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

Introduction

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  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; Bloomgarden³, ; 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 Phatak²,  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.at el. ^7-10 and 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.

EXPERIMENT

Ligand-Metal Ratio

a)      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 (L₂M).

b)      Formation of 2:1 (L₂M) ratio was further confirmed by Job’s method¹⁹(Table-1 fig. 1a,b)  of continuous variation as modified by Turner and Anderson²⁰spectrophotometric studies were conducted using absorbance as index property, from these values the stability constant (log k) and free energy change (-DF), were also calculated (Irving and Rossotti)^21-22 .

 

Table-1  Gliclazide with zinc  Click here to View table

Fig.1a, Fig.1b  Click here to View figure

 

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.

RESULT AND DISCUSSION

Physico-chemical Characterstics of “Glicazide-zinc complex

Molecular formula (C₁₅H₂₁N₃O₃S)₂ Zn, Mol.wt: 443.92; Colour:white;6 Yield:53.16%;       m.p86:1^oC, –ΔF: 16.00; LogK: 10.75; Anal. Calcd (%) for (C₁₅H₂₁N₃O₃S)₂ Zn_, : C,51.03; H,6.03; N,11.64;  S,9.01;  Zn,6.0; Found (%): C,50.83; H, 5.63; N,11.86;  S,9.03; Zn,8.74.

Infra-red Spectra Studies

The IR spectra (Fig 2) of ligand and isolated complex  was recorded within the range 4000-400 cm^-1. Assignments of the infrared spectral bands are based on literature IR spectrum shows important bands due to n(M-O) 400-600 cm^-1, n(Ar-S) 700-800 cm^-1,     n(-S-N) 1085±20 cm^-1, n(SO₂-N) 1120±20 cm^-1, n(C-N) 1210±20 cm^-1, n(S=O)       1340±20 cm^-1, n(C=O) 1710 cm^-1( present only in pure drug and variation in complex),1600±20 cm-¹vs, (C-H Stretch)2800-2900cm^-1 ,v(Ar-H) 3150±20 cm-¹ ,n(NH-stretch) 3274±20 cm^-1.The proposed structure (I) for the isolated complex is also supported by IR absorption , C.N.Rao²³. , Bellamy ²⁴,and  Weissberger²⁵.

 

Fig. 2. Ir spectra of gliclazide-zinc complex  Click here to View figure

 

X-RAY DIFFRACTION STUDIES

X-ray diffraction studies also confirm the complex and formation due to new bonds ^26-33. The number of peaks in gliclazide are 22 while (GLZ)₂Zn 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 Sin²θ 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, β is the half-maximum line width, and λ 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 =  weight / volume is found 0.050 g/cm³. Space group is Pmmm and α=90°, β=90°, γ =90°, crystal system- orthorhombic. Cullity³⁴(1978), Duval³⁵    Bragg and Bragg³⁶ , Guinier³⁷(1952), Henry et al³⁸, (1951),  Kidwai³⁹(2011).

 

Fig.3: X-ray diffactrogram of gliclazide-zinc complex  Click here to View figure

 

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. All other chemicals used for this study were of analytical grade.

Induction of diabetes by alloxan ^41-43

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.

Experimental 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.

Group III: Rats received gliclazide (2mg/kg, p.o.) for 4 days and alloxan 120g/kg, i.p.  on 1^st  day.

Group IV Rats received gliclazide-zinc complex, (2mg/kg, p.o.) once daily for 4 days and Alloxan 120g/kg, i.p.  on 1^st day.

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.

 

Table.2. HYPOGLYCEMIC ACTIVITY OF GLICLAZIDE AND ITS COMPLEXES AT 3th DAY(mg/dl)  Click here to View table

Table.3. HYPOGLYCEMIC ACTIVITY OF GLICLAZIDE AND ITS COMPLEXES AT 7th DAY(mg/dl)  Click here to View table

 

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 (C₁₅H₂₀N₃O₃S)₂Zn , (L₂M) .

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 metal-complex of oral sulphonylureas used as antidiabetic agents for lowering blood sugar concentration may likely substantiate the use of these complexes after extensive clinical studies.

 

Structure (I)  Click here to View figure

 

ACKNOWLEDGEMENT

The author is thankful to the principal of Saifia Science College, Bhopal India, Principal of Crescent College of Technology, Bhopal for providing all necessary facilities and IIT, Bombay ,P.U Chandigarh, India for providing IR spectra  and X-Ray data.

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