SYNTHESIS AND IN- VIVO PHARMACOLOGICAL EVALUATION OF SOME NOVEL 4(3H)- QUINAZOLINONE DERIVATIVES AS POTENTIAL ANTI-MALARIAL AGENTS

In this work six 3-aryl-2-(substitutedstyryl)-4(3H)-quinazolinones derivatives were synthesized by the reaction of 3-aryl-2-methyl-4(3H)-quinazolinone (intermediate products) with different substituted aromatic aldehydes. Three intermediate products were synthesized by reacting 2-methyl-3, 1-benzoxazin-4-one, which was initially prepared by cyclizing anthranilic acid using acetic anhydride, with three aromatic amines. Their structures were confirmed using IR, 1HNMR, 13CNMR spectroscopic methods and elemental microanalyses. The synthesized compounds were evaluated for their in vivo antimalarial activity against P. berghei. Four of the synthesized compounds (IIIc, IVa, IVb and IVf) exhibited activity against the parasite. Among these compound IVa was found to be the most active compound. Results of acute toxicity study showed that oral administration of the synthesized compounds in single doses (100, 250 and 500mg/kg) had no adverse effects, indicating that the compounds have high safety margin and their LD50 is higher than 500 mg/kg. In general this study indicates that 4(3H)-quinazolinones derivatives are potential sources of lead compounds for antimalarial drugs.


INTRODUCTION
Malaria is one of the oldest recorded diseases in the world. Ancient Chinese and Sanskrit medical texts described its symptoms and Hippocrates referred to the disease in the 4th Century BC [1]. It is estimated to account for 300 million to 500 million illnesses and nearly 1 million deaths each year [2]. Malaria is a protozoal disease caused by parasites of the genus Plasmodium [3]. Four identified species of this parasite exist, which cause different types of human malaria, namely; Plasmodium vivax, Plasmodium falciparum, antimicrobial, anesthetic, anticancer, antihypertensive, antiinflammatory, antimalarial, diuretic and muscle relaxant properties [13][14][15][16][17][18][19][20][21][22]. Increased efforts in antimalarial drug discovery are urgent to develop safe and affordable new drugs to counter the spread of malaria parasites that are resistant to existing agents. Furthermore, quinazolinones substituted at 2 and 3-position play a pivotal role in the antimalarial activity [13].
Several bio-active natural products such as febrifugine and isofebrifugine contain quinazolinone moieties with potential antimalarial activity [11,12]. Therefore, 2,3-disubstituted-4(3H)-quinazolinones, are point of interest to seek for new drugs that act against the malarial pathogen in order to combat and reduce its tremendous prevalence. Hence, in this work compounds containing 4(3H)-quinazolinone moiety were designed to study their antimalarial activities. The simple synthesis and antmalarial results of these newly synthesized compounds are reported in this paper. plate of 0.25 mm thickness using benzene: methanol (9:1) as a solvent system. Iodine chamber was used as a developing chamber. All the reagents used were AR grade.

4(3H)-quinazolinones (III a-c)
A mixture of acetanthranil II, (0.1 mol) and an equimolar amount of the appropriate aromatic amine was heated under reflux for 5-7 hrs. The dark sticky mass formed was cooled and recrystallized from ethanol. General procedure for the preparation of 3-aryl-2-

In vivo antimalarial activity
In vivo antimalarial activity test of the synthesized compounds was performed using a 4-day standard suppressive test [26]. On day 0, the test mice were injected with 0.2 ml of 2X107 parasitized erythrocytes, (P. berghei Untreated control mice typically die in about one week after infection [29]. For treated mice the survival-time (in days) was recorded and the mean survival time was calculated in comparison with that of the negative group [30].

In vivo acute toxicity
Oral acute toxicity study was done for the synthesized compounds. Four groups of mice, each group consisting of six male mice were used for testing acute toxicity. The mice in each group were fasted over night and weighed before test.
Test compounds were dissolved in 70% Tween 80 and 30% ethanol. This solution was further diluted 10-fold with sterile distilled water to give a stock solution containing 7% Tween 80 and 3% ethanol [31]. Mice in groups one, two and three were given 100, 250 and 500 mg/kg/day of the synthesized compounds respectively with a maximum dose volume of 1 ml/100 g of body weight orally while mice in the control group (group four) were treated with the vehicle.
After administration of the substance food was withheld for a further 2 hr period [32]. Toxicity signs such as changes in skin, eyes (blinking), tremors, convulsion, lacrimation, muscle weakness, sedation, urination, salivation, diarrhea, lethargy, sleep, coma and also death were observed for 72 hrs.
Twenty-four hours later, the % mortality and weight of mice in each group and for each test compound at each dose level were recorded [27].

Data analysis
Results of the study were expressed as mean ± standard deviation. Statistical significance for suppressive test was determined by one-way ANOVA at 95% confidence limits

In vivo antimalarial activity
To ascertain their antimalarial activity, compounds IIIc, IVa, IVb, IVc, IVd, IVe and IVf were assayed in vivo against P.
berghei, a rodent malaria parasite, using a 4-day standard suppressive test [26]. The synthesized compounds were given at dose levels of 20 mg/kg and then 40 mg/kg to see if there is a dose-response relationship ( The data indicated that the test compounds did not show any significant change in weight and the slight variation observed was not found to be dose dependent (Table 2). and their LD50 could be higher than 500 mg/kg. Since the 4(3H)-quinazolinone derivatives have not been used as antimalarial agents the existence of resistance against these compounds is barely true. Therefore these 4(3H)-quinazolinone derivatives are expected to have activity against plasmodium species resistant to existing drugs.

Recommendation
In this work only limited numbers of compounds were synthesized and their antimalarial activity was tested. Thus more compounds should be synthesised and tested to exploit the possible most active 4(3H)-quinazolinone derivatives.
Additional toxicity studies need to be done to prove the subacute and chronic safety of the synthesized compounds. The antimalarial mechanism of action of 4(3H)-quinazolinone derivatives need to be determined and docking study be performed.