Cancer has become the leading cause of death worldwide among people under 70 years old [1]. In addition, premature deaths due to various types of cancer are also rising across countries [2]. In 2020, the estimated number of cancer cases was almost 20 million, and 10 million deaths related to cancer. Among the most common and fatal cancers include breast, prostate, lung, colon, and leukemia [3]. Cancer cells are constantly evolving which results in a high tumor heterogeneity with a subpopulation of cells displaying a different molecular landscape [4]. This characteristic leads to drug resistance due to genetic, epigenetic, and metabolic changes that hinder anticancer activity [5, 6]. Another important factor that limits cancer treatment is chemotherapy-related toxicities that affect healthy cells leading to acute toxicities, such as hematological and gastrointestinal [7, 8]. Therefore, there is an urgent need for the development of new anticancer compounds to overcome these limitations.

Among the most promising scaffolds for addressing challenges in cancer therapy are 1,2,3-triazoles, a class of heterocycles known for their exceptional stability and diverse pharmacological properties. Their mechanisms of action include the inhibition of cancer cell proliferation, cell cycle arrest, and induction of apoptosis, making them effective against both drug-sensitive and multidrug-resistant cancers [9].

In recent years, triazole-based hybrid compounds have emerged as promising candidates for cancer treatment, demonstrating remarkable therapeutic potential [10]. These hybrids often exhibit dual or multiple mechanisms of action, addressing the limitations of conventional chemotherapeutics by improving selectivity and reducing side effects. Furthermore, their ability to overcome drug resistance and their promising in vivo activities against various cancer types have solidified their role as valuable frameworks for next-generation anticancer agents. By integrating triazoles into hybrid molecules, researchers aim to unlock new possibilities for more effective and safer cancer therapies [11].

Aryl(1-aryl-1H-1,2,3-triazole-4-yl)methanols, commonly known as hydroxy-1,2,3-triazoles, are a promising and versatile scaffold with significant potential for novel drug development. Our research group has extensive expertise in their synthesis, utilizing both copper-catalyzed alkyne-azide cycloaddition (CuAAC) between propargylic alcohols and aryl azides [12], as well as the metal-free and solvent-free enaminone-azide cycloaddition (EACA) employed in this study to synthesize 4-acyl-1,2,3-triazoles [13]. The EACA method provided a simpler and more efficient route to generate bioactive hydroxy-1,2,3-triazoles through acyl reduction. These compounds presented antileishmanial [14], antiviral [15], and cystic fibrosis activity [13].

9H-Fluorene-based compounds have emerged in past years due to their unique chemical and physical properties, which allow their application in a wide range of scientific disciplines, such as optoelectronics [16, 17], solar cells [18], nanostructures [19, 20], and others. Its great prominence is seen in the field of medicinal chemistry where this scaffold is being used in various bioactive compounds [21–24].

Given their structural characteristics (conformational rigidity with significant aromaticity structure), which intensify their pharmacophoric characteristics [25, 26], it is possible to combine them with other structures, such as 1,2,3-triazoles. These synthetic heterocycles have great potential and applicability, mainly due to their trans-amides bioisosteric proprieties that possess broad-spectrum biological activity, low cytotoxicity, and significant structural versatility. These properties allow triazoles to act both as pharmacophores, directly engaging biological targets, and as linkers in hybrid molecules, tethering different pharmacophores to enhance efficacy.

The development of fluorene-triazole hybrid compounds is a little-explored field with great potential. In literature, some examples are present so far, mainly in functional materials chemistry [27–31]. With a view to biological applications, examples can be found, however, most of them bonded together by fluorene’s aromatic rings [32–35].

Fewer examples are found when it comes to 9H-fluorenes-triazole hybrids. Yang et al. [36] proposed the synthesis of fluorene-triazoles containing the pentafluorosulfanyl group, with emphasis on the CuAAC between 3,5-bis(pentafluorosulfanyl)phenyl azide (1a) and 9-ethynyl-9H-fluoren-9-ol (2) as presented in Figure 1a. The authors verified the ability of compound 3 to trigger cell death in human leukemic monocyte lymphoma U937 cells obtaining a half-maximal inhibitory concentration (IC₅₀) value of 6.29. Compound 3 was also compared to Caspase-3, an important enzyme responsible for apoptosis, which has been proven to be 3.4 times more efficient than this enzyme. These results demonstrate the antitumor potential of fluorene-triazole hybrids.

Looking for analogs of structures reported in the literature as Mycobacterium tuberculosis (M. tb) InhA (NADH-dependent 2-trans-enoyl-acyl carrier protein reductase) inhibitors, Suresh et al. [37] rationalized such molecular skeletons as essential elements for InhA inhibition. In this way, the authors presented three 9H-fluorene structures derived from fluorene-triazoles (5) with a good variety of substituents by reacting fluorene-based alkynes (4) and azidobenzenes (1) [37] (Figure 1b).

More recently in 2020, our research group reported 3 examples of 9H-fluorenes-triazole hybrids employing a Friedel-Crafts alkylation. Starting from substituted biaryl hydroxy-1,2,3-triazoles (6) (previously synthesized by CuAAC between biaryl-propargyl alcohols and azidobenzenes), in the presence of Pd(OAc)₂, Na₂CO₃, PhI(TFA)₂ in dichloroethane, 100°C for 36 hours, with yields of 25 to 40% [38] (Figure 1c). Since the metal-free and solvent-free EACA to obtain 4-acyl-1,2,3-triazoles is a simpler and more effective alternative than CuAAC, our research group decided to employ this methodology for obtaining biaryl hydroxy-1,2,3-triazoles (6) as precursors of 9H-fluorene-triazole (7) (Figure 1d). Given the biological potential of these compounds, another goal was to evaluate the antitumor potential by maximum growth inhibition (MGI) of the new biaryl hydroxy-1,2,3-triazoles and fluorene-triazole hybrids against different cell lines.

Among the fluorene-triazole compounds evaluated, LSO258 and LSO272, both containing bromine at the blue ring exhibited selective cytotoxicity against the MOLM-13 cell line, with IC₅₀ values of 25.5 µM and 12.5 µM, respectively. In contrast, the flexible biaryl hydroxy-triazole ligands, LSO232 and LSO280, were inactive. In this case, the rigid scaffolds may minimize the entropic loss associated with the ligand adopting a preferred binding conformation, resulting in enhanced potency, improved selectivity, and reduced potential for drug metabolism [25, 26].

Considering the flexible biaryl hydroxy-triazole ligands, compound LSO278, which contains two bromine groups on the blue ring, was the only one to exhibit significant cytotoxicity across three cell lines (HCT-116, MDA-MB-231, and MOLM-13), with IC₅₀ values of 23.4 µM, 34.3 µM, and 18.7 µM, respectively. In contrast, the fluorene-triazole compound LSO275 was inactive even with these two bromine groups, and the conformational rigidity doesn't corroborate the antitumoral activity.

The only instance where both a biaryl hydroxy-triazole (flexible ligand) and a fluorene-triazole (rigid scaffold) demonstrated activity was observed with LSO258 and LSO276, both of which feature bromine in the same position in the blue ring. Compound LSO276 showed cell line-dependent activity, with an IC₅₀ of 24.8 µM in the MOLM-13 acute leukemia cell line and LSO258 with IC₅₀ values of 25.5 µM, both with same potency.

Overall, most of the compounds evaluated in this study were more potent against MOLM-13, even when IC₅₀ values were not achieved. For instance, LSO259, LSO234, and LSO280 demonstrated an MGI above 55% for MOLM-13, while for cell lines derived from solid tumors, their MGI values were below 35. In addition, most of the compounds evaluated in this study were more potent towards MOLM-13, even those that did not reach an IC₅₀ value. For example, LSO259, LSO234, and LSO280 displayed an MGI above 55% for MOLM-13, while for the cell lines from solid tumors, only an MGI below 35%.

The MOLM-13 cell line represents a subtype of AML, specifically M5a, characterized by wild-type TP53 and a mutated FLT3 gene [52–54]. This FLT3-ITD mutation leads to the constitutive activation of the FLT3 tyrosine kinase receptor, driving uncontrolled cell proliferation and resistance to apoptosis [55, 56]. As a result, AML patients with this mutation often experience poor survival rates. First-generation tyrosine kinase inhibitors targeting the FLT3 pathway have been evaluated in clinical trials; however, their low specificity limited their therapeutic efficacy [57, 58]. To overcome this, second-generation FLT3 inhibitors, such as gilteritinib and quizartinib, were developed and demonstrated significant clinical benefits, leading to their approval for AML treatment in the USA, Japan, and Europe. Nevertheless, treatment resistance remains a challenge, as AML cells can acquire additional mutations in FLT3 or related genes, ultimately reducing the effectiveness of FLT3 inhibitors [56, 59, 60].

Different studies have shown the antiproliferative effect of 1,2,3-triazoles-hybrids against leukemia cell lines. Ashwini et al. [54] synthesized a series of 1,2-benzisoxazole tethered 1,2,3-triazoles with preeminent anticancer activity in vitro. The lead compound PTB presented IC₅₀ ranging from 1–2.5 μM after 96 h exposure in the following cell lines, MOLM-14, MOLM-14 and MV4-11. In another study, novels 1,4-diaryl-1,2,3-triazolo-based ureas compounds were developed as FLT3-ITD inhibitors and tested against cancer cell lines FLT3-ITD dependent (BaF3, MV4-11, and MOM-13) and FLT3-WT cell line. The results showed that different compounds were highly cytotoxic and selective towards FLT3-ITD cell lines compared to the wild-type one. In addition, the molecular docking evaluation indicated that the binding of one of the derivatives was mainly due to the triazole ring [55].

The observed cytotoxicity of bromine-containing fluorene-triazole and biaryl hydroxy-triazole derivatives, particularly LSO258, LSO272, LSO276, and LSO278, aligns with the well-established role of bromine in enhancing drug-target interactions through halogen bonding (X-bonding) [56]. Bromine’s moderate electronegativity, polarizability, and ability to form directional halogen bonds likely contributed to the increased potency of these compounds, particularly against MOLM-13 cells. Halogen bonds with carbonyl oxygens, amides, and π-stacking with aromatic residues may have improved ligand-protein interactions, enhancing selectivity and binding affinity to biological targets [57, 58]. Furthermore, bromine’s lipophilic nature may have facilitated better membrane permeability and cellular uptake, contributing to the observed cytotoxic effects [59].

Additionally, the structural influence of bromine within rigid fluorene-triazole scaffolds suggests that the conformational constraints imposed by this framework may have minimized entropic losses during binding, resulting in more favorable protein-ligand interactions. Interestingly, LSO278, a flexible biaryl hydroxy-triazole bearing two bromine atoms, demonstrated broad activity across multiple cancer cell lines, suggesting that in certain cases, enhanced electronic interactions provided by multiple bromine substituents can compensate for flexibility-related entropic penalties. These findings reinforce the importance of bromine in medicinal chemistry, highlighting its potential to fine-tune molecular recognition, optimize pharmacokinetic properties, and enhance cytotoxicity in drug discovery efforts [60, 61].

In conclusion, we successfully synthesized 13 new biaryl hydroxy-1,2,3-triazoles using a one-pot reaction that eliminated the need for isolating intermediates, achieving yields ranging from 16% to 89%. These triazoles served as precursors for 9H-fluorene-triazole hybrids, resulting in the formation of novel compounds with yields between 20% and 97%. The synthesized structures demonstrated significant potential for inhibiting the growth of several cancer cell lines. Among the compounds evaluated, most exhibited higher potency against MOLM-1, with LSO258, LSO272, LSO276, and LSO278 showing IC₅₀ values of 25.5, 12.5, 24.8, and 18.7 µM, respectively. Notably, LSO278 displayed versatility, exhibiting additional activity against HCT-116 (IC₅₀ = 23.4 µM) and MDA-MB-231 (IC₅₀ = 34.3 µM). While a direct correlation between flexible ligands and rigid scaffolds could not be established, the presence of a triazole moiety and bromine substitution on the blue ring (derived from aryl azide) consistently emerged as key features, as all active compounds shared this structure.