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. 2021 Apr 29;6(18):12361–12374. doi: 10.1021/acsomega.1c01604

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

Ashraf S Hassan †, Gaber O Moustafa ‡, Hanem M Awad §, Eman S Nossier ∥, Mohamed F Mady ⊥,#,*
PMCID: PMC8154124  PMID: 34056388

Abstract

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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.

1. 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 ATP-binding 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 inhibitor-based 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, antimicrobial, 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).14Figure 1 presents some of the novel potential antitumor and cytotoxic agent-based indole derivatives, such as semaxanib (III) and sunitinib (IV).15,16

Figure 1.

Figure 1

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Schematic representation of the designed bioactive scaffold containing indole and pyrazole moieties (5a–j and 7a–e).

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. 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.

2. Results and Discussion

2.1. Chemistry

The synthetic pathways of new pyrazole–indole hybrids (5a–j and 7a–e) are outlined in Schemes 1 and 2. The starting materials, 5-aminopyrazoles 2a–e, were prepared by the reaction of N-aryl-3-(arylamino)-2-cyano-3-(methylthio)acrylamide derivative 1a–e with hydrazine hydrate in refluxed ethanol in the presence of a catalytic amount of triethylamine.45−48 Also, N-substituted isatin 4a,b were prepared by the reaction of isatin with alkyl iodide in dimethylformamide (DMF) in the presence of K2CO3.49 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.

Scheme 1. Schematic Representation of the Synthesis of Pyrazole–Oxindole Hybrids 5a–j.

Scheme 1

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Scheme 2. Synthesis of Pyrazole–Indole Hybrids 7a–e.

Scheme 2

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The structure of pyrazole–oxindole hybrids 5a–j was confirmed based on their spectral data. The 1H NMR spectrum of 5-(1-ethyl-2-oxoindolin-3-ylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (5i) revealed one triplet at δ 1.24 (3H, J = 7.2 and 7.1 Hz), one singlet signal at 3.76 (s, 3H), and one quartet at 3.85 (q, 2H, J = 7.2 Hz) attributed to methyl (−NCH2CH3), methoxy (−OCH3), and methylene (−NCH2CH3) protons, respectively. The three protons of 3NH appear as three signals at δ 8.81, 11.15, and 13.18 ppm. Furthermore, the nine protons of the two aromatic rings, phenyl (5H) and 4-methoxyphenyl (4H), appear as one doublet at 6.96 (2H, J = 8.8 Hz), one triplet at 7.06 (1H, J = 7.4 and 7.4 Hz), one doublet at 7.29 (2H, J = 8.6 Hz), one triplet at 7.36 (2H, J = 8.0 and 7.7 Hz), and one doublet at 7.95 (2H, J = 7.9 Hz), while the four protons of the isatin ring appear as one triplet at 7.12 (1H, J = 7.7 and 7.6 Hz), one doublet at 7.21 (1H, J = 7.8 Hz), one triplet at 7.58 (1H, J = 7.7 and 7.8 Hz), and one doublet at 9.15 (1H, J = 7.6 Hz). The 13C NMR spectrum of 5i afforded five characteristic signals at δ 12.52, 34.46, 55.32, 162.28, and 163.52 ppm for the methyl, −NCH2, OCH3, C=O, and C=O carbon atoms, respectively.

Finally, 1H-indole-3-carbaldehyde (6) was refluxed with 5-aminopyrazoles 2a–e in ethanol as a solvent and a catalytic amount of AcOH acid, giving pyrazole–indole hybrids 7a–e, as shown in Scheme 2.

The structures of the target products 7a–e were confirmed by spectroscopic techniques. For example, the 1H NMR spectrum of 5-((1H-indol-3-yl)methyleneamino)-3-(4-methoxyphenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (7e) showed two signals at 2.26 and 3.72 for the protons of methyl (s, 3H, CH3) and methoxy (s, 3H, OCH3) groups, respectively. Three signals at 9.05 (1NH), 9.96 (1NH), and 12.26 (2NH) are assigned to the four NH protons. Two typical signals were observed at 8.31 and 8.78 for the protons of indole (1H) and the azomethine function (1H, −N=CH−), respectively. The protons of para-substitution phenyl rings (8H) appeared as four doublets at 6.89 (2H), 7.13 (2H), 7.48 (2H), and 7.53 (2H) with the coupling contents of 9.0 Hz, 8.3 Hz, 8.5 Hz, and 8.4 Hz, respectively. The four protons of the indole ring appeared as two triplets and two doublets. The two triplets were observed at 7.25 (1H, J = 7.8 and 7.9 Hz) and 7.34 (1H, J = 8.2 and 8.1 Hz), and the two doublets were found at 7.59 (1H, J = 8.1 Hz) and 8.34 (1H, J = 7.7 Hz).

Furthermore, the 13C NMR spectrum of the pyrazole–indole hybrid 7e showed distinct signals of CH3 and OCH3 at 20.37 and 55.22 ppm, respectively. In addition, 13C NMR displayed a distinct singlet signal at 163.12 ppm corresponding to the C=O group.

2.2. Biological Evaluation

2.2.1. In Vitro Anticancer Screening

Fifteen compounds (5a–j and 7a–e) were examined in vitro for their activities on HCT-116, MCF-7, HepG2, and A549 human cancer cells using the MTT assay.50−52

The percentages of intact cells were calculated and compared to those of the control. Activities of these compounds against the four cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the four human cells in a dose-dependent manner (Table 1). To study the efficacy of the synthesized compounds (5a–j and 7a–e), a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follows.

  • In the case of HCT-116 human colorectal carcinoma cells, 11 compounds, 5a, 5b, 5f, 5g, 5h, 5j, 7a, 7b, 7c, 7d, and 7e, with a range from IC50 = 17.4 ± 3.2 to 38.9 ± 4.1 μM, showed significantly more potent anticancer activities. Two compounds 5c (IC50 = 39.5 ± 4.5 μM) and 5e (IC50 = 39.9 ± 4.2 μM) showed an equipotent anticancer effect compared to doxorubicin. In addition, two compounds 5d (IC50 = 54.2 ± 5.5 μM) and 5i (IC50 = 53.9 ± 5.7 μM) afforded weak anticancer activities in comparison with doxorubicin (IC50 = 40.0 ± 3.9 μM).

  • In the case of MCF-7 human breast cancer cells, all of the synthesized compounds (5a–j and 7a–e) afforded excellent anticancer activities with a range from IC50 = 10.6 ± 2.3 to 63.7 ± 5.5 μM compared to doxorubicin (IC50 = 64.8 ± 4.1 μM).

  • In the case of HepG2 human liver cancer cells, three compounds 5f (IC50 = 23.7 ± 3.1 μM), 7a (IC50 = 6.1 ± 1.9 μM), and 7b (IC50 = 7.9 ± 1.9 μM) displayed potential inhibition performance compared to the cytotoxic reference compound [doxorubicin (IC50 = 24.7 ± 3.2 μM)]. Compound 5j (IC50 = 24.4 ± 2.9 μM) showed equipotent activity. It was also found that the remainder of the tested compounds (5a–e, 5h, 5i, 7c, 7d, and 7e) showed poor inhibition performance compared to doxorubicin (IC50 = 24.7 ± 3.2 μM).

  • In the case of A549 human lung cancer cells, 13 compounds (5a–d, 5f–5j, 7a–7c, and 7e) provided powerful anticancer activities. Compound 7d (IC50 = 58.7 ± 4.2 μM) had equipotent activity. On the contrary, compound 5e (IC50 = 77.7 ± 5.6 μM) exhibited low anticancer activity compared to doxorubicin (IC50 = 58.1 ± 4.1 μM).

Table 1. IC50 (μM) of the 15 Compounds (5a–j and 7a–e) against the Four Cancer Cell Lines Using the MTT Assay.

2.2.1.

        IC50 (μM) ± SD
compounds Ar Ar1 R HCT-116 MCF-7 HepG2 A549
5a C6H5 C6H5 CH3 25.7 ± 3.5 28.3 ± 3.5 27.6 ± 3.1 42.8 ± 4.2
5b 4-CH3-C6H4 C6H5 CH3 28.1 ± 3.5 48.0 ± 5.1 37.1 ± 4.3 46.7 ± 4.9
5c H 4-CH3O-C6H4 CH3 39.5 ± 4.5 22.0 ± 3.5 32.8 ± 3.8 40.7 ± 4.1
5d C6H5 4-CH3O-C6H4 CH3 54.2 ± 5.5 46.4 ± 4.5 34.8 ± 3.9 56.0 ± 4.1
5e 4-CH3-C6H4 4-CH3O-C6H4 CH3 39.9 ± 4.2 61.8 ± 5.1 32.0 ± 3.9 77.7 ± 5.6
5f C6H5 C6H5 C2H5 28.9 ± 3.9 42.1 ± 4.7 23.7 ± 3.1 52.9 ± 4.5
5g 4-CH3-C6H4 C6H5 C2H5 35.7 ± 3.9 54.0 ± 4.9 27.8 ± 3.5 46.8 ± 5.3
5h H 4-CH3O-C6H4 C2H5 25.7 ± 4.3 25.4 ± 3.9 28.2 ± 3.5 41.9 ± 3.9
5i C6H5 4-CH3O-C6H4 C2H5 53.9 ± 5.7 30.1 ± 4.2 26.7 ± 3.1 47.8 ± 4.5
5j 4-CH3-C6H4 4-CH3O-C6H4 C2H5 38.9 ± 4.1 63.7 ± 5.5 24.4 ± 2.9 57.0 ± 4.9
7aa C6H5 C6H5   17.4 ± 3.2 10.6 ± 2.3 6.1 ± 1.9 23.7 ± 3.1
7ba 4-CH3-C6H4 C6H5   19.6 ± 3.5 14.5 ± 2.5 7.9 ± 1.9 14.1 ± 2.1
7c H 4-CH3O-C6H4   31.9 ± 3.8 22.2 ± 3.3 35.8 ± 3.9 43.4 ± 4.2
7d C6H5 4-CH3O-C6H4   25.3 ± 3.5 17.4 ± 2.3 27.2 ± 3.5 58.7 ± 4.2
7e 4-CH3-C6H4 4-CH3O-C6H4   37.4 ± 4.1 16.2 ± 2.3 25.8 ± 3.5 40.8 ± 4.3
doxorubicin       40.0 ± 3.9 64.8 ± 4.1 24.7 ± 3.2 58.1 ± 4.1
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a

The most potent compound as new anticancer agents.

We can conclude from the above results that both compounds (7a and 7b) showed an excellent cancer inhibition performance. They could be proposed as candidate drugs for human colon cancer, breast cancer, liver cancer, and lung cancer types.

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.

Figure 2.

Figure 2

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Flow cytometry apoptotic status on HepG2 cancer cells for the negative control, DMSO, and compounds (7a,b), respectively.

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.

Figure 3.

Figure 3

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Flow cytometry cancer cell cycle distribution on HepG2 cancer cells for the negative control, DMSO, and compounds (7a,b), respectively.

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 and 6.1 μM, respectively, for 24 h. As shown in 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.

Table 2. Results of Caspase-3/Bax/BCL-2 Analysis after Treatment of Cells with Two Compounds 7a and 7b.
compounds caspase-3 (Pg/mL) Bcl-2 (ng/mL) Bax (Pg/mL)
7a 388.7 ± 7 3.123 ± 0.1 211.3 ± 5.9
7b 469.8 ± 10 2.479 ± 0.07 272.6 ± 11.5
cont. HepG2 67.3 ± 2.8 6.222 ± 0.14 5.762 ± 1.18
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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 (IC50) 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 (IC50 = 0.074 ± 0.15, 0.095 ± 0.10, and 0.100 ± 0.25 μM, respectively).

Table 3. Inhibitory Assessment (IC50 in μM) of Compounds 7a and 7b on CDK-2 Enzyme Performed Using Enzyme-Linked Immunosorbent Assay (ELISA).
compounds CDK-2 (IC50, μM)
7a 0.074 ± 0.15
7b 0.095 ± 0.10
roscovitine 0.100 ± 0.25
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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.

Figure 4.

Figure 4

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(A, B) Two-dimensional (2D) and three-dimensional (3D) images of the native ligand (roscovitine) redocked in the ATP active site of CDK-2 (PDB ID: 2A4L) using MOE software. (C) 3D image of the superimposition of the docking pose (yellow) and the co-crystallized inhibitor pose (red) of roscovitine with an RMSD of 0.72 Å.

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.

Figure 5.

Figure 5

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(A, B) Two-dimensional (2D) and three-dimensional (3D) interaction diagrams of docked compound 7a with CDK-2 (PDB code: 2A4L). (C, D) 2D and 3D interaction diagrams of compound 7b with CDK-2 (PDB code: 2A4L). Hydrogen bonds are illustrated as arrows. Carbon atoms are labeled in gray, nitrogen atoms in blue, and oxygen atoms in red.

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).

Figure 6.

Figure 6

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3D representation of docked roscovitine (red) in superimposition with compounds 7a (yellow) and 7b (blue) in the active site of CDK-2 (PDB code: 2A4L).

3. 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.

4. Experimental Section

4.1. Chemistry

All melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. The IR spectra were recorded (KBr disk) on a Perkin Elmer 1650 FT-IR instrument. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Varian spectrometer using DMSO-d6 as a solvent and TMS as an internal standard. Chemical shifts are reported in ppm. Mass spectra were recorded on a Varian MAT 112 spectrometer at 70 eV. Elemental analyses were performed at the Microanalytical Center, Cairo University, Egypt.

The progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F254 (Merck, Darmstadt, Germany), with detection under ultraviolet light in the range of 254-360 nm. All evaporations were carried out under reduced pressure at 40 °C.

4.1.1. General Procedure for the Preparation of Pyrazole–Oxindole Hybrids (5a–j)

A mixture of compounds 2a–e (0.01 mol) and 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.

4.1.1.1. 5-(1-Methyl-2-oxoindolin-3-ylideneamino)-N-phenyl-3-(phenylamino)-1H-pyrazole-4-carboxamide (5a)

Black crystals, mp 270–272 °C, yield (74%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.25 (s, 3H, NCH3), 6.85 (t, 1H, J = 7.0 and 6.8 Hz, ArH), 7.04–7.15 (m, 4H, ArH), 7.34–7.38 (m, 4H, ArH), 7.58 (d, 2H, J = 7.7 and 7.7 Hz, ArH), 7.93 (d, 2H, J = 6.4 Hz, ArH), 8.99 (s, 1H, NH), 9.11 (d, 1H, J = 7.3 Hz, ArH), 11.16 (s, 1H, NH), 13.43 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 26.30 (C, NCH3), 95.28, 109.75, 116.55, 118.31, 118.56, 122.41, 122.97, 128.89, 129.01, 129.50, 135.77, 138.13, 139.10, 147.94, 148.99, 158.86, 159.01, 162.14 (22C), 162.29, 163.81 (2C, 2C=O). Anal. calcd. (%) for C25H20N6O2 (436.47): C, 68.80; H, 4.62; N, 19.25. Found: C, 68.92; H, 4.55; N, 19.30%.

4.1.1.2. 5-(1-Methyl-2-oxoindolin-3-ylideneamino)-3-(phenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (5b)

Brown crystals, mp > 300 °C, yield (70%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.27 (s, 3H, CH3), 3.25 (s, 3H, NCH3), 7.05 (t, 1H, J = 6.8 and 6 Hz, ArH), 7.10–7.16 (m, 4H, ArH), 7.30–7.38 (m, 4H, ArH), 7.59 (t, 1H, J = 7.6 and 6.5 Hz, ArH), 7.82 (d, 2H, J = 6.0 Hz, ArH), 9.06 (s, 1H, NH), 9.11 (d, 1H, J = 7.2 Hz, ArH), 11.07 (s, 1H, NH), 13.44 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 20.47 (C, CH3), 26.30 (C, NCH3), 96.52, 109.73, 118.29, 118.50, 119.53, 122.41, 129.27, 129.39, 129.51, 131.86, 135.75, 136.62, 139.53, 147.16, 148.99, 153.11, 153.51 (22C), 161.96, 162.75 (2C, 2C=O). Anal. calcd. (%) for C26H22N6O2 (450.49): C, 69.32; H, 4.92; N, 18.66. Found: C, 69.25; H, 5.00; N, 18.60%.

4.1.1.3. 3-(4-Methoxyphenylamino)-5-(1-methyl-2-oxoindolin-3-ylideneamino)-1H-pyrazole-4-carboxamide (5c)

Dark brown crystals, mp 265–267 °C, yield (71%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.24 (s, 3H, NCH3), 3.74 (s, 3H, OCH3), 6.87 (d, 2H, J = 8.9 Hz, ArH), 7.06 (t, 1H, J = 7.6 and 7.5 Hz, ArH), 7.13 (d, 2H, J = 7.9 Hz, ArH), 7.26, 7.38 (2s, 2H, NH2), 7.48 (t, 1H, J = 7.7 and 7.9 Hz, ArH), 7.57 (d, 1H, J = 7.8 Hz, ArH), 8.90 (s, 1H, NH), 8.98 (d, 1H, J = 7.7 Hz, ArH), 12.96 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 26.19 (C, NCH3), 55.29 (C, OCH3), 94.92, 110.05, 114.26, 114.67, 121.23, 122.38, 123.44, 134.92, 141.56, 146.25, 148.65, 152.97, 154.93, 159.42 (16C), 163.24, 163.36 (2C, 2C=O). Anal. calcd. (%) for C20H18N6O3 (390.40): C, 61.53; H, 4.65; N, 21.53. Found: C, 61.60; H, 4.58; N, 21.45%.

4.1.1.4. 3-(4-Methoxyphenylamino)-5-(1-methyl-2-oxoindolin-3-ylideneamino)-N-phenyl-1H-pyrazole-4-carboxamide (5d)

Dark green crystals, mp 271–273 °C, yield (74%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.24 (s, 3H, NCH3), 3.76 (s, 3H, OCH3), 6.96 (d, 2H, J = 8.8 Hz, ArH), 7.05 (t, 1H, J = 7.3 and 8.8 Hz, ArH), 7.11 (t, 1H, J = 6.6 and 7.0 Hz, ArH), 7.13 (d, 1H, J = 7.1 Hz, ArH), 7.28 (d, 2H, J = 8.6 Hz, ArH), 7.35 (t, 2H, J = 8.2 and 7.9 Hz, ArH), 7.56 (t, 1H, J = 7.8 and 7.7 Hz, ArH), 7.94 (d, 2H, J = 7.8 Hz, ArH), 8.80 (s, 1H, NH), 9.10 (d, 1H, J = 7.5 Hz, ArH), 11.12 (s, 1H, NH), 13.15 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 26.27 (C, NCH3), 55.31 (C, OCH3), 94.17, 109.65, 114.74, 116.60, 118.44, 121.80, 122.34, 122.81, 128.87, 129.34, 132.05, 135.62, 139.27, 148.56, 148.91, 149.45, 149.57, 155.66 (22C), 162.27, 163.88 (2C, 2C=O). Anal. calcd. (%) for C26H22N6O3 (466.49): C, 66.94; H, 4.75; N, 18.02. Found: C, 67.00; H, 4.70; N, 18.00%.

4.1.1.5. 3-(4-Methoxyphenylamino)-5-(1-methyl-2-oxoindolin-3-ylideneamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (5e)

Dark green crystals, mp 262–263 °C, yield (76%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.27 (s, 3H, CH3), 3.25 (s, 3H, NCH3), 3.76 (s, 3H, OCH3), 6.96 (d, 2H, J = 8.9 Hz, ArH), 7.09–7.16 (m, 4H, ArH), 7.28 (d, 2H, J = 8.2 Hz, ArH), 7.58 (t, 1H, J = 7.8 and 7.7 Hz, ArH), 7.82 (d, 2H, J = 7.5 Hz, ArH), 8.81 (s, 1H, NH), 9.10 (d, 1H, J = 6.7 Hz, ArH), 11.03 (s, 1H, NH), 13.15 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 20.47 (C, CH3), 26.29 (C, NCH3), 55.32 (C, OCH3), 94.20, 109.68, 114.74, 116.60, 118.41, 119.45, 121.67, 122.36, 129.26, 131.70, 132.16, 135.63, 136.75, 148.50, 148.91, 149.39, 149.53, 155.48 (22C), 162.10, 163.86 (2C, 2C=O). MS (m/z, %): 480 (M+, 100). Anal. calcd. (%) for C27H24N6O3 (480.52): C, 67.49; H, 5.03; N, 17.49. Found: C, 67.40; H, 5.10; N, 17.55%.

4.1.1.6. 5-(1-Ethyl-2-oxoindolin-3-ylideneamino)-N-phenyl-3-(phenylamino)-1H-pyrazole-4-carboxamide (5f)

Dark brown crystals, mp 274–276 °C, yield (73%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.24 (t, 3H, J = 7.2 and 7.1 Hz, CH2CH3), 3.83 (q, 2H, J = 7.2 Hz, CH2CH3), 6.86 (t, 1H, J = 7.6 and 6.6 Hz, ArH), 7.03–7.14 (m, 3H, ArH), 7.19 (d, 1H, J = 7.8 Hz, ArH), 7.24 (t, 1H, J = 8.3 and 7.3 Hz, ArH), 7.35–7.39 (m, 4H, ArH), 7.57 (t, 1H, J = 7.7 and 7.7 Hz, ArH), 7.93 (d, 2H, J = 7.7 Hz, ArH), 9.05 (s, 1H, NH), 9.10 (d, 1H, J = 7.2 Hz, ArH), 11.98 (s, 2H, 2NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 14.10 (C, CH3), 34.50 (C, NCH2), 95.27, 109.80, 116.72, 118.31, 119.60, 122.36, 123.02, 128.93, 129.54, 130.93, 135.83, 139.13, 141.54, 147.99, 148.44, 158.98, 159.81, 162.21 (22C), 162.60, 163.41 (2C, 2C=O). Anal. calcd. (%) for C26H22N6O2 (450.49): C, 69.32; H, 4.92; N, 18.66. Found: C, 69.25; H, 5.00; N, 18.60%.

4.1.1.7. 5-(1-Ethyl-2-oxoindolin-3-ylideneamino)-3-(phenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (5g)

Black crystals, mp 288–290 °C, yield (71%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.24 (t, 3H, J = 7.2 and 7.1 Hz, CH2CH3), 2.27 (s, 3H, CH3), 3.83 (q, 2H, J = 7.2 Hz, CH2CH3), 7.06–7.38 (m, 8H, ArH), 7.59 (t, 2H, J = 6.4 and 7.7 Hz, ArH), 7.83 (d, 2H, J = 7.6 Hz, ArH), 9.07 (s, 1H, NH), 9.15 (d, 1H, J = 7.2 Hz, ArH), 11.09 (s, 1H, NH), 13.45 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 12.51 (C, CH3), 20.47 (C, CH3), 34.45 (C, NCH2), 95.40, 109.73, 118.48, 119.51, 122.49, 128.92, 129.27, 129.53, 131.83, 135.74, 136.65, 139.50, 141.10, 147.07, 147.95, 159.00, 159.86, 161.97 (22C), 162.12, 163.46 (2C, 2C=O). Anal. calcd. (%) for C27H24N6O2 (464.52): C, 69.81; H, 5.21; N, 18.09. Found: C, 69.75; H, 5.27; N, 18.00%.

4.1.1.8. 5-(1-Ethyl-2-oxoindolin-3-ylideneamino)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (5h)

Dark green crystals, mp 269–271 °C, yield (75%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.21 (t, 3H, J = 6.9 and 6.5 Hz, CH2CH3), 3.71 (s, 3H, OCH3), 3.78 (q, 2H, J = 7.0 Hz, CH2CH3), 6.87 (d, 2H, J = 8.7 Hz, ArH), 7.06 (t, 1H, J = 6.8 and 6.1 Hz, ArH), 7.21 (d, 2H, J = 8.8 Hz, ArH), 7.47 (s, 2H, NH2), 7.54 (t, 1H, J = 8.0 and 8.0 Hz, ArH), 7.75 (d, 1H, J = 7.4 Hz, ArH), 8.90 (s, 1H, NH), 8.98 (d, 1H, J = 7.6 Hz, ArH), 13.00 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 12.47 (C, CH3), 34.36 (C, NCH2), 55.30 (C, OCH3), 94.90, 109.64, 114.77, 117.52, 121.23, 123.30, 128.97, 132.35, 135.40, 139.68, 145.26, 148.34, 150.58, 155.42 (16C), 162.51, 163.07 (2C, 2C=O). MS (m/z, %): 404 (M+, 11.16). Anal. calcd. (%) for C21H20N6O3 (404.42): C, 62.37; H, 4.98; N, 20.78. Found: C, 62.30; H, 5.00; N, 20.70%.

4.1.1.9. 5-(1-Ethyl-2-oxoindolin-3-ylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (5i)

Dark brown crystals, mp 269–271 °C, yield (77%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.24 (t, 3H, J = 7.2 and 7.1 Hz, CH2CH3), 3.76 (s, 3H, OCH3), 3.85 (q, 2H, J = 7.2 Hz, CH2CH3), 6.96 (d, 2H, J = 8.8 Hz, ArH), 7.06 (t, 1H, J = 7.4 and 7.4 Hz, ArH), 7.12 (t, 1H, J = 7.7 and 7.6 Hz, ArH), 7.21 (d, 1H, J = 7.8 Hz, ArH), 7.29 (d, 2H, J = 8.6 Hz, ArH), 7.36 (t, 2H, J = 8.0 and 7.7 Hz, ArH), 7.58 (t, 1H, J = 7.7 and 7.8 Hz, ArH), 7.95 (d, 2H, J = 7.9 Hz, ArH), 8.81 (s, 1H, NH), 9.15 (d, 1H, J = 7.6 Hz, ArH), 11.15 (s, 1H, NH), 13.18 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 12.52 (C, CH3), 34.46 (C, NCH2), 55.32 (C, OCH3), 94.19, 109.75, 114.76, 116.75, 181.45, 121.88, 122.30, 122.84, 128.90, 129.63, 132.04, 135.73, 139.25, 147.92, 148.63, 149.48, 149.63, 155.69 (22C), 162.28, 163.52 (2C, 2C=O). MS (m/z, %): 480 (M+, 13.67). Anal. calcd. (%) for C27H24N6O3 (480.52): C, 67.49; H, 5.03; N, 17.49. Found: C, 67.40; H, 5.10; N, 17.55%.

4.1.1.10. 5-(1-Ethyl-2-oxoindolin-3-ylideneamino)-3-(4-methoxyphenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (5j)

Dark brown crystals, mp 240–241 °C, yield (79%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.24 (t, 3H, J = 7.2 and 7.1 Hz, CH2CH3), 2.27 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.84 (q, 2H, J = 7.1 Hz, CH2CH3), 6.95 (d, 2H, J = 8.8 Hz, ArH), 7.11 (t, 1H, J = 7.6 and 7.8 Hz, ArH), 7.15 (d, 2H, J = 8.3 Hz, ArH), 7.20 (d, 1H, J = 7.9 Hz, ArH), 7.27 (d, 2H, J = 8.6 Hz, ArH), 7.57 (t, 1H, J = 7.7 and 7.7 Hz, ArH), 7.83 (d, 2H, J = 8.0 Hz, ArH), 8.82 (s, 1H, NH), 9.14 (d, 1H, J = 7.6 Hz, ArH), 11.05 (s, 1H, NH), 13.17 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 12.52 (C, CH3), 20.47 (C, CH3), 34.45 (C, NCH2), 55.31 (C, OCH3), 94.24, 109.71, 114.75, 116.76, 181.41, 121.72, 122.27, 129.27, 129.62, 131.68, 132.10, 135.67, 136.76, 147.89, 148.50, 149.46, 149.57, 155.63 (22C), 162.12, 163.50 (2C, 2C=O). MS (m/z, %): 493 (M+ – 1, 45.05), 494 (M+, 9.75), 273 (100). Anal. calcd. (%) for C28H26N6O3 (494.54): C, 68.00; H, 5.30; N, 16.99. Found: C, 68.10; H, 5.25; N, 16.90%.

4.1.2. General Procedure for the Preparation of 5-((1H-Indol-3-yl)methyleneamino)-N-aryl-3-(arylamino)-1H-pyrazole-4-carboxamide (7a–e)

A mixture of compounds 2a–e (0.01 mol) and 1H-indole-3-carbaldehyde 6 (0.01 mol, 1.45 g) 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 compounds 7a–e.

4.1.2.1. 5-((1H-Indol-3-yl)methyleneamino)-N-phenyl-3-(phenylamino)-1H-pyrazole-4-carboxamide (7a)

Yellow crystals, mp 266–268 °C, yield (73%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 6.87 (t, 1H, J = 7.3 and 7.3 Hz, ArH), 7.07 (t, 1H, J = 7.4 and 7.4 Hz, ArH), 7.24–7.37 (m, 6H, ArH), 7.57–7.65 (m, 5H, ArH), 8.33 (s, 1H, indole), 8.35 (d, 1H, J = 7.9 Hz, ArH), 9.00 (s, 1H, −N=CH−), 9.06 (s, 1H, NH), 10.03 (s, 1H, NH), 12.30 (s, 2H, 2NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 92.05, 112.99, 114.34, 116.44, 119.42, 119.82, 121.23, 122.07, 123.33, 123.90, 124.35, 128.63, 128.98, 129.02, 137.73, 138.57, 141.36, 149.74, 152.03, 158.89 (24C), 163.29 (C, C=O). Anal. calcd. (%) for C25H20N6O (420.47): C, 71.41; H, 4.79; N, 19.99. Found: C, 71.35; H, 4.85; N, 20.05%.

4.1.2.2. 5-((1H-Indol-3-yl)methyleneamino)-3-(phenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (7b)

Yellow crystals, mp 276–278 °C, yield (75%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.27 (s, 3H, CH3), 6.87 (t, 1H, J = 7.3 and 7.3 Hz, ArH), 7.14 (d, 2H, J = 8.3 Hz, ArH), 7.24–7.31 (m, 3H, ArH), 7.35 (t, 1H, J = 7.1 and 7.0 Hz, ArH), 7.53 (d, 2H, J = 8.4 Hz, ArH), 7.57 (d, 2H, J = 8.2 Hz, ArH), 7.60 (d, 1H, J = 8.1 Hz, ArH), 8.33 (s, 1H, indole), 8.34 (d, 1H, J = 7.6 Hz, ArH), 9.00 (s, 1H, −N=CH−), 9.05 (s, 1H, NH), 9.96 (s, 1H, NH), 12.29 (s, 2H, 2NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 20.38 (C, CH3), 92.02, 112.94, 114.28, 116.34, 119.35, 121.18, 122.02, 123.84, 124.27, 128.98, 129.31, 132.27, 135.99, 137.66, 139.39, 141.34, 149.44, 153.15, 155.63, 158.66 (24C), 163.09 (C, C=O). Anal. calcd. (%) for C26H22N6O (434.49): C, 71.87; H, 5.10; N, 19.34. Found: C, 71.95; H, 5.00; N, 19.40%.

4.1.2.3. 5-((1H-Indol-3-yl)methyleneamino)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (7c)

Buff crystals, mp 260–262 °C, yield (75%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.71 (s, 3H, OCH3), 6.86 (d, 2H, J = 8.6 Hz, ArH), 7.23–7.56 (m, 7H, NH2 + ArH), 8.14 (d, 1H, J = 7.7 Hz, ArH), 8.24 (s, 1H, indole), 8.85 (s, 1H, −N=CH−), 8.94 (s, 1H, NH), 12.18 (s, 1H, NH), 12.38 (s, H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 55.21 (C, OCH3), 86.37, 112.72, 114.30, 117.06, 120.77, 121.98, 123.55, 124.31, 128.16, 131.61, 134.87, 137.56, 147.18, 153.04, 158.45, 163.70 (18C), 166.70 (C, C=O). Anal. calcd. (%) for C20H18N6O2 (374.40): C, 64.16; H, 4.85; N, 22.45. Found: C, 64.10; H, 4.90; N, 22.39%.

4.1.2.4. 5-((1H-Indol-3-yl)methyleneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (7d)

Yellow crystals, mp 272–274 °C, yield (77%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.73 (s, 3H, OCH3), 6.90 (d, 2H, J = 8.2 Hz, ArH), 7.07 (t, 1H, J = 7.4 and 7.4 Hz, ArH), 7.26 (t, 1H, J = 7.2 and 7.5 Hz, ArH), 7.30–7.36 (m, 4H, ArH), 7.60 (d, 1H, J = 8.1 Hz, ArH), 7.64 (d, 2H, J = 7.6 Hz, ArH), 8.33 (s, 1H, indole), 8.35 (d, 2H, J = 7.9 Hz, ArH), 8.77 (s, 1H, −N=CH−), 9.03 (s, 1H, NH), 9.98 (s, 1H, NH), 12.30 (s, H, NH), 12.60 (s, H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 55.21 (C, OCH3), 91.67, 112.92, 114.29, 117.42, 119.30, 121.18, 122.03, 123.19, 123.82, 124.31, 128.93, 137.65, 138.62, 152.83, 155.31, 156.20, 156.60, 158.92 (24C), 163.27 (C, C=O). Anal. calcd. (%) for C26H22N6O2 (450.49): C, 69.32; H, 4.92; N, 18.66. Found: C, 69.25; H, 5.00; N, 18.60%.

4.1.2.5. 5-((1H-Indol-3-yl)methyleneamino)-3-(4-methoxyphenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (7e)

Yellow crystals, mp 258–260 °C, yield (77%). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.26 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 6.89 (d, 2H, J = 9.0 Hz, ArH), 7.13 (d, 2H, J = 8.3 Hz, ArH), 7.25 (t, 1H, J = 7.8 and 7.9 Hz, ArH), 7.34 (t, 1H, J = 8.2 and 8.1 Hz, ArH), 7.48 (d, 2H, J = 8.5 Hz, ArH), 7.53 (d, 2H, J = 8.4 Hz, ArH), 7.59 (d, 1H, J = 8.1 Hz, ArH), 8.31 (s, 1H, indole), 8.34 (d, 1H, J = 7.7 Hz, ArH), 8.78 (s, 1H, −N=CH−), 9.05 (s, 1H, NH), 9.96 (s, 1H, NH), 12.26 (s, 2H, 2NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 20.37 (C, CH3), 55.22 (C, OCH3), 91.54, 112.90, 114.28, 144.33, 118.23, 119.28, 121.18, 121.94, 123.78, 124.28, 129.30, 132.16, 136.09, 137.52, 137.64, 150.02, 153.20, 153.24, 153.31, 158.37 (24C), 163.12 (C, C=O). MS (m/z, %): 464 (M+, 43.73), 337 (100). Anal. calcd. (%) for C27H24N6O2 (464.52): C, 69.81; H, 5.21; N, 18.09. Found: C, 69.75; H, 5.25; N, 18.00%.

4.2. Biological Evaluation

4.2.1. In Vitro Anticancer Activity

4.2.1.1. Cell Culture Conditions

The cells of human liver carcinoma (HepG2), human breast adenocarcinoma (MCF-7), human colorectal carcinoma (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 heat-inactivated 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% CO2.

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)-2,5-diphenyl-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 × 104 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 serum-free 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 IC50s were determined by the SPSS probit analysis software program (SPSS Inc., Chicago, IL).

4.2.2. 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 × 104 and incubated at 37 °C and 5% CO2 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 IC50 of compounds 7a and 7b.

4.2.3. Enzymatic Assay

4.2.3.1. Caspase-3 Assay

Activities of caspase-3 were measured using the Invitrogen caspase-3 (Active) (human) ELISA kit, Catalog # KHO1091 (96 tests) (Invitrogen Corporation) according to the manufacturer’s instructions.

4.2.3.2. Bcl-2 ELISA Assay

Activities of Bcl-2 were measured using the Invitrogen Zymed Bcl-2 ELISA Kit, Catalog # 99–0042 (96 tests) (Invitrogen Corporation) according to the manufacturer’s instructions.

4.2.3.3. Bax ELISA Assay

Activities of Bax were measured using the Human Bax ELISA kit (DRG Human Bax ELISA (EIA-4487) DRG International, Inc.) according to the manufacturer’s instructions.

4.2.3.4. 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

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01604.

  • 1H and 13C NMR spectra of the synthesized pyrazole–indole hybrids (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c01604_si_001.pdf (1.4MB, pdf)

References

  1. Ibrahim H. S.; Albakri M. E.; Mahmoud W. R.; Allam H. A.; Reda A. M.; Abdel-Aziz H. A. Synthesis and biological evaluation of some novel thiobenzimidazole derivatives as anti-renal cancer agents through inhibition of c-MET kinase. Bioorg. Chem. 2019, 85, 337–348. 10.1016/j.bioorg.2019.01.006. [DOI] [PubMed] [Google Scholar]
  2. Tadesse S.; Anshabo A. T.; Portman N.; Lim E.; Tilley W.; Caldon C. E.; Wang S. Targeting CDK2 in cancer: challenges and opportunities for therapy. Drug Discovery Today 2020, 25, 406–413. 10.1016/j.drudis.2019.12.001. [DOI] [PubMed] [Google Scholar]
  3. Davies T. G.; Tunnah P.; Meijer L.; Marko D.; Eisenbrand G.; Endicott J. A.; Noble M. E. M. Inhibitor Binding to Active and Inactive CDK2: The Crystal Structure of CDK2-Cyclin A/Indirubin-5-Sulphonate. Structure 2001, 9, 389–397. 10.1016/S0969-2126(01)00598-6. [DOI] [PubMed] [Google Scholar]
  4. Bačević K.; Lossaint G.; Achour T. N.; Georget V.; Fisher D.; Dulić V. Cdk2 strengthens the intra-S checkpoint and counteracts cell cycle exit induced by DNA damage. Sci. Rep. 2017, 7, 13429 10.1038/s41598-017-12868-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Xu Z.; Zhao S.-J.; Lv Z.-S.; Gao F.; Wang Y.; Zhang F.; Bai L.; Deng J.-L. Fluoroquinolone-isatin hybrids and their biological activities. Eur. J. Med. Chem. 2019, 162, 396–406. 10.1016/j.ejmech.2018.11.032. [DOI] [PubMed] [Google Scholar]
  6. Mayur Y. C.; Peters G. J.; Prasad V. V.; Lemo C.; Sathish N. K. Design of new drug molecules to be used in reversing multidrug resistance in cancer cells. Curr. Cancer Drug Targets 2009, 9, 298–306. 10.2174/156800909788166619. [DOI] [PubMed] [Google Scholar]
  7. Ke S.; Shi L.; Yang Z. Discovery of novel isatin-dehydroepiandrosterone conjugates as potential anticancer agents. Bioorg. Med. Chem. Lett. 2015, 25, 4628–4631. 10.1016/j.bmcl.2015.08.041. [DOI] [PubMed] [Google Scholar]
  8. Pandeya S. N.; Smitha S.; Jyoti M.; Sridhar S. K. Biological activities of isatin and its derivatives. Acta Pharm. 2005, 55, 27–46. [PubMed] [Google Scholar]
  9. Xu Z.; Zhang S.; Gao C.; Fan J.; Zhao F.; Lv Z.-S.; Feng L.-S. Isatin hybrids and their anti-tuberculosis activity. Chin. Chem. Lett. 2017, 28, 159–167. 10.1016/j.cclet.2016.07.032. [DOI] [Google Scholar]
  10. Ganim M. A.; Baloglu M. C.; Aygun A.; Altunoglu Y. C.; Sayiner H. S.; Kandemirli F.; Sen F. Analysis of DNA protection, interaction and antimicrobial activity of isatin derivatives. Int. J. Biol. Macromol. 2019, 122, 1271–1278. 10.1016/j.ijbiomac.2018.09.084. [DOI] [PubMed] [Google Scholar]
  11. Varun; Sonam; Kakkar R. Isatin and its derivatives: a survey of recent syntheses, reactions, and applications. MedChemComm 2019, 10, 351–368. 10.1039/C8MD00585K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Panga S.; Podila N. K.; Ciddi V. Design, Synthesis, Characterization, and In Vitro Evaluation of Isatin-Pomalidomide Hybrids for Cytotoxicity against Multiple Myeloma Cell Lines. J. Heterocycl. Chem. 2018, 55, 2919–2928. 10.1002/jhet.3365. [DOI] [Google Scholar]
  13. Jaiswal S.; Tripathi R. K. P.; Ayyannan S. R. Scaffold hopping-guided design of some isatin based rigid analogs as fatty acid amide hydrolase inhibitors: Synthesis and evaluation. Biomed. Pharmacother. 2018, 107, 1611–1623. 10.1016/j.biopha.2018.08.125. [DOI] [PubMed] [Google Scholar]
  14. El-Sharief A. M. S.; Ammar Y. A.; Belal A.; El-Sharief M. A. M. Sh.; Mohamed Y. A.; Mehany A. B. M.; Ali G. A. M. E.; Ragab A. Design, synthesis, molecular docking and biological activity evaluation of some novel indole derivatives as potent anticancer active agents and apoptosis inducers. Bioorg. Chem. 2019, 85, 399–412. 10.1016/j.bioorg.2019.01.016. [DOI] [PubMed] [Google Scholar]
  15. Alafeefy A. M.; Ashour A. E.; Prasad O.; Sinha L.; Pathak S.; Alasmari F. A.; Rishi A. K.; Abdel-Aziz H. A. Development of certain novel N-(2-(2-(2-oxoindolin-3-ylidene)hydrazinecarbonyl)phenyl)-benzamides and 3-(2-oxoindolin-3-ylideneamino)-2-substituted quinazolin-4(3H)-ones as CFM-1 analogs: Design, synthesis, QSAR analysis and anticancer activity. Eur. J. Med. Chem. 2015, 92, 191–201. 10.1016/j.ejmech.2014.12.048. [DOI] [PubMed] [Google Scholar]
  16. Fares M.; Eldehna W. M.; Abou-Seri S. M.; Abdel-Aziz H. A.; Aly M. H.; Tolba M. F. Design, Synthesis and In Vitro Antiproliferative Activity of Novel Isatin-Quinazoline Hybrids. Arch. Pharm. 2015, 348, 144–154. 10.1002/ardp.201400337. [DOI] [PubMed] [Google Scholar]
  17. Abu-Melha S.; Edrees M. M.; Salem H. H.; Kheder N. A.; Gomha S. M.; Abdelaziz M. R. Synthesis and Biological Evaluation of Some Novel Thiazole-Based Heterocycles as Potential Anticancer and Antimicrobial Agents. Molecules 2019, 24, 539 10.3390/molecules24030539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Bekhit A. A.; Ashour H. M. A.; Guemei A. A. Novel Pyrazole Derivatives as Potential Promising Anti-inflammatory Antimicrobial Agents. Arch. Pharm. 2005, 338, 167–174. 10.1002/ardp.200400940. [DOI] [PubMed] [Google Scholar]
  19. Nagaraju B.; Kovvuri J.; Kumar C. G.; Routhu S. R.; Shareef M. A.; Kadagathur M.; Adiyala P. R.; Alavala S.; Nagesh N.; Kamal A. Synthesis and biological evaluation of pyrazole linked benzothiazole-β-naphthol derivatives as topoisomerase I inhibitors with DNA binding ability. Bioorg. Med. Chem. 2019, 27, 708–720. 10.1016/j.bmc.2019.01.011. [DOI] [PubMed] [Google Scholar]
  20. Madhavilatha B.; Fatima N.; Sabitha G.; Subba Reddy B. V.; Yadav J. S.; Bhattacharjee D.; Jain N. Synthesis of 1,2,3-triazole and isoxazole-linked pyrazole hybrids and their cytotoxic activity. Med. Chem. Res. 2017, 26, 1753–1763. 10.1007/s00044-017-1884-z. [DOI] [Google Scholar]
  21. Dai H.; Huang M.; Qian J.; Liu J.; Meng C.; Li Y.; Ming G.; Zhang T.; Wang S.; Shi Y.; Yao Y.; Ge S.; Zhang Y.; Ling Y. Excellent antitumor and antimetastatic activities based on novel coumarin/pyrazole oxime hybrids. Eur. J. Med. Chem. 2019, 166, 470–479. 10.1016/j.ejmech.2019.01.070. [DOI] [PubMed] [Google Scholar]
  22. Chaudhry F.; Naureen S.; Ashraf M.; Al-Rashida M.; Jahan B.; Munawar M. A.; Khan M. A. Imidazole-pyrazole hybrids: Synthesis, characterization and in-vitro bioevaluation against α-glucosidase enzyme with molecular docking studies. Bioorg. Chem. 2019, 82, 267–273. 10.1016/j.bioorg.2018.10.047. [DOI] [PubMed] [Google Scholar]
  23. Hassan A. S.; Moustafa G. O.; Askar A. A.; Naglah A. M.; Al-Omar M. A. Synthesis and antibacterial evaluation of fused pyrazoles and Schiff bases. Synth. Commun. 2018, 48, 2761–2772. 10.1080/00397911.2018.1524492. [DOI] [Google Scholar]
  24. Magd-El-Din A. A.; Mousa H. A.; Labib A. A.; Hassan A. S.; Abd El-All A. S.; Ali M. M.; El-Rashedy A. A.; El-Desoky A. H. Benzimidazole-Schiff bases and their complexes: synthesis, anticancer activity and molecular modeling as Aurora kinase inhibitor. Z. Naturforsch., C 2018, 73, 465–478. 10.1515/znc-2018-0010. [DOI] [PubMed] [Google Scholar]
  25. Hassan A. S.; Hafez T. S. Antimicrobial Activities of Ferrocenyl Complexes: A Review. J. Appl. Pharm. Sci 2018, 8, 156–165. 10.7324/JAPS.2018.8522. [DOI] [Google Scholar]
  26. El-Naggar M.; Hassan A. S.; Awad H. M.; Mady M. F. Design, Synthesis and Antitumor Evaluation of Novel Pyrazolopyrimidines and Pyrazoloquinazolines. Molecules 2018, 23, 1249 10.3390/molecules23061249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hassan A. S.; Moustafa G. O.; Awad H. M. Synthesis and in vitro anticancer activity of pyrazolo[1,5-a]pyrimidines and pyrazolo[3,4-d][1,2,3]triazines. Synth. Commun. 2017, 47, 1963–1972. 10.1080/00397911.2017.1358368. [DOI] [Google Scholar]
  28. Hassan A. S.; Masoud D. M.; Sroor F. M.; Askar A. A. Synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine-3-carboxamide as antimicrobial agents. Med. Chem. Res. 2017, 26, 2909–2919. 10.1007/s00044-017-1990-y. [DOI] [Google Scholar]
  29. Hassan A. S.; Hafez T. S.; Ali M. M.; Khatab T. K. Design, synthesis and cytotoxic activity of some new pyrazolines bearing benzofuran and pyrazole moieties. Res. J. Pharm., Biol. Chem. Sci. 2016, 7, 417–429. [Google Scholar]
  30. Abd El-All A. S.; Hassan A. S.; Osman S. A.; Yosef H. A. A.; Abdel-Hady W. H.; El-Hashash M. A.; Atta-Allah S. R.; Ali M. M.; El Rashedy A. A. Synthesis, characterization and biological evaluation of new fused triazine derivatives based on 6-methyl-3-thioxo-1,2,4-triazin-5-one. Acta Pol. Pharm. 2016, 73, 79–92. [PubMed] [Google Scholar]
  31. Hassan A. S.; Hafez T. S.; Osman S. A.; Ali M. M. Synthesis and in vitro cytotoxic activity of novel pyrazolo[1,5-a]pyrimidines and related Schiff bases. Turk. J. Chem. 2015, 39, 1102–1113. 10.3906/kim-1504-12. [DOI] [Google Scholar]
  32. Hassan A. S.; Osman S. A.; Hafez T. S. 5-Phenyl-2-furaldehyde: synthesis, reactions and biological activities. Egypt. J. Chem. 2015, 58, 113–139. 10.21608/ejchem.2015.978. [DOI] [Google Scholar]
  33. Osman S. A.; Mousa H. A.; Yosef H. A. A.; Hafez T. S.; El-Sawy A. A.; Abdallah M. M.; Hassan A. S. Synthesis, characterization and cytotoxicity of mixed ligand Mn(II), Co(II) and Ni(II) complexes. J. Serb. Chem. Soc. 2014, 79, 953–964. 10.2298/JSC130813134O. [DOI] [Google Scholar]
  34. Osman S. A.; Yosef H. A. A.; Hafez T. S.; El-Sawy A. A.; Mousa H. A.; Hassan A. S. Synthesis and antibacterial activity of some novel chalcones, pyrazoline and 3-cyanopyridine derivatives based on khellinone as well as Ni(II), Co(II) and Zn(II) complexes. Aust. J. Basic Appl. Sci. 2012, 6, 852–863. [Google Scholar]
  35. Elgemeie G. H.; Elsayed S. H.; Hassan A. S. Design and synthesis of the first thiophene thioglycosides. Synth. Commun. 2009, 39, 1781–1792. 10.1080/00397910802590928. [DOI] [Google Scholar]
  36. Moustafa G. O.; Younis A.; Al-Yousef S. A.; Mahmoud S. Y. Design, synthesis of novel cyclic pentapeptide derivatives based on 1, 2-benzenedicarbonyl chloride with expected anticancer activity. J. Comput. Theor. Nanosci. 2019, 16, 1733–1739. 10.1166/jctn.2019.8114. [DOI] [Google Scholar]
  37. Kassem A. F.; Moustafa G. O.; Nossier E. S.; Khalaf H. S.; Mounier M. M.; Al-Yousef S. A.; Mahmoud S. Y. In vitro anticancer potentiality and molecular modelling study of novel amino acid derivatives based on N1, N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide. J. Enzyme Inhib. Med. Chem. 2019, 34, 1247–1258. 10.1080/14756366.2019.1613390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mohamed F. H.; Shalaby A. M.; Soliman H. A.; Abdelazem A. Z.; Mounier M. M.; Nossier E. S.; Moustafa G. O. Design, Synthesis and Molecular Docking Studies of Novel Cyclic Pentapeptides Based on Phthaloyl Chloride with Expected Anticancer Activity. Egypt. J. Chem. 2020, 63, 1723–1736. 10.21608/ejchem.2019.18452.2137. [DOI] [Google Scholar]
  39. Abo-Ghalia M. H.; Moustafa G. O.; Amr A. E.; Naglah A. M.; Elsayed E. A.; Bakheit A. H. Anticancer activities of newly synthesized chiral macrocyclic heptapeptide candidates. Molecules 2020, 25, 1253 10.3390/molecules25051253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kalmouch A.; Radwan M. A. A.; Omran M. M.; Sharaky M.; Moustafa G. O. Synthesis of novel 2, 3’-bipyrrole derivatives from chalcone and amino acids as antitumor agents. Egypt. J. Chem. 2020, 63, 4409–4421. 10.21608/ejchem.2020.27117.2560. [DOI] [Google Scholar]
  41. Moustafa G. O.; Al-Wasidi A. S.; Naglah A. M.; Refat M. S. Synthesis of Dibenzofuran Derivatives Possessing Anticancer Activities: A Review. Egypt. J. Chem. 2020, 63, 2355–2367. 10.21608/ejchem.2020.21937.2310. [DOI] [Google Scholar]
  42. Elhenawy A. A.; Al-Harbi L. M.; Moustafa G. O.; El-Gazzar M. A.; Abdel-Rahman R. F.; Salim A. E. Synthesis, comparative docking, and pharmacological activity of naproxen amino acid derivatives as possible anti-inflammatory and analgesic agents. Drug Des., Dev. Ther. 2019, 13, 1773–1790. 10.2147/DDDT.S196276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mady M. F.; Awad G. E. A.; Jørgensen K. B. Ultrasound-assisted synthesis of novel 1,2,3-triazoles coupled diaryl sulfone moieties by the CuAAC reaction, and biological evaluation of them as antioxidant and antimicrobial agents. Eur. J. Med. Chem. 2014, 84, 433–443. 10.1016/j.ejmech.2014.07.042. [DOI] [PubMed] [Google Scholar]
  44. Mady M. F.; Saleh T. S.; El-Kateb A. A.; El-Rahman N. M. A.; El-Moez S. I. A. Microwave-assisted synthesis of novel pyrazole and pyrazolo[3,4-d]pyridazine derivatives incorporating diaryl sulfone moiety as potential antimicrobial agents. Res. Chem. Intermed. 2016, 42, 753–769. 10.1007/s11164-015-2054-x. [DOI] [Google Scholar]
  45. Elgemeie G. H.; Elsayed S. H.; Hassan A. S. Direct route to a new class of acrylamide thioglycosides and their conversions to pyrazole derivatives. Synth. Commun. 2008, 38, 2700–2706. 10.1080/00397910802222605. [DOI] [Google Scholar]
  46. Hafez T. S.; Osman S. A.; Yosef H. A. A.; Abd El-All A. S.; Hassan A. S.; El-Sawy A. A.; Abdallah M. M.; Youns M. Synthesis, structural elucidation and in vitro antitumor activities of some pyrazolopyrimidines and Schiff bases derived from 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides. Sci. Pharm. 2013, 81, 339–357. 10.3797/scipharm.1211-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Hassan A. S.; Hafez T. S.; Osman S. A. Synthesis, characterization, and cytotoxicity of some new 5-aminopyrazole and pyrazolo[1,5-a]pyrimidine derivatives. Sci. Pharm. 2015, 83, 27–39. 10.3797/scipharm.1409-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Khatab T. K.; Hassan A. S.; Hafez T. S. V2O5/SiO2 as an efficient catalyst in the synthesis of 5-aminopyrazole derivatives under solvent free condition. Bull. Chem. Soc. Ethiop. 2019, 33, 135–142. 10.4314/bcse.v33i1.13. [DOI] [Google Scholar]
  49. Shmidt M. S.; Reverdito A. M.; Kremenchuzky L.; Perillo I. A.; Blanco M. M. Simple and Efficient Microwave Assisted N-Alkylation of Isatin. Molecules 2008, 13, 831–840. 10.3390/molecules13040831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Kassem A. F.; Nassar I. F.; Abdel-Aal M. T.; Awad H. M.; El-Sayed W. A. Synthesis and Anticancer Activity of New ((Furan-2-yl)-1,3,4-thiadiazolyl)-1,3,4-oxadiazole Acyclic Sugar Derivatives. Chem. Pharm. Bull. 2019, 67, 888–895. 10.1248/cpb.c19-00280. [DOI] [PubMed] [Google Scholar]
  51. Alminderej H. F. M.; Elganzory H. H.; El-Bayaa M. N.; Awad H. M.; El-Sayed W. A. Synthesis and Cytotoxic Activity of New 1,3,4-Thiadiazole Thioglycosides and 1,2,3-Triazolyl-1,3,4-Thiadiazole N-glycosides. Molecules 2019, 24, 3738 10.3390/molecules24203738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Haiba M. E.; Al-Abdullah E. S.; Ahmed N. S.; Ghabbour H. A.; Awad H. M. Efficient and easy synthesis of new Benzo[h]chromene and Benzo[h]quinoline derivatives as a new class of cytotoxic agents. J. Mol. Struct. 2019, 1195, 702–711. 10.1016/j.molstruc.2019.05.081. [DOI] [Google Scholar]
  53. Gao W.; Fenglian X.; Wang X.; Chen T. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen speciesmediated amplification activation loop among caspase-9, -8 and -3. Apoptosis 2013, 18, 1201–1213. 10.1007/s10495-013-0857-z. [DOI] [PubMed] [Google Scholar]
  54. Nicholson D. W.; Nicotera P.; Melino G.. Caspases and Cell Death, Encyclopedia of Biological Chemistry, 1st ed; Elsevier, 2004; pp 319–327. [Google Scholar]
  55. Mace P. D.; Ried S. J.; Salvesen G. S. Chapter Seven-Caspase enzymology and activation mechanisms. Methods Enzymol. 2014, 544, 161–178. 10.1016/B978-0-12-417158-9.00007-8. [DOI] [PubMed] [Google Scholar]
  56. Lu Q. L.; Abel P.; Foster C. S.; Lalani E. N. bcl-2: role in epithelial differentiation and oncogenesis. Hum. Pathol. 1996, 27, 102–110. 10.1016/S0046-8177(96)90362-7. [DOI] [PubMed] [Google Scholar]
  57. Oltval Z. N.; Milliman C. L.; Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993, 74, 609–619. 10.1016/0092-8674(93)90509-O. [DOI] [PubMed] [Google Scholar]
  58. Shimizu S.; Narita M.; Tsujimoto Y.; Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 1999, 399, 483–487. 10.1038/20959. [DOI] [PubMed] [Google Scholar]
  59. Bettaieb A.; Dubrez-Daloz L.; Launay S.; Plenchette S.; Rébé C.; Cathelin S.; Solary E. Bcl-2 proteins: targets and tools for chemosensitisation of tumor cells. Curr. Med. Chem.: Anti-Cancer Agents 2003, 3, 307–318. 10.2174/1568011033482396. [DOI] [PubMed] [Google Scholar]
  60. Kirkin V.; Joos S.; Zörnig M. The role of Bcl-2 family members in tumorigenesis. Biochim. Biophys. Acta, Mol. Cell Res. 2004, 1644, 229–249. 10.1016/j.bbamcr.2003.08.009. [DOI] [PubMed] [Google Scholar]
  61. De Azevedo W. F.; Leclerc S.; Meijer L.; Havlicek L.; Strnad M.; Kim S. H. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur. J. Biochem. 1997, 243, 518–526. 10.1111/j.1432-1033.1997.0518a.x. [DOI] [PubMed] [Google Scholar]
  62. Thornton T. M.; Rincon M. Non-classical P38 map kinase functions: cell cycle checkpoints and survival. Int. J. Biol. Sci. 2009, 5, 44–52. 10.7150/ijbs.5.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Hassan A. S.; Askar A. A.; Nossier E. S.; Naglah A. M.; Moustafa G. O.; Al-Omar M. A. Antibacterial evaluation, in silico characters and molecular docking of Schiff bases derived from 5-aminopyrazoles. Molecules 2019, 24, 3130 10.3390/molecules24173130. [DOI] [PMC free article] [PubMed] [Google Scholar]

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