A MAJOR APPROACH- 2-CHLOROPYRIDINE-5-TRIFLUOROMETHYL DERIVATIVE AS ANTIAMNESIC AGENTS

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3.3.1 PASS PREDICTION-RESULTS-
The PASS predictions can be interpreted and used in a flexible manner—(i) only activities with Pa > Pi are considered as possible for a particular compound. (ii) If Pa > 0.7—the chance to find the activity experimentally is high. But, in many cases the compound may occur to be a close analogue of known pharmaceutical agents. (iii) If 0.5 < Pa < 0.7—the chance to find the activity experimentally is less, but the compound is probably not so similar to known pharmaceutical agents.(iv) If Pa < 0.5—the chance to find the activity experimentally is even less, but the chance to find a structurally new compound, that is, NCEs increases33.

COMPOUND CODE & No.

Pa

Pi

RST-1, (25)

0.478

0.015

RST-2, (26)

0.482

0.106

RST-3, (27)

0.600

0.055

RST-4, (28)

0.592

0.058

Table 4:The probabilities of being active for nootropic activity (Pa) of the synthesized compounds (25-28) on the basis of PASS prediction.

PASS prediction results showed that for all the synthesized compounds (25-28) the probability of being active was observed in the range of 0.600 > Pa > 0.478 for nootropic activity (table 4). This showed that for compounds (27,28) with values 0.7 < Pa > 0.5 the chance of finding the activity experimentally is less but the chance of finding a structurally new compound was greater. Compound (25, 26) is having Pa value less than 0.5, so the chance of finding the activity experimentally is even lesser but the chance of finding a structurally new compound is maximum.

3.4 PHARMACOLOGICAL EVALUATION

3.4.1 ANTIAMNESIC ACTIVITY34

ELEVATED PLUS MAZE
The test measures the transfer latency (TL) i.e. the time in which the mouse moves from open arm to the enclosed arm.TL was recorded on the first day. If the animal did not enter into one of the covered arms within 90 s, it was gently pushed into one of the two covered arms and the TL was assigned as 90 s. The mouse was allowed to explore the maze for 10 s and then returned to its home cage. Immediately after the training, the animals were administered the test compounds intraperitonially (i.p.). On the second day i.e. 24 h after the first exposure, TL was again noted. The decrease in TL was taken as index of antiamnesic activity.The results are expressed as % retention (Mean ±S.E.M) calculated as:

     TL on 1st day - TL on 2nd day   Χ 100
      -----------------------------------
                   TL on 1st day

3.5 BIOCHEMICAL EVALUATION
The biochemical study to be carried out in two phases, the estimation of acetylcholinesterase activity and IC50 and second is the estimation of protein Content.

3.5.1 ASSAY OF ACETYLCHOLINESTERASE
The activity of acetylcholinesterase to be determined in the brain homogenate according to the Ellman et al (1961) method35.

Principle
The substrate used in the assay system is acetylthiocholine iodide, the ester of thiocholine and acetic acid. The substrate, acetylthiocholine is hydrolyzed into thiocholine and acetate by the enzyme AChE. Thiocholine forms mercaptan, which reacts with the oxidizing agent 5, 5’-dithio-bis-2-nitrobenzoic acid (DTNB) to form 5-thio-2-nitrobenzoate, which has a maximum absorption at 412 nm. Thus the activity of AChE can be measured by following an increase in absorbance at 412 nm.


Reagents
·         0.1 M Sodium phosphate buffer (pH 8.0)
·         10 mM DTNB (Ellman’s reagent)
·         14.9 mM Acetylthiocholine iodide

Calculations
AChE activity to be calculated using molar extinction coefficient of 5-thio-2-nitrobenzoic acid (14.15 ´103 M-1cm-1).  The results to be expressed as nmols of acetylthiocholine iodide hydrolyzed/min/mg protein.

Change in absorbance per minute            Volume of assay
     ------------------------       ?      ------------------------
             14150                         mg of protein by protein estimation

3.5.2 ESTIMATION OF PROTEIN CONTENT
The protein content to be estimated according to the method of Lowry36et al, 1951.

Principle37, 38
This method is based on the colour reactions of amino acids tryptophan and tyrosine with the Folin’s phenol reagent. These amino acids react with phosphomolybdic acid and phosphotungstic acid (present in Folin’s reagent) to give blue colour, which is estimated colourimetrically. This colour is the result of reduction of phosphomolybdic acid and phosphotungstic acid and biuret reaction of proteins with Cu2+ ions in alkaline medium.

Reagents
·         Reagent A: 1 % (w/v) Copper sulphate solution.
·         Reagent B: 2 % (w/v) Sodium potassium tartarate.
·         Reagent C: 2 % (w/v) Sodium carbonate in 0.1 N Sodium hydroxide.
·         Lowry’s reagent: It was prepared just before use by mixing reagents A, B and C in the ratio of 1:1:98.
·         Folin-Ciocalteau reagent: It was prepared fresh by diluting the commercial 2N Folin’s reagent with double distilled water (1:1, v/v).
·         Standard bovine serum albumin (BSA) solution (1 mg/ml).

Calculations
The protein concentration was calculated from the standard curve made by taking different concentrations of the BSA standard.                 

 Volume of homogenate X sample OD        X
   ------------------------------       -------    = mg/ml protein
           0.372 (Standard)                    1000 

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