Crimean-Congo hemorrhagic fever virus (CCHFV) is an enveloped negative-sense RNA virus that belongs to the order Hareavirales, family Nairoviridae, and genus Orthonairovirus [1]. As an important member of the bunyaviruses, CCHFV possesses a characteristic tri-segmented genome (L, M, and S segments), each of which is tightly bound by the L protein and encodes essential viral proteins [[2], [3], [4]]. CCHFV is mainly transmitted through ticks, as its principal vectors, and it is recognized as one of the most widely distributed tick-borne viruses known to date [5,6]. Epidemiological reports and serological surveys indicate that CCHFV is mainly distributed across Africa, Southeast Asia, the Middle East, and Eastern and Southern Europe, with its geographical range largely overlapping with the distribution of Hyalomma asiaticum [[7], [8], [9], [10]]. The virus circulates naturally between animals and humans. Livestock typically develop transient viremia without clinical disease, while human infections usually occur through tick bites or direct contact with infected animals [11,12]. Following infection, humans initially present with nonspecific febrile illness that may progress to severe hemorrhagic fever, with case fatality rates ranging from 10 % to 40 % [13]. Given its high transmissibility and considerable mortality, the World Health Organization (WHO) has classified CCHFV as a priority pathogen in a series of documents since 2015, including Prioritizing diseases for research and development in emergency contexts, the annual review of diseases prioritized under the R&D Blueprint [[14], [15], [16]]. Currently, no specific antiviral drugs or licensed vaccines are available for the treatment of CCHFV infection, and clinical management primarily relies on supportive care measures, such as fluid replacement, correction of electrolyte imbalance, hemorrhage control, and prevention of secondary infections [17,18].

Drug repositioning, or drug repurposing, has emerged as an attractive and efficient strategy to respond to public health emergencies caused by viral outbreaks [19,20]. Through repurposed drugs that are already approved or in clinical development for new indications, this approach circumvents the lengthy and high-risk early phases of drug discovery, such as target identification and lead optimization, thereby significantly shortening the development timeline, reducing costs, and increasing the likelihood of success [21,22]. Comparative analyses of the bunyavirus L protein and the influenza virus polymerase complex have revealed notable structural and functional similarities [[23], [24], [25]]. The linear L protein of bunyaviruses contains key functional domains, including an endonuclease (EN), an RNA-dependent RNA polymerase (RdRp), and a cap-binding domain (CBD) [26,27] (Fig. 1).

Owing to its essential catalytic roles in the viral life cycle and its high degree of conservation among diverse bunyaviruses, the L protein is considered an ideal target for anti-bunyavirus drug development. Similar to influenza virus, bunyaviruses employ a “cap-snatching” mechanism during transcription, which is mediated by the L protein and coordinated by its core domains: the cap-binding domain (CBD) recognizes host mRNA, the endonuclease (EN) cleaves a capped oligonucleotides, and the RNA-dependent RNA polymerase (RdRp) core uses this fragment to prime viral transcription. Therefore, inhibiting any critical step in this cytoplasmic process—which is functionally analogous to the nuclear cap-snatching of influenza viruses—would effectively disrupt the viral life cycle. A notable example is baloxavir marboxil (BXM), a novel influenza therapeutic first approved in Japan in 2018 for the treatment of influenza A and B virus infections [28,29]. BXM is an orally available prodrug that is rapidly converted into its metabolite, baloxavir acid (BXA) [30]. BXA specifically targets the cap-dependent endonuclease activity within the PA subunit of the influenza virus RNA polymerase complex, thereby blocking the “cap-snatching” process essential for viral mRNA synthesis [31,32].

In enzymatic assays, BXA inhibits the influenza endonuclease at low nanomolar concentrations, with IC50 values of 1.4–3.1 nM for influenza A and 4.5–8.9 nM for influenza B [30]. According to the latest official prescribing information of baloxavir marboxil tablets (Roche, May 9, 2025), the antiviral activity of baloxavir against both laboratory strains and clinical isolates of influenza A and B viruses was determined using a plaque reduction assay in MDCK cells. The median 50 % effective concentrations (EC50) of baloxavir were 0.73 nM (n = 31; range: 0.20–1.85 nM) for influenza A(H1N1), 0.83 nM (n = 33; range: 0.35–2.63 nM) for influenza A(H3N2), and 5.97 nM(n = 30; range: 2.67–14.23 nM) for influenza B virus [33].

Following a single oral dose of 50 mg of BXM in humans, BXA achieves plasma Cmax values of 284 ng/mL (∼0.50 μΜ) and AUClast values achieves 1580 h ng/mL [34]. In vitro studies have reported that the EC50 for BXA against CCHFV is approximately 0.60 μΜ, which is several hundred-fold higher than that for influenza viruses (Table 1) [34].Since baloxavir is classified as a time-dependent antiviral agent, its pharmacological efficacy is better correlated with the overall drug exposure rather than the peak plasma concentration. Accordingly, the ratio of area under the plasma concentration-time curve (AUC) to the EC50 is commonly employed as a key pharmacodynamic index to evaluate whether systemic exposure is sufficient to achieve antiviral activity. A higher AUC/EC50 ratio generally indicates that the achieved plasma levels are more likely to maintain inhibitory concentrations for an extended period, which is particularly relevant for time-dependent drugs such as baloxavir. Based on the above approach, the AUC/EC50 ratio of baloxavir were calculated for influenza virus and CCHFV. The values reached approximately 3243-3688-fold for influenza, whereas only about 4.5-fold was obtained for CCHFV. Assuming linear exposure-response and similar protein binding, the ratio suggests insufficient coverage against CCHFV. This striking difference in exposure-response relationships provides a reasonable explanation for the distinct clinical outcomes: baloxavir achieves sufficient systemic exposure to exert potent antiviral activity against influenza following oral administration, while the markedly lower AUC/EC50 ratio against CCHFV indicates inadequate drug coverage, thereby accounting for the lack of oral efficacy observed in CCHFV studies (Fig. 2).

Notably, animal studies have demonstrated that modified formulations of BXA with improved solubility and systemic exposure can partially restore antiviral activity against CCHFV, underscoring that the limitation lies in pharmacokinetic coverage rather than inaccessibility of the viral target. These observations highlight the need for structural optimization of baloxavir analogues to enhance potency against the CCHFV endonuclease and thereby overcome the pharmacodynamic barrier that prevents oral efficacy [35].

The present study utilized BXA (1), the active form of BXM, as the lead compound. Guided by molecular docking analyses with the CCHFV L protein, we pursued structural optimization to design and synthesize target compounds exhibiting enhanced inhibitory activity against CCHFV [36]. Our design strategy retained the essential carbamoyl pyridone bicycle (CAB) pharmacophore, known to be critical for activity [37,38]. At the N-1 position hydrophobic moiety, we introduced bulky aliphatic polycyclic structures, anticipating potential hydrophobic interactions with the protein. Conversely, at the N-3 position, smaller aliphatic side chains were employed as substituents to optimize the physicochemical profile of the molecules. Based on this rationale, we synthesized 20 novel compounds (Z01-Z20). This effort aims to identify potent CCHFV inhibitors with optimized antiviral efficacy and favorable drug-like properties (Fig. 3).

We aimed to identify novel CCHFV L protein inhibitors with improved solubility, enhanced antiviral activity, low toxicity and favorable PK properties through structural design and optimization of the lead compound. Through antiviral activity assays of the synthesized compounds, Z10, featuring a novel open-ring non-rigid substitution structure, was identified as having high antiviral activity. Given that Z10 was almost insoluble, it was therefore converted to its sodium salt form to improve its solubility [39]. In vivo PK property measurements suggest that its PK properties were enhanced compared to the lead compound BXA, demonstrating preliminary antiviral potential and warranting further investigation.