Eclet. Quim. 49 | e-1526, 2024 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 ISSN 1678-4618 page 1/20 1Sana’a University, Faculty of Science, Sana’a, Yemen. +Corresponding author: Yasmin Mosa’d Saeed Jamil, Phone: +967771952842, Email address: y.jamil@su.edu.ye Original Article Evaluation of the effectiveness of new mixed ligand complexes against the vector of Dengue fever Aedes aegypti (Diptera; Culicidae) Nedhal Abdulmawla Al–Selwi1 , Abbas Mohamed Al–Azab1 , Yasmin Mosa’d Saeed Jamil1+ , Fathi Mohamed Al– Azab1 , Anwar Mohamed Al–Ryami1 Abstract Chloroquine phosphate with ibuprofen and sulphamethoxazole were used as mixed ligands with Ni(II), Co(II), Cu(II), and Zn(II) to prepare new transition metal complexes. Metal analysis, IR, and X-ray diffraction (XRD) spectrum analyzes were used to describe them, with some modifications to the World Health Organization (WHO) standard susceptibility test under laboratory conditions. The biological effects of ligands and new complexes against Aedes aegypti mosquito larvae were evaluated at various concentrations. To assess larvicidal efficacy, late third or early fourth instar larvae were exposed to multiple concentrations of the examined compounds, ranging from 50 to 1000 ppm. Cu(II) complexes revealed significant high activity (LC50 = 100 ppm against Ae. Aegypti was compared with the rest of the metal complexes. On the other hand, the Co(II) complex showed no activity against Ae. aegypti. These mixed ligand complexes seemed to be an alternative method for manufactured insecticides to control larvae of this medically important mosquito vector Ae. aegypti. Further research on other metal complex compounds responsible for larvicidal efficacy will be required. Article History Received October 20, 2023 Accepted May 10, 2024 Published August 21, 2024 Keywords 1. complex; 2. mixed ligands; 3. XRD; 4. Aedes aegypti; 5. dengue fever. Section Editor Rogéria Rocha Gonçalves Highlights Chloroquine phosphate was used with ibuprofen and sulphamethoxazole as mixed ligands. The mixed ligands were complexed with Ni(II), Co(II), Cu(II), and Zn(II). The biological effects against Aedes aegypti mosquito larvae were evaluated. The Cu(II) complexes revealed higher activity against Aedes aegypti than others. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://ror.org/04hcvaf32 mailto:y.jamil@su.edu.ye https://orcid.org/0000-0003-1575-3673 https://orcid.org/0000-0002-4203-7536 https://orcid.org/0000-0003-1585-3770 https://orcid.org/0000-0002-5578-2981 https://orcid.org/0009-0005-8232-5101 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 2/20 1. Introduction Mosquitoes belong to the order Diptera presented worldwide in most ecosystems. They are widely distributed around the world and can transmit viral, protozoal, and helminth diseases to human beings, such as dengue fever, chikungunya, West Nile virus, yellow fever, Rift Valley fever, malaria and elephantiasis in almost all tropical and subtropical countries, as well as many other parts of the world (Chew et al., 2019; Netoi et al., 2018). Aedes aegypti Linnaeus was described in 1762 (Bar et al., 2013). In most of the world, it is the primary vector for dengue (WHO, 2009), yellow fever (Al–Azab et al., 2020), zika (Fauci and Morens, 2016; Marcondes and Ximenes, 2015; Schrauf et al., 2020), chikungunya (Charrel et al., 2014). Due to its strong ties to people, Aedes aegypti is significant. Unlike other animals, females attack people. They lay their eggs near human habitations in man- made containers, typically breeding grounds that are not completely covered with many organic resources for larval nutrition. Aedes aegypti has become more adapted to urban surroundings all over the world due to unchecked urbanization, rising temperatures, and a lack of long–term vector control measures (David et al., 2021; Leandro et al., 2023; Maciel-de- Freitas et al., 2007; Scott et al., 1993). In some tropical and subtropical countries in the past 30 years, dengue fever outbreaks have grown ten times (Simmons and Farrar, 2009; WHO, 2009). The dengue virus has infected more than 3 billion people around the world and is responsible for more than 20,000 deaths annually. A total of 128 countries are in danger (Bhatt et al., 2013; Rajaganesh et al., 2016; Suresh et al., 2015). Dengue fever (DF) is a common local endemic infectious disease that mainly affects underdeveloped areas of Yemen and has high mortality and severity rates. This prevalence and geographic expansion of epidemic dengue have hurt Yemen, and the number of reported cases has increased in lockstep with the nation’s social upheaval and civil war (Alyousefi et al., 2016; Gutu et al., 2021). Since 2000, there have been regular outbreaks of DF in Yemen, where it was initially identified in the Shabwah governorate in 1994. Aden and Taiz (2010; 2020), Hadramout and Mulalla (2005), and Al-Hudeidah Governorate, Yemen (1994, 2000, 2004, and 2005), outbreaks have been detected in Shabwah Governorate in 2001, 2002, 2005, 2018, 2019, and 2020 (unpublished reports) (Bin Ghouth et al., 2012; Gutu et al., 2021). Chloroquine (CQ), an antimalarial drug, was first developed in the 1930s. It is produced by a convergent synthesis in which the aliphatic components (novaldiamine, 2–amino–5– dimethylaminopentane) and the quinoline parts (4,7– dichloroquinoline) are combined in the final stage of aromatic nucleophilic displacement. Although affordable and widely available, chloroquine is no longer highly effective against P. falciparum, the most lethal strain of the malaria parasite, due to drug resistance (Kucharski et al., 2022; V. Singh, 2019). The bacteriostatic agent sulfamethoxazole (SMX) is an antibiotic ften used for the treatment of many infections. Due to its common use, humans and animals can develop active and unmetabolized metabolites that have a variety of biological effects (Jasim et al., 2022). In both human and veterinary medicine, is a frequently prescribed antibiotic that is also used as a sulfonamide in aquatic and terrestrial environments. In a European assessment of medicine and personal hygiene items, it was also included in the top 10 lists of essential drugs (Rauseo et al., 2019). One non–steroidal anti-inflammatory medicine (NSAID) that has therapeutic effects is Ibuprofen, which inhibits both the COX1 and COX2 isoforms of the cyclooxygenase enzyme. The production of prostaglandins, which are endogenous mediators implicated in the start of pain, inflammation, and fever, is catalyzed by this enzyme (Singh, 2019; Kucharski et al., 2022). Metals have always attracted researchers in modern medicine because of their significant involvement in physiological and pathological processes, and their antibacterial, antifungal, anti-inflammatory, and antimalarial capabilities. In many fields as molecular biology, photochemistry, bioinorganic chemistry, medicine, and chemistry, mixed ligand–metal complexes are essential (Muthuppalani et al., 2022; A. Singh et al., 2020). The biological features of these compounds and their inherent chemical value as multidentate ligands have been influenced by specific central metals, particularly Cu(II), Zn(II), Fe(III), Ni(II), Co(II), etc. This has led to a substantial improvement in the research on their coordination behavior (Kumar et al., 2021). One of the main goals of modern inorganic coordination chemists and pharmaceutical research is to find and produce more effective pharmaceuticals to treat ailments. This has led to several investigations of drug–metal complexes (Adediji et al., 2018), and researchers are exploring the therapeutic potential of metal-based antimalarial agents, especially since the discovery of ferroquine, once the most potent organometallic antimalarial drug (Biot et al., 2011). In this work, the potential larvicidal activities of these compounds are evaluated together with the production and characterization of complexes from chloroquine phosphate, ibuprofen and sulphamethoxazole with transition–metal ions. 2. Experimental 2.1. Materials High–purity chemicals were used by many companies. Chloroquine phosphate, ibuprofen (Ibu), and sulphamethoxazole were obtained from Shaphaco Pharmaceutical Company. Other chemicals, solvents, indicators, and metal (II) chlorides (nickel chloride hexahydrate, cobalt chloride hexahydrate, copper chloride dehydrate, and zinc chloride hexahydrate) from BDH were used without further purification. 2.2. Preparation of metal complexes In general, aqueous solutions of hydrated metal chlorides, chloroquine phosphate, and (10% ammonia solution of ibuprofen or 10% potassium hydroxide solution of sulphamethoxazole) in 1:1:1 mole ratio are prepared. The combination of the corresponding three solutions was refluxed on a hot plate for 3 h at 60–80 °C with constant stirring to obtain colored precipitates. Filters were used to obtain the solid precipitates, then carefully cleaned in hot water and dried by air (Muthuppalani et al., 2022). 2.3. Physical Measurements The Central Laboratory of the Faculty of Science of Cairo University, Giza, Egypt, used a Vario EL Fab. CHNS Nr to conduct the C, H, N, and S element analysis. At the Global Pharmaceutical Company, the metal content was evaluated using an atomic absorption spectrophotometer with standard solutions of metal chlorides used for calibration. A definite weight of the solid complexes was digested with 10 mL of concentrated HNO3. This was repeated several times until all the organic matter was completely decomposed. The reaction mixture was evaporated just to dryness. After being cooled, the residue complex was dissolved in distilled water (Bader, 2011). At the Global https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 3/20 Pharmaceutical Company, a Sartorius conductivity meter model pp20 was used to measure the molar conductance of 10–3 mol L–1 solutions of metal complexes in 10% DMSO (dimethylsulfoxide) solvent. For the freshly produced solutions, all measurements were made at room temperature. At Sana’a University, the infrared spectra of the complexes were measured using an FT/IR– 140 spectrophotometer with a KBr disc (4000–400 cm–1) from Jasco, Japan. A magnetic susceptibility balance from the Johnson– Metthey and Sherwood model was used to measure magnetic susceptibilities using Gouy’s approach (Szafran et al., 1991). The effective magnetic moment (μeff) values were obtained using the Eqs. 1–3). Xg= 𝑪𝑩𝒂𝒍𝑳 (𝑹−𝑹𝟎) 𝟏𝟎𝟗𝑴 (1) where Ro=Reading of empty tube, L=Sample length (cm), M=Sample mass (g), R=Reading for the tube with sample, CBal=balance calibration constant=2.086 XM= Xg. MWt (2) The values of XM as calculated from Eq. 2 are corrected for the diamagnetism of the ligands using Pascal’s constants, then used in Curie’s Eq. 3. μeff = 2.84√𝑋𝑀. 𝑇 (3) where T = t (°C) + 273 All melting points for the compounds are given in degrees Celsius and are measured in glass capillary tubes. Silver nitrate was used to determine chloride using a gravimetric method (Jamil et al., 2022a). The weight loss approach allowed us to gravimetrically calculate the number of coordinated and uncoordinated water molecules (Refat et al., 2014). TLC was carried out on Silica Gel GF254 plates (mn–kieselgel G., 0.2 mm thickness) with acetone solution as mobile eluent at room temperature (25 oC), plates were scanned using an ultraviolet 254 nm lamp. XRD patterns were obtained using an XD–2 powder X- ray diffractometer (Shimadzu ED–720) at a voltage of 35 kV and a current of mA using CuK(α) radiation in the range of 5o ˂ 2θ ˂ 70o at 1o min–1 scanning rate and a wavelength 1.54056 Å, in Yemen Geological Survey and Mineral Resources Board. 2.4. Crystallinity and particle size from XRD The percentage of crystallinity, XC (%) was calculated based on the integrated peak areas of the principal peaks (Jamil et al., 2023a). The crystallinity of the complexes is calculated relative to the crystallinity of the ligands as a ratio (Eq. 4): 𝑋𝐶 (%) = [ (𝐴𝑙𝑖𝑔𝑎𝑛𝑑) (𝐴𝑐𝑜𝑚𝑝𝑙𝑒𝑥) ] × 100 (4) where Acomplex and Aligand are the areas under the principal peaks of the complex and ligand sample, respectively. XRD was also used to determine the average particle size (D), which was estimated by the Scherrer equation (Eq. 5) (Patterson, 1939; Saeed et al., 2015). 𝐷 = 𝐾𝜆 𝛽 𝑐𝑜𝑠𝜃 (5) where K is Scherrer constant and equals 0.94, λ is the X-ray wavelength of Cu–Kα radiations (1.5405 Å), β is the full width at half maximum (FWHM) and θ is the Bragg diffraction angle in degrees. 2.5. Source of mosquito strain and rearing 2.5.1. Insects: mosquitoes Aedes aegypti (Diptera; Culicidae) were obtained from untreated sites using insecticides from Sana’a city. 2.5.2. Mosquito strain rearing The larvae of Ae. aegypti were reared under laboratory conditions of medical entomology, Department of Biological Sciences, Faculty of Science – Sana’a University for over several generations using fish food diet medium, larvae from culture. 2.5.3. Bioassay of mixed ligand complexes against Ae. aegypti (L.) The larvicidal bioassay followed the WHO (2008) standard protocols with some modifications. Individual ligands and metal complexes were dissolved in 3% DMSO and the results were recorded 24 h later. 3. Results and discussion After being washed with hot water, the Ni(II), Co(II), Cu(II), and Zn(II) complexes produced with the ligands were collected. Because of this, the complexes on their thin–layer chromatography displayed single spots with acceptable Rf values, indicating their pure condition. Insoluble in water, ethanol, methanol, acetone, CHCl3, 0.1 mol/L NaOH, and dimethylformamide (DMF), all complexes were pigmented and stable in the presence of air but were all soluble in DMSO 10% and 0.1 mol/L HCl. The new complexes have melting points higher than those of the pure ligands. The melting points of Ni(II), Co(II) and Cu(II) complexes with the complex (CQ)(Ibu) and [Zn(CQ)(SMX)Cl3] were (> 350 °C). The melting temperatures of the other materials ranged from 204 to 254 °C. The complexes of Ni(II), Co(II), Cu(II) and Zn(II) exhibited molar conductivity values between 7 and 15 Ω–1 mol–1 cm2, indicating the non– electrolytic nature of these complexes (Jamil et al., 2022b). Some physical properties and analytical data of these complexes are listed in Tables 1 and 2. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 4/20 Table 1. Some physical properties of the complexes. Compound Color (Yield%) M.P. (˚C) Ʌm (Ω–1 cm2 mol–1) µeff (B.M.) F.Wt (g/mol) TLC No. of Spots Rf* [Ni(CQ)(Ibu)(H2O)2Cl] Light yellow (21) >350 9 3.09 655.343 One 0.81 [Co(CQ)(Ibu)(H2O)2Cl] Dark purple (26) >350 7 4.75 655.583 One 0.71 [Cu(CQ)(Ibu)(H2O)2Cl] Light green (20) >350 8 1.68 660.199 One 0.54 [Zn (CQ)(Ibu)(H2O)2Cl] White (22) 240 7 – 662.033 One 0.67 [Ni (CQ)(SMX )(H2O)Cl2] Light green (36) 204 15 3.01 720.791 One 0.85 [Co (CQ)(SMX)(H2O)Cl2] Pink (22) 254 14 5.05 721.001 One 0.74 [Cu (CQ)(SMX)Cl3] Sky blue (25) 220 12 1.73 743.055 One 0.49 [Zn (CQ)(SMX)Cl3] White (28) >350 14 – 744.889 One 0.61 Note: *Rf = retention factor in Thin Layer Chromatography Table 2. Elemental analysis of the complexes. Complex (Molecular formula) Element Analysis, Found (Calculated)% C H N S M Cl [Ni(CQ)(Ibu)(H2O)2Cl] 56.82 7.21 6.42 – 8.98 10.83 (C31H47Cl2NiN3O4) (56.81) (7.23) (6.41) – (8.96) (10.82) [Co(CQ)(Ibu)(H2O)2Cl] 56.79 7.25 6.44 – 8.94 10.85 (C31H47Cl2CoN3O4) (56.75) (7.23) (6.41) – (8.99) (10.82) [Cu(CQ)(Ibu)(H2O)2Cl] 56.43 7.16 6.38 – 9.67 10.75 (C31H47Cl2CuN3O4) (56.40) (7.18) (6.36) – (9.63) (10.74) [Zn(CQ)(Ibu)(H2O)3] 56.28 7.14 6.37 – 9.86 10.73 (C31H47Cl2ZnN3O4) (56.24) (7.16) (6.35) – (9.88) (10.71) [Ni(CQ)(SMX)(H2O)Cl2] 46.61 5.40 11.58 4.47 8.10 14.72 (C28H37Cl3CuO4N6S) (46.66) (5.45) (11.66) (4.45) (8.14) (14.76) [Co(CQ)(SMX)(H2O)Cl2] 46.61 5.43 11.67 4.42 8.19 14.79 (C28H39Cl3CoN6SO4) (46.64) (5.45) (11.66) (4.45) (8.17) (14.75) [Cu(CQ)(SMX)Cl3] 45.31 5.09 11.27 4.30 8.60 19.01 (C28H37Cl4CuO3N6S) (45.25) (5.02) (11.31) (4.32) (8.55) (19.06) [Zn(CQ)(SMX)Cl3] 45.21 4.97 11.62 4.33 8.81 14.71 (C28H37Cl4ZnN6O4S) (45.15) (5.01) (11.66) (4.30) (8.78) (14.76) 3.1. The infrared spectra The infrared spectrum of chloroquine was compared with that of the metal complex. It is reported (Otuokere et al., 2019) that the vibration frequencies of NH and C=N of chloroquine occur at 3260 and 1580 cm–1. Chloroquine in these complexes behaves as a neutral monodentate molecule, due to the nitrogen atom in the quinoline ring. 3.1.1. Infrared spectra of nickel (II), cobalt (II), copper (II) and zinc (II) complexes with chloroquine and ibuprofen Ibuprofen was bidentate through the oxygen atoms in the carboxyl group. As a result, in these complexes (Figs. S1–S4), a metal ion is coordinated with each of the two ligands, ibuprofen and chloroquine (Table S1) summarizes the bonding site assignments, which are based on the following pieces of evidence. A wide band that was present in all complexes within the 3467– 3195 cm–1 range has been assigned to ν(OH) and v(NH), with ν(OH) appearing at 3388–3467 cm–1 and ν(NH) appearing at 3195–3220 cm–1 (Otuokere et al., 2019). At 3451 cm–1, ν(OH) in the ibuprofen–free ligand is visible. The coordination is confirmed by the absence of ν(OH) vibration (Abbas et al., 2022). According to (Bamigboye et al., 2020), coordinated water can be distinguished by the appearance of 880 and 522 cm–1, respectively. The peak at 1709 cm–1, previously ascribed to the free ibuprofen band of C=O stretching (Abbas et al., 2022), has been shifted to 1722 cm–1, indicating that coordination occurred at this position. The stretch of C=N in free chloroquine is linked to the peak at 1580 cm–1 (Mohammad and Abdullah, et al., 1984). This peak moved to 1458 cm–1, indicating that the C=N functional group was involved in coordination. The C=N bond length grew because of the falling electron density, which in turn caused the vibration frequency to decrease. This change implies that the location of the quinoline ring in chloroquine caused coordination. For each of the M–O vibrations at 416–445 cm–1, the Ni(II), Co(II), Cu(II), and Zn(II) complexes displayed a single weak band. The current evidence of υ(M–N) could not be brought into the IR data due to instrumental limitations in the 219–297 cm–1 region (Nakamoto, 1978). 3.1.2. Infrared spectra of nickel(II), cobalt(II), copper(II), and zinc(II) complexes with chloroquine and sulfamethoxazole Using KBr discs to examine the infrared bands of free sulfamethoxazole, three distinct and strong bands were found at 3466, 3377, and 3298 cm–1. These bands are the result of symmetric and asymmetric stretching vibrations of the aromatic amino group (NH2) and sulfonamide (NH), respectively (Al–Noor et al., 2014; Alosaimi, 2022; Chavda et al., 2021). According to Torre et al. (2005) and Jamil et al. (2017), stretching vibrations of the S=O group were found at 1365 cm–1 and 1091 cm–1, respectively. By stretching the phenyl ring (C=C) strong and sharp bands are generated at 1596 cm–1 and 1503 cm–1. As shown in Figs. S5 and S6, the complexes have the same bands in the same location with the same wave number without shifting. However, the complex in Fig. S7 shows only one band at 3443 cm–1, with the other bands disappearing. Three bands overlap, resulting in the formation of a broadband and the elimination of the other bands. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 5/20 The complex in Fig. S8 contains a single broadband at 3453cm–1 for the same reason. The S=O group stretching vibrations are asymmetric and symmetric at 1365 and 1091 cm–1, respectively. Asymmetric and symmetric were found at 1383 cm–1 and 1076 cm–1, respectively, for the molecule shown in Fig. S5. Table S2 contains the remaining items. The M–O stretching vibrations in the 421–483 cm–1 region are responsible for the new bands in the complex spectra. Current evidence of υ(M–N) was not included in IR data due to an experimental limitation in the 219–297 cm–1 range (Nakamoto, 1978). 3.2. X–ray diffraction 3.2.1. X–ray diffraction of the chloroquine, ibuprofen ligands, and their complexes Figure S9–S14 represent the XRD patterns for chloroquine, ibuprofen, and its complexes. From these figures, a notable shifting of the main peak toward higher diffraction angles (Table S3) is observed for the complexes, suggesting the reduction of the unit cell dimensions and consequently contracting the crystal lattice (Aroyo, 2016). In addition, significant changes in intensities of the main peaks of ligands and their complexes are observed in these figures, which are attributed to the reduction in crystallinity. The crystallinity calculations are based on the ratio of the principal peak area of the complex sample to that of the ligand sample, obtaining a relative crystallinity (Shah et al., 2006). The results in Table S3 show significant changes in crystallinity between chloroquine, ibuprofen, and its complexes that exhibit low relative crystallinity (3.201, 4.600, 23.017 and 24.366%). In the literature, the range between 1–100 nm is reported to be of nanoparticle size (Boverhof et al., 2015). The particle size of the complexes obtained from XRD shows effects on their nanoparticle size (4.422, 2.99, 4.821 and 4.4054 nm). 3.2.2. X–ray diffraction of the chloroquine, sulphamethoxazol ligands, and their complexes The XRD patterns for chloroquine, sulfamethoxazole, and their compounds are shown in Fig. S15– S19. According to these figures, a notable shift of the primary peak for the complexes towards higher diffraction angles (Table S4) suggests that the dimensions of the unit cells have decreased, which has caused the crystal lattice to constrict (Aroyo, 2016). Calculations of relative crystallinity are also based on the ratio of the primary peak area of the complex sample to that of the ligand sample (Shah et al., 2006). The findings shown in Table S4 demonstrate considerable variations in the relative crystallinity of chloroquine, sulfamethoxazole and their complexes (61.386, 23.513, 92.978 and 32.538%). The literature reports that the sizes of nanoparticles fall within the range of 1 to 100 nm (Boverhof et al., 2015). The complexes discovered by XRD had nanoparticle–sized particles (5.058, 4.789, 3.5754 and 4.9042 nm). The proposed structure of the complexes in Figs. 1 and 2. 3.3 Biological effect of metal complexes against Aedes aegypti larvae The larvicidal bioassay adhered to the established guidelines by the WHO (Abbott, 1925). Concentrations of 50, 100, 150, 300, 500, and 1000 ppm were prepared in 3% DMSO for each chemical. The late third or early fourth instar larvae were constantly exposed to different concentrations of chemicals for the duration of the treatments. Following these tests, the larvae were given their regular diet. The fatality percentage was reported after a 24–hour exposure period. The compounds were carefully stored according to the recommended conditions by periodic measurements to assess their pH, solubility, and dissolution stability. DMSO solvent was employed to ensure the integrity of the compounds, avoiding any solvent that could potentially affect their properties. The compounds’ physical appearance, including color and odor, were examined in addition to the physicochemical assessments. Comprehensive biological tests took place to evaluate their properties during the storage period. These experiments involved a wide range of concentrations at the beginning of the evaluation, and to determine the stability and shelf–life of these compounds high (1000 ppm) and low (50 ppm) concentrations were utilized. These concentrations were chosen after four months of storage, reflecting real–life conditions. By employing this approach, the compounds’ long–term stability could be assessed and established their suitability for practical applications. Figure 1. Proposed structure of (CQ)(Ibu) complexes. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 6/20 Figure 2. Proposed structure of (CQ)(SMX)complexes. 3.3.1 Statistical analysis A complete randomized design (CRD) was used in a factorial experiment design. Using the Minitab application, the statistical analysis of the data was performed using analysis of variance (ANOVA) tools, and the averages were compared by Least Significance Difference LSD at p ≤ 0.05. According to the Probit analysis program, LC50 (concentration that kills 50% of mosquito larvae) and LC95 (concentration that kills 95% of mosquito larvae) were determined (Abbott, 1925; Tisgratog et al., 2016). According to an automated log–probit analysis, the 95% confidence interval. 3.3.2. Biological effect of the complexes nickel (II), cobalt (II), copper (II) and zinc (II) with chloroquine phosphate, and ibuprofen against larvae mosquito (Aedes aegypti) As given in Table S5 chloroquine, ibuprofen and their complexes with nickel (II), cobalt (II), copper (II) and zinc (II) were less effective in larvae mortality compared to the ibuprofen compound at concentrations of 1000 ppm and 300 ppm. For copper and zinc metal complexes, the percentage of mosquito larvae mortality was higher than that of the nickel (II) complex, and the cobalt (II) complex did not affect mosquito larvae. The acidic effect of the chloroquine diphosphate ligand resulting from the two phosphate groups is the main reason for the effectiveness, where the pH is (3.8 to 4.3). The effect of free Ibuprofen may be due to the carboxylic acid group of the acidic effect. It was also assumed that complexes with endogenous metals (Ni, Co, Cu, and Zn) could be less toxic than those with nonessential metals (WHO, 1999). Copper–containing coordination compounds were found to be promising therapeutic agents in a diverse range of diseases, including malaria, due to their ability to act by various mechanisms, such as inhibition of proteasome activity (WHO, 2003), telomerase activity (Meshnick and Dobson, 2001), and formation of reactive oxygen species (ROS) (Wells, 2011), DNA degradation (Stork et al., 2001), and DNA intercalation (Wongsrichanalai et al., 2002). All complexes are less acidic than their ligands due to chelation and changing of the –COOH group in ibuprofen into the –COO– group but, may have a high effect of some complexes against Ae. aegypti. depend on the toxicity of the metal (Rayms–Keller et al., 1998). The effectiveness of CQ,Ibu–Cu complexes increases with concentration (Table 3). The high effect of this complex against Ae. aegypti depends on the toxicity of the metal (Rayms–Keller et al., 1998). Then, the toxicity line was drowned to calculate LC50 and LC95 (Table 4 and Fig. S20). Table 3. Toxicity effect of CQ,Ibu–Cu complex against Ae. aegypti larvae. Compound Conc. (ppm) Larval Mortality (%) CQ,Ibu–Cu complex 50 10 100 17 150 26 300 70 500 88 1000 100 Control 3% DMSO 0 Table 4. Statistical parameters of CQ,Ibu –Cu complex. Statistical parameters CQ, Ibu–Cu complex LC50 (ppm) 209.73 95% (*F. L) 198.48 – 221.09 LC95 (ppm) 609.12 95% (*F. L) 565.26 – 662.08 Slope 1.37 R2 0.98 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 7/20 3.3.3. Biological effect of nickel (II), cobalt (II), copper (II) and zinc (II) complexes with chloroquine phosphate and sulfamethoxazole against larvae mosquito (Aedes aegypti) Chloroquine, sulfamethoxazole and their complexes with nickel (II), cobalt (II), copper (II) and zinc (II) were less efficient in causing mortality in larvae than their complexes at concentrations of 1000 and 300 ppm, as shown in Table S6. Sulphamethoxazole ligand may have a basic effect in situations where it was not effective against larvae, and the effects of all subsequent ligands and complexes rely on acidity. The effectiveness of the CQ,SMX–Cu complex increased with concentration (Table 5). Depending on the metal’s toxicity, this combination has a strong anti–Aedes aegypti action (Wongsrichanalai et al., 2002). Then, the toxicity line was drowned to calculate LC50 and LC95 (Jamil et al., 2023b) (Table 6 and Fig. S21). Table 5. Toxicity effect of CQ,SMX–Cu complex against Ae. aegypti larvae. Compound Concentration (ppm) Larval Mortality (%) CQ SMX–Cu Complex 50 30 100 50 150 68 300 80 500 85 1000 100 Control 3% DMSO 0 Table 6. Statistical parameters of CQ,SMX–Cu complex. Statistical parameters CQ,SMX–Cu complex LC50 (ppm) 103.12 95% (*F. L) 92.56 – 113.51 LC95 (ppm) 699.39 95% (*F. L) 622.64 – 799.36 Slope 0.76 R2 0.94 4. Conclusions The novel compounds have been produced by mixing chloroquine phosphate with ibuprofen and sulfamethoxazole, individually with the metals Ni(II), Co(II), Cu(II) and Zn(II). This research has looked at how the ligands and their complexes affect Ae. aegypti mosquito larvae biologically. Based on these findings, it was determined that the mixed ligand complexes have beneficial effects against the larval stage of Ae. aegypti. The most effective compounds were Cu(II) complexes, followed by Ni(II), Co(II), and Zn(II) complexes. The Cu(II) complexes may offer other strategies for limiting the Ae. aegypti mosquito, the dengue vector, according to this study’s findings. These findings could motivate researchers to look for new active substances that could be effective substitutes for the present pesticides. Authors’ contributions Conceptualization: Jamil, Y. M.; Al–Azab, F. M.; Al–Ryami, A. M.; Data curation: Al–Ryami, A. M.; Formal Analysis: Jamil, Y. M.; Al– Ryami, A. M.; Al–Selwi, N. A.; Funding acquisition: Not applicable; Investigation: Jamil, Y. M.; Al–Azab, A. M.; Al–Ryami, A. M.; Methodology: Jamil, Y. M.; Al–Azab, A. M.; Al–Ryami, A. M.; Project administration: Jamil, Y. M.; Resources: Jamil, Y. M.; Al–Azab, F. M.; Al–Azab, A. M.; Software: Jamil, Y. M.; Al–Azab, A. M.; Al–Ryami, A. M.; Supervision: Jamil, Y. M.; Al–Azab, F. M.; Validation: Jamil, Y. M.; Al–Azab, F. M.; Visualization: Jamil, Y. M.; Al–Azab, F. M.; Al–Selwi, N. A.; Writing – original draft: Al–Ryami, A. M.; Al–Selwi, N. 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Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 10/20 Supplementary Material Figure S1. Infrared Spectrum of [Ni(CQ)(Ibu)(H2O)2Cl]complex. Figure S2. Infrared Spectrum of [Co(CQ)(Ibu)(H2O)2Cl]complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 11/20 Figure S3. Infrared Spectrum of [Cu(CQ)(Ibu)(H2O)2Cl]complex. Figure S4. Infrared Spectrum of [Zn(CQ)(Ibu)(H2O)2Cl]complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 12/20 Figure S5. Infrared Spectrum of [Ni(CQ)( SMX)(H2O)Cl2] complex. Figure S6. Infrared Spectrum of [Co(CQ)(SMX)(H2O)Cl2] complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 13/20 Figure S7. Infrared Spectrum of [Cu(CQ)(SMX)Cl3] complex. Figure S8. Infrared Spectrum of [Zn(CQ)(SMX)Cl3] complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 14/20 Figure S9. XRD pattern for chloroquine phosphate. Figure S10. XRD pattern for ibuprofen. Figure S11. XRD pattern for CQ,Ibu-Ni complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 15/20 Figure S12. XRD pattern for CQ,Ibu-Co.complex. Figure S13. XRD pattern for CQ,Ibu-Cu.complex. Figure S14. XRD pattern for CQ,Ibu-Zn.complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 16/20 Figure S15. XRD pattern for CQ,SMX-Co complex. Figure S16. XRD pattern for CQ,SMX-Cu complex. Figure S17. XRD pattern for CQ,SMX-Zn complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 17/20 Figure S18. XRD pattern for sulphamethoxazole. Figure S19. XRD pattern for CQ,SMX-Ni complex. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 18/20 Figure S20. The relationship between concentration of CQ,Ibu-Cu complex and mortality percentage of Ae. aegypti larvae. Figure S21. The relationship between concentration of CQ,SMX-Cu complex and mortality percentage of Ae. aegypti larvae. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 19/20 Table S1. Significant IR spectral bands (cm–1) of complexes with chloroquine and ibuprofen. [Ni(CQ)(Ibu)(H2O)2Cl] [Co(CQ)(Ibu)(H2O)2Cl] [Cu(CQ)(Ibu)(H2O)2Cl] [Zn(CQ)(Ibu)(H2O)2Cl] Assignment 3215 br &w 3210 br & w 3195 br & w 3220 br & w ν(NH) 3388 br &s 3448 br & w 3467 br & w 3422 br & w ν(OH) of H2O 1626 sh&w 1633 sh & w 1637 sh & w 1638 sh & w Benzene ring +pyridine ring stretch 2959 sh&w 3064 sh & w 2954 sh & m 2955 sh & w νCH–arom 2859 sh&w 2859 sh & w 2867 br & w 2853 sh & w νCH–aliph 1101 sh&m 1102sh & m 1053 sh & m 1115 sh & m ν(C–N) 1722 sh&m 1723 sh & m 1720 sh & w 1720 sh & m ν(C=O) 1467 sh&w 1459 sh & w 1462 sh & w 1458 sh & w ν(C=N) 416 sh&w 420 sh & w 445 sh & w 419 sh & w ν(M–O) Note: w = weak; m = medium; s = strong; br = broad; sh= short. Table S2. Significant IR spectral bands (cm–1) of complexes with chloroquine and sulfamethoxazole. [Ni(CQ)(SMX)(H2O)Cl2] [Co(CQ)(SMX)(H2O)Cl2] [Cu(CQ)(SMX)Cl3] [Zn(CQ)(SMX)Cl3] Assignment Overlab with ν(OH) of H2O Overlab with ν(OH) of H2O 3298 sh & s 3298 sh & s ν(NH) 3440 br & m 3453 br & m – – ν(OH) of H2O 1591 br & m 1578 sh & w 1596 sh & w 1502 sh & m Benzene ring +pyridine ring stretch 3147 br & s 3187 br & s 3239 sh & s 3143 sh & m νCH–arom 3030 br & w 3068 br & s 3066 sh & s 2958 sh & s νCH–aliph 1076 sh & w 1068 sh & w 1091 sh & s 1091 br & w ν(C–N) 1383 sh & w 1383 sh & w 1382 sh & s 1366 sh & s ν(O=S=O) 1483 sh & w 1490 sh & w 1502 sh & w 1502 sh & w ν(C=N) 483sh & w 410sh & w 419 sh & w 421 sh & w ν(M–O) Note: w = weak; m = medium; s = strong; br = broad; sh= short. Table S3. XRD spectra data of the principal intensity values of the ligands chloroquine, ibuprofen, and their complexes with (Ni,Co,Cu, and Zn). Compound 2θ β D (nm) Mean D XC (%) CQ. 19.581 0.223 6.5895 7.056 100 26.259 0.231 6.4369 21.321 0.181 8.1409 Ibu 12.16 0.422 3.451 3.658 100 16.70 0.389 3.762 20.16 0.301 4.886 22.30 0.397 3.718 24.64 0.600 2.470 CQ,Ibu–Ni complex 10.16 0.357 4.0725 4.422 3.201 32.26 0.316 4.7704 CQ,Ibu–Co complex 7.121 0.622 2.3327 2.998 4.600 7.379 0.620 2.3405 7.62 0.620 2.3407 8.40 0.387 3.7519 13.101 0.345 4.2247 CQ,Ibu–Cu complex 14.841 0.216 6.761 4.821 23.017 18.04 0.379 3.869 18.30 0.422 3.476 30.459 0.174 8.625 33.60 0.331 4.569 33.88 0.408 3.710 34.101 0.553 2.739 CQ,Ibu–Zn complex 9.780 0.364 3.9929 4.4054 24.366 16.740 0.298 4.9119 19.539 0.314 4.6795 20.319 0.303 4.8553 22.440 0.408 3.6183 31.56 0.344 4.3744 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 Eclet. Quim. 49 | e-1526, 2024 ISSN 1678-4618 page 20/20 Table S4. XRD spectra data of the principal intensity values of the ligand’s chloroquine, sulphamethoxazole and their complexes with (Ni, Co, Cu, and Zn). Compound 2θ β D (nm) Mean D XC (%) CQ. 19.581 0.223 6.5895 7.056 100 26.259 0.231 6.4369 21.321 0.181 8.1409 SMX. 13.98 0.247 5.9066 6.570 100 20.98 0.205 7.1839 24.22 0.253 5.8539 29.121 0.204 7.3339 CQ, SMX –Ni complex 11.4 0.334 4.3571 5.058 61.386 13.46 0.332 4.3919 18.5 0.216 6.7924 22.259 0.261 5.6545 28.342 0.271 5.5111 30.682 0.248 6.0547 33.602 0.403 3.7534 36.22 0.351 4.3408 37.837 0.203 7.5409 39.699 0.28 5.4983 44.484 0.895 1.7480 CQ, SMX –Co complex 13.1 0.244 5.9737 4.789 23.513 21.9 0.279 5.2864 26.34 1.111 1.3386 33.339 0.323 4.6798 55.24 0.245 6.6706 CQ, SMX –Cu complex 9.3 0.696 2.0874 3.5754 92.978 20.88 0.237 6.2128 28.98 0.341 4.3860 29.52 0.927 1.6154 CQ, SMX –Zn complex 14.279 0.308 4.7382 4.9042 32.538 20.4 0.505 2.9135 20.759 0.2 7.3608 24.00 0.274 5.4029 28.24 0.269 5.5509 28.901 0.325 4.6011 32.58 0.401 3.7621 Table S5. Date mortality of larvae after 24 h for chloroquine, Ibuprofen, and their complex. Compound Percentage of mortality of Aedes aegypti after 24 h Water 0 DMSO 3% 0 CQ1000ppm 50 Ibu 1000ppm 100 CQ,Ibu–Ni 1000ppm 50 CQ,Ibu–Co 1000ppm 0 CQ,Ibu–Cu 1000ppm 100 CQ,Ibu–Zn 1000ppm 80 CQ300ppm 20 Ibu 300 ppm 70 CQ,Ibu–Ni 300ppm 40 CQ,Ibu–Co 300ppm 0 CQ,Ibu–Cu 300ppm 70 CQ,Ibu–Zn 300ppm 20 Table S6. Date mortality of larvae after 24 h for chloroquine, sulphamethoxazole and their complex. Compound Percent mortality of larvae after 24 h DMSO 3% 0 CQ1000 ppm 50 CQ300 ppm 20 SMX 1000 ppm 0 SMX 300 ppm 0 CQ,SMX–Ni 1000ppm 0 CQ,SMX–Co 1000ppm 0 CQ,SMX–Cu 1000ppm 100 CQ,SMX–Zn 1000ppm 40 CQ,SMX–Ni 300ppm 0 CQ,SMX–Co 300ppm 0 CQ,SMX–Cu 300ppm 80 CQ,SMX–Zn 300ppm 20 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1526