Vector-borne diseases constitute more than 17 % of all infectious diseases and are caused by parasites, viruses and bacteria. Their distribution is influenced by environmental and social factors, with recent global trends such as globalization, unplanned urbanization, and environmental issues like climate change impacting on their prevalence [1,2]. Dengue virus (DENV) and the closely related Zika virus (ZIKV) belong to the Orthoflavivirus genus within the Flaviviridae virus family, which also includes other significant human pathogens like the yellow fever, Japanese encephalitis, and West Nile viruses. Aedes mosquitoes transmit DENV and ZIKV in tropical and subtropical regions, where approximately 40 % of the global population (around 3 billion people) resides and is susceptible to these infections [3]. DENV has been a significant human pathogen for decades, infecting approximately 400 million people annually, of which, around 100 million present a symptomatic disease. While most cases resolve spontaneously, approximately 500,000 cases per year progress to severe dengue, with symptoms such as hemorrhagic fever and shock syndrome, causing around 22,000 deaths annually, primarily among children [4,5]. The existence of four different DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) complicates the diagnosis, and individuals who recover from one serotype remain susceptible to the others, heightening the risk of severe disease [6].

ZIKV has caused large outbreaks, such as those in Yap Island (with approximately 7000 cases in 2007), in French Polynesia (around 28,000 cases in 2013) and in Brazil and other American countries (in the season spanning 2015–2016), causing significant repercussions [7]. In 48 Pan-American countries and territories, millions of people have been infected, exhibiting symptoms such as fever, rashes, and conjunctivitis. Although most individuals recover within days, ZIKV infection has been linked to a 20-fold increase in severe neurological diseases, including Guillain-Barré syndrome. Over 4000 cases of microcephaly and other neurological disorders in newborns have been reported [8]. The prospects for these infants to achieve normal brain function are minimal, prompting the World Health Organization to declare ZIKV a “Public Health Emergency”. Moreover, ZIKV transmission can occur through sexual contact or body fluids, even in asymptomatic individuals, presenting additional challenges for containment [9].

Besides, the efforts made in mosquito control programs have proved to be not totally efficacious; and currently, there are no antiviral drugs available for the prevention and treatment of neither ZIKV nor DENV infections. For DENV, existing vaccines have limitations. One key hurdle in vaccine development is the existence of four DENV serotypes, which hinders the development of fully protective vaccines. While two dengue vaccines have received regulatory approvals, one of them is recommended only for individuals with prior dengue exposure, and the other has shown reduced efficacy against DENV-3, with inconclusive results for DENV-4 [10].

Currently, the only clinical trial assessing a specific antiviral drug against dengue is the one carried out by Janssen. The compound (JNJ642401802) under evaluation inhibits the interaction between the NS3-NS4B viral proteins of all dengue serotypes. JNJ642401802 is the first antiviral drug of its kind to demonstrate safety and good tolerance in phase 1 clinical study [11].

Viral entry stands as a critical target process for antiviral interventions, potentially achievable by small molecule inhibitors, as underscored by Zhou and Simmons [12]. The E protein plays a pivotal role in the entry process, engaging in interactions with host cell receptors and undergoing low pH-induced conformational changes during viral host membrane fusion. In the mature Flavivirus particle, the ectodomain of the E protein adopts a dimeric form with three domains [Domain I (DI), Domain II (DII), and Domain III (DIII)], playing diverse roles in the viral entry into host cells. DIII interacts with receptors on the host cell surface, initiating virion internalization through clathrin-mediated endocytosis [13,14]. Within the acidic environment of endosomes, the E protein undergoes distinct rearrangements, exposing the fusion loop in DII, and undergoing the folding back event of DIII during viral-host membrane fusion, leading to the formation of the trimeric form of the E protein. The crystal structure of DENV-2 E protein reveals a hydrophobic pocket occupied by a molecule of the detergent n-octyl-β-D-glucoside (β-OG), at the hinge region between DI and DII [15]. This β-OG pocket is a crucial region where major conformational changes occur during membrane fusion. Various authors have proposed that small molecules interacting with the E protein can block viral entry [[16], [17], [18], [19]]. Strong pharmacological evidence supporting the pocket as a viable target has been presented by Wispelaere et al. [16], who demonstrated that pyrimidine analogs can bind extracellularly to the E protein, preventing infection by blocking E-mediated membrane fusion during viral entry. The resistance mutation M196V, located adjacent to the β-OG pocket, diminishes the affinity of the evaluated compound, restoring viral infectivity [16].

In previous studies, we identified novel molecules capable of inhibiting DENV entry for all four serotypes. Utilizing de novo design together with lead optimization strategies, we successfully identified two compounds, 3e (1) and 3h (2) (see Fig. 1), both having low EC50 values within the micromolar range [18]. The pre-clinical study of these molecules encountered setbacks due to limited water solubility, and other pharmacokinetic and safety concerns.

In an effort to identify novel small molecules acting as DENV and/or ZIKV inhibitors, a low-throughput phenotypic antiviral screening using a virus yield reduction assay with DENV-2 (Thailand/16681/1984 strain) and ZIKV (Argentine strain INEVH116141) was performed with the synthesized compounds (5a-i; 10a-b; 14, 16a-i; 18 and 21). These experiments led to the identification of compound 16a as a novel DENV and ZIKV entry inhibitor. Using a relative viral RNA quantification (qPCR) and an immunofluorescence assay as a confirmatory test, we demonstrated that compound 16a had a potent in vitro antiviral activity against DENV-2 and ZIKV (EC50 = 1.4 μM and 2.4 μM, respectively). This study was aimed at designing new molecules with improved selectivity and pharmacokinetic properties to obtain enhanced antiviral efficacy. Solubility, permeability and stability assays proved that compound 16a presents an adequate pharmacokinetic profile.