Synthesis and study effects of 3,4-Dihydro-4-(p-anisyl)-6-phenyl pyrimidine-2(1H)-one on growth and morphology of Leishmania tropica promastigotes in vitro

Most of the drugs used in the treatment of leishmaniasis have side effects, there is a need for a safe and effective drugs, therefore, in the present study heterocyclic compound containing pyrimidine ring ( 3,4-Dihydro-4-(p-anisyl)-6-phenyl pyramidine-2-(1H)-one ) was synthesized, then tested for there antileishmanial activity against Leishmania tropica promastigotes in vitro. The effect of the compound on the growth, generation number and generation time of the parasite was determined using concentrations ranging from 1 to 10 μg/ml. The effect of the compound concentration was found to be important to the parasite growth as the more the concentration the less the growth, generation number and increased the generation time. At the log phase the IC50 and IC90 were 2.5 and 10 μg/ml respectively and caused morphological distortions. The toxicity of the synthesized compound was determined using Balb/c mice model. Dixon's up and down method ( 1980 ) was found to have an LD50 of 642.5 mg / Kg of body weight. Key word: Phenyl pyrimidine , Leishmania , Growth. Introduction: Leishmaniasis is a vector – born disease that is transmitted by sand flies and caused by obligate intracellular protozoa of the genus Leishmania. Leishmaniasis is prevalent in 88 countries from Tropical to Mediterranean regions, where 12 million people are infected and approximately 350 million people are at risk, 1 – 2 million new cases registered annually [1]. At least there are four species of genus Leishmania, similar in morphology, but different in many aspects: cultural, characteristics, clinical manifestation, geographic distribution and sand fly vectors, can cause the disease in humans. Leishmania donovani, the etiologic agent of visceral leishmaniasis, or Kala-azar ; Leishmania tropica, of old world cutaneous leishmaniasis, or oriental sore ; the Leishmania braziliensis, complex of cutaneous and mucocutaneous disease in America and the Leishmania maxicana, complex are associated mainly with cutaneous lesions. Within each major group are strains and possibly subspecies with their own distinctive characteristics [2]. Cutaneous leishmaniasis is prevalent in some of the Middle Eastern, Tropical African and Asian countries including Iraq [3]. The drugs available for the treatment of leishmaniasis are in general, toxic, expensive and require long – term treatment. Large scale clinical resistance against the most commonly used antimonial agent, have been reported [4]. The usual form of treatment of leishmaniasis is still intravenously using highly poisonous antimony compounds, e.g., in pentostam 15% of the patients die from these injections, i.e. the therapy causes death in the advanced stage of the disease. In addition, patients are increasingly not reacting to the conventional poisonous antimonials. According to rough estimates, about 40% of the infected persons in India are already resistant to this therapy. Similar observations in Sudan supports this report [5]. In the present work, particular pyrimidinonic compounds were synthesized and tested for their antiparasitic activity against Leishmania tropica promastigotes in vitro. Since pyrimidine bases are major constituents of nucleic acids, the chemistry of pyrimidines have been the subjects of many researches owing to their applications in molecular biology and medicine. They show many activities including antibacterial [6], antiviral activities [7], and antifungal effects [8]. Some pyrimidinone derivatives have anticancer activities [9], antidiabetic effects [10], immuonomodulator [11]. Thus, the present study drives at the synthesis of heterocyclic compound that contain pyrimidine ring and have a considerable inhibitory effect on the growth of Leishmania tropica promastigotes, and low toxicity in vivo. Material and Methods: 1. Synthesis of 3,4-Dihydro-4-(p-anisyl) -6-phenyl pyrimidine-2(1H)-one A. Preparation of 1-phenyl-3-(p-methoxy phenyl )-2propen l-one: 1-phenyl-3-(p-methoxy phenyl )-2-propen l-one were prepared according to [12]. A mixture of 0.05 mol of sodium hydroxide pellets, 20 ml of water and 0.2 mol of ethanol was magnetically stirred in a 100 ml round bottomed flask which immersed in an ice – bath. A 0.043 mol of freshly distilled acetophenone was poured on stirred mixture followed by 0.043 mol freshly distilled anis aldehyde with vigorous stirring, the temperature was kept at 20 – 25 C o for ( 2 – 3 ) hrs. until the mixture became thick. The thick mixture was kept in a refrigerator over night. The product filtered then, vacuum and washed with water until the neutralization of filtrates then washed with 20 ml ice – cold ethanol ( See paragraph A of results ). B. Preparation of 3,4-Dihydro-4-(p-anisyl) -6-phenyl pyrimidine-2(1H)-one: This was prepared applied according to [13]. In a 100 ml round bottomed flask, a mixture of 5 mmol of 1-phenyl3-( p-methoxy phenyl) -2-propen l-one and 50 mmol of sodium hydroxide dissolved in 25 ml absolute ethanol Tikrit Journal of Pure Science Vol. 13 No.(3) 2008 was stirred magnetically for 10 minutes. 5 mmol of urea which dissolved in 25 mmol absolute ethanol was added to the stirred mixture then cooled at room temperature and the solvent was separated to give the solid product of 3,4-Dihydro-4-(p-anisyl) -6-phenyl pyrimidine-2(1H)-one ( See paragraph B of results ). 2. Biological activity of 3,4-Dihydro-4-(p-anisyl)-6phenyl pyrimidine-2(1H)-one A. Leishmania used: MHOM / IQ / 1992 / MREC3 Leishmania tropica stock culture was used. The culture was obtained from the College of Medicine AL-Nahreen University, which has been characterized using isoenzyme method according to [14]. The obtained culture was cultivated in Tobie`s medium [15]. B. Cultivation and estimation of numbers of parasites: 1.9 ml of liquid phase was added to McCantry vials that contained 5 ml solid phase slants, then 0.1 ml of Leishmania promastigotes inoculums was taken from stock culture during logarithmic phase, so that the initial density of the organism was 2 x 10 5 / ml, then the number of organisms of new culture incubated at 26 C o for 4 days, was counted directly using a haemocytometer. C. Effect of the synthesized pyrimidinone on growth, generation number and time of Leishmania tropica promastigotes: Effect of the compound on growth was studied in vitro in comparison with untreated groups. The compound was dissolved in 2% dimethyl sulfoxide (DMSO). Five concentrations (1 , 2.5 , 5 , 7.5 , 10 ) μg / ml were used to determined the ( IC50 ) and the ( IC90 ) of the cultivated organisms. Numbers of promastigotes were determined at different time intervals ( 24 , 48 , 72 , 96 ) hours, then generation number and time of each culture at each time intervals were estimated using the following laws [16]: Log N – Log N0 n= ــــــــــــ Log 2 whereas, n = generation number , N = number of promastigotes at time (t) N0 = initial number at zero time