Design, Synthesis, and Evaluation of a New Series of Hydrazones as Small-Molecule Akt Inhibitors for NSCLC Therapy

In an endeavor to identify small molecules for the management of non-small-cell lung carcinoma, 10 new hydrazone derivatives (3a–j) were synthesized. MTT test was conducted to examine their cytotoxic activities against human lung adenocarcinoma (A549) and mouse embryonic fibroblast (L929) cells. Compounds 3a, 3e, 3g, and 3i were determined as selective antitumor agents on A549 cell line. Further studies were conducted to figure out their mode of action. Compounds 3a and 3g markedly induced apoptosis in A549 cells. However, both compounds did not show any significant inhibitory effect on Akt. On the other hand, in vitro experiments suggest that compounds 3e and 3i are potential anti-NSCLC agents acting through Akt inhibition. Furthermore, molecular docking studies revealed a unique binding mode for compound 3i (the strongest Akt inhibitor in this series), which interacts with both hinge region and acidic pocket of Akt2. However, it is understood that compounds 3a and 3g exert their cytotoxic and apoptotic effects on A549 cells via different pathway(s).


INTRODUCTION
Cancer remains a deadly global health concern with more than 200 different types, affecting over 60 human organs. 1 Among all cancers, lung cancer is the primary cause of cancer-related morbidity and mortality in males, while, in females, it ranks second for mortality, after breast cancer, and third for incidence, after breast and colorectal cancer. 2 The disease is notorious for its exceptional potency to spread to distant parts of the body (metastasis), as well as its ability to progress rapidly in its early stages. 3 There are two groups of lung cancer, namely, small cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). 1 85% of lung cancer cases are attributed to NSCLC. 4 Surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy are currently available approaches for NSCLC therapy. 5 The best treatment strategy for patients with earlystage NSCLC is still surgical intervention. However, surgery is no longer an option for patients with advanced or metastatic NSCLC. 6,7 Major treatment approaches for unresectable NSCLC are chemotherapy and radiotherapy. 7 Platinumbased chemotherapeutics, taxanes, and other chemotherapeutics (e.g., vinorelbine, gemcitabine, and pemetrexed) are used either alone or in combination. 5 Despite their benefits in NSCLC therapy, these chemotherapeutics damage healthy cells as well as cancer cells and therefore they cause severe adverse effects and toxicity. 8 Resistance to radio(chemo)therapy is also a significant barrier to NSCLC therapy resulting in tumor recurrence and disease progression. 9 The current challenges in the diagnosis and treatment of NSCLC lead to poor prognosis and low survival rate. 10 Akt belongs to the family of serine/threonine-specific protein kinases essential for regulating crucial cellular processes including cell survival, proliferation, growth, apoptosis, and glucose metabolism. The abnormal overexpression or activation of Akt is involved in a variety of human malignancies and therefore inhibiting Akt has emerged as a pivotal strategy for the treatment of various types of cancer, particularly B-cell malignancies, NSCLC, and breast cancer. 11−13 Despite the large number of Akt inhibitors developed to date, the U.S. Food and Drug Administration has not approved any Akt inhibitors yet. 11 Hydrazides-hydrazones are frequently occurring eligible motifs in druglike small molecules due to their distinctive characteristics and several pharmaceutical applications for the treatment of many diseases, particularly severe bacterial infections, cancer, and inflammation. 14−18 Hydrazones have been reported to exert striking antitumor action via induction of apoptosis, cell cycle arrest, inhibition of angiogenesis, and a plethora of cancer-related biological targets (e.g., Akt). 19−26 Benzoxazoles are privileged building blocks for the synthesis of biologically active ligands targeting a plethora of crucial targets/pathways due to their unique features allowing them to effectively bind to diverse biological targets with distinct affinities. 27−31 Several studies have revealed that benzoxazoles exert marked cytotoxic activity against a variety of cancer cell lines through diverse mechanisms including induction of apoptosis, inhibition of Akt phosphorylation, and so on. 32−39 The publications related to hydrazones 19−26 and benzoxazoles 32−39 exerting pronounced anticancer activity through inhibition of target enzymes involved in the pathogenesis of lung cancer motivated us to design novel anti-NSCLC agents by means of the molecular hybridization of the benzoxazole core with the hydrazide group, which was conjugated with the benzylidene moiety substituted at the para position with dialkylamino groups (dimethylamino, diethylamino), nitrogencontaining electron-withdrawing groups (nitro substituent), five-(pyrrolidine) or six-membered heterocyclic motifs (piperidine, morpholine and piperazine), heteroaromatic rings (imidazole, triazole) based on our previous work ( Figure  1), 21 and the structure−activity relationships of existing Akt inhibitors 11 together with the bioisosteric replacement. In this context, the designed compounds were synthesized readily and assessed for their cytotoxic properties on human lung adenocarcinoma (A549) and mouse embryonic fibroblast (L929) cells. In vitro experimental studies were carried out for promising anti-NSCLC agents to shed light on their mechanism of action.
In the IR spectra of compounds 3a−j, the N−H stretching band of the hydrazone group was detected at 3207.62− 3159.40 cm −1 , while the C�O stretching band was detected at 1705.07−1654.92 cm −1 . In the 1 H NMR spectra of compounds 3a−j, S-CH 2 protons gave rise to two singlets in the range of 4.25−4.73 ppm. The peaks at 11.47−12.09 ppm were attributed to the N−H proton of the hydrazone moiety, while the peaks in the region 7.88−8.26 ppm were assigned to the CH�N proton. In the 13  According to the data presented in Table 1, the replacement of the dimethylamino group (compound 3a) with the diethylamino substituent (compound 3b) led to a significant decline in anticancer activity. This outcome indicated that the elongation of the alkyl chains reduced the cytotoxic effect on A549 cells. The bis(2-chloroethyl) and the nitro groups caused the loss of anticancer activity.
Among the six-membered heterocyclic rings (piperidine, morpholine, and piperazine) attached to the 4th position of the benzylidene motif, the piperazine ring gave rise to a significant increase in anticancer activity against A549 cells. On the contrary, the introduction of the pyrrolidine scaffold into the 4th position of the benzylidene group (compound 3h) resulted in the loss of anticancer activity. Taking into account the IC 50 values of compounds 3i and 3j (Table 1), the 1H-imidazole core increased the cytotoxic effect on A549 cell line, while the 1H-1,2,4-triazole ring caused the loss of anticancer activity.
A colorimetric method was used to assess the inhibitory effects of compounds 3a, 3e, 3g, and 3i on Akt in A549 cells. Compound 3e carrying a piperidine ring in the 4th position of the benzylidene motif and compound 3i bearing an imidazole ring in the 4th position of the benzylidene moiety inhibited Akt in A549 cells with IC 50 values of 105.88 ± 53.71 and 69.45 ± 1.48 μM, respectively, compared to GSK690693 (IC 50 = 5.93 ± 1.20 μM), a well-known Akt inhibitor. According to the data indicated in Table 3, it can be concluded that compounds Dimethylamino-substituted compound 3a caused 35.40 ± 13.36% Akt inhibition at 91.35 μM. Compound 3g, which carries the 4-methylpiperazine motif in the 4th position of the benzylidene moiety, did not exert any inhibitory effect on Akt. Accordingly, it is obvious that both compounds cause cytotoxicity and apoptosis in A549 cell line via different pathway(s).

In Silico Studies.
Computer-aided drug design tools provide a detailed picture of the biologically active molecules at the molecular level. Our recent successful in silico studies 40−42 prompted us to confer molecular docking simulations as a tool to enlighten binding conformations of the ligands.
Before moving forward to in silico calculations, internal validation 43 was carried out to test how well our docking method performs. The conformation of co-crystallized ligand (GSK690693) was successfully reproduced with Glide XP   method yielding a root-mean-square deviation (RMSD) of 0.436. Molecular docking studies showed the binding mode of compound 3i within Akt2 binding site (Figure 3). The imidazole nitrogen engages in a key hydrogen bond with the backbone amine of ALA232 in the hinge region. The sulfur linker seems to facilitate compound 3i to adopt a unique binding mode. This folded mode that resembles a V-shaped conformation brings the benzoxazole ring to the vicinity of the acidic pocket. A new hydrogen bond is observed between ASP293 and the N−H of the hydrazide spacer. The interaction of a ligand with this acidic pocket was reported to be important for potent inhibition. 44 Certain descriptors related to absorption, distribution, metabolism, and excretion (ADME) were also predicted. Blood−brain barrier parameter was also calculated that could be useful in central nervous system-targeted studies. Of note is that compounds 3a, 3e, 3g, and 3i were estimated to have good pharmacokinetic profiles and druglike properties (Table  4).

CONCLUSIONS
This paper describes the synthesis of new hydrazones (3a−j) designed as small molecules for NSCLC therapy. Taking into account in vitro cytotoxicity data, selective anticancer agents in this series on A549 cells were determined as compounds 3a (IC 50 = 91.35 ± 21.89 μM), 3e (IC 50 = 176.23 ± 56.50 μM), 3g (IC 50 = 46.60 ± 6.15 μM), and 3i (IC 50 = 83.59 ± 7.30 μM). Among them, compounds 3a and 3g caused induction of apoptosis in A549 cells stronger than cisplatin. Both compounds did not exert any significant Akt inhibitory activity in A549 cells, while compounds 3e and 3i caused Akt inhibition in A549 cells. It can be concluded that compounds 3e and 3i exert their cytotoxic action against A549 cell line via the inhibition of Akt. Molecular docking simulations disclosed a unique binding mode for compound 3i (the most active Akt inhibitor in this series) which interacts with the hinge region and the acidic pocket of Akt2. On the other hand, further research is required to shed light on the mechanism of action underlying the cytotoxic and apoptotic effects of compounds 3a and 3g on A549 cells.

4.3.
In Silico Studies. The crystal structure of human Akt2 in complex with GSK690693 was downloaded from Protein Data Bank (PDB ID: 3D0E at 2.00 Å resolution). This raw protein structure was initially prepared 49 by adding missing hydrogens, correcting bond orders, and removing all heteroatoms except the native ligand. The structure was then optimized to address any overlapping hydrogens, and finally, restrained minimization was carried out.
The binding site of Akt2 was defined by computing grid file using GSK690693 as a reference agent. The size of enclosing box that represents grid was extended to allow docking of the ligands with length <20 Å. Glide XP module 50 was selected for molecular docking. LigPrep module 51 was used to prepare ligands for in silico calculations to consider all possible geometries, tautomers, and also protonation states at physiological conditions. Important molecular descriptors related to ADME were predicted using QikProp module. 52 ■ ASSOCIATED CONTENT