Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
  • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
  • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

• the present invention relates to the development of novel substituted 4,5-dihydro-2H- benzo[e]indazole-9-carboxylates, which can be used as therapeutic agents for the treatment and prevention of metabolic disorders, and a process of preparing said novel compounds. More particularly, the present invention relates to substituted 4,5-dihydro- 2H- benzo[e]indazole-9-carboxylates and their related compounds, processes for preparing the said compounds and to their use in the treatment of diabetes and related metabolic disorders.
• Diabetes mellitus A disease of all age group practically in all parts of the world, the Diabetes mellitus (Madhumeha). Diabetes mellitus is a chronic, incurable disease which altered the metabolism of lipids, carbohydrates and proteins, it is one of the common endocrine disorders with increased risk of complications from artery diseases, myocardial infarction, hypertension, and dyslipidemia, and clinically characterized by peripheral hyperglycemia. Diabetes can be classified clinically as insulin-dependent diabetes mellitus (IDDM, or type-1 diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, or type-2 diabetes). Type-2 diabetes is common and found in >90% patient characterized by either normal/abnormal insuhn secretion or function.
• IDDM insulin-dependent diabetes mellitus
• NIDDM non-insulin-dependent diabetes mellitus
• T2DM diabetes mellitus
• the main object of the present invention is to provide substituted 4,5-dihydro-2H- benzo[e]indazole-9-carboxylates and related compounds for the treatment of diabetes and related disorders.
• Another object of the present invention is to obtain/prepare therapeutically effective compound pharmaceutically acceptable formulations/dehvery systems such as tablets, capsules, suppository, beads, aerosols, etc. for the treatment and prevention of diabetes and other related metabolic disorders.
• Still another object of the present invention relates to the synthesis of the compounds which requires easily accessible precursors and mild reaction conditions in a single step at room temperature.
• R 1 is independently selected from the groups consisting of optionally substituted aryl, optionally substituted heteroaryl;
• R 2 , R 3 are independently selected from the groups consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkenyl;
• R 4 is independently selected from the groups consisting of optionally substituted alkylthio, optionally substituted alkylsulfonyl, optionally substituted amines;
• R is independently selected from the groups consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl groups;
• Y is selected from the groups consisting of one methylene group, two methylene groups, or optionally substituted methylene group;
• the compounds are useful for the treatment of diabetes and related disorders.
• anoher embodiment of the present invention also provides a process for the synthesis of 4,5-dihydro-2H-benzo[e]indazole-9-carboxylates having the general formula I, which comprises;
• step (a) reacting 2H-pyran-2-ones with a cyclic ketone in presence of an alkali base for a period upto 24 hours at a temperature ranging between 0 to 50 degree centigrade;
• step (b) pouring the reaction mixture to an ice cold water and neutralized with an acid; step(c) purifying the product by chromatographic method, LLS 267 step (d) reacting the compound obtained in above step with m-chloroperbenzoic acid in dichloromethane at a temperature ranging between 25 to 28 degree C for a period ranging between 1-2 hrs, treating the reaction mixture with sodium bicarbonate solution to obtain the desired compound.
• substituted 2H-pyran-2-ones is used to react with substituted or unsubstituted cyclic ketones such as 2-phenyl-6,7-dihydro-2H-indazol- 4(5H)-one as described in examples 1-13.
• the organic solvent used is selected from the group consisting of acetone, N,N-dimethyl formamide, N,N-dimethyl acetamide, dimethyl sulfoxide, benzene, toluene, xylene, chlorobenzene, tetrahydrofuran.
• base used is selected from a group consisting of NaOH, KOH, NaH, t-BuOK, K 2 C0 3 , and NaOEt,
• a pharmaceutical composition comprising an effective amount of compound of formula I optionally along with pharmaceutically acceptable, excipients, carrier/s, lubricants, and diluents.
• the effective amount of the compound in the composition is ranging lOmg to 500mg.
• the pharmaceutically acceptable excipient may be selected from the group consisting of starch, lactose, cellulose.
• the diluents used may be selected from the group consisting of starch, lactose, dicalcium phosphate.
• the lubricant may be selected from the group comprising of talc, magnesium stearate.
• a method of treating diabetes in a subject comprising the step of administering to the subject suffering from diabeties, a pharmaceutical composition an effective amount of compound of formula I optionally along with pharmaceutically acceptable, excipients, carrier/s, lubricants, diluents. LLS 267
• Sheet 1 illustrates the general formula I for the novel 4,5-dihydro-2H-benzo[e]indazole-9- carboxylates compounds of the invention and illustrates the reaction sequence (Scheme 1) resulting in the preparation of various 4,5-dihydro-2H-benzo[e]indazole derivatives and related compounds.
• Sheet 2 illustrates Effect of the synthetic compounds (Figure 2) and standard drug Januvia (at 50 mg/kg) on the blood glucose levels of the sucrose loaded normoglycaemic rats at various time intervals and Effect of the synthetic compounds ( Figure 3) and standard drug Januvia (at 50 mg/kg) on the blood glucose levels of the sucrose challenged STZ induced diabetic rats at various time intervals.
• Sheet 3 illustrates Effect of the synthetic compounds ( Figure 4) and standard drug Januvia (at 50 mg/kg) on the blood glucose levels of the Low dosed STZ induced diabetic rats at various time intervals.
• R 1 is independently selected from the groups consisting of optionally substituted aryl, optionally substituted heteroaryl; LLS 267
• R 2 , R 3 are independently selected from the groups consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkenyl,
• R 4 is independently selected from the groups consisting of optionally substituted alkylthio, optionally substituted alkylsulfonyl, optionally substituted amines etc,
• R is independently selected from the groups consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl groups
• Y is selected from the groups consisting of one methylene group, two methylene groups, or optionally substituted methylene group.
• the starting material 2H-pyran-2-ones are known and has been prepared by the reaction of methyl 2-cyano/methoxycarbonyl-3,3-di(methylsulfanyl)acrylate with substituted acetophenones under alkaline conditions in dry DMSO in high yields.
• the 2H-pyran-2- ones is treated with cyclic ketone preferably dissolved either in a volatile solvents such as acetone, THF or in a high boiling solvents such as dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide in presence of an alkali such as K 2 C0 3 , KOH, t-BuOK, NaH.
• a volatile solvents such as acetone, THF or in a high boiling solvents
• an alkali such as K 2 C0 3 , KOH, t-BuOK, NaH.
• equimolar amounts of the reagents are used and the reaction takes place within a period of 24 hours at the reaction temperature between 25
• Streptozotocin a most widely used chemical diabetogen was procured from Sigma-Aldrich chemicals Co., St Louis, MO, USA. All the chemicals and reagents used were obtained from local sources and were of analytical grade.
• the crude LLS 267 mixture was purified on a silicagel column using 40% chloroform in hexane as eluent to yield 78% of methyl 6-(4-fluorophenyl)-8-(methylsulfonyl)-2-phenyl-4,5-(iihydro-2H- benzo[e]indazole-9-carboxylate.
• the crude mixture was purified on a silicagel column using 40% chloroform in hexane as eluent to yield 80% of methyl 8-(methylsulfonyl)-6-(naphthalen-l-yl)-2-phenyl-4,5-dihydro-2f]r- benzo[e]indazole-9-carboxylate.
• the crude mixture was purified on a silicagel column using 40% chloroform in hexane as eluent to yield 80% of methyl 8-(memylsulfonyl)-2-phenyl-6-( -tolyl)-4,5-(H3 ⁇ 4ydro-2H-benzo[e]indazole-9- carboxylate.
• mice Male albino rats of Sprague Dawley strain of 8 to 10 weeks of age and 160 + 20 g of body weight were procured from the animal colony of the Institute. The work with these animals was cleared by institutional ethics committee for animal study and was conducted in accordance with the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) formed by the Government of India in 1964. The institute has taken permission from the animal ethical committee for the work (No. 129/07/Biocmem/IAEC). The animals were housed four or five in a polypropylene cage in the animal house.
• test compounds and standard antidiabetic drag Januvia were prepared in 1.0 % freshly prepared Gum acacia.
• the diabetic animals were divided into four groups (Group 1 to 4) each consisting 6 rats.
• Group 1 received 0.4 ml vehicle (1% gum acacia).
• Group 2 received the test compound 5 at an oral dose of 100 mg/kg/body weight.
• Group 3 was given compound 6 at an oral dose of 100 mg/kg/body weight.
• the standard antidiabetic drug Januvia was given to the animals of Group 4 at an oral dose of 50 mg/kg body weight.
• the blood glucose levels of each animal were again measured by glucostrips at 1 h, 2 h, 3 h, 4 h, 5 h and 24 h post treatment with the test sample/standard drug by glucometer. Food but not water was withheld from the cages during the course of experimentation. Percent hyperglycemic activity was determined by comparing the AUC (area under curve) of experimental groups with that of the vehicle treated control group.
• the diabetic rats with fasting blood glucose values (baseline at 0 min) from 140 to 270 mg/dl were included in this study. All the diabetic animals were again divided into groups with five animals in each group. Animals of one group were regarded as the control group (orally administered 1% gum acacia) and the others were treated as the experimental groups. Rats in the experimental groups were orally administered suspension of the test compounds (lOOmg/kg) prepared in 1.0% gum acacia. Animals of the control group were given an equal amount of 1.0% gum acacia. The standard antidiabetic drug Januvia was given to the animals at an oral dose of 50 mg/kg body weight.
• sucrose load (2.5 g/kg) was given to each animal orally exacdy after 30 min post administration of the test sample/ LLS 267 vehicle. Blood glucose levels were checked at lh, 2h, 3h, 4h, 5h and 24h post- administration of sucrose by glucometer. Food but not water was withheld from the cages during the course of experimentation. Percent hyperglycemic activity was determined by comparing the AUC (area under curve) of experimental groups with that of the vehicle treated control group.
• Protein tyrosine phosphatase- IB assay- Protein tyrosine phosphatase- IB inhibitory activity of the compounds were evaluated at 10 ⁇ concentration using colorimetric, non-radioactive PTP1B tyrosine phosphatase drug discovery kit BML-AK 822 from Enzo Life Sciences, USA.
• Components of the kit include human recombinant PTP1B enzyme, substrate (IR5 insulin receptor residues), biomol red (phosphate determining reagent), assay buffer, suramin (PTP1B inhibitor) and calibration standards.
• Assay was done according to the Kit manufacturers protocol, in brief the reaction was carried out in 96 well flat bottomed microliter plate by the addition of assay buffer, solution of test compounds and diluted PTP1B enzyme. Enzyme reaction was initiated by addition of 50 ⁇ 1 of warmed 2x substrate then incubated the plate at 30°c for 30min. After incubation for 30 min. Reaction was terminated by addition of 25 ⁇ of biomol red reagent and mixed thoroughly by repeated pipetting. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and solution of ⁇ concentration was prepared of which 10 ⁇ solution was added in each reaction well to achieve final concentration of 10 ⁇ in reaction mixture. Volumes and dilution of other component were accordingly as instructed in the manual provided in the kit.
• DMSO dimethyl sulfoxide
• PTP1B phosphatase acting on the phosphopeptide substrate and release phosphate The detection of free phosphate released is based on classic Malachite green assay. After adding biomol red to reaction wells after 30 minutes of incubation as described earlier the plate was incubated for another 20 min to develop the colour. Absorbance was recorded at 620 nm. on a microplate reader. The percentage inhibition of PTPlb enzyme by test compounds was calculated based on activity in the control tube (without inhibitor) taking as 100 % from three independent set of experiments. LLS 267
• IC 5 o value a series of dose response data ie. % inhibition of PTP IB at different concentration of inhibitor is required.
• compound 6 % inhibition of at 5 different concentrations was evaluated using PTP IB tyrosine phosphatase drug discovery kit by the method as described above.
• Five different 5 compound concentrations selected for IC 5 o calculation were 0.3 ⁇ , ⁇ . ⁇ , 3.0 ⁇ , 5.0 ⁇ , ⁇ . ⁇ and accordingly calculated the IC 5 o value.
• Quantitative glucose tolerance of each animal was calculated by the area under the curve (AUC) method using Prism Software. The area under the curve of the control group and the 10 experimental group was compared to determine the percent antihyperglycaemic activity.
• Results- Table 1 Effect of synthetic compounds (100 mg kg) and standard drug Januvia (50 mg/kg) on the blood glucose levels at various time intervals.
• Table-4 Effect of the synthetic compounds (at 100 mg/kg) and standard drug Januvia (at 50 mg/kg) on the blood glucose levels of the low dosed STZ induced diabetic rats at various time intervals.
• LLS 267 Effect of the synthetic compounds (at 100 mg/kg) and standard drug Januvia (at 50 mg/kg) on the blood glucose levels of the low dosed STZ induced diabetic rats at various time intervals.
• Tlie effect exerted by compound 5 and 6 at the dose level of 100 mg/kg is comparable with the standard drug Januvia at the dose level of 50 mg/kg.
• Table 1 ,2 and Figure 1 shows the effect of compound 5 and 6 (100 mg/kg b.w.) and Januvia (50 mg/kg b.w.) on sucrose loaded normoglycemic rats. It is evident from the results that compound 5 and 6 show significant improvement in glucose tolerance.
• the fasting blood glucose levels (meant SEM) of the test compounds, standard drug Januvia and control groups were 66.2 ⁇ 3.05, 66.2 ⁇ 2.63, 66.3 ⁇ 3.12 and 66.0 ⁇ 0.78 mg/dl (Table 2) respectively.
• the decrease in the blood glucose level at 2 hrs in the test group compared with that of the control was 35.8% and 23.5% whereas the decrease in the Januvia treated group was 35.0%.
• Table 1,3 and Figure 2 represent the average antihyperglycaemic activity profile of compound 5 and 6 and standard drug Januvia in the sucrose challenged streptozotocin- induced diabetic rats.
• the fasting blood glucose levels (mean + SEM) of the test compounds, standard drug and control groups were 222.3 ⁇ 9.39,222.6 +6.55, 222.3 ⁇ 16.2 and 222.6 ⁇ 14.1 mg/dl (Table 3) respectively.
• Table 4 and Figure 3 represent the average antihyperglycaemic activity profile of compound 5 and 6 and standard drug Januvia in the low dosed stteptozotocin-induced diabetic rats .
• the fasting blood glucose levels (mean + SEM) of the test, standard drug and control groups were 346.6 ⁇ 6.19,344.0 ⁇ 3.91, 341.4 ⁇ 3.53 and 343.4 ⁇ 3.15 mg/dl (Table 4) respectively.
• Advantages of the present invention are the synthesis and identification of a novel series of 4,5-dihydro-2H-benzo[e]indazole compounds prepared through innovative route under mild reaction conditions and their useful anti-hyperglycemic activity in both in vitro (PTPIB inhibitors) and in vivo (SLM, STZ, STZ-S animal models). These compounds hold therapeutic potential for the treatment of diabetes and related metabolic disorders.

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