{"paper_id":"307978e0-e401-4581-a356-44fa8eaca62f","body_text":"Synthesis of Pyrazolyl Acrylamide-Chalcone Conjugates with Sub-Micromolar Antitrypanosomal Activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of Pyrazolyl Acrylamide-Chalcone Conjugates with Sub-Micromolar Antitrypanosomal Activities Devesh S Agarwal, Karol R. Francisco, Richard M. Beteck, Yashpreet Kaur, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6869476/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract A series of compounds was synthesized in 4-5 steps and studied for their activity against Trypanosoma brucei, the causative agent of human African trypanosomiasis. These compounds were also evaluated for their cytotoxicity against the HEK293 human embryonic kidney cell line. Among the synthesized analogues, 5m (R = 3-pyridyl) was the most active with an EC 50 value of 0.245 ± 0.067 µM against T. brucei , but was also cytotoxic with a CC 50 value of 0.285 ± 0.062 µM. Compounds 5g (R = 4-ClC 6 H 4 ), 5i (R = 3-NO 2 C 6 H 4 ) and 5j (R = 4- NO 2 C 6 H 4 ) were also active with similar EC 50 values of 0.278 ± 0.081, 0.256 ± 0.043 and 0.268 ± 0.061 µM, respectively. Interestingly, the same three compounds were apparently not cytotoxic to human HEK293 cells with CC 50 values >20 µM. Pyrazole Acrylamide Chalcone Trypanosoma brucei drug discovery Figures Figure 1 Figure 2 1. Introduction Human African trypanosomiasis (HAT; sleeping sickness) is a vector borne and neglected tropical disease (NTD) that is caused by the protozoan parasite, Trypanosoma brucei [ ]. Transmitted by the bite of the infected tsetse fly [ ], HAT is endemic in 36 sub-Saharan countries with a total of 70 million people, mainly in rural areas, at risk [ ]. HAT is caused by subspecies of T. brucei , specifically, T. brucei gambiense and T. brucei rhodesiense [ ] Infection caused by T. b. gambiense is chronic in nature, and accounts for 98% of the reported HAT cases with the highest prevalence in central and western Africa [4]. In contrast, infection by T. b. rhodesiense is acute and is mainly found in east and southern Africa; this sub-species can also infect animals [ ]. A third sub-species, T. b. brucei , does not infect humans, and is used as a laboratory model for the species as a whole [5]. The clinical progression of HAT is divided into two stages: hemolymphatic and meningo-encephalitic [ ]. The first stage of disease is characterized by the presence of the parasite in lymph and blood with symptoms such as headache and intermittent fever [ ]. The second stage manifests when the parasite enters and proliferates in the CNS, with associated, and progressively severe, neurological and neuropsychiatric symptoms that terminate in coma and death [ ]. First stage disease, due to T. b. gambiense and T. b. rhodesiense , is treated with pentamidine and suramin [ ]. Second stage disease caused by T. b. gambiense is treated with eflornithine monotherapy or a nifurtimox-eflornithine combination therapy (NECT) [ ] whereas for T. b. rhodesiense , the organoarsenical, is used [ ]. Although these drugs are effective and have been in use for decades, they suffer from several shortcomings such as emergence of drug resistant parasites, life-threatening adverse effects, poor oral bioavailability, limited spectrum of activity and poor efficacy [ ]. In the absence of a vaccine, the treatment of HAT has relied on different drugs that are administered depending on the stage of the disease and the sub-species of parasite involved [ ]. Treatment is further complicated by the lengthy, often parenteral, administration of drugs and that can exhibit serious side effects [ ]. The recent registration of the oral, nitroimidazole drug, fexinidazole [ ], and the continued clinical progression of the trifluoromethylbenzene, acoziborole [ ], will greatly simplify therapy. Nonetheless cross-resistance between drugs has been documented which encourages the search for new treatment options [ ]. In this context, one line of research in the discovery of new treatments for HAT and other kinetoplastid diseases, has involved 1 H -pyrazole derivatives. For example, Bernardino et al . reported the synthesis of 1 H -pyrazole-4-carbohydrazides and their leishmanicidal activity in vitro . The most active compound ( I , Fig. 1 ) generated an EC 50 value (the concentration at which parasite growth is inhibited by 50%) of 50 µM against promastigotes (insect stage) of Leishmania amazonensis [ ]. In 2014, Bekhit et al. reported the synthesis of 1 H -pyrazole derivatives and their antileishmanial (and antimalarial) activities. The most potent derivative ( II , Fig. 1 ) exhibited an EC 50 value of 0.079 µg/mL against promastigotes of Leishmania aethiopica [ ]. In 2021, Rosa et al . reported the synthesis of 1,3,4,5-tetrasubstituted pyrazoles. Against L . amazonensis promastigotes and Trypanosoma cruzi insect-stage epimastigotes, the most active derivative ( III , Fig. 1 ) generated an EC 50 value of 4.5 ± 0.7 µM [ ]. In 2023, Khan et al . reported acrylamide derivatives with activity against of Leishmania major promastigotes; specifically, the most active derivative ( IV , Fig. 1 ) elicited an EC 50 value of 4.6 µM [ ]. In 2024, our group reported the synthesis of imidazo[1,2- a ]pyridine-chalcone amides and studied their anti-trypanosomal activity in vitro. The most active compound ( V , Fig. 1 ) generated an EC 50 value of 1.13 µM against T. b. rhodesiense [ ]. Finally, our group synthesized pyrazolyl amidechalcones conjugates, the most potent of which demonstrated an EC 50 value of 0.46 µM against T. b. rhodesiense [ ]. In our efforts to develop potent antitrypanosomal agents [22, ], we replaced the amide linkage between the pyrazole and chalcone moieties previously reported [23], with an acrylamide functionality, because compounds that contain an acrylamide moiety are reported to show antiparasitic activity [21] (Fig. 2 ). The synthesis of 13 pyrazolyl acrylamide-chalcone conjugates is reported (Scheme 1 , 5a-m). These conjugates were evaluated for their antitrypanosomal activity against T. b. brucei and their cytotoxicity profile against a human embryonic kidney cell line (HEK293). 2. Result and discussion 2.1. Chemistry A series of pyrazolyl acrylamide-chalcone conjugates ( 5a - m ) were synthesized via multiple steps. Initially, ( E )-3-(1,3-diphenyl-1 H -pyrazol-4-yl)acrylic acid ( 1 ) was synthesized as previously reported []. In the second step, 1 was coupled with 1-(4-aminophenyl)ethan-1-one ( 2 ) via TBTU-mediated coupling in the presence of DMAP to give ( E )- N -(4-acetylphenyl)-3-(1,3-diphenyl-1 H -pyrazol-4-yl)acrylamide ( 3 ). Successful synthesis of 3 was confirmed by 1 H NMR, which showed an amide proton (-NH-) at δ 11.05 ppm, and a methyl group (-COCH 3 ) proton at δ 2.44 ppm. Finally, aldol condensation of compound 3 with different aldehydes ( 4a - m ) in the presence of 2N NaOH in EtOH at room temperature (rt) provided pyrazolyl acrylamide-chalcone conjugates ( 5a-m ) in good to excellent yields (50–81%) (Scheme 1). Compounds 3 , 5a - m were characterized using 1 H NMR, 13 C NMR and HRMS. 2.2. Pharmacology Compounds 3 and 5a-m were evaluated for their antitrypanosomal and cytotoxic activities using T. b. brucei Lister 427 and HEK293 cells, respectively. All compounds were initially tested at 8 µM. Compounds that inhibited parasite growth by > 70% were subjected to eight-point dose-response analysis (three biological replicates each in duplicate). EC 50 values were calculated with Prism GraphPad and a sigmoidal four parameter logistic curve, and the data are summarized in Table 1. Compound 3 was inactive against T. b. brucei (Table 1). However, derivatives 5a - m were active with EC 50 values in the range of 0.245 to 1.352 µM. Compounds with electron donating group substituents, i.e ., 5a (R = 2-OCH 3 C 6 H 4 ), 5b (R = 2-OCH 3 C 6 H 4 ) and 5c (R = 2,4,5-tri-OCH 3 C 6 H 2 ) resulted in EC 50 values of 0.356, 0.983 and 1.352 µM, respectively. Compound 5a was apparently non-cytotoxic (CC 50 value > 20 µM, where CC 50 measures the concentration at which cell growth is inhibited by 50%), whereas 5b and 5c exhibited some cytotoxicity with CC 50 values of 6.524 and 5.810 µM, respectively. All of the halogen substituted derivatives ( 5d - h ) were active with similar sub-micromolar EC 50 values (Table 1). This included the mono brominated compound 5d (R = 4-BrC 6 H 4 ), the mono chloro-substituted derivatives, 5e (R = 2-ClC 6 H 4 ), 5f (R = 3-ClC 6 H 4 ) and 5g (R = 4-ClC 6 H 4 ), and the di-chloro substituted derivative 5h (R = 3,4-di-ClC 6 H 3 ). Interestingly, all five halogenated derivatives were apparently non-toxic to the HEK293 cells with CC 50 values > 20 µM. In the case of the nitro-substituted derivatives 5i (R = 3-NO 2 C 6 H 4 ) and 5j (R = 4-NO 2 C 6 H 4 ), similar sub-micromolar EC 50 values were measured, and, again, the compounds were seemingly non-cytotoxic (Table 1). The heterocyclic-substituted derivatives, 5k (R = 2-furyl), 5l (R = 2- thiophenyl) and 5m (3-pyridyl) were all active with similar sub-micromolar EC 50 values and an antiparasitic activity order of 5m (0.245 µM) > 5k (0.339 µM) > 5l (0.520 µM). Whereas 5m was cytotoxic (CC 50 = 0.285 µM), 5k and 5l were apparently non-toxic. The replacement of the amide linkage from our previous work [23] with an acrylamide functionality generally led to an improved growth inhibition of T. b. brucei . For instance, compound VIa with a 3-pyridyl substitution was inactive, whereas its acrylamide analogue 5m was active with an EC 50 value of 0.245 µM. Similar results were observed with the -OCH 3 substitution, whereby the amide, VIb , was inactive but its acrylamide analogue, 5a , was active with an EC 50 value of 0.356 µM. The same trend was observed with the nitro-substituted derivative, VId , which was weakly active (EC 50 = 6.69 µM) whereas the corresponding compound, 5i , had an EC 50 value of 0.256 µM (Fig. 2). 3. Conclusions We designed and synthesized a series of 13 pyrazolyl acrylamide-chalcone conjugates, 5a - m , and measured their activities against T. brucei and HEK293 cells in vitro. All but one of the compounds demonstrated sub-micromolar anti-trypanosomal activity, and all but three were seemingly not cytotoxic. Compared to analogues containing an amide linkage, the current acrylamide-linked compounds, in general, had improved activities against T. brucei. Identifying further differentially substituted derivatives will provide lead molecule(s) for pharmacokinetic studies and in vivo efficacy testing. 4. Materials and methods 4.1. General Reagents and solvents were purchased from various chemical vendors such as Sigma-Aldrich (Pty) Ltd. (Johannesburg, South Africa), Merck (Pty) Ltd. (Johannesburg, South Africa). Reactions were monitored by thin layer chromatography (TLC) using Merck 60F254 silica gel plates (Merck, Johannesburg, South Africa) supported on aluminium sheets. Developed TLC plates were visualized under ultraviolet (UV254 and 366 nm) light or stained with iodine vapour. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Biospin 600 MHz spectrometer at 600 MHz (for 1 H) and 150 MHz (for 13 C). The 1 H NMR chemical shifts ( δ ) were reported in parts per million (ppm) and were measured relative to residual deuterochloroform (CDCl 3 ) (7.26 ppm) or DMSO- d 6 (2.5 ppm). The 13 C NMR chemical shifts ( δ ) were reported in ppm relative to deuterochloroform (CDCl 3 ) (77.0 ppm) or DMSO- d 6 (39.5 ppm). All coupling constants J were reported in Hz. The following abbreviations were used to describe peak splitting patterns: s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet and brs = broad singlet. High resolution mass spectra were recorded on an Agilent Technologies micrOTOF-Q II 2010390 by using atmospheric pressure chemical ionization (APCI) in positive ion mode. Melting points were obtained using Buchi B-545 melting point apparatus and are uncorrected. 4.2. Chemistry 4.2.1. General procedure for the synthesis of (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid ( 1 ) Off-white solid, 3.15 g (90% yield), m.p.: 185–186°C (Lit. m.p.: 184–186°C)[19]. 4.2.2. General procedure for the synthesis of (E)-N-(4-acetylphenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 3 ) To a stirred solution of ( E )-3-(1,3-diphenyl-1 H -pyrazol-4-yl)acrylic acid ( 1 , 3g, 0.0103 mol) in DMF (10 mL), DMAP (1.38g, 0.011 mol) was added at room temperature (rt) and subsequently, TBTU (4.96g, 0.0154 mol) was added. The reaction was stirred at rt for 0.5 hrs, after that 1-(4-aminophenyl)ethan-1-one ( 2 , 1.67g, 0.0124 mol) was added and the reaction was further stirred at rt for 8 h. The progress of the reaction was monitored by TLC. After the completion of the reaction, it was quenched by adding water and the aqueous layer was extracted using ethyl acetate (200 mL) twice. The combined organic layer was concentrated under reduced pressure to give crude product. The crude product was purified by recrystallized using EtOH to give pure 3 as off-white solid. Off-white solid, 3.53 g (84% yield), m.p.: 201–203°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.05 (s, 1H), 9.12 (s, 1H), 8.93 (s, 1H), 8.00–90 (m, 2H), 7.95 (d, J = 5.1 Hz, 2H), 7.83 (d, J = 15.6 Hz, 1H), 7.68 (d, J = 7.1 Hz, 2H), 7.64 (s, 1H), 7.58–7.48 (m, 4H), 7.28 (d, J = 7.1 Hz, 2H), 6.93 (d, J = 15.6 Hz, 1H), 2.44 (s, 3H). 4.2.3. General procedure for the synthesis of pyrazolyl acrylamide chalcone conjugates ( 5a - m ) To a stirred solution of ( E )- N -(4-acetylphenyl)-3-(1,3-diphenyl-1 H -pyrazol-4-yl)acrylamide ( 3 , 0.200 g, 0.490 mmol) in ethanol (5 mL), 2N NaOH (2 mL) was added at rt and the reaction was stirred for 10 minutes. Subsequently, different aldehydes ( 4 , 0.490 mmol) were added, and the reaction was stirred at rt for 8 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched by adding water and the resulted precipitate is filtered under suction. The resulted precipitate is consequently washed with water and finally recrystallized with ethanol to obtain pure products 5a - m . 4.2.3.1. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(3-methoxyphenyl)acryloyl)phenyl)acrylamide ( 5a ) Off-white solid, 0.209 g (81% yield), m.p.: 195–196°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.60 (s, 1H), 9.06 (s, 1H), 8.19 (d, J = 8.3 Hz, 2H), 7.99–7.88 (m, 5H), 7.75–7.63 (m, 4H), 7.59–7.48 (m, 6H), 7.46–7.36 (m, 3H), 7.03 (d, J = 8.2 Hz, 1H), 6.72 (d, J = 15.6 Hz, 1H), 3.84 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 188.0, 164.8, 160.2, 152.6, 144.3, 143.8, 139.5, 136.7, 132.6, 130.4, 130.2, 129.4, 129.1, 128.9, 128.5, 122.8, 122.1, 121.8, 119.3, 119.2, 117.8, 117.1, 113.8, 55.8. HRMS (APCI): m/z calcd for C 34 H 29 N 3 O 3 + 526.2125 [M + H] + , found 526.2115 [M + H] + . 4.2.3.2. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)acrylamide ( 5b ) Off-white solid, 0.209 g (81% yield), m.p.: 201–202°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.67 (s, 1H), 9.07 (s, 1H), 8.17 (s, 1H), 7.98–7.94 (m, 3H), 7.90 (d, J = 6 Hz,1H), 7.86–7.83 (m, 2H), 7.70–7.67 (m, 3H), 7.61–7.55 (m, 6H), 7.53–7.49 (m, 2H), 7.41–7.38 (m, 2H), 7.04 (s, 1H), 6.78 (d, J = 8.3 Hz, 1H), 3.83 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 196.7, 167.2, 164.8, 152.6, 144.2, 139.5, 132.6, 131.8, 131.2, 130.2, 129.9, 129.3, 129.1, 128.8, 128.5, 127.5, 121.9, 119.3, 119.1, 117.9, 114.9, 55.9. HRMS (APCI): m/z calcd for C 34 H 29 N 3 O 3 + 526.2125 [M + H] + , found 526.2107 [M + H] + . 4.2.3.3. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(2,4,5-trimethoxyphenyl)acryloyl)phenyl)acrylamide ( 5c ) Off-white solid, 0.227 g (79% yield), m.p.: 187–188°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.20 (s, 1H), 9.06 (s, 1H), 7.98–7.92 (m, 3H), 7.91–7.84 (m, 2H), 7.68 (d, J = 7.4 Hz, 2H), 7.64–7.54 (m, 6H), 7.53–7.47 (m, 2H), 7.40 (t, J = 7.4 Hz, 1H), 7.17 (s, 2H), 6.80 (d, J = 1.9 Hz, 2H), 3.92 (d, J = 5.3 Hz, 6H), 3.74 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6 ) δ . HRMS (APCI): m/z calcd for C 36 H 32 N 3 O 5 + 586.2336 [M + H] + , found 586.2314 [M + H] + . 4.2.3.4. (E)-N-(4-((E)-3-(4-bromophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 5d ) Off-white solid, 0.222 g (79% yield), m.p.: 197–198°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.86 (s, 1H), 9.11 (s, 1H), 8.95 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.00–7.95 (m, 2H), 7.94–7.91 (m, 3H), 7.72–7.62 (m, 4H), 7.58–7.46 (m, 8H), 7.38–7.31 (m, 2H), 6.85 (d, J = 15.6 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.9, 164.9, 152.6, 151.7, 144.5, 142.4, 139.7, 134.6, 133.1, 132.6, 132.3, 131.2, 130.4, 130.1, 130.0, 129.3, 129.1, 128.8, 128.6, 127.4, 127.1, 124.3, 123.3, 122.1, 119.2, 118.9, 117.9. HRMS (APCI): m/z calcd for C 33 H 25 BrN 3 O 2 + 574.1125, 576.1169 [M + H] + , found 574.1130, 576.1136 [M + H] + . 4.2.3.5. (E)-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 5e ) Off-white solid, 0.205 g (79% yield), m.p.: 196–197°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.78 (s, 1H), 9.09 (s, 1H), 8.25–8.18 (m, 2H), 8.03 (d, J = 4.2 Hz, 1H), 7.97 (d, J = 7.8 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 7.5 Hz, 2H), 7.64 (d, J = 7.8 Hz, 2H), 7.59–7.44 (m, 8H), 7.39 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 6.80 (d, J = 15.7 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.8, 164.9, 152.6, 144.6, 139.5, 138.4, 132.9, 132.3, 131.9, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.2, 127.5, 125.3, 121.9, 119.3, 118.9, 117.9. 4.2.3.6. (E)-N-(4-((E)-3-(3-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 5f ) Off-white solid, 0.200 g (50% yield), m.p.: 189–190°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.79 (s, 1H), 9.10 (s, 1H), 8.21 (d, J = 8.5 Hz, 2H), 8.11–8.03 (m, 2H), 7.97 (d, J = 7.9 Hz, 2H), 7.94–7.90 (m, 3H), 7.68 (d, J = 8.1 Hz, 2H), 7.65–7.62 (m, 1H), 7.59–7.55 (m, 3H), 7.53–7.48 (m, 4H), 7.39 (t, J = 7.4 Hz, 1H), 7.36–7.32 (m, 1H), 6.81 (d, J = 15.5 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.9, 164.9, 152.6, 144.5, 142.1, 139.6, 137.6, 134.3, 132.6, 131.8, 131.2, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.6, 128.3, 127.5, 124.1, 122.0, 119.3, 118.9, 117.9. HRMS (APCI): m/z calcd for C 33 H 25 ClN 3 O 2 + 530.1630 [M + H] + , found 530.1609 [M + H] + . 4.2.3.7. (E)-N-(4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 5g ) Off-white solid, 0.210 g (81% yield), m.p.: 204–205°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.61 (s, 1H), 9.07 (s, 1H), 8.21–8.14 (m, 2H), 8.03–7.92 (m, 5H), 7.92–7.89 (m, 2H), 7.73 (d, J = 15.6 Hz, 1H), 7.70–7.67 (m, 2H), 7.63 (d, J = 15.5 Hz, 1H), 7.60–7.56 (m, 4H), 7.53–7.50 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.31 (t, J = 8.8 Hz, 2H), 6.73 (d, J = 15.5 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.9, 164.8, 152.6, 144.3, 142.6, 139.6, 132.8, 132.6, 131.9, 131.6, 130.4, 130.2, 129.3, 129.1, 128.9, 128.5, 127.5, 122.4, 121.8, 119.3, 119.2, 117.8, 116.5, 116.3. 4.2.3.8. (E)-N-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide ( 5h ) Off-white solid, 0.210 g (76% yield), m.p.: 198–199°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.20 (s, 1H), 9.15 (s, 1H), 8.93 (s, 1H), 8.20 (d, J = 8.4 Hz, 2H), 7.99–7.95 (m, 2H), 7.94–7.90 (m, 3H), 7.76–7.70 (m, 1H), 7.68–7.64 (m, 2H), 7.55–7.46 (m, 7H), 7.36–7.30 (m, 2H), 6.99 (d, J = 15.6 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.8, 164.9, 151.6, 140.9, 139.7, 139.5, 133.2, 132.6, 132.3, 131.5, 131.4, 130.6, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.6, 127.3, 126.9, 119.2, 118.9. HRMS (APCI): m/z calcd for C 33 H 24 Cl 2 N 3 O 2 + 564.1240 [M + H] + , found 564.1256 [M + H] + . 4.2.3.9. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(3-nitrophenyl)acryloyl)phenyl)acrylamide ( 5i ) Off-white solid, 0.214 g (81% yield), m.p.: 201–202°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.78 (s, 1H), 9.08 (s, 1H), 8.79 (s, 1H), 8.34 (d, J = 7.7 Hz, 1H), 8.27 (dd, J = 8.2, 2.3 Hz, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 15.6 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.68 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 15.6 Hz, 1H), 7.59–7.54 (m, 4H), 7.52 (d, J = 7.3 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 6.79 (d, J = 15.6 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.8, 164.8, 152.6, 148.9, 144.6, 141.2, 139.5, 137.2, 135.5, 132.6, 132.4, 131.9, 130.8, 130.6, 130.1, 129.3, 128.5, 127.5, 125.3, 125.0, 123.4, 121.9, 119.3, 119.2, 117.9. HRMS (APCI): m/z calcd for C 33 H 25 N 4 O 4 + 541.1870 [M + H] + , found 541.1849 [M + H] + . 4.2.3.10. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(4-nitrophenyl)acryloyl)phenyl)acrylamide ( 5j ) Off-white solid, 0.217 g (82% yield), m.p.: 215–216°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.68 (s, 1H), 9.07 (s, 1H), 8.35–8.25 (m, 2H), 8.23–8.14 (m, 5H), 7.99–7.95 (m, 2H), 7.94–7.90 (m, 2H), 7.81 (d, J = 15.6 Hz, 1H), 7.72–7.66 (m, 2H), 7.63 (d, J = 15.5 Hz, 1H), 7.60–7.55 (m, 4H), 7.53–7.50 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 6.74 (d, J = 15.7 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.8, 164.8, 152.6, 148.5, 144.6, 141.8, 140.9, 139.5, 132.6, 132.4, 132.0, 131.1, 130.6, 130.3, 130.2, 129.4, 129.1, 128.9, 128.5, 127.5, 126.6, 124.7, 124.4, 121.8, 119.3, 119.2, 117.8. HRMS (APCI): m/z calcd for C 33 H 25 N 4 O 4 + 541.1858 [M + H] + , found 541.1870 [M + H] + . 4.2.3.11. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(thiophen-2-yl)acryloyl)phenyl)acrylamide ( 5k ) Off-white solid, 0.194 g (79% yield), m.p.: 189–190°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.75 (s, 1H), 9.09 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.0 Hz, 2H), 7.94–7.88 (m, 3H), 7.79 (d, J = 5.0 Hz, 1H), 7.70–7.67 (m, 2H), 7.66–7.62 (m, 2H), 7.58–7.49 (m, 6H), 7.39 (t, J = 7.3 Hz, 1H), 7.20 (t, J = 4.3 Hz, 1H), 6.80 (d, J = 15.6 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.5, 164.8, 152.6, 144.3, 140.3, 139.5, 136.6, 133.0, 132.6, 131.8, 130.7, 130.2, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.6, 127.5, 122.0, 120.8, 119.3, 118.9, 117.9. HRMS (APCI): m/z calcd for C 31 H 24 N 3 O 2 S + 502.1584 [M + H] + , found 502.1559 [M + H] + . 4.2.3.12. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(furan-2-yl)acryloyl)phenyl)acrylamide ( 5l ) Off-white solid, 0.193 g (81% yield), m.p.: 205–206°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.60 (s, 1H), 9.06 (s, 1H), 8.09 (d, J = 8.7 Hz, 2H), 7.99–7.95 (m, 2H), 7.92 (d, J = 1.8 Hz, 1H), 7.91–7.87 (m, 2H), 7.70–7.67 (m, 2H), 7.63 (d, J = 15.6 Hz, 1H), 7.60–7.56 (m, 6H), 7.53–7.49 (m, 1H), 7.42–7.38 (m, 1H), 7.10 (d, J = 3.4 Hz, 1H), 6.75–6.68 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.5, 164.8, 152.6, 151.7, 146.5, 144.2, 139.5, 132.7, 132.6, 131.9, 130.4, 130.1,129.3, 129.1, 128.9, 128.5, 127.5, 121.8, 119.3, 119.2, 117.8, 117.1, 113.6. 4.2.3.13. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)acrylamide ( 5m ) Off-white solid, 0.197 g (81% yield), m.p.: 210–211°C, 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.61 (s, 1H), 9.12–8.94 (m, 2H), 8.62 (d, J = 4.6 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.10 (d, J = 15.7 Hz, 1H), 8.00–7.90 (m, 4H), 7.76 (d, J = 15.6 Hz, 1H), 7.71–7.62 (m, 3H), 7.60–7.53 (m, 5H), 7.52–7.46 (m, 2H), 7.40 (t, J = 7.3 Hz, 1H), 6.72 (d, J = 15.5 Hz, 1H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 187.8, 164.8, 152.6, 151.4, 150.7, 144.5, 140.3, 139.6, 135.5, 132.6, 132.5, 131.9, 130.5, 130.1, 129.3, 129.1, 128.9, 128.8, 128.5, 127.5, 124.4, 121.8, 119.3, 119.2, 117.8. HRMS (APCI): m/z calcd for C 32 H 25 N 4 O 2 + 497.1972 [M + H] + , found 497.1979 [M + H] + . 4.3. Biology 4.3.1 T. brucei Cell Culture and Viability Assay Bloodstream forms of T. b. brucei (Lister 427) were cultivated in HMI-9 medium at 37°C in 5% CO 2 , and passaged every 48–72 h. Antitrypanosomal activity was determined using a resazurin viability assay in a 96-well microplate format. Parasites in exponential growth phase were suspended at 2x10 5 parasites/mL in medium. Compounds were diluted in DMSO and were added (1 µL/well) to 96-well polystyrene assay plates. Fresh medium (99 µL/well) was added, followed by the addition of suspended parasites (100 µL/well) to a total density of 2x10 4 parasites/well. Assay plates were incubated at 37°C and 5% CO 2 for 72 h, followed by addition of 20 µL 0.5 mM resazurin (AlfaAesar, Cat. B21187). Assay plates were incubated for 2 h at 37°C. Fluorescence was measured at 531 nm and 595 nm excitation and emission wavelengths, respectively, using a 2104 EnVision® multilabel plate reader. Test compounds were first tested at 8 µM in two separate assays each conducted in quadruplicate (n = 8). Compounds that were active, i.e ., inhibited T. brucei growth by > 70%, were tested in eight-point dose-response assays (two or three separate assays each performed in duplicate; n = 4 or 6) EC 50 values were calculated using Prism GraphPad Version 8.0.1 and a sigmoidal four parameter logistic curve [ ]. 4.3.2 HEK293 Cell Culture and Cytotoxicity Assay HEK293 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37°C and 5% CO 2 , and were sub-cultured when 60–90% confluent. Cytotoxicity against HEK293 cells was determined using a resazurin viability assay in a 96-well microplate format. HEK293 cells were suspended in medium at 4x10 4 cells/mL. Test compounds in DMSO were added to 96-well polystyrene assay plates. Fresh medium (49 µL/well) was added, followed by addition suspended cells (50 µL/well) for a total density of 2x10 4 cells/well. Assay plates were incubated at 37°C and 5% CO 2 for 72 h, followed by addition of 20 µL 0.5 mM resazurin. Test compounds were first tested at 20 µM in two assays each conducted in quadruplicate (n = 8). Compounds that were cytotoxic ( i.e. , > 50% inhibition) were tested in eight-point dose-response assays (two separate assays each performed in duplicate; n = 4). The 50% cytotoxic concentration (CC 50 ) was calculated using Prism GraphPad Version 8.0.1 and a sigmoidal four parameter logistic curve [ ]. Declarations Clinical trial number not applicable. Declarations : Ethical approval : Project was approved by North-West University ethics committee. Consent to participate : Not applicable Consent to publish : Not applicable Conflict of Interest: The authors declare no conflicts of interest. Author Contribution DSA synthesized the compounds and produced the first draft, KF, YK, and AC carried out in vitro biological evaluation. CRC supervised the in vitro biological evaluation. RMB supervised compounds yntheses and compilation of the manuscript. LJL designed and coordinated the studies. All authors read through the draft manuscript. Acknowledgements DS Agarwal acknowledges North-West University, Potchefstroom Campus, South Africa for the post-doctoral funding. The in vitro maintenance and assay of T. brucei and HEK293 cells were, in part, supported by NIH R21AI171824 to CRC. Data Availability All data generated during this study are included in this published article and its supplementary information files. References (a) Altamura F, et al. The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets. Drug Dev Res. 2022; 83 : 225–252; (b) Brun R, et al. Human african trypanosomiasis. The Lancet, 2010; 375 : 148–159; (c) Brun R, et al. The phenomenon of treatment failures in human African trypanosomiasis. Trop Med Int Health. 2001; 6 (11): 906–914; (d) Gao J.-M, et al. Human African trypanosomiasis: the current situation in endemic regions and the risks for non-endemic regions from imported cases. Parasitology, 2020: 147 : 922–931. (a) Steverding D. The history of African trypanosomiasis. Parasit. Vectors. 2008; 1:1–8; (b) Kennedy P,. Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 2013; 12 (2): 186–194. (a) World Health Organization. Trypanosomiasis, Human African (sleeping sickness). Fact sheet 259. World Health Organization. Available : http://www.who.int/mediacentre/factsheets/fs259/en/ . Accessed on 2024, 11; (b) Kennedy P. Update on human African trypanosomiasis (sleeping sickness). J. Neurol. 2019; 266; 2334–2337; (c) De Koning H. The drugs of sleeping sickness: their mechanisms of action and resistance, and a brief history. Trop. med. infect. dis. 2020; 5: 14; (d) Berninger M, et al. Novel lead compounds in pre-clinical development against African sleeping sickness. MedChemComm. 2017; 8: 1872–1890. (a) Márquez-Contreras M. Mechanisms of immune evasion by Trypanosoma brucei. Microbiol. Curr. Res. 2018; 2: 39–44; (b) Wang X, et al. Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei. Protein Expr Purif. 2017, 138, 56–62. (a) Barrett M, et al. The trypanosomiases. The Lancet. 2003; 362: 1469–1480; (b) Franco J, et al. Epidemiology of human African trypanosomiasis. Clin. Epidemiol. 2014; 257–275. Amin D, et al. Identification of stage biomarkers for human African trypanosomiasis. Am. J. Trop. Med. Hyg. 2010; 82: 983. MacLean L, et al. Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response. PLoS Neglected Trop Dis. 2012; 6: e1857. (a) Idro R, et al. Neuroimmunology of common parasitic infections in Africa. Front. Immunol. 2022; 13: 791488; (b) Natuva D, et al. 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Current treatments to control African trypanosomiasis and one health perspective. Microorganisms, 2022; 10: 1298. Kennedy P. G and Rodgers J. Clinical and neuropathogenetic aspects of human African trypanosomiasis. Front Immunol. 2019; 10:39. Kennedy P. G. Human African trypanosomiasis of the CNS: current issues and challenges. J Clin Invest. 2004; 113: 496–504. Bernhard S, et al. Fexinidazole for human African trypanosomiasis, the fruit of a successful public-private partnership. Diseases, 2022; 10: 90. Tarral A, et al. Determination of the optimal single dose treatment for acoziborole, a novel drug for the treatment of human African trypanosomiasis: First-in-Human Study. Clin Pharmacokinet. 2023; 62: 481–491. Baker N, et al. Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 2013; 29: 110–118. Bernardino A, et al. Synthesis and leishmanicidal activities of 1-(4-X-phenyl)-N′-[(4-Y-phenyl) methylene]-1H-pyrazole-4-carbohydrazides. Eur J Med Chem. 2006; 41: 80–87. Bekhit A, et al. Evaluation of some 1H-pyrazole derivatives as a dual acting antimalarial and anti-leishmanial agents. Pak J Pharm Sci. 2014; 27: 1767–1773. da Silva M, et al. Discovery of 1, 3, 4, 5-tetrasubstituted pyrazoles as anti-trypanosomatid agents: Identification of alterations in flagellar structure of L. amazonensis. Bioorg Chem. 2021; 114: 105082. Khan T, et al. Evaluation of the antiparasitic and antifungal activities of synthetic piperlongumine-type cinnamide derivatives: booster effect by halogen substituents. ChemMedChem, 2023; 18: e202300132. Agarwal D, et al Design and synthesis of imidazo [1, 2‐a] pyridine‐chalcone conjugates as antikinetoplastid agents. Chem. Biol. Drug Des. 2024; 103: e14400. Agarwal D, et al. Pyrazolyl amide-chalcones conjugates: Synthesis and antikinetoplastid activity. N-S Arch. Pharmacol. 2024; 398: 4199–4210. (a) Chirwa KA, et al. Tractable Quinolone Hydrazides Exhibiting Sub-Micromolar and Broad Spectrum Antitrypanosomal Activities. ChemMedChem, 2024; 19: e202300667; (b) Beteck RM, et al. In vitro anti-trypanosomal activities of indanone-based chalcones. Drug Res (Stuttg). 2019; 69:337–341. Verma G, et al. Targeting malaria and leishmaniasis: Synthesis and pharmacological evaluation of novel pyrazole-1, 3, 4-oxadiazole hybrids. Part II. Bioorg Chem. 2019; 89: 102986. Lucero B, et al. Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies. ChemMedChem, 2024; 19: e202300656. Francisco, K et al. Structure-activity relationship of dibenzylideneacetone analogs against the neglected disease pathogen, Trypanosoma brucei. Bioorg Med Chem Lett. 2023; 81: 129123. Tables Table 1 is available in the Supplementary Files section. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Table1Antitrypanosomalandcytotoxicactivitiesofcompounds3and5a.docx Scheme1.png Scheme 1. Synthesis of pyrazolyl acrylamide-chalcone conjugates 5a-m. 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Francisco\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California San Diego\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Karol\",\"middleName\":\"R.\",\"lastName\":\"Francisco\",\"suffix\":\"\"},{\"id\":486450264,\"identity\":\"f88d4a5e-72d4-49fa-9b11-03fb8e8893f4\",\"order_by\":2,\"name\":\"Richard M. Beteck\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"North-West University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Richard\",\"middleName\":\"M.\",\"lastName\":\"Beteck\",\"suffix\":\"\"},{\"id\":486450265,\"identity\":\"9b96e032-78bf-4318-918c-f958d5db70cc\",\"order_by\":3,\"name\":\"Yashpreet Kaur\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California San Diego\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yashpreet\",\"middleName\":\"\",\"lastName\":\"Kaur\",\"suffix\":\"\"},{\"id\":486450268,\"identity\":\"6eb95879-ea85-484d-9d14-7a7da7e5bb42\",\"order_by\":4,\"name\":\"Adeline Y. Cheng\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California San Diego\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Adeline\",\"middleName\":\"Y.\",\"lastName\":\"Cheng\",\"suffix\":\"\"},{\"id\":486450269,\"identity\":\"76c4113d-07b1-4c3f-8559-3c46f1b9689c\",\"order_by\":5,\"name\":\"Conor R. Caffrey\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California San Diego\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Conor\",\"middleName\":\"R.\",\"lastName\":\"Caffrey\",\"suffix\":\"\"},{\"id\":486450271,\"identity\":\"1c311a46-4dd9-4b46-9455-6f03fae4f491\",\"order_by\":6,\"name\":\"Lesetja J. Legoabe\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYBACPjDJA2UwNjDIEdTCBiIO8EAZQC3GRGphQGhJbCCohb394eMPMnYgxsUHP3fYpG+XyDFg+FHDYM+PQzMbzxljgwM8ySBGsWHvmbTcnTNyDBh7jjEwSxzAoUUih03iAA8ziJEmwdt2OHfDbaAtvA1Al+LSIv/8+Y8DPPVAxps0yb9t/9MNgFoY/zYw8MjjtIXBDBhih4EM9mPSvG0HEkBamIG2SBjg0sKTYyxxhuc4D5DBbCx7Jtlww/1nBYdljkkYGOLQws9+/OGHyp5qORDj4dsddvIGZw5vfPimxsZeDocWMGDsAcU/jwFcAKhYAo96EPgBItgfEFA1CkbBKBgFIxUAABwjU+wyiXvVAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"North-West University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Lesetja\",\"middleName\":\"J.\",\"lastName\":\"Legoabe\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-06-11 08:23:37\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6869476/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6869476/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":87064395,\"identity\":\"91c02e91-b7c0-4182-980d-5f1526fb5baf\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 17:55:24\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":117714,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSample pyrazolyl acrylamide-chalcone conjugates with anti-kinetoplastid activities\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/31e50f6dabcd5944b4e697b0.jpg\"},{\"id\":87064399,\"identity\":\"0b9e6bd3-3e44-482f-b1d9-5bae079d734a\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 17:55:24\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":105467,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eComparison of pyrazolyl amide-chalcone and pyrazolyl acrylamide-chalcone conjugates and their activity against \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/a51694a7561b8bb3137e638a.jpg\"},{\"id\":87065608,\"identity\":\"3d81b4bf-f587-450b-89e5-2e0fcff7abbc\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 18:19:24\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1692524,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/fd41f803-a5b3-4282-b280-31b0386d995b.pdf\"},{\"id\":87064401,\"identity\":\"c4a629b2-541b-4519-9be7-f675e43d59d3\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 17:55:24\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1615122,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supportinginformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/beb17cff3aab9ab77931e242.docx\"},{\"id\":87064829,\"identity\":\"b4def1ff-7c19-41ea-928e-fb845ec0e527\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 18:03:24\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":116161,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table1Antitrypanosomalandcytotoxicactivitiesofcompounds3and5a.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/4f4ada152d6621aa0b71a4d8.docx\"},{\"id\":87064396,\"identity\":\"9e4e2788-7f66-4381-a331-13507ccaa979\",\"added_by\":\"auto\",\"created_at\":\"2025-07-18 17:55:24\",\"extension\":\"png\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":18389,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eScheme 1.\\u003c/strong\\u003e Synthesis of pyrazolyl acrylamide-chalcone conjugates \\u003cstrong\\u003e5a-m\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Scheme1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6869476/v1/8fea5c8084e583aaafa6ca1f.png\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Synthesis of Pyrazolyl Acrylamide-Chalcone Conjugates with Sub-Micromolar Antitrypanosomal Activities\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eHuman African trypanosomiasis (HAT; sleeping sickness) is a vector borne and neglected tropical disease (NTD) that is caused by the protozoan parasite, \\u003cem\\u003eTrypanosoma brucei\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn1\\\" id=\\\"#FNLinkFn1\\\"\\u003e\\u003c/a\\u003e]. Transmitted by the bite of the infected tsetse fly [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn2\\\" id=\\\"#FNLinkFn2\\\"\\u003e\\u003c/a\\u003e], HAT is endemic in 36 sub-Saharan countries with a total of 70\\u0026nbsp;million people, mainly in rural areas, at risk [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn3\\\" id=\\\"#FNLinkFn3\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003cp\\u003eHAT is caused by subspecies of \\u003cem\\u003eT. brucei\\u003c/em\\u003e, specifically, \\u003cem\\u003eT. brucei gambiense\\u003c/em\\u003e and \\u003cem\\u003eT. brucei rhodesiense\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn4\\\" id=\\\"#FNLinkFn4\\\"\\u003e\\u003c/a\\u003e] Infection caused by \\u003cem\\u003eT. b. gambiense\\u003c/em\\u003e is chronic in nature, and accounts for 98% of the reported HAT cases with the highest prevalence in central and western Africa [4]. In contrast, infection by \\u003cem\\u003eT. b. rhodesiense\\u003c/em\\u003e is acute and is mainly found in east and southern Africa; this sub-species can also infect animals [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn5\\\" id=\\\"#FNLinkFn5\\\"\\u003e\\u003c/a\\u003e]. A third sub-species, \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e, does not infect humans, and is used as a laboratory model for the species as a whole [5].\\u003c/p\\u003e\\u003cp\\u003eThe clinical progression of HAT is divided into two stages: hemolymphatic and meningo-encephalitic [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn6\\\" id=\\\"#FNLinkFn6\\\"\\u003e\\u003c/a\\u003e]. The first stage of disease is characterized by the presence of the parasite in lymph and blood with symptoms such as headache and intermittent fever [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn7\\\" id=\\\"#FNLinkFn7\\\"\\u003e\\u003c/a\\u003e]. The second stage manifests when the parasite enters and proliferates in the CNS, with associated, and progressively severe, neurological and neuropsychiatric symptoms that terminate in coma and death [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn8\\\" id=\\\"#FNLinkFn8\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003cp\\u003eFirst stage disease, due to \\u003cem\\u003eT. b. gambiense\\u003c/em\\u003e and \\u003cem\\u003eT. b. rhodesiense\\u003c/em\\u003e, is treated with pentamidine and suramin [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn9\\\" id=\\\"#FNLinkFn9\\\"\\u003e\\u003c/a\\u003e]. Second stage disease caused by \\u003cem\\u003eT. b. gambiense\\u003c/em\\u003e is treated with eflornithine monotherapy or a nifurtimox-eflornithine combination therapy (NECT) [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn10\\\" id=\\\"#FNLinkFn10\\\"\\u003e\\u003c/a\\u003e] whereas for \\u003cem\\u003eT. b. rhodesiense\\u003c/em\\u003e, the organoarsenical, is used [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn11\\\" id=\\\"#FNLinkFn11\\\"\\u003e\\u003c/a\\u003e]. Although these drugs are effective and have been in use for decades, they suffer from several shortcomings such as emergence of drug resistant parasites, life-threatening adverse effects, poor oral bioavailability, limited spectrum of activity and poor efficacy [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn12\\\" id=\\\"#FNLinkFn12\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn the absence of a vaccine, the treatment of HAT has relied on different drugs that are administered depending on the stage of the disease and the sub-species of parasite involved [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn13\\\" id=\\\"#FNLinkFn13\\\"\\u003e\\u003c/a\\u003e]. Treatment is further complicated by the lengthy, often parenteral, administration of drugs and that can exhibit serious side effects [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn14\\\" id=\\\"#FNLinkFn14\\\"\\u003e\\u003c/a\\u003e]. The recent registration of the oral, nitroimidazole drug, fexinidazole [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn15\\\" id=\\\"#FNLinkFn15\\\"\\u003e\\u003c/a\\u003e], and the continued clinical progression of the trifluoromethylbenzene, acoziborole [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn16\\\" id=\\\"#FNLinkFn16\\\"\\u003e\\u003c/a\\u003e], will greatly simplify therapy. Nonetheless cross-resistance between drugs has been documented which encourages the search for new treatment options [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn17\\\" id=\\\"#FNLinkFn17\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn this context, one line of research in the discovery of new treatments for HAT and other kinetoplastid diseases, has involved 1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazole derivatives. For example, Bernardino \\u003cem\\u003eet al\\u003c/em\\u003e. reported the synthesis of 1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazole-4-carbohydrazides and their leishmanicidal activity \\u003cem\\u003ein vitro\\u003c/em\\u003e. The most active compound (\\u003cb\\u003eI\\u003c/b\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) generated an EC\\u003csub\\u003e50\\u003c/sub\\u003e value (the concentration at which parasite growth is inhibited by 50%) of 50 \\u0026micro;M against promastigotes (insect stage) of \\u003cem\\u003eLeishmania amazonensis\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn18\\\" id=\\\"#FNLinkFn18\\\"\\u003e\\u003c/a\\u003e]. In 2014, Bekhit \\u003cem\\u003eet al.\\u003c/em\\u003e reported the synthesis of 1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazole derivatives and their antileishmanial (and antimalarial) activities. The most potent derivative (\\u003cb\\u003eII\\u003c/b\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) exhibited an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.079 \\u0026micro;g/mL against promastigotes of \\u003cem\\u003eLeishmania aethiopica\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn19\\\" id=\\\"#FNLinkFn19\\\"\\u003e\\u003c/a\\u003e]. In 2021, Rosa \\u003cem\\u003eet al\\u003c/em\\u003e. reported the synthesis of 1,3,4,5-tetrasubstituted pyrazoles. Against \\u003cem\\u003eL\\u003c/em\\u003e. \\u003cem\\u003eamazonensis\\u003c/em\\u003e promastigotes and \\u003cem\\u003eTrypanosoma cruzi\\u003c/em\\u003e insect-stage epimastigotes, the most active derivative (\\u003cb\\u003eIII\\u003c/b\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) generated an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 4.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.7 \\u0026micro;M [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn20\\\" id=\\\"#FNLinkFn20\\\"\\u003e\\u003c/a\\u003e]. In 2023, Khan \\u003cem\\u003eet al\\u003c/em\\u003e. reported acrylamide derivatives with activity against of \\u003cem\\u003eLeishmania major\\u003c/em\\u003e promastigotes; specifically, the most active derivative (\\u003cb\\u003eIV\\u003c/b\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) elicited an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 4.6 \\u0026micro;M [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn21\\\" id=\\\"#FNLinkFn21\\\"\\u003e\\u003c/a\\u003e]. In 2024, our group reported the synthesis of imidazo[1,2-\\u003cem\\u003ea\\u003c/em\\u003e]pyridine-chalcone amides and studied their anti-trypanosomal activity \\u003cem\\u003ein vitro.\\u003c/em\\u003e The most active compound (\\u003cb\\u003eV\\u003c/b\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) generated an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 1.13 \\u0026micro;M against \\u003cem\\u003eT. b. rhodesiense\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn22\\\" id=\\\"#FNLinkFn22\\\"\\u003e\\u003c/a\\u003e]. Finally, our group synthesized pyrazolyl amidechalcones conjugates, the most potent of which demonstrated an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.46 \\u0026micro;M against \\u003cem\\u003eT. b. rhodesiense\\u003c/em\\u003e [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn23\\\" id=\\\"#FNLinkFn23\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn our efforts to develop potent antitrypanosomal agents [22, \\u003ca class=\\\"FNLink\\\" href=\\\"#Fn24\\\" id=\\\"#FNLinkFn24\\\"\\u003e\\u003c/a\\u003e], we replaced the amide linkage between the pyrazole and chalcone moieties previously reported [23], with an acrylamide functionality, because compounds that contain an acrylamide moiety are reported to show antiparasitic activity [21] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The synthesis of 13 pyrazolyl acrylamide-chalcone conjugates is reported (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, 5a-m). These conjugates were evaluated for their antitrypanosomal activity against \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e and their cytotoxicity profile against a human embryonic kidney cell line (HEK293).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"2. Result and discussion\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\"\\u003e\\n \\u003ch2\\u003e2.1. Chemistry\\u003c/h2\\u003e\\n \\u003cp\\u003eA series of pyrazolyl acrylamide-chalcone conjugates (\\u003cstrong\\u003e5a\\u003c/strong\\u003e-\\u003cstrong\\u003em\\u003c/strong\\u003e) were synthesized \\u003cem\\u003evia\\u003c/em\\u003e multiple steps. Initially, (\\u003cem\\u003eE\\u003c/em\\u003e)-3-(1,3-diphenyl-1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazol-4-yl)acrylic acid (\\u003cstrong\\u003e1\\u003c/strong\\u003e) was synthesized as previously reported []. In the second step, \\u003cstrong\\u003e1\\u003c/strong\\u003e was coupled with 1-(4-aminophenyl)ethan-1-one (\\u003cstrong\\u003e2\\u003c/strong\\u003e) via TBTU-mediated coupling in the presence of DMAP to give (\\u003cem\\u003eE\\u003c/em\\u003e)-\\u003cem\\u003eN\\u003c/em\\u003e-(4-acetylphenyl)-3-(1,3-diphenyl-1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazol-4-yl)acrylamide (\\u003cstrong\\u003e3\\u003c/strong\\u003e). Successful synthesis of \\u003cstrong\\u003e3\\u003c/strong\\u003e was confirmed by \\u003csup\\u003e1\\u003c/sup\\u003eH NMR, which showed an amide proton (-NH-) at \\u003cem\\u003e\\u0026delta;\\u003c/em\\u003e 11.05 ppm, and a methyl group (-COCH\\u003csub\\u003e3\\u003c/sub\\u003e) proton at \\u003cem\\u003e\\u0026delta;\\u003c/em\\u003e 2.44 ppm.\\u003c/p\\u003e\\n \\u003cp\\u003eFinally, aldol condensation of compound \\u003cstrong\\u003e3\\u003c/strong\\u003e with different aldehydes (\\u003cstrong\\u003e4a\\u003c/strong\\u003e-\\u003cstrong\\u003em\\u003c/strong\\u003e) in the presence of 2N NaOH in EtOH at room temperature (rt) provided pyrazolyl acrylamide-chalcone conjugates (\\u003cstrong\\u003e5a-m\\u003c/strong\\u003e) in good to excellent yields (50\\u0026ndash;81%) (Scheme 1). Compounds \\u003cstrong\\u003e3\\u003c/strong\\u003e, \\u003cstrong\\u003e5a\\u003c/strong\\u003e-\\u003cstrong\\u003em\\u003c/strong\\u003e were characterized using \\u003csup\\u003e1\\u003c/sup\\u003eH NMR, \\u003csup\\u003e13\\u003c/sup\\u003eC NMR and HRMS.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec4\\\"\\u003e\\n \\u003ch2\\u003e2.2. Pharmacology\\u003c/h2\\u003e\\n \\u003cp\\u003eCompounds \\u003cstrong\\u003e3\\u003c/strong\\u003e and \\u003cstrong\\u003e5a-m\\u003c/strong\\u003e were evaluated for their antitrypanosomal and cytotoxic activities using \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e Lister 427 and HEK293 cells, respectively. All compounds were initially tested at 8 \\u0026micro;M. Compounds that inhibited parasite growth by \\u0026gt;\\u0026thinsp;70% were subjected to eight-point dose-response analysis (three biological replicates each in duplicate). EC\\u003csub\\u003e50\\u003c/sub\\u003e values were calculated with Prism GraphPad and a sigmoidal four parameter logistic curve, and the data are summarized in Table 1.\\u003c/p\\u003e\\n \\u003cp\\u003eCompound \\u003cstrong\\u003e3\\u003c/strong\\u003e was inactive against \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e (Table 1). However, derivatives \\u003cstrong\\u003e5a\\u003c/strong\\u003e-\\u003cstrong\\u003em\\u003c/strong\\u003e were active with EC\\u003csub\\u003e50\\u003c/sub\\u003e values in the range of 0.245 to 1.352 \\u0026micro;M. Compounds with electron donating group substituents, \\u003cem\\u003ei.e\\u003c/em\\u003e., \\u003cstrong\\u003e5a\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2-OCH\\u003csub\\u003e3\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), \\u003cstrong\\u003e5b\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2-OCH\\u003csub\\u003e3\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e) and \\u003cstrong\\u003e5c\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2,4,5-tri-OCH\\u003csub\\u003e3\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e2\\u003c/sub\\u003e) resulted in EC\\u003csub\\u003e50\\u003c/sub\\u003e values of 0.356, 0.983 and 1.352 \\u0026micro;M, respectively. Compound \\u003cstrong\\u003e5a\\u003c/strong\\u003e was apparently non-cytotoxic (CC\\u003csub\\u003e50\\u003c/sub\\u003e value\\u0026thinsp;\\u0026gt;\\u0026thinsp;20 \\u0026micro;M, where CC\\u003csub\\u003e50\\u003c/sub\\u003e measures the concentration at which cell growth is inhibited by 50%), whereas \\u003cstrong\\u003e5b\\u003c/strong\\u003e and \\u003cstrong\\u003e5c\\u003c/strong\\u003e exhibited some cytotoxicity with CC\\u003csub\\u003e50\\u003c/sub\\u003e values of 6.524 and 5.810 \\u0026micro;M, respectively. All of the halogen substituted derivatives (\\u003cstrong\\u003e5d\\u003c/strong\\u003e-\\u003cstrong\\u003eh\\u003c/strong\\u003e) were active with similar sub-micromolar EC\\u003csub\\u003e50\\u003c/sub\\u003e values (Table 1). This included the mono brominated compound \\u003cstrong\\u003e5d\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;4-BrC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), the mono chloro-substituted derivatives, \\u003cstrong\\u003e5e\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2-ClC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), \\u003cstrong\\u003e5f\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;3-ClC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e) and \\u003cstrong\\u003e5g\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;4-ClC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), and the di-chloro substituted derivative \\u003cstrong\\u003e5h\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;3,4-di-ClC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e3\\u003c/sub\\u003e). Interestingly, all five halogenated derivatives were apparently non-toxic to the HEK293 cells with CC\\u003csub\\u003e50\\u003c/sub\\u003e values\\u0026thinsp;\\u0026gt;\\u0026thinsp;20 \\u0026micro;M.\\u003c/p\\u003e\\n \\u003cp\\u003eIn the case of the nitro-substituted derivatives \\u003cstrong\\u003e5i\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;3-NO\\u003csub\\u003e2\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e) and \\u003cstrong\\u003e5j\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;4-NO\\u003csub\\u003e2\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), similar sub-micromolar EC\\u003csub\\u003e50\\u003c/sub\\u003e values were measured, and, again, the compounds were seemingly non-cytotoxic (Table 1).\\u003c/p\\u003e\\n \\u003cp\\u003eThe heterocyclic-substituted derivatives, \\u003cstrong\\u003e5k\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2-furyl), \\u003cstrong\\u003e5l\\u003c/strong\\u003e (R\\u0026thinsp;=\\u0026thinsp;2- thiophenyl) and \\u003cstrong\\u003e5m\\u003c/strong\\u003e (3-pyridyl) were all active with similar sub-micromolar EC\\u003csub\\u003e50\\u003c/sub\\u003e values and an antiparasitic activity order of \\u003cstrong\\u003e5m\\u003c/strong\\u003e (0.245 \\u0026micro;M)\\u0026thinsp;\\u0026gt;\\u0026thinsp;\\u003cstrong\\u003e5k\\u003c/strong\\u003e (0.339 \\u0026micro;M)\\u0026thinsp;\\u0026gt;\\u0026thinsp;\\u003cstrong\\u003e5l\\u003c/strong\\u003e (0.520 \\u0026micro;M). Whereas \\u003cstrong\\u003e5m\\u003c/strong\\u003e was cytotoxic (CC\\u003csub\\u003e50\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;0.285 \\u0026micro;M), \\u003cstrong\\u003e5k\\u003c/strong\\u003e and \\u003cstrong\\u003e5l\\u003c/strong\\u003e were apparently non-toxic.\\u003c/p\\u003e\\n \\u003cdiv\\u003e\\n \\u003ctable id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003eThe replacement of the amide linkage from our previous work [23] with an acrylamide functionality generally led to an improved growth inhibition of \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e. For instance, compound \\u003cstrong\\u003eVIa\\u003c/strong\\u003e with a 3-pyridyl substitution was inactive, whereas its acrylamide analogue \\u003cstrong\\u003e5m\\u003c/strong\\u003e was active with an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.245 \\u0026micro;M. Similar results were observed with the -OCH\\u003csub\\u003e3\\u003c/sub\\u003e substitution, whereby the amide, \\u003cstrong\\u003eVIb\\u003c/strong\\u003e, was inactive but its acrylamide analogue, \\u003cstrong\\u003e5a\\u003c/strong\\u003e, was active with \\u003cstrong\\u003ean\\u003c/strong\\u003e EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.356 \\u0026micro;M. The same trend was observed with the nitro-substituted derivative, \\u003cstrong\\u003eVId\\u003c/strong\\u003e, which was weakly active (EC\\u003csub\\u003e50\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;6.69 \\u0026micro;M) whereas the corresponding compound, \\u003cstrong\\u003e5i\\u003c/strong\\u003e, had an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.256 \\u0026micro;M (Fig. 2).\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"3. Conclusions\",\"content\":\"\\u003cp\\u003eWe designed and synthesized a series of 13 pyrazolyl acrylamide-chalcone conjugates, \\u003cb\\u003e5a\\u003c/b\\u003e-\\u003cb\\u003em\\u003c/b\\u003e, and measured their activities against \\u003cem\\u003eT. brucei\\u003c/em\\u003e and HEK293 cells \\u003cem\\u003ein vitro.\\u003c/em\\u003e All but one of the compounds demonstrated sub-micromolar anti-trypanosomal activity, and all but three were seemingly not cytotoxic. Compared to analogues containing an amide linkage, the current acrylamide-linked compounds, in general, had improved activities against \\u003cem\\u003eT. brucei.\\u003c/em\\u003e Identifying further differentially substituted derivatives will provide lead molecule(s) for pharmacokinetic studies and \\u003cem\\u003ein vivo\\u003c/em\\u003e efficacy testing.\\u003c/p\\u003e\"},{\"header\":\"4. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.1. General\\u003c/h2\\u003e\\u003cp\\u003eReagents and solvents were purchased from various chemical vendors such as Sigma-Aldrich (Pty) Ltd. (Johannesburg, South Africa), Merck (Pty) Ltd. (Johannesburg, South Africa). Reactions were monitored by thin layer chromatography (TLC) using Merck 60F254 silica gel plates (Merck, Johannesburg, South Africa) supported on aluminium sheets. Developed TLC plates were visualized under ultraviolet (UV254 and 366 nm) light or stained with iodine vapour. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Biospin 600 MHz spectrometer at 600 MHz (for \\u003csup\\u003e1\\u003c/sup\\u003eH) and 150 MHz (for \\u003csup\\u003e13\\u003c/sup\\u003eC). The \\u003csup\\u003e1\\u003c/sup\\u003eH NMR chemical shifts (\\u003cem\\u003eδ\\u003c/em\\u003e) were reported in parts per million (ppm) and were measured relative to residual deuterochloroform (CDCl\\u003csub\\u003e3\\u003c/sub\\u003e) (7.26 ppm) or DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e (2.5 ppm). The \\u003csup\\u003e13\\u003c/sup\\u003eC NMR chemical shifts (\\u003cem\\u003eδ\\u003c/em\\u003e) were reported in ppm relative to deuterochloroform (CDCl\\u003csub\\u003e3\\u003c/sub\\u003e) (77.0 ppm) or DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e (39.5 ppm). All coupling constants \\u003cem\\u003eJ\\u003c/em\\u003e were reported in Hz. The following abbreviations were used to describe peak splitting patterns: s\\u0026thinsp;=\\u0026thinsp;singlet, d\\u0026thinsp;=\\u0026thinsp;doublet, t\\u0026thinsp;=\\u0026thinsp;triplet, dd\\u0026thinsp;=\\u0026thinsp;doublet of doublet, m\\u0026thinsp;=\\u0026thinsp;multiplet and brs\\u0026thinsp;=\\u0026thinsp;broad singlet. High resolution mass spectra were recorded on an Agilent Technologies micrOTOF-Q II 2010390 by using atmospheric pressure chemical ionization (APCI) in positive ion mode. Melting points were obtained using Buchi B-545 melting point apparatus and are uncorrected.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.2. Chemistry\\u003c/h2\\u003e\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e4.2.1. General procedure for the synthesis of (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid (\\u003cb\\u003e1\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 3.15 g (90% yield), m.p.: 185\\u0026ndash;186\\u0026deg;C (Lit. m.p.: 184\\u0026ndash;186\\u0026deg;C)[19].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e4.2.2. General procedure for the synthesis of (E)-N-(4-acetylphenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e3\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eTo a stirred solution of (\\u003cem\\u003eE\\u003c/em\\u003e)-3-(1,3-diphenyl-1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazol-4-yl)acrylic acid (\\u003cb\\u003e1\\u003c/b\\u003e, 3g, 0.0103 mol) in DMF (10 mL), DMAP (1.38g, 0.011 mol) was added at room temperature (rt) and subsequently, TBTU (4.96g, 0.0154 mol) was added. The reaction was stirred at rt for 0.5 hrs, after that 1-(4-aminophenyl)ethan-1-one (\\u003cb\\u003e2\\u003c/b\\u003e, 1.67g, 0.0124 mol) was added and the reaction was further stirred at rt for 8 h. The progress of the reaction was monitored by TLC. After the completion of the reaction, it was quenched by adding water and the aqueous layer was extracted using ethyl acetate (200 mL) twice. The combined organic layer was concentrated under reduced pressure to give crude product. The crude product was purified by recrystallized using EtOH to give pure \\u003cb\\u003e3\\u003c/b\\u003e as off-white solid.\\u003c/p\\u003e\\u003cp\\u003eOff-white solid, 3.53 g (84% yield), m.p.: 201\\u0026ndash;203\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 11.05 (s, 1H), 9.12 (s, 1H), 8.93 (s, 1H), 8.00\\u0026ndash;90 (m, 2H), 7.95 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;5.1 Hz, 2H), 7.83 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.68 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.1 Hz, 2H), 7.64 (s, 1H), 7.58\\u0026ndash;7.48 (m, 4H), 7.28 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.1 Hz, 2H), 6.93 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 2.44 (s, 3H).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e4.2.3. General procedure for the synthesis of pyrazolyl acrylamide chalcone conjugates (\\u003cb\\u003e5a\\u003c/b\\u003e-\\u003cb\\u003em\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eTo a stirred solution of (\\u003cem\\u003eE\\u003c/em\\u003e)-\\u003cem\\u003eN\\u003c/em\\u003e-(4-acetylphenyl)-3-(1,3-diphenyl-1\\u003cem\\u003eH\\u003c/em\\u003e-pyrazol-4-yl)acrylamide (\\u003cb\\u003e3\\u003c/b\\u003e, 0.200 g, 0.490 mmol) in ethanol (5 mL), 2N NaOH (2 mL) was added at rt and the reaction was stirred for 10 minutes. Subsequently, different aldehydes (\\u003cb\\u003e4\\u003c/b\\u003e, 0.490 mmol) were added, and the reaction was stirred at rt for 8 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched by adding water and the resulted precipitate is filtered under suction. The resulted precipitate is consequently washed with water and finally recrystallized with ethanol to obtain pure products \\u003cb\\u003e5a\\u003c/b\\u003e-\\u003cb\\u003em\\u003c/b\\u003e.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.1. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(3-methoxyphenyl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5a\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.209 g (81% yield), m.p.: 195\\u0026ndash;196\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.60 (s, 1H), 9.06 (s, 1H), 8.19 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.3 Hz, 2H), 7.99\\u0026ndash;7.88 (m, 5H), 7.75\\u0026ndash;7.63 (m, 4H), 7.59\\u0026ndash;7.48 (m, 6H), 7.46\\u0026ndash;7.36 (m, 3H), 7.03 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.2 Hz, 1H), 6.72 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 3.84 (s, 3H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 188.0, 164.8, 160.2, 152.6, 144.3, 143.8, 139.5, 136.7, 132.6, 130.4, 130.2, 129.4, 129.1, 128.9, 128.5, 122.8, 122.1, 121.8, 119.3, 119.2, 117.8, 117.1, 113.8, 55.8. HRMS (APCI): m/z calcd for C\\u003csub\\u003e34\\u003c/sub\\u003eH\\u003csub\\u003e29\\u003c/sub\\u003eN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e3\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 526.2125 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 526.2115 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.2. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5b\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.209 g (81% yield), m.p.: 201\\u0026ndash;202\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.67 (s, 1H), 9.07 (s, 1H), 8.17 (s, 1H), 7.98\\u0026ndash;7.94 (m, 3H), 7.90 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;6 Hz,1H), 7.86\\u0026ndash;7.83 (m, 2H), 7.70\\u0026ndash;7.67 (m, 3H), 7.61\\u0026ndash;7.55 (m, 6H), 7.53\\u0026ndash;7.49 (m, 2H), 7.41\\u0026ndash;7.38 (m, 2H), 7.04 (s, 1H), 6.78 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.3 Hz, 1H), 3.83 (s, 3H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 196.7, 167.2, 164.8, 152.6, 144.2, 139.5, 132.6, 131.8, 131.2, 130.2, 129.9, 129.3, 129.1, 128.8, 128.5, 127.5, 121.9, 119.3, 119.1, 117.9, 114.9, 55.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e34\\u003c/sub\\u003eH\\u003csub\\u003e29\\u003c/sub\\u003eN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e3\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 526.2125 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 526.2107 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.3. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(2,4,5-trimethoxyphenyl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5c\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.227 g (79% yield), m.p.: 187\\u0026ndash;188\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.20 (s, 1H), 9.06 (s, 1H), 7.98\\u0026ndash;7.92 (m, 3H), 7.91\\u0026ndash;7.84 (m, 2H), 7.68 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 2H), 7.64\\u0026ndash;7.54 (m, 6H), 7.53\\u0026ndash;7.47 (m, 2H), 7.40 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 7.17 (s, 2H), 6.80 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;1.9 Hz, 2H), 3.92 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;5.3 Hz, 6H), 3.74 (s, 3H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e. HRMS (APCI): m/z calcd for C\\u003csub\\u003e36\\u003c/sub\\u003eH\\u003csub\\u003e32\\u003c/sub\\u003eN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e5\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 586.2336 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 586.2314 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.4. (E)-N-(4-((E)-3-(4-bromophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e5d\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.222 g (79% yield), m.p.: 197\\u0026ndash;198\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.86 (s, 1H), 9.11 (s, 1H), 8.95 (s, 1H), 8.18 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.4 Hz, 1H), 8.00\\u0026ndash;7.95 (m, 2H), 7.94\\u0026ndash;7.91 (m, 3H), 7.72\\u0026ndash;7.62 (m, 4H), 7.58\\u0026ndash;7.46 (m, 8H), 7.38\\u0026ndash;7.31 (m, 2H), 6.85 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.9, 164.9, 152.6, 151.7, 144.5, 142.4, 139.7, 134.6, 133.1, 132.6, 132.3, 131.2, 130.4, 130.1, 130.0, 129.3, 129.1, 128.8, 128.6, 127.4, 127.1, 124.3, 123.3, 122.1, 119.2, 118.9, 117.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e33\\u003c/sub\\u003eH\\u003csub\\u003e25\\u003c/sub\\u003eBrN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 574.1125, 576.1169 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 574.1130, 576.1136 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.5. (E)-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e5e\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.205 g (79% yield), m.p.: 196\\u0026ndash;197\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.78 (s, 1H), 9.09 (s, 1H), 8.25\\u0026ndash;8.18 (m, 2H), 8.03 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;4.2 Hz, 1H), 7.97 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.8 Hz, 2H), 7.92 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.4 Hz, 2H), 7.68 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.5 Hz, 2H), 7.64 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.8 Hz, 2H), 7.59\\u0026ndash;7.44 (m, 8H), 7.39 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 7.34 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.3 Hz, 1H), 6.80 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.7 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.8, 164.9, 152.6, 144.6, 139.5, 138.4, 132.9, 132.3, 131.9, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.2, 127.5, 125.3, 121.9, 119.3, 118.9, 117.9.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.6. (E)-N-(4-((E)-3-(3-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e5f\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.200 g (50% yield), m.p.: 189\\u0026ndash;190\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.79 (s, 1H), 9.10 (s, 1H), 8.21 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.5 Hz, 2H), 8.11\\u0026ndash;8.03 (m, 2H), 7.97 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.9 Hz, 2H), 7.94\\u0026ndash;7.90 (m, 3H), 7.68 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.1 Hz, 2H), 7.65\\u0026ndash;7.62 (m, 1H), 7.59\\u0026ndash;7.55 (m, 3H), 7.53\\u0026ndash;7.48 (m, 4H), 7.39 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 7.36\\u0026ndash;7.32 (m, 1H), 6.81 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.5 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.9, 164.9, 152.6, 144.5, 142.1, 139.6, 137.6, 134.3, 132.6, 131.8, 131.2, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.6, 128.3, 127.5, 124.1, 122.0, 119.3, 118.9, 117.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e33\\u003c/sub\\u003eH\\u003csub\\u003e25\\u003c/sub\\u003eClN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 530.1630 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 530.1609 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.7. (E)-N-(4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e5g\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.210 g (81% yield), m.p.: 204\\u0026ndash;205\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.61 (s, 1H), 9.07 (s, 1H), 8.21\\u0026ndash;8.14 (m, 2H), 8.03\\u0026ndash;7.92 (m, 5H), 7.92\\u0026ndash;7.89 (m, 2H), 7.73 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.70\\u0026ndash;7.67 (m, 2H), 7.63 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.5 Hz, 1H), 7.60\\u0026ndash;7.56 (m, 4H), 7.53\\u0026ndash;7.50 (m, 1H), 7.40 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 7.31 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.8 Hz, 2H), 6.73 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.5 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.9, 164.8, 152.6, 144.3, 142.6, 139.6, 132.8, 132.6, 131.9, 131.6, 130.4, 130.2, 129.3, 129.1, 128.9, 128.5, 127.5, 122.4, 121.8, 119.3, 119.2, 117.8, 116.5, 116.3.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.8. (E)-N-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide (\\u003cb\\u003e5h\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.210 g (76% yield), m.p.: 198\\u0026ndash;199\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 11.20 (s, 1H), 9.15 (s, 1H), 8.93 (s, 1H), 8.20 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.4 Hz, 2H), 7.99\\u0026ndash;7.95 (m, 2H), 7.94\\u0026ndash;7.90 (m, 3H), 7.76\\u0026ndash;7.70 (m, 1H), 7.68\\u0026ndash;7.64 (m, 2H), 7.55\\u0026ndash;7.46 (m, 7H), 7.36\\u0026ndash;7.30 (m, 2H), 6.99 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.8, 164.9, 151.6, 140.9, 139.7, 139.5, 133.2, 132.6, 132.3, 131.5, 131.4, 130.6, 130.5, 130.1, 130.0, 129.3, 129.1, 128.8, 128.6, 127.3, 126.9, 119.2, 118.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e33\\u003c/sub\\u003eH\\u003csub\\u003e24\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003eN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 564.1240 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 564.1256 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.9. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(3-nitrophenyl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5i\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.214 g (81% yield), m.p.: 201\\u0026ndash;202\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e6\\u003c/sub\\u003e) δ 10.78 (s, 1H), 9.08 (s, 1H), 8.79 (s, 1H), 8.34 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.7 Hz, 1H), 8.27 (dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.2, 2.3 Hz, 1H), 8.23 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.5 Hz, 2H), 8.18 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.97 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.0 Hz, 2H), 7.93 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.5 Hz, 2H), 7.84 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.75 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.9 Hz, 1H), 7.68 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.2 Hz, 2H), 7.62 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.59\\u0026ndash;7.54 (m, 4H), 7.52 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.3 Hz, 1H), 7.40 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 6.79 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.8, 164.8, 152.6, 148.9, 144.6, 141.2, 139.5, 137.2, 135.5, 132.6, 132.4, 131.9, 130.8, 130.6, 130.1, 129.3, 128.5, 127.5, 125.3, 125.0, 123.4, 121.9, 119.3, 119.2, 117.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e33\\u003c/sub\\u003eH\\u003csub\\u003e25\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003eO\\u003csub\\u003e4\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 541.1870 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 541.1849 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.10. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(4-nitrophenyl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5j\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.217 g (82% yield), m.p.: 215\\u0026ndash;216\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.68 (s, 1H), 9.07 (s, 1H), 8.35\\u0026ndash;8.25 (m, 2H), 8.23\\u0026ndash;8.14 (m, 5H), 7.99\\u0026ndash;7.95 (m, 2H), 7.94\\u0026ndash;7.90 (m, 2H), 7.81 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.72\\u0026ndash;7.66 (m, 2H), 7.63 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.5 Hz, 1H), 7.60\\u0026ndash;7.55 (m, 4H), 7.53\\u0026ndash;7.50 (m, 1H), 7.40 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 1H), 6.74 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.7 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.8, 164.8, 152.6, 148.5, 144.6, 141.8, 140.9, 139.5, 132.6, 132.4, 132.0, 131.1, 130.6, 130.3, 130.2, 129.4, 129.1, 128.9, 128.5, 127.5, 126.6, 124.7, 124.4, 121.8, 119.3, 119.2, 117.8. HRMS (APCI): m/z calcd for C\\u003csub\\u003e33\\u003c/sub\\u003eH\\u003csub\\u003e25\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003eO\\u003csub\\u003e4\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 541.1858 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 541.1870 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec22\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.11. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(thiophen-2-yl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5k\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.194 g (79% yield), m.p.: 189\\u0026ndash;190\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.75 (s, 1H), 9.09 (s, 1H), 8.11 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.4 Hz, 2H), 7.97 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.0 Hz, 2H), 7.94\\u0026ndash;7.88 (m, 3H), 7.79 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;5.0 Hz, 1H), 7.70\\u0026ndash;7.67 (m, 2H), 7.66\\u0026ndash;7.62 (m, 2H), 7.58\\u0026ndash;7.49 (m, 6H), 7.39 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.3 Hz, 1H), 7.20 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;4.3 Hz, 1H), 6.80 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.5, 164.8, 152.6, 144.3, 140.3, 139.5, 136.6, 133.0, 132.6, 131.8, 130.7, 130.2, 130.1, 130.0, 129.3, 129.1, 128.8, 128.7, 128.6, 127.5, 122.0, 120.8, 119.3, 118.9, 117.9. HRMS (APCI): m/z calcd for C\\u003csub\\u003e31\\u003c/sub\\u003eH\\u003csub\\u003e24\\u003c/sub\\u003eN\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003eS\\u003csup\\u003e+\\u003c/sup\\u003e 502.1584 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 502.1559 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec23\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.12. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(furan-2-yl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5l\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.193 g (81% yield), m.p.: 205\\u0026ndash;206\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.60 (s, 1H), 9.06 (s, 1H), 8.09 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.7 Hz, 2H), 7.99\\u0026ndash;7.95 (m, 2H), 7.92 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;1.8 Hz, 1H), 7.91\\u0026ndash;7.87 (m, 2H), 7.70\\u0026ndash;7.67 (m, 2H), 7.63 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.60\\u0026ndash;7.56 (m, 6H), 7.53\\u0026ndash;7.49 (m, 1H), 7.42\\u0026ndash;7.38 (m, 1H), 7.10 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;3.4 Hz, 1H), 6.75\\u0026ndash;6.68 (m, 2H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.5, 164.8, 152.6, 151.7, 146.5, 144.2, 139.5, 132.7, 132.6, 131.9, 130.4, 130.1,129.3, 129.1, 128.9, 128.5, 127.5, 121.8, 119.3, 119.2, 117.8, 117.1, 113.6.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec24\\\" class=\\\"Section4\\\"\\u003e\\u003ch2\\u003e4.2.3.13. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)acrylamide (\\u003cb\\u003e5m\\u003c/b\\u003e)\\u003c/h2\\u003e\\u003cp\\u003eOff-white solid, 0.197 g (81% yield), m.p.: 210\\u0026ndash;211\\u0026deg;C, \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (600 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 10.61 (s, 1H), 9.12\\u0026ndash;8.94 (m, 2H), 8.62 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;4.6 Hz, 1H), 8.36 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.0 Hz, 1H), 8.21 (s, 1H), 8.10 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.7 Hz, 1H), 8.00\\u0026ndash;7.90 (m, 4H), 7.76 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.6 Hz, 1H), 7.71\\u0026ndash;7.62 (m, 3H), 7.60\\u0026ndash;7.53 (m, 5H), 7.52\\u0026ndash;7.46 (m, 2H), 7.40 (t, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.3 Hz, 1H), 6.72 (d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;15.5 Hz, 1H). \\u003csup\\u003e13\\u003c/sup\\u003eC NMR (150 MHz, DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e6\\u003c/em\\u003e\\u003c/sub\\u003e) \\u003cem\\u003eδ\\u003c/em\\u003e 187.8, 164.8, 152.6, 151.4, 150.7, 144.5, 140.3, 139.6, 135.5, 132.6, 132.5, 131.9, 130.5, 130.1, 129.3, 129.1, 128.9, 128.8, 128.5, 127.5, 124.4, 121.8, 119.3, 119.2, 117.8. HRMS (APCI): m/z calcd for C\\u003csub\\u003e32\\u003c/sub\\u003eH\\u003csub\\u003e25\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e 497.1972 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e, found 497.1979 [M\\u0026thinsp;+\\u0026thinsp;H]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec25\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.3. Biology\\u003c/h2\\u003e\\u003cdiv id=\\\"Sec26\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e4.3.1 \\u003cem\\u003eT. brucei\\u003c/em\\u003e Cell Culture and Viability Assay\\u003c/h2\\u003e\\u003cp\\u003eBloodstream forms of \\u003cem\\u003eT. b. brucei\\u003c/em\\u003e (Lister 427) were cultivated in HMI-9 medium at 37\\u0026deg;C in 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e, and passaged every 48\\u0026ndash;72 h. Antitrypanosomal activity was determined using a resazurin viability assay in a 96-well microplate format. Parasites in exponential growth phase were suspended at 2x10\\u003csup\\u003e5\\u003c/sup\\u003e parasites/mL in medium. Compounds were diluted in DMSO and were added (1 \\u0026micro;L/well) to 96-well polystyrene assay plates. Fresh medium (99 \\u0026micro;L/well) was added, followed by the addition of suspended parasites (100 \\u0026micro;L/well) to a total density of 2x10\\u003csup\\u003e4\\u003c/sup\\u003e parasites/well. Assay plates were incubated at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 72 h, followed by addition of 20 \\u0026micro;L 0.5 mM resazurin (AlfaAesar, Cat. B21187). Assay plates were incubated for 2 h at 37\\u0026deg;C. Fluorescence was measured at 531 nm and 595 nm excitation and emission wavelengths, respectively, using a 2104 EnVision\\u0026reg; multilabel plate reader. Test compounds were first tested at 8 \\u0026micro;M in two separate assays each conducted in quadruplicate (n\\u0026thinsp;=\\u0026thinsp;8). Compounds that were active, \\u003cem\\u003ei.e\\u003c/em\\u003e., inhibited \\u003cem\\u003eT. brucei\\u003c/em\\u003e growth by \\u0026gt;\\u0026thinsp;70%, were tested in eight-point dose-response assays (two or three separate assays each performed in duplicate; n\\u0026thinsp;=\\u0026thinsp;4 or 6) EC\\u003csub\\u003e50\\u003c/sub\\u003e values were calculated using Prism GraphPad Version 8.0.1 and a sigmoidal four parameter logistic curve [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn26\\\" id=\\\"#FNLinkFn26\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec27\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e4.3.2 HEK293 Cell Culture and Cytotoxicity Assay\\u003c/h2\\u003e\\u003cp\\u003eHEK293 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e, and were sub-cultured when 60\\u0026ndash;90% confluent. Cytotoxicity against HEK293 cells was determined using a resazurin viability assay in a 96-well microplate format. HEK293 cells were suspended in medium at 4x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/mL. Test compounds in DMSO were added to 96-well polystyrene assay plates. Fresh medium (49 \\u0026micro;L/well) was added, followed by addition suspended cells (50 \\u0026micro;L/well) for a total density of 2x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well. Assay plates were incubated at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 72 h, followed by addition of 20 \\u0026micro;L 0.5 mM resazurin. Test compounds were first tested at 20 \\u0026micro;M in two assays each conducted in quadruplicate (n\\u0026thinsp;=\\u0026thinsp;8). Compounds that were cytotoxic (\\u003cem\\u003ei.e.\\u003c/em\\u003e, \\u0026gt;\\u0026thinsp;50% inhibition) were tested in eight-point dose-response assays (two separate assays each performed in duplicate; n\\u0026thinsp;=\\u0026thinsp;4). The 50% cytotoxic concentration (CC\\u003csub\\u003e50\\u003c/sub\\u003e) was calculated using Prism GraphPad Version 8.0.1 and a sigmoidal four parameter logistic curve [\\u003ca class=\\\"FNLink\\\" href=\\\"#Fn27\\\" id=\\\"#FNLinkFn27\\\"\\u003e\\u003c/a\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eClinical trial number\\u003c/h2\\u003e\\u003cp\\u003enot applicable.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cdiv class=\\\"Heading\\\"\\u003e\\u003cb\\u003eDeclarations\\u003c/b\\u003e:\\u003c/div\\u003e\\u003cp\\u003e\\u003cstrong\\u003e\\u003cb\\u003eEthical approval\\u003c/b\\u003e:\\u003c/strong\\u003e\\u003cp\\u003eProject was approved by North-West University ethics committee.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003e\\u003cb\\u003eConsent to participate\\u003c/b\\u003e:\\u003c/strong\\u003e\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003e\\u003cb\\u003eConsent to publish\\u003c/b\\u003e:\\u003c/strong\\u003e\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest:\\u003c/strong\\u003e\\u003cp\\u003eThe authors declare no conflicts of interest.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eDSA synthesized the compounds and produced the first draft, KF, YK, and AC carried out in vitro biological evaluation. CRC supervised the in vitro biological evaluation. RMB supervised compounds yntheses and compilation of the manuscript. LJL designed and coordinated the studies. All authors read through the draft manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e\\u003cp\\u003eDS Agarwal acknowledges North-West University, Potchefstroom Campus, South Africa for the post-doctoral funding. The \\u003cem\\u003ein vitro\\u003c/em\\u003e maintenance and assay of \\u003cem\\u003eT. brucei\\u003c/em\\u003e and HEK293 cells were, in part, supported by NIH R21AI171824 to CRC.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eAll data generated during this study are included in this published article and its supplementary information files.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Altamura F, et al. The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets. Drug Dev Res. 2022; 83 : 225\\u0026ndash;252; (b) Brun R, et al. Human african trypanosomiasis. The Lancet, 2010; 375 : 148\\u0026ndash;159; (c) Brun R, et al. The phenomenon of treatment failures in human African trypanosomiasis. Trop Med Int Health. 2001; \\u003cem\\u003e6\\u003c/em\\u003e (11): 906\\u0026ndash;914; (d) Gao J.-M, et al. Human African trypanosomiasis: the current situation in endemic regions and the risks for non-endemic regions from imported cases. Parasitology, 2020: 147 : 922\\u0026ndash;931.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Steverding D. The history of African trypanosomiasis. Parasit. Vectors. 2008; 1:1\\u0026ndash;8; (b) Kennedy P,. Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 2013; 12 (2): 186\\u0026ndash;194.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) World Health Organization. Trypanosomiasis, Human African (sleeping sickness). Fact sheet 259. World Health Organization. \\u003cem\\u003eAvailable\\u003c/em\\u003e: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.who.int/mediacentre/factsheets/fs259/en/\\u003c/span\\u003e\\u003cspan address=\\\"http://www.who.int/mediacentre/factsheets/fs259/en/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. \\u003cem\\u003eAccessed on\\u003c/em\\u003e 2024, 11; (b) Kennedy P. Update on human African trypanosomiasis (sleeping sickness). J. Neurol. 2019; 266; 2334\\u0026ndash;2337; (c) De Koning H. The drugs of sleeping sickness: their mechanisms of action and resistance, and a brief history. Trop. med. infect. dis. 2020; 5: 14; (d) Berninger M, et al. Novel lead compounds in pre-clinical development against African sleeping sickness. MedChemComm. 2017; 8: 1872\\u0026ndash;1890.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) M\\u0026aacute;rquez-Contreras M. Mechanisms of immune evasion by Trypanosoma brucei. Microbiol. Curr. Res. 2018; 2: 39\\u0026ndash;44; (b) Wang X, et al. Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei. Protein Expr Purif. 2017, 138, 56\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Barrett M, et al. The trypanosomiases. The Lancet. 2003; 362: 1469\\u0026ndash;1480; (b) Franco J, et al. Epidemiology of human African trypanosomiasis. Clin. Epidemiol. 2014; 257\\u0026ndash;275.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Amin D, et al. Identification of stage biomarkers for human African trypanosomiasis. Am. J. Trop. Med. Hyg. 2010; 82: 983.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e MacLean L, et al. Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response. PLoS Neglected Trop Dis. 2012; 6: e1857.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Idro R, et al. Neuroimmunology of common parasitic infections in Africa. Front. Immunol. 2022; 13: 791488; (b) Natuva D, et al. Molecular mechanisms and therapeutic approaches to the treatment of African Trypanosomiasis. DIT. 2012; 4: (c) Kennedy P. G. The continuing problem of human African trypanosomiasis (sleeping sickness). Ann Neurol. 2008; 64 (2): 116\\u0026ndash;126.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Babokhov P, et al. A current analysis of chemotherapy strategies for the treatment of human African trypanosomiasis. Pathogens and global health. 2013; 107 (5): 242\\u0026ndash;252; (b) Voogd T, et al. Recent research on the biological activity of suramin. Pharmacol Rev. 1993; 45: 177\\u0026ndash;203; (c) Steverding, D. The development of drugs for treatment of sleeping sickness: a historical review. Parasit Vectors. 2010; 3: 1\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e riotto G, et al. Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. The Lancet. 2009; 374; 56\\u0026ndash;64.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e B\\u0026uuml;scher P, et al. Human African trypanosomiasis. The Lancet. 2017; 390; 2397\\u0026ndash;2409.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Venturelli A, et al. Current treatments to control African trypanosomiasis and one health perspective. Microorganisms, 2022; 10: 1298.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Kennedy P. G and Rodgers J. Clinical and neuropathogenetic aspects of human African trypanosomiasis. Front Immunol. 2019; 10:39.\\u003c/span\\u003e \\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Kennedy P. G. Human African trypanosomiasis of the CNS: current issues and challenges. J Clin Invest. 2004; 113: 496\\u0026ndash;504.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Bernhard S, et al. Fexinidazole for human African trypanosomiasis, the fruit of a successful public-private partnership. Diseases, 2022; 10: 90.\\u003c/span\\u003e \\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Tarral A, et al. Determination of the optimal single dose treatment for acoziborole, a novel drug for the treatment of human African trypanosomiasis: First-in-Human Study. Clin Pharmacokinet. 2023; 62: 481\\u0026ndash;491.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Baker N, et al. Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 2013; 29: 110\\u0026ndash;118.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Bernardino A, et al. Synthesis and leishmanicidal activities of 1-(4-X-phenyl)-N\\u0026prime;-[(4-Y-phenyl) methylene]-1H-pyrazole-4-carbohydrazides. Eur J Med Chem. 2006; 41: 80\\u0026ndash;87.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Bekhit A, et al. Evaluation of some 1H-pyrazole derivatives as a dual acting antimalarial and anti-leishmanial agents. Pak J Pharm Sci. 2014; 27: 1767\\u0026ndash;1773.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e da Silva M, et al. Discovery of 1, 3, 4, 5-tetrasubstituted pyrazoles as anti-trypanosomatid agents: Identification of alterations in flagellar structure of L. amazonensis. Bioorg Chem. 2021; 114: 105082.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Khan T, et al. Evaluation of the antiparasitic and antifungal activities of synthetic piperlongumine-type cinnamide derivatives: booster effect by halogen substituents. ChemMedChem, 2023; 18: e202300132.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Agarwal D, et al Design and synthesis of imidazo [1, 2‐a] pyridine‐chalcone conjugates as antikinetoplastid agents. Chem. Biol. Drug Des. 2024; 103: e14400.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Agarwal D, et al. Pyrazolyl amide-chalcones conjugates: Synthesis and antikinetoplastid activity. N-S Arch. Pharmacol. 2024; 398: 4199\\u0026ndash;4210.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e (a) Chirwa KA, et al. Tractable Quinolone Hydrazides Exhibiting Sub-Micromolar and Broad Spectrum Antitrypanosomal Activities. ChemMedChem, 2024; 19: e202300667; (b) Beteck RM, et al. In vitro anti-trypanosomal activities of indanone-based chalcones. Drug Res (Stuttg). 2019; 69:337\\u0026ndash;341.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Verma G, et al. Targeting malaria and leishmaniasis: Synthesis and pharmacological evaluation of novel pyrazole-1, 3, 4-oxadiazole hybrids. Part II. Bioorg Chem. 2019; 89: 102986.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Lucero B, et al. Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies. ChemMedChem, 2024; 19: e202300656.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003e Francisco, K et al. Structure-activity relationship of dibenzylideneacetone analogs against the neglected disease pathogen, Trypanosoma brucei. Bioorg Med Chem Lett. 2023; 81: 129123.\\u003c/span\\u003e \\u003c/li\\u003e\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTable 1 is available in the Supplementary Files section.\\u003c/p\\u003e\"},{\"header\":\"Scheme \",\"content\":\"\\u003cp\\u003eScheme 1 is available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)\",\"snPcode\":\"44371\",\"submissionUrl\":\"https://submission.nature.com/new-submission/44371/3\",\"title\":\"Discover Chemistry\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Pyrazole, Acrylamide, Chalcone, Trypanosoma brucei, drug discovery\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6869476/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6869476/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eA series of compounds was synthesized in 4-5 steps and studied for their activity against \\u003cem\\u003eTrypanosoma brucei, \\u003c/em\\u003ethe causative agent of human African trypanosomiasis. These compounds were also evaluated for their cytotoxicity against the HEK293 human embryonic kidney cell line. Among the synthesized analogues, \\u003cstrong\\u003e5m\\u003c/strong\\u003e (R = 3-pyridyl) was the most active with an EC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.245 ± 0.067 µM against \\u003cem\\u003eT. brucei\\u003c/em\\u003e, but was also cytotoxic with a CC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.285 ± 0.062 µM. Compounds \\u003cstrong\\u003e5g \\u003c/strong\\u003e(R = 4-ClC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e), \\u003cstrong\\u003e5i \\u003c/strong\\u003e(R = 3-NO\\u003csub\\u003e2\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e) and \\u003cstrong\\u003e5j\\u003c/strong\\u003e (R = 4- NO\\u003csub\\u003e2\\u003c/sub\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e4\\u003c/sub\\u003e) were also active with similar EC\\u003csub\\u003e50\\u003c/sub\\u003e values of 0.278 ± 0.081, 0.256 ± 0.043 and 0.268 ± 0.061 µM, respectively. Interestingly, the same three compounds were apparently not cytotoxic to human HEK293 cells with CC\\u003csub\\u003e50\\u003c/sub\\u003e values \\u0026gt;20 µM.\\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\",\"manuscriptTitle\":\"Synthesis of Pyrazolyl Acrylamide-Chalcone Conjugates with Sub-Micromolar Antitrypanosomal Activities\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-07-18 17:55:19\",\"doi\":\"10.21203/rs.3.rs-6869476/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-08-27T07:43:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-29T05:10:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-27T16:25:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-22T16:24:45+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-20T08:07:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"221488604884409033193582946388102938027\",\"date\":\"2025-07-16T15:57:18+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"101000257963311729776163956243980120751\",\"date\":\"2025-07-15T05:00:42+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"115696171922300711639818140442353077139\",\"date\":\"2025-07-15T01:56:46+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"328027886206257686360515268206443920193\",\"date\":\"2025-07-14T08:15:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-07-14T07:25:05+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-07-07T15:57:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-07-04T08:24:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Discover Chemistry\",\"date\":\"2025-07-04T08:21:44+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)\",\"snPcode\":\"44371\",\"submissionUrl\":\"https://submission.nature.com/new-submission/44371/3\",\"title\":\"Discover Chemistry\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"e1f894e6-00d8-49ea-b64a-cfbc142e0b6d\",\"owner\":[],\"postedDate\":\"July 18th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-11-18T09:54:17+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-07-18 17:55:19\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6869476\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6869476\",\"identity\":\"rs-6869476\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}