The aza-spirocyclic compounds were synthesized using the recently developed ipso-annulation reactions [4, 5]. In the first case, radical-promoted ipso-annulation using aryl sulfonates or AgSCN or AgSCF₃ in the presence of ceric ammonium nitrate (CAN) as an oxidant, provided thio-functionalized (SO₂Ar or SCN or SCF₃) spirocycles in good yields [4]. Next, selenylated aza-spirocycles were prepared via electrophilic ipso-annulation using diphenyl diselenide/FeCl₃ reaction conditions. Accordingly, spirocycles AS1 to AS6 were obtained from the reaction of N-benzylacrylamides induced by S-centered radicals (SCN/SCF₃/SO₂Ar) in the presence of CAN as an oxidant. Next, the chalcogenated (SCN/SCF₃/SePh) benzimidazo-fused azaspiro[5,5]undecatri-enones AS7 to AS12 were synthesized by the reaction of N-propiolyl-2-arylbenzimidazoles involving both the radical-based and electrophilic reactions. All the general synthetic strategies (Scheme S1 & S2) are provided in the Supplementary Information along with the analytical data (¹H NMR, ¹³C NMR & mass) along with the HPLC data for the representative compounds.

The cell line was obtained from the National Centre for Cell Science (NCCS, Pune, India). All cell lines supplied by NCCS were authenticated using Short Tandem Repeat (STR) profiling and tested for mycoplasma contamination [6]. RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (AT241-1L, Grand Island, NY, USA) medium supplemented with 10% fetal bovine serum (FBS) (Gibco-16170078) and 1% Penicillin-Streptomycin (Pen-Strep) (Gibco-15140-122, Thermo Fisher Scientific, Grand Island, NY, USA) solution under standard conditions of 37 °C and 5% CO₂ for 24 hours. Cells were cultured in T75 flasks containing 10 mL of media. Then, cells were counted using a haematocytometer. Ten thousand cells per well were cultured in 96-well plates for 24 hours under optimum conditions. Also, compounds were serially diluted in culture media starting with 100 µM up to 0.8 µM concentration. Then, cells were treated with decreasing concentrations of the compounds and incubated for 24 hours at 37 °C and 5% CO₂. Control wells contained only untreated cells. Following 24 hours of incubation, 50 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, M5655, Milwaukee, WI, USA) solution (5 mg/mL concentration in PBS) was added into each well and incubated for 4 hours at 37 °C and 5% CO₂ for 24 hours. Later, ~100 µL of media was aspirated and discarded, and formazan crystals were dissolved in 50 µL of dimethyl sulfoxide (DMSO) and incubated for 15 minutes. Absorbance (O.D.) was then measured at 570 nm using a microplate reader (MEGALLAN, Tecan Life Sciences, Männedorf, Switzerland). Percentage cell viability was then calculated based on the compound treatment’s absorbance relative to the cell control’s absorbance [7].

RAW264.7 cells were maintained as mentioned in the above section. Cells were counted using a haematocytometer, approximately 0.5 $\times$ 10⁶ cells per well were seeded in 1 mL in 24-well plates for 24 hours under optimum conditions. After incubation, 100 µM, 10 µM, and 1 µM of each compound and 100 ng/mL of Lipopolysaccharide (LPS) were added to the cells and incubated for 24 hours at 37 °C with 5% CO₂. Supernatant from the cultures was collected and estimated for NO production. The standard was prepared by serial dilution of NaNO₂ starting from 100 µM up to 8 times in autoclaved water. 100 µL Griess reagent (Sigma, G4410-10G) was then added to a 96-well plate and incubated for 30 minutes, then absorbance (O.D.) was measured at 545 nm using a microplate reader (MEGALLAN, Tecan Life Sciences). NO production was then calculated based on the compounds’ treatment absorbance relative to the standard’s absorbance via the linear curve method [8].

Animal experiments were approved by the Institutional Animal Ethics Committee (IEAC) of the Council of Scientific & Industrial Research, Indian Institute of Chemical Technology (CSIR-IICT). Three C57BL/6 mice were acquired from the internal animal house facility (CSIR-IICT, Hyderabad) and used for isolating spleens for immunological study (IAEC approval number: IICT-IAEC-069-2025). Mice (6–8 weeks old, female) were sacrificed with cervical dislocation, and the spleen was dissected carefully. Splenocytes were isolated by using Ammonium-Chloride-Potassium (ACK) lysis buffer [prepared in the lab]. Later, cells were counted, and approximately 1 $\times$ 10⁶ cells per well were seeded in 1 mL in 24-well plates and treated with 100 µM and 10 µM compound, and incubated at 37 °C with 5% CO₂ for 24 hours under optimum conditions. Cell supernatant was then collected for cytokine estimation [9].

Secreted cytokines were measured by sandwich enzyme-linked immunosorbent assay (ELISA). For this, RAW264.7 cells and Splenocytes (0.5 $\times$ 10⁶ and 1 $\times$ 10⁶ cells, respectively) were cultured in a 24-well tissue culture plate in a complete medium (as mentioned earlier) and incubated for 24 hours under standard conditions. Cells were then treated with immunomodulatory compounds at the desired concentration and incubated at 37 °C with 5% CO₂ for 24 hours. In some experiments, cells were pretreated with LPS (100 ng/mL) for 2 hours to induce an inflammatory condition prior to compound treatment. Culture supernatants were then collected and centrifuged at 1500 rpm for 5 minutes to remove any cell debris. IL-6 [BD OPt ELISA, Cat No.- 555240, San Diego, CA, USA], IL-10 [Invitrogen, 88-7105-88, Grand Island, NY, USA], and TNF-$\alpha$ [BD OPt ELISA, Cat No.- 555268, San Diego, CA, USA] in supernatants were then measured using the ELISA kits following the manufacturer’s protocol [10].

Molecular docking was performed using AutoDock Vina (Center for Computational Structural Biology, Scripps Research, La Jolla, CA, USA; https://vina.scripps.edu/) [11, 12], which employs a scoring function that combines empirical and knowledge-based terms to estimate the binding affinity between ligands and the protein. It utilizes a gradient-based optimization algorithm to efficiently explore the ligand’s binding conformations. The 2D structures of the 12 compounds were obtained using ChemDraw (Revvity Signals Software, Inc., Waltham, MA, USA), and their 3D structures were generated with OpenBabel (http://openbabel.org). The experimental crystal structures of JAK1 kinase (PDB: 6N7A) [13], an isoquinoline derivative inhibitor with JAK2 kinase (PDB: 2B7A) [14], and JAK3 (PDB: 5LWM) [15] in complex with a pyrazolopyridine inhibitor and TYK2 (PDB: 6AAM) in complex with peficitinib were considered as starting structures for docking. The protein structures were processed by modeling missing residues, removing all heteroatoms and co-crystal ligands, and converting them to the required PDBQT format using Mgltools. All compounds were also converted to PDBQT format. The binding site was defined by obtaining the center of mass of the respective co-crystal ligand for each JAK isoform. The exhaustiveness parameter was set to the default value of 8. The docking results were visualized with UCSF Chimera, and interaction analysis was performed using BIOVIA Discovery Studio (2024, Dassault Systèmes BIOVIA, San Diego, CA, USA).

The mouse macrophages (RAW264.7) cells were seeded in a 12-well plate at a density of 1 $\times$ 10⁶ cell/well. After overnight incubation, cells were treated with 100 µM potential JAK inhibitors (AS1, AS2, AS8, and AS10), LPS (100 ng/mL), Filgotinib (100 µM), and Momelotinib (50 µM), either with or without LPS stimulation for 2 hours. After 24 hours, cells were lysed using 1$\times$ RIPA buffer containing protease and phosphatase inhibitors to extract the protein. The protein concentration was estimated using a BCA kit, and an equal amount (30 µg) of protein was loaded and resolved in 10% SDS-PAGE. The separated proteins were transferred to the nitrocellulose membrane and blocked with 5% BSA for 1 hour. Following blocking, the membrane was incubated with appropriate primary antibodies overnight at 4 °C. After washing three times with 1$\times$ TBST, an HRP-conjugated secondary antibody was added and incubated for 2 hours. The signals were developed using an ECL solution, and the bands were visualized and quantified using the ChemiDoc imaging system.

Data were analysed using GraphPad Prism 8 (GraphPad Software, LLC, San Diego, CA, USA). Comparison of two groups was performed by Student’s t-test, an unpaired, non-parametric test, and the Mann-Whitney test.

The azaspirocyclic framework is a structural motif found in various natural and synthetic molecules known for their significant pharmacological activities [16]. In addition, thio- and seleno-functionalized molecules are valuable in medicinal chemistry because sulfur and selenium introduce distinctive electronic and redox properties that considerably modify the pharmaceutical properties. These groups can improve lipophilicity, metabolic stability, and target interactions, while offering useful bioisosteric options for tuning potency and selectivity [17]. In light of the favorable properties associated with this class of molecules, we have developed an efficient protocol to access a series of thio-functionalized azaspiro[4,5]decatrienones via the dearomative ipso-annulation of unactivated N-benzyl acrylamides, induced by S-centered radicals (SCN/SCF₃/SO₂Ar) in the presence of CAN as an oxidant [4]. Later, we reported an ipso-carbocyclization of N-propiolyl-2-arylbenzimidazoles leading to chalcogenated (SCN/SCF₃/SePh) benzimidazo-fused azaspiro[5,5]undecatrienones in good yields, involving both radical-based and electrophilic pathways [4, 5]. Having developed these two ipso-annulation strategies to synthesize both the azaspiro[4,5]decatrienone and azaspiro[5,5]undecatrienone frameworks bearing chalcogenated (S/Se) functionalities, we became interested in investigating their immunomodulatory effects. In particular, the benzimidazole-fused azaspiro[5,5]undecatrienones incorporate two high-value pharmacophoric motifs within a single molecular framework. These motifs are widely represented in bioactive small molecules allied with diverse pharmacological activities, thereby underscoring the potential relevance of these hybrid structures. In addition, the chalcogen-functionalized azaspirocycles obtained through our synthetic strategy provide promising structural templates for medicinal chemistry optimization. Their unique combination of rigidity, three-dimensionality, and heteroatom incorporation positions them as attractive candidates for future drug-discovery and lead-generation efforts (Supplementary Table 1).

The cytotoxic potential of the spirocyclic compounds (Supplementary Table 1) was evaluated in murine macrophages using a colorimetric assay based on the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) by mitochondrial dehydrogenases, leading to the formation of blue formazan crystals. All tested compounds preserved cell viability above 90% across concentrations up to 100 µM. None of the compounds exhibited measurable cytotoxicity within this range, and the half-maximal inhibitory concentration (IC₅₀) values were estimated to be greater than 1000 µM (Supplementary Table 2), indicating a favorable safety profile for in vitro studies.

The impact of spirocyclic compounds on reactive nitrogen species (RNS) production was assessed using a nitric oxide (NO) assay in RAW264.7 macrophages. None of the tested compounds induced NO production, however, a few of the compounds showed slightly higher NO, although it was not significant. These findings indicate that the spirocyclic compounds do not function as danger-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) inducers (Fig. 1A). To further investigate their immunomodulatory potential, culture supernatants from treated macrophages were analyzed for inflammatory cytokine secretion, specifically interleukin-6 (IL-6) and interleukin-10 (IL-10) (Fig. 1B,C), using a sandwich ELISA. IL-6 production remained largely unchanged across treatments, with the exception of compound 1, which elicited a modest increase compared to the cell control. In contrast, IL-10 secretion was significantly elevated in a dose-dependent manner following treatment with the spirocyclic compounds (AS1, AS2, AS3, AS4, AS5, AS6, AS8, AS9, and AS10), suggesting an anti-inflammatory skewing of the compound on macrophage response. Also, LPS induced the significant IL-10 secretion, which may be due to a response of anti-inflammatory macrophages that usually appear at the late stage of LPS stimulation (after 24 hours) [18].

Fig. 1.

Immunomodulatory activity of spirocyclic compounds—cytokine production analysis. (A) Nitric oxide (NO) production was assessed using the Griess reagent assay. RAW264.7 macrophages (0.5 $\times$ 10⁶ cells/well) were incubated with the indicated compounds at 100 µM, 10 µM, or 1 µM for 24 hours (n = 3, technical replicates). (B,C) Cytokine quantification in naïve macrophages. Interleukin-6 (IL-6) (n = 3, technical replicates) and interleukin-10 (IL-10) (Biological replicates, n = 2; technical replicates, n = 3) levels in culture supernatants were determined by indirect sandwich enzyme-linked immunosorbent assay (ELISA), following 24 hours of treatment with compounds. lipopolysaccharide (LPS) (100 ng/mL) served as a positive control, and cell control (CC; untreated cells) as a negative control. Data are expressed as mean $\pm$ SD. Statistical analysis was performed between CC and compound-treated groups using an unpaired, non-parametric Student’s t-test; * p 0.05, ** p 0.01, *** p 0.001, and **** p 0.0001 indicate the level of significance.

The impact of spirocyclic compounds on cytokine secretion by murine splenocytes was assessed by quantifying IL-6 and tumor necrosis factor-alpha (TNF-$\alpha$) levels following treatment. Overall, compound exposure did not enhance pro-inflammatory cytokine production; instead, both IL-6 and TNF-$\alpha$ levels were reduced in a dose-dependent manner, comparable to the suppression observed in LPS-treated cells. Notably, among all compounds tested, compounds 1–12 inhibited IL-6 levels compared to the untreated control. In contrast, TNF-$\alpha$ secretion remained largely unchanged relative to the cell control, however, a few compounds, such as AS2, AS4, AS5, and AS6, did exhibit a slight increase, which could be explained by the heterogeneous cell population of the splenocytes (Fig. 2A,B). Given the above observations indicating that spirocyclic compounds do not exert pro-inflammatory effects, their potential anti-inflammatory activity was next evaluated using an in vitro inflammatory model. RAW264.7 macrophages were stimulated with LPS prior to compound exposure (Fig. 2C). ELISA analysis revealed that AS2, AS4, AS5, AS6, AS7, AS8, AS9, and AS10 compounds significantly inhibited LPS-induced IL-6 production at 100 µM, suggesting an anti-inflammatory and inhibitory activity on macrophage responses.

Fig. 2.

Anti-inflammatory activity of spirocyclic compounds on murine splenocytes and RAW264.7 macrophages. (A,B) Quantification of pro-inflammatory cytokines IL-6 and tumor necrosis factor-alpha (TNF-$\alpha$) in murine splenocyte cultures by indirect sandwich ELISA. Splenocytes (0.5 $\times$ 10⁶ cells/well) were incubated with the indicated compounds at 100 µM or 10 µM for 24 hours (n = 3 biological replicates). LPS (100 ng/mL) served as a positive control, and cell control (CC; untreated cells) as a negative control. (C) Decrease of IL-6 secretion in RAW264.7 macrophages following LPS stimulation and compound treatment. Cells (0.5 $\times$ 10⁶/well) were pre-incubated with LPS (100 ng/mL) for 2 hours, followed by treatment with spirocyclic compounds at 100 or 10 µM for 24 hours. Data represent two independent experiments (biological replicate = 2) performed in triplicate (n = 3, technical replicates). Data are expressed as mean $\pm$ SD. Statistical analysis was performed between CC and compound-treated groups (A,B) using an unpaired, non-parametric student’s t-test; also statistical comparisons were made between LPS-treated and LPS + compound-treated groups (C) using an unpaired, non-parametric Student’s t-test; * p 0.05; ** p 0.01; *** p 0.001, and **** p 0.0001 indicate the level of significance.

The compounds induced significant IL-10 production in RAW264.7 macrophages, while some also inhibited IL-6 production (200 pg/mL), thereby warranting further investigation. Previous studies have shown that spirocyclic salts can inhibit immune responses and, specifically, the JAK2/STAT3 pathway. The regulation of PD-L1 expression is known to be mediated by the JAK/STAT pathway, activated by cytokines released from immune cells. The JAK proteins are multi-domain proteins consisting of seven domains: the kinase domain (JH1), the pseudo-kinase domain (JH2), the Src-homology-2 domain (JH3–JH4), and the FERM domain (JH5–JH7) [19]. The four JAK isoforms (JAK1, JAK2, JAK3, and TYK2) are observed to play a crucial role in the transduction of cytokine-mediated signals through the JAK-STAT pathway and are expressed in nearly all cells. The specific JAK-STAT combinations determine the cytokine signaling pathways that mediate various immunological functions. It has been observed that JAK1 and JAK3 are critical for the signaling pathways of several interleukins, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Conversely, JAK2 modulates IL-6 and gp130 cytokine signalling. To this end, our experimentally verified, non-toxic series of 12 benzimidazole-based spirocyclic compounds was selected for further investigation of their binding interactions with JAK proteins. Molecular docking was performed to gain molecular-level insights into how these compounds interact with different JAK isoforms, which is crucial for understanding their potential inhibitory mechanisms [3].

Molecular docking was performed on all 12 compounds with JAK1, JAK2, JAK3 and TYK2 proteins to elucidate their binding interactions. The top ten scoring molecules for each protein were identified, and the common candidates with the potential to bind strongly to all JAK isoforms were shortlisted. These included compounds AS1, AS2, AS7, AS8, AS10, AS11, and AS12. Supplementary Table 3 provides the docking scores of these seven molecules with all JAK isoforms. It is evident that these compounds bind to JAK proteins with varying affinity. The binding scores for these top seven molecules ranged from –6.9 kcal/mol to –9.3 kcal/mol. Specifically, it is observed that all these molecules exhibited binding scores $\le$–8.0 kcal/mol for JAK1 and JAK3 proteins, whereas compounds 11 and 12 have binding scores of –6.9 kcal/mol and –7.6 kcal/mol for JAK2 and TYK2 proteins, which are relatively low compared to the binding affinities of other compounds toward these proteins. Compounds AS8 and AS10 were consistently ranked among the top leads with high binding affinities ($\le$–8.0 kcal/mol), while AS1 and AS2 also demonstrated favorable interactions ($\le$–7.7 kcal/mol) across all four JAK isoforms [20]. To understand the molecular basis of these findings, the docking poses of these four compounds were further analyzed to identify specific hydrogen bonding and hydrophobic interactions.

The interaction analysis reveals that while both hydrogen, hydrophobic and pi interactions are present, the binding is majorly driven by hydrophobic interactions. A full summary of each of the compound AS1, AS2, AS8, and AS10 interactions with all four isoforms is provided in the Supplementary Table 4. Moreover, Fig. 3A shows the binding pose for compounds AS8 and AS10 with the JAK2 protein, along with a 2D interaction map [20].

Fig. 3.

In silico molecular docking and in vitro evaluation of the interaction between JAK kinase and the spirocyclic compounds. (A) The binding poses of compounds AS8 and AS10 in JAK2 proteins, along with corresponding 2D interaction maps. (B,C) Bar graph representation after quantification of JAK2 protein expression level in the murine macrophage cell line (All western blots, including uncropped, are available in the Supplementary Information). 0.5 $\times$ 10⁶ cells/well were incubated with the indicated compounds at 100 µM for 24 hours with and without LPS stimulation for 2 hours (n = 2 biological replicates). Bar graph representation after quantification of JAK2 protein expression level. All data are expressed as mean $\pm$ SD. Statistical analysis was performed between CC and compound-treated groups for no LPS stimulation (B) and between LPS and compound-treated groups for LPS stimulation (C); using an unpaired, parametric Student’s t-test; * p 0.05 and ** p 0.01 indicate the level of significance.

In silico docking, supported by prior inhibitory activity data, indicated that AS1, AS2, AS8, and AS10 are potential inhibitors of the JAK/STAT pathway. To validate these findings, the expression of key genes and proteins involved in this pathway was evaluated following compound treatment. No significant changes were observed at the mRNA level (Supplementary Fig. 1). However, a pronounced reduction in total JAK2 protein expression was observed upon treatment with AS8 and AS10 (Fig. 3B,C and Supplementary Fig. 2). However, the phosphorylation status of JAK2 following compound treatment remains to be evaluated. In summary, AS8 and AS10 showed no cytotoxicity and significantly increased IL-10 while suppressing LPS-induced IL-6 in macrophages and splenocytes, indicating potent anti-inflammatory activity. Both compounds exhibited strong binding affinity to JAK isoforms ($\le$–8 kcal/mol) and reduced JAK2 protein expression. These findings suggest that selenium-containing scaffolds modulate inflammatory signaling pathways effectively.