Design, Synthesis, Anticancer Evaluation, Enzymatic Assays, and a Molecular Modeling Study of Novel Pyrazole–Indole Hybrids

The molecular hybridization concept has recently emerged as a powerful approach in drug discovery. A series of novel indole derivatives linked to the pyrazole moiety were designed and developed via a molecular hybridization protocol as antitumor agents. The target compounds (5a–j and 7a–e) were prepared by the reaction of 5-aminopyrazoles (1a–e) with N-substituted isatin (4a,b) and 1H-indole-3-carbaldehyde (6), respectively. All products were characterized via several analytical and spectroscopic techniques. Compounds (5a–j and 7a–e) were screened for their cytotoxicity activities in vitro against four human cancer types [human colorectal carcinoma (HCT-116), human breast adenocarcinoma (MCF-7), human liver carcinoma (HepG2), and human lung carcinoma (A549)] using the MTT assay. The obtained results showed that the newly synthesized compounds displayed good-to-excellent antitumor activity. For example, 5-((1H-indol-3-yl)methyleneamino)-N-phenyl-3-(phenylamino)-1H-pyrazole-4-carboxamide (7a) and 5-((1H-indol-3-yl)methyleneamino)-3-(phenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (7b) provided excellent anticancer inhibition performance against the HepG2 cancer cell line with IC50 values of 6.1 ± 1.9 and 7.9 ± 1.9 μM, respectively, compared to the standard reference drug, doxorubicin (IC50 = 24.7 ± 3.2 μM). The two powerful anticancer compounds (7a and 7b) were further subjected to cell cycle analysis and apoptosis investigation in HepG2 using flow cytometry. We have also studied the enzymatic assay of these two compounds against some enzymes, namely, caspase-3, Bcl-2, Bax, and CDK-2. Interestingly, the molecular docking study revealed that compounds 7a and 7b could well embed in the active pocket of the CDK-2 enzyme via different interactions. Overall, the prepared pyrazole–indole hybrids (7a and 7b) can be proposed as strong anticancer candidate drugs against various cancer cell lines.


INTRODUCTION
Cancer is one of the significant health problems and the second reason for deaths globally. Liver, breast, and lung are among the most common types of cancer diseases. Several ways have been discovered and reported for inhibiting cancer diseases, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, biological therapy, and photodynamic therapy. 1 More recently, targeted therapy has shown great potential in addressing drugs toward cancer cells of specific genes and proteins without attacking the healthy cells. It is well known that protein kinases play a vital role in regulating cell function. Therefore, these proteins can be used as a molecular target for designing new cancer inhibitors. For example, it was found that most human cancers are associated with the deregulation of cyclin-dependent kinases (CDKs). CDKs are a family of serine-threonine kinases that regulate cell cycle progression via the phosphorylation process. CDKs play an essential role in the inactivation of the retinoblastoma tumor suppressor gene (Rb) and the G2/M damage checkpoint. However, designing CDK selective inhibitors is still the main drawback because the ATPbinding site of the CDKs is highly protected across the enzyme. CDK-2 is an S/T-protein kinase required for the cell cycle G1/S transition. The inhibition of CDK-2 modulates siRNA and generates cell cycle arrest and apoptosis, leading to decreased proliferation of several cancer cells. This class of enzymes has attracted great attention for the designing and preparation of selective cancer inhibitors. Several inhibitorbased CDK-2s have been developed and progressed into clinical evaluation, such as roscovitine, dinaciclib, and milciclib. Therefore, there is a clear need to design and synthesize novel, selective, and less-toxic bioactive antitumor agents. 2−4 Recently, a molecular hybridization strategy based on incorporating two or more bioactive fragments into a single molecule has shown a simple, effective, and promising approach to discovering new drugs and could be beneficial for the treatment of cancer diseases. 5−7 In the last few decades, isatin (indoline-2,3-dione) derivatives have been widely used as a vital privileged scaffold in medical applications such as antitumor, antiviral, antimicro-bial, antituberculosis, and enzyme inhibitors. 8−12 1H-Benzo-[d]imidazol-2-ylimino-isatin ( Figure 1I) showed an excellent inhibition performance against the human FAAH enzyme. 13 Also, sulfonyl-isatin derivative afforded potent inhibitory activity against EGFR ( Figure 1II). 14 Figure 1 presents some of the novel potential antitumor and cytotoxic agent-based indole derivatives, such as semaxanib (III) and sunitinib (IV). 15,16 The pyrazole moiety displayed interesting biological activities for cancer treatment. 17−19 For example, the pyrazole compound ( Figure 1V) showed significant antitumor activity against the breast (MCF-7) and the liver (HepG2). 20 1,3-Dimethyl-1H-pyrazole derivative ( Figure 1VI) demonstrated low acute toxicity and a potent antitumor property against SMMC-7721 cell line in vivo. 21 Furthermore, pyrazole compounds play an essential role as potent enzyme inhibitors.

ACS Omega
http://pubs.acs.org/journal/acsodf Article For example, 1-phenyl-1H-pyrazole derivatives can be used as an inhibitor of α-glucosidase ( Figure 1VII). 22 Based on the aforementioned considerations and in continuation of our research program aimed to develop bioactive candidates, 23−44 we have designed and synthesized a series of novel pyrazole−indole hybrids (5a−j and 7a−e) and evaluated their anticancer activity in vitro against four human cancer cells [HCT-116, MCF-7, HepG2, and A549] using the MTT assay. Moreover, the two most potent target compounds (7a and 7b) have been selected to investigate their mechanism of action (cell cycle analysis and apoptosis investigation), enzymatic assays against caspase-3, Bcl-2, Bax, and CDK-2 kinase enzymes. Besides, we have studied the molecular modeling for both chemicals to understand the interactions with the active site of the proteins. The schematic diagram of the design strategy of the new anticancer agents is depicted in Figure 1. were prepared by the reaction of isatin with alkyl iodide in dimethylformamide (DMF) in the presence of K 2 CO 3 . 49 The target products, pyrazole−oxindole hybrids (5a−j), were prepared via the direct condensation of 5-aminopyrazoles 2a−e with N-substituted isatin 4a,b in refluxing EtOH in the presence of a catalytic amount of AcOH acid, as shown in Scheme 1.
2.2.2. Cell Cycle Analysis and Apoptosis Detection. Compounds 7a and 7b showed the best cytotoxic activities compared to the commercial cytotoxic reference compound, as well as other synthesized pyrazole derivatives. These results encouraged us to study the cellular mechanistic action of both compounds on the progression of the cell cycle and induction of apoptosis on the HepG2 cell line. The induction of apoptosis has been investigated using the annexin V/ propidium iodide (PI) staining assay for both compounds 7a and 7b on HepG2. It was found that compounds 7a and 7b induced more apoptotic cells (annexin V+/PI− and annexin V +/PI+), producing total necrosis and apoptosis (early and late) percentages of 22.18 and 27.51%, respectively, compared to the negative control dimethyl sulfoxide (DMSO) (1.49%), as presented in Figure 2.
To elucidate whether the cytotoxic activity is due to suppression of cell cycle progression, HepG2 cells were exposed to compounds 7a and 7b at concentrations of 7.9 and 6.1 μM, respectively, for 24 h and analyzed using flow cytometry. The obtained results revealed that compounds 7a and 7b induced significant accumulation of cells at the Pre G1 phase by 14.9-and 18.5-fold comparing to the control, showing a significant reduction in the percentage of cells at the G2/M phase by 2.4-and 29.4-fold, respectively. These compounds also provided a slight increase in S phases by 0.1-and 0.14-fold, respectively, compared to the reference control, as shown in Figure 3.
2.2.3. Enzymatic Assay. 2.2.3.1. Effect of Compounds 7a and 7b on the Levels of Caspase-3, Bcl-2, and Bax. It has been reported that caspases cascade through either intrinsic or extrinsic pathways that mediate the induction of apoptosis, which may lead to apoptotic cell death. 53−55 Caspase-3 is involved in cell shrinkage, chromatin condensation, and DNA fragmentation inside the cells, causing apoptosis induction. In this study, the bioluminescent intensities of caspase-3 for both compounds 7a and 7b indicated that caspase-3 activation has been measured in HepG2 cells, treated at concentrations of 7.9  Table 2, a significant increase in caspase-3 activities was detected for both compounds 7a and 7b compared to the negative control. They showed 7-and 5.8-fold higher activation, respectively. Furthermore, it is well known that the antiapoptotic Bcl-2 protein plays a critical role in cancer resistance therapy. 56 Therefore, we have studied the effect of both compounds 7a and 7b on Bcl-2 protein expression levels. It was found that 7a and 7b caused significant downregulation of the Bcl-2 protein level, as tabulated in Table 2. They provided a 0.5-and 0.4-fold decrease in the Bcl-2 concentration, respectively. These results agree with the cell cycle and apoptosis results, which indicated that both compounds could induce apoptosis by cell cycle arrest and/or by inhibition of Bcl-2.
In addition, the pro-apoptotic protein (Bax) is a protein that accelerates apoptosis by binding to and antagonizing the death repressor activity of Bcl-2. 57 Following any apoptotic stimuli, Bax causes activation of caspase-3 and perpetuates the apoptotic cascade. 58 The Bax protein expression level is altered in various human malignancies. 59,60 Therefore, the effect of both compounds 7a and 7b on the Bax expression level has been studied. The obtained results showed that both compounds 7a and 7b caused significant upregulation of the Bax protein level as they showed an 8.2-and 10.6-fold increase in the Bax concentration, respectively ( Table 2).  Overall, the above results may indicate that the stimulation of the apoptotic pathway by both compounds 7a and 7b further affects the upregulation of Bax protein, leading to stimulation of caspase-3 upregulation and Bcl-2 downregulation.
2.2.3.2. In Vitro CDK-2 Kinase Assessment. The promising antiproliferative impact of the conjugates 7a and 7b, besides their cell cycle disruption and pro-apoptotic effects, pushed for additional exploration for their inhibitory activities against the cell cycle regulator CDK-2 enzyme. Table 3 summarizes the inhibitory assessment (IC 50 ) of compounds 7a and 7b compared to the reference control roscovitine. The analyzed results showed that compounds 7a and 7b demonstrated superior inhibitory activity toward CDK-2 in comparison with roscovitine (IC 50 = 0.074 ± 0.15, 0.095 ± 0.10, and 0.100 ± 0.25 μM, respectively).
2.3. Molecular Docking Study. This molecular docking study aims to understand the possible binding modes of the potential anticancer compounds 7a and 7b with the key amino acids (hot spots) in the active site of the CDK-2 enzyme. This study was performed using Molecular Operating Environment (MOE) 2008. 10. The X-ray crystal structure of CDK-2 (PDB code: 2A4L) 61 was downloaded from the Protein Data Bank.
Validation of the docking protocol was first performed by redocking of the co-crystallized ligand roscovitine in the CDK-2 active site. The redocking validation step confirmed that the docking protocol used is suitable for the subsequent docking study. This is illustrated by the score energy of −11.25 kcal/ mol and the small root mean standard deviation (RMSD)  between the docked pose and the co-crystallized inhibitor pose of 0.72 Å and the highly observed superimposition between them ( Figure 4C). The benzyl moiety of the co-crystallized ligand (roscovitine) interacts with the active site of CDK-2 by arene−cation interaction with the essential amino acid Lys89. In addition, roscovitine formed many hydrophobic interactions with other amino acid residues, Ala31, Lys33, Phe80, Glu81, Leu83, His84, and Leu134, as shown in Figure 4A,B. Subsequently, the docking procedure for both compounds 7a and 7b was investigated, as shown in Figure 5. The corresponding 2D and 3D diagrams of the binding modes of both inhibitors with higher negative energy scores of −13.68 and −12.55 kcal/mol denote higher predicted binding affinity than that of the native ligand.
It was found that the docked derivatives 7a and 7b were fitted within the active site of the enzyme using the same crucial amino acid residue Lys89 via two arene−cation interactions with the centroids of indole and H-bonding with the N2 of the pyrazole moiety (distance: 2.92 and 2.97 Å, respectively). Upon investigation, it was also found that the N1 of pyrazole 7a supported the binding through another hydrogen bond donor with the side chain of Lys89 (distance: 2.91 Å).
Finally, we anticipated that the two compounds (7a and 7b), including indole and pyrazole moieties, could well embed in the active pocket of CDK-2 via different interactions with the key amino acid Lys89. This is confirmed by the superimposition phenomenon, as explained in Figure 6. Moreover, the achieved binding pattern explored the superior CDK-2 inhibitory activity of these compounds than the co-crystalized inhibitor (roscovitine).

CONCLUSIONS
In this study, we have designed and synthesized for the first time a series of novel pyrazole−indole hybrids via a molecular hybridization protocol as anticancer agents. The target compounds (5a−j and 7a−e) were screened against four types of human cancers [HCT-116, MCF-7, HepG2, and A549] using the MTT assay. The antiproliferative activity results showed that most synthesized compounds showed a moderate-to-excellent inhibition performance compared to the standard reference drug, doxorubicin. Interestingly, compounds 7a and 7b incorporating pyrazole−indole itself, and not the oxindole ring, displayed powerful inhibition against HepG2 and MCF-7 cancer cell lines. Moreover, these two compounds demonstrated significant inhibitory activity toward cyclin-dependent kinase 2 (CDK-2). Also, cell cycle experiments for compounds 7a,b revealed significant accumulation of cells at the Pre G1 phase, as well as a late apoptotic induction effect, as demonstrated from the annexin V FTIC study. These two compounds induced a significant increase in the caspase-3 activities, remarkable downregulation of the Bcl-2 protein level, and significant upregulation of the Bax protein level.
Finally, the obtained results were supported by a molecular docking study of these two compounds bearing indole and pyrazole moieties, which revealed that these two compounds could fit well and interact with the active pocket of CDK-2 via different interactions. Overall, the results indicate that both compounds 7a and 7b can be proposed as promising CDK-2 inhibitors and anticancer candidate drugs.  N-substituted isatin 4a,b (0.01 mol) {namely, 1-methylindoline-2,3-dione (4a) and 1-ethylindoline-2,3-dione (4b)} with a catalytic amount of glacial acetic acid (0.5 mL) in absolute ethanol (25 mL) was refluxed for 1 h and then left to cool. The solid product was filtered off, dried, and finally recrystallized from ethanol to afford target products 5a− j.    10.03 (s, 1H, NH), 12.30 (s, 2H, 2NH). 13 s, 2H, 2NH). 13 13 (HCT-116), and human lung carcinoma (A549) were purchased from the American Type Culture Collection (Rockville, MD). All cells were maintained in a Dulbecco's modified Eagle's medium (DMEM), which was supplemented with 10% of heatinactivated fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin each. The cells were grown at 37°C in a humidified atmosphere of 5% CO 2 .
4.2.1.2. MTT Cytotoxicity Assay. The cytotoxicity activities on the human liver carcinoma (HepG2), human breast adenocarcinoma (MCF-7), human colorectal carcinoma (HCT-116), and human lung carcinoma (A549) cell lines were estimated employing the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay, which was grounded on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in viable cells. 51,52 The cells were dispensed in a 96-well sterile microplate (3 × 10 4 cells/ well), followed by their incubation at 37°C with a series of different concentrations of 10 μL of each compound or doxorubicin (positive control, in DMSO) for 48 h in a serumfree medium prior to the MTT assay. Subsequently, the media were carefully removed, and 40 μL of MTT (2.5 mg/mL) was added to each well and then incubated for an additional 4 h. Purple formazan dye crystals were solubilized by the addition of 200 μL of DMSO. The absorbance was measured at 570 nm using a SpectraMax Paradigm Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells relative to the untreated control cells. All experiments were conducted in triplicate and were repeated on three different days. All of the values were represented as mean ± standard deviation (SD). The IC 50 s were determined by the SPSS probit analysis software program (SPSS Inc., Chicago, IL).

Cell Cycle Analysis and Apoptosis Detection.
Cell cycle analysis and apoptosis detection were carried out using flow cytometry. 62 Both HepG2 and MCF-7 cells were seeded at 8 × 10 4 and incubated at 37°C and 5% CO 2 overnight. After treatment with the tested compounds for 24 h, cell pellets were collected and centrifuged (300g, 5 min). For cell cycle analysis, the cell pellets were fixed with 70% ethanol on ice for 15 min and collected again. The collected pellets were incubated with propidium iodide (PI) staining solution (50 μg/mL PI, 0.1 mg/mL RNaseA, 0.05% Triton X-100) at room temperature for 1 h and analyzed using a Gallios flow cytometer (Beckman Coulter, Brea, CA). Apoptosis detection was performed using a FITC annexin V/PI commercial kit (Becton Dickenson, Franklin Lakes, NJ) following the manufacturer's protocol. The samples were analyzed using fluorescence-activated cell sorting (FACS) with a Gallios flow cytometer (Beckman Coulter, Brea, CA) within 1 h after staining. Data were analyzed using Kaluza v1.2 (Beckman Coulter). All monolayers of cells were treated separately for 48 h with DMSO or the IC 50  In Vitro CDK-2 Enzyme Inhibitory Assessment. Estimation of CDK-2 was performed using ELISA through an affinity tag labeled capture antibody and a reporter conjugated detector antibody, which immunocapture the sample analyte in solution. The addition of the standard and samples to the wells is carried out, followed by the addition of the antibody mix. After the incubation period is completed, the wells are washed, and the unrestrained substance is discarded. Then, TMB (3,3′,5,5′-tetramethylbenzidine) substrate is added, and prompted by horseradish peroxidase (HRP), blue coloration appeared. The reaction was stopped by the addition of a stop solution, completely changing the color from blue to yellow. Signals were created equivalently to the quantity of the bound analyte, and the intensity was recorded at a certain wavelength (450 nm) using a Robonik P2000 ELISA reader. The concentrations of the tested compounds were calculated from the plotted curve.
4.3. Molecular Docking Study. Molecular docking studies were carried out using Molecular Operating Environment (MOE, 10.2008) software. The X-ray crystal structure of CDK-2 (PDB code: 2A4L) 61 was complexed with roscovitine, which was retrieved from the RCSB Protein Data Bank. All structure minimizations were performed with MOE until an RMSD gradient of 0.05 kcal/(mol Å) with an MMFF94x force field and the partial charges were automatically calculated. The structure of the CDK-2 enzyme was prepared for molecular docking using Protonate 3D protocol in MOE with the default options. The Triangle Matcher placement method and the London dG scoring function were applied in the docking protocol. First, the validation process was confirmed by redocking the native ligand, followed by docking of the compounds 7a and 7b into the active site after removing the co-crystallized ligand following the reported procedure. 63 ■ ASSOCIATED CONTENT