Antiparkinsonian activity and behavioural effects of newer quinazolinones

Pharmacological Research Communications. Vol. 19, No. 9, 1987

ANTIPARKINSONIAN ACTIVITY AND BEHAVIOURAL EFFECTS OF NEWER QUINAZOLINONES*

Vijai K. Srivastava, G. Palit, A.K. Agarwal and K. Shanker**

Department of Pharmacology and Therapeutics, King George's Medical College, Lucknow - 226003,

INDIA.

Received in final form 2 1 September 1987

Sixteen new compounds P-methylamino substituted phenyl-3-substituted

anilino 4(3H) quinazolinong (3-18) were prepared. All the compounds were

evaluated for their antiparkinsonian activity and compared with bromo-

criptine. Compounds lo,15 and 18 showed better activity. These compounds

also bind with the dopamine receptors in striatal membrane preparations

of rat brain. Key Mords : Antiparkinsonian; Behavioural; Quinazolinones.

Introduction :

Quinazolinones, possess antiparkinsonian activity (Parmar and

Singh, 1979). Our earlier studies have shown that incorporation of bulky

groups in quinazolinone nucleus at position-2 is beneficial for activity

(Kumar et al, 1982; Srivastava et al, 1986). Thus with a view to gain

further insight into the antiparkinsonian activity of quinazolinone

congeners, we have synthesized-2-methyl amino substituted phenyl-3-

substituted anilino 4(3H) quinazolinones which contain aryl amino groups

at position-3 of quinazolinone and bulky amino groups linked via a ----_---------------------------------------------------------------------

* Part of Ph.D. Thesis of Vijai K. Srivastava; ** For correspondence.

0031-6989/87/090617-12/$03.00/O 0 1987 The Italian Pharmacological Society

618 Pharmacological Research Communicatrons. Vol. 19. No 9, 1987

methylene bridge at position-Z of quinazolinone nucleus. The compounds

were evaluated for their antiparkinsonian activity against tremor,

rigidity, hypokinesia and catatonia.Theywerecompared with bromocriptine.

The mode of action of active compounds was studied using dopamine

receptor binding technique.

Materials and Methods

The compounds were synthesized and characterized by their

elemental analysis, infra red spectra, nuclear magnetic spectra and

mass spectra. The compounds were checked for their purity by thin layer

chromatography (TLC) on silica gel G.

2-Bromomethyl-3-(substituted anilinoj-4(3H) quinazolinone(l-2)

To 2-methyl-3tsubstituted anilinol-4(3H) quinazolinone (0.01

mole), prepared from benzoxamine and substituted phenyl hydrazine, was

added bromine (0.01 mole) portionwise in acetic acid (40 ml). The reaction

mixture was refluxed for 4-5 hours. It was poured into NaOH solution,

a solid separated out which was recrystallized from acetic acid/water.

Compound No. 1 (Table I) molecular formula C15H12N30Br requires : %C 54.54,

XH 3.63, %N 12.72 Found %C 54.50, %H 3.81, XN 12.68.

IR(KBr) 1680 cm-' C = 0 Cyclic, 3400 cm-l -NH, 3100 cm-l Arc-H.

P.m.r.(CiiC1316 2.55(s) , 2H CH2Br, 6 8.15 (b) 1H. NHAr;e 7.2-7.8 (m) 7H, Ar-H;

6 6.15 (b)2H..:Ar&Hadjacent to NH.

2-Substituted amino methyl-3-(substituted anilinoj-4(3H)-

quinazolinone(3-18)

Appropriate 2-bromo methyl-3-(substituted anilinoj-4(3H) quinazoli-

none (0.1 mole) was added slowly to an excess of substituted appropriate

Pharmacological Research Communications, Vol. 19. No. 9, 1987 619

Table I

E-Methyl amino substituted phenyl-3-(substituted anilin&-4(3H) quinazolinones

R

Sl. R No.

M.P. C

Yield %

Molecular formula

1 H

2 4-NO2

3 H

4 H

5 H

6 H

7 H

8 H

9 H

10 H

lf H

12 H

13 H

14 H

15 H

16 4-NO2

17 4-NO2

18 4-NO2

Br

Br

'sH5 Morpheline

3-C1.C6H4

2-OCH3.C6H4

Piperidine

2-CH3.C6H4

3-CH3.C6H4

4-C1.C6H4

CH2.C6H5

2-C1.C6H4

2,4-C12C6H3

4-OCH3.C6H4

CH2.CH2-C6H5

4-C1.C6H4

2,5-C12.C6H3

2,4-C12.C6H3

176 70

102 72

162 70

157 72

135 62

164 71

156 76

156 78

150 75

164 70

155 70

130 70

160 72

160 68

139 70

95 71

120 71

195 70

All the compounds were analysed for N and found within the limit of + 0.4% of the theoritical values.

Melting points were taken in open capillary tubes and are uncorrected.

620 Pharmacological Research Communications, Vol. 19, No. 9, 1987

amine (0.5 mole). It was heated at 55-60°C for one hour. The reaction

mixture was poured into cold water. A solid separated out which was

recrystallized from benzene/pet. ether.

Compound No. 3 (Table I) Molecular formula C2IHI8N40, requires

%C 73.68, %H 5.28, XN 16.37, Found : %C 73.65, %H 5.22, %N 15.30. IR(KBr)

1680 cm-l C=O, 3100 cm-l Arc-H, 3380 cm-l -NH. P.m.r. (CdC13) : 6- 2.60(s)

2H, CH,2-NH; 6 3.60 (bs) 1H CH2-NH;.6 8.15 (b) 1H NH Ar; 6 6.4-6.7 (m)

4H, aromatic 0, O-position with respect to the NH groups; 6 7.10-7.45 (m)

6H aromatic protons, 6 7.55-7.80 (m) 4H, Aromatic protonof Qz ring. Mass

spectrum of this compound exhibited (M) at m/z 342, and other fragments

at m/z 328 (loo%), mlz 250, m/z 173, m/z 146, m/z 115, m/z 191, m/z 172

respectively.

Behavioural Studies

The behavioural observations were made on albino mice, weighing

20-30 gm of either sex on various motor, sensory and autonomic behavioural

patterns according to the method of Nodine and Siegel (1964) after intra-

peritoneal injection (i.p.1 (100 mg/kg) of test compounds. The study

of motor behavioural pattern consisted of spontaneous motor activity,

gait, muscular tone, posture, shivering, convulsions and stereotypg. In

sensory behaviour, awareness to subject or sound, touch and pain reflexes

and for assessment of autonomic behaviour, respiration, salivation, assess-

ment of urination, deafecation, piloerection, pupil size, righting reflex,

restlessness or any other behavioural changes were observed.

The effect of compounds was observed on above behavioural

responses for the period of four hours after compound administration.

Careful observations were recorded for each group of animals at 5 min.

intervals for the first 30 min. and then 15 min. intervals in the next


4 hrs. The animals were fed and allowed water ad-libitum, the number of

animals in each group was five. These behavioural patterns were also

quantitated by arbitary scoring i.e. Normal = 0, Mild (slight increase or

decrease in behaviour pattern as compared to normal) = 1, Moderate

(increase or decrease in behavioural patterns regularly present but

interrupted) = 2 and Marked = 3.

2. Antiparkinsonian activity:

The study was carried out on albino rats weighing 100-200

g and mice weighing 20-30 g of either sex. The animals were fed and

allowed water ad-libitum. The number of animals in each group was 5.

All the compounds were administered in dose of 100 mg/kg (i.p.1.

(i)

(ii 1

Tremor: Tremors were induced by oxotremorine (OT) (0.5 mg/kg

i.p.1 in mice 45 minutes after pretreatment with test

compounts. After 5 minutes of OT injection, tremors were

assessed visually and scored as: 0 = no tremor; 1 = occa-

sional tremor; 2 = intermittent tremors; 3 = continuous

tremors (Coward et al., 1977). Each animal of a group was

scored and tremor index (mean score for each group) was

determined.

Rigidity: Reserpine (5 mg/kg i.p.1 was administered in rats

to produce rigidity and after 15 minutes test compounds were

injected. Rigidity was measured 1 hour after reserpine. To

measure rigidity, rats were grasped immediately below fore-

limbs and slight pressure was applied upward against the hind

limbs. The degree of resistance was scored according to

(Goldstein et al., 1975) 0 = no resistance; 1 = normal resis-

tance; 2 = complete resistance. A score of 2 was selected as

622

(iii)

(iv)

Pharmacological Research Communications, Vol. 19, No. 9, 1987

criterian for rigidity and expressed as percentage of animals

showing rigidity in a group.

Hypokinesia: It was produced by administering reserpine (5 mg/

kg i.p.1 in rats. Locomotor activity was measured after 2 hrs

by placing each group of rats in photoactometer for 15 minutes

and total counts were recorded. The test compounds were

administered 15 minutes after reserpine. The perce-nt increase

or decrease in counts was calculated on the basis of counts of

untreated groups.

Catatonia: Reserpine (5 rag/kg i.p.1 was administered in rats

and after 4 hrs, catatonia was observed and scored according

to (Morpurgo 1962). The test compounds were administered

after 15 minutes of reserpineadministration.

3. Toxicity studies:

Acute neurological toxicity was observed according to Swinyard

et al (19521. ALD5D values were determined according to Smith (1950).

4. Biochemical studies

Dopamine receptor binding study

Compounds which exhibited significant effect on various

behavioural parameters were also studied for their effect on dopamine

receptor binding using 3H spiroperidol as specific ligand to label

dopamine receptors in corpus striatal membrane preparations of rat brain

(Leysen et al, 1978). Corpus striatum from adult rat brain was dissected

out using the method of Glowinski and Iverson (1966) and crude synaptic

membrane from corpus striatum was prepared according to the method

described earlier (Agarwal et al, 1981; Bennett, 1978.). Binding assay was


performed using glass fiber disc filteration method as described earlier

(Seth et al, 1981). The incubation mixture of 1.00 ml. consisted of crude

synaptic membrane equivalent to 5 mg wet weight of tissue containing

300-35OAg protein alongwith 1mM of l-phenyl-4-3H spiroperidol (Sp. acti-

vity 22 Ci/m mol NEN) in 50 mM Tris HCl pH 7.4 in presence of different

concentrations (10m4 to 10-5 MI of 2-methyl amino substituted phenyl-3-

substituted anilino 4(3H) quinazolinones. A parallel incubation was

carried out simultaneously in presence of 10e6 M haloperidol to obtain

non-specific binding (L20% of specific binding). Reaction mixture was

incubated for 15 minutes at 37'C and filtered using glass disc (25 mm

diameter, 0.2 Urn pore size, Gelman Inc. Ann Arbor MI) and washed rapidly

with 2 x 5 ml cold Tris HCl buffer and radioactivity was counted in

scintillation counter LKB Rack B II with an efficiency of 50% for tritium.

Specific binding was calculated by substracting non specific from total

binding obtained in absence of lo-’ M haloperidol. Results are expressed

in terms of p mole bound/g protein. Protein was estimated following the

method of Lowry et al, (1951).

5. Statistical analysis

Mean score was calculated for the groups and significance of difference

from control was determined by the mean - Whitney U test (non parametric

analysis) as described by Siegel (1956). Percentage change in hypokinesia

was analysed by Chi-square (Yates correction).

Results and Discussion

The effect of sixteen compounds (3-18) was studied on motor, sensory,

autonomic behavioural parameters on albino mice. Compound No. 5 showed a

marked decrease in spontaneous motor activity. The effect started within

15-20 minutes. The peak response was observed at 60-75 min and the effect

624 Pharmacological Research Communications, Vol. 19. No. 9. 1987

persisted upto 3 hrs, after that the animal returned back to its normal

behaviour, similarly there was decrease in awareness to sound, light, touch

and pain responses whereas other behavioural parameters were more or less

normal as compared to the control group of animals. Compound 11, 13 and 14

also showed a decrease in spontaneous motor activity but the effects were

not so marked as compared to compound No. 5.

The antiparkinsonian profile of these compounds is reported in Table

II. Compound 15 showed significant inhibition of oxotremorine induced tremors.

Compounds No. 10, 11, 12, 15 and 18 showed significant anti-rigidity activity

which was better in comparison to bromocriptine. The locomotor activity

counts in untreated group was 106. Reserpine alone reduced the locomotor

activity to 8.24 (percent counts) of the compounds of untreated rats.

Compounds 10, 15 and 18 increased significantly the locomotor activity in

reserpinised rats. A significant reduction in catatonia was observed with

compounds12, 15 and 18 which was similar to bromocriptine.

The active compounds lo,15 and 18 were evaluated at three

dose levels 5Omgjkg., 2ODmg/kgand 400 @kg to see the dose response of these

compounds (Table III).

It is interesting to point out that at 50 mg/kg dose the

compounds were active against all the parameters. Furthermore the compounds

showed significant increase in their antiparkinsonian activity against

all the parameters at the higher doses tested.

It is evident from the results of in vitro effect of Z-methyl

amino substituted phenyl J-substituted anilino 4(3H) quinazolinones 10, 15

and 18 on dopamine receptor binding (Fig. 1) that compound No. 10 and 15

showed significant inhibition of binding of 3H-spiroperidol at concentra-

tions ranging from 0.1 m mole to 1.0.~ mole, where as compound No. 18 did

Pharmacological Research Communications, Vol. 19. No. 9. 1987 625

Table II

Antiparkinsonian activity of E-methyl amino substituted phenyl-3- (substituted anilinol-4(3H) quinazolinones (3-18)

Test R R, Oxotre- Reserpine (5 mg/kg i.p. Compounds 100 mg/kg,:

n=5

I morine inrats (O-5 mgl Rigi- kg i.p.) Hypoki- Catatonia

ditv nesia Mean %- % score at

course 4 hrs.

Control -

3 H 'sH5

4 H Morpholino

5 H 3-C1.C6H4 I

6 H 2-OCH3.C6H4 7 H Piperidino

8 H 2-CH3.C6H4

9 H 3-CH3.C6H4 10 H 4-C1.C6H4

11 H CH2.C6H5 12 H 2-C1.C6H4

13 H 2,4-C12.C6H3

14 H 4-OCH3.C6H4 15 H CH2CH2-C6H5

16 4-NO2 4-C1.C6H4 17 4-NO2 2,5-C12C6H3

18 4-NO2 2,4-C12C6H3

Bromocriptine - (10 mg/kg)

3.09

3.0+0

2.75~0.4

2.75~0.5 3.0+0

3.0+0

3.0+0

3.0+0

2.5to.3

3.0+0

3.0+0 - 3.0+0

3.0+0

2.3+0.3x

3.0+0 - 3.0+0 - 3.0+0 - 3.0+0 -

100 6.67

100 9.64

50 22.42

75 9.21

100 4.62

100 4.83

50 9.17

62 8.61

12.5* 44.12"

25* 10.06

25* 36.12"

100 6.08

50 7.31

25* 48:13**

100 6.50

75 14.01

12.5* 32.5*

20 71.92**

3.03

3.020

2.0*+0.2 - 2.6220.4

3.020

2.75iO.4

2.5020.3

2.25*+0.2

2.50~0.3

2.8720.5

1.25**+0 2 -- 3.0+0 - 2.oto.2 - 1.12*+0.2

3.0+0

3.0+0

1.37**+0 2 -* 1.4*+0.2 -

*(P / 0.05) and **(P/0.01) denote significant difference from control. -

ALD53 7 1000 mg/kg

626 Pharmacological Research Communications. Vol. 19, No. 9, 1987

Table III Dose Response of antiparkinsonian active Z-methyl amino-substituted

phenyl 3-substituted anilino-4(3H) quinazolinones (lo,15 and 18)

Compd. Dose Oxotremorine (0.5 mg/kg)

Reserpine (5 mg/kg.)

Rigidity Hypokinesia Catatonia % % counts mean score

at 4 hrs.

10 50 mglkg

10 200 mglkg

10 400 mglkg

15 50 mg/kg

15 200 mglkg

15 400 mg/kg

18 50 mg/kg

18 200 mglkg

18 400 mglkg

2.6+0.5

2.0+0.2

1.8+0.2 -

2.5+0.5 -

2.0+0.2

2.oto.2

3.oto.o

2.6tO.5

2.5to.4

25 24.12

10 76.16

10 78.00

50 26.17

12.5 67.18

25 74.14

25 22.18

12.5 42.18

10 70.16

2.6tO.5 -

2.4to.4

2.oto.2 -

2.oto.2 -

l.OfO.2

1.oto.2

1.6tO.2

1.2ul.2

1.oto.2 -

-----

not show significant inhibition. These observations suggest that compounds

No. 10 and 15 bind with DA receptors. Neverthless, a possibility exists

that the compounds (10 and 15) may also bind to 5-HT2 receptor,since spiro-

peridol has also been reported to be a 5-HT2 receptor antagonist (Goodman,

1985). However these compounds also exhibited significant reversal of

reserpine induced rigidity, hypokinesia and catatonia, it is quite probable

that these compounds act as DA receptor agonist.

Acknowledgement

The authors are grateful to Neuropharmacology Unit (C.D.R.I.,

Lucknow) and Mr. D.N. Bhalla for his technical assistance.


80

60

5 i= 40 z T z 20 -s

0

o-----o Compound

A-A. Compound

10

15

18

-4 -‘5 I

IO IO -‘s IO IO lo8 -7

M-Concentration of Compounds

IN VITRO EFFECT OF 2-METHYLAMINO 'JJBSTITUTED PHENYL

3-SUBSTITUTED ANILINO 4(3H) QUIYAZOLINONESlO, 15 & 18 ON

3H SPIROPERIDOL BINDING TO STRIXTAL MEMBRANE

(Fig. 1)

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Antiparkinsonian activity and behavioural effects of newer quinazolinones

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