Nafimidone Synthesis Essay

1. Introduction

Cancer has become one of the most serious diseases which represents a great threat to human health around the world [1]. Although there are currently a large number of anticancer drugs, more and more people continue to die of cancer [2]. Epidermal growth factor receptor (EGFR) plays important roles in human cancer. As a member of the HER family, EGFR is a tyrosine kinase receptor which plays an essential role in normal cell growth and differentiation, and is involved in tumor proliferation and survival [3,4]. The HER family comprises four members: EGFR (HER1/ErbB-1), ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4 (HER4) [5]. EGFR is one of the most important targets in current cancer research and its over-expression or abnormal activation often causes cell malignant transformation [6]. The over-expression of EGFR has been observed in many solid tumors, such as colon [7], ovarian [8], breast and non-small cell lung cancer (NSCLC) [9]. Therefore, EGFR inhibition has been developed as one of the most efficient strategies for cancer therapy.

Dihydropyrazole, a small bioactive molecule, is a prominent structural motif found in numerous pharmaceutically active compounds. Some representatives had been demonstrated to have important biological activities such as antiviral/antitumor [10], antibacterial [11], fungistatic [12], antihyperglycemic activity [13], antimalarial [14] and antitubercular effects [15]. Biological evaluation indicated that some of the synthesized compounds were potent inhibitors of EGFR. Some novel compounds containing the dihydropyrazole skeletons were reported as potent anticancer agents against EGFR tyrosine kinase. It had been reported that anticancer compounds such as 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines analogues (3A-1-PP, Figure 1) showed potent EGFR kinase inhibitory activity, with IC50 values reaching single digit nanomolar values [16]. Compounds a, b, c and d (Figure 1) displayed the most potent EGFR inhibitory activity, with IC50 values of 0.07, 0.24, 0.06 and 0.26 μM respectively, which were comparable to the positive control erlotinib (IC50 = 0.03 μM) [17,18,19,20]. Compound e containing the dihydropyrazole and naphthalene ring showed the highest inhibitory EGFR activity (IC50 = 0.12 μM) [21].

Some naphthalene derivatives have been reported as potent microtubule inhibitors, apoptosis inducers, glutamamide analogues or P-glycoprotein inhibitors [22,23]. On the other hand, the naphthalene ring can be recognized in various biologically active compounds with clinical applications. Naftifine and terbinafine, allylamine antifungal agents, are widely used for the treatment of fungal infections [24,25,26]. Propranolol is an anti-angina drug, duloxetine is used for the treatment of depression, nafimidone and its oxime ester derivative can be further developed for the treatment of epilepsy [27]. Therefore, preparation and extensive biological evaluations on naphthalene derivatives have continuously attracted our attention [28]. Besides, benzohydrazides are reported to possess a wide variety of biological activities like antiglycation [29], antioxidant [30], antileishmanial [22], antibacterial [31], antifungal [23], antitumor [32] and anticonvulsant [24]. Benzothiazole Schiff bases with diverse biological activities have also been reported [26].

The three combined substructures—the dihydropyrazole along with the naphthalene ring and benzohydrazides—might exhibit synergistic anticancer effects. All of these facts encouraged us to integrate these three moieties and screen new benzohydrazide derivatives containing dihydropyrazoles as potential EGFR inhibitory agents. Herein we disclose the design and synthesis of some novel benzohydrazide derivatives containing dihydropyrazoles as potential EGFR kinase inhibitors, and their antitumor activity against the A549 (human lung cancer), MCF-7 (human breast cancer), HeLa (human cervical cancer) and HepG2 (human hepatocellular cancer) cancer cell lines.

2. Results and Discussion

2.1. Chemistry

A series of novel benzohydrazide derivatives containing dihydropyrazole moieties were synthesized by the routes outlined in Scheme 1. The structures of the compounds are listed in Table 1. Methyl 4-hydrazinylbenzoate (B) was synthesized from 4-hydrazinylbenzoic acid in methanol at room temperature. The diverse substituted chalcones E1E16 were obtained by stirring substituted acetophenones and naphthaldehyde in ethanol at room temperature for 6–8 h. The cyclization different chalcones and methyl 4-hydrazinylbenzoate in refluxing ethanol gives compounds F1F16.

Compounds F1F16 further reacted with excess hydrazine hydrate (80%) to give the requisite intermediate G1G16. The desired compounds H1H32 were generated by the reaction of compounds G1G16 with substituted benzaldehydes in ethanol for 6–8 h. Purified compounds H1H32 were finally obtained by chromatography. All synthesized compounds H1H32 gave satisfactory analytical and spectroscopic data in full accordance with their depicted structures, and other data published in a Chinese patent [33].

2.2. Bioassays

2.2.1. Antiproliferative Assay

The antiproliferative activities of the newly synthesized derivatives H1H32 were evaluated under identical conditions by the MTT assay against four cultured cell lines (A549, MCF-7, HeLa and HepG2) with erlotinib as control. The IC50 values of the compounds against these human cancer cells are summarized in Table 2. As shown, the results revealed that all of the target compounds exhibited significant antiproliferative activities, ranging from 0.15 to 100 μM.

Among them, compound H20, which had IC50 values of 0.46, 0.29, 0.15 and 0.21 μM against A549, MCF-7, HeLa and HepG2, respectively, displayed the most potent antiproliferative activity, comparable to that of the positive control drug erlotinib (with corresponding IC50 values of 0.20, 0.12, 0.17 and 0.10 μM). These results suggested that compound H20 is more potent than the other compounds overall. Modification of substituents such as methyl, halogen, methoxyl, ethyl and ethoxy was performed to explore the structure-activity relationships of these compounds. Compounds bearing one methyl group at the para-position in the A ring showed stronger antiproliferative activity (the IC50 values of H4 and H20: 0.45 and 0.21 μM against HeLa cells) than those with hydrogen (H1 and H17), fluorine (H11 and H27), chlorine (H14 and H30), bromine (H16 and H32), ethyl (H5 and H21), ethoxy (H8 and H24) and methoxyl (H6 and H22) substituents, in the order of Me > H > Br > Cl > F, Me > OMe and Et > OEt. This means that compounds with electron-donating groups at the para-position of the A ring had better antiproliferative activity than those with electron-withdrawing groups. A comparison of the composition on the A ring was as follows: for compounds H2H4, H9H11, H13H14, H18H20, H25H27 and H29H30, the potency order of substituent groups on the A ring was found to be para > ortho > meta. Compounds H9H15 and H25H31 with halogen groups on the A ring exhibited antiproliferative activities in the order of disubstituted > monosubstituted. Interestingly, compounds H6 and H22 with one methoxyl group on the A ring showed stronger antiproliferative activities than compounds H7 and H23 with three methoxyl groups, so we concluded that more electron-donating groups could reduce the electron density of the A ring and make it difficult to form π-π bonds with amino acids containing aromatic groups.

In the case of constant A ring substituents, changes of substituents on the B ring can also affect the activities of these compounds. Contrary to the A ring, derivatives H17H32 with a methoxyl at the para-position displayed better activities than at the meta-position (compounds H1H16). These indicated that the position of the substituent had an influence on the antitumoral activity.

2.2.2. Kinase Inhibitory Activity

All these newly synthesized compounds were evaluated for their inhibitory activities against EGFR kinases using a solid-phase ELISA assay. The approved EGFR inhibitor drug erlotinib was used as a positive control. The inhibition constants (IC50) of the compounds were summarized in Table 3.

The EGFR kinase inhibitory activity results of the tested compounds correlated with the structural relationships (SAR) of their inhibitory effects on the cell proliferation assay. Among the tested compounds, compound H20 showed potent anticancer activity with IC50 of 0.08 μM, which was comparable to the positive control erlotinib (IC50 = 0.03 μM). Other tested compounds displayed moderate inhibitory activities, with IC50 values ranging from 0.35 μM to 24.58 μM. This suggests that the potent inhibitory effects of the synthetic compounds on the cell proliferation assay were basically related to their kinase inhibitory activities.

2.2.3. Cytotoxicity Test

All the target compounds H1H32 were evaluated for their toxicity against 293T human kidney epithelial cells at the median cytotoxic concentration (CC50) value of the tested compounds determined by the MTT assay. As displayed in Table 4, these compounds were tested at multiple doses to study the viability of 293T cells. Judging from the median cytotoxic concentration (CC50) data, all of the tested compounds demonstrated similar cytotoxic activities as erlotinib in vitro against human kidney epithelial 293T cell.

2.2.4. Analysis of Apoptosis

To verify whether the inhibition of cell growth of HeLa cell lines was related to cell apoptosis, we decided to use flow cytometry with an Annexin V-FITC/PI Apoptosis Detection Kit to induce the HeLa cell apoptosis with compound H20. The uptake of Annexin V-FITC/PI markedly increased, and the uptake of normal cells was significantly decreased in a time-dependent manner. According to the annotated data, the percentage of apoptotic cells was significantly elevated directly and in a dose-dependent manner. The results are shown in Figure 2 and Figure 3. As can be seen, the percentages of cell apoptosis were 9.01%, 28.7%, 34.9%, 47.7%, 58.12%, in response to 0, 2.0, 4.0, 6.0, 8.0 μM concentrations of compound H20, respectively. This leads to the conclusion that the percentage of apoptotic cells significantly increased after treatment with high doses of H20.

2.3. Molecular Docking

To gain better understanding of the potency of the 32 compounds and guide further SAR studies, we examined the interaction of these compounds with EGFR (PDB code: 1M17) by molecular docking. A simulation of binding between the compounds and the ATP binding sites in EGFR was performed. All docking runs used the DS 4.5 software (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China). The obtained results, presented in Figure 4 and Figure 5, show the optimal binding mode of compound H20 interacting with the 1M17 protein. The amino acid residues of EGFR which had interactions with compound H20 were labeled. In the proposed binding mode, compound H20 was nicely bound to the ATP binding pocket of EGFR through hydrogen bond interactions and hydrophobic interactions.

In the binding model, compound H20 was well bound to the EGFR protein with Gln767, Thr766, Arg752, Val702, Leu694, Leu820, Ala719, the seven amino acids located in the binding pocket of the protein, playing an important role in the combination with compound H20. As we can see from Figure 4 and Figure 5, Gln767 formed conventional hydrogen bonds and Thr766 formed Van der Waals bonds with the nitrogen atom of the Schiff base group, which enhanced the combination activity of compound H20. Moreover, a carbon–hydrogen bond and π-sulfur bonds were found between Arg828 and Cys751 with a benzene ring. Meanwhile the Leu694 formed three alkyl bonds with the methyl, benzene ring and dihydropyrazole ring. Alkyl and π-alkyl bonds were displayed in presence of Leu820 and dihydropyrazole ring and benzene ring, respectively. Moreover, Ala719 also formed π-alkyl bonds with the benzene ring. Naphthalene was also bonded with Val702 by a π-alkyl bond. In molecular docking 3D modeling, compound H20 was nicely bound to the ATP binding site through the hydrogen bond with the backbone of Gln767 (distance = 2.4 Å) which increased the binding affinity dramatically in theory, as displayed in Figure 5.

In addition, the predicted binding interaction energy was used as the criterion for ranking; the estimated interaction energies of other synthesized compounds ranged from −64.95 to −52.99 kcal/mol, as displayed in Figure 6 with a histogram. The selected compound of H20 had a best estimated binding free energy of −64.95 kcal/mol for EGFR. These molecular docking results, along with the biological assay data, suggested that compound H20 is a potential inhibitor of EGFR.

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