Synthesis, Characterization, and Antiplasmodial Evaluation of Novel 2-Pyrazoline Carboxamide Derivatives Against Plasmodium berghei in Mice

Synthesis, Characterization, and Antiplasmodial Evaluation of Novel 2-Pyrazoline Carboxamide Derivatives Against Plasmodium berghei in Mice | 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, Characterization, and Antiplasmodial Evaluation of Novel 2-Pyrazoline Carboxamide Derivatives Against Plasmodium berghei in Mice Yusuf Jimoh, Abdullahi Yunusa Idris, Asmau Nasir Hamza, Maryam Abdullahi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7603860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Malaria, caused by Plasmodium species, remains a global health challenge, exacerbated by drug resistance, necessitating novel therapeutic agents. Pyrazoline derivatives have shown promise as antimalarial candidates due to their heterocyclic structure and pharmacological versatility. Two novel 2-pyrazoline carboxamide derivatives, P5 and P13, were synthesized via a base-catalyzed one-pot reaction using substituted acetophenones, benzaldehydes, and semicarbazide in ethanol. Compounds were characterized using FT-IR, ¹H NMR, and ¹³C NMR. Acute oral toxicity was assessed in mice per OECD Test No. 423, and antiplasmodial activity was evaluated against Plasmodium berghei in a curative test. Mice were treated with 25, 50, and 100 mg/kg of P5 and P13, with chloroquine (5 mg/kg) as the positive control. Parasitaemia suppression was calculated, and data analyzed using ANOVA with Dunnett’s post hoc test. P5 and P13 were synthesized with yields of 75.6% and 87.3% respectively and spectroscopic data confirmed their pyrazoline structures. They exhibited no toxicity up to 5000 mg/kg. Both compounds significantly reduced parasitaemia in a dose-dependent manner, with P13 achieving 71.43% chemo suppression and P5 61.52% at 100 mg/kg, compared to chloroquine’s 93.70%. P5 and P13 demonstrate promising antiplasmodial activity and safety, with P13 showing superior efficacy, suggesting their potential as novel antimalarial agents warranting further development. 2-Pyrazoline carboxamide Plasmodium berghei antiplasmodial activity one-pot synthesis spectroscopic characterization acute toxicity malaria treatment Figures Figure 1 Figure 2 1. Introduction Malaria remains a significant global health burden, with over 263 million cases and 597,000 deaths reported in 2023, predominantly caused by Plasmodium species, particularly Plasmodium falciparum and Plasmodium berghei in rodent models [ 1 ]. The emergence of drug-resistant strains, including resistance to artemisinin-based combination therapies, necessitates the development of novel antimalarial agents with distinct mechanisms of action [ 2 – 7 ]. Heterocyclic compounds, such as pyrazolines, have garnered attention due to their diverse pharmacological properties, including antimalarial, antimicrobial, and anti-inflammatory activities [ 8 , 9 ]. The pyrazoline scaffold, characterized by a five-membered ring with two adjacent nitrogen atoms, offers a versatile platform for chemical modification, enhancing its therapeutic potential [ 10 ]. Recent studies have highlighted the efficacy of pyrazoline derivatives as protease inhibitors against Plasmodium parasites, suggesting their role in disrupting parasite lifecycle processes [ 11 ]. One-pot synthesis approaches have emerged as efficient strategies for generating such heterocycles, offering advantages in terms of reduced reaction time, high yields, and environmental sustainability [ 12 ]. Despite these advances, the antimalarial potential of 2-pyrazoline carboxamide derivatives remains underexplored, particularly in vivo models like Plasmodium berghei -infected mice, which closely mimic human malaria pathology [ 13 ]. This study aims to synthesize, characterize, and evaluate the antiplasmodial activity of novel 2-pyrazoline carboxamide derivatives (P5 and P13) using a one-pot reaction protocol, with the goal of identifying promising candidates for malaria treatment. By integrating spectroscopic characterization and in - vivo curative testing, this work seeks to contribute to the growing body of evidence supporting pyrazoline-based therapeutics 2. Materials and methods 2.1 Materials 2.1.1 Equipment and glassware The melting points of the compounds were determined using Gallenkamp melting point apparatus and were uncorrected, Proton Nuclear Magnetic Resonance and Carbon-13 Nuclear Magnetic Resonance were carried-out at the Jodrell Laboratory, NCPH, Royal Botanical Gardens, Kew, UK, Using 400MHz Brucker. The Nuclear Magnetic Resonance data were recorded in chemical shift(δ) in ppm downfield from Tetra-Methyl Silane (as the internal standard). Also the Fourier Transform Infared (FTIR) spectra were carried out at the Multiuser Science Research Laboratories, Ahmadu Bello University, Zaria, recorded on Agilent spectrophotometer as wave numbers (cm − 1 ). Some of the appliances and glassware used include; Erlenmeyer flasks, sapatulas, testubes, beakers, measuring cylinder, glass rod, funnel, pipette, watch glass, microscope slide, syringes, capillary tubes, TLC Tank, magnetic stirrer, stir bar, refrigerator, Gallenkamp melting point device, microtiter plate, analytical balance microscope. 2.1.2 Reagents, solvents, and standard drugs All starting reagents and solvents used were of analytical grade which include: acetophenone, 2,4,5-trimethoxyacetophenone, 2,4-dichlorobenzaldehyde, piperonaldehyde, semicarbazide hydrochloride, ethanol, methanol, hexane, ethylacetate, sodium hydroxide and Chloroquine Sodium. All the reagents were purchased from Sigma Aldrich, Germany. 2.1.3 Experimental animals Fifty mice weighing 18-22g were bought from the Animal House, Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria and kept under standard laboratory conditions. The animals were kept in clean polypropylene cages at the Animal House of the Department of Pharmacology and Therapeutics. All experimental protocols were in accordance with Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals as accepted internationally. 2.1.4 Malarial parasite Chloroquine-sensitive malaria parasites ( Plasmodium berghei NK65) were obtained from the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. The mice were inoculated intraperitoneally, with 0.2ml standard inoculum containing approximately 1 x 10 7 parasitized red blood cells. 2.2 Methods 2.2.1 Procedure for the one-port synthesis of the compound P5 In a round bottom flask, a mixture of 2,4,5-trimethoxyacetophenone (7 mmol), 2,4-dichlorobenzaldehyde (7 mmol), and semicarbazide hydrochloride (10 mmol) were dissolved in absolute ethanol (30 ml). To this mixture a catalytic amount of sodium hydroxide (12 mmol) was added and refluxed for 6hrs with stirring as shown in scheme 1 below. The completion of reaction was monitored by thin layer chromatography (TLC) in methanol/ethyl acetate (9:1) as a solvent system. The solution was cooled to room temperature, filtered the obtained solid, washed with cold ethanol, and dried. The pure target compounds (P5) was obtained by recrystallization from methanol. 2.2.2 Procedure for the one-port synthesis of the compound P13 For P13, the synthesis involved a different set of starting materials. A mixture of 7 mmol of acetophenone, 7 mmol of piperonal, and 10 mmol of semicarbazide hydrochloride was dissolved in 30 ml of absolute ethanol in a round-bottom flask. Again, a catalytic amount of sodium hydroxide (12 mmol) was added to the mixture as shown in scheme in 2. The mixture was refluxed with stirring, and the progress was tracked using TLC under the same solvent system of Methanol/ethyl acetate (9:1). After the reaction reached completion, the solution was cooled to room temperature. The solid product was filtered, washed with cold ethanol, and dried. The crude solid was then recrystallized from methanol to yield the pure compound P13. 2.2.3 Melting point determination All melting point were determined using a Gallenkamp melting point apparatus and were uncorrected. 2.2.4 Spectroscopic analysis Detailed structural analysis of the synthesized compounds was performed using Fourier Transformed Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) which include; proton ( 1 H) NMR, and carbon-13 ( 13 C) NMR. FT-IR data are reported in terms of frequency of absorption cm − 1 . Data for 1 H NMR and 13 C NMR spectra are reported as: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublet, m = multiplet), integration (J). 2.2.5 Acute oral toxicity test in mice In this study, the acute oral toxicity of the test compound was assessed in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines, specifically Test No. 423, which outlines the limit test method for evaluating acute toxicity. Three mice were utilized for the experiment, each receiving a different dose of the test compound, followed by a 24hour observation period. The first mouse was administered an oral dose of 1600 mg/kg body weight. After observing the first mouse for 24 hours for any signs of toxicity or adverse effects, the second mouse was given a dose of 2000 mg/kg body weight. This mouse was also monitored for 24hours for any clinical signs of toxicity. Following the observation of the second mouse, the third mouse was administered an oral dose of 5000 mg/kg body weight. Throughout the 24hour observation periods for each mouse, various parameters were monitored, including clinical signs of toxicity, body weight changes, and mortality. Observations were recorded meticulously to evaluate the acute effects of the test compound and to determine the dose-response relationship. After the initial 24hours observation period for each mouse, the animals were monitored for a total of 14 days to assess any delayed toxic effects that may arise. This extended observation period is crucial for identifying any long-term consequences of the exposure to the test compound. All procedures were conducted in compliance with ethical guidelines to ensure the humane treatment of the animals involved [ 14 ]. 2.2.6 Innoculation of plasmodium berghei parasite Infected blood was collected from a donor mouse with a parasitemia level of approximately 20–25% using heparinized capillary tubes through the tail vein. The collected blood was then transferred into a sterile plain beaker. Two milliliters of the infected blood were diluted with 10 milliliters of normal saline, resulting in a suspension where 0.2 milliliters contained approximately 1 x 10 7 infected red blood cells. Each mouse was subsequently inoculated intraperitoneally with 0.2 milliliters of this blood suspension. 2.2.7 Curative test in mice The curative efficacy of the synthesized compounds against established Plasmodium berghei infection was evaluated using the method described by Ryley and Peters (1970). Mice were inoculated intraperitoneally with 1 × 10 6 Plasmodium berghei -infected red blood cells, prepared from a donor mouse with a parasitemia level of 20–25%, as previously detailed. After 72 hours, allowing for the establishment of the infection, the parasitemia levels were assessed, and the mice were randomly divided into 5 groups (n = 5 per group). Group 1 received the negative control intraperitoneally (1% w/v acacia). Groups 2 to 3 were treated with 25 mg/kg, 50 mg/kg, and 100 mg/kg of the test compound (2-pyrazoline carboxamide; P5 and P13), respectively. Group 5 received the positive control (5 mg/kg of chloroquine). All treatments were administered intraperitoneally for four consecutive days. 2.2.8 Parasitemia determination On the fifth day post-treatment, blood was obtained from the tail vein of the treated mice in all groups. A thin blood film was prepared by smearing the samples onto microscopic slides. The slides were then fixed in absolute methanol and stained with a 3% Giemsa solution at pH 7.2. The average parasitemia was calculated from six different fields of view under the microscope. The percentage average suppression of the parasite by the compounds, relative to the negative control, was calculated for each group using the following formula below $$\:\mathbf{P}\mathbf{e}\mathbf{r}\mathbf{c}\mathbf{e}\mathbf{n}\mathbf{t}\mathbf{a}\mathbf{g}\mathbf{e}\:\mathbf{C}\mathbf{h}\mathbf{e}\mathbf{m}\mathbf{o}\mathbf{s}\mathbf{u}\mathbf{p}\mathbf{r}\mathbf{e}\mathbf{s}\mathbf{s}\mathbf{i}\mathbf{o}\mathbf{n}=\frac{A-B}{A}\:x\:100$$ A = Average parasitaemia of negative control B = Average Parasitaemia in each treated group 2.2.9 Euthanization method At the end of the experiment, mice were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg/kg), in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020 Edition) and as approved by the Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals. Death was confirmed by absence of heartbeat and respiratory movement. 2.2.10 Statistical analysis Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 25.0 and presented as Mean ± SEM (Standard Error of the Mean). One-way ANOVA was conducted to compare the means between the control group and the treated group. This was followed by Dunnett's post hoc test for multiple comparisons. P-values less than 0.05 were considered statistically significant. 3 Results and discussion Table 1 Yield and Some Physical Properties of the synthesized 2-pyrazoline carboxamide (P5 and P13) Compound ID Color/Appearance Melting Point ( o C) Yield (%) P5 White crystal 163–165 75.6% P13 White crystal 142–146 87.3% Table 2 Retention factor Values of P5 and P13 Compound code Solvent system No. of spots Rf. Value Solubility P5 EA(1):H(1) 1 Methanol and ethyl acetate EA(9):M(1) 1 0.84 P13 EA(1):H(1) 1 Methanol and ethyl acetate EA(9):M(1) 1 0.78 EA = Ethyl acetate, M = Methanol, H = Hexane, Rf = Retention factor and No.= Number Table 3 1 HNMR, 13 CNMR and IR assignment of P5 Carbon Position 13 C δ(ppm) 1 H δ(ppm) FT-IR 3 151.62 C = N stretching (1654 cm − 1 ), N–H stretching (3157 cm − 1 ) and C = O stretching (1591 cm − 1 ) bands 4 38.26 3.14 (d, J = 4 Hz,1H)4a 3.48 (d, J = 4 Hz,1H)4b 5 63.99 5.45 (dd, J = 4.1Hz, 4.1Hz 1H) 6 155.26 9 118.91 10 153.33 11 99.28 8.09 (d, J = 12Hz, 1Hz) 12 155.13 13 144.55 14 112.30 16 56.79 3.81 (s, 3H) 18 56.79 3.81 (s, 3H) 20 56.79 3.81 (s, 3H) 21 135.30 22 135.27 23 130.92 8.28 (s, 1H) 24 132.55 25 127.74 7.49 (d, J = 4Hz, 1H) 26 131.12 7.36 (d, J = 12Hz, 1H) Table 4 1 HNMR, 13 CNMR and FT-IR assignment of P13 Carbon Position 13 C δ(ppm) 1 H δ(ppm) FT-IR 3 154.08 C = N stretching (1587 cm − 1 ), N–H stretching (3459 cm − 1 ) and C = O stretching (1662 cm − 1 ) bands. 4 39.04 3.12 (d, J = 4 Hz, 1H)4a 3.47 (d, J = 4 Hz, 1H)4b 5 63.73 5.30 (dd J = 4 Hz, 4Hz, 1H) 6 155.26 9 132.76 10 125.81 7.75 (d, J = 4 Hz, 1H) 11 128.56 6.84 (m, 1H) 12 128.64 6.83 (m, 1H) 13 128.56 6.82 (m, 1H 14 125.81 7.75 (d, J = 4 Hz, 1H) 16 102.12 18 147.81 19 147.81 20 109.52 7.02 (d, J = 4 Hz, 1H) 21 122.96 7.04 (d, J = 4Hz, 1H) 22 132.82 23 107.91 7.36 (s, 1H) Table 5 Effect of compounds P5 and P13 on curative activity in plasmodium bergei infected mice Treatment Dosage (mg/kg) Average parasitaemia % Chemo suppression NS 10 ml/kg 27.00 ± 0.82 - Compound P5 25 16.50 ± 0.96* 38.89 50 14.34 ± 0.24* 46.90 100 10.39 ± 0.15* 61.52 Compound P13 25 15.92 ± 0.89* 43.65 50 8.50 ± 0.29* 68.52 100 7.43 ± 0.14* 71.43 CQ 5 2.05 ± 0.19* 93.70 Values are presented as Mean ± SEM; Data analysed by one-way ANOVA followed by Dunnett’s post hoc test, n = 5, *, P < 0.05 versus NS, NS = Normal Saline, CQ = Chloroquine; route = IP (Intraperitoneal) 3.1 The synthesize 2-pyrazoiline carboxamide The yield and some physical properties of the synthesized compounds are presented in Table 1 . The compounds appeared as crystals after purification. The thin layer chromatography (TLC) was used to monitor the reaction progress of the synthesized 2-pyrazoine carboxamide. The TLC was visualized under Ultraviolet light at 254nm. The retention factor (Rf) values were calculated and recorded as shown in Table 2 . 3.2 Synthesis and characterization of 2-pyrazoline carboxamide derivatives The designed compounds (P5 and P13) [ 11 ] were synthesized via base catalyzed one-pot three components condensation reaction, as this became preferred protocol and was used extensively for synthesizing new heterocyclic compounds due to its simple setup procedure, reduced reaction time, solvent management, excellent yields and reduced pollutant production [ 12 ]. The reaction progress was monitored using a TLC plate. The percentage yield of the synthesized P5 and P13 were calculated to be 75.6% and 87.3%, both having white crystal color appearance respectively. 3.3 Characterization of 5-(2,4-dichlorophenyl)-3-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (P5) The FT-IR spectra (Table 3 ) of compound P5 showed characteristic absorption frequencies of pyrazoline; C = N stretching (1654 cm − 1 ), N–H stretching (3157 cm − 1 ) and C = O stretching (1591 cm − 1 ) bands [ 12 , 15 , 16 ]. The 1 H NMR and 13 C NMR spectra confirmed the structure of P5 (Table 3 ). The CH 2 protons of the pyrazoline ring resonated as a pair of doublets of doublets at 3.14ppm (HA), 3.48ppm (HB) with coupling constant of 4Hz for both protons (2H). The CH (C5) proton appeared as a triplet at 5.44–5.46 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring [ 12 , 15 , 16 ]. The rest of the protons appear in their expected regions with their usual coupling constants. A noticeable observation is the appearance of the methoxy protons as triplet with resonance ranging from 3.82–3.79 ppm. The resonance observed at chemical shift(δ) of 155.26 ppm is a typical of carbon attached to oxygen of carboxamide (C = O) [ 12 , 15 , 16 ]. The carbon atoms at position C3 (C = N), C4 (CH 2 ) and C5 (CH) give rise to characteristic signals at 151.62 ppm, 38.26 ppm and 63.99 ppm respectively. These are all evidence that the pyrazoline have been formed. 3.4 Characterization of 5-(benzo[d][ 1 , 3 ]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (P13) The FT-IR spectra (Table 4 ) of compound P13 showed characteristic absorption frequencies of pyrazoline C = N stretching (1587 cm − 1 ), N–H stretching (3459 cm − 1 ) and C = O stretching (1662 cm − 1 ) bands [ 12 , 15 , 16 ]. The 1 H NMR and 13 C NMR spectra confirmed the structure of P13 (Table 4 ). The CH 2 protons of the pyrazoline ring resonated as a pair of doublets of doublets at 3.12ppm (HA), 3.47ppm (HB) with coupling constant of 4Hz for both protons (2H). The CH (C5) proton appeared as a triplet at 5.29–5.32 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring [ 12 , 15 , 16 ]. The rest of the protons appear in their expected regions with their usual coupling constants. A noticeable observation is the appearance of C16 (methylenedioxy) protons resonating as a singlet at 5.98 ppm. The resonance observed at chemical shift(δ) of 155.26 ppm is a typical of carbon attached to oxygen of carboxamide (C = O) [ 12 , 15 , 16 ]. The carbon atoms at position C3, C4 and C5 give rise to characteristic signals at 154.08 ppm, 39.04 ppm and 63.73 ppm respectively. These are all evidence that the pyrazoline have been formed. 3.5 Acute toxicity study The oral median lethal dose of the synthesized compounds P5 and P13 was found to be greater than 5000 mg/kg in mice in line with the OECD Test No. 423. Limit Test. 3.6 In-vivo Antiplasmodial Studies (Curative Test) Curative method for the evaluation of parasitaemia established by [ 13 ] was adopted as mentioned earlier in the methods. The antiplasmodial evaluation studies are presented in Table 5 showing the antiplasmodial value of compound P5 and P13 at dose of 25, 50 and 100 mg/kg and the percentage chemo suppression in infected plasmodium bergei mice. The results from the study were analysed using oneway ANOVA followed by post-hoc Dunnet test and the results were recorded as mean ± SEM. From the results on the effects of compounds P5 and P13 on curative activity in Plasmodium berghei -infected mice, the obtained data indicate that both compounds exhibit significant antimalarial activity, as evidenced by the reduction in average parasitaemia levels and the percentage of chemo suppression observed at various dosages. These findings reveal a clear dose-dependent relationship, where increasing the dosage of both compounds leads to a marked decrease in parasitaemia level, suggesting their potential efficacy as therapeutic agents against malaria. The results show that at a dosage of 100 mg/kg, compound P5 reduced average parasitaemia to 10.39 ± 0.15, while compound P13 demonstrated an even greater reduction to 7.43 ± 0.14. This indicates that compound P13 may be more effective than P5 at higher doses. Also, the percentage of chemo suppression achieved by compound P5 at 100 mg/kg was 61.52%, whereas compound P13 achieved a higher chemo suppression rate of 71.43%. These results highlight the superior efficacy of compound P13 in combating Plasmodium berghei infections. When compared to the normal saline control group, which exhibited an average parasitaemia of 27.00 ± 0.82, both compounds demonstrated significant antimalarial effects. The standard treatment, chloroquine, at a dosage of 5 mg/kg, resulted in a remarkable chemo suppression of 93.70% with an average parasitaemia of 2.05 ± 0.19. While both compounds P5 and P13 showed a promising results. The statistical significance of the results, indicated by the asterisk (*) next to the values (P < 0.05), reinforces the conclusion that both compounds have a meaningful impact on reducing parasitaemia in infected mice and are considered as active when reduction in parasitaemia is ≥ 30% [ 17 ]. This finding is crucial as it establishes a foundation for the potential development of these compounds into viable antimalarial therapies 4. Conclusion The present study successfully synthesized and characterized two novel 2-pyrazoline carboxamide derivatives, P5 and P13, via a base-catalyzed one-pot synthesis method, achieving yields of 75.6% and 87.3%, respectively. Spectroscopic analyses (FT-IR, ¹H NMR, and ¹³C NMR) confirmed the structural integrity of these compounds, revealing characteristic pyrazoline features. Acute toxicity studies indicated that both compounds are safe, with an oral median lethal dose exceeding 5000 mg/kg in mice. In - vivo antiplasmodial evaluation against Plasmodium berghei demonstrated significant curative effects, with P13 exhibiting superior efficacy (71.43% chemo suppression at 100 mg/kg) compared to P5 (61.52% at 100 mg/kg), though both were less potent than chloroquine (93.70% at 5 mg/kg). The dose-dependent reduction in parasitaemia underscores their potential as antimalarial agents. These findings suggest that P5 and P13 are promising candidates for further optimization and development as novel therapeutics against malaria, contributing to the ongoing efforts to combat this global health challenge Declarations Acknowledgments Thanks to the synthesis team of Ahmadu Bello University Zaria, Department of Pharmaceutical and Medicinal Chemistry for guidance and moral support. Author contributions Yusuf Jimoh: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft. Abdullahi Yunusa Idris, Asmau Nasir Hamza, Mariam Abdullahi: Supervision, Validation, Writing – review & editing. Lukman Hassan Ali, Ibrahim Gidado: Writing – review & editing. Dauda Garba: Formal analysis, Writing – review & editing. Funding No external funding Data availability All data generated or analyzed during this study are included in this published article. Competing interest The authors declare no competing interest Ethics approval and consent to participate Ethical approval for the use of laboratory animals was sought from Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) with ethical approval number of ABUCAUC/2024/156. Accordance statement All experimental protocols were in accordance with Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals as accepted internationally. Consent to participate Not Applicable Consent to publish Not Applicable References WHO. World malaria World malaria report report. 2024. Rasmussen C, Alonso P, Ringwald P. Current and emerging strategies to combat antimalarial resistance. Expert Rev Anti Infect Ther 2022;20:353–72. https://doi.org/10.1080/14787210.2021.1962291. Ouji M, Augereau J-M, Paloque L, Benoit-Vical F. Plasmodium falciparum resistance to artemisinin-based combination therapies: A sword of Damocles in the path toward malaria elimination. 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Parasite Immunol 2014;36:131–9. https://doi.org/https://doi.org/10.1111/pim.12086. Schemes Schemes 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Schemes.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7603860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":527101403,"identity":"e7eeec59-3f6d-40ac-ad35-4a5a3799f040","order_by":0,"name":"Yusuf Jimoh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACxmYwJSEHIg88IEGLhTFYSwIJllUkNoAoorQwt3OnSfP8kUifH3b4IdAWOzndBoIO490mzdsmkbvxdpoBUEuysdkBorQ0ALXMTgBpOZC4jSgtIIcZzk7/QIoWNokEeekc4m3ZbDm3TcJwg3ROwYEEAyL8Yth/duONN3/q5OVnp2/+8KHCTo6wlgYGFgkQwwCs0oCAchCQB0bNBzCjgQjVo2AUjIJRMDIBAMl/QjAWDSdUAAAAAElFTkSuQmCC","orcid":"","institution":"Ahmadu Bello University","correspondingAuthor":true,"prefix":"","firstName":"Yusuf","middleName":"","lastName":"Jimoh","suffix":""},{"id":527101404,"identity":"2cec80e8-17cf-4e66-ae68-a33e68050c4a","order_by":1,"name":"Abdullahi Yunusa Idris","email":"","orcid":"","institution":"Ahmadu Bello University","correspondingAuthor":false,"prefix":"","firstName":"Abdullahi","middleName":"Yunusa","lastName":"Idris","suffix":""},{"id":527101405,"identity":"654692cf-d61c-4bc7-9a26-7508eb8362a5","order_by":2,"name":"Asmau Nasir 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Education","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Gidado","suffix":""},{"id":527101409,"identity":"5d6c7984-06d1-43dd-aa79-cbdade57ce71","order_by":6,"name":"Dauda Garba","email":"","orcid":"","institution":"University of Abuja","correspondingAuthor":false,"prefix":"","firstName":"Dauda","middleName":"","lastName":"Garba","suffix":""}],"badges":[],"createdAt":"2025-09-13 00:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7603860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7603860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93245702,"identity":"b875739e-3f64-45a6-a5f4-303723752fab","added_by":"auto","created_at":"2025-10-10 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2","display":"","copyAsset":false,"role":"figure","size":139576,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectral of compound P13\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7603860/v1/65cd43aef0f079a83e91cb52.png"},{"id":97664790,"identity":"b5b86b70-f188-44d1-82c6-50b692d58621","added_by":"auto","created_at":"2025-12-08 09:14:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1402540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7603860/v1/280d1af5-5a0e-4ecd-8f35-6da2acc40eaf.pdf"},{"id":93246717,"identity":"8935c3c0-21c5-4493-9218-de7be9157b05","added_by":"auto","created_at":"2025-10-10 15:18:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":65324,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-7603860/v1/4d60298a7860c45bcf937566.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis, Characterization, and Antiplasmodial Evaluation of Novel 2-Pyrazoline Carboxamide Derivatives Against Plasmodium berghei in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMalaria remains a significant global health burden, with over 263\u0026nbsp;million cases and 597,000 deaths reported in 2023, predominantly caused by \u003cem\u003ePlasmodium\u003c/em\u003e species, particularly \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium berghei\u003c/em\u003e in rodent models [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The emergence of drug-resistant strains, including resistance to artemisinin-based combination therapies, necessitates the development of novel antimalarial agents with distinct mechanisms of action [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Heterocyclic compounds, such as pyrazolines, have garnered attention due to their diverse pharmacological properties, including antimalarial, antimicrobial, and anti-inflammatory activities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The pyrazoline scaffold, characterized by a five-membered ring with two adjacent nitrogen atoms, offers a versatile platform for chemical modification, enhancing its therapeutic potential [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the efficacy of pyrazoline derivatives as protease inhibitors against Plasmodium parasites, suggesting their role in disrupting parasite lifecycle processes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. One-pot synthesis approaches have emerged as efficient strategies for generating such heterocycles, offering advantages in terms of reduced reaction time, high yields, and environmental sustainability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite these advances, the antimalarial potential of 2-pyrazoline carboxamide derivatives remains underexplored, particularly \u003cem\u003ein vivo\u003c/em\u003e models like \u003cem\u003ePlasmodium berghei\u003c/em\u003e-infected mice, which closely mimic human malaria pathology [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This study aims to synthesize, characterize, and evaluate the antiplasmodial activity of novel 2-pyrazoline carboxamide derivatives (P5 and P13) using a one-pot reaction protocol, with the goal of identifying promising candidates for malaria treatment. By integrating spectroscopic characterization and \u003cem\u003ein\u003c/em\u003e-\u003cem\u003evivo\u003c/em\u003e curative testing, this work seeks to contribute to the growing body of evidence supporting pyrazoline-based therapeutics\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1 Equipment and glassware\u003c/h2\u003e\u003cp\u003eThe melting points of the compounds were determined using Gallenkamp melting point apparatus and were uncorrected, Proton Nuclear Magnetic Resonance and Carbon-13 Nuclear Magnetic Resonance were carried-out at the Jodrell Laboratory, NCPH, Royal Botanical Gardens, Kew, UK, Using 400MHz Brucker. The Nuclear Magnetic Resonance data were recorded in chemical shift(δ) in ppm downfield from Tetra-Methyl Silane (as the internal standard). Also the Fourier Transform Infared (FTIR) spectra were carried out at the Multiuser Science Research Laboratories, Ahmadu Bello University, Zaria, recorded on Agilent spectrophotometer as wave numbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eSome of the appliances and glassware used include; Erlenmeyer flasks, sapatulas, testubes, beakers, measuring cylinder, glass rod, funnel, pipette, watch glass, microscope slide, syringes, capillary tubes, TLC Tank, magnetic stirrer, stir bar, refrigerator, Gallenkamp melting point device, microtiter plate, analytical balance microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2 Reagents, solvents, and standard drugs\u003c/h2\u003e\u003cp\u003eAll starting reagents and solvents used were of analytical grade which include: acetophenone, 2,4,5-trimethoxyacetophenone, 2,4-dichlorobenzaldehyde, piperonaldehyde, semicarbazide hydrochloride, ethanol, methanol, hexane, ethylacetate, sodium hydroxide and Chloroquine Sodium. All the reagents were purchased from Sigma Aldrich, Germany.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3 Experimental animals\u003c/h2\u003e\u003cp\u003eFifty mice weighing 18-22g were bought from the Animal House, Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria and kept under standard laboratory conditions. The animals were kept in clean polypropylene cages at the Animal House of the Department of Pharmacology and Therapeutics. All experimental protocols were in accordance with Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals as accepted internationally.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.1.4 Malarial parasite\u003c/h2\u003e\u003cp\u003eChloroquine-sensitive malaria parasites (\u003cem\u003ePlasmodium berghei\u003c/em\u003e NK65) were obtained from the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. The mice were inoculated intraperitoneally, with 0.2ml standard inoculum containing approximately 1 x 10\u003csup\u003e7\u003c/sup\u003e parasitized red blood cells.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Methods\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Procedure for the one-port synthesis of the compound P5\u003c/h2\u003e\u003cp\u003eIn a round bottom flask, a mixture of 2,4,5-trimethoxyacetophenone (7 mmol), 2,4-dichlorobenzaldehyde (7 mmol), and semicarbazide hydrochloride (10 mmol) were dissolved in absolute ethanol (30 ml). To this mixture a catalytic amount of sodium hydroxide (12 mmol) was added and refluxed for 6hrs with stirring as shown in scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below. The completion of reaction was monitored by thin layer chromatography (TLC) in methanol/ethyl acetate (9:1) as a solvent system. The solution was cooled to room temperature, filtered the obtained solid, washed with cold ethanol, and dried. The pure target compounds (P5) was obtained by recrystallization from methanol.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Procedure for the one-port synthesis of the compound P13\u003c/h2\u003e\u003cp\u003eFor P13, the synthesis involved a different set of starting materials. A mixture of 7 mmol of acetophenone, 7 mmol of piperonal, and 10 mmol of semicarbazide hydrochloride was dissolved in 30 ml of absolute ethanol in a round-bottom flask. Again, a catalytic amount of sodium hydroxide (12 mmol) was added to the mixture as shown in scheme in 2. The mixture was refluxed with stirring, and the progress was tracked using TLC under the same solvent system of Methanol/ethyl acetate (9:1). After the reaction reached completion, the solution was cooled to room temperature. The solid product was filtered, washed with cold ethanol, and dried. The crude solid was then recrystallized from methanol to yield the pure compound P13.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Melting point determination\u003c/h2\u003e\u003cp\u003eAll melting point were determined using a Gallenkamp melting point apparatus and were uncorrected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Spectroscopic analysis\u003c/h2\u003e\u003cp\u003eDetailed structural analysis of the synthesized compounds was performed using Fourier Transformed Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) which include; proton (\u003csup\u003e1\u003c/sup\u003eH) NMR, and carbon-13 (\u003csup\u003e13\u003c/sup\u003eC) NMR. FT-IR data are reported in terms of frequency of absorption cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Data for \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra are reported as: chemical shift (δ ppm), multiplicity (s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, dd\u0026thinsp;=\u0026thinsp;doublet of doublet, m\u0026thinsp;=\u0026thinsp;multiplet), integration (J).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5 Acute oral toxicity test in mice\u003c/h2\u003e\u003cp\u003eIn this study, the acute oral toxicity of the test compound was assessed in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines, specifically Test No. 423, which outlines the limit test method for evaluating acute toxicity. Three mice were utilized for the experiment, each receiving a different dose of the test compound, followed by a 24hour observation period. The first mouse was administered an oral dose of 1600 mg/kg body weight. After observing the first mouse for 24 hours for any signs of toxicity or adverse effects, the second mouse was given a dose of 2000 mg/kg body weight. This mouse was also monitored for 24hours for any clinical signs of toxicity. Following the observation of the second mouse, the third mouse was administered an oral dose of 5000 mg/kg body weight. Throughout the 24hour observation periods for each mouse, various parameters were monitored, including clinical signs of toxicity, body weight changes, and mortality. Observations were recorded meticulously to evaluate the acute effects of the test compound and to determine the dose-response relationship. After the initial 24hours observation period for each mouse, the animals were monitored for a total of 14 days to assess any delayed toxic effects that may arise. This extended observation period is crucial for identifying any long-term consequences of the exposure to the test compound. All procedures were conducted in compliance with ethical guidelines to ensure the humane treatment of the animals involved [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6 Innoculation of plasmodium berghei parasite\u003c/h2\u003e\u003cp\u003eInfected blood was collected from a donor mouse with a parasitemia level of approximately 20\u0026ndash;25% using heparinized capillary tubes through the tail vein. The collected blood was then transferred into a sterile plain beaker. Two milliliters of the infected blood were diluted with 10 milliliters of normal saline, resulting in a suspension where 0.2 milliliters contained approximately 1 x 10\u003csup\u003e7\u003c/sup\u003e infected red blood cells. Each mouse was subsequently inoculated intraperitoneally with 0.2 milliliters of this blood suspension.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7 Curative test in mice\u003c/h2\u003e\u003cp\u003eThe curative efficacy of the synthesized compounds against established \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection was evaluated using the method described by Ryley and Peters (1970). Mice were inoculated intraperitoneally with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003ePlasmodium berghei\u003c/em\u003e-infected red blood cells, prepared from a donor mouse with a parasitemia level of 20\u0026ndash;25%, as previously detailed. After 72 hours, allowing for the establishment of the infection, the parasitemia levels were assessed, and the mice were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;5 per group). Group 1 received the negative control intraperitoneally (1% w/v acacia). Groups 2 to 3 were treated with 25 mg/kg, 50 mg/kg, and 100 mg/kg of the test compound (2-pyrazoline carboxamide; P5 and P13), respectively. Group 5 received the positive control (5 mg/kg of chloroquine). All treatments were administered intraperitoneally for four consecutive days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8 Parasitemia determination\u003c/h2\u003e\u003cp\u003eOn the fifth day post-treatment, blood was obtained from the tail vein of the treated mice in all groups. A thin blood film was prepared by smearing the samples onto microscopic slides. The slides were then fixed in absolute methanol and stained with a 3% Giemsa solution at pH 7.2. The average parasitemia was calculated from six different fields of view under the microscope. The percentage average suppression of the parasite by the compounds, relative to the negative control, was calculated for each group using the following formula below\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{P}\\mathbf{e}\\mathbf{r}\\mathbf{c}\\mathbf{e}\\mathbf{n}\\mathbf{t}\\mathbf{a}\\mathbf{g}\\mathbf{e}\\:\\mathbf{C}\\mathbf{h}\\mathbf{e}\\mathbf{m}\\mathbf{o}\\mathbf{s}\\mathbf{u}\\mathbf{p}\\mathbf{r}\\mathbf{e}\\mathbf{s}\\mathbf{s}\\mathbf{i}\\mathbf{o}\\mathbf{n}=\\frac{A-B}{A}\\:x\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA\u0026thinsp;=\u0026thinsp;Average parasitaemia of negative control\u003c/p\u003e\u003cp\u003eB\u0026thinsp;=\u0026thinsp;Average Parasitaemia in each treated group\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.2.9 Euthanization method\u003c/h2\u003e\u003cp\u003e At the end of the experiment, mice were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg/kg), in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020 Edition) and as approved by the Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals. Death was confirmed by absence of heartbeat and respiratory movement.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.2.10 Statistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using the Statistical Package for the Social Sciences (SPSS) version 25.0 and presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (Standard Error of the Mean). One-way ANOVA was conducted to compare the means between the control group and the treated group. This was followed by Dunnett's post hoc test for multiple comparisons. P-values less than 0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eYield and Some Physical Properties of the synthesized 2-pyrazoline carboxamide (P5 and P13)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColor/Appearance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMelting Point (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYield (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite crystal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e163\u0026ndash;165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e75.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite crystal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e142\u0026ndash;146\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e87.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRetention factor Values of P5 and P13\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolvent system\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo. of spots\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRf. Value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSolubility\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEA(1):H(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMethanol and ethyl acetate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEA(9):M(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEA(1):H(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMethanol and ethyl acetate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEA(9):M(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eEA\u0026thinsp;=\u0026thinsp;Ethyl acetate, M\u0026thinsp;=\u0026thinsp;Methanol, H\u0026thinsp;=\u0026thinsp;Hexane, Rf\u0026thinsp;=\u0026thinsp;Retention factor and No.= Number\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13\u003c/sup\u003eCNMR and IR assignment of P5\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbon\u003c/p\u003e\u003cp\u003ePosition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC δ(ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH δ(ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFT-IR\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e151.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"18\" rowspan=\"19\"\u003e\u003cp\u003eC\u0026thinsp;=\u0026thinsp;N stretching (1654 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), N\u0026ndash;H stretching (3157 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026thinsp;=\u0026thinsp;O stretching (1591 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) bands\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.14 (d, J\u0026thinsp;=\u0026thinsp;4 Hz,1H)4a\u003c/p\u003e\u003cp\u003e3.48 (d, J\u0026thinsp;=\u0026thinsp;4 Hz,1H)4b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.45 (dd, J\u0026thinsp;=\u0026thinsp;4.1Hz, 4.1Hz 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e118.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e99.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.09 (d, J\u0026thinsp;=\u0026thinsp;12Hz, 1Hz)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e144.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e112.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.81 (s, 3H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.81 (s, 3H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.81 (s, 3H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e135.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e135.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e130.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.28 (s, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e132.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e127.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.49 (d, J\u0026thinsp;=\u0026thinsp;4Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e131.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.36 (d, J\u0026thinsp;=\u0026thinsp;12Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13\u003c/sup\u003eCNMR and FT-IR assignment of P13\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbon\u003c/p\u003e\u003cp\u003ePosition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC δ(ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH δ(ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFT-IR\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"16\" rowspan=\"17\"\u003e\u003cp\u003eC\u0026thinsp;=\u0026thinsp;N stretching (1587 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), N\u0026ndash;H stretching (3459 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026thinsp;=\u0026thinsp;O stretching (1662 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) bands.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e39.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.12 (d, J\u0026thinsp;=\u0026thinsp;4 Hz, 1H)4a\u003c/p\u003e\u003cp\u003e3.47 (d, J\u0026thinsp;=\u0026thinsp;4 Hz, 1H)4b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.30 (dd J\u0026thinsp;=\u0026thinsp;4 Hz, 4Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e132.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e125.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.75 (d, J\u0026thinsp;=\u0026thinsp;4 Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e128.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.84 (m, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e128.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.83 (m, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e128.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.82 (m, 1H\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e125.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.75 (d, J\u0026thinsp;=\u0026thinsp;4 Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e102.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e147.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e147.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e109.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.02 (d, J\u0026thinsp;=\u0026thinsp;4 Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e122.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.04 (d, J\u0026thinsp;=\u0026thinsp;4Hz, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e132.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e107.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.36 (s, 1H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of compounds P5 and P13 on curative activity in \u003cem\u003eplasmodium bergei\u003c/em\u003e infected mice\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDosage (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage parasitaemia\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e% Chemo suppression\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 ml/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCompound P5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCompound P13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e71.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eValues are presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; Data analysed by one-way ANOVA followed by Dunnett\u0026rsquo;s post hoc test, n\u0026thinsp;=\u0026thinsp;5, *, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 versus NS, NS\u0026thinsp;=\u0026thinsp;Normal Saline, CQ\u0026thinsp;=\u0026thinsp;Chloroquine; route\u0026thinsp;=\u0026thinsp;IP (Intraperitoneal)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1 The synthesize 2-pyrazoiline carboxamide\u003c/h2\u003e\u003cp\u003eThe yield and some physical properties of the synthesized compounds are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The compounds appeared as crystals after purification. The thin layer chromatography (TLC) was used to monitor the reaction progress of the synthesized 2-pyrazoine carboxamide. The TLC was visualized under Ultraviolet light at 254nm. The retention factor (Rf) values were calculated and recorded as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Synthesis and characterization of 2-pyrazoline carboxamide derivatives\u003c/h2\u003e\u003cp\u003eThe designed compounds (P5 and P13) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] were synthesized via base catalyzed one-pot three components condensation reaction, as this became preferred protocol and was used extensively for synthesizing new heterocyclic compounds due to its simple setup procedure, reduced reaction time, solvent management, excellent yields and reduced pollutant production [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The reaction progress was monitored using a TLC plate. The percentage yield of the synthesized P5 and P13 were calculated to be 75.6% and 87.3%, both having white crystal color appearance respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Characterization of 5-(2,4-dichlorophenyl)-3-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (P5)\u003c/h2\u003e\u003cp\u003eThe FT-IR spectra (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) of compound P5 showed characteristic absorption frequencies of pyrazoline; C\u0026thinsp;=\u0026thinsp;N stretching (1654 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), N\u0026ndash;H stretching (3157 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026thinsp;=\u0026thinsp;O stretching (1591 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) bands [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra confirmed the structure of P5 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The CH\u003csub\u003e2\u003c/sub\u003e protons of the pyrazoline ring resonated as a pair of doublets of doublets at 3.14ppm (HA), 3.48ppm (HB) with coupling constant of 4Hz for both protons (2H). The CH (C5) proton appeared as a triplet at 5.44\u0026ndash;5.46 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The rest of the protons appear in their expected regions with their usual coupling constants. A noticeable observation is the appearance of the methoxy protons as triplet with resonance ranging from 3.82\u0026ndash;3.79 ppm. The resonance observed at chemical shift(δ) of 155.26 ppm is a typical of carbon attached to oxygen of carboxamide (C\u0026thinsp;=\u0026thinsp;O) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The carbon atoms at position C3 (C\u0026thinsp;=\u0026thinsp;N), C4 (CH\u003csub\u003e2\u003c/sub\u003e) and C5 (CH) give rise to characteristic signals at 151.62 ppm, 38.26 ppm and 63.99 ppm respectively. These are all evidence that the pyrazoline have been formed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Characterization of 5-(benzo[d][\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (P13)\u003c/h2\u003e\u003cp\u003eThe FT-IR spectra (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of compound P13 showed characteristic absorption frequencies of pyrazoline C\u0026thinsp;=\u0026thinsp;N stretching (1587 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), N\u0026ndash;H stretching (3459 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026thinsp;=\u0026thinsp;O stretching (1662 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) bands [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra confirmed the structure of P13 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The CH\u003csub\u003e2\u003c/sub\u003e protons of the pyrazoline ring resonated as a pair of doublets of doublets at 3.12ppm (HA), 3.47ppm (HB) with coupling constant of 4Hz for both protons (2H). The CH (C5) proton appeared as a triplet at 5.29\u0026ndash;5.32 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The rest of the protons appear in their expected regions with their usual coupling constants. A noticeable observation is the appearance of C16 (methylenedioxy) protons resonating as a singlet at 5.98 ppm. The resonance observed at chemical shift(δ) of 155.26 ppm is a typical of carbon attached to oxygen of carboxamide (C\u0026thinsp;=\u0026thinsp;O) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The carbon atoms at position C3, C4 and C5 give rise to characteristic signals at 154.08 ppm, 39.04 ppm and 63.73 ppm respectively. These are all evidence that the pyrazoline have been formed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Acute toxicity study\u003c/h2\u003e\u003cp\u003eThe oral median lethal dose of the synthesized compounds P5 and P13 was found to be greater than 5000 mg/kg in mice in line with the OECD Test No. 423. Limit Test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.6 In-vivo Antiplasmodial Studies (Curative Test)\u003c/h2\u003e\u003cp\u003eCurative method for the evaluation of parasitaemia established by [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] was adopted as mentioned earlier in the methods. The antiplasmodial evaluation studies are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e showing the antiplasmodial value of compound P5 and P13 at dose of 25, 50 and 100 mg/kg and the percentage chemo suppression in infected \u003cem\u003eplasmodium bergei\u003c/em\u003e mice. The results from the study were analysed using oneway ANOVA followed by post-hoc Dunnet test and the results were recorded as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. From the results on the effects of compounds P5 and P13 on curative activity in \u003cem\u003ePlasmodium berghei\u003c/em\u003e-infected mice, the obtained data indicate that both compounds exhibit significant antimalarial activity, as evidenced by the reduction in average parasitaemia levels and the percentage of chemo suppression observed at various dosages. These findings reveal a clear dose-dependent relationship, where increasing the dosage of both compounds leads to a marked decrease in parasitaemia level, suggesting their potential efficacy as therapeutic agents against malaria. The results show that at a dosage of 100 mg/kg, compound P5 reduced average parasitaemia to 10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, while compound P13 demonstrated an even greater reduction to 7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14. This indicates that compound P13 may be more effective than P5 at higher doses. Also, the percentage of chemo suppression achieved by compound P5 at 100 mg/kg was 61.52%, whereas compound P13 achieved a higher chemo suppression rate of 71.43%. These results highlight the superior efficacy of compound P13 in combating \u003cem\u003ePlasmodium berghei\u003c/em\u003e infections. When compared to the normal saline control group, which exhibited an average parasitaemia of 27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82, both compounds demonstrated significant antimalarial effects. The standard treatment, chloroquine, at a dosage of 5 mg/kg, resulted in a remarkable chemo suppression of 93.70% with an average parasitaemia of 2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19. While both compounds P5 and P13 showed a promising results. The statistical significance of the results, indicated by the asterisk (*) next to the values (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reinforces the conclusion that both compounds have a meaningful impact on reducing parasitaemia in infected mice and are considered as active when reduction in parasitaemia is \u0026ge;\u0026thinsp;30% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This finding is crucial as it establishes a foundation for the potential development of these compounds into viable antimalarial therapies\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe present study successfully synthesized and characterized two novel 2-pyrazoline carboxamide derivatives, P5 and P13, via a base-catalyzed one-pot synthesis method, achieving yields of 75.6% and 87.3%, respectively. Spectroscopic analyses (FT-IR, \u0026sup1;H NMR, and \u0026sup1;\u0026sup3;C NMR) confirmed the structural integrity of these compounds, revealing characteristic pyrazoline features. Acute toxicity studies indicated that both compounds are safe, with an oral median lethal dose exceeding 5000 mg/kg in mice. \u003cem\u003eIn\u003c/em\u003e-\u003cem\u003evivo\u003c/em\u003e antiplasmodial evaluation against \u003cem\u003ePlasmodium berghei\u003c/em\u003e demonstrated significant curative effects, with P13 exhibiting superior efficacy (71.43% chemo suppression at 100 mg/kg) compared to P5 (61.52% at 100 mg/kg), though both were less potent than chloroquine (93.70% at 5 mg/kg). The dose-dependent reduction in parasitaemia underscores their potential as antimalarial agents. These findings suggest that P5 and P13 are promising candidates for further optimization and development as novel therapeutics against malaria, contributing to the ongoing efforts to combat this global health challenge\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the synthesis team of Ahmadu Bello University Zaria, Department of Pharmaceutical and Medicinal Chemistry for guidance and moral support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYusuf Jimoh: Conceptualization, Methodology, Investigation, Data curation, Writing \u0026ndash; original draft. Abdullahi Yunusa Idris, Asmau Nasir Hamza, Mariam Abdullahi: Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing. Lukman Hassan Ali, Ibrahim Gidado: Writing \u0026ndash; review \u0026amp; editing. Dauda Garba: Formal analysis, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u003c/strong\u003e \u003cstrong\u003eavailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the use of laboratory animals was sought from Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) with ethical approval number of ABUCAUC/2024/156.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccordance statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were in accordance with Ahmadu Bello University Research Policy and guides for the use and care of laboratory animals as accepted internationally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. World malaria World malaria report report. 2024.\u003c/li\u003e\n\u003cli\u003eRasmussen C, Alonso P, Ringwald P. Current and emerging strategies to combat antimalarial resistance. Expert Rev Anti Infect Ther 2022;20:353\u0026ndash;72. https://doi.org/10.1080/14787210.2021.1962291.\u003c/li\u003e\n\u003cli\u003eOuji M, Augereau J-M, Paloque L, Benoit-Vical F. Plasmodium falciparum resistance to artemisinin-based combination therapies: A sword of Damocles in the path toward malaria elimination. Parasite 2018;25.\u003c/li\u003e\n\u003cli\u003eNureye D, Assefa S. Old and Recent Advances in Life Cycle, Pathogenesis, Diagnosis, Prevention, and Treatment of Malaria including Perspectives in Ethiopia. Sci World J 2020;2020. https://doi.org/10.1155/2020/1295381.\u003c/li\u003e\n\u003cli\u003eAssefa A, Fola AA, Tasew G. Emergence of Plasmodium falciparum strains with artemisinin partial resistance in East Africa and the Horn of Africa: is there a need to panic? Malar J 2024;23:34. https://doi.org/10.1186/s12936-024-04848-8.\u003c/li\u003e\n\u003cli\u003eSiddiqui FA, Liang X, Cui L. Plasmodium falciparum resistance to ACTs: Emergence, mechanisms, and outlook. Int J Parasitol Drugs Drug Resist 2021;16:102\u0026ndash;18. https://doi.org/10.1016/j.ijpddr.2021.05.007.\u003c/li\u003e\n\u003cli\u003evan der Pluijm RW, Amaratunga C, Dhorda M, Dondorp AM. Triple Artemisinin-Based Combination Therapies for Malaria \u0026ndash; A New Paradigm? Trends Parasitol 2021;37:15\u0026ndash;24. https://doi.org/https://doi.org/10.1016/j.pt.2020.09.011.\u003c/li\u003e\n\u003cli\u003eKerru N, Gummidi L, Maddila S, Gangu KK, Jonnalagadda SB. A review on recent advances in nitrogen-containing molecules and their biological applications. Molecules 2020;25. https://doi.org/10.3390/molecules25081909.\u003c/li\u003e\n\u003cli\u003eWiratama M, Satria S, Waskitha W, Haryadi W, Wahyuningsih TD. Synthesis, antimalarial activity assay and molecular docking study of N-substituted chloro-pyrazolines. Trop J Pharm Res 2022;21:1255\u0026ndash;61. https://doi.org/10.4314/tjpr.v21i6.18.\u003c/li\u003e\n\u003cli\u003eAggarwal S, Paliwal D, Kaushik D, Gupta GK, Kumar A. Synthesis, Antimalarial Evaluation and SAR Study of Some 1,3,5-Trisubstituted Pyrazoline Derivatives. Lett Org Chem 2019;16:807\u0026ndash;17. https://doi.org/10.2174/1570178616666190212145754.\u003c/li\u003e\n\u003cli\u003eJimoh Y, Abdullah IY, Hamza AN, Abdullahi M, Ahmadu J. In Silico Evaluation of Novel 2- Pyrazoline Carboxamide Derivatives as Potential Protease Inhibitors Against Plasmodium Parasites Derivatives as Potential Protease Inhibitors Against Plasmodium. Chem Proceeding 2024;16.\u003c/li\u003e\n\u003cli\u003eSalih RHH, Hasan AH, Hussein AJ, Samad MK, Shakya S, Jamalis J, et al. One-pot synthesis, molecular docking, ADMET, and DFT studies of novel pyrazolines as promising SARS-CoV-2 main protease inhibitors. Res Chem Intermed 2022;48:4729\u0026ndash;51. https://doi.org/10.1007/s11164-022-04831-5.\u003c/li\u003e\n\u003cli\u003eRyley JF, Peters W. The antimalarial activity of some quinolone esters. Ann Trop Med Parasitol 1970;64:209\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eOECD. Test No. 423: Acute Oral toxicity - Acute Toxic Class Method. Oecd Guidel Test Chem 2002:1\u0026ndash;14. https://doi.org/10.1787/9789264071001-en.\u003c/li\u003e\n\u003cli\u003eBeyhan N, Kocyigit-Kaymakcioglu B, G\u0026uuml;mr\u0026uuml; S, Aricioglu F. Synthesis and anticonvulsant activity of some 2-pyrazolines derived from chalcones. Arab J Chem 2017;10:S2073\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eBoudou F, Sehmi A, Belakredar A, Zaoui O. Synthesis, characterization, antimicrobial activity, and in silico assessment of a novel pyrazoline carboxamide heterocyclic compound. Bangladesh J Pharmacol 2023;18:152\u0026ndash;61. https://doi.org/10.3329/bjp.v18i4.69267.\u003c/li\u003e\n\u003cli\u003ede Souza JB. Protective immunity against malaria after vaccination. Parasite Immunol 2014;36:131\u0026ndash;9. https://doi.org/https://doi.org/10.1111/pim.12086.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"2-Pyrazoline carboxamide, Plasmodium berghei, antiplasmodial activity, one-pot synthesis, spectroscopic characterization, acute toxicity, malaria treatment","lastPublishedDoi":"10.21203/rs.3.rs-7603860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7603860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalaria, caused by \u003cem\u003ePlasmodium\u003c/em\u003e species, remains a global health challenge, exacerbated by drug resistance, necessitating novel therapeutic agents. Pyrazoline derivatives have shown promise as antimalarial candidates due to their heterocyclic structure and pharmacological versatility. Two novel 2-pyrazoline carboxamide derivatives, P5 and P13, were synthesized via a base-catalyzed one-pot reaction using substituted acetophenones, benzaldehydes, and semicarbazide in ethanol. Compounds were characterized using FT-IR, ¹H NMR, and ¹³C NMR. Acute oral toxicity was assessed in mice per OECD Test No. 423, and antiplasmodial activity was evaluated against \u003cem\u003ePlasmodium\u003c/em\u003e \u003cem\u003eberghei\u003c/em\u003e in a curative test. Mice were treated with 25, 50, and 100 mg/kg of P5 and P13, with chloroquine (5 mg/kg) as the positive control. Parasitaemia suppression was calculated, and data analyzed using ANOVA with Dunnett’s post hoc test. P5 and P13 were synthesized with yields of 75.6% and 87.3% respectively and spectroscopic data confirmed their pyrazoline structures. They exhibited no toxicity up to 5000 mg/kg. Both compounds significantly reduced parasitaemia in a dose-dependent manner, with P13 achieving 71.43% chemo suppression and P5 61.52% at 100 mg/kg, compared to chloroquine’s 93.70%. P5 and P13 demonstrate promising antiplasmodial activity and safety, with P13 showing superior efficacy, suggesting their potential as novel antimalarial agents warranting further development.\u003c/p\u003e","manuscriptTitle":"Synthesis, Characterization, and Antiplasmodial Evaluation of Novel 2-Pyrazoline Carboxamide Derivatives Against Plasmodium berghei in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 15:10:25","doi":"10.21203/rs.3.rs-7603860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"147e89b9-2cca-48d5-967a-12c1164ae6a4","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T14:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 15:10:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7603860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7603860","identity":"rs-7603860","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}