Synthesis and Pharmacological Evaluation of Novel Indolo TriazineDerivatives

INTRODUCTION

Diabetes mellitus is one of the most important non infective disease to hit the globe in the present millennium. Diabetes mellitus is a metabolic disorder characterized by high blood glucose level.

The pancreatic β-cells and its secretary hormone i.e. insulin are central in the pathophysiology of Diabetes. In type 2 or non-insulin dependent Diabetes mellitus, muscle and fat cells are ‘resistant’ to the action of the insulin and compensatory mechanisms that are activated in the β-cell to secrete more insulin are not sufficient to maintain blood glucose levels within a normal physiological range. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidemia, hypertension and atherosclerosis. The epidemic of type 2 Diabetes and impaired glucose tolerance are main causes of morbidity and mortality worldwide, which result from defects in insulin secretion, or action, or both.

Antibiotics fight against bacterial infections but due to emergence of drug-resistant bacteria they have become less effective against certain illness hence it is essential to investigate new drugs with less resistance. The harmful microorganisms can be controlled with drugs and these results in the emergence of multiple drug-resistant bacteria and it has created alarming clinical situations in the treatment of infections. The pharmacological industries have produced a number of new antibiotics; resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to synthetic drugs which are utilized as therapeutic agents. Therefore development of efficient and selective antimicrobial agents is one of the fundamental challenges of researchers working in the field of pharmaceutical sciences.

The literature survey undoubtedly indicates the biological significance of indole and its substituted nucleus. The indole nucleus exhibits extensive spectrum of biological activities such as anticancer, antitubercular, antioxidant,³ antimicrobial,^4,5 antidepressant, antiepileptic⁶ etc. The objective of present work is to synthesize libraries of substituted indolo triazines with high purity and screen the synthesized compounds for antihyperglycemic⁷ and antimicrobial activity. Therefore, it is necessary to search for and develop potent antimicrobial and antihyperglycemic agents to meet the increasing demand.

EXPERIMENTAL

Materials and methods:

Synthetic starting material, reagents and solvents were of analytical reagent grade. The melting points were taken in open capillary tube and are uncorrected. IR spectra were recorded with KBr pellets (Shimadzu IR Affinity-1 FTIR). Proton 1 H-NMR spectra (Bruker 400 NMR spectrometer) were recorded with TMS as internal reference. A quadrupol mass spectrometer was used to record mass spectral data with (Shimadzu GC MS QP 5000). The purity of the compounds was checked by TLC on pre-coated SiO2 gel (HF254, 200 mesh) aluminium plates (E.Merck) using ethyl acetate: benzene: hexane and visualized in UV chamber. IR, 1 H-NMR, mass spectral data was used to confirm the structure.

The Synthesis of title compounds 1-(8-fluoro5H-1,2,4 triazino[5,6-b] indol-3-yl))-2-substituted benzylidene cyclohexylidene)hydrazine MC1-15 wasby the following method

General Procedure^8,9

The equimolar quantities of each (0.1 mol) of 5-fluoroindoline-2,3-dione and thiosemicarbazide.¹⁰ (0.1 mol) were taken in a beaker, to this 10% sodium hydroxide solution was added to make the solution alkaline, this was shaken and kept aside 1h. The solid 8-fluoro-5H-1,2,4 triazino [5, 6-b] indole3-thiol (1) thus obtained, was filtered, washed with water and re-crystallized from absolute ethanol. A cyclic condensed ring of 8-fluoro-5H-1,2,4 triazino [5,6-b] indole-3-thiol 1 (0.039 mol) hydrazine hydrate (0.03 mol) in ethanol was heated under reflux for 1 h. The solid thus obtained, was filtered, washed with water and recrystallized from ethanol to give 1-(8-fluoro-5H-1,2,4 triazino [5, 6-b] indol3-yl) hydrazine.²

Preparation of cyclohexanone derivatives (MC 1-15)^11,12

The 1-(8-fluoro-5H-[1,2,4] triazino [5,6-b] indol3-yl)hydrazine 2 (0.1 mol) was melted on a water bath and cyclohexanone (0.1 mol) was added to it portion wise to maintain its homogeneity. The homogeneous mixture was further heated on a water bath for 30min and kept overnight. The solid thus obtained was washed with water until neutralized and crystallized from ethanol to give 1-(8-fluoro-5H-[1,2,4] triazino [5,6-b] indol-3-yl)) cyclohexylidenehy-drazine.(3a)

A mixture of 3a (0.002 mol), treated with substituted benzaldehyde (0.002 mol) in a beaker, to this 20 ml of 10% sodium hydroxide solution was added to make the solution alkaline, this was shaken and kept aside 2 h. The solid 1-(8-fluoro5H-[1, 2, 4] triazino[5,6-b]indol-3-yl))-2-substituted benzylidene cyclohexylidene) hydrazine MC1-15 thus obtained, was filtered, washed with water and recrystallized from absolute ethanol.

Spectral data:

Compound code: MC1 (2-Hydroxy)

IR: 3561 (OH), 3373 & 3269 (NH), 3067 (ArCH), 2925 (CH2-CH), 1654 (C=N), 1622 (C=C), 1138 (C-F) NMR: 1.54-2.28 (m, 8H, CH2 of cyclohexane), 5.64 (s, 1H, OH), 6.65 (s, 1H, =CH- ), 7.10-8.06 (m, 7H, Ar-H), 8.99 (s, 1H, -NH-N=), 9.41 (s, 1H, NH of indole) Mass: C22H19FN6 O; 402 [M+]

Compound code: MC2 (3-Hydroxy)

IR: 3583 (OH), 3347 & 3273 (NH), 3020 (Ar-CH), 2922 (CH2-CH), 1643 (C=N), 1631 (C=C), 1145 (CF) NMR: 1.39-1.82 (m, 8H, CH2 of cyclohexane), 4.59 (s, 1H, OH), 6.67 (s, 1H, =CH-), 7.08-7.69 (m, 7H, Ar-H), 8.51 (s, 1H, -NH-N=), 9.54 (s, 1H, NH of indole) Mass: C22H19FN6 O; 402 [M+]

Compound code: MC3 (4-Hydroxy)

IR: 3563 (OH), 3372 & 3257 (NH), 3055 (Ar-CH), 2924 (CH2 -CH), 1636 (C=N), 1602 (C=C), 1130 (C-F) NMR: 1.46-1.92 (m, 8H, CH2 of cyclohexane), 4.73 (s, 1H, OH), 6.30 (s, 1H,=CH), 6.82-7.84 (m, 7H, Ar-H), 8.59 (s, 1H, -NH-N=), 9.10 (s, 1H, NH of indole) Mass: C22H19FN6 O; 402 [M+]

Compound code: MC4 (2-Methoxy)

IR: 3339 & 3285 (NH), 3067 (Ar-CH), 2941 (CH2-CH), 1640 (C=N), 1617 (C=C), 1129 (C-F) NMR: 1.19-2.04 (m, 8H, CH2 of cyclohexane), 2.60 (s, 3H, OCH3 ), 6.43 (s, 1 H, =CH-), 7.06-8.11 (m, 7H, Ar-H), 8.97 (s, 1H, -NH-N=), 9.46 (s,1H, NH of indole) Mass: C23H21FN6 O; 416 [M+]

Compound code: MC5 (3-Methoxy)

IR: 3319 & 3274 (NH), 3063 (Ar-CH), 2946 (CH2-CH), 1652 (C=N), 1626 (C=C), 1127 (C-F) NMR: 1.44-2.03 (m, 8H, CH2 of cyclohexane), 2.59 (s, 3H, OCH3 ), 6.13 (s, 1H, =CH-), 6.77-7.59 (m, 7H, Ar-H), 8.53 (s, 1H, -NH-N=), 9.42 (s, 1H, NH of indole) Mass: C23H21FN6 O; 416 [M+]

Compound code: MC6 (4-Methoxy)

IR: 3317 & 3256 (NH), 3003 (Ar-CH), 2921 (CH2-CH), 1641 (C=N), 1611 (C=C), 1144 (CF) NMR: 1.78-2.20 (m, 8H, CH2 of cyclohexane), 3.11 (s, 3H, OCH3 ), 5.70 (s, 1H, =CH-), 6.89-8.01 (m, 7H, Ar-H), 8.32 (s, 1H, -NH-N=), 8.86 (s, 1H, NH of indole) Mass: C23H21FN6 O; 416 [M+]

Compound code: MC7 (2-Methyl)

IR: 3317 & 3244 (NH), 3002 (Ar-CH), 2941 (CH2 -CH), 1643 (C=N), 1610 (C=C), 1140 (C-F) NMR: 1.49-2.36 (m, 8H, CH2 of cyclohexane), 3.03 (s, 3H, CH3), 6.27 (s, 1H, =CH-), 7.06-8.09 (m, 7H, Ar-H), 8.80 (s, 1H, -NH-N=), 9.42 (s, 1H, NH of indole) Mass: C23H21FN6 ; 400 [M+]

Compound code: MC8 (3-Methyl)

IR: 3349 & 3265 (NH), 3082 (Ar-CH), 2936 (CH2 - CH), 1630 (C=N), 1601 (C=C), 1143 (C-F) NMR: 1.32-2.07 (m, 8H, CH2 of cyclohexane), 2.46 (s, 3H, CH3 ), 6.52 (s, 1H, =CH-), 6.92-7.80 (m, 7H, Ar-H), 8.90 (s, 1H, -NH-N=), 9.46 (s, 1H, NH of indole) Mass: C23H21FN6 ; 400 [M+]

Compound code: MC9 (4-Methyl)

IR: 3360 & 3257 (NH), 3039 (Ar-CH), 2976 (CH2 - CH), 1645 (C=N), 1620 (C=C), 1137 (C-F) NMR: 1.43-2.10 (m, 8H, CH2 of cyclohexane), 2.68 (s, 3H, CH3 ), 6.46 (s, 1H, =CH-), 6.91-7.88 (m, 7H, Ar-H), 8.91 (s, 1H, -NH-N=), 9.56 (s, 1H, NH of indole) Mass: C23H21FN6 ; 400 [M+]

Compound code: MC10 (2-Amino)

IR: 3351 & 3270 (NH), 3043 (Ar-CH), 2967 (CH2 - CH), 1649 (C=N), 1611 (C=C), 1124 (C-F) NMR: 1.54-2.27 (m, 8H, CH2 of cyclohexane), 4.69 (s, 2H, NH2 ), 6.08 (s, 1H, =CH-), 6.93-7.99 (m, 7H, Ar-H), 8.50 (s, 1H, -NH-N=), 9.67 (s, 1H, NH of indole) Mass: C22H20FN7 ; 401 [M+]

Compound code: MC11 (3-Amino)

IR: 3315 & 3283 (NH), 3049 (Ar-CH), 2923 (CH2 - CH), 1647 (C=N), 1615 (C=C), 1144 (C-F) NMR: 1.40-1.99 (m, 8H, CH2 of cyclohexane), 4.58 (s, 2H, NH2 ), 6.16 (s, 1H, =CH-), 6.83-7.97 (m, 7H, Ar-H), 9.00 (s, 1H, -NH-N=), 9.28 (s, 1H, NH of indole) Mass: C22H20FN7 ; 401 [M+]

Compound code: MC12 (4-Amino)

IR: 3390 & 3237 (NH), 3046 (Ar-CH), 2945 (CH2 - CH), 1655 (C=N), 1602 (C=C), 1138 (C-F) NMR: 1.26-2.05 (m, 8H, CH2 of cyclohexane), 4.51 (s, 2H, NH2 ), 6.47 (s, 1H, =CH-), 7.04-8.16 (m, 7H, Ar-H), 8.90 (s, 1H, -NH-N=), 9.43 (s, 1H, NH of indole) Mass: C22H20FN7 ; 401 [M+]

Compound code: MC13 (2-Nitro)

IR: 3313 & 3272 (NH), 3050 (Ar-CH), 2949 (CH2 - CH), 1645 (C=N), 1616 (C=C), 1576 & 1334 (NO2), 1124 (C-F) NMR: 1.64-2.32 (m, 8H, CH2 of cyclohexane), 6.07 (s, 1H, =CH-), 7.02-7.94 (m, 7H, Ar-H), 8.83 (s, 1H, -NH-N=), 9.34 (s, 1H, NH of indole) Mass: C22H18FN7 O2 ; 431 [M+]

Compound code: MC14 (3-Nitro)

IR: 3331 & 3219 (NH), 3053 (Ar-CH), 2978 (CH2 - CH), 1651 (C=N), 1617 (C=C), 1573 & 1313 (NO2 ), 1148 (C-F) NMR: 1.29-1.98 (m, 8H, CH2 of cyclohexane), 6.24 (s, 1H, =CH-), 7.00-8.36 (m, 7H, Ar-H), 8.94 (s, 1H, -NH-N=), 9.60 (s, 1H, NH of indole) Mass: C22H18FN7 O2 ; 431 [M+]

Compound code: MC15 (4-Nitro)

IR: 3331 & 3257 (NH), 3022 (Ar-CH), 2931 (CH2 - CH), 1645 (C=N), 1622 (C=C), 1550 & 1339 (NO2 ), 1138 (C-F) NMR: 1.41-2.04 (m, 8H, CH2 of cyclohexane), 5.87 (s, 1H, =CH-), 7.61-8.22 (m, 7H, Ar-H), 8.79 (s, 1H, -NH-N=), 9.45 (s, 1H, NH of indole) Mass: C22H18FN7 O2 ; 431 [M+]

ANTIHYPERGLYCEMIC ACTIVITY

Streptozotocin (STZ) model^13-16

A solution of streptozotocin (60mg/kg) in 100 mm citrate buffer, pH 4.5 was prepared and calculated amount of the fresh solution was dosed to overnight fasted rats (60mg/kg) intraperitoneally. The blood sugar level was measured after 48 h by glucometer. Animals showing 200–400mg/dL were selected for antidiabetic screening.^17,18 The diabetic animals were divided into groups of six animals each. Rats of experimental group were administered a suspension of the desired test sample (prepared in 1% gum acacia) orally (100 mg/kg body weight). Controlled group animals were also fed with 1% gum acacia. The blood glucose levels were measured at 1, 2, 3, 4, 5, 6, 7 and 24 h intervals. The % fall in blood glucose from 1 to 24 h by test sample was calculated according to the area under curve (AUC) method. The average fall in AUC in experimental group compared to control group provided % antihyperglycemic activity.

Sucrose-loaded (SLM) model¹⁹

Overnight fasted male Sprague–Dawleyrats were used for sucrose-loaded experiment. Blood was collected initially and there after test compounds were given to the test group consisting of five rats by oral gavage at a dose of 100mg/kg body weight. After half an hour post-test treatment, a sucrose load of 10 gm/kg body weight was given to each rat. Blood was collected at 30, 60, 90 and 120 min post sucrose load. The % fall in blood glucose level was calculated according to the AUC method.

Antimicrobial Screening:

All the synthesized compounds were screened for antibacterial^20,21 and antifungal activities by paper disc diffusion technique. The antibacterial activity of the compounds were evaluated against four gram positive bacteria (Staphylococcus aureus ATCC 9144, Staphylococcus epidermidis ATCC 155, Micrococcus luteus ATCC 4698 and Bacillus cereus ATCC 11778) and three gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 2853 and Klebsiella pneumoniae ATCC 11298). The antifungal activities of the synthesized compounds were evaluated against two fungi (Aspergillus niger ATCC 9029and Aspergillus fumigates ATCC 2091).

Paper Disc Diffusion Technique:

The sterilized1 (autoclaved at 120°C for 30 minutes) medium (40-50°C) was inoculated (1 mL/100 mL of medium) with the suspension (105 cfu mL-1) of the microorganism (Matched to McFarland barium sulphate standard) and poured into a petri dish to give a depth of 3-4 mm. The paper impregnated with the test compounds (µg ml-1 in dimethyl formamide) was placed on the solidified medium. The plates were pre-incubated for 1h at room temperature and incubated at 37°C for 24 and 48 h for antibacterial and antifungal activities, respectively. Ciprofloxacin (Dr. Reddy’s Laboratories, Batch No: IC666E04, India) and Ketoconazole (Wuhan Shengmao Corporation, Batch No: SBML/403, China) were used as standard for antibacterial and antifungal activities, respectively.

Minimum Inhibitory Concentration :( MIC)

MIC of the compound was determined by agar streak dilution method. A stock solution of the synthesized compound (100 µg ml-1) in dimethyl formamide was prepared and graded quantities of the test compounds were incorporated in specified quantity of molten sterile agar (nutrient agar for antibacterial activity and sabouraud dextrose agar medium for antifungal activity). A specified quantity of the medium (40-50°C) containing the compound was poured into a petri dish to give a depth of 3-4 mm and allowed to solidify. Suspension of the microorganism were prepared to contain approximately 105 cfu ml-1 and applied to plates with serially diluted compounds in dimethyl formamide to be tested and incubated at 37°C for 24 h and 48 h for bacteria and fungi, respectively.

The MIC was considered to be the lowest concentration of the test substance exhibiting no visible growth of bacteria or fungi on the plate.

Statistical Analysis

Student’s t-test was used to determine a significant difference between the controls.

RESULTS AND DISCUSSION

Antihyperglycemic activity:

Among the 12 screened compounds from MC 1-15 series of compounds, MC6, MC12, MC11, MC14 and MC9reduced blood glucose level in STZ and SLM models. The other compounds such as MC1, MC4, MC2, and MC 8 displayed inactive anti hyperglycemic activity in both STZ model and SLM model, this is possibly due to its slow transformation than other highly active metabolites. A compound with fluorine substitution displayed a significant blood glucose lowering activity in both the SLM and STZ model. The compound with methoxy derivative MC6 possessing reduced blood glucose level by (68.0%) in STZ model while latter demonstrated significant activities (54.4%) in SLM model and Metformin, Glybenclamide were used as standard antidiabetic drug in both the models, the observed values are represented in Table 2.

Antibacterial screening:

Among the compounds tested, MC6 and MC12,were found to possess significant antibacterial and antifungal activity when compared to standard drug Ciprofloxacin and Ketoconazole. Compounds MC 9 displayed moderate antimicrobial activity whereas the remaining compounds showed mild activity

CONCLUSION

A library of novel triazinoindole derivatives were synthesized and the purity of the synthesized compounds checked by thin layer chromatography. The structures were elucidated by IR and NMR spectral studies. Insulin deficiency leads to various metabolic aberrations in animals like increased blood pressure level, decreased protein content, increased level of cholesterol and triglycerides were reported. It was observed that Indolo triazines exhibited good antihyperglycemic and antimicrobial activity. The screening data reveals that the presence of electron donating substituent on indole ring enhances the activity. All the synthesized compounds were screened for their antibacterial activity and few of the derivatives were found to possess significant antibacterial and antifungal activity.

In the view of the above findings research done to identify new candidates that may value in designing novelty, selectivity, less toxicity and versatile therapeutic agents who might serve as novel analogue. Therefore, because of the side effects associated with the present antidiabetic drugs, there is need to develop effective, safe and cheap drugs for diabetes management. These antihyperglycemic agents can be explored for development of antidiabetic lead molecules and further studies carried out in this direction to find out effective treatment for control of diabetes.