**1. Introduction**

Cancer is a life-threatening disease; one of the major challenges for relieving its burden is to develop highly e ffective drugs with specificity for cancer, but few to no side e ffects on normal mammalian cells [1]. Gefitinib, the epidermal growth factor receptor (EGFR) inhibitor, was added recently to the

Food and Drug Administration (FDA) list of the recommended first-line treatments for lung cancer [2]. Gefitinib is particularly recommended for non-small cell lung cancer (NSCLC) type [3]. Although that erlotinib and afatinib are also included in this list, gefitinib may be the most tolerable [4]. The most common adverse effects of this group of drugs were gastrointestinal diarrhea, nausea and/or vomiting which is due to the toxic effect of the drug on normal flora [5,6]. The absence of antibacterial activity of the anticancer drug removes its detrimental effect against intestinal flora, suggesting a highly promising new strategy for the development of anticancer drugs with reduced side effects [7]. In 2008, O'Shea and Moser reported [8] that molecular weight is an important factor for antimicrobial activity. They did experiment on 147 antibacterial and 4623 non-antibacterial compounds and found average molecular weight for an antibacterial compound were 812 Da (Gram '+' positive) and 414 Da (Gram ' −' negative). In addition, another important factor for becoming an antimicrobially active compound is polarity. According to the Lipinski's rule of five, also known as Pfizer's rule of five or simply the rule of five (RO5) [9], 70.4% of the antibacterial active compounds showed logP values ranges from 0–5. Therefore, in order to target a compound with no/little antimicrobial activity, lower molecular weight and with more logP values should be taken into consideration, isatin and its derivatives with low molecular weight could be a choice of interests [10–12]. Isatin, a natural compound, is known for more than a century and still being used extensively in medicinal compound synthesis [13–19]. It has been reported that, various substituents on isatin nucleus displayed numerous biological activities [12,20]. In recent years, number of isatin derivatives were reported with extensive biological activities [21–25], included EGFR activity [26]. *N-*benzylisatin hydrazones of fluorescein had showed antiproliferative activity as well as topo II inhibitory activity [27]. Moreover, two series of hydrazone derivatives has been reported recently with antiproliferative activity [28,29]. A number of marketed drugs and potential anticancer agents having isatin moiety, depicted in Figure 1, inspired us to synthesize a series of isatin hydrazones having *N-*benzyl protection at 1-position of isatin and hydrazone formation at 3-position with various aryl substituents. We thus anticipating that simplification of isatin molecule would prevent its inhibitory effect on different microbes, providing more selectivity in the action on cancerous cell lines with less/no toxicity on the gastrointestinal tract (GIT) lining cells. Considering the above points and the importance of the development of anticancer therapeutics with no/few side effects, we therefore designed and synthesized a series of *N-*protected *N-*benzylisatin-aryl hydrazones with a lower molecular weight (353–418 Da), evaluated their antibacterial activity against two Gram-positive, four Gram-negative bacterial strains and antifungal activity against *Candida albicans* NRRL Y-477, and antiproliferative activities against non-small cell lung cancer cell lines 'A549', as well as human cervical cancer cells lines 'HeLa.' In addition, for comparing the potency of the synthesized compounds, "gefitinib" was used as a positive control for antiproliferative activity evaluation.

In order to consider a compound as drug molecule, it is necessary to test their drug likeness properties as well as the analysis of physiological descriptors such as absorption, distribution, metabolism and excretion (ADME). ADME are important physiological descriptors of chemical compounds for selecting highly potential drug targets. However, testing a wide range of compounds directly in clinical or pre-clinical phase is extensively time consuming and costly. Moreover, ADME was considered as the last step of drug development where many drugs (approximately 60%) were failed after all the procedures. To solve these problems, recent experiments utilizes in silico ADME tools as the first step to shorten the amount of target compounds, by calculating predicted ADME properties and by discarding the compounds with unsatisfactory ADME values from the drug designing pipe line [30]. This prediction enabled us to identify potent drug candidate by analyzing the properties of the designed compounds. Therefore, we studied the ADME predicted parameters of the synthesized compounds (**6a**–**j**) using in silico ADME tools and compared them with that of "gefitinib".

**Figure 1.** Isatin moiety containing active & potential drugs and gefitinib.

### **2. Materials and Methods**

*2.1. Chemicals and Solvents Were of Commercial Reagent Grade (Sigma-Aldrich, St. Louis, MO, USA) and Used without Further Purification*

The progress of reactions and purity of reactants and products were checked using pre-coated silica gel 60 aluminum TLC sheets with fluorescent indicator UV254 of Macherey-Nagel, and detection was carried out with ultraviolet light (254 nm). Melting points were determined on a Fisher ScientificTM digital melting point apparatus (model number IA9100) and are uncorrected. Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed using an Agilent high performance liquid chromatography (HPLC) 1200 connected to an Agilent 6320 ion trap mass spectrometer fitted with an electrospray ionization (ESI) ion source (Agilent Technologies, Palo Alto, CA, USA). Infrared spectra were recorded as KBr disks using the Fourier Transform Infrared Spectrophotometer of Shimadzu; model: IR affinity-1S (Shimadzu, Tokyo, Japan). NMR spectra were taken on Agilent-NMR-VNMRS 600 MHz spectrometer (Agilent Technologies, Palo Alto, CA, USA) and DMSO-d6 was used as solvent.

### *2.2. Preparation and Characterization of Target Compounds*

2.2.1. 1-Benzylindoline-2,3-dione (**3**)

> Orange powder (95%) mp = 130–131 ◦C (Lit. [31] mp. = 125–126 ◦C).

2.2.2. (Z)-1-Benzyl-3-hydrazonoindolin-2-one (**4**)

Yellow powder (90%) mp = 124.5–126 ◦C (Lit. [32,33] mp. = 125–126 ◦C). ESI mass m/z = 252 [M + H]<sup>+</sup>.

#### 2.2.3. General Procedure for the Synthesis of **6a–j**

To a mixture of isatin monohydrazone (1 mmol) and substituted aryl aldehyde (1 mmol) in 10 mL ethanol, a few drops of glacial acetic acid was added. The reaction mixture was refluxed for 4 h. The precipitate solid was filtered, washed with cold ethanol and air dried to obtain the target compounds (**6a**–**j**), which was then further purified by recrystallization using methanol. 13CNMR spectra for **6a-j** is available in the Supplementary Materials file.

1-Benzyl-3-((4-(dimethylamino) benzylidene)hydrazono)indolin-2-one (**6a**)

Dark red solid (53%) mp = 183–184 ◦C. IR (KBr): νmax (cm−1): 2926 (C-H aliphatic), 1707 (C=O), 1606 (C=N), 1558 (C=N). 1H NMR (DMSO-d6, 600 MHz: δ 3.06 (s, 6H, N(CH3)2), 4.97 (s, 2H, N-CH2-Ph), 6.84 (d, *J* = 7.8, 2H, ArH), 6.98 (d, *J* = 7.2, 1H, ArH), 7.15 (t, 1H, ArH), 7.26 (s, 1H, ArH), 7.34 (m, 5H, ArH), 7.85 (d, *J* = 7.8, 2H, ArH), 8.30 (d, *J* = 7.2, 1H, ArH), 8.67 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6, 150 MHz: δ 166.41, 164.43, 153.76, 149.56, 144.80, 136.70, 133.09, 131.97, 129.18, 129.08, 127.99, 127.68, 123.35, 120.76, 117.04, 112.30, 110.22, 43.11 and 40.14 ppm. ESI mass m/z = 383 [M + H]<sup>+</sup>.
