Synthesis and biological evaluation of certain hydrazonoindolin-2-one derivatives as new potent anti-proliferative agents

Abstract In connection with our research program on the development of novel indolin-2-one-based anticancer candidates, herein we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. The synthesised derivatives were in vitro evaluated for their anti-proliferative activity towards lung A-549, colon HT-29, and breast ZR-75 human cancer cell lines. Compounds 5b, 5c, 7b, and 10e emerged as the most potent derivatives with average IC50 values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC50 = 8.11 µM). Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC50 = 16 µM. In addition, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).


Introduction
Cancer is a major health burden worldwide and it is deemed to be the second leading cause of mankind mortality after cardiovascular diseases. Most of the clinically available anticancer chemotherapeutic agents are not able to discriminate between cancer cells and the rapidly dividing healthy cells 1 . Moreover, the growing increase in drug resistance and undesired side effects of the clinically available cancer chemotherapeutic agents aroused the necessity to search for newer more potent and safer cancer chemotherapeutic candidates 2,3 . Subsequently, there is an urgent need to pay much attention to modify and update drug leads from the point of view of medicinal chemistry and drug design to fulfill more effective and potent anticancer agents.
Isatin (1H-indole-2,3-dione) is a privileged scaffold that is present endogenously in both human and other mammalian fluids and tissues, in addition to its presence in diverse naturally occurring components such as marine natural products and alkaloids 4 . Isatin constitutes a leading class of heterocycles that is endowed with interesting biological activities [4][5][6][7][8] , chiefly anticancer activity. Nintedanib (Ofev V R ) I (Figure 1), an orally available triple angiokinase inhibitor, is approved in Europe in combination with docetaxel for patients with advanced non-small cell lung cancer of adenocarcinoma who have progressed to first-line chemotherapy 9 . Currently, Nintedanib is being investigated in patients with various solid tumors including phase III studies in colorectal cancer 10,11 and phase I studies in breast cancer 12 . Sunitinib (Sutent V R ) II (Figure 1), a 5-fluoro-3-substituted isatin derivative, has been approved by the FDA for the treatment of gastrointestinal stromal tumors (GIST) and advanced renal cell carcinoma (RCC) 13 . It is a multikinase inhibitor that targets VEGFR-1, VEGFR-2, PDGFRb, and c-Kit.
Based on the aforementioned findings and in connection with our research program on the development of indolin-2-one-based anticancer candidates [14][15][16][17][18][19][20][21][22] , it was thought worthwhile to extend our investigations to probe certain hydrazonoindolin-2-ones displaying promising anti-proliferative activity. In this study, we reported the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l, adopting three distinctive strategies to develop such derivatives ( Figure 2). The first one focused on grafting of large lipophilic ester or acetyl groups on the pendant phenyl ring of lead structure III to furnish target compounds 3a-e ( Figure 2). In the second strategy, a bioisosteric tactic was utilised to replace the pendant substituted phenyl ring in compounds 3a-e with 2-thiazolyl ring, as in compounds 5a-e, or 5-pyrazolyl ring, as in compounds 7a-c (Figure 2), to carry out further elaboration of the hydrazonoindolin-2-ones scaffold and to investigate a valuable SAR. Finally, the hybridisation approach was adopted to conjugate 4-arylthiazoles with indolin-2-one moiety, compounds 10a-l ( Figure 2).
The synthesised hydrazonoindolin-2-ones (3a-e, 5a-e, 7a-c, and 10a-l) were in vitro evaluated for their anti-proliferative activity towards three human cancer cell lines, namely lung cancer A-549, human colon cancer HT-29, and breast cancer ZR-75 cell lines. Furthermore, the most active anti-proliferative congeners were further evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cells. In addition, their effect on multidrug-resistant lung cancer NCI-H69AR cell line was assessed. Finally, the cytotoxic activities of the active members were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss 3t3) to estimate their selectivity for tumor cells.

Chemistry
Melting points were measured with a Stuart melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and were uncorrected. Mass spectra were recorded using Agilent Quadrupole 6120 LC/MS with ESI (Electrospray ionisation) source (Agilent Technologies, Palo Alto, CA). NMR spectra were recorded on a Bruker NMR spectrometer (Bruker, Karlsruhe, Germany). 1 H spectra were run at 500 MHz and 13 C spectra were run at 125 MHz in deuterated dimethyl sulfoxide (DMSO-d 6 ). Chemical shifts are expressed in values d (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in Hertz. The abbreviations used are: "s" for singlet, "d" for doublet, and "m" for multiplet. Elemental analyses were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques.
General procedure for the synthesis of hydrazones 3a-e, 5a-e, and 7a-c A mixture of equimolar quantities of previously reported hydrazones 1a-c 19 (2 mmol) and aldehydes 2a,b, 4a,b, or 6 (2 mmol) in methyl alcohol (30 ml) containing catalytic amount of glacial acetic acid was refluxed for 4 h, and then cooled to room temperature. The precipitates formed were collected by filtration, dried and crystallised from EtOH/DMF to afford compounds 3a-e, 5a-e, and 7a-c, respectively.       General procedure for the synthesis of thiazoles 10a-l A mixture of previously reported thiosemicarbazones 8a-c 23 (2 mmol) and bromoethanones (9a-d) (2 mmol) in absolute ethanol (20 ml) was heated under reflux for 3 h, then left to cool. The precipitated solid was collected by filtration, washed with ethanol, dried and recrystallised from EtOH/DMF to yield the corresponding thiazole derivatives 10a-l, respectively. Compounds 10a-e and 10g-l 24-29 .

Biological evaluation
In vitro anti-proliferative activity. The anti-proliferative activity of the title compounds 3a-e, 5a-e, 7a-c, and 10a-l towards cancer cell lines, normal cell lines, and multidrug-resistant lung cancer NCI-H69AR cell line was determined using CellTiter-Glo TM Luminescent cell viability assay according to the previously published procedures 14,15 .
Cell cycle effects. Cell cycle effects of the synthesised compounds 7b and 10e were determined adopting the reported procedures 14,15 .
The assigned structures of compounds 3a-e, 5a-e, and 7a-c were confirmed by IR, 1   of the indolin-2-one moiety, in addition to the absorption bands of (C¼O) groups in the region of 1660-1751 cm À1 . 1 H NMR spectra of compounds 3a-e, 5a-e, and 7a-c revealed the presence of a singlet signal in the region d 10.93-11.11 corresponding to NH proton of isatin moiety, in addition to the singlet signal of the methine (-CH¼N-) proton in the region d 8.55-8.82. Also, compounds 7a-c displayed additional signal attributable to the NH proton of the pyrazole ring at d 13.17-13.62. Moreover, the 13 C NMR spectra of these compounds displayed signals resonating around d 164 corresponding to the amidic (C¼O) carbons.
On the other hand, the condensation of indoline-2,3-diones with thiosemicarbazide in refluxing ethanol containing a catalytic amount of acetic acid yielded the corresponding thiosemicarbazones 8a-c. The second targeted isatin-based derivatives 10a-l were obtained in good yields (67-90%) through the reaction of the key intermediates 8a-c with the appropriate phenacyl bromide 9a-d in refluxing absolute ethyl alcohol (Scheme 2).
IR spectra of the compounds 10a-l displayed the characteristic absorption bands in the regions 3136-3315 cm À1 and 1685-1707 cm À1 corresponding to the (NH) and (C¼O) groups, respectively, while their 1 H NMR spectra were characterised by the presence of three singlets in the regions d 11.24-11.38, 13.32-13.36, and 7.58-7.75 which were assigned to two NH groups and H-5 of the thiazole ring, respectively. Besides, 13 C NMR spectra of these compounds showed characteristic signals resonating around d 166 which was attributed to (C¼O) carbon. It is worth noting that some derivatives are existing in E/Z geometrical isomerism in the DMSOsolution state, such as compounds 5e, 10f, and 10j 17 .

Biological evaluation
In vitro anti-proliferative activity The cancer cell growth inhibitory activity of the synthesised 25 compounds 3a-e, 5a-e, 7a-c, and 10a-l was evaluated in vitro towards three human cancer cell lines, namely lung cancer A-549, human colon cancer HT-29, and breast cancer ZR-75 cell lines using CellTiter-Glo TM Luminescent cell viability assay 30 . Sunitinib was included in this assay as a reference drug.
The prepared hydrazonoindolin-2-ones were first evaluated at preliminary anti-proliferative assay against the three cancer cell lines in quadruplicate at a single concentration of 30 mM. The data presented as percentage growth inhibition (GI%) caused by the test compounds (Table 1).
The tested hydrazonoindolin-2-ones displayed different levels of anti-proliferative activity and possessed a distinctive pattern of selectivity against the tested cancer cell lines with average growth inhibition in the range of 9.0-97.7%. Close examination of the GI% values in Table 1, revealed that compounds 3b, 3c, 5b-e, 7b, 10e, 10i, and 10j are the most active analogs in this study, showing broad spectrum activity toward the tested cell lines with average GI% values >80%. On the other hand, compounds 3a, 5a, 7c, 10d, 10h, and 10l displayed modest growth inhibitory activity (GI% values <35%). It is noteworthy to mention that compounds 3e and 10l stimulated the growth of the A-549 cell line and compound 10h stimulated the growth of both A-549 and HT-29 cell lines.
The quantitative inhibitory concentration 50% (IC 50 ) values were evaluated for the active members that displayed effective growth inhibition toward the three cancer cell lines (GI% values >35%) in the preliminary single high dose (30 mM) screening (Table 2). From the displayed results in Table 2, it was obvious that several of the synthesised hydrazonoindolin-2-ones possessed excellent to moderate growth inhibitory activity against the tested cancer cell lines.
Compound 7b was the most active congener in the synthesised Schiff bases 3a-e, 5a-e, and 7a-c with an average IC 50 value of 2.14 mM toward the tested human cancer cell lines being nearly four-fold more potent than the positive control, Sunitinib (average IC 50 value ¼8.11 mM). Whereas, compound 10e was the most active candidate in the arylthizole derivatives 10a-l with an apparent average IC 50 value of 4.66 mM against the tested cell lines.

Structure activity relationship
Observing the results, we could deduce valuable data about the structure activity relationship (SAR). Firstly, investigation of the impact of the substitution on the pendant phenyl group in compounds 3a-e revealed that grafting an ester group (compound 3b; average IC 50 ¼ 14.01 mM) is more beneficial for activity than acetyl group (compound 3a; average IC 50 > 30 mM). The effect of bioisosteric replacement of the pendant phenyl ring in compounds 3a-e with 2-thiazolyl ring, compounds 5a-e, or 5-pyrazolyl ring, compounds 7a-c was explored. Both 2-thiazolyl moiety (compound 5c; average IC 50 ¼ 2.53 mM) and 5-pyrazolyl moiety (compound 7b; average IC 50 ¼ 2.14 mM) resulted in remarkable increase in the activity in comparison to compounds 3a-e (compound 3c; average IC 50 ¼ 6.25 mM). Also, substitution of the 2-thiazolyl ring in compounds 5a-e with 4-methyl group resulted in an increase of the anti-proliferative activity (compound 5b; average IC 50 ¼ 4.37 mM vs. compound 5c; average IC 50 ¼ 2.53 mM).
Regarding phenylthiazolyl series, compounds 10a-l, the impact of the substitution on the 4-phenyl group was examined. The decreased average IC 50 values of compounds 10a, 10e, and 10i (11.07, 4.66 and 5.45 mM, respectively) which incorporated an unsubstituted phenyl moiety than that of their corresponding members 10b, 10f, and 10j (>28.19, >25.38, and 12.99 mM, respectively), with 4-methyl substituted phenyl moiety, and their corresponding members 10c, 10g, and 10k (>17.36, >12.65, and 14.49 mM, respectively), with 4-chloro substituted phenyl moiety, indicated that unsubstitution of the phenyl moiety is advantageous. On the other hand, substitution with 4-bromo substituent (compounds 10d, 10h, and 10l) diminished the activity. Finally, we explored the effect of the substitution of the indolin-2-one moiety. Interestingly, grafting 5-chloro substituent was found to be more beneficial for the activity rather than unsubstituted or 5bromo substituted indolin-2-one moieties throughout the study.
In conclusion, we can deduce that replacing the phenyl moiety of compounds 3a-e with 2-thiazolyl ring, 5-pyrazolyl ring, or its substitution with alipophilic ester group greatly enhances the antiproliferative activity. In addition, hybridisation of the indolin-2-one moiety with 4-phenylthiazolylmoiety, compounds 10a-l seems to be a prosperous approach.

Cell cycle effects
The cell cycle consists of a number of complex biochemical pathways and controlled by numerous mechanisms to ensure correct cell division. An important role is played by cyclin/cyclin-dependent kinase (CDK) complexes, which are critical regulators of cell cycle progression and RNA transcription. Deregulation of the cell cycle underlies the aberrant cell proliferation which characterises loss of cell cycle checkpoint control and cancer 31,32 . Retinoblastoma protein (pRb), a cell cycle regulatory molecule, plays a critical role in control of the G1-to-S phase checkpoint of the cell cycle. pRb suppresses the activity of E2F transcription factors thereby inhibiting transcription of cell cycle promoting genes, in its hypophosphorylated state. Upon phosphorylation, mainly via CDKs, phosphorylated pRb dissociates from E2F and permits cell cycle progression 33,34 . In the current medical era, therapeutic strategies directed against critical cell cycle regulators have stood out as an attractive approach for discovering therapies for many human malignancies. In this study, cancer cells derived from lung adenocarcinoma (A-549) were used to evaluate the effects of compounds 7b and 10e on various aspects of the cell cycle progression. Both immunofluorescent imaging of phosphorylated Rb protein and total DNA content of each cell were explored to determine the activity of compounds 7b and 10e over a range of concentrations less than 100 nM to 50 mM (Figures 3-5).
Compound 7b caused a dose-dependent reduction in the total cell number after 48 h of treatment with IC 50 value ¼8.08 mM, while compound 10e caused a dose-dependent reduction in the total cell number after 24 h of treatment with IC 50 value ¼7.38 mM (Table 3).
Moreover, compound 7b caused a decrease in the percentage of cells in the G1 phase of the cell cycle with corresponding increase in the G2/M-phases. Arrest in G2 may represent a checkpoint blockade whereas mitotic arrest may, in some cases, lead to mitotic catastrophe and subsequent programed death of cells with multiple or aberrant nuclei. In contrast, compound 10e caused an increase in the percentage of cells in the G1 phase of the cell cycle with corresponding decreases in S-and G2/M phases. This suggests that part of the compound effects on growth may be attributable to a decreased rate of progression through the cell cycle and corresponding decreases in proliferation.
However, levels of phosphorylated Rb protein were significantly reduced in a dose-dependent manner by the control and the test compounds 7b and 10e (Figure 4). The IC 50 values were consistent with the IC 50 values for reductions in cell number caused by each compound after 24 h of treatment while the correlation is less apparent after 48 h of treatment (Table 3). This may support the hypothesis that inhibition of cyclin-dependent kinases by isatin compounds plays a vital role in their growth inhibitory activity.

Selectivity
The cell growth inhibitory activity of compounds 7b and 10e was examined towards three non-tumorigenic cell lines to estimate their selectivity for tumor cells. A-549 human non-small cell lung cancer (NSCLC) cell line was included in the assay for comparison. Cultures derived from human fibrocystic mammary tissue (MCF-10A) are non-tumorigenic and exhibit features of primary cultures of breast tissue including dome formation 35 . IEC-6 cells derived from rat intestine show morphologic and karyotypic features of normal intestinal epithelial cells 36 . Fibroblasts derived from embryonic tissue from mice (Swiss-3t3) are both non-tumorigenic and contact inhibited 37 . Compounds 7b and 10e were tested quadruplicate at maximum concentrations of 25 mM, with subsequent 10 serially diluted concentrations.
The results were displayed in Figure 6, while the IC 50 values and mean selectivity index were listed in Table 4. Both compounds 7b and 10e displayed mean tumor selectivity index (1.6 and 1.8, respectively) higher than that of Sunitinib (1.4).

Multidrug resistant lung cancer cell line (NCI-H69AR)
The anti-proliferative activity of compounds 7b and 10e was evaluated towards multidrug resistant lung cancer cell line (NCI-H69AR), which expresses the ABCC1 efflux pump protein. Table 5 and Figure 7 revealed that both 7b and 10e displayed growth inhibitory activity towards NCI-H69AR cells with IC 50 values of 16.0 mM. Accordingly, the NCI-H69AR cells were about four-fold less sensitive than A-549 cells, suggesting that these compounds might be subjected to efflux by ABCC1. Furthermore, the H69AR cells manifested a lesser degree of resistance to the reference drug, Sunitinib (1.9-fold less sensitive).

Conclusion
In summary, herein, we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. All the prepared hydrazonoindolin-2-ones were examined for their   anti-proliferative activity towards three cell lines, namely lung A-549, colon r HT-29, and breast ZR-75 human cancer cell lines using CellTiter-Glo TM Luminescent cell viability assay. Compounds 5b, 5c, 7b, and 10e were the most potent members with average IC 50 values of 4.37, 2.53, 2.14, and 4.66 mM, respectively, which are superior to Sunitinib (average IC 50 ¼ 8.11 mM). The SAR study suggested that replacing the phenyl moiety of compounds 3a-e with 2-thiazolyl ring, 5-pyrazolyl ring, or its substitution with a lipophilic ester group greatly enhances their anti-proliferative activity. In addition, hybridisation of the indolin-2-one moiety with 4-phenylthiazolyl moiety, compounds 10a-l, seems to be a prosperous approach. Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 human cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC 50 ¼ 16 mM. Finally, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).