyperglycemia in type 2 diabetes mellitus occurs when the sensitivity of insulin-dependent tissues (skeletal muscle, adipose tissue and liver) to insulin is impaired, which leads to a significant decrease in their glucose uptake and hyperinsulinemia. In a feedback loop, the pancreas produces more insulin to lower the blood glucose concentration, which in later stages of the disease leads to depletion of the beta cells of the islets of Langerhans and a lack of insulin. Often, carbohydrate metabolism disorders occur in parallel with lipid metabolism disorders, characterized by dyslipidemia with elevated concentrations of low-density lipoproteins and triglycerides in the blood. Such metabolic disorders cause rapid progression of atherosclerosis and damage to the nervous system [Sonksen, P.; Sonksen, J. Insulin: Understanding its action in health and disease. Br. J. Anaesth. 2000 , 85 , 69-79]. Complications are the main cause of death and high disability in patients with diabetes mellitus. Hypoglycemic drugs are needed to control glycemia and lipid levels [American Diabetes Association. 9. Pharmacologic approaches to glycemic treatment: Standards of medical care in diabetes-2019. Diabetes Care 2019 , 42 , 90-102].

Currently, there is not a wide variety of therapeutic agents for increasing tissue sensitivity to insulin. Only metformin and thiazolidinediones have this effect, the action of all other drugs used is associated with an increase in insulin secretion (sulfonylurea derivatives, DPP4 inhibitors), a decrease in glucose absorption in the gastrointestinal tract (acarbose) or with its increased excretion by the kidneys (SGLT2 inhibitors) [Pappachan, J.M.; Fernandez, C.J.; Chacko, E.C. Diabesity and antidiabetic drugs, Mol. Asp. Med. 2019, 66, 3-12, doi: 10.1016/j.mam.2018.10.004].

The main side effects of current oral antidiabetic agents include hypoglycemia, lactic acidosis, fluid retention, osteoporosis, and heart failure, which limits their clinical use. Therefore, the development of new candidate compounds for the control of various parameters of metabolic disorders (hyperglycemia, hyperinsulinemia and hypertriglyceridemia) with minimization of side effects remains the first and most important task in the treatment of metabolic disorders [Vieira, R.; Souto, S. B.; Sánchez-López, E.; Machado, A.L.; Severino, P.; Joseph, S.; Santini, A.; Fortuna, A.; Garcia, ML; Silva, A. M.; et al. Sugar-Lowering Drugs for Type 2 Diabetes Mellitus and Metabolic Syndrome-Review of Classical and New Compounds: Part-I. Pharmaceuticals 2019 , 12 , 152].

The invention relates to the field of organic chemistry and medicine, namely, to the hypoglycemic activity of the compounds of formula 1 .

Analogues of the proposed means of pharmacological action are hypoglycemic pharmaceuticals of the class of biguanides (metformin) and thiazolidinediones (rosiglitazone). These drugs often have pronounced side effects on the part of the digestive system (abdominal pain, nausea, vomiting, diarrhea), and can also lead to an increase in the incidence of infections of the upper respiratory tract, urinary system, etc. Since resistance may develop during long-term therapy with oral drugs ( a condition in which the drugs no longer cause the proper therapeutic response in the body, thereby leading to an increase in the dose to achieve the desired therapeutic effect) or the impossibility of proper control of carbohydrate metabolism, fundamentally new drugs are needed for the treatment of type 2 diabetes mellitus.

Medicines based on natural compounds are widely distributed in the pharmaceutical market [Newman, DJ; Cragg, GM Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J. Nat. Prod. 2020 , 83 , 770-803] often have a good safety profile and a milder (often prolonged) effect and usually do not cause serious side effects and drug tolerance. Natural compounds are considered by researchers as potential therapeutic agents for diabetes mellitus [Vieira, R.; Souto, S. B.; Sánchez-López, E.; Machado, A.L.; Severino, P.; Joseph, S.; Santini, A.; Fortuna, A.; Garcia, ML; Silva, A. M.; et al. Sugar-Lowering Drugs for Type 2 Diabetes Mellitus and Metabolic Syndrome-Review of Classical and New Compounds: Part-I. Pharmaceuticals 2019 , 12 , 152]; however, only a small part of the published research in the field of hypoglycemic agents is associated with targeted synthetic modification of natural metabolites.

Closest to the claimed drug for pharmacological action - the prototype is berberine.

Berberine 2 is a plant alkaloid of the isoquinoline series. Berberine has been used in traditional Chinese medicine for centuries to treat a variety of ailments, one of its strongest properties being its anti-diabetic activity. The clinical use of berberine 2 in type 2 diabetes mellitus has been reported since 1988, the mechanism of its hypoglycemic action is multitarget [Advances in Structural Modifications and Biological Activities of Berberine: An Active Compound in Traditional Chinese Medicine Z.-J. Huang, Y. Zeng, P. Lan, P.-H. Sun and W.-M. Chen Mini-Reviews in Medicinal Chemistry, 2011, 11, 1122-1129].

The hypoglycemic effect of berberine is pronounced, but the effective dose of berberine is relatively high (380 mg/kg). [Xia, X., Yan, J., Shen, Y., Tang, K., Yin, J., Zhang, Y., ... Weng, J. (2011). Berberine Improves Glucose Metabolism in Diabetic Rats by Inhibition of Hepatic Gluconeogenesis. PLoS ONE, 6(2), e16556. doi:10.1371/journal.pone.0016556]. The main reason is that the bioavailability of berberine is extremely low, not exceeding 1% [An insight into the medicinal attributes of berberine derivatives: A review Sobhi Gaba, Anjali Saini, Gurpreet Singh, Vikramdeep Monga Bioorg. Med. Chem. 38 (2021) 116143].

The literature describes analogues of berberine, obtained by its chemical modification, or by the method of molecular assembly, which in the model of diabetes mellitus induced by alloxan in mice showed a hypoglycemic effect at a dose of 200 mg/kg (N-substituted derivative of berberine 3 ) [Bian, X. , He, L., & Yang, G. (2006). Synthesis and antihyperglycemic evaluation of various protoberberine derivatives. Bioorganic & Medicinal Chemistry Letters, 16(5), 1380-1383. doi:10.1016/j.bmcl.2005.11.045], in a rat model of streptozotocin-induced diabetes mellitus at a dose of 100 mg/kg (8-oxoberberine 4 ) [Yaoxing Dou, Ronglei Huang, Qiaoping Li, Yuhong Liu, Yucui Li, Hanbin Chen, Gaoxiang Ai, Jianhui Xie, Huifang Zeng, Jiannan Chen, Chaodan Luo, Ziren Su Oxyberberine, an absorbed metabolite of berberine, possess super-perior hypoglycemic effect via regulating the PI3K/Akt and Nrf2 signaling pathways Biomedicine & Pharmacotherapy 137 (2021 ) 111312], in a 100 mg/kg (9-O-acyl derivative 5 ) murine model of diabetes mellitus [Berberine: a Promising Natural Isoquinoline Alkaloid for Development of Hypolipidemic Drug Article in Current Topics in Medicinal Chemistry September 2020 DOI: 10.2174/ 1568026620666200908165913].

The problem to be solved by the invention is to create a new effective agent based on berberine with hypoglycemic activity. The problem is solved by using compounds of formula 1 as a hypoglycemic agent, where R=n-pentyl, n-hexyl, n-heptyl, n-decyl, n-dodecyl..

Compounds 1a - e can be synthesized according to Scheme 1.

Scheme 1

For the study of compounds 1a - e used mice C57BL/6, and C57BL/6 ^Ay (mice AY), which are characterized by the presence of obesity, hyperinsulinemia, impaired glucose tolerance and concomitant non-alcoholic fatty liver disease [S. Kuranov, O. Luzina, M. Khvostov, D. Baev, D. Kuznetsova, N. Zhukova, P. Vassiliev, A. Kochetkov, T. Tolstikova, N. Salakhutdinov. Bornyl Derivatives of p-(Benzyloxy)Phenylpropionic Acid: In Vivo Evaluation of Antidiabetic Activity Pharmaceuticals 2020, 13(11), 404]. In the experiment on AY mice, metformin (MF) was used as a positive control, the effective dose of which in animals, according to the literature, varies in the range of 200-250 mg/kg [Guo WR, Liu J, Cheng LD, Liu ZY, Zheng XB , Liang H, Xu F. Metformin Alleviates Steatohepatitis in Diet-Induced Obese Mice in a SIRT1-Dependent Way. Front Pharmacol. 2021 Aug 18;12:704112. doi: 10.3389/fphar.2021.704112.]. The introduction of compound 1b at a dose of 15 mg/kg for three weeks significantly affected the condition of mice AY. All the main markers of the metabolic syndrome inherent in these animals were normalized: reduced body weight (figure 1), reduced concentrations of glucose (figure 2 and 3) and insulin (figure 4) on an empty stomach, improved glucose tolerance. The decrease in body weight occurred, among other things, due to a decrease in the mass of gonadal and interscapular fat, including brown fat (Table 1), which indicates an increase in the activity of the latter [Carolline Santos Miranda, Flavia Silva-Veiga, Fabiane Ferreira Martins, Tamiris Lima Rachid, Carlos Alberto Mandarim-De-Lacerda, Vanessa Souza-Mello, PPAR-α activation counters brown adipose tissue whitening: a comparative study between high-fat- and high-fructose-fed mice, Nutrition, Volume 78, 2020, 110791 ]. In a biochemical study (Table 2), a decrease in the level of lactate in the blood of animals treated with compound 1b was found. This is an additional confirmation of the improvement in the sensitivity of mouse tissues (liver, muscle, fat) to insulin [J. Lovejoy, FD Newby, SSP Gebhart, M. DiGirolamo, Insulin resistance in obesity is associated with elevated basal lactate levels and diminished lactate appearance following intravenous glucose and insulin, Metabolism, Volume 41, Issue 1, 1992, Pages 22-27]. The levels of triglycerides and alkaline phosphatase in the group of mice treated with compound 1b have a slight tendency to decrease compared with AY mice in the negative control group. The levels of total cholesterol, total protein and alanine aminotransferase were the same as in AY mice. This indicates the absence of hepatotoxicity in compound 1b at a dose of 15 mg/kg.

The study of analogs of compound 1b with shorter (C5) and longer (C7, C10, C12) aliphatic fragments in order to determine their effect on the severity of the hypoglycemic effect was carried out using the oral glucose tolerance test (OGTT) in C57BL/6 mice, where as vildagliptin (VLD) was used as a positive control. Studies were carried out at a dose of 15 mg/kg. It was shown that the length of the aliphatic fragment affects the severity of the hypoglycemic effect, namely, with a decrease in the number of carbon atoms, it increases, while an increase in the chain length leads to its decrease (Fig.4-7 ).

Thus, berberine derivatives 9-(alkylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1a - e are able to improve carbohydrate metabolism at a dose of 15 mg/kg and thus are promising candidates for the treatment of hyperglycemia and metabolic syndrome.

Spectral studies were carried out at the Chemical Service Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences.

The invention is illustrated by the following examples.

Example 1 . Synthesis of 9-(pentylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1a.

Berberine chloride 2 was dried in an oven for 2 hours at 95°C. To dried berberine chloride 2 (2 g, 5.4 mmol) was added 1-pentylamine (3.1 ml, 26.9 mmol). The reaction mixture was heated to 125°C and stirred for 4 hours. Acetone was added to the reaction mixture cooled to room temperature, and the formed burgundy precipitate was filtered off. The residue was purified by column chromatography to give compound 1a (1.01 g, 44%) as a burgundy solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.10 (s, 1H, H-8), 8.68 (s, 1H, H-13), 7.88 (d, J=8.6 Hz, 1H, H-9 *), 7.75 (s, 1H, H-1), 7.46 (d, J=8.6 Hz, 1H, H-10*), 7.06 (s, 1H, H-4), 6.42 (t, J=5.8 Hz , 1H, NH), 6.15 (s, 2H, OCH ₂ O), 4.79 (t, J=6.1 Hz, 2H, H-6), 3.95 (s, 3H, OCH ₃ ), 3.54-3.60 (m, 2H , NH CH ₂ ), 3.19 (t, J=5.9 Hz, 2H, H-5), 1.57-1.65 (m, 2H, NHCH ₂ CH ₂ ), 1.27-1.35 (m, 4H, NHCH ₂ CH ₂ CH ₂ CH ₂ ), 0.87 (t, J=6.9 Hz, 3H, CH ₂ CH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 149.80, 147.93, 146.92, 137.56, 135.93, 133.34, (C-2, C-3, C-4a, C-10, C-12a, C-13a ), 146.67 (C-8), 130.55, 120.91, 117.17 (C-8a, C-9, C-13b), 124.80 (C-13), 119.97 (C-12), 116.30 (C-11), 108.74 (C-4), 105.52 (C-1), 102.29 (OCH ₂ O), 57.28 (OCH ₃ ), 55.13 (C-6), 47.60 (NHCH ₂ ), 30.49, 28.90, 26.96, 22.25 (NHCH ₂ CH ₂ CH ₂ CH ₂ , C-5), 14.28 (CH ₂ CH ₃ ) . MS (ESI): m/z (M+) calcd for C ₂₅ H ₂₉ N ₂ O ₃ , 391.202 found: 391.202.

Example 2 . Synthesis of 9-(hexylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1b.

Berberine chloride 2 was dried in an oven for 2 hours at 95°C. To dried berberine chloride 2 (2 g, 5.4 mmol) was added 1-hexylamine (2.5 ml, 19.0 mmol). The reaction mixture was heated to 125°C and stirred for 4 hours. Acetone was added to the reaction mixture cooled to room temperature, and the formed burgundy precipitate was filtered off. The residue was purified by column chromatography to give compound 1b (1.23 g, 52%) as a burgundy solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.10 (s, 1H, H-8), 8.68 (s, 1H, H-13), 7.88 (d, J=8.7 Hz, 1H, H-9 *), 7.75 (s, 1H, H-1), 7.46 (d, J=8.7 Hz, 1H, H-10*), 7.06 (s, 1H, H-4), 6.40 (t, J=6.07 Hz , 1H, NH), 6.15 (s, 2H, OCH ₂ O), 4.79 (t, J=6.1 Hz, 2H, H-6), 3.95 (s, 3H, OCH ₃ ), 3.54-3.60 (m, 2H , NH CH ₂ ), 3.18 (t, J=6.1 Hz, 2H, H-5), 1.57-1.64 (m, 2H, NHCH ₂ CH ₂ ), 1.26-1.35 (m, 6H, NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ), 0.85 (t, J=6.9 Hz, 3H, CH ₂ CH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 149.45, 147.58, 146.54, 137.24, 135.85, 133.00, (C-2, C-3, C-4a, C-10, C-12a, C-13a ), 146.38 (C-8), 130.20, 120.57, 116.83 (C-8a, C-9, C-13b), 124.45 (C-13), 119.62 (C-12), 115.91 (C-11), 108.40 (C-4), 105.17 (C-1), 101.95 (OCH ₂ O), 56.92 (OCH ₃ ), 54.74 (C-6), 47.24 (NHCH ₂ ), 31.03, 30.43, 26.62, 26.00, 22.04 (NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ , C-5), 13.88 (CH ₂ CH ₃ ) . MS (ESI): m/z (M+) calcd for C ₂₅ H ₂₉ N ₂ O ₃ , 405.217 found: 405.217.

Example 3 . Synthesis of 9-(heptylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1c.

Berberine chloride 2 was dried in an oven for 2 hours at 95°C. To dried berberine chloride 2 (1.5 g, 4.0 mmol) was added 1-heptylamine (2.98 ml, 20.1 mmol). The reaction mixture was heated to 125°C and stirred for 4 hours. Acetone was added to the reaction mixture cooled to room temperature, and the formed burgundy precipitate was filtered off. The residue was purified by column chromatography to give compound 1c (0.5 g, 27%) as a burgundy solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.17 (s, 1H, H-8), 8.68 (s, 1H, H-13), 7.88 (d, J=8.8 Hz, 1H, H-9 *), 7.75 (s, 1H, H-1), 7.47 (d, J=8.8 Hz, 1H, H-10*), 7.07 (s, 1H, H-4), 6.48 (t, J=6.1 Hz , 1H, NH), 6.15 (s, 2H, OCH ₂ O), 4.79 (t, J=6.0 Hz, 2H, H-6), 3.95 (s, 3H, OCH ₃ ), 3.53-3.61 (m, 2H , NH CH ₂ ), 3.18 (t, J=6.0 Hz, 2H, H-5), 1.55-1.65 (m, 2H, NHCH ₂ CH ₂ ), 1.20-1.37 (m, 8H, NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ), 0.83 (t, J=6.8 Hz, 3H, CH ₂ CH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 149.78, 147.91, 146.83, 137.59, 135.90, 133.33, (C-2, C-3, C-4a, C-10, C-12a, C-13a ), 146.74 (C-8), 130.53, 120.92, 117.16 (C-8a, C-9, C-13b), 124.80 (C-13), 119.94 (C-12), 116.18 (C-11), 108.74 (C-4), 105.51 (C-1), 102.29 (OCH ₂ O), 57.24 (OCH ₃ ), 55.04 (C-6), 47.50 (NHCH ₂ ), 31.57, 30.82, 28.83, 26.96, 26.65, 22.39 (NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ , C-5), 14.27 (CH ₂ CH ₃ ) . MS (ESI): m/z (M+) calcd for C ₂₅ H ₂₉ N ₂ O ₃ , 419.233 found: 419.233.

Example 4 . Synthesis of 9-(decylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1d.

Berberine chloride 2 was dried in an oven for 2 hours at 95°C. To dried berberine chloride 2 (2 g, 5.4 mmol) was added 1-decylamine (5.35 ml, 26.9 mmol). The reaction mixture was heated to 125°C and stirred for 4 hours. Acetone was added to the reaction mixture cooled to room temperature, and the formed burgundy precipitate was filtered off. The residue was purified by column chromatography to give compound 1d (1.07 g, 40%) as a burgundy solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.00 (s, 1H, H-8), 8.69 (s, 1H, H-13), 7.89 (d, J=8.6 Hz, 1H, H-9 *), 7.76 (s, 1H, H-1), 7.47 (d, J=8.6 Hz, 1H, H-10*), 7.07 (s, 1H, H-4), 6.31 (t, J=6.1 Hz , 1H, NH), 6.15 (s, 2H, OCH ₂ O), 4.78 (t, J=6.2 Hz, 2H, H-6), 3.96 (s, 3H, OCH ₃ ), 3.53-3.60 (m, 2H , NH CH ₂ ), 3.15-3.20 (t, J=6.2 Hz, 2H, H-5), 1.55-1.63 (m, 2H, NHCH ₂ CH ₂ ), 1.18-1.34 (m, 14H, NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ), 0.83 (t, J=6.8 Hz, 3H, CH ₂ CH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 149.76, 147.90, 146.92, 137.48, 135.89, 133.26 (C-2, C-3, C-4a, C-10, C-12a, C-13a) , 146.68 (C-8), 130.52, 120.89, 117.10 (C-8a, C-9, C-13b), 124.59 (C-13), 119.98 (C-12), 116.30 (C-11), 108.73 ( C-4), 105.49 (C-1), 102.28 (OCH ₂ O), 57.19 (OCH ₃ ), 55.14 (C-6), 47.65 (NHCH ₂ ), 31.61, 30.75, 29.31, 29.25, 29.11, 29.02, 26.91, 26.61 , 22.41 (NHCH ₂ CH ₂ CH ₂ CH ₂ CH 2 CH ₂ CH ₂ CH ₂ CH ₂ , C-5), 13.88 (CH ₂ CH _{3 )} . MS (ESI): m/z (M+) calcd for C ₂₅ H ₂₉ N ₂ O ₃ , 461.280 found: 461.280.

Example 5 . Synthesis of 9-(dodecylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride 1e.

Berberine chloride 2 was dried in an oven for 2 hours at 95°C. To dried berberine chloride 2 (2 g, 5.4 mmol) was added 1-dodecylamine (5 g, 26.9 mmol). The reaction mixture was heated to 125°C and stirred for 4 hours. Acetone was added to the reaction mixture cooled to room temperature, and the formed burgundy precipitate was filtered off. The residue was purified by column chromatography to give compound 1e (0.96 g, 34%) as a burgundy solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.00 (s, 1H, H-8), 8.70 (s, 1H, H-13), 7.89 (d, J=8.6 Hz, 1H, H-9 *), 7.76 (s, 1H, H-1), 7.47 (d, J=8.6 Hz, 1H, H-10*), 7.07 (s, 1H, H-4), 6.32 (s, 1H, NH) , 6.16 (s, 2H, OCH ₂ O), 4.79 (t, J=5.9 Hz, 2H, H-6), 3.95 (s, 3H, OCH ₃ ), 3.54-3.59 (m, 2H, NH CH ₂ ) , 3.17-3.21 (t, J=5.9 Hz, 2H, H-5), 1.57-1.62 (m, 2H, NHCH ₂ CH ₂ ), 1.18-1.36 (m, 18H, NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ), 0.84 (t, J=6.9 Hz, 3H, CH ₂ CH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 149.79, 147.92, 146.83, 137.60, 135.90, 133.34, (C-2, C-3, C-4a, C-10, C-12a, C-13a ), 146.74 (C-8), 130.52, 120.92, 116.17 (C-8a, C-9, C-13b), 124.81 (C-13), 119.95 (C-12), 116.17 (C-11), 108.74 (C-4), 105.51 (C-1), 102.30 (OCH ₂ O), 57.24 (OCH ₃ ), 55.04 (C-6), 47.48 (NHCH ₂ ), 31.62, 30.79, 29.38, 29.36, 29.32, 29.31 , 29.13, 29.05, 26.96, 26.65, 22.42 (NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ , C-5), 14.29 (CH ₂ CH ₃ ) . MS (ESI): m/z (M+) calcd for C ₂₅ H ₂₉ N ₂ O ₃ , 489.311 found: 489.311.

Example 6 . Biological testing of compound 1b (9-(hexylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride).

Animals.

Male AY mice weighing 30-33 g and CD-1 mice weighing 20-25 g were obtained from the SPF vivarium of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. The animals were kept under standard conditions with free access to water and food in rooms with controlled humidity and temperature at a light-dark cycle of 12/12 h. 199n of 4 January 2016 and Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

AY mouse experiment design

Mice were fed a standard granulated chow supplemented with lard and biscuits ad libitum for 30 days until they gained a body weight of more than 35 g. After that, the following groups were formed: 1) mice (n=5) AY+solvent (water+2 Tween 80 drops), 2) mice (n=5) AY+SHE-196 15 mg/kg, 3) mice (n=5) AY+metformin (MF) 250 mg/kg and 4) mice (n=5) C57BL/6 + thinner (water + 2 drops of Tween 80). Each group consisted of 6 animals. The diet remained unchanged until the end of the experiment. All compounds were administered intragastrically once a day. OGTT was performed on the 14th day of the experiment. At the end of the experiment (22 days), blood was taken for biochemical analysis and measurement of insulin.

Oral glucose tolerance test (OGTT).

The test was performed after a 12-hour fast. Oral glucose loading (2.5 g/kg) was performed in all groups of mice. All compounds were mixed with two drops of Tween 80 prior to dissolution in water. Test compounds were administered via gavage 30 minutes prior to glucose loading. Blood glucose levels were quantified using a ONE TOUCH Select glucometer (LIFESCAN Inc., Milpitas, CA, USA) before administration (point 0) and 30, 60, 90, and 120 minutes after glucose loading. The area under the glycemic curve was calculated using the Tai model [Tai, M. A Mathematical Model for the Determination of Total Area Under Glucose Tolerance and Other Metabolic Curves. Diabetes Care, 1994, 17(2), 152-154].

Biochemical parameters

After 22 days of compound 1b administration, mice were decapitated, blood was collected and serum was separated by centrifugation at 1640 xg for 15 min. The levels of total cholesterol, triglycerides, total protein, alkaline phosphatase, alanine aminotransferase, and lactate in blood serum were quantified in all groups using standard diagnostic kits (Vector-Best, Novosibirsk, Russia) and a Stat Fax 3300 spectrophotometer (Awareness Technology Inc., USA) .

Evaluation of the concentration of insulin in the blood after a single injection.

CD-1 mice (n=5), fasted for 4 hours, were administered orally (through a gastric tube) compound 1b at a dose of 15 mg/kg or water with 2 drops of Tween 80 once. The experiment was carried out on the same animals every other day . Blood samples of 0.1 ml were taken from the tail of the animals before administration (0 min) and 30, 45 and 60 minutes after administration. After coagulation (at least 30 min), blood samples were centrifuged for 15 min at 1640 × g for 15 min to separate the serum. Serum samples were frozen for further evaluation of insulin by ELISA. The rat/mouse insulin ELISA kit (Catalog No. EZRMI-13K, Millipore) was used to analyze serum insulin concentrations. Sample preparation and all procedures were performed in accordance with the manufacturer's guidelines. A Multiscan Ascent photometer (Thermo Labsystems, Finland) was used for analysis.

Results:

During the three weeks of the experiment, the dynamics of weight loss in animals treated with metformin and 1b (Fig 1).

Two weeks after the start of the experiment, an OGTT was performed and a pronounced hypoglycemic effect of compound 1b was found (Fig. 2 and 3).

It was shown that compound 1b at a dose of 15 mg/kg after a single oral administration can increase the sensitivity of tissues to insulin, which is reflected in a decrease in the concentration of insulin in the blood (figure 4).

Example 7 . Biological testing of compounds 1a - e in C57BL/6 mice.

Animals.

Male C57BL/6 mice weighing 22–25 g were obtained from the SPF vivarium of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences. The animals were kept under standard conditions with free access to water and food in rooms with controlled humidity and temperature at a light-dark cycle of 12/12 h. 199n of 4 January 2016 and Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

Oral glucose tolerance test (OGTT).

The test was performed on animals after a 12-hour fast (n=6 in each group). Oral glucose loading (2.5 g/kg) was performed in all groups of mice. Before dissolving in water, all compounds were mixed with two drops of Tween 80. Vildagliptin (VLD) positive control tablets (Galvus, Novartis Farmaceu- ca SA, Barcelona, Spain) were dissolved in water. Test compounds were administered by gavage 30 minutes prior to glucose loading. Blood glucose levels were quantified using a ONE TOUCH Select glucometer (LIFESCAN Inc., Milpitas, CA, USA) before administration (point 0) and 30, 60, 90, and 120 minutes after glucose loading. The area under the glycemic curve was calculated using the Tai model [Tai, M. A Mathematical Model for the Determination of Total Area Under Glucose Tolerance and Other Metabolic Curves. Diabetes Care, 1994, 17(2), 152-154].

As a result of the studies, it was found that the length of the aliphatic fragment, with the introduction of compounds 1a - e at a dose of 15 mg/kg, significantly affects the severity of the hypoglycemic effect (Figure 5-8), namely, with a decrease in the number of carbon atoms, it is strengthened , whereas an increase in the chain length leads to its decrease.

List of figures:

Fig. 1. Change in body weight of AY mice during three weeks of compound 1b at 15 mg/kg and metformin (MF) at 250 mg/kg.*p<0.05 compared to AY mice (control).

Fig. 2 . OGTT was performed after 2 weeks of compound 1b at 15 mg/kg and metformin (MF) at 250 mg/kg.* p<0.05 compared to AY mice.

Fig. 3 . Area under the glycemic curve calculated from OGTT data after 2 weeks of administration of compound 1b at 15 mg/kg and metformin (MF) at 250 mg/kg to AY mice. a, b, c: p<0.05 compared to AY mice; d: p<0.05 compared to MF.

Fig. 4. Change in blood insulin concentration after a single oral administration of compound 1b at a dose of 15 mg/kg to CD-1 mice. *p<0.05 compared to control animals.

Fig. 5 . Results of OGTT, 1a, 1b, 1c at 15 mg/kg. *p<0.05 1a vs. glucose, ^#p <0.05 1b and 1c vs. glucose.

Fig. 6 . Results of OGTT (area under the glycemic curve), 1a, 1b, 1c at a dose of 15 mg/kg. *p<0.05 in relation to glucose.

Fig. 7 . Results of OGTT, 1e, 1d at 15 mg/kg. *p<0.05 with respect to glucose.

Fig. 8 . OGTT results (area under the glycemic curve), 1e, 1d at 15 mg/kg.*p<0.05 relative to glucose.

Table 1 Mass of gonadal fat, interscapular fat and interscapular brown fat in AY mice treated for three weeks with compound 1b at a dose of 15 mg/kg and metformin at a dose of 250 mg/kg. Gonad fat, g Interscapular
fat, g Interscapular
brown fat, g Mice AY 2.51±0.18 1.21±0.02 0.21±0.02 Metformin 1.91±0.17 0.87±0.09* 0.13±0.02* 1b 2.14±0.27 0.92±0.19* 0.12±0.03* C57Bl/6 0.29±0.07 - 0.03±0.003*

*p<0.05 compared to AY mice.

Table 2 . Blood biochemical parameters of AY mice treated for three weeks with compound 1b at a dose of 15 mg/kg and metformin at a dose of 250 mg/kg.*p<0.05 compared to AY mice. Total cholesterol, mmol/l tg,
mmol/l Total protein, g/l Lactate, mmol/l SHF,
U/l ALT,
U/l Mice AY 2.54±0.11 0.28±0.03 63.49±3.65 8.41±0.54 98.50±5.94 45.38±3.36 Metformin 2.52±0.14 0.25±0.02 59.70±3.64 5.88±0.41* 94.60±94.68 46.20±2.65 1b 2.65±0.28 0.21±0.01 59.93±3.49 5.15±0.24* 85.50±1.55 41.00±3.49 Mice C57Bl/6 1.90±0.05* 0.18±0.01* 59.60±1.72 4.40±0.49* 87.71±4.92 38.43±2.10

Claims (2)

The use of compounds 1a-e of the following structure as an agent for increasing the sensitivity of tissues to insulin in type 2 diabetes mellitus

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