A comparative study of central nervous system depressant effects of some of the disubstituted piperazine compounds-part INK Dadkar, Vaishali N Dadkar, UK Sheth
Department of Pharmacology and Pharmacology Research Unit C.S.I.R., Seth G. S. Medical College, Parel, Bombay 400 012, India
Comparison of central nervous system depressant activity of R-1230, R-1945 and R-1946 (disubstituted piperazine derivatives) employing various pharmacological tests revealed that R-1230 (Orthomethoxyphenyl derivative) and R-1946 (Parafluorophenyl derivative) were more potent than R-1945 (Orthof luorophenyl derivative) as CNS depressants and the CNS depression produced by these agents could be classified pharmacologically in same group as major tranquillizers like chlorpromazine. It also signified, that presence of methoxy or fluoro group within the drug molecule does not necessarily determine the activity but the position o f these groups in drug molecule is mainly responsible for determining degree of CNS depressant activity exerted by the compound.
After the preliminary pharmacological evaluation of various members of disubstituted piperazine derivative (Structure activity relationship has been described by Dadkar et al  ) , three compounds were selected for more detailed study-Compound R-1230 pos sessing pronounced adrenolytic, hypotensive and CNS depressant activity, R-1945 possessing pronounced hypotensive and adrenolytic activity but poor CNS depressant activity and R-1946 possessing pronounced CNS depressant activity with hypotensive effects but poor adrenolytic activity.[Figure 1].
The present study was undertaken to correlate and compare central nervous system effects of these three compounds with their molecular structure.
For study of CNS depressant action of these compounds male albino mice weighing 20-25 gm were selected. Hydrochlorides of R:.1230, R-1945 and R-1946 were employed and vehicle was distilled water. When concentration of solution employed was more than 20 mg/ml, 1%gum acacia was used as vehicle.
Acute toxicity studies in mice
(LO 50 determination)
Acute toxicity of test compounds in mice (20-25 gm) was determined by the i.p. route of administration. Six doses ranging from 10-800 mg/kg were administered to group of 6 mice; 1 % gum acacia was used as a vehicle. The group treated with vehicle alone served as control. Approximate LD 50 was determined by recording the percentage mortality registered during a 72 hr. observation period at each dose level by using Litchfield-Wilcoxon's probit method. 
Motor activity in mice
Motor activity in mice was evaluated quantitatively by the photocell method of Dews  using Actophotometer (Metro Industry). Six mice were used in each group and 10 minutes counts were registered at ˝, 1, 1˝, 2, 3 and 4 hrs. after the treatment with test compounds (R-1230:5 mg/kg i.p., R-1945:10 mg/kg i.p. and R-1946:5 mg/kg i.p.). Mice treated with saline served as control. CPZ was used as the reference compound.
Dose response studies were done at the time of peak drug action. Log dose response curves were plotted and the ED 50 value was determined from the calculated regression line.
The effect on forced co-ordinated motor activity was studied on rotardo.  Percentage of mice falling off the rotarod (12 revolutions per min) at the peak drug effect was computed and ED 50 values were determined.
Effect on d-amphetamine induced hyperactivity
The effects of various doses of test compounds (R-1230:5.0 and 10.0 mg/kg i.p., R-1945:10.0, 15.0 and 20.0 mg/kg i.p. and R-1946:5.0, 10.0 and 15.0 mg/kg i.p.) given 1 hr. prior to amphetamine (4.5 mg/kg i.p.) were studied by using the Actophotometer. Group treated with saline served as control. Activity of each group (6 mice/group) was recorded for 10 minutes, at intervals of half an hour after the administration of amphetamine.
Effect on toxicity of d-amphetamine or aggregated mice
The effect of 1 hr. prior treatment of different doses of R-1230: 1, 2, 5 and 10 mg/kg, R-1945:4, 6, 8 and 10 mg,/kg and R-1946:1, 2.5, 5 and 10mg/kg on amphetamine (14.5 mg/kg i.p. 100;; mortality in control) toxicity in aggregated mice was determined by the method described by Burn and Hobbs.  Deaths were recorded after 24 hours and ED 50 was calculated by a method of Litchfield and Wilcoxon. 
Effect on conditioned avoidance response (CAR)
The effect on CAR was studied in male albino rats (130-160 gm) employing the method of Cook and Weidley.  The ED50 value for each test compound was determined using the LitchfieldWilcoxon's probit method.  CPZ was used as the reference compound.
LD 50 determination in mice Approximate LD 50 of R-1230 was 250 mg/kg i.p., it was 500 mg/kg i.p. for compound R-1945 and 400 mg/kg i.p. for R-1946.
All the three compounds depressed locomotor activity in mice and action was seen within half an hour.. Peak inhibitory effect was seen at 1 hr. for all compounds (R-1230 at dose of 5 mg/kg i.p., R-1245 at 10 mg/kg i.p. and R-1946 at 5 mg/kg i.p.) and action largely disappeared by 4 hours.
The ED 50 was found to be 2.2 mg/kg for R-1230, 6.0 mg/kg for R-1945, 1.95 mg/kg for R-1946 and 1 mg/kg for chlorpromazine, as determined from the regression line [Figure 2].
All the three compounds, at the various doses studied, reduced the induced activity in mice. With R-1230 peak drug effect was observed at 1 hr. and it lasted for 3-4 hrs. With R-1945, peak effect was evident at 2 hrs. and disappeared by 3 hrs. except for higher doses when effect lasted for more than 4 hrs. and with R-1946, effects were similar to those of R-1230.
The ED 50 of R-1230 was found to be 12.0 (9.5-15.0) mg/kg i.p., it was 25.0 (16.7-37.7) mg/kg i.p. for R-1945 and 28.0 (22.1-38.0) mg/kg i.p. for R-1946 (95% confidence limits). Evaluation of the results by the method of Kinnard and Carr  revealed that the activity ratio lines for R-1230 and R-1946 ran parallel to each other fairly closely. Actophotometer studies demonstrated a greater degree of depression at comparable points on the two lines as compared to the rotarod method [Figure 3],[Figure 4].
All the three compounds showed a marked inhibition of amphetamine induced hyperactivity. Inhibition was evident with R-1230 at a dose of 5 mg/kg i.p. and almost complete inhibition was seen at a dose of 10 mg/kg i.p. Comparable doses of R-1945 and R-1946 were 10.0 and 20.0 mg/kg and 10.0 and 15.0 mg/kg respectively.
Effect on toxicity of d-amphetamine on aggregated mice [Table 2]
All the compounds studied greatly attenuated the increased toxicity of amphetamine for grouped mice. The ED 50 for R-1230 was 2.6 (1.53-4.42) mg/kg, 6.0 (4.28-7.2) mg/kg for R-1945 and 1.7 (1.0-2.89) mg/kg for R-1946. Complete protection (100%) was achieved at 10 mg/kg i.p. dose for all compounds.
R-1230 in a dose of 8.0 mg/kg and R1946 in a dose of 10.0 mg/kg inhibited avoidance response (80% inhibition) without any effect on the ability of the rats to escape. The action commenced at 30 minutes after injection and continued for 3 to 4 hours. A comparable degree of inhibition was achieved with R-1945 in a dose of 25.0 mg/kg, its action commenced at 30 min and continued for 2 to 3 hours. No ataxia was evident at these dose levels of test compounds.
The ED 50 values of R-1230, R-1945 and R-1946 were 2.83 (1.55-5.2) mg/kg, 18.0 (15-22) mg/kg and 5.7 (4.2-6.7) mg/kg respectively (95% confidence limits). ED 50 of chlorpromazine was 4.7 (3.98-6.45) mg/kg (95% confidence limits) [Figure 5].
Structurally compounds R-1230, R-1945 and R-1946 resemble reserpine and phenothiazines which are the agents used in psychiatric disorders. Like reserpine, these compounds possess trimethoxy group and like phenothiazines, they have a piperazine group in their chemical structure. Since trimethoxy group is associated with CNS depressant activity  , the test compounds were assessed for CNS depressant activity using chlorpromazine as a reference compound.
Effect of compounds on orientation hyperactivity, a form of the so called unlearned reactivity  measured by actophotometer studies. This orientation hyperactivity is more affected by rauwolfia alkaloids and phenothiazines than by minor tranquillizers like meprobamate.  It was evident that compounds R-1230 and R-1946 caused inhibition of motor activity almost comparable to that of chlorpromazine, their ED 50 values being almost of the same magnitude as that of chlorpromazine. R-1945 required much larger doses to produce similar inhibition.
Inhibition of motor activity as assessed by rotarod studies is affected to a greater extent by hypnotic-sedatives (like barbiturates) and minor tranquillizers (like mephrobamate) as compared to the major tranquillizers like chlorpromazine.  In this study, it was revealed that doses of R1230, R-1945 and R-1946 required to produce 50% diminution of the animal's performance on a rotarod were much greater than those required for inhibition of orientation activity revealed by actophotometer studies. Less doses were required for R-1230 and R-1946 as compared to R-1945.
It has also been shown that major tranquillizers are specific depressants of CAR while minor tranquillizers and hypnotics suppress this response only in larger atonic doses.  ED 50 values of R-1230 and R-1946 in suppressing the CAR were more or less similar to that of chlorpromazine, while R-1945 had an ED 50 value several fold greater than these compounds. So compounds R-1230 and R1N6 appeared more potent than R-1945 as CNS depressant and the character of CNS depression induced by these compounds could be classified pharmacologically in same group as the major tranquillizers.
These compounds in the doses tested could inhibit amphetamine induced hyperactivity, R-1230, R-1946 being more potent inhibitors than R-1945.
A significant degree of protection was provided by R-1230 and R-1946 against aggregation toxicity in amphetamine treated mice. This protective effect and reduction in mortality is observed selectively with major tranquillizers and not with minor tranquillizers.  Compound R-1945 required larger doses to exert an equivalent action to the other two compounds. As far as protection against amphetamine induced aggregation toxicity is concerned, Moore  has reported that chlorpromazine and phenoxybenzamine could afford protection; with former, no significant alteration with brain catecholamines was seen while with latter, no alteration in central excitatory effect of amphetamine was observed. Thus it appears that both central and peripheral mechanisms may be involved in causation of amphetamine toxicity and the test compounds we have studied exert central activity like chlorpromazine and possibly some peripheral activity like phenoxybenzamine.
Since it was shown that R-1230 (Orthomethoxyphenyl derivative) and R-1946 (Parafluorophenyl derivative) were more potent CNS depressants than R-1945 (Orthofluorophenyl derivative) it signifies that presence of methoxy or fluoro group within the drug molecule does not determine the activity but the position of these groups in drug molecule is responsible for determining the degree of CNS depressant activity exerted by the compound.[Table 1]
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]