Thiophene-triazole hybrids inhibit the major toxic activities of Bothrops jararacussu venom

Abstract

Snakebite envenomation (SBE) is a neglected disease that causes approximately 140,000 deaths annually worldwide. Antivenoms save lives; however, the muscle damage and physical sequelae associated with SBE are not effectively treated by this therapy. This study assessed the effect of 16 thiophene-triazole hybrids (6a-h and 7a-h) against the in vitro toxic activities of Bothrops jararacussu venom, which is responsible for most SBE events in Brazil. The compounds were incubated with venom, and the proteolytic, plasma coagulation, and phospholipase A₂ (PLA₂) activities were assessed. Regarding proteolytic activity, compound 7g inhibited 97%, 7a and 7h inhibited ~85%, 6a, 6c, 6d, 7b, and 7c inhibited 58%-65%, 6b, 6e, 6f, and 7f inhibited 30%-40%, and 6g and 7f inhibited 25%. Compounds 6e, 6g, 7b, 7c, and 7e delayed venom-induced plasma coagulation. Regarding PLA₂ activity, 6a, 6b, 6h, 7a, 7c, and 7h achieved 100% of inhibition, 6d, 6e, 6f, 6g, 7b, 7d, 7e, 7f, and 7g achieved 80%-90% and 6c achieved approximately 30%. None of the compounds inhibited all of the assessed toxic effects. These findings suggest that triazole-thiophene hybrids are promising candidates for the development of complementary treatments for B. jararacussu envenomation.

Key words:
antivenom; medicinal chemistry; neutralization; thiophene; triazole; snake venom

Resumo

O envenenamento por serpentes é uma doença negligenciada que causa aproximadamente 140.000 mortes anualmente no mundo. Apesar do antiveneno salvar vidas, o dano muscular e sequelas físicas não são eficazmente revertidos. Este estudo avaliou o efeito de 16 moléculas híbridas contendo tiofeno e triazol (6a-h and 7a-h) sobre atividades tóxicas in vitro do veneno de Bothrops jararacussu, que é endêmica no Brasil. Os compostos foram incubados com o veneno e, em seguida as atividades coagulante, proteolítica e fosfolipasica A₂ (PLA₂) do veneno de B. jararacussu foram realizadas. O composto 7g inibiu 97%, 7a e 7g inibiram 80%, 6a, 6c, 6d, 7b e 7c inibiram 58%-65%, 6b, 6e, 6f e 7f inibiram 30%-40%, 6g e 7f inibiram 25% a atividade proteolítica; os compostos 6e, 6g, 7b, 7c e 7e inibiram a

Palavras-chave:
antiveneno; química medicinal; neutralização; tiofeno; triazol; veneno de serpente

Introduction

Animal venom including that of snakes consists of a complex mixture of proteic and non-proteic components that serve primarily to immobilize, kill, and initiate the digestion of prey. Snakebite envenomation (SBE) is a neglected tropical disease (NTD) that causes 2.7 million accidents, 138,000 deaths, and 400,000 permanent physical sequelae annually (WHO 2024). Furthermore, mental health conditions such as depression and posttraumatic stress should be considered as consequences of SBE (Costa et al. 2019).

In Brazil, 99% of the incidents are caused by the family Viperidae which has three genera: Bothrops (jararacas), Lachesis (bushmaster), and Crotalus (rattlesnakes). Bothrops is the largest genus with 47 species, and is responsible for the highest frequency of SBE in Brazil (approximately 87%); thus, it is the most medically genus in Brazil (SINAN 2024). Bothrops envenomation causes a wide range of toxic effects, including disturbances in blood coagulation, inflammation, acute pain, hemorrhage, edema, blister formation, massive tissue necrosis, renal and cardiac failure, hypotension, and eventually death (Kishimoto et al. 2010; Castañeda et al. 2019). Among Bothrops species, B. jararacussu is the largest (capable of growing up to 2.2 meters), extremely feared, endemic to South America (Lopes et al. 2009), and can inject larger volumes of venom (averaging 300-500 mg). In Brazil, B. jararacussu is found in the coastal areas of Bahia to Santa Catarina (Medeiros et al. 1992) and is included on the International Union for Conservation of Nature and Natural Resources (IUCN) red list of threatened species, mainly due to the destruction of its forest habitat (Scott et al. 2019); however, B. jararacussu is currently in the Least Concern category. The active protein components of snake venoms are classified into 42 families, and the most abundant are snake venom serinoproteases (SVSPs), snake venom metaloproteinases (SVMPs), phospholipases A₂ (PLA₂s), L-aminoacid oxidase (LAAO), C-type lectins (CTL), and three-finger toxins (3-FTX) (Bhattacharjee et al. 2017). SVSPs and SVMPs cause disturbances in the blood coagulation cascade, resulting in hemorrhage and intravascular coagulation, whereas PLA₂ enzymes contribute to hemolysis, muscle damage, neurotoxicity, inflammation, edema, and pain (Pinto et al. 2013).

The only treatment for SBE recommended by the World Health Organization (WHO) is antivenom, which is currently produced through the hyperimmunization of equines (Gutiérrez et al. 2017). Antivenom treatment is effective in saving lives; however, it has some drawbacks, including high production costs, limited distribution in remote areas, and the potential to induce side effects such as anaphylaxis and fever (Marcussi et al. 2019). Other medicines have been used in the clinic to complement antivenom therapy, such as the corticosteroid dexamethasone (Brustolin et al. 2007). Nevertheless, antivenom insufficiently neutralizes local effects, such as muscle damage, which is the most critical issue that may lead to physical sequelae in victims (Amorim et al. 2022; Soares et al. 2025). Thus, seeking complementary treatment for SBE is of substantial importance, particularly in countries that have long experienced negative effects from this NTD.

Plants have been used by healers, indigenous people, and those from the Orient and Occident continents to alleviate a wide range of illness, including SBE. This practice is frequently employed in areas of limited access to commercial antivenoms (Ferreira et al. 2013). In Brazil and other tropical countries, communities frequently use medicines known as “garrafadas”, which are mixtures of macerated plants diluted in alcohol, to alleviate SBE symptoms (Silva et al. 2020). Ethnobotanical studies have identified plants with antivenom properties and compounds such as flavonoids, terpenes, phenolic acids, which have showed inhibitory effect against the toxic effects of snake venoms (Martz 1992; Mors et al. 2000). Therefore, the plant kingdom is a promising source of biologically active compounds with antivenom potential. However, few prospecting studies have assessed the use of organic synthesis-derived compounds as antivenom.

Molecular scaffolds derived from triazoles are regularly used in the pharmaceutical industry and medicinal chemistry due to their wide range of pharmacological effects, demonstrating their potential in drug discovery (Bozorov et al. 2019; Malik et al. 2020). The structure of triazole comprises of a five-membered heterocyclic compound with three nitrogen atoms in the ring; it is heat-stable, which facilitates handling, and its production is cost-effective (Caldeira et al. 2024; Malik et al. 2020). Moreover, 1,2,3-triazole has been utilized as a framework for synthesizing potential pharmacophores in commercial pharmaceuticals (Raman et al. 2025; Bozorov et al. 2019). The triazole ring is biologically active at various positions, enabling it to interact with a broad range of key chemical groups, such as naphthoquinone and thiophene (Bozorov et al. 2019; Chazin et al. 2022).

Thiophene derivatives are characterized by a five-membered aromatic ring containing a sulfur atom. These derivatives are chemically stable and possess a strong ability to form conjugated systems (Thakur et al. 2024). Pharmacological effects of medical interest have been described, including antifungal, antimicrobial, antidepressant, and antileishmanial properties (Chen et al. 2014; Kovačević et al. 2020). While triazole- and thiophene-containing molecules have previously shown inhibitory effects against snake venom enzymes, no specific combination of these moieties as a hybrid compound has been investigated against B. jararacussu venom. Thus, this study addresses a research gap by evaluating the inhibitory effect of 16 triazole-thiophene hybrid compounds against the toxic activities of B. jararacussu venom in vitro, including proteolytic, plasma coagulant, and PLA₂ activities. We hypothesized that some of these compounds would display selective or broad inhibitory effects, offering a potential tool to develop novel complement therapy in addition to commercial antivenom.

Therefore, this study aimed to assess for the first time, the effect of 16 thiophene-triazole hybrid compounds as inhibitors of the plasma coagulant, proteolytic, and PLA₂ activities of B. jararacussu venom.

Material and Methods

Preparation and collection of B. jararacussu venom

Lyophilized crude venom from B. jararacussu was kindly supplied by Dr. Eladio Flores Sanchez from Ezequiel Dias Foundation (FUNED), Belo Horizonte, MG, and stored at -20 °C prior to use. The venom was dissolved in physiological saline solution to a concentration of 1 mg/mL. Snake venom collection was authorized by the Brazilian System for Genetic Management of Heritage and Associated Traditional Knowledge (SISGEN); process number A39CD4E.

Synthesis and preparation of thiophene-triazole hybrid compounds

Ornellas et al. 2024 previously synthesized a series of thiophene-triazole hybrids and determined their chemical structural features (including infrared, nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) and purity. The 16 compounds (6a-h and 7a-h) were kindly provided by Dr. Vitor Francisco Ferreira and Dr. Fernando de Carvalho da Silva from the Institute of Chemistry of Federal Fluminense University. The derivatives were dissolved in 100% dimethylsulfoxide (DMSO, from Sigma Chemical Co., USA) at a concentration of 10 mg/mL and stored at -20 °C prior to use.

Effect of thiophene-triazole hybrid compounds on the proteolytic activity of B. jararacussu venom

The proteolytic activity of B. jararacussu venom was determined using azocasein (Sigma Chemical Co., USA) as the substrate (Garcia et al. 1978). Different concentrations of B. jararacussu venom (20-60 μg/mL) were added to a reaction mixture containing 400 μL of 0.2% azocasein (from Sigma Chemical Co., USA) dissolved in 20 mM CaCl₂ and 200 mM Tris-HCl at pH 8.8 (final volume of 1,200 mL) for 90 min at 37 °C. The enzymatic reaction was stopped by adding 0.4 mL of 10% trichloroacetic acid (Sigma Chemical Co., USA), followed by centrifugation of the tubes for 3 min at 16,740 relative centrifugal force (RCF) (Centrifuge Ultra Speed, Model NT 835, SN 15030153, Novatecnica, Piracicaba, São Paulo, Brazil). Then, 1.0 mL of the supernatant was transferred to tubes containing 0.5 mL 2 M NaOH (Sigma Chemical, Co., USA) and the samples were measured at an absorbance of 420 nm using a spectrophotometer (Kasvi, model K37-VIS, bandwidth 4 nm, wavelength of 320-1,020 nm, São José dos Pinhais, Curitiba, Brazil). The concentration of B. jararacussu venom (μg/mL) that produces an absorbance value of ~0.2 at 420 nm, corresponding to the maximal velocity of the reaction, was defined as the arbitrary unit, enzymatic unit (EU). One EU of B. jararacussu venom (20 μg/mL) was incubated with saline solution (0.9% v/v), DMSO, or 80 μg/mL of the compounds (6a-h and 7a-h) for 30 min at 25 ^oC in a final volume of 220 μL, adjusted with the Tris-HCl buffer (pH 8.8). After incubation, 50 μL of each mixture was added to medium and the proteolytic method was performed as described above. Negative controls contained compounds mixed with the solvent DMSO (0.9% v/v, final concentration) and saline solution (0.9% v/v) or solvents alone in the absence of venom. Assays were performed in two individual experiments with three replicates, yielding a total of n = 6.

Effect of thiophene-triazole hybrid compounds on the plasma coagulation of B. jararacussu venom

The coagulation activity of B. jararacussu venom was assessed using a pool of human plasma obtained from healthy donors at the blood bank of the Antônio Pedro Hospital of Federal Fluminense University (HUAP), under the authorization of the Committee for Ethical in Experimentation (CEP-UFF, CAAE: 28941314.0.0000.5243). Plasma (200 μL) previously diluted 1:1 (v/v) in 0.9% saline solution was incubated for 1 min at 37 °C, followed by the addition of different concentrations of B. jararacussu venom (10-80 μg/mL) to a final volume of 250 μL. Coagulation was monitored using a digital coagulometer (Amelung KC4A, AN Mikro Coagulation Analyzer, GmbH, SN 00N50754, Germany), and the concentration of B. jararacussu venom that clotted the plasma in approximately 60 seconds was defined as the arbitrary unit (minimum coagulant concentration (MCC)). One MCC of B. jararacussu venom (50 μg/mL) was incubated with saline solution, DMSO (0.9% v/v, final concentration), or 200 μg/mL of the compounds (6a-h and 7a-h) for 30 min at 25 °C in a final volume of 220 μL, adjusted with 20 mM Tris-HCl, pH 7.5. Then, an aliquot of this mixture (50 μL) was added to 200 μL of plasma, and coagulation was monitored as described. As negative controls, compounds or solvents without venom were added to the plasma, and coagulation was monitored until 800 s. Each experimental group was performed with three replicates of two individual experiments, yielding n = 6.

Effect of thiophene-triazole hybrid compounds on the PLA ₂ activity of B. jararacussu venom

PLA₂ activity of B. jararacussu venom was determined as described by Marinetti et al. (1965) using hen’s egg yolk as the substrate. Fresh egg yolk (average 15 mL) was obtained from a local supermarket, filtered, and diluted in saline solution to a final volume of 100 mL. The tubes were homogenized and centrifuged at 16,740 RCF (Centrifuge Ultra Speed, Model NT 835, SN 15030153, Novatecnica, Piracicaba, São Paulo, Brazil) for 60 min at 10 ^oC. The supernatant was then diluted in nine parts of saline solution to obtain the working solution of egg yolk. B. jararacussu venom (10 µg) was incubated with solvents (DMSO or saline) and 40 µg/mL of the compounds (6a-h and 7a-h) for 30 min at 25 ^oC in a final volume of 170 μL, adjusted with 20 mM Tris-HCl, pH 7.5. Aliquots of 50 μL of each mixture were added to a reaction mixture containing 580 µL saline, 25 µL sodium taurocholate 0.4% (Sigma Chemical Co., USA), 25 µL Tris-HCl (200 mM), pH 7.5, and 20 µL CaCl₂ (0.5 M). The enzymatic reaction was initiated by adding 300 µL of the working solution of egg yolk. After 30 min of reaction at 37 ^oC, absorbance was measured at 740 nm using a spectrophotometer (Kasvi, model K37-VIS). Incubating B. jararacussu venom with saline in the absence of compounds yielded 100% of PLA₂ activity; 0% of PLA₂ activity was observed when saline solution was incubated without venom. Negative controls contained DMSO (0.9% v/v, final concentration) or compounds without venom. Each experimental group was performed with three replicates of three individual experiments, yielding n = 9.

Statistical analysis

The results are expressed as the means ± standard deviation (SD) analyzed via ANOVA, followed by Dunnett’s post hoc test; p < 0.05 was considered to indicate significance. Statistical analyses and graphs construction were performed using GraphPad Prism® version 9.0 (Software Inc., San Diego, CA, USA).

Results

Chemistry of the thiophene-triazole hybrid compounds

Thiophene-triazole hybrid compounds were synthesized as described by Ornellas et al. (2024). This process yielded 16 compounds (6a-h and 7a-h), the chemical structures of which are shown in Figure 1. The effects of these compounds on the proteolytic, plasma coagulant, and PLA₂ toxic activities of B. jararacussu venom were then assessed. Moreover, the structural elucidation of compounds was performed using infrared spectroscopy, ¹H and ¹³C NMR, and HRMS (Ornellas et al. 2024).

Figure 1 -
Chemical structure of the thiophene-triazole hybrid compounds 6a-h and 7a-h.

Antiproteolytic effect of thiophene-triazole hybrid compounds

As shown in Figure 2, all 16 compounds (6a-h and 7a-h) at the concentration of 80 µg/mL inhibited the proteolytic activity of 20 µg/mL of B. jararacussu venom, albeit at varying percentages. Compound 7g inhibited 97% of venom-induced proteolysis, whereas compounds 6h, 7a, 7e, and 7h inhibited 85%-88%, 6a, 6c, 6d, 7b, and 7c inhibited 58%-65%, 6b, 6e, 6f, and 7f inhibited 30%-39%, and 6g inhibited 25% (Fig. 2). None of the compounds without venom hydrolyzed azocasein under the experimental conditions (data not shown).

Figure 2 -
Effect of compounds on the proteolytic activity of B. jararacussu venom - B. jararacussu venom (20 µg/mL) was incubated with saline, DMSO, or 80 µg/mL of the derivatives (6a-h and 7a-h) for 30 min at 25 ^oC. The results are expressed the percentage of proteolytic activity, with means ± SD (n = 6) of two individual experiments. *, p < 0.05 compared with B. jararacussu + saline and DMSO. The raw values are shown in the supplementary material (Table S2, available on supplementary material <10.6084/m9.figshare.30379603>).

Anticoagulant effect of thiophene-triazole hybrid compounds

The MCC of B. jararacussu venom (50 µg/mL) mixed with saline solution and DMSO (0.9% v/v, final concentration) clotted plasma at approximately 60 s (Fig. 3). B. jararacussu-induced coagulation was delayed to 98 s in the presence of 6g and 7e, 107 s for 6e and 7b, and 125 s for 7c; thus, these compounds were able to inhibit the venom-induced plasma coagulation. However, such plasma coagulation was not inhibited by compounds 6a, 6b, 6c, 6d, 6f, 6h, 7a, 7d, 7f, 7g, and 7h (Fig. 3). In the control groups, none of the compounds (6a-h and 7a-h) clotted plasma in the absence of B. jararacussu venom, indicating the absence of procoagulant effects of all compounds under the experimental conditions (data not shown).

Figure 3 -
Effect of the compounds on B. jararacussu venom-induced plasma coagulation - B. jararacussu venom (50 µg/mL) was incubated with saline, DMSO, or 200 µg/mL of the derivatives (6a-h and 7a-h) for 30 min at 25 ^oC. Then, an aliquot of each mixture was added to plasma, and the coagulation time was monitored as described in the methods section. The results are expressed as mean ± SD (n = 6) of two individual experiments. *, p < 0.05 compared with B. jararacussu + saline and DMSO. The raw values are shown in the supplementary material (Table S1. available on supplementary material <10.6084/m9.figshare.30379603>).

AntiPLA ₂ effect of thiophene-triazole hybrid compounds

A sample of 10 µg/mL B. jararacussu venom mixed with saline solution was considered to indicate 100% of PLA₂ activity (Fig. 4). The incubation of this venom concentration with 40 µg/mL of compounds (6a-h and 7a-h) inhibited the PLA₂ activity of venom with different efficacies. Compounds 6a, 6b, 6h, 7a, 7c, and 7h inhibited 100% of the PLA₂ activity of B. jararacussu venom, whereas 6d, 6e, 6f, 6g, 7b, 7d, 7e, 7f, and 7g inhibited 80%-90%, and 6c inhibited with the lowest efficacy (~30%) (Fig. 4). Compounds (6a-h and 7a-h) were unable to hydrolyze egg yolk in the absence of venom, demonstrating a lack of PLA₂ activity of them under the experimental conditions (data not shown).

Figure 4 -
Effect of the compounds on the phospholipase activity of B. jararacussu venom - B. jararacussu venom (10 µg/mL) was incubated with saline, DMSO, or 40 µg/mL of the derivatives (6a-h and 7a-h) for 30 min at 25 ^oC. PLA₂ activity was determined, as described in the methods section. The results are expressed the percentage of PLA₂ activity, with means ± SD (n = 9) of three individual experiments. *, p < 0.05 compared with B. jararacussu + saline and DMSO. The raw values are shown in the supplementary material (Table S3, available on supplementary material <10.6084/m9.figshare.30379603>).

None of the compounds could inhibit all the in vitro activities of B. jararacussu venom, including proteolytic, plasma coagulant, and PLA₂. Compound 7 g inhibited the proteolytic and PLA₂ activity up to 90%, but failed to inhibit venom-induced coagulation, in which SVSPs and SVMPs are involved. Therefore, it can be assumed that this compound binds to the PLA₂s family, rather than to SVSPs and SVMPs. Moreover, all of the compounds appear to bind more selectively to PLA₂ enzymes versus SVSPs and SVMPs, since all compounds inhibited the PLA₂ activity, whereas only five interfered with venom-induced plasma coagulation. Undoubtedly, the compounds exhibit a structure-activity relationship (SAR), since the presence of electronegative or bulk groups in the compounds may favor the inhibitory effect against venom PLA₂ enzymes due to the presence of positively charged amino acid residues (as histidine at position 48) at the active site of such enzymes. Moreover, PLA₂ enzymes need Ca⁺² to exert their activity, and compounds could bind to this divalent metal and inhibit the enzymatic activity. Compound 6a has the simplest structure and is suitable for SAR comparisons among compounds. A molecular docking investigation should be conducted to further explore the mechanism of action of these compounds. Experiments using purified enzymes from B. jararacussu venom could reveal the type of inhibition observed.

Discussion

The WHO has encouraged researchers, industries, and governments to seek complementary treatments that reduce the main sequelae by SBE (Battellino et al. 2023). Currently, antivenom is the only effective treatment to prevent death; however, this therapy is ineffective in treating the devastating local effects of SBE, including hemorrhage, myotoxicity, and edema (Estevão-Costa et al. 2016). Thus, research has been conducted to develop compounds that can bind active proteins and peptides, or cofactors from venoms, leading to complete inhibition of toxic effects.

Our research group has assessed triazoles (Amorim et al. 2022; Domingos et al. 2013; Ornellas et al. 2024) and 1,3-benzoxathiol-2-one sulfonamides (Chazin et al. 2022) for their ability to inhibit the in vitro and in vivo toxic activities of B. jararaca, B. neuwiedi, and Lachesis muta venom. Recently, Ornellas et al. (2024) described the antivenom effect of thiophene-triazole hybrids (6a-h and 7a-h) against the in vitro toxic activities of B. jararaca and B. neuwiedi venom and demonstrated that these compounds were not toxic to red blood cells at concentration up to 2,000 µg/mL. In this work, these 16 compounds (6a-h and 7a-h) were assessed for the first time as inhibitors of the major toxic activities of B. jararacussu venom, including plasma coagulant, proteolytic, and PLA₂. Notably, the species B. jararaca, B. neuwiedi, and B. jararacussu are responsible for 87% of SBE in Brazil and are also of medical interest in other South American countries, such as Argentina, Paraguay, and Bolivia (Castro-Pinheiro et al. 2024). In this scenario, molecules capable of blocking the toxic effects of venoms from different species are needed, as the composition of snake venoms varies both intra- and interspecifically, impacting the efficacy of antivenom (Mora-Obando et al. 2023).

Overall, as seen in this work, the 16 thiophene-triazole hybrid compounds (6a-h and 7a-h) inhibited the proteolytic, plasma coagulant, and PLA₂ activities caused by B. jararacussu venom; however, none of them fully achieved 100% inhibition of these activities. These compounds were also effective against the proteolytic, plasma coagulant, and PLA₂ activities induced by B. jararaca and B. neuwiedi venom, but again did not achieve fully inhibition (Ornellas et al. 2024). The inhibitory profile of compounds 6a-h and 7a-h against the venom of B. jararaca, B. neuwiedi, and B. jararacussu was assessed using the same venom:compound ratio of 1:4 (wt/wt). The use of a similar ratio venom:compound is important for comparing the results of such compounds against the toxic in vitro activities of venom from different species. However, depending on the in vitro toxic activity of venom, slight differences in the inhibitory percentages were observed, which can be explained by the fact that sex, age, diet, geographical location, hydration, and prey type can alter the venom composition of individuals of a species (Hussain et al. 2024). These inter- and intraspecific variations in snake venom may alter the efficacy of antivenoms; thus, developing a molecule that can neutralize the major group of enzymes found in venoms is challenging.

Using a mixture of compounds containing a low concentration of each compound could be a good strategy to enhance the inhibitory profile against these toxic activities. However, this hypothesis requires further assessment through additional experiments. The findings of our study reinforce the potential of rationally designed compounds to complement commercial antivenoms. The toxic activities assessed in this work are caused by SVSPs, SVMPs, and PLA₂, which are the major family of active proteins in pit vipers and are considered the most important toxin families of snake venoms, due to the wide range of toxic effects induced (Gutiérrez et al. 2017). The family of SVSPs, SVMPs, and PLA₂s comprises approximately 70%-85% of Viperidae, and the associated SBE symptoms in humans include myotoxicity, hemorrhage, renal and cardiac failure, neurotoxicity, cytotoxicity, disturbances in blood coagulation, and death (Faria et al. 2002; Caldeira et al. 2024).

As previously mentioned in the introduction, compounds containing triazoles and thiophene have been utilized as anti-inflammatory and leishmanicidal agents, fungicides, antiviral, and anticonvulsant (Lima et al. 2021; Santos et al. 2020). As known from literature, thiophene-triazole hybrids are heat-resistant, have low toxicity, and are easily synthesized with low cost, which are attractive features for drug development (Bozorov et al. 2019; Thakur et al. 2024). Thus, such molecules should be considered candidates to enhance the efficacy of commercial antivenom in neutralizing the toxic effects of venoms.

Several 1,2,3-triazole-containing hybrids, such as cefatrizine (1,2,3-triazole-β-lactam hybrid), radezolid (1,2,3-triazole-oxazolidone hybrid), and tazobactam (1,2,3-triazole-β-lactam hybrid), are already applied in clinics to treat infections caused by diverse organisms, including ESKAPE pathogens and their drug-resistant forms, reflecting the importance of these compounds in the development of novel anti-ESKAPE agents (Cui et al. 2022). Given the results of this study and previous reports, it is anticipated that a hybrid triazole-thiophene molecule may exhibit a synergistic antivenom effect. The combination of triazole and thiophene structures was successfully achieved, as confirmed by the inhibitory effects of compounds 6a-h and 7a-h.

The 16 thiophene-triazole hybrid compounds 6a-h and 7a-h belong to the same chemical group; however, different substituents are attached at different positions. Thus, slight modifications of the structure of such compounds may alter their stability, molecular weight, and ability to bind molecules, including SVMPs, SVSPs, PLA₂, and metals (such as Ca⁺² and Zn⁺²), affecting their inhibitory efficacy. Moreover, it is worth noting that snake venoms contain multiple isoforms, and some of them have distinct and intriguing mechanisms of action. Therefore, the inhibition efficacy of the 16 compounds can be attributed to their distinct chemical structures. Soares et al. (2025) demonstrated that the varying concentrations of SVSPs and SVMPs in the venoms of eight Bothrops species directly impacted the pattern of fibrinolytic activity and the efficacy of commercial antivenom.

In conclusion, this study demonstrated the efficacy of thiophene-triazole hybrid compounds in inhibiting the major toxic activities of B. jararacussu venom, highlighting their potential as a valuable strategy for treating SBE caused by this species.

Acknowledgements

This work was supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grants 304719/201212-9, 309823/2021-8; Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), E-26/201.163/2014, E-26/211.058/2021 and E-26/01.001918/2015; and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Grants AUC-00022-16, APQ-01858-15. EFS and ALF are Research Members of CNPq.

Data availability statement

In accordance with Open Science communication practices, the authors inform that all data are available within the manuscript.

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• See supplementary material at <10.6084/m9.figshare.30379603>
• Cite as:
  Oliveira BB, Sanchez EF, Ferreira VF, Silva FC, Gonzaga DTG & Fuly AL (2025) Thiophene-triazole hybrids inhibit the major toxic activities of Bothrops jararacussu venom. Rodriguésia 76: e00282025. DOI: 10.1590/2175-7860202576051

Edited by

• Area Editor:
  Dr. Davyson Moreira

Publication Dates

• Publication in this collection
  28 Nov 2025
• Date of issue
  2025

History

• Received
  27 Mar 2025
• Accepted
  13 July 2025