Title of Invention

"NAPHTHOQUINONES AS ANTIDIABETIC AGENTS"

Abstract

The present invention describes naphthoquinones of synthetic as well as natural origin, designated to have significant anti-hyperglycaemic activity in vivo, as potential antidiabetic agents. The structure of these compounds is depicted by the general formula as given in Fig 1 where R1, R2, R3, R4 and R5 are herein described; wherein R1 is H, OH , alkyl, aryl, alkylarly; R2 represents H, OH, alkyl, aryl; R3 is H, OH, alkyl, alkoxy, aryl; R4 represents H, OH, alkyl, aryl and R5 is H, OH, alkyl, alkoxy, ary1 etc.

Full Text

Field of invention The present invention relates to antihyperglycaemic activity of some synthetic as well as naturally occurring naphthoquinone compounds and their derivatives or solvates in vivo, useful as potential antidiabetic agents. Background of invention Diabetes is associated with a variety of metabolic abnormalities, principle among them being hyperglycaemia. This syndrome includes hypertriglyceridemia, reduced HDL, cholesterol and abnormal postprandial lipidemia (Diabetes, 1988, 37, 1597-1607). Type 1 diabetes or insulin dependent diabetes mellitus (IDDM) is a complex, multifactorial disease involving severe destruction of the insulin producing pancreatic (3 cells. Type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM) typically occurs at older age and obesity adds greater risk in its development (J. Clin. Endocrinol. metab. 1999, 84, 2329-2335). Although glycemic control, insulin treatment and other chemical therapies can control many aspects of diabetes, numerous complications are common and diverse. Diabetic patients have an increased risk of developing various clinical complications that are due to microvascular or macrovascular diseases that includes nephropathy, retinopathy and neuropathy. Insulin resistance is manifested in type 2 diabetes mellitus. Type 2 diabetic patients have elevated levels of fatty acids. It appears that control of non esterified fatty acids in the blood could be an important approach to reduce insulin resistance. Studies on the molecular actions of compounds indicate that thiazolidinediones (potent glucose lowering agents) and fibrates (hypolipidaemic agents) exert their action by activating distinct transcriptional factors of the peroxisome proliferator activated receptors (PPAR) family (JMC, 2001. 44, 2675-2678, JMC, 2003,4(5, 1306-1317). A reduced autophosphorylation of the insulin receptor from skeletal muscle and adipose tissue of type 2 diabetic patients has been described in literature. This impaired receptor autophosphorylation might contribute to impaired insulin signalling and insulin resistance of skeletal muscle and adipose tissue in diabetic patients. Thus, insulin receptor kinase activation is necessary in insulin signalling pathway (Nature 2001, 414, 799). The discovery of a non-peptidyl fungal metabolite, L-783,281 (a quinone compound) which induced activation of human insulin receptor tyrosine kinase and acted as an insulin mimetic in several biochemical and cellular assays, stimulated research on quinone related compounds as possible anti-diabetic agents. Oral administration of L-783,281 to mouse models of diabetes resulted in significant lowering in blood glucose levels (Science 1999, 284, 974; J. Biol. Chem. 2000, 275, 36590). Terpenoid quinones isolated from Pycanthus angolensis, lowered plasma glucose concentrations when given orally to ob/ob mice, which are hyperglycaemic, and hyperinsulinemic (J. Pharmacol. Exp. Therap. 1999, 9, 185). It has been shown that diabetic subjects exhibit high lipid peroxides and other forms of oxidative stress. (Bratisl Lek Listy, 2000, 101(10) 541-551). In diabetes, oxidative stress seems to be caused by increased production of reactive oxygen species (ROS), sharp reduction in antioxidant defenses and altered cellular redox status. Consequences of oxidative stress are damage to DNA, lipids, proteins, disruption in cellular homeostasis and accumulation of vascular damages. It is known that supplementation with antioxidant is a promising complementary treatment which exerts beneficial effects in diabetes and provides further support for implication of oxidative stress in beta cell dysfunction in diabetes (Diabetes, 1999, 48(12) 2398-2406). Targeting therapy to specific tissues and organs of diabetes by specific antioxidants or combined drug preparations could become a relevant adjuvant pharmacotherapy with improved glycemic control, blood pressure control and management of dyslipidaemia for the treatment or prevention of progression of micro and macro vascular diabetic complications. The molecular mechanism supports that prolonged oxidative stress impairs insulin induced GLUT4 translocation in 3T3-L1 adipocytes (Diabetes, 1998,47, 1562-1569). Plumbagin (5-hydroxy-2-methyl-l,4-naphthoquinone) and related compounds have been reported to exhibit several biological activities such as antileishmanial, antimalarial, anti-tumour, anti-inflammatory, antifungal, antibacterial etc. (Planta Med., 1998, 64, 237-241; J. Pharm.Sci., 1996, 55, 194-196 ; Biol.Pharm.Bull., 1997, 20,1250-1256 ; Chem.Pharm.Bull., 2002, 50, 672-674 ; Indian J.Exp.Biol., 1980, 18, 876-877). Plumbagin has been reported to reduce serum cholesterol and LDL-cholesterol by 53-S6°o and 61 to 91% respectively, when administered to hyperlipidaemic rabbits (Indian.!. Physiol.Pharmacol., 1991,35, 10-14). With this literature information, we focussed our attention on naphthoquinone class of compounds, which have not been described in literature so far to possess anti-diabetic activity. The Applicant started their work with naturally occurring naphthoquinone compound, plumbagin and synthesized some naphthoquinone derivatives. Detailed description The present invention provides compounds of general structure as given in Fig. 1 Fig. 1 wherein R, represents H, OH, alkyl, aryl, alkylaryl R2 is H, OH, alkyl, aryl R3 represents H, OH, alkyl, alkoxy, aryl R4 is H, OH, alkyl, aryl and Rj represents H, OH, alkyl, alkoxy, aryl and their pharmaceutically acceptable salts, derivatives or solvates or any tautomeric forms. Further, in the said compounds i) alkyl group is selected from C1.5 alky ii) aryl group is selected from (un)substituted 6 membered cyclic ring iii) alkylaryl group selected from C1.5 alkyl and aryl selected from (un)substituted 6 membered cyclic ring iv) alkoxy group selected from C1.5 alkyl The said compounds of the invention, are useful as anti-diabetic compounds. The compounds are derived from naturally occurring naphthoquinone; plumbagin was isolated and characterized from Plumbago zeylanica. The said compounds with alkyl substituents showed good antihyperglycaemic activity. All the synthesized and isolated compounds showed medium to weak antioxidant activity. Further, the present invention relates to the preparation of pharmaceutically acceptable salts, intermediates and all the tautomeric forms of compounds of general structure shown in Fig. 1. The invention also provides pharmaceutical compositions prepared using the compounds shown in shown and described hereinabove together with pharmaceutically acceptable additives. All the compounds were subjected to antihyperglycaemic activity in diabetic rats and the results were obtained by measuring their ability to decrease the plasma glucose levels (PGL) by using metformin as positive control. The effect of naphthoquinone compounds on oral glucose tolerance test (OGTT) was also studied in normal rats. Most of the compounds studied gave medium to weak antihyperglycaemic activity in diabetic rats. Some gave high activity comparable to metformin. The results of the representative compounds are given in the Table. The evaluation of antioxidant activity was done by using DPPH free radical scavenging in vitro assay. Example 1: Isolation of plumbagin from Plumbago zeylanica Powdered roots (250 g) of Plumbago zeylanica were extracted with chloroform in soxhlet apparatus. The chloroform extract after drying and evaporation in vacuo gave a semi-solid residue (1.6 g). The chloroform extract, after adsorption onto silica gel, was chromatographed on silica gel (50 gm). The sample was eluted from the column using a step-wise gradient of hexane containing increasing amounts of ethyl acetate (95:5, 9:1, 85:15, 8.2, 7:3, 6:4, 5:5 and 0:1). The plumbagin containing fractions which gave positive color reaction with 10% alcoholic K.OH, were combined, evaporated and recrystallized from methanol to afford 80 mg of plumbagin. Mp 77-78°C (reported: 78°C) Example 2: Synthesis of 2,6-dimethyl-l,4-naphthoquinone A solution of 0.7 g (4.48 mM) of 2,6-dimethylnapthalene in 2.25 g (22.5 raM) of chromium trioxide was added to 20 ml of glacial acetic acid while cooling the reaction mixture to maintain the temperature of 20 - 25°C for 24 hours. The resulting mixture was poured over 100 ml of crushed ice with continuous stirring. After 1 hour, the crystals were separated by filtration and recrystallized from methanol to give 0.292 g (35%) of yellow needles, mp 98°C (reported: 98°C) IR(KBr, cm"'): 1662 'H NMR 8 (CDC13): 7.99 (d, 1H), 7.85 (s, 1H), 7.52 (d, 1H), 6.80 (s, 1H), 2.49 (s, 3H), 2.18 (s,3H) Example 3: Synthesis of menadione (2-methyl-l,4-naphthoquinone) It was synthesized using 2-methylnaphthalene as starting compound, as per the procedure described in example 2 Example 4: Synthesis of 2,3-diethyl-l,4-naphthoquinone Synthesis was carried out using 2,3-diethylnaphthalene as the starting compound as per the procedure described in example 2 Example 5: Synthesis of 2,6-diethyl-l,4-naphthoquinone Synthesis was carried out using 2,6-diethylnaphthalen as the starting compound as per procedure described in example 2. Example 6: Synthesis of 5-phenyl-l,4-naphthoquinone Synthesis was done using 1-phenylnaphthalene as the starting compound as per the procedure described in example 2. Example 7: Synthesis of 5,8-dihydroxy-l,4-naphthoquinone A solution of 0.5 g of sulphur in 7.5 ml of cone, sulphuric acid was added dropwise with stirring to a suspension of 1 g (0.046 mol) of 1,5-dinitronaphthalene in 4.6 ml of cone. sulphuric acid. The reaction mixture was maintained between 50-60°C for 2 hours. The dark red solution was poured over 100 ml of crushed ice. This solution was filtered and heated until it became dark red and a thick mass formed. The red solid was separated by filtration and dried. The product was obtained by repeated extraction of the red solid with benzene followed by evaporation of solvent to give 0.13 g (15%) of red crystalline solid. mp 234°C (dec.) (reported: 234°C) IR(KBr, cm"1): 1614 'H NMR S (CDC13): 715 (s, 4H), 12.4 (s, 2 OH) Example 8 Synthesis of 5,8-dimethoxy-l,4-naphthoquinone A solution of 0.5 g (0.0052 mol) of 5,8-dihyroxy-l,4-naphthoquinone, 2ml of methyl iodide, 18 ml of chloroform was refluxed with 0.5 g of silver oxide for 48 hours until an aliquot from the reaction mixture failed to give a violet colour with 5% sodium hydoxide. Additions of 1 ml of methyl iodide and 0.5 g of silver oxide were made after 2, 6 and 24 hours. When the reaction was completed, the silver salt was removed by filtration and the filtrate was passed through alumina column using petroleum ether (40-60°C) as an eluent. Evaporation of the solvent from the fraction containing the second band gave orange needles of 5,8-dimethoxy-l,4-naphthoquinone (25%). mp 155°C (reported: 155°C) IR(KBr, cm"1): 1650 'H NMR 5 (CDCI3): 3.97 (s, 6H), 6.78 (s, 2H), 7.32 (s, 2H). Example 9: Synthesis of 5,8-diisopropoxy-l,4-naphthoquinone It was synthesized using isopropyl iodide as the alkylating agent, as described in example 8. Example 10 Antihyperglycaemic activity of naphthoquinone compounds Animals Female Sprague-Dawley rats weighing 130-150 g were procured from central animal facility (CAF) of the institute. The animals were maintained under controlled room temperature (22 ± 2°C) and humidity (55 ± 5 %) with 12h light and dark cycle. All the animals were housed in polypropylene cages in groups of 3 per cage and provided with standard pellet diet and water ad libitum. All the animals were acclimatized for a minimum period of one week prior to the experiment. The protocol of this experiment was approved by the Institutional Animal Ethics Committee (IAEC) and the experiments were earned out in accordance with the guidelines of IAEC given on animal experimentation. Induction of diabetes by streptozotocin in rats Female Sprague-Dawley rats were rendered diabetic by an intraperitoneal injection of streptozotocin (65mg/Kg, Sigma chemicals, MO, USA) reconstituted in cold citrate buffer (0.1M, pH 4.4). The development of hyperglycaemia was confirmed by the estimation of plasma glucose level 48h post STZ injection. The rats were used for the experiment two weeks after the STZ injection. Those rats with fasting (3h) plasma glucose level of > 300mg/dl were considered diabetic and included in the study. Blood collection and biochemical measurement Blood was collected from retro orbital plexus of rats using capillary tubes into eppendorf tubes containing heparin solution, gently mixed and centrifuged at 5000 RPM for 5 min to separate plasma. The plasma samples were then analyzed for glucose by glucose oxidase- peroxidase method using commercial GOD-POD diagnostic kit (Qualigens, Mumbai). The diabetic rats post 2 weeks of STZ injection were randomly divided in to different groups consisting each of 6 rats based on fasting plasma glucose level. The animals were grouped as negative control group, treated groups and positive control group. The negative controls, given vehicle 1 % Na-CMC (5 ml/kg). The positive controls group was given standard antihyperglycemic agent metformin (100 mg/kg). The treated groups were given test samples (napthoquinone compounds). All above doses were singly administered to the diabetic animals. All the rats were fasted for 3 h initially and the blood was collected 3 h after the administration of compounds. All the compounds were administered at the dose of 100 mg/kg and menadione, 2,6-dimethyl-1.4-naphthoquinone were also administered at 50 mg/Kg dose. The percentage reduction in plasma glucose levels in treated groups was calculated in comparison to control group. Effect of single administration of some naphthoquinone compounds on plasma glucose levels (PGL) in diabetic rats. s.No. Groupa PGL (mg/dl) (Mean ± SEM, n = 6) % PGLb Reduction 14 days (Oh) After 3 h 1 Vehicle (2ml/Kg) 473.57 ±15.77 463.36 ± 14.96 2.15 -y menadione (100 mg/Kg) 434.37 ±36.29 225 ±46.69* 48.2 menadione (50 mg/Kg) 441.14 ±17.83 325.65 ±29.92* 26.18 3 2,6-dimethyl-l,4-napthoquinone (100 mg/Kg) 448.38 ±24.09 228.75 ±33.86* 48.98 2,6-dimethyl-1,4-napthoquinone (50 mg/Kg) 471.87 ±19.12 391.87 ± 14.31 16.95 4 5,8-dihydroxy-1,4-napthoquinone(100 mg/Kg) 476.56 ±32.75 418.13 ±39.21 12.26 5 5,8-dimethoxy-l,4-napthoquinone (100 mg/Kg) 468.33 ±25.30 396.90 ± 18.64 15.25 6 Metformin (100 mg/Kg) 452.08 ±20.91 286.45 ±39.62* 36.63 * p ■' All the test compounds singly administered orally ' reduction in % PGL calculated from 0 h value of respective groups Example 11 Antioxidant activity The free radical scavenging activity was evaluated by the DPPH method. The samples (0.1 ml) were added to aliquots (3.9 ml) of a solution made up with 4.8 mg DPPH in 200 ml of methanol and the mixture incubated for 1 h at room temperature. The absorbance at 517 nm was measured. The percentage inhibition was calculated by the degree of absorbance disappearance at 517 nm [(1-A/A(1)x 100].

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