Introduction

Human African Trypanosomiasis (HAT), sometimes referred to as sleeping sickness is a vector-borne parasitic disease. It is caused by protozoans of the genus Trypanosoma and spread by the bite of the blood-sucking tsetse fly (genus Glossina) [1]. There are two types of the disease: the slower-progressing form, which is caused by Trypanosoma brucei gambiense (T. b. gambiense) and is endemic in Western and Central Africa; and the faster-progressing form, which is brought on by Trypanosoma brucei rhodesiense (T. b. rhodesiense) which is prevalent in Eastern and Southern Africa [2,3,4]. The parasite causing HAT are endemic in 30 sub-Saharan African countries, wherein 65 million people are at risk of contracting the disease [5].

HAT progresses through two stages: haemolympathic and meningo-encephalitic stages. The haemolympathic stage is characterized by less severe symptoms, including fever and headache. In the meningo-encephalitic stage, the parasites penetrate the blood-brain barrier, causing neuropsychiatric disorders. If left untreated, this stage eventually leads to death [6].

Over the years, a limited number of medications has been authorized for treating sleeping sickness. Pentamidine is registered for treating the first stage of gambiense-HAT, while nifurtimox-eflornithine combination therapy (NECT) is registered for the treatment of second stage gambiense-HAT. NECT is a shorter and safer treatment compared to nifurtimox, or eflornithine monotherapy [7]. A newly registered oral drug, fexinidazole, is recommended for the treatment of both first and second stages of gambiense-HAT, but its use require medical supervision [8]. In rhodesiense-HAT, the first stage is managed using suramin, while the second stage is treated with melarsoprol, an arsenic derivative. Melarsoprol causes fatal reactive encephalopathy in 3–10% of people receiving it [6] Trials for the use of fexinidazole for the treatment of T. b. rhodesiense-infected patients are ongoing and a recent report indicates that the drug can be successfully used [9].

Chagas disease (CD), also referred to as American Trypanosomiasis (AT), is a potentially fatal zoonotic disease resulting from infection with the parasite Trypanosoma cruzi (T. cruzi). This illness is predominantly observed in Central and South America, and the southern regions of the United States [10, 11]. It impacts around 6–8 million individuals globally, leading to roughly 50,000 fatalities annually. Additionally, an estimated 65–100 million people worldwide are at risk of infection [12].

CD is transmitted by triatomine insects, commonly referred to as ‘kissing bugs’ [13]. The primary mode of transmission occurs when these insects deposit infected fecal material into a wound during/after feeding [13]. Additionally, transmission can take place when hosts consume the infected triatomine or its fecal material [14]. Other routes of transmission include vertical transmission, either through the placenta or maternal milk, direct contact with infected body secretions or blood, transfusions, organ transplantation, contaminated food/drinks and laboratory accidents [12, 15].

Despite more than a century since its identification, CD continues to pose health and economic burden for the majority of Latin American nations [16]. For example, over half a million Disability-Adjusted Life Years (DALY) are lost to CD annually [17,18,19,20,21]. The World Bank and the World Health Organization (WHO) classify CD as the 4th most significant infectious disease, following malaria, tuberculosis, and schistosomiasis [22].

At present, only two drugs, benznidazole and nifurtimox, are available for the treatment of CD [23, 24]. Both benznidazole and nifurtimox can result in diverse adverse side effects which sometimes result in abrupt treatment termination [25]. Benznidazole, favored for its superior safety profile, is typically the primary choice [26]. It is administered orally with daily doses of 5 mg/kg for adults and 7.5 mg/kg for children over 60 days [27].

Visceral leishmaniasis (VL), also known as kala-azar, is a severe sand fly (Lutzomyia and Phlebotomus sp.)-transmitted illness that poses a significant public health threat. According to the WHO, it is classified as a neglected disease, reaching the second and seventh positions among tropical diseases in terms of mortality and DALY, respectively [28]. Two species of Leishmania linked to VL have been identified in West Africa: Leishmania infatum, is primarily found in dogs, and causes sporadic human cases and Leishmania donovani, which is an anthroponotic [29].

Approximately 13,000 cases of VL were reported in 2020 by the WHO [30, 31]. VL is prevalent in over 70 nations spanning all continents and poses a potential risk to approximately 200 million people. Nonetheless, the distribution of VL is concentrated primarily in seven countries: Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan, where over 90% of global VL cases are documented [28, 32,33,34]. Every year, 500,000 new cases of VL and 50,000 associated deaths are estimated to occur [32].

Chalcones, also known as 1,3-diphenyl-2-propene, are intermediates in the synthesis of several biologically important classes of compounds such as flavones, pyrimidines, pyrazoles, indoles [35]. In several cases, chalcone and its derivatives have been identified from numerous medicinal plants like Dracaena cinnabari, Medicago sativa, and Angelica keiskei [36]. There have been reports of chalcones exhibiting a variety of biological and pharmacological properties such as anti-inflammatory, analgesic, anti-oxidant, anti-bacterial, anti-fungal, anti-viral, anti-tumor, and anti-parasitic activity [37,38,39,40]. Figure 1 shows the biological activities of different chalcone derivatives.

Fig. 1
figure 1

Biological activities of different chalcone derivatives [36, 43,44,45]

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Results and discussion

Synthetic chemistry

The compounds of interest were synthesized as outlined in Scheme 1. Amidation reaction between the anhydride (1), and aniline (2) afforded an amide intermediate (3). Intermediate 3 underwent base (NaOH) mediated aldol condensation reaction with appropriate aldehydes to form compound K1, K3-K12, K15-K23, and K25 in 50–86% yield.

Scheme 1
scheme 1

Synthesis of target compounds

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Target compounds were characterized using 1H-NMR, 13C-NMR, HRMS, HPLC and FTIR. The 1H-NMR spectrum displayed a peak at approximately 10.39 ppm, which is characteristics of amide (-CONH-) proton. The singlet peak at ca 2.82 ppm integrating to three protons confirms the presence of N-methyl (NCH3) group. In the 13C NMR, the amide carbon appears ca 168.33 ppm, and the peak ca 29.37 ppm is assigned to NCH3. The IR spectra showed distinctive absorption bands between 1640–1660 cm−1 (C=O), 1570–1605 cm−1 (C=C), 675–995 cm−1 (C-H bending). The chalcone carbonyl carbon appears ca 188.73 ppm. The high-resolution mass spectrometry (HRMS) established the molecular weight of target compounds, which are consistent with their molecular formulae.

In vitro anti-protozoal activities and cell toxicity

Table 1 below, displays the anti-parasitic activity, cytotoxicity, and selectivity index of the hit compounds. The study examined the anti-kinetoplastid efficacy of the synthesized compounds K1, K3-K12, K15-K23, and K25 against T. cruzi, T. b. brucei, T. b. rhodesiense, and L. infantum. Furthermore, all the synthesized compounds were subjected to counter-screening against the human fetal lung fibroblast (MRC-5) cell line, to explore potential toxicity to human cells. The minimum concentration needed to achieve a 50% (CC50) reduction in cell viability was determined for all compounds.

Table 1 This table displays the anti-parasitic activity, cytotoxicity, and Selectivity Index of compounds
Full size table

From the 21 compounds investigated, five antitrypanosomal hit compounds were identified. Compound K9 showed modest to potent activity against various parasites deployed in this study. Compound K4 exhibited sub-micromolar IC50 values of 0.31 ± 0.27 µM and 0.96 ± 0.86 µM against T. b. brucei and T. b. rhodesiense, respectively. Compound K9 also demonstrated significant activity of 0.45 ± 0.14 µM against T. b. brucei and 0.93 ± 0.51 µM against T. b. rhodesiense. Compound K12 demonstrated low micromolar antitrypanosomal activity, with IC50 values of 2.49 ± 2.93 μM against T. b. brucei and 2.11 ± 1.77 μM against T. b. rhodesiense. Compounds K19 and K22 demonstrated modest activity in the range of 8–15 μM against both T. b. rhodesiense and T. b. brucei.

Compounds K4 and K9 demonstrated modest and equipotent activity (IC50 = 8 μM) against L. infantum, while the rest of the compounds exhibited no activity (>64 μM). None of the compounds in this study proved to be active against T. cruzi.

Regarding cytotoxicity against MRC-5, compounds in this study were generally not cytotoxic. They exhibited CC50 values of >64 µM. Most of the hit compounds exhibit a selectivity index (SI) that exceeds 20 and are thus considered to demonstrate intrinsic and selective anti-T. brucei activity. For example, compound K4 exhibited excellent selectivity, with an SI of 160.

Although the number of active compounds is limited to permit an extensive structure-activity relationship (SARs) analyses, it could generally be observed that the nature and position of substituents on the styrenyl moiety influence antiparasitic activity. m-NO2, and p-Cl substituted derivatives showed the best antiparasitic activities, while ortho substitution, and derivatives with electron donating substituents were generally not active.

Material and methods

Chemicals and instrumentation

Reagents and solvents were bought from Merck (Pty) Ltd and were utilized as provided. Reactions were monitored through thin layer chromatography (TLC), which used Merck 60F254 silica gel plates supported on 0.20 mm thick aluminum sheets. Developed plates were visualized with ultraviolet (UV) light at wavelengths of 254 nm and 366 nm or stained in iodine chamber. A Bruker Biospin 600 MHz spectrometer was used to record the 1H and 13C NMR spectra. Chemical shifts are reported relative to residual solvent peaks (DMSO d6: 2.5 ppm for 1H and 39.5 ppm for 13C). The spectra were processed using MestReNova software. Chemical shifts were quantified in parts per million (ppm), whereas J-coupling constants were recorded in hertz (Hz). Signal multiplicities are expressed 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 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. The melting points (m. pt.) of the target compounds were ascertained using a Büchi® melting point equipment (Model B-545, AC/DC input 230 V AC). For the Fourier-transform infrared spectroscopy (FTIR), a Bruker ALPHA FTIR spectrometer equipped with a Diamond Crystal ATR (Attenuated Total Internal Reflectance) accessory was employed. High- performance liquid chromatography (HPLC) using an Agilent 1200 series HPLC system equipped with a quaternary pump and an Agilent 1200 series diode array detector was used to ascertain the purity of the compound. A Venusil XBP C18 column (4.60 × 150 mm, 5 µm) was used for separation, and the mobile phase consisted of 10% methanol and 90% MilliQ water at a flow rate of 1 mL/min. The compounds were prepared in HPLC grade acetonitrile (ACN), and 10 µL of the sample was injected. The samples were examined at 280 nm and 400 nm wavelengths with a flow rate adjusted to 1 mL/min.

Synthesis procedure for target compounds

Amide formations

General procedure for synthetic route for N-(4-acetylphenyl)-2-(methylamino)benzamide

A round-bottomed flask was charged with p-aminoacetophenone (1 g, 5.64 mmol), 1.5 equiv of N-methylisatoic anhydride (1.14 g, 8.47 mmol) and 10 mL of glacial acetic acid. The mixture was heated at 100 °C overnight. After the reaction was completed, the content was poured into water. The resulting precipitate was then filtered, dried to give intermediate N-(4-acetylphenyl)-2-(methylamino)benzamide, which was used in the next step without further purification.

Aldol reaction

A mixture of N-(4-acetylphenyl)-2-(methylamino)benzamide, 1.5 equiv of benzaldehyde, aqueous solution of sodium hydroxide (NaOH; 20%, 10 mL), and 30 mL of ethanol was stirred at room temperature for 15 h. The reaction progress was monitored by TLC. After the completion of the reaction, the mixture was poured over crushed ice, acidified with diluted HCl and the precipitated chalcone was filtered and dried. The crude compound was purified through recrystallization from 95% ethanol to obtain the desired product of 50.16–80.21% yield.

Compounds characterization

(E)-N-(4-(3-(3-fluorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K1)

Brown powder, yield of 50.2% and m. pt. 151.0 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.20 (d, J = 8.2 Hz, 2H), 8.03 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 9.9 Hz, 1H), 7.73 (s, 1H), 7.72–7.68 (m, 2H), 7.53–7.47 (m, 1H), 7.38 (t, J = 7.1 Hz, 1H), 7.28 (t, J = 8.1 Hz, 2H), 6.71 (d, J = 8.2 Hz, 1H), 6.66 (t, J = 7.1 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.43, 168.33, 161.67, 150.21, 143.97, 141.77, 137.34, 133.15, 132.15, 130.80, 130.74, 129.68, 129.07, 125.44, 123.48, 119.60, 116.98, 115.07, 114.03, 110.74, 29.37. IR (ATR) Vmax cm−1: 3319 (N-H stretching), 1681 (C=O stretching), 1649 (C=C stretching), 1218 (C-F stretching) and 736 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20FN2O2 375.1503; found 375.1506. Purity (HPLC): 97%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)benzamide (K3)

Mustard powder, yield of 53.47% and m. pt. 189.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.29 (d, J = 7.8 Hz, 2H), 8.21 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 7.8 Hz, 3H), 8.14 (s, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 15.6 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 6.66 (t, J = 7.1 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.31, 168.33, 150.20, 147.97, 144.16, 141.29, 140.41, 133.17, 131.92, 129.79, 129.73, 129.08, 126.09, 123.85, 119.61, 115.04, 114.05, 110.76, 29.38. IR (ATR) Vmax cm−1: 3378 (N-H stretching), 1653 (C=O stretching), 1594 (C=C stretching), 1516 (N-O stretching) and 732 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20N3O4 402.1448; found 402.1440. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)benzamide (K4)

Mustard powder, yield of 80.21% and m. pt. 102.1–102.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.78 (s, 1H), 8.33 (d, J = 7.1 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.23 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 15.7 Hz, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.1 Hz, 1H), 7.34 (s, 1H), 6.73–6.64 (m, 2H), 3.31 (s, 3H).13 C NMR (151 MHz, DMSO-d6) δ 187.36, 168.34, 150.23, 148.41, 144.09, 140.73, 136.70, 134.98, 133.16, 132.01, 130.27, 129.80, 129.08, 124.79, 124.47, 122.86, 119.58, 115.04, 114.01, 110.72, 29.36. IR (ATR) Vmax cm−1: 3347 (N-H stretching), 1649 (C=O stretching), 1609 (C=C stretching), 1509 (N-O stretching) and 740 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20N3O4 402.1448; found 402.1443. Purity (HPLC): 97%. Rt: 2.2 min.

(E)-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K5)

Brown powder, yield of 86% and m. pt. 171.7–174.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.18–8.08 (m, 2H), 7.97 (d, J = 4.4 Hz, 2H), 7.94 (t, J = 10.7 Hz, 3H), 7.78–7.68 (m, 2H), 7.38 (d, J = 5.6 Hz, 1H), 7.30 (d, J = 4.4 Hz, 3H), 6.73–6.68 (m, 1H), 6.66 (d, J = 5.0 Hz, 1H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.46, 168.32, 164.12, 150.22, 143.83, 142.03, 133.14, 132.31, 131.45, 131.05, 129.57, 129.06, 121.93, 119.60, 115.92, 115.08, 114.02, 110.72, 29.36. IR (ATR) Vmax cm−1: 3356 (N-H stretching), 1648 (C=O stretching), 1608 (C=C stretching), 1334 (C-F stretching) and 740 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20FN2O2 375.1503; found 375.1496. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(3-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K6)

Dark brown powder, yield of 50.80% and m. pt. 157.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.21 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 11.0 Hz, 2H), 8.04 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 5.3 Hz, 1H), 7.71 (t, J = 11.1 Hz, 2H), 7.53–7.46 (m, 2H), 7.38 (t, J = 7.0 Hz, 1H), 6.76–6.63 (m, 2H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.39, 168.33, 150.21, 143.98, 141.54, 137.05, 133.76, 133.15, 132.14, 130.61, 129.94, 129.71, 129.07, 127.81, 127.78, 123.55, 119.59, 115.08, 114.04, 110.74, 29.37. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1651 (C=O stretching), 1592 (C=C stretching), 737 (C-Cl stretching) and 737 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1195. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(o-tolyl)acryloyl)phenyl)benzamide (K7)

Lime green powder, yield of 66.64% and m. pt. 185.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.17 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 6.1 Hz, 2H), 7.98–7.94 (m, 3H), 7.83 (d, J = 15.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.37–7.32 (m, 1H), 7.29 (s, 2H), 6.80 (d, J = 8.1 Hz, 1H), 6.74 (t, J = 7.0 Hz, 1H), 2.84 (s, 3H), 2.45 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.62, 168.07, 149.03, 143.77, 140.39, 137.85, 133.39, 133.12, 132.41, 130.73, 130.20, 129.58, 129.16, 126.81, 126.32, 122.94, 119.69, 116.25, 115.30, 111.87, 30.04, 19.31. IR (ATR) Vmax cm−1: 3306 (N-H stretching), 1657 (C=O stretching), 1597 (C=C stretching), 741 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O2 371.1754; found 371.1736. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-N-(4-(3-(4-bromophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K8)

Light brown powder, yield of 80.13% and m. pt. 190.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 8.01 (s, 1H), 7.98 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 7.9 Hz, 2H), 7.76 (d, J = 7.4 Hz, 1H), 7.71 (s, 1H), 7.69–7.65 (m, 2H), 7.41 (t, J = 7.3 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.75 (t, J = 6.9 Hz, 1H), 2.84 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.47, 168.07, 148.99, 143.87, 141.92, 134.10, 133.15, 132.29, 131.83, 130.69, 129.64, 129.18, 123.77, 122.84, 119.68, 116.27, 115.40, 111.96, 30.10. IR (ATR) Vmax cm−1: 3380 (N-H stretching), 1656 (C=O stretching), 1605 (C=C stretching), 673 (C-Br stretching) and 749 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20BrN2O2 435.0703, found 435.0705. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K9)

Mustard powder, yield of 54.92% and m. pt. 167.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.00 (s, 1H), 7.96 (d, J = 10.9 Hz, 2H), 7.93 (d, J = 8.2 Hz, 3H), 7.74–7.70 (m, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.38 (t, J = 7.7 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.66 (t, J = 7.4 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.43, 168.33, 150.22, 143.91, 141.78, 134.89, 133.76, 133.15, 132.22, 130.44, 129.62, 129.07, 128.88, 122.78, 119.60, 115.07, 114.02, 110.73, 29.36. IR (ATR) Vmax cm−1: 3321 (N-H stretching), 1652 (C=O stretching), 1590 (C=C stretching), 744 (C-Cl stretching) and 744 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1214. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K10)

Lime green powder, yield of 68.65% and m. pt. 175.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.22 (d, J = 7.4 Hz, 1H), 8.19 (d, J = 7.9 Hz, 2H), 8.02 (d, J = 6.1 Hz, 2H), 7.97 (t, J = 9.4 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.47 (dd, J = 9.4, 4.3 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 6.82 (d, J = 8.3 Hz, 1H), 6.76 (t, J = 7.3 Hz, 1H), 2.84 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.38, 168.04, 148.79, 144.01, 137.90, 134.25, 133.15, 132.40, 132.11, 131.83, 129.98, 129.74, 129.20, 128.54, 127.65, 124.81, 119.71, 116.47, 115.62, 112.15, 30.21. IR (ATR) Vmax cm−1: 3314 (N-H stretching), 1658 (C=O stretching), 1602 (C=C stretching) 758 (C-Cl stretching) and 768 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1194. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-N-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K11)

Brown powder, yield of 51.54% and m. pt. 188.7 °C 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.18 (d, J = 7.4 Hz, 2H), 7.95 (d, J = 7.3 Hz, 2H), 7.86 – 7.82 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 15.4 Hz, 1H), 7.54 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.38 (s, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.77 (t, J = 7.1 Hz, 1H), 3.87 (s, 3H), 3.82 (s, 3H), 2.84 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.48, 168.01, 151.20, 149.04, 148.76, 143.84, 143.56, 133.10, 132.73, 129.46, 129.19, 127.63, 123.79, 119.66, 119.57, 116.59, 115.65, 112.17, 111.61, 110.83, 55.78, 55.61, 30.22. IR (ATR) Vmax cm−1: 3357 (N-H stretching), 1652 (C=O stretching), 1594 (C=C stretching), 1256 (C-C stretching), and 730 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C25H25N2O4 417.1778; found 417.1778. Purity (HPLC): 96%. Rt: 2.2 min.

(E)-N-(4-(3-(3,4-dichlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K12)

Mustard powder, yield of 71.92% and m. pt. 195 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.28 (s, 1H), 8.21 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 2H), 7.68 (s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.37 (s, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.28, 168.33, 150.23, 144.04, 140.44, 135.69, 133.16, 132.51, 132.06, 131.77, 130.91, 130.02, 129.73, 129.07, 129.03, 124.08, 119.58, 115.05, 114.02, 110.73, 29.36. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1647 (C=O stretching), 1593 (C=C stretching), 834 (C-Cl stretching) and 741 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H19Cl2N2O2 425.0801; found 425.0801. Purity (HPLC): 95%. Rt: 2.2 min.

E)-2-(methylamino)-N-(4-(3-(4-(trifluoromethyl)phenyl)acryloyl)phenyl)benzamide (K15)

Yellow powder, yield of 63.22% and m. pt. 202.1 °C. 1H NMR (600 MHz, DMSO) δ 10.42 (s, 1H), 8.22 (s, 1H), 8.20 (s, 1H), 8.12 (d, J = 7.8 Hz, 2H), 8.10 (s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.84 – 7.79 (m, 2H), 7.77 (s, 1H), 7.73–7.71 (m, 1H), 7.40–7.32 (m, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.66 (t, J = 7.5 Hz, 1H), 2.81 (d, J = 4.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.46, 168.42, 150.28, 144.18, 141.34, 138.86, 133.24, 132.05, 129.82, 129.39, 129.16, 125.71, 125.69, 124.73, 119.67, 119.53, 115.08, 114.07, 110.77, 29.41. IR (ATR) Vmax cm−1: 3364 (N-H stretching), 1653 (C=O stretching), 1592 (C=C stretching), 1319 (C-F stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H20F3N2O2 425.1456; found 425.1456. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(2,4,6-trimethoxyphenyl)acryloyl)phenyl)benzamide (K16)

Maroon powder, yield of 54.08% and m. pt. 132.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.08 (t, J = 12.9 Hz, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.94–7.88 (m, 3H), 7.71 (d, J = 7.2 Hz, 1H), 7.38 (t, J = 7.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.66 (t, J = 6.8 Hz, 1H), 6.32 (d, J = 7.5 Hz, 3H), 3.93 (s, 6H), 3.86 (s, 3H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 188.54, 168.25, 163.16, 161.31, 150.05, 143.20, 134.56, 133.25, 133.05, 129.05, 128.91, 120.35, 119.72, 115.33, 114.14, 110.78, 105.21, 91.04, 56.06, 55.52, 29.42. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1647 (C=O stretching), 1590 (C=C stretching), 1294 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C26H27N2O5 447.1908; found 447.1908. Purity (HPLC): 100%. Rt: 2.2 min.

(E)-N-(4-(3-(3-methoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K17)

Grey powder, yield of 51.38% and m. pt. 130.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.19 (d, J = 6.3 Hz, 2H), 7.99 (s, 1H), 7.95 (d, J = 7.8 Hz, 2H), 7.72 (s, 1H), 7.70 (s, 1H), 7.49 (s, 1H), 7.44 (s, 1H), 7.41–7.31 (m, 3H), 7.03 (s, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 3.84 (s, 3H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.56, 168.32, 159.63, 150.21, 143.84, 143.27, 136.17, 133.13, 132.30, 129.85, 129.59, 129.06, 122.27, 121.54, 119.59, 116.50, 115.09, 114.01, 113.31, 110.71, 55.28, 29.35. IR (ATR) Vmax cm−1: 3371 (N-H stretching), 1652 (C=O stretching), 1602 (C=C stretching), 1289 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O3 387.1695; found 387.1695. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(2-bromophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K18)

Mustard powder, yield of 61.64% and m. pt. 130.7–134.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.22–8.18 (m, 3H), 7.99 (s, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 7.3 Hz, 1H), 7.73–7.71 (m, 1H), 7.50 (t, J = 7.3 Hz, 1H), 7.41–7.39 (m, 1H), 7.38 (d, J = 7.0 Hz, 1H), 7.34 (d, J = 5.2 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.81 (d, J = 4.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.34, 168.42, 150.27, 144.18, 140.66, 134.10, 133.30, 133.24, 132.07, 129.82, 129.16, 128.74, 128.24, 125.33, 124.93, 119.67, 115.08, 114.07, 110.77, 29.42. IR (ATR) Vmax cm-1: 3359 (N-H stretching), 1650 (C=O stretching), 1590 (C=C stretching), 754 (C-Br stretching) and 736 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20BrN2O2 435.0686; found 435.0703. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K19)

Brown powder, yield of 50.25% and m. pt. 102.2–102.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.18 (s, 2H), 7.98 (d, J = 43.7 Hz, 3H), 7.72 (s, 1H), 7.57 (s, 2H), 7.51–7.28 (m, 2H), 7.05 (s, 2H), 6.70 (s, 1H), 6.67 (s, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.66, 168.31, 153.25, 152.64, 150.20, 143.74, 137.56, 133.11, 132.45, 129.52, 129.06, 123.63, 121.99, 119.59, 117.93, 115.11, 114.01, 113.02, 112.56, 110.70, 56.14, 55.68, 29.35. IR (ATR) Vmax cm−1: 3311 (N-H stretching), 1648 (C=O stretching), 1591 (C=C stretching), 1233 (C-O stretching) and 742 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C25H25N2O4 417.1799; found 417.1799. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(thiophen-3-yl)acryloyl)phenyl)benzamide (K20)

Light mustard powder, yield of 51.82% and m. pt. 114.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.10 (d, J = 1.7 Hz, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 15.4 Hz, 2H), 7.76 (s, 1H), 7.72 (dd, J = 11.1, 4.2 Hz, 1H), 7.67 (dd, J = 4.7, 2.9 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 4.5 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 2.81 (d, J = 4.8 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.80, 168.39, 150.25, 143.86, 138.34, 137.20, 133.19, 132.42, 130.34, 129.52, 129.14, 127.66, 126.19, 121.49, 119.64, 115.16, 114.07, 110.75, 29.42. IR (ATR) Vmax cm−1: 3339 (N-H stretching), 1650 (C=O stretching), 1592 (C=C stretching), 2813 (S-H stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C21H19N2O5S 363.1162; found 363.1139. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(thiophen-2-yl)acryloyl)phenyl)benzamide (K21)

Mustard powder, yield of 51.82% and m. pt. 146.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.11 (d, J = 8.0 Hz, 2H), 7.95–7.91 (m, 3H), 7.89 (s, 1H), 7.78 (d, J = 4.3 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.68 (s, 1H), 7.59 (d, J = 15.3 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.20 (s, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.01, 168.30, 150.19, 143.76, 139.79, 136.03, 133.11, 132.48, 132.23, 130.12, 129.39, 129.06, 128.61, 120.32, 119.64, 115.10, 114.02, 110.71, 29.36. IR (ATR) Vmax cm−1: 3322 (N-H stretching), 1645 (C=O stretching), 1593 (C=C stretching), 2360 (S-H stretching) and 735 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C21H19N2O2S 363.1135; found 363.1135. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)phenyl)-2-(methylamino)benzamide (K22)

Light yellow powder, yield of 67.01% and m. pt. 135.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.17 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 8.7 Hz, 2H), 7.85 (d, J = 15.5 Hz, 1H), 7.74–7.70 (m, 1H), 7.69–7.65 (m, 2H), 7.40–7.31 (m, 3H), 7.00 (d, J = 8.0 Hz, 1H), 6.70 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.4 Hz, 1H), 6.11 (s, 2H), 2.81 (d, J = 4.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.38, 168.38, 150.25, 149.47, 148.11, 143.83, 143.37, 133.18, 132.53, 129.54, 129.36, 129.14, 125.82, 119.96, 119.63, 115.18, 114.06, 110.75, 108.54, 106.94, 101.65, 29.42. IR (ATR) Vmax cm−1: 3420 (N-H stretching), 1656 (C=O stretching), 1588 (C=C stretching), 1217 (C-O stretching) and 746 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H21N2O4 401.1472; found 401.1472. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(2,4,5-trimethoxyphenyl)acryloyl)phenyl)benzamide (K23)

Yellow powder, yield of 60.09% and m. pt. 121.3–123.0 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 15.6 Hz, 1H), 7.96–7.93 (m, 1H), 7.92 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.78 (t, J = 11.9 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.37 (dd, J = 16.1, 9.0 Hz, 1H), 7.33 (s, 1H), 6.75 (s, 1H), 6.70 (dd, J = 8.2, 3.8 Hz, 1H), 6.66 (t, J = 7.0 Hz, 1H), 3.92–3.89 (m, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 2.81 (t, J = 4.5 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 186.83, 168.37, 154.19, 152.78, 150.23, 143.11, 137.87, 133.17, 132.85, 129.42, 129.19, 129.13, 119.61, 119.53, 118.64, 115.22, 114.08, 110.97, 110.74, 97.58, 56.44, 55.84, 29.42, 26.46. IR (ATR) Vmax cm−1: 3337 (N-H stretching), 1651 (C=O stretching), 1591 (C=C stretching) 1266 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C26H27N2O5 447.1898; found 447.1898. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(2-methoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K25)

Light yellow powder, yield of 69.43% and m. pt. 143.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.05 (d, J = 15.7 Hz, 1H), 7.99 (d, J = 7.1 Hz, 1H), 7.95–7.92 (m, 2H), 7.90 (s, 1H), 7.72 (d, J = 7.1 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 4.6 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 3.90 (s, 3H), 2.81 (d, J = 4.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.73, 168.38, 158.21, 150.25, 143.82, 137.85, 133.20, 132.50, 132.19, 129.54, 129.14, 128.39, 123.07, 121.79, 120.70, 119.68, 115.15, 114.07, 111.80, 110.76, 55.74, 29.42. IR (ATR) Vmax cm−1: 3322 (N-H stretching), 1647 (C=O stretching), 1590 (C=C stretching), 1126 (C-O stretching) and 738 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O3 387.1683; found 387.1683. Purity (HPLC): 98%. Rt: 2.2 min.

In vitro anti-parasitic assay

Anti-parasitic assays were performed as described elsewhere [41, 42]. Briefly, to evaluate anti-Leishmania activity, L. infantum [MHOM/ MA (BE)/67] was used with primary peritoneal mouse macrophages as host cells. 3 × 104 macrophages were infected with 4.5 × 105 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, β-galactosidase strain (nifurtimox-sensitive) was used and maintained on MRC-5SV2 (human lung fibroblast). 4 × 103 cells were infected with 4 × 104 parasites per well. Parasite burdens were assessed after adding the substrate CPRG (chlorophenol red β-d- 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 × 104 parasites/well and T. b. rhodesiense at 4 × 103 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 (λex 550 nm, λem 590 nm).

In all assays, parasite growth was compared to untreated-infected controls (100% growth) and noninfected controls (0% growth). Results were expressed as % parasite reduction at the different drug concentrations and used to calculate IC50 values from the dose–response curves.

In vitro cytotoxicity

MRC-5SV2 cell cytotoxicity was evaluated as described elsewhere [41]. Briefly, 1.5 × 105 cells/ mL cells were cultured with compound dilutions at 37 °C and with 5% CO2. Cell growth was compared to untreated-control wells (100% cell growth) and medium-control 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. The results were expressed as a % reduction in cell growth/ viability compared to control wells and an IC50 value was determined.

Conclusion

Parasitic diseases remain a critical global health issue. Aside from their potential fatality, several presently available medications have inherent drawbacks, such as toxicity, questionable efficacy, extended treatment durations, and the emergence of resistance. Twenty-one new chalcone derivatives were synthesized, characterized utilizing variety of spectrometry/spectroscopy techniques such as 1H and 13C NMR & HRMS, and evaluated for their in vitro antiparasitic activities. Two compounds, K4 and K9, stand out as particularly promising, demonstrating significant activity in the low micromolar range against both T. b. brucei and T. b. rhodesiense. These compounds demonstrated no inhibitory impact on human cells (MRC-5). Specifically, K4 exhibited a selectivity index (SI) of 160 for T. b. brucei and 52 for T. b. rhodesiense, respectively. These compounds deserve further preclinical exploration of their therapeutic activity against African trypanosomes.