Synthesis , Characterization and Spectral Study of Zinc Complex with Gliclazide ( N-( hexahydrocyclopenta [ c ] pyrrol-2 ( 1 H )-ylcarbamoyl )-4-methylbenzenesulfonamide ) and its Hypoglycemic Activity

Metal ion is required for many critical functions in humans. Scarcity of some metal ions can leads to disease1. Well – known example can leads to pernicious anaemia resulting from iron deficiency; growth retardation arising from insufficient dietary zinc, and heart disease in infants owing to copper deficiency. The ability to recognize, to understand at the molecular level, and to the diseases caused by inadequate metalion function constitutes an important aspect of medicinal bioinorganic chemistry. Understanding the biochemistry and molecular biology of natural detoxification mechanisms and ORIENTAL JOURNAL OF CHEMISTRY


INTRODUCTION
Metal ion is required for many critical functions in humans.Scarcity of some metal ions can leads to disease 1 .Well -known example can leads to pernicious anaemia resulting from iron deficiency; growth retardation arising from insufficient dietary zinc, and heart disease in infants owing to copper deficiency.The ability to recognize, to understand at the molecular level, and to the diseases caused by inadequate metal-ion function constitutes an important aspect of medicinal bioinorganic chemistry.Understanding the biochemistry and molecular biology of natural detoxification mechanisms and designing and applying ion -specific chelating agents to treat metal over -loads are the two components of a second major aspect of the new science that is evolving at the interface of bioinorganic chemistry and medicine.
Diabetes is a deceptive disease and if not detected in early stage may cause even death.It is considered hereditary but actual genetic disorder is still a mystery.Several million people are suffering from this disease all over the world [Sadilot and Phatak, 2 ; Bloomgarden 3 , ; Sanger and Thompson, 4 .Zinc-insulin was discovered as early as in 1921 and later it proved to be a very efficacious medicine in the treatment of diabetes mellitus.To avoid the daily pricks of hypodermic syringe, oral hypoglycemic agents were discovered which has revolutionized the treatment of diabetes .It is worthwhile to mention here that the majority of the essential metallic elements of biological importance are transition metals ,whose ability to form coordination complexes and chelates is the characteristic aspects of their chemistry.
In recent years much attention is given to the use of sulphonylureas because of their high complexing nature with essential metals .Sulphonylureas are effective for non-insulin dependent diabetes mellitus, [Sadilot and Phatak 2 , Bioomgarden, 3 Sanger and Thompson, 4 .These compounds are completely absorbed on oral administration.They are metabolized by liver and are excreted predominantly through urine.
Complexation of sulphonylureas with lighter transition metals has been studied in detail by yoshinaga and yamamotto, 5-6 Iqbal.atel. 7-10and other author synthesis the complexes of various metals with compound 11-18 . .It is interesting to have an insight in to the synthesis of zinc complex with gliclazide and to diagnose various structural aspects of the isolated complex and compare its hypoglycemic activity to parent drug.
Here the synthesis and characterization of zinc with gliclazide have been described.

Ligand-Metal Ratio
Pure Gliclazide m.p. 180°C (Lit.179.5-180.5),0.005 M, were diluted to 100 ml as required and titrated conductometrically against zinc chloride at 30±1°C.Results were plotted in the form of a graph which indicate ligand metal ratio as 2:1 (L 2 M).Formation of 2:1 (L 2 M) ratio was further confirmed by Job's method 19 (Table-1 fig.1a,b) of continuous variation as modified by Turner and Anderson 20 spectrophotometric studies were conducted using absorbance as index property, from these values the stability constant (log k) and free energy change (-F), were also calculated (Irving and Rossotti ) [21][22] .

Synthesis of Complexes
The chemicals used in this synthesis were all of analar grade Hi-media.A weighed quantity of Gliclazide (2 mol) (supplied by Zim laboratory Nagpur) was dissolved separately in minimum quantity of 90% ethanol.The Zinc chloride solution was prepared by dissolving (1 mol) separately in the same solvent.Ligand solution was added slowly with stirring into the metallic salt solution at room temperature; maintain the pH between 6.0 to 6.5 by adding dilute NaOH solution.On refluxing the mixture for 3 hours and on cooling the complexes separated out.Which were filtered off, washed well with ethanol and finally dried in vacuum and weighed.

X-ray diffraction studies
X-ray diffraction studies also confirm the complex and formation due to new bonds [26][27][28][29][30][31][32][33] .The number of peaks in gliclazide are 22 while (GLZ) 2 Zn are 11 (Fig- 3) .Thus indicating the something new formation i.e complex formed is a well kit one.All the reflections present are new ones and the patterns are fairly strong.On comparing the pattern obtained with available literature.It is evident that its pattern  is not in good agreement with available information and thus confirms the formation of totally new complexes.The X-ray pattern have been indexed by using computer software(FPSUIT 2.0V) and applying interactive trial and error method keeping in mind the characteristics of the various symmetry system, till a good fit was obtained between the observed and the calculated Sin 2 è value.The unit cell parameters were calculated from the indexed data, from cell data and crystal lattice parameters of Zn(II) indicates complex attributed to orthorhombic crystal system.The particle size is 7.28 nm which is calculated from x-ray line broadening using the Scherrer formula Dhkl =κλ/βhkl cosθ, where D is the particle diameter in ångstroms, κ is a coefficient and is equal to 0.89 here, λ is the half-maximum line width, and ë is the wavelength of x-rays, porosity is 6.84 calculated by formula and volume of the unit cell is 14065 is calculated by Volume(Å), = abc where a, b and c are lattice parameters .Density = is found 0.050 g/ cm 3 .Space group is Pmmm and α=90°, β=90°, γ =90°, crystal system-orthorhombic.Cullity 34 (1978), Duval 35 Bragg and Bragg 36 , Guinier 37

Hypoglycemic Studies
Pharmacotherapy includes the treatment of the sick with drugs and therefore is of prime importance in practice of medicine.It is fundamental to the health-economy of the people.A compound or a complex which is to be recommended as a drug of utility must be capable of easy absorption and excretion.It is also essential that neither the absence itself nor the metabolic products thereof should exercise toxicity or any adverse side effect to the patient.

1.
Where necessary such tests should be carried out on animals as rats, rabbits and dogs.
When a substance has given satisfactory results for the aforesaid animals then only it may be tried on monkeys and men. 2.
In present study we analyze the hypoglycemic activity of pure drug and its complexes with zinc on albino rats.

Animal care and handling
The anti-diabetic activity was carried out on Wistar albino rats of 4 months of both sexes, weighing between 140 to 200 gm.They were provided from Sapience Bio-analytical Research Laboratory, Bhopal, (M.P.).The animals were acclimatized to the standard laboratory conditions in cross ventilated animal house at temperature 25±2°C relative humidity 44 -56% and light and dark cycles of 12:12 hours, fed with standard pallet diet and water ad libitum during experiment.The experiment was approved by the institutional ethics committee and as per CPCSEA guidelines (approval no.1413/PO/a/11/CPCSEA).

Chemicals
Alloxan monohydrate was purchased from Central Drug House (P) LTD.Gliclazide was provided as gift sample by Zim laboratories, Nagpur.][43] The diabetes was induced by a single intraperitoenal injection of a freshly prepared solution of Alloxan monohydrate (120 mg/kg b.w.).Hyperglycemia was confirmed on the third day of Alloxan-injection.Rats with moderate diabetes having hyperglycemia (with blood glucose above150 mg/dl) were taken for the experiment.

Design
In the experiment, a total of 24 rats were used.The rats were divided into 5 groups.Comprising of 6 animals in each group as follows:-

Group I
Normal control rats received 1ml/100gm of 0.5% CMC (carboxy methyl cellulose) using an intragastric tube for 4 days.

Group II
Negative control rats received Alloxan 120g/kg, i.p. for inducing diabetes.

Sample collection
At the end of study(7 th day), blood samples were collected through tail vein and blood glucose levels were estimated using an electronic glucometer and result is given in table 2, 3.

DISCUSSION
For supporting the proposed structure of gliclazide-zinc complex , initially Job , s method of continuous variation as modified by Turner and Anderson was conducted which indicate 2:1 ligand:metal ratio of the complex , moreover stability constant and free energy change was also calculated .Analytical data agrees to the molecular formula (C 15 H 20 N 3 O 3 S) 2 Zn , (L 2 M) .
For determining the proposed structure on the basis of stoichiometry and analysis of the complexes.Advance spectroscopic methods like IR was conducted which suggest the coordination of metal atom with enolic oxygen of the carbonyl group on one side and oxygen of the sulphonyl group from the other side.A detailed study of X-Ray also supports the complexes formation and the various values like particle size , porosity , volume of unit cell , density as well as crystal system was evaluated and discussed .Moreover looking to the higher electronegativity of oxygen as compared to nitrogen and to enolization is strongly supported.Zinc complex of gliclazide was evaluated for their hypoglycemic effect on experimental rats using alloxan induced diabetes model.The effect of zinc complex of gliclazide on blood glucose level at 3 rd and 7 th day of alloxan induced diabetes in rats shown in table-3, 4 respectively.So from table we can conclude that zinc complex of gliclazide have more hypoglycemic activity as compared to parent drug.These interesting observations on metalcomplex of oral sulphonylureas used as antidiabetic agents for lowering blood sugar concentration may likely substantiate the use of these complexes after extensive clinical studies.