The PI3K/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR, PAM) pathway has drawn significant pharmacological investment in the search for inhibitors to treat human cancers. Phosphoinositide 3 kinases (PI3Ks) are a lipid kinase family that includes PI3K1, PI3K2, and PI3K3. PI3K1 is a well-studied PI3K that consists of a catalytic subunit (p110α, p110β, p110γ, and p110δ) [1], [2], [3], [4]. PI3Kδ, one of these four PI3K isoforms, is predominantly expressed in B-cells and catalyzes the phosphorylation of phosphatidylinositol-4,5-biphosphate to phosphatidylinositol 3,4,5-triphosphate via the PI3K/Akt signaling downstream, making it essential for B-cell proliferation, development, and survival [5]. As a result, PI3Kδ inhibition is thought to be therapeutically advantageous for hematological malignancies. On the other hand, mTOR, the downstream signaling effector in the PI3K/Akt/mTOR cascades, is a kinase that belongs to the phosphatidylinositol 3-kinase family. Phosphorylation of PI3K causes phosphorylation of various downstream effectors, including mTOR and protein kinase B (PKB/Akt), resulting in cell cycle progression, proliferation, survival, and migration [6], [7], [8], [9]. Inhibiting PI3Ks or mTOR alone can slow cancer growth; however, the negative feedback loop of mTOR can reactivate PI3K via S6K1 and IRS-1, restarting cancer progression and mediating treatment resistance. In light of this, simultaneous inhibition of PI3Kδ and the downstream effector mTOR with a single therapeutic drug is favourable for avoiding or delaying such resistance responses, as well as providing two-spot PAM pathway disruption and creating synergism. As seen by the clinical development of many medicines targeting PI3Kδ and mTOR, both PI3Kδ and mTOR are intriguing anti-cancer targets with great therapeutic potential. Furthermore, because mTOR and PI3K have the same catalytic domain, many PI3K inhibitors also inhibit mTOR. Several PI3K/mTOR dual inhibitors have moved into clinical trials, including BGT226, GSK1059615 [10], [11], dactolisib [11], omipalisib [12], and others [13] (Fig. 1).

Quinoline is a well-known scaffold for developing PI3K/mTOR dual inhibitors. The SAR of dual PI3K/mTOR inhibitors reveals that activity requires a parent six-member heterocyclic ring. Furthermore, the amide branch at C-3 of quinoline analogues conferred greater activity against PI3K/mTOR due to an H-bond with Ser854 at the entrance of the binding pocket [14]. Also, the pyrazole (diazole) ring and aromatic amine at the C-2 and C-3 positions of quinoline derivatives, respectively, enhanced PI3kδ inhibition [13]. The nitrogen of quinoline in BGT226, GSK1059615, omipalisib, and dactolisib (Fig. 1) established an H-bond with Val882 at the hinge region of PI3Ks [10], [11], [13], and [15]. Furthermore, the quinoline ring of omipalisib formed H-bond and pi-pi interactions with Val2240 and Tyr2239 with mTOR, respectively [12]. Compounds 5 and 6 (Fig. 1) containing diazole rings demonstrated significant IC50s of 0.15 µM and 0.45 µM against PI3Kδ [13], [16]. In light of the foregoing, compounds (D) and (E) were created using the lead (A), (B), and (C) templates. The addition of diazole (pyrazole at C-2) and amine side chains (purine at C-3) in lead (A) made it more PI3Kδ-friendly, with an IC50 of 0.0026 μM [13]. Furthermore, diazole (indazole at C-2) in lead (C) increased IC50 against mTOR and PI3Kδ to 0.58 μM and 0.003 μM, respectively [17]. Pyrazole and indazole, respectively, increased the efficacy of (A) and (C) via H-bonds to Asp911, Val828, Ile825, Gln2167, and Ser2165. Taking these facts into account, we continued to investigate lead (A), where pyrazole was bioisoterically swapped for triazole to create (D) and (E) while retaining a similar pattern of connections. Furthermore, the amino-purine side chain was substituted with aromatic amines for 5h with the idea of interaction in enlarged lipophilic pockets. Also, fluorine in lead (B) enhanced the inhibition against PI3Kδ by 0.0024 µM and 0.0046 µM in biochemical and cellular investigations. The pyridine ring improved selectivity against α, β, and γ with IC50s of 7.9 µM, 2.8 µM, and 0.85 µM, respectively, with low clearance (0.057 l/h/kg) and oral bioavailability (%F = 51) [18]. Moreover, quinoline analogues with methoxy groups at C-7 and C-8 effectively inhibited PI3K with IC50s of 0.3 µM and 48 µM, respectively [19], [20], [21]. Furthermore, an in silico investigation revealed that the methoxy group at C-7 of quinolinone analogues inhibited PI3K via lipophilic and H-bond with Glu259 and Arg662, respectively. It also improved quinolinone binding to Phe666 in the hinge region of the kinase domain [22]. Furthermore, the size of the methoxy group has been found to confer strong binding affinity within the hinge area of the kinase domain, facilitating accommodation of the ligand within the ATP binding pocket, thus contributing to the maintenance of the desired kinase binding mode [21], [22]. Additionally, both fluorine and methoxy groups, acting as bioisosteres of each other, possess hydrogen bond acceptor properties, which play a pivotal role in modulating intermolecular interactions within ligand–protein complexes [23], [24]. By bioisosterically replacing fluorine with methoxy in designs (D) and (E), we aimed to retain both the H-bond acceptor tendency and the crucial binding interactions within the hinge region, thereby supporting the proposed kinase binding mode. (Fig. 2).

In this study, we developed and evaluated new 6-methoxy-2-(1H-1,2,4-triazol-1-yl)-quinolin-3-methylene analogues for cytotoxicity, PI3Ks/mTOR inhibition, and apoptosis. The target of PI3Kδ/mTOR was then confirmed using a western blot assay. In addition, the apoptosis pathway was examined in the ELISA immunoassay. Also, the PI3Kδ/mTOR target was validated using docking interactions. Finally, the in silico ADMET/drug-likeness parameters and liver microsomal stability were investigated.