Effects of Synthetic Ligustrazine-Based Chalcone Derivatives on Trypanosoma brucei brucei and Leishmania spp. Promastigotes

Current medication therapy for leishmaniasis and trypanosomiasis remains a major challenge due to its limited efficacy, significant adverse effects, and inaccessibility. Consequently, locating affordable and effective medications is a pressing concern. Because of their easy-to-understand structure and high functionalization potential, chalcones are promising candidates for use as bioactive agents. Thirteen synthetic ligustrazine-containing chalcones were evaluated for their ability to inhibit the growth of leishmaniasis and trypanosomiasis in etiologic agents. The tetramethylpyrazine (TMP) analogue ligustrazine was chosen as the central moiety for the synthesis of these chalcone compounds. The most effective compound (EC50 = 2.59 µM) was the chalcone derivative 2c, which featured a pyrazin-2-yl amino on the ketone ring and a methyl substitution. Multiple actions were observed for certain derivatives, including 1c, 2a–c, 4b, and 5b, against all strains tested. Eflornithine served as a positive control, and three ligustrazine-based chalcone derivatives, including 1c, 2c, and 4b, had a higher relative potency. Compounds 1c and 2c are particularly efficacious; even more potent than the positive control, they are therefore promising candidates for the treatment of trypanosomiasis and leishmaniasis.


Introduction
There are a number of major neglected tropical diseases (NTDs), but three of the most important are leishmaniasis, human African trypanosomiasis (HAT) (also known as sleeping sickness), and American trypanosomiasis (also known as Chagas disease). In South America, leishmaniasis and Chagas disease are caused by similar trypanosomatid protozoan parasites, and in Africa, leishmaniasis and human African trypanosomiasis (HAT) are co-endemic [1][2][3]. Human and animal populations continue to be threatened by African trypanosomiasis. The lack of effective treatments, drug toxicity, and drug resistance have kept the disease at bay, making it imperative to learn more about the parasite's biology and develop new treatments. HAT and animal African trypanosomiasis (AAT) are two forms of the same neglected tropical disease that pose serious risks to both human and animal populations in endemic areas [4,5]. Between 2016 and 2020, it was predicted that 55 million people in 36 countries across sub-Saharan Africa will be susceptible to HAT [6], which is fatal unless treated. From approximately 40,000 cases in 1998, there were only 992 and 663 new cases recorded in 2019 and 2020, respectively [6]. This represents a substantial drop in the number of cases that has been brought about by multiple control techniques. Even though the illness has not been completely eliminated, its prevalence has been drastically cut down [7]. Given that most patients live in rural areas and conflict zones, these numbers are likely to be low. However, African trypanosomiasis in animals is still a major problem in sub-Saharan Africa [8]. The annual mortality toll from this disease is estimated to be around 3 million, and it is responsible for a yearly economic loss in cow output of around USD 1.0-1.2 billion [7]. Cattle can contract nagana, also known as animal African trypanosomiasis, from the parasite Trypanosoma brucei brucei. Three million cattle are lost every year to the disease, which threatens about 55 million [9]. There is currently no vaccination available for trypanosomes due to the antigenic diversity displayed by the parasites; hence, chemotherapy is the major form of treatment [10]. Current drugs are dangerous because they are poisonous, have undesirable side effects, and are losing their efficacy as a result of drug resistance. As a result, there is an urgent demand for the research and development of new treatments for Trypanosoma that are not only efficient but also risk-free.
Leishmaniasis is an often fatal, parasitic protozoal illness spread mostly via the bite of an infected insect in warm, humid environments [11]. Leishmania spp., a genus of flagellated protozoa, contains over 20 species that are accountable for the illness [12]. The clinical manifestations of leishmaniasis can be divided into two categories. The cutaneous form of leishmaniasis (CL) is by far the most common form of the disease. Because of its catastrophic effects on the skin, which make it difficult for many patients to go about their normal activities, it has become a serious issue in international healthcare. L. major and L. tropica are the species that are responsible for the majority of CL cases across the globe. The presence of the sand fly Phlebotomus papatasi in regions where L. major infections are common is likely to blame for the vast majority of reported cases of the disease in Saudi Arabia and other Arabian countries [13]. The zoonotic form of CL evolved as a result of the global expansion of leishmaniasis in the 20th century, with an estimated yearly incidence in Saudi Arabia of more than 4000 [14,15]. The widespread prevalence of desert rodents (reservoir animals) and sand flies is largely responsible for the endemic nature of CL in several regions in Saudi Arabia. [16,17]. Recent research into the incidence of leishmaniasis in Central Saudi Arabia's Qassim Province found that L. major accounts for 50% of cases, L. tropica 29%, and L. infantum 4% [12].
For the treatment of trypanosomiasis and leishmaniasis, only a handful of antiquated medications are available; unfortunately, these treatments all have similar low safety, effectiveness, and pharmacokinetic profiles. There has been no new clinical drug introduced in decades, despite public and private attempts to speed up the drug discovery process in recent years. In order to effectively combat these illnesses, new and better medications must be developed. These drugs should have high pharmacological activity and bioavailability, low toxicity, a suitable dosing regimen, and parasite resistance bypassing [18].
In light of our prior findings [19], I have gone above and beyond the scope of our previously reported research in an effort to identify novel compounds with antiprotozoal activities [20], which included ligustrazine (tetramethylpyrazine)-based chalcones with new pyrazin-2-yl amino and quinazolin-4-yl amino moieties. In traditional Chinese medicine, TMP is found in the chuanxiong plant (Ligusticum chuanxiong Hort). Based on their high antiparasitic efficacy and minimal toxicity, synthesized oxime and cyclohexanone compounds were identified in a prior investigation as having potential applications against trypanosomiasis and leishmaniasis. Because of their activity against both strains of trypanosomiasis, the compounds identified in that investigation provide promising leads for further structure-based optimization and development against T. brucei brucei [19]. Several strains of Leishmania spp. and Trypanosoma were tested in this investigation of novel α,β-unsaturated carbonyl-based compounds coupled to TMP moiety.

Results and Discussion
In the present study, I demonstrated the effect of synthetic compounds on the growth of promastigotes of L. major, L. mexicana, and a wild-type strain of Trypanosoma brucei brucei (s427-WT) in vitro. The antitrypanosomal and leishmanicidal activities of the compounds are summarized in Table 1.

Antitrypanosomal Activity of Tetramethylpyrazine Containing Chalcone Derivatives
In vitro trypanocidal activity against wild-type (WT) Trypanosoma brucei brucei was tested for all 13 synthetic α,β-unsaturated carbonyl-based tetramethylpyrazine containing chalcone derivatives (Table 1). Ten compounds inhibited the strain, and their EC 50 values were quite high. The chalcone derivative 2c showed the strongest inhibitory activity (EC 50 = 2.59 µM), followed by the chalcone derivative 4b with EC 50 = 22.8 µM, which was nearly as active as 1c with EC 50 = 23.3 µM.
In this study, eflornithine and pentamidine served as positive controls, and it was found that three compounds were significantly more potent than the positive control eflornithine: 1c exhibited the same activity as eflornithine, 2c was ten times more potent, and 4b was also more effective than the positive control. Despite eflornithine's original development for oncology purposes, it was later found to be an efficient antitrypanosomal therapy. It is on the WHO's proposed template for a roster of essential medicines.
All of these compounds feature a central α,β-unsaturated carbonyl-based linker that has been variously replaced with functional groups. Based on the functional moieties that are connected, these compounds fall into one of five categories. In the first category are some recently discovered chalcone derivatives based on ligustrazine that include a quinazolin-4yl amino substitution in the ketone ring's position 4 (1a-c). The second class consists of unheard-of pyrazin-2-yl amino which substituted ketone-4 ligustrazine-based chalcone derivatives (2a-c). The next three classes are simple chalcones; all of these compounds contain ligustrazine in their aldehyde moiety, while different classes of ketones on the other side of 3a-c bear methyl-substituted ketones, 4a-b bear 1-Pyridin-3-yl-ethanone as a ketone moiety, and lastly 5a-b have pyrazine in ketone moiety.
There are three compounds in this first class of ligustrazine-based chalcone derivatives (1a-c): compound 1a, which has no substitutions; compound 1b, which has a chlorine at R 2 ; and compound 1c, which has trimethoxy replacements at R 2 , R 3 , and R 4 . By comparing the trypanocidal activities of the tested compounds (Table 1), it was found that the substitution pattern had a significant impact on the results, with compounds 1a and 1b containing no substitution and 1c containing trimethoxy substitutions at R 2 , R 3 , and R 4 demonstrating three times the potency with an EC 50 = 23.3 µM.
The most interesting and potent effects were exhibited by the second type of ligustrazinebased chalcone derivatives with pyrazin-2-yl amino substitution at position 4 on the ketone ring (2a-c). Among these three compounds (2a-c)were one derivative 2a without any substitution and one derivative 2b in the group bearing methyl substitution at R 1 position which exhibited the same trypanocidal activity, but the third derivative 2c in the group bearing methyl substitution at R 2 position on the pyrazin-2-yl amino moiety showed the highest inhibition as compared to all test compounds in this study with EC 50 = 2.59 µM.
All other tested compounds were simple chalcone derivatives without any extension on ketone moiety as compared to 1a-c and 2a-c. Among these seven chalcone derivatives were three compounds (3a-c) bearing methyl-substituted ketones, two derivatives (4a-b) bearing 1-Pyridin-3-yl-ethanone as a ketone moiety, and two derivatives 5a-b having pyrazine in ketone moiety which showed inhibition of Trypanosoma brucei brucei with EC 50 ranging from 22.0 to above 100.0 µM. On the other hand, one derivative 4b with 1-Pyridin-3-yl-ethanone was found to be a strong inhibitor for the tested strain.
Previously, we [19] found that cyclohexanone derivatives, with a tetramethylpyrazine moiety on both sides of the central cyclohexanone linker, were more active than the respective oximes against the protozoa of the Trypanosoma and Leishmania spp. species when tested in a series of 34 synthetic ligustrazine-containing, unsaturated carbonyl-based compounds and oximes [19]. Hicks and co-workers synthesized and characterized a series of chalcones with a prenyloxy or geranyloxy unit, and they discovered that the position of the substituents in the molecule, whether on the ketone or aldehyde ring of the chalcone system, affected the biological activity and the selectivity index of these compounds [21].

Antileishmanial Activity of Synthesized Chalcone Derivatives
The same 13 synthetic ligustrazine chalcone derivatives were tested on promastigotes of L. major and L. mexicana (Table 1) for 1c and 2a-c, while 1a-b were found to be inactive for L. major.
The remaining three groups of chalcones with ligustrazine on the aldehyde portion; 3a-c bearing methyl-substituted ketones, 4a-b bearing 1-Pyridin-3-yl-ethanone as a ketone moiety, and lastly 5a-b having pyrazine in ketone moiety; were found to be almost inactive or far less effective against both L. mexicana and L. major.
Twenty-five new prenylated chalcones [22] with antiparasitic efficacy against Leishmania mexicana were recently synthesized. The selectivity index (SI) values for these chalcones were also the highest. Antileishmanial action may be affected by the substitution pattern, as shown by the fact that two chalcone derivatives with a meta position substituent in the B ring have been found to exist. In addition, homology modeling was used to generate a three-dimensional structure of L. mexicana fumarate reductase. According to docking studies, prenylated chalcones have the potential to regulate fumarate reductase activity by binding to two essential binding sites [22].

Materials
Compounds of synthetic origin that were tested in this research were ones that we had previously synthesized, characterized, and reported on [20] (Table 2). Each compound was used to prepare a stock solution in 100% DMSO; afterward, the concentration of that standard solution was adjusted appropriately for the experiment by diluting it with complete medium, with the final DMSO content kept below 1%.

Materials
Compounds of synthetic origin that were tested in this research were ones that we had previously synthesized, characterized, and reported on [20] (Table 2). Each compound was used to prepare a stock solution in 100% DMSO; afterward, the concentration of that standard solution was adjusted appropriately for the experiment by diluting it with complete medium, with the final DMSO content kept below 1%. Table 2. Chemical structures of tested synthetic chalcone derivatives [20].

Trypanosoma brucei brucei s427-WT
Trypanosoma brucei brucei s427-WT, the wild-type strain, was employed. According to the method reported by Hirumi and Hirumi, the optimal conditions for the development

Trypanosoma brucei brucei s427-WT
Trypanosoma brucei brucei s427-WT, the wild-type strain, was employed. According to the method reported by Hirumi and Hirumi, the optimal conditions for the development of this strain were a pH 7.4 HMI-9 medium supplemented with 10% heat-inactivated fetal calf serum (FCS, BioSera) and 14 µL/L of 13.4 M -mercaptoethanol (Sigma-Aldrich, St. Louis, CA, USA) [23]. In a flow cabinet, the medium was filtered at 0.22 m (Millipore, Burlington, MA, USA) to remove any bacteria. T. brucei brucei was grown in an incubator and passed three times a week in vented flasks at 37 • C and 5% carbon dioxide.

Leishmania spp. Promastigotes
In essential medium (HOMEM) at 25 • C, pH 7.4, and 10% FCS in polyethylene flasks, we cultivated Leishmania major Friedlin (LmjF) and Leishmania mexicana (MNYC/BZ/62/M379). All of the Leishmania spp. strains were grown as promastigotes in a standard HOMEM (GIBCO, Life Technologies, Paisley, UK) medium that was augmented with 10% heatinactivated fetal bovine serum (FBS; PAA Laboratories, Linz, Austria) and 1% of a penicillinstreptomycin solution (Life Technologies, Carlsbad, CA, USA) and incubated at a temperature of 25 • C. The cultures were swapped out for new media three times a week.

Alamar Blue Assay
As an indication of metabolic function in cells, resazurin sodium salt (Alamar Blue) is widely utilized. To assess the in vitro susceptibility of African trypanosomes or Leishmania spp. cells to test substances, a nonfluorescent blue dye was added to cell cultures with different drugs doses [24,25]. In the absence of drug-induced toxicity, live cells glow a bright red instead of a dull blue. The Alamar Blue solution was made by dissolving 12.5 mg of resazurin sodium salt (Sigma) in 100 mL of phosphate-buffered saline (PBS) at pH 7.4, filter-sterilizing the solution, and then storing it in the dark at 4 • C.

Drug Sensitivity
Using a 20 mM stock solution in DMSO, a solution of 200 µM in HMI-9 medium + 10% FCS was made, and 200 µL of this was put into the first well of a 96-well plate for each test chemical. One hundred microliters of this solution was transferred to a well that already had one hundred microliters of the same medium in it, creating a 1:1 dilution and setting in motion a doubling dilution series across two rows of the plate. In all experiments involving Trypanosoma brucei brucei, eflornithine and pentamidine served as positive controls, whereas for Leishmania spp. promastigotes, only pentamidine served as a positive control, and HMI-9 medium was used as a negative, drug-free control in the final well for each compound. Each well received 100 µL of a 2 × 10 5 cells/mL cell solution, for a final cell density of 1 × 10 5 cells/mL. After 48 h incubation at 37 • C and 5% carbon dioxide, 20 µL of the Alamar Blue solution was added and the plate was put back in the incubator for another 24 h. The plate's fluorescence was measured using a fluorimeter (FluoStar Optima) at excitation and emission wavelengths of 590 nm and 530 nm, respectively; the data were then analyzed using the GraphPad Prism 5 software package, and the EC 50 value was calculated by fitting the fluorescence to a sigmoid curve with a variable slope. Using Leishmania spp. promastigotes, a very similar procedure was performed [26].

Conclusions
Large numbers of individuals in underdeveloped nations are afflicted by neglected tropical diseases including leishmaniasis and trypanosomiasis, both of which are prevalent in tropical locations. The antileishmanial and antitrypanosomal actions of a panel of 13 ligustrazine-based chalcones were tested in vitro. Some chalcones with quinazolin-4yl amino substitution at position 4 on the ketone ring (1c) and with pyrazin-2-yl amino substitution at position 4 on the ketone ring (2a-c) displayed a strong growth inhibition of the promastigotes of L. major, L. mexicana, and a wild-type strain of Trypanosoma brucei brucei (s427-WT) in vitro. Trypanosoma brucei brucei growth suppression was enhanced by compounds 1c, 2c, and 4b compared to the positive control eflornithine. These findings provide evidence for the therapeutic potential of ligustrazine-based chalcone derivatives. Generally speaking, ligustrazine-based chalcone derivatives with pyrazin-2-yl amino and quinazolin-4-yl amino substituents may be promising for the creation of new therapeutic medications against parasite illnesses.
Funding: This research received no external funding.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data has been provided in this article.