Synthesis and antileishmanial evaluation of some 2,3-disubstituted-4(3H)-quinazolinone derivatives

Background Leishmaniasis is a neglected tropical parasitic diseases affecting millions of people around the globe. Quinazolines are a group of compounds with diverse pharmacological activities. Owing to their promising antileishmanial activities, some 3-aryl-2-(substitutedstyryl)-4(3H)-quinazolinones were synthesized in good yields (65.2% to 86.4%). Results The target compounds were synthesized by using cyclization, condensation, and hydrolysis reactions. The structures of the synthesized compounds were determined using elemental microanalysis, infrared (IR), and proton nuclear magnetic resonance (1H NMR). The in vitro antileishmanial activities of the synthesized compounds were evaluated using Leishmania donovani strain. All the synthesized compounds displayed appreciable antileishmanial activities (IC50 values, 0.0128 to 3.1085 μg/ml) as compared to the standard drug miltefosine (IC50 = 3.1911 μg/ml). (E)-2-(4-chlorostyryl)-3-p-tolyl-4(3H)-quinazolinone (7) is the compound with the most promising antileishmanial activities (IC50 = 0.0128 μg/ml) which is approximately 4 and 250 times more active than the standard drugs amphotericin B deoxycholate (IC50 = 0.0460 μg/ml) and miltefosine (IC50 = 3.1911 μg/ml), respectively. Conclusions The results obtained from this investigation indicate that the synthesized and biologically evaluated quinazoline compounds showed promising antileishmanial activities and are good scaffolds for the synthesis of different antileishmanial agents. Electronic supplementary material The online version of this article (doi:10.1186/s13588-014-0010-1) contains supplementary material, which is available to authorized users.


Background
Leishmanisis is a neglected tropical disease resulting from infection of macrophages by obligate intracellular parasites of the genus Leishmania [1][2][3]. It is a public health problem in at least 88 countries with an estimated 350 million people at risk. The estimated global prevalence of all forms of the disease is 12 million. Every year, 1.5 to 2 million new cases and 70,000 deaths occur due to cutaneous leishmaniasis (CL). In addition, 500,000 new cases and 59,000 deaths from visceral leishmaniasis (VL) occur annually [4]. The number of cases of leishmaniasis is increasing globally due to Leishmania/HIV co-infection [5,6], international travel, and migration of immigrants and refugees from endemic regions [7,8].
The prophylactic treatment of leishmaniasis mainly rely on vector and reservoir control [9][10][11]. Control of reservoir host and vector is difficult due to high coast, operational difficulties, and frequent relapses in the host [12]. Although considerable effort has been made to produce vaccine candidates for the treatment of leishmaniasis, there is no vaccine against any form of human leishmaniasis yet [13][14][15][16][17].
Pentavalent antimonials (Sb V ) have been used for the treatment of leishmania infections. Unfortunately, in many parts of the world, the parasite has become resistant to Sb V [18]. Treatment failure to sodium stibogluconate (SSG) is observed in Eastern Sudan [19] and in Tigray, Northern Ethiopia [20]. Recent reports showed that pentamidine also developed resistance as well as difficulties in treating patients with Leishmania/HIV co-infection [21].
Combination chemotherapy has improved prospects for decreasing the emergence of drug resistance, increasing activity, and reducing required doses and thereby toxic side effects. In the previous study, WR 279,396 (a topical formulation containing 15% paromomycin and 0.5% gentamicin) was found to be safe and effective against CL caused by Leishmania major [22]. In addition, AmBisomeparomomycin is the most cost-effective combination among miltefosine-paromomycin and AmBisome-miltefosine [23]. So far, no combination chemotherapy has been used in treatment programs, except paromomycin/SSG [24].

Instruments and apparatuses
Melting points were determined in open capillaries using electro-thermal 9100 melting point apparatus and were uncorrected. Infrared (IR) spectra in nujol were recorded with the SHIMADZU 8400SP FT-IR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), and proton nuclear magnetic resonance ( 1 H NMR) spectral data were performed on Bruker Avance DMX400 FT-NMR spectrometer (Bruker, Billerica, MA, USA) using tetramethyl silane (TMS) as internal standard. Silica gel TLC plates of 0.25-mm thickness were used in the study.

Experimental animals and strains
Swiss albino male mice of weight 20 to 32 g and age 6 to 8 weeks (for acute toxicity test) were obtained from Biomedical Laboratory, Department of Biology, Faculty of Science, AAU. Leishmania donovani isolate used in this study was obtained from Leishmania Diagnosis and Research Laboratory (LDRL) culture bank, School of Medicine, AAU.

Reference drugs
Miltefosine/hexadecylphosphocholine (AG Scientific, San Diego, CA, USA) and amphotericin B deoxyhcholate (Fungizone®, ER Squibb, Middlesex, UK) were employed as reference drugs in the in vitro antileishmanial activity testing of the synthesized compounds.

Preparation of stock and working solutions
Stock solutions of 10 mg/ml of the synthesized compounds were prepared by dissolving each compound in DMSO. Stock solutions were diluted using complete RPMI to obtain aliquots of 10 μg/ml. Then, threefold serial dilution with complete RPMI gave the final six working concentrations (10, 3.33, 1.11, 0.37, 0.12, and 0.04 μg/ml) of each of the synthesized compounds. Amphotericin B deoxycholate and miltefosine, which were used as a positive control for comparison of the antileishmanial activities of the test compounds, were also made in threefold serial dilutions. All the prepared drugs were stored at −20°C and retrieved only during use [41].

In vitro antileishmanial activity
In a 96-well microtiter plate, 100 μl of each of the seven threefold serial dilutions of synthesized compounds were added in triplicate wells. Then, 100 μl of suspension of parasites (3.0 × 10 6 promastigotes/ml of L. donovani) was added in duplicate. Some of the wells contained only the parasites which served as a positive control. The media and DMSO alone acted as a negative control. The contents of the plates were then maintained in humidified atmosphere at 22°C under 5% CO 2 .
After 68 h of incubation, 10 μl of fluorochrome resazurin solution (12.5 mg dissolved in 100 ml of distilled water) was added into each well. The fluorescence intensity was measured after a total incubation period of 72 h using Victor3 Multilabel Counter (PerkinElmer, Waltham, MA, USA), at an excitation wavelength of 530 nm and emission wavelength of 590 nm [42]. The IC 50 values were evaluated from sigmoidal dose-response curves using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA).

In vivo acute toxicity test
The oral acute toxicity of compound 7 that exhibited promising antileishmanial activity was investigated using male Swiss albino mice (approximately 20 g each) following reported methods [43]. The experimental animals were divided into six groups (containing six mice per group) and fasted overnight. Groups 1-5 received compound 7 suspended in a vehicle containing 1% gum acacia, in doses of 10, 50, 100, 200, and 300 mg/kg, respectively. The sixth group received vehicle containing 1% gum acacia (served as a control group) at a maximum dose of 1 ml/100 g of body weight by oral route. The mice were observed closely for 24 h with special attention to the first 4 h. Acute toxicity signs were checked in the test mice.

Statistical analysis
The IC 50 values for in vitro promastigotes assay of synthesized compounds were evaluated from sigmoidal dose-response curves using computer software Graph-Pad Prism 5.0.

Results and discussion
Chemistry of the synthesized compounds Synthesis of the target compounds involved the formation of 2-5 and 10 as intermediates. It was accomplished using nucleophilic reaction, nucleophilic with ring opening and closing, condensation reaction, and hydrolysis reactions. The target compounds are synthesized in a good yield, which ranged from 65.2% to 86.4% (Table 1). All the synthesized compounds were readily soluble in DMSO and chloroform except compound 12 which is readily soluble in acetone. Spectral data (IR and 1 H NMR) of the synthesized compounds were in full agreement with the proposed structures.

Biological activity testing results
In vitro antileishmanial activity of the synthesized compounds The antipromastigote activities of the synthesized compounds and the standard antileishmanial drugs (amphotericin B deoxycholate and miltefosine) were evaluated using the clinical isolate of L. donovani strain. The IC 50 of the synthesized and reference drugs were evaluated from fluorescence characteristic of AlamarBlue® (resazurin) (Trek Diagnostic Systems, Inc., Cleveland, OH, USA) which is soluble, stable in culture medium, non-toxic to cells, and does not affect the secretary abilities of cells [44]. The test works as a cell viability and proliferation indicator through the conversion of resazurin to resorufin via reduction. The amount of fluorescence produced is proportional to the number of living cells [45,46]. The quinazolinone derivatives synthesized were shown to have good antileishmanial activity which was in line with the previous reports [37][38][39][40]. All the tested compounds exhibited better antileishmanial activity than the standard drug miltefosine as shown in Table 2. Among them, compound 7 was found to have a very promising antileishmanial activity with an IC 50 value of 0.0128 μg/ml which was 250 times superior than miltefosine (3.1911 μg/ ml). Compounds 8 and 11 were 30 times more active than miltefosine. Compounds 6 and 12 were 10 times and twice more active than miltefosine, respectively. Compounds 9 and 13 were as active as miltefosine.

General procedure for the synthesis of 2-methyl-3,1-benzoxazin-4-one (2)
A solution of anthranillic acid (1) (10 g, 0.073 mol) in acetic anhydride (25 ml) was heated under reflux for 1 h. The precipitate formed on cooling was filtered and the excess acetic anhydride was washed with anhydrous petroleum ether, where upon a solid mass is obtained. This solid mass (2), without purification, was used for subsequent reaction [47].