INTRODUCTION:

The present work has been centered upon the study of metal complexes specrophotometrically. HT(HMCPT) are very good chelating agents^1-2. Their metal complexes have been shown excellent biological activities such as anti-inflammatory, insecticidal, antibacterial and antifungal^3-7.

EXPERIMENTAL:

The ligand HMCPT has been screened for biological activity on the basis of PASS^11-12 (http://www.195.172.207.233/PASS). Computer aided program PASS provides large no. of possible biological activities with their percent activity and percent inactivity, which helps to design drug in huge chemical pharmacological space. Probable activities predicted by PASS are validating by experimental bioassay, which would have been a way to CADD.

HMCPT have been synthesized as per the standard method^8-10. The overall process has been completed in three steps. The compound has been characterized by IR studies, CHN-analysis, and m.p. determination (AR grade chemicals were used for synthesis and crystallization).

Materials and Methods –

For the synthesis of HMCPT, AR grade chemicals were used . The process is completed in three steps. The general method of synthesis has been given by the following three step reaction procedure

Step-1- Reduction of p-nitrotoluene-

0.25 mole of p-nitrotoluene, 15 gm. of NH4Cl were taken in one ltr beaker containing 400 ml H₂O and 100 ml of rectified spirit. The contents were stirred mechanically and the temp. of the reaction mixture remain between 55-60⁰C. 40gm. Zn dust was added in small lots till one hour. The resulting mix. was filtered under suction and the filterate was taken into another beaker and kept it in freezer to cool to 0⁰C.

Step-2- Diazzotization of 3-Chloro-2-Methyl Aniline-

0.2 mole of 3-Chloro-2-Methyl-Aniline was dissolved in a warm mix. of 50 ml. conc. HCl and 50 ml of H₂O in a 500 ml beaker . This mix. was kept in freezer to cool after stirring. In an another beaker 13 gm of NaNO₂ was dissolved in H₂O and it was kept in freezer to cool. The beaker containing 3-Chloro-2-Methyl-Aniline solution, was kept in an ice bath and to this NaNO₂ solution was added slowly with continuous stirring. This resulted in the formation of diazotized product, used for coupling in the next step.

Step-3- Coupling-

p-tolyl hydroxylamine prepared in step(1) was coupled with the diazotized product obtained in step(2) at 0-50⁰C to maintain the pH between 5-6 by using Na –acetate solution as buffer. When the addition was completed, the resultant mix. was crystallized with appropriate solvent. This result the formation of HMCPT in the form of yellow crystals.

Step-1 Reduction of p-nitro toluene.

Step-2 Diazzotization of 3-Chloro,2-Methyl Aniline

Step- 3 Coupling

Table 1. Physical Characteristics, M.P., C H N Values of the reagent

Molecular Formula	Melting Point (^oC)	Colour and shape of the crystal	% Analysis
			Calculated			Experimental
			C %	H %	N %	C %	H %	N %
C₁₄H₁₄N₃OCl	110	Light yellow needle	60.98	5.08	15.24	61.24	5.24	15.13

Resultant compound was subjected to four spot tests detection as described by Purohit^13-16. This compound gave positive test with all the four reagents proving that the synthesized compound is a hydroxytriazene.

To check the purity of the compound CHN-analysis and IR studies have been done. CHN data has been given in table 1. IR-data of this compound has been given in table 2, showing the characteristic peaks of (O-H str.) (C-H str. Ar) (C-H str. CH₃) (N=N str.) (N-N str.) and (C-Cl str.) vibrations.

Table 1.2 IR data of the reagent(Cm^-1)

O-H str.	C-H str. Ar	C-H str. CH₃	N=N str.	N-N str.	C-Cl str.
3618	3028	2920	1581	1454	682

Spectrophotometric Study of the Complex-

(A) Preparation of Solutions-

(i)Reagent solution-

A fresh stock solution of 1.0*10^-2 M of the reagent 3-hydroxy-3(4-methyl, phenyl)-1-(3-chloro, 2-methyl phenyl) triazene was prepared by dissolving requisite quantity of the reagent in ethanol. Dilute solution were prepared from this stock solution as and when required.

(ii)Standard solution of Copper(II)-

A 1.0*10^-2 M stock solution of Cu(II) was prepared by dissolving the requisite quantity of A.R. grade Copper sulphate and making it up to the required volume with double distilled water. It was standardized with standard 1.0*10^-2 EDTA solution at pH 2.5-3.0 using sulphosalicylic acid as an indicator. Dilute solution of different concentration were prepared from stock solution by proper dilution with double distilled water.

(iii) Solutions for pH adjustment:- (a)Tris buffer solution :- A 1.0% of tris buffer solution was prepared by dissolving 1.0 gm of the tris buffer in minimum quantity of double distilled water and then making it up to 100 ml with distilled water.

(b) Perchloric acid solution:

A 1.0% Perchloric acid solution was prepared by dissolving 1.0 ml of the Perchloric acid in minimum quantity of double distilled water and then making up to 100 ml with distilled water.

(iv) Instruments:

The spectrophotometric study have been carried out on Systronic-108 UV-VIS spectrophotometer and Systronic-324 was used for pH measurement.

(B) Selection of suitable working wavelength:

5ml (1*10^-5M) Cu(II) solution and 3ml (1*10^-4M) reagent solution was taken in 10ml volumetric flask and then make upto the mark with acetone. Absorbance of solution against its reagent blank was measured selected in a region where the absorption of Cu(II) complex was maximum. For working wavelength, maximum absorbance was found at 404 nm.

(C) Effect of pH on absorbance:

Absorbance of the solutions at various pH values containing Cu(II) and reagent solutions in the ratio of 1:6 was taken at working wavelength 404 nm against reagent blank . The optimum pH range for constant maximum absorption was selected.

(D) Composition of the Copper(II) complex:

The composition of the Cu(II) complex was determined using Job’s method , mole ratio method of Yoe and Jone’s and slope ratio method.

(a) Job’s method:

The composition of Cu(II) complex was determined at two different concentration with Job’s method . For each concentration, set of solution was prepared by varying the volume of equimolar Cu(II) and reagent solution from 0 to 6 ml. After pH adjustment, the solutions were marked (10ml) with ethanol. The absorbance of solution was measured at suitable working wavelength against reagent blank. The second set of this method differed from the first set only in the concentration used. By this method the composition was found to be 1:2 [Cu:R].

(b) Mole ratio method of Yoe and Jone’s:

In this method Cu(II) concentration was kept constant and reagent concentration was varied. A series of solutions having Cu(II) to regent ratio 1:0.4 to 1:10 were prepared with maintaining the pH of constant absorbance. Absorbance of each solution of a set was measured at working wavelength against the reagent blank . By this method the composition was found 1:2 [Cu:R].

(E) Beer’s Law:

A set of solution, having to ligand ratio 1:10 was prepared . The studies were performed under optimum condition of pH, concentration and solvent at corresponding working wavelength . The absorbance was measured for the complex against the reagent blank. The results are shown in Table 2.

(F) Sandell’s sensitivity:

The molar absorptivity of the Cu(II) complex was calculated from the Beer’s law graph and it was found to be €= 51,875 L/mol.cm. The value thus obtained was used for determining Sandell’s sensitivity of the complex that are obtained 1.22 ng/cm². This value shows that the method used is quite sensitive and satisfactory for the determination of Cu(II).

Prediction of the spectrum of biological activities:-

Brief description of PASS:-

The computer system for the prediction of the spectrum of biological activity according to the structural formula (PASS) was used to predict the spectrum of biological activity of the test selection compounds. The functioning of the PASS system is based on a training selection of chemical substances with known biological activities. The PASS system involves the following basic elements: description of the chemical structure, representation of the biological activity, training selection, and mathematical search algorithm for the structure-activity relationship. The biological activity in the PASS system is predicted qualitatively (presence or absence). The general number of the predicted activities (the spectrum of activity) involves more than 400 pharmacological effects and mechanisms of action, as well as carcinogenicity, probabilities that a definite activity would be or would not be exhibited (Pa and Pi, respectively) are calculated for each predicted type of activity with the use of a specially developed mathematical algorithm.

Activity prediction:-

The biological activity spectra of the 3-hydroxy-3-(4-methyl, phenyl)-1-(3- chloro, 2-methyl phenyl) triazene were obtained by PASS software. The predictions were carried out on the basis of analysis of training set containing about 10000 drugs and biologically active compounds.

This set consider as reference compounds for known chemical compounds as well as different biological activities. Percent activity (Pa) and inactivity (Pi) of compound have been represented in Table- 3.

Table 3: Predictions of Percent activity (Pa) and inactivity (Pi) of compound

0.805	0.004	Mutagenic
0.786	0.004	Mutagenic, Salmonella
0.744	0.018	Glutamyl endopeptidase II inhibitor
0.719	0.046	Neutrophilic dermatosis (Sweet's syndrome)
0.670	0.009	Carcinogenic
0.686	0.026	5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase inhibitor
0.647	0.003	Papillary necrosis
0.668	0.034	Occult bleeding
0.673	0.060	CYP2J substrate
0.669	0.074	Aspulvinone dimethylallyltransferase inhibitor
0.598	0.004	Thiosulfate dehydrogenase inhibitor
0.617	0.027	Pseudoporphyria
0.600	0.011	Carcinogenic, rat, male
0.626	0.037	Omptin inhibitor
0.636	0.057	Polyporopepsin inhibitor
0.628	0.052	Nail discoloration
0.645	0.074	Testosterone 17beta-dehydrogenase (NADP+) inhibitor
0.579	0.011	Plastoquinol-plastocyanin reductase inhibitor

RESULT AND DISCUSSION:-

As described in the Table-2, 3-hydroxy-3-(4-methyl, phenyl )-1-(3- chloro, 2-methyl phenyl) triazene forms complex in the ratio of 1:2 . The conditional stability constants have also been given in the table, determined by using two different methods. Hydroxytriazenes act as, bidentate ligand and in the present case, the reagent has been found to form a 1:2 complex with 3-hydroxy-3-(4-methyl, phenyl )-1-(3- chloro, 2-methyl phenyl) triazene, which indicates penta coordinated Copper(II) complex with a probable geometry being trigonal bipyramidal.

Biological activity spectra were predicted for title compound with PASS computer program. The result of prediction is presented in table 4 as the list of activities with appropriate Pa and Pi (Pa-Pi) > 0.PASS is based on a robust analysis of structure–activity relationship in a heterogeneous training set currently including about sixty thousand of biologically active compounds from different chemical series with about four thousand five hundred types of biological activity . It can be seen from the results of PASS that most probable activities are Hematotoxic, Antineurotoxic, Anti inflammatory and Analgesic.

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Received on 01.04.2014 Modified on 25.04.2014

Asian J. Research Chem. 7(5): May 2014; Page 487-491