*3.2. Biological Evaluation*

### 3.2.1. Antimicrobial Evaluation of **6a–j**

Synthesized *N-*benzylisatin-aryl hydrazones (**6a**–**j**) were evaluated for their antibacterial activity against two Gram-positive Staphylococcus aureus ATCC 29,213 and *Bacillus subtilis* ATCC 3366, four Gram-negative, *Escherichia coli* ATCC 25922, *Klebsiella pneumoniae* ATCC 13883, *Salmonella typhi* ATCC 25,566 and *Pseudomonas aeruginosa* ATCC 27,853 bacterial strains. As expected, the result of the antibacterial evaluation revealed no effect of these compounds (**6a**–**j**) on selected strains of bacteria in concentration up to 100 μg/disc (where no significant inhibition zones were detected). In case of antifungal activity, no inhibition zone was detected against *Candida albicans* NRRL Y-477. Excitingly, our hypothesis matched with the antimicrobial data of the evaluated compounds **6a**–**j**, which are all having low molecular weight (353–418 Da), conforming to O'Shea and Moser's findings. In addition, the antimicrobial data of **6a**–**j** also meet the rule of five (RO5) with logP value of 9.36–11.8 (please see Section 3.4), which is far more than to have anti-microbial activity. In case of antifungal activity, there were no inhibition zones detected against *Candida albicans* NRRL Y-477 either.

### 3.2.2. Antiproliferative Activity of **6a**–**j**

The antiproliferative activity of compounds **6a**–**j** are listed in Table 1 as values of IC50. All the tested compounds displayed excellent antiproliferative activity against non-small cell lung cancer cell lines 'A549' and human cervical cancer cells lines 'HeLa' tested. Among them, compound **6c** displayed the highest antiproliferative activity against the cell lines 'A549' (4.35 μM) and 'Hela' (4.09 μM). Whereas positive control gefitinib showed less antiproliferative activity against the cell lines 'A549' (15.23 μM) and 'Hela' (7.35 μM). Besides compound **6c**, another three bromo derivatives of *N-*benzylisatin-aryl hydrazones (**6d**, **6f** and **6g**) showed excellent antiproliferative activity, which is comparable to the control gefitinib. Detail explanations and SARs study are given below.


**Table 1.** Antiproliferative activity of *N-*benzylisatin-aryl hydrazones (**6a–j**).

Data represent means ± SD obtained from three different experiments. Cell lines used as follows, non-small cell lung cancer cell lines 'A549' and human cervical cancer cells lines 'HeLa'. Gefitinib was used as positive controls.

### *3.3. Structure Activity Relationships (SARs) of 6a–j*

The compound, namely (E)-1-benzyl-3-hydrazonoindolin-2-one (**4**) showed moderate antiproliferative activity (14.15 μM) against the non-small cell lung cancer cell lines 'A549' similar to that of gefitinib (15.23 μM), but six-fold less active against human cervical cancer cells lines 'HeLa' (46.03 μM). Compound **4** reacted with 4-substituted aryl aldehydes forming compound **6a** having 4-(*N*,*N-*)-dimethyl group (**6a**), chloro group (**6b**) and methyl group (**6i**) decreased the antiproliferative activity than the gefitinib against both cell lines tested. While substituted by methylthio group (**6j**) shows similar antiproliferative activity comparing gefitinib. Interestingly, as depicted in Table 2, entry **6c**, substituted by hydroxy group at 4-position showed higher antiproliferative activity which is around four fold than the gefitinib against non-small cell lung cancer cell lines 'A549' (4.35 μM) and two fold higher than the HeLa cell lines (4.09 μM). In case of bromo substituents at the 2- and 4-positions of the aryl ring, the compounds **6d** and **6f**, respectively, the antiproliferative activity dramatically increased more than two-fold for A549, and similar for HeLa cell lines in comparison to the gefitinib. While addition at the 3-position by a bromo group (**6e**) and by a methyl group (**6h**) of the aryl ring showed similar or less antiproliferative activity comparing to gefitinib. Interestingly, the methyl group at 2-position (**6g**) increased the activity by two-fold compared to gefitinib, against A549 and similarly against HeLa cell lines. In conclusion, ortho and para substitution by bromo and hydroxy group proved to be the most active potential agents for non-small cell lung cancer cell lines 'A549' among the compound tested.



b Molecular weight in Daltons (acceptable range: <500); c Hydrogen bond donor (acceptable range: ≤5); d Hydrogen bond acceptor (acceptable range: ≤10); **e** Predicted octanol/water partition coefficient (acceptable range: −2–6.5); **f** Predicted aqueous solubility, S in mol/dm−<sup>3</sup> (acceptable range: −6.5–0.5); **g** Predicted IC50 value for blockage of hERG K+ channels (concern: below −5); h Caco−2 value, permeability to Caco−2 (human colorectal carcinoma) cells in vitro; **I** Blood−brain barrier permeability (acceptable range: <sup>~</sup>−0.4); j Predicted apparent MDCK cell permeability in nm/sec, QPPMDCK= >500 is great, <25 is poor; k Predicted human oral absorption on 0% to 100% scale (<25% is poor and >80% is high); l G = Gefitinib.

### *3.4. In Silico Drug Likeness Property Analysis*

In modern drug discovery approaches, evaluation of absorption, distribution, metabolism and excretion (ADME) of drug candidates impose significant value to the rational drug design. In vitro and in vivo ADME prediction has now become faster and more accurate with the computational chemistry tools which has been developed recently and aids the pharmaceutical industries to screen many compounds within a very short time [38]. In this experiment, we analyzed the predicted ADME values of the designed compounds (**6a**–**j**) and are summarized in Table 2. Since high molecular weight compounds are always less effective in terms of intestinal absorption, our synthesized compounds' (**6a**–**j**) molecular weight (353–418Da) of this study are less than the established drug gefitinib (447Da), which supports the established parameters [9,39]. Subsequently, the studied compounds showed recommended values for hydrogen bond donor (<5) and acceptor (<10). Gefitinib showed HBD value of 1 and HBA value of 7.7 whereas the studied compounds showed HBD values of 0 (except **6c** having HBD 1) and HBA values of maximum 6.5 (compound **6a**), 6.25 (compound **6c**), 6.0 (compound **6j**)

and other compounds **6b**, **6d**–**6i** having HBA values of 5.5, which indicates superior values to the gefitinib (HBA values is 7.7). In 2002, Jorgensen and Duffy established a parameter to check the bioavailability of the drug compound which can be determined by octanol/water partition coefficient and solubility scoring where the recommended values are −2–6.5 and −6.5–0.5 mol/dm−3, respectively [40]. Compounds of this study showed scoring within the reference values including gefitinib. Octanol/water partition coefficient and solubility score in between 3.82–5.01 and −5.93–−5.07, respectively. The hERG K+ channel blockers are potentially toxic for the heart and thus the recommended range for predicted log IC50 values for blockage of hERG K+ channels (loghERG) is > −5 [41]. Intriguingly, all the compounds of this study showed higher value for loghERG score (<sup>&</sup>gt;−7.13) than gefitinib (−7.12) which proved their less toxicity than gefitinib. The Caco-2 cell, considered as the reliable in vitro models to estimate oral drug absorption and transdermal delivery [42], was high for all compounds except gefitinib and compound **6c**, which signifies the improved and well oral drug absorption and transdermal delivery efficiency of the studied compounds than gefitinib. The blood–brain barrier separates CNS from blood and a successful compound must pass through the blood stream [43] which depends on several factors such as molecular weight which must be below 480. Since our synthesized compounds have less molecular weight than the recommended values therefore its showed significant result. Madin–Darby canine kidney (MDCK) cell permeability is considered as the measurement of the blood–brain barrier permeability where greater than 500 is of grea<sup>t</sup> value and less than 25 indicates very poor result according to Jorgensen's rule of 3 [44]. Compounds **6b**, **6d**, **6e**, **6f** and **6j** gave much higher MDCK value than gefitinib while others showed around similar values except **6c**. The synthesized compounds also gave a predicted human oral absorption rate of 100%. Taken together, all the designed compounds of this study showed higher predicted ADME values than gefitinib which can be tested further by in vitro and in vivo experiments to establish successful drug candidates.
