2‐Aroyl quinazolinone: Synthesis and in vitro anti‐parasitic activity

Trypanosomes and Leishmania are parasitic protozoans that affect millions of people globally. Herein we report the synthesis of 2‐aroyl quinazolinones and their antiprotozoal efficacy against Trypanosoma brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania infantum. These compounds were counter‐screened against a human cell line for cytotoxicity. Thirteen of the twenty target compounds in this study inhibited the growth of these parasites, with compounds KJ1, and KJ10 exhibiting IC50 values of 4.7 μM (T. b. brucei) and 1.1 μM (T. b. rhodesiense), respectively.


| INTRODUCTION
Chagas disease (CD), which is also referred to as American trypanosomiasis (AT) is a potentially fatal condition that is caused by Trypanosoma cruzi. This parasite is transmitted through the bite of an infected insect known as a "kissing bug" (Rassi & de Rezende, 2012). Up to seven million people worldwide have a T. cruzi infection, and about 75 million people living in endemic regions (Latin America) are at risk of contracting the disease (Rodriguez & Loaiza, 2017). Annually, there are about 12,000 deaths attributed to AT. The incidence of AT is rising in both endemic and non-endemic regions of the world due to a variety of variables, including climate change, and international travel (Campos et al., 2017;Velázquez-Ramírez et al., 2022).
Nifurtimox and benznidazole are the only two medications currently available for the treatment of CD and have been in use since the 1970s. These medications have side effects and are only useful in the acute or early stages of the infection. Some detrimental effects of these drugs include anorexia, nausea, vomiting, seizures, vertigo, headaches, and dermatitis; consequently causing 10%-30% of patients to stop taking their medications (García-Huertas & Cardona-Castro, 2021). According to estimates, AT results in an annual global spending of USD 627.5 million, mostly in the form of medical expenses. The cost per AT patient ranges from $200 in the early stages of the disease to $6000 in the chronic stage. Due to this cost, AT has been identified as a parasitic disease with the highest global burden (Gómez-Ochoa et al., 2022).
Human African trypanosomiasis (HAT), also referred to as sleeping sickness, is a neglected disease solely found in Africa, and it is caused by Trypanosoma brucei-a parasitic protozoan transmitted to mammals through the bite of an infected tsetse fly (Dunn et al., 2021). Two subspecies of T. brucei, T. b. gambiense and T. b. rhodesiense, cause HAT in humans (Magez et al., 2021). If untreated, the illness is almost always lethal (Beteck et al., 2019).
Sleeping sickness progresses through two distinct stages. In the first or early stage of the disease, trypanosomes are limited to the blood and lymphatic systems. In the second or late stage of the disease, parasites invade the central nervous system (Steverding, 2008).
Depending on whether or not parasites crossed the blood-brain barrier (determined by examination of cerebrospinal fluid obtained by lumbar puncture), the few current HAT medications are split into two categories. Suramin or pentamidine injections are given intramuscularly (IM) for Stage 1 HAT. Melarsoprol or eflornithine/nifurtimox combination are used for the treatment of Stage 2 HAT. Due to the toxicity of the molecules used to treat Stage 2 and/or the challenges associated with administering them, patients in Stage 2 are typically treated in hospitals (Eperon et al., 2014;Venturelli et al., 2022).
Visceral leishmaniasis (VL) is predominantly caused by Leishmania donovani and L. infantum parasites-which have phlebotomine sand flies as their vector. The risk factors for VL include malnutrition, human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV-AIDS), and other immunosuppressive diseases/conditions (Adriaensen et al., 2018;Lindoso et al., 2016). According to World Health Organization (WHO) reports, leishmaniasis poses a threat to 350 million people in 98 countries. In 2020, nearly 90% of all new VL cases reported to the WHO came from 10 countries: Brazil, China, Ethiopia, Eritrea, India, Kenya, Somalia, South Sudan, Sudan, and Yemen (Yimer et al., 2022). East Africa is currently the most affected region in the world, accounting for 45% of all cases reported to the WHO in 2018. In the Mediterranean region, Central Asia, and South America, VL is extensively dispersed (Carvalho et al., 2021;Maroli et al., 2013). Around 700 cases of autochthonous VL in Southern Europe have been reported annually in nine European Union nations (Dujardin et al., 2008). Each year, 130 cases on average are reported in Morocco (Echchakery et al., 2018) and almost 90% of VL cases can result in mortality if appropriate treatment is not started promptly (van Griensven & Diro, 2012). Few therapeutic options are available, but none of them are currently the ideal option because of high toxicity, exorbitant cost, a lengthy course of therapy, unsuitable mode of administration, and patient's none-compliance which is precipitating the emergence of resistance (Freitas-Junior et al., 2012).

| Chemistry
We synthesized 19 new compounds with various substituents at position 2 of the quinazoline scaffold (Scheme 1).

| In vitro antiprotozoal activities and cell toxicity
Nineteen compounds were synthesized and tested for activity against four parasitic protozoans. Table 1 compiles the IC 50 values of 12 compounds showing activity against one or more parasitic protozoans deployed in this study.
Compounds KJ1 and KJ10 had the best antitrypanosomal activity in this study, exhibiting low micromolar activity of ~5 μM, and ~1 μM against T. b. rhodesiense. The activity data suggest that quinazolinones wherein the aroyl moiety at position 2 bears Br, or a NO 2 unit promote activity against T. b. rhodesiense. KJ10 also demonstrated low micromolar activity against T. b. brucei, exhibiting an IC 50 value of ~4 μM. Compounds in this study were generally not active against T. cruzi, and L. infantum.

F I G U R E 1 Structures of 2-and 4-quinazolinone.
All 19 compounds were also screened against the human fetal lung fibroblast (MRC-5) cell line to investigate potential toxicity to normal human cells. The minimum concentration required to reduce cell viability by 50% (CC 50 ) was determined for all compounds. Only KJ25 and KJ26 exhibited moderate cell toxicity with CC 50 values of 15 μM and 12 μM, respectively. Quinazolinone derivatives investigated in this study generally showed weak to little toxicity against the MRC-5 cell line.
As most of the molecules investigated were more active against T. b. rhodesiense, activity data against this target was used to generate a selectivity index (SI) in relation to the MRC-5 cell line. SI is the ratio of cytotoxicity against MRC-5 cells and activity against T. b. rhodesiense. Compounds with SI values >10 are presumed to have intrinsic activity against the target, with minimal risk to mammalian or human cells. The calculated SI for T. b. rhodesiense ranged from 0.5 to >55.

| Chemical and instrumentation
Reagents and solvents were purchased from Merck (Pty) Ltd and were used as supplied. Thin layer chromatography (TLC), making use of Merck 60F 254 silica gel plates supported on 0.20 mm thick aluminum sheets, was used to monitor reactions. After development, plates were visualized under ultraviolet (UV) light at 254 nm and 366 nm and/or in an iodine chamber. Where necessary, the crude compounds were purified by silica gel column chromatography using Merck Kieselgel 60 Å: 70-230 (0.068-0.2 mm) silica gel mesh. 1 H and 13 C NMR spectra were recorded on a Bruker Biospin 600 MHz spectrometer. Chemical shift data are reported relative to the residual solvent peaks (DMSO d 6 : 2.5 ppm for 1 H and 39.5 ppm for 13 C). MestReNova software was used to process the spectra. The J-coupling constants and chemical shifts were measured in Hertz (Hz) and parts per million (ppm) respectively. Signal multiplicities are denoted as follows: s for a singlet, d for a doublet, dd for the doublet of doublet, ddd for the doublet of the doublet of the doublets, t for a triplet and m for a multiplet. High-resolution mass spectra (HRMS) were recorded on Bruker micrOTOF-Q II mass spectrometer using atmospheric pressure chemical ionization (APCI) technique in positive ion mode. A Büchi® melting point apparatus (Model B-545, AC/DC input 230 V AC) was used to determine the melting points of the target compounds. A Bruker ALPHA FTIR spectrometer with a Diamond Crystal ATR (Attenuated Total Internal Reflectance) accessory was used for the Fourier-transform infrared spectroscopy (FTIR).

| In vitro anti-parasitic assay
Anti-parasitic assays were performed as described elsewhere (Bouton et al., 2021;Hulpia et al., 2019). Briefly, to evaluate anti-Leishmania activity, L. infantum [MHOM/ MA (BE)/67] was used with primary peritoneal mouse macrophages as host cells. 3 × 10 4 macrophages were infected with 4.5 × 10 5 parasites per well. Compound dilutions were added after 2 h of infection. After 5 days of incubation, parasite burdens (mean number of amastigotes/ macrophage) were assessed microscopically after staining with a 10% Giemsa solution. For T. cruzi, the Tulahuen CL2, βgalactosidase strain (nifurtimox-sensitive) was used and maintained on MRC-5SV2 (human lung fibroblast) 4 × 10 3 cells were infected with 4 × 10 4 parasites per well. Parasite burdens were assessed after adding the substrate CPRG (chlorophenol red βd-galactopyranoside). The change in color was measured spectrophotometrically at 540 nm after 4 h incubation at 37°C. Drug susceptibility tests for T. brucei were performed using a resazurin assay. Susceptibility assays were performed with T. brucei Squib 42749 or T. b. rhodesiense STIB-90050. T. brucei Squib 427 was seeded at 1.5 × 10 4 parasites/well and T. b. rhodesiense at 4 × 10 3 parasites per well, followed by the addition of resazurin after 24 h (T. brucei) or 6 h (T. b. rhodesiense). After the addition of resazurin, plates were incubated for another 24 h followed by fluorescence detection.
In all assays, parasite growth was compared to untreatedinfected controls (100% growth) and noninfected controls (0% growth). Results were expressed as % parasite reduction at the different drug concentrations and used to calculate IC 50 values from the dose-response curves.

| In vitro cytotoxicity assay
MRC-5SV2 cell cytotoxicity was evaluated as described elsewhere (Bouton et al., 2021). Briefly, 1.5 × 105 cells/ mL cells were cultured with compound dilutions at 37°C and with 5% CO 2 . Cell growth was compared to untreated-control wells (100% cell growth) and mediumcontrol wells (0% cell growth). After 3 days of incubation, cell viability was assessed fluorimetrically after the addition of 50 μL resazurin per well. After 4 h at 37°C, fluorescence was measured (λ ex 550 nm, λ em 590 nm). The results were expressed as a % reduction in cell growth/ viability compared to control wells and an IC 50 value was determined.

| CONCLUSION
Nineteen 2-aroylquinazolin-4(3H)-ones derivatives were synthesized, characterized, and evaluated in vitro for antiparasitic activities. Two compounds KJ1 and KJ10 are the most intriguing anti-parasitic hits, exhibiting low micromolar activity against T. b. rhodesiense, and T. b. brucei. These compounds showed no inhibitory effect on human cells (MRC-5), with compound KJ10 having a T. b. rhodesiense selectivity index of >55. Compounds in this study possess a low molecular weight and are easy to synthesize. This allows for further analogues, and/or derivatives with drug-like properties to be explored in search of more potent anti-parasitic compounds.