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Article

Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity

by
Wagdy M. Eldehna
1,*,†,
Haytham O. Tawfik
2,†,
Denisa Veselá
3,
Veronika Vojáčková
3,
Ahmed T. Negmeldin
4,5,*,
Zainab M. Elsayed
6,
Taghreed A. Majrashi
7,
Petra Krňávková
3,
Mostafa M. Elbadawi
1,
Moataz A. Shaldam
1,
Ghada H. Al-Ansary
8,
Vladimír Kryštof
3,9 and
Hatem A. Abdel-Aziz
10,*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta University, Tanta 31527, Egypt
3
Department of Experimental Biology, Faculty of Science, Palacký University Olomouc, Šlechtitelů 27, 77900 Olomouc, Czech Republic
4
Department of Pharmaceutical Sciences, College of Pharmacy and Thumbay Research Institute for Precision Medicine, Gulf Medical University, Ajman 4184, United Arab Emirates
5
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo 12613, Egypt
6
Scientific Research and Innovation Support Unit, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh 6860404, Egypt
7
Department of Pharmacognosy, College of Pharmacy, King Khalid University, Asir 62521, Saudi Arabia
8
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abassia, Cairo 11566, Egypt
9
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 5, 77900 Olomouc, Czech Republic
10
Applied Organic Chemistry Department, National Research Center, Dokki, Cairo 12622, Egypt
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pharmaceuticals 2025, 18(11), 1770; https://doi.org/10.3390/ph18111770 (registering DOI)
Submission received: 16 October 2025 / Revised: 3 November 2025 / Accepted: 7 November 2025 / Published: 20 November 2025
(This article belongs to the Special Issue Structural Insights into Protein Kinases-Targeted Therapies: Overcoming Resistance and Advancing Precision Oncology)
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Abstract

Background/Objectives: In the current medical era, Topoisomerase II is recognized as an essential enzyme that regulates DNA topology during critical biological processes such as DNA replication, transcription, and repair. This study aimed to design, synthesize, and biologically evaluate a new series of pyrazolo[3,4-b]pyridines (8a–g, 10a–g, and 12) as potential anticancer agents and Topoisomerase II inhibitors. Methods: The synthesized compounds were subjected to in vitro anticancer screening at the National Cancer Institute (NCI, USA). Active derivatives were further evaluated through a five-dose screening to determine their antiproliferative potency. Selected compounds were examined for their effects on leukemia cell lines (K562 and MV4-11), and mechanistic studies were performed to assess DNA damage, cell cycle distribution, and apoptosis-related protein modulation. Additionally, enzyme inhibition assays were conducted to determine Topoisomerase IIα (TOPIIα) inhibition. Results: Initial single-dose screening identified several active compounds, notably 8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f. Among these, compound 8c exhibited potent and broad-spectrum antiproliferative activity across the NCI cancer cell line panel, with a GI50 MG-MID value of 1.33 µM (range: 0.54–2.08 µM). The synthesized molecules showed moderate to good anti-leukemic efficacy against K562 and MV4-11 cells. Mechanistic investigations revealed that compound 8c induced DNA damage and S-phase cell cycle arrest, leading to apoptosis as evidenced by the modulation of PARP-1, Bax, XIAP, and Caspases. Furthermore, target-based assays confirmed that compound 8c significantly inhibited the DNA relaxation activity of TOPIIα in a dose-dependent manner, comparable to etoposide. Conclusions: The study highlights compound 8c as a promising pyrazolo[3,4-b]pyridine derivative with potent antiproliferative activity and effective inhibition of Topoisomerase IIα. These findings suggest its potential as a lead scaffold for further optimization in anticancer drug development..

1. Introduction

Cancer is still one of the leading causes of death worldwide and remains a serious public health concern [1,2]. By 2050, the global burden of cancer is anticipated to reach 19 million cancer diagnoses worldwide, with an estimated 10.5 million cancer-related deaths [3]. Among the diverse types of cancer, leukemia stands out as one of the most aggressive and potentially fatal hematological malignancies [4]. Acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL) are the four primary subtypes of leukemia that are clinically distinguished based on cell lineage and the rate of disease progression [5]. Each subtype exhibits distinct pathological, genetic, and clinical features, which require individualized treatment approaches. Although leukemia often responds well to chemotherapy, the range of safe and durable treatment options remains limited [6]. As a result, the development of novel, more effective therapies for leukemia remains a pressing and continuous task. Pyrazolo [3,4-b]pyridine is a privileged fused heterocyclic scaffold commonly found in pharmaceuticals and drug-like substances [7]. Research has shown that derivatives of pyrazolo [3,4-b]pyridine (Figure 1) exhibit anticancer properties through various mechanisms, including inhibition of several targets such as cyclin-dependent kinases (I–IV) [8,9,10], tubulin polymerization (V) [11], glycogen synthase kinase-3 (VI) [12], hematopoietic cell kinase (VII) [13], fibroblast growth factor receptor (VIII) [14], tropomyosin receptor kinases (IX) [15] and TOPII (X–XI) [16,17].
Indole is a simple chemical scaffold with a bicyclic planar structure found in many natural products [18]. The biological actions of indole derivatives include antibacterial [19], antiinflammatory [20], anticancer [21,22], anticonvulsant [23], antiviral [24], antidiabetic [25], and antioxidant [26] properties. Regarding cancer proliferation inhibition, indole derivatives are highly effective because they strongly target multiple pathways (Figure 2), including PIM (XII) [27], CDK (XIII) [28], EGFR (XIV) [29], PI3Kα (XV) [30], Bcl-2/Mcl-1 (XVI) [31], TOPII (XV–XVI) [32], etc. [33].
The endonuclease class enzymes known as DNA topoisomerases (TOPs) are widely distributed throughout all life spheres and play essential roles in fundamental cellular processes [34,35]. TOP enzymes carry out a variety of tasks linked to the maintenance of DNA metabolism and regulate DNA topology during transcription and replication [36,37]. Based on their structure, amino acid sequence, catalytic activity, and reaction mechanism, TOPs can be divided into type I and type II [38,39]. Numerous investigations have demonstrated that TOPs are overexpressed in tumor cells, and their inhibition suppressed tumor cell growth, establishing TOPs as valuable targets for the development of anticancer therapeutics [40,41]. Furthermore, leukemia’s higher topoisomerase IIα expression levels have been associated with increased sensitivity to agents targeting this enzyme [42]. TOPII inhibitors, such as doxorubicin and etoposide, have been extensively employed in cancer therapy. Although these compounds demonstrate clinical efficacy, their application is often limited by toxicity and adverse side effects [43,44]. Doxorubicin, for instance, is known to induce dose-dependent cardiotoxicity primarily through the generation of reactive oxygen species (ROS) and mitochondrial dysfunction, leading to cardiomyocyte apoptosis [45,46]. Etoposide, on the other hand, is associated with myelosuppression due to its interference with DNA synthesis and repair in rapidly dividing hematopoietic cells [47]. Moreover, resistance to TOPII inhibitors in leukemia patients remains a significant clinical challenge. Mechanisms contributing to drug resistance include the deletion or downregulation of the TOPIIα gene, upregulation of DNA repair enzymes such as PARP and RAD51, and enhanced drug efflux via ATP-binding cassette (ABC) transporters [48]. These adaptations reduce drug efficacy and complicate treatment outcomes, thus, the development of novel and more efficient TOPII inhibitors is the subject of extensive research [41,49].
TOPII inhibitors are known for their inflexible, coplanar aromatic structures, which serve as templates for additional structural optimization to facilitate the intercalation of stable ternary complexes with DNA. The molecular scope of inhibitor design tactics has been expanded beyond “poison inhibition” to encompass “catalytic inhibition”, “dual-targeting”, and “structure-induced blockade” [50]. As a result, new TOPII inhibitors with non-classical structures are appearing in preclinical research. A tendency towards structural and mechanistic diversity is reflected in these structures, which extend beyond conventional scaffolds and incorporate a variety of chemotypes [50]. Furthermore, the creation of dual-target compounds that integrate TOPII inhibitors with other target inhibitors, such as PARP [51], HDAC [52], and tubulin [53], results in long-lasting lethal stress and reduces resistance.
Previous studies have highlighted planar structures as a key pharmacophoric feature of TOPII inhibitors such as the scaffolds found in compounds X [16] and XI [17], both of which contain pyrazolo [3,4-b]pyridine in their structure. Additionally, the indole ring, due to its planar structure, can act as an intercalation site for DNA interaction, as demonstrated by compounds XV and XVI, which showed promising TOPII inhibition activities. All things considered, the compounds in this study were based on a hybridization strategy involving pyrazolo [3,4-b]pyridine as the main scaffold bearing the indole moiety at three varying positions to achieve promising activity against TOPII. As a part of the structure, phenyl rings were also grafted to afford binding interaction with the lipophilic DNA groove binding regions (Figure 3). The obtained compounds were tested in a preliminary anticancer screening at the National Cancer Institute (NCI), and the most promising were subjected to biological mechanistic studies and molecular docking to explore their potential anticancer mechanisms.

2. Results and Discussion

2.1. Chemistry

Scheme 1 and Scheme 2 show the synthetic routes taken to produce the target pyrazolo [3,4-b]pyridine derivatives (8a–g, 10a–g, and 12). To obtain 3-(1H-indol-3-yl)-3-oxopropanenitrile (2), indole (1) was heated with cyanoacetic acid, and acetic anhydride was involved [54].
Additionally, 3-oxo-3-phenylpropanenitrile (4) and 3-(1H-indol-3-yl)-3-oxopropanenitrile (2) were condensed with phenylhydrazine in refluxing absolute ethanol to create the 3-substituted-1-phenyl-1H-pyrazol-5-amines (5 and 9) [55,56,57,58,59,60]. As shown in Scheme 1 and Scheme 2, the target pyrazolo [3,4-b]pyridine derivatives (8a–g, 10a–g, and 12) were then prepared using a one-pot, three-component reaction] that included the relevant 3-substituted-1-phenyl-1H-pyrazol-5-amine (5 or 9), an equimolar amount of the corresponding 3-oxo-3-arylpropanenitrile (2 or 4), and the appropriate aldehyde (3a-g and 11).
Intermediates 6a–g are produced as part of the reaction mechanism, and they are cyclized and then water-eliminated to produce intermediates 7a–g. The required compounds 8a–g are subsequently obtained through a final oxidative dehydrogenation process, in which atmospheric oxygen acts as the oxidant. Nuclear magnetic resonance (NMR) spectroscopy (1H and 13C) (Figures S1–S37) and high-resolution mass spectrometry (HRMS) (Figures S38–S51) validated the structures of the synthesized compounds, which were in agreement with the proposed structures.

2.2. Structure Elucidation of the Target Compounds

The 1H NMR spectra of target compounds 8a–g, 10a–g, and 12 confirmed their structures. In particular, the absence of the aldehydic proton (CH=O) signals from aldehydes (3a–g and 11) and the active methylene protons (CH2) from nitriles (2 and 4) around δ 3.5 ppm, as well as the disappearance of characteristic signals around δ 5.0 ppm, which correspond to the two NH2 protons of the precursor 3-substituted-1-phenyl-1H-pyrazol-5-amines (5 and 9). The addition of aromatic moieties throughout the process was further supported by the spectra, which showed an increase in aromatic proton signals.
The suggested structures were further supported by the 13C NMR spectra, which revealed the elimination of carbon signals associated with nitrile and aldehyde carbonyl groups, typically detected at δ 190 ppm. These modifications verified that the initial materials had completely changed into the final pyrazolo [3,4-b]pyridine derivatives.
With variances falling within the permissible range of ±0.4%, the elemental analysis findings showed good agreement with the estimated values for the target compounds’ molecular formulae. The structures were further validated by molecular ion peaks obtained from high-resolution mass spectrometry (HRMS) that matched the predicted molecular weights. The effectiveness and selectivity of the synthetic processes were further demonstrated by high-performance liquid chromatography (HPLC) analysis, which verified that all synthesized compounds had a purity higher than 95.00% (Figures S52–S66).

2.3. Biological Evaluation

The NCI 60 human cancer cell line panel was used for preliminary in vitro anticancer screening at a single dose of 10 μM.
The United States’ National Cancer Institute (NCI) conducted the first anticancer assessment of the recently synthesized indole-conjugated pyrazolo [3,4-b]pyridine derivatives (8a–g, 10a–g, and 12). The comprehensive NCI 60 human tumor cell line panel, which comprises leukemia, non-small cell lung, melanoma, central nervous system (CNS), ovarian, renal, prostate, and breast cancer cell lines, was first screened in vitro at a fixed concentration of 10 µM. The findings, presented as the mean percentage growth (G%) of treated cells compared to untreated controls, shed light on the cytotoxic effects (G% less than 0) as well as the cytostatic activity (G% between 0 and 100).
The single-dose activity profiles of compounds 8a–g, 10a–g, and 12 were examined using the COMPARE algorithm on 60 distinct cancer cell lines. The pyrazolo [3,4-b]pyridines assessed exhibited a broad range of cytotoxic effects on various cancer types, with anticancer activities ranging from weak to very potent [61]. Table 1 summarizes the growth inhibition percentages (GI%) for each target molecule, which are computed as (100–G%).
According to the NCI criteria, derivatives 8d, 8g, and 10d (mean GI% values below 20) did not exhibit significant cytotoxicity against the examined cell lines (except for 8d towards NCI-H226 and 10d towards CCRF-CEM, HL-60(TB), and MOLT-4) [62]. Compounds 8a (mean GI% = 24), 10a (mean GI% = 43), 10g (mean GI% = 29), and 12 (mean GI% = 23) exposed promising sensitivity towards limited representative cell lines (leukemia; K-562 and renal cancer; RXF 393) (Table 1).
The remaining eight compounds; 3-hydroxyphenyl derivatives (8b and its counterpart 10b), 4-hydroxyphenyl derivatives (8c and its counterpart 10c), 4-hydroxy-3-methoxyphenyl derivatives (8e and its counterpart 10e) and 3-hydroxy-4-methoxyphenyl derivatives (8f and its counterpart 10f) showed auspicious results in NCI preliminary screening with mean GI% = 120, 154, 138, 125, 126, 118, 138 and 122 (Table 1).
The antiproliferative activity of compounds 8a (mean GI% = 24) and 10a (mean GI% = 43) was considerably increased by adding a hydroxyl group to the meta position of one of the phenyl rings. This resulted in 8b and 10b derivatives, with mean growth inhibition (GI%) values of 120% and 126%, respectively. Among the 60 cell lines examined, both compounds showed the most potent anticancer effects. According to Table 1, each demonstrated fatal activity against 42 cell lines, 33 of which were shared by both, and inhibitory effects on the other lines. The GI% values for these lines ranged from 46 to 99.
When the hydroxyl group in compound 8b was shifted from the meta to the para position, obtaining compound 8c, the antiproliferative activity increased. Both derivatives exerted a lethal effect on 47 cell lines. The same structure modification of 10b, yielding compound 10c, led to a modest decrease in activity, and 10c exerted a lethal effect only on 37 cell lines. The mean GI% values for 8c and 10c were 154% and 118%, respectively (Table 1).
Introducing a methoxy group at the para position adjacent to the hydroxyl group in compounds 8b and 10b resulted in enhanced antiproliferative activity, as observed in the obtained derivatives 8e and 10e, both of which showed a mean GI% of 138. These compounds exerted lethal effects on 39 common cancer cell lines. Lastly, compounds 8e and 10e showed enhanced anticancer activity with mean GI% values of 125 and 122, respectively, when the hydroxyl and methoxy groups were repositioned, with the hydroxyl in the para position and the methoxy in the meta position, as in compounds 8f and 10f with 34 and 35 cancer cell lines were fatally affected by these compounds, respectively.
Based on their in vitro cytotoxic characteristics against the NCI-60 human cancer cell line panel, the structure–activity relationship (SAR) of the synthesized compounds is depicted in Figure 4. The mean growth inhibition percentages (GI%) presented in Table 1 serve as the basis for this analysis. Important pharmacophoric characteristics that contribute to potency and selectivity across several cancer cell types were identified by establishing a correlation between the observed anticancer activity and the structural alterations within the pyrazolo [3,4-b]pyridine scaffold. This SAR analysis lays the groundwork for future structural optimization in the development of more potent anticancer drugs, providing insightful information on how specific substituents affect biological activity.
In the next step, the most potent derivatives, including 3-hydroxyphenyl derivatives (8b and its counterpart 10b), 4-hydroxyphenyl derivatives (8c and its counterpart 10c), 4-hydroxy-3-methoxyphenyl derivatives (8e and its counterpart 10e) and 3-hydroxy-4-methoxyphenyl derivatives (8f and its counterpart 10f), were screened against the 60 cancer cell lines at 10-fold dilutions and five different concentrations (0.01, 0.1, 1, 10 and 100 μM) [61]. Following established experimental procedures, the sulforhodamine B (SRB) colorimetric assay was used to evaluate cell viability [63]. Cell density and vitality after chemical treatment are indirectly assessed by measuring cellular protein content in this experiment. The molar concentrations of the substances needed to inhibit 50% of cell proliferation (GI50) and induce 50% cell death (LC50) are represented by the GI50 and LC50 values for each cancer cell line that is tested. Table 2 summarizes these important pharmacological parameters, providing information on the cytotoxic and antiproliferative efficacy of the investigated agents [64,65]. The NCI 60 cancer cell line panel demonstrated strong antiproliferative activity across all chosen compounds (8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f), with efficacy ranging from sub-micromolar to low micromolar concentrations, according to the results. These results demonstrate compounds’ strong growth inhibition against a wide range of cancer cell types, underscoring their considerable potential as potent anticancer agents.
The results showed that most compounds had significant inhibitory effects, with GI50 values ranging from 0.15 µM to 5 µM. Only a few cell lines showed reduced sensitivity, with GI50 values not exceeding 15 µM. Among the tested compounds, 8c (a 4-hydroxyphenyl derivative) and 8e (a 4-hydroxy-3-methoxyphenyl derivative) demonstrated remarkable potency, particularly against leukemia, followed by CNS, renal, and breast cancer cell lines.
Compound 8c displayed outstanding activity with GI50 values ranging from 0.15 to 4.43 μM among the tested NCI panel, including 15 cell lines with GI50 below 1.00 μM. Interestingly, it showed sub-micromolar inhibitory activity towards all leukemia, all CNS (except SF-268 and SNB-19), renal (786-0, RXF 393, and UO-31), and breast cancer cell lines (MCF7 and HS 578T). Regarding compound 8e, it displayed remarkable activity with GI50 values ranging from 0.23 to 4.68 μM (with 10 cancer cell lines with GI50 below 1.00 μM). Compound 8e showed sub-micromolar inhibitory activity towards all leukemia (except K-562 and MOLT-4), all CNS (except SF-268 and SNB-19), and renal cancer cell lines (786-0 and RXF 393) (Table 2).
Additionally, an average activity metric for all cell lines is provided by the mean graph midpoint (MG-MID). The compounds 8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f showed MID (average sensitivity of all cell lines in µM) values of 2.43, 1.33, 1.93, 3.04, 3.37, 4.23, 2.65, and 2.49 µM, respectively (Table 3).
The ratio of the entire panel mean graph midpoint (MID) to the individual subpanel MID was used to calculate the selectivity of these substances. The subpanel MID shows the average sensitivity of cell lines within a particular tumor subpanel, whereas the whole panel MID shows the average sensitivity of all cell lines to the test agent. Compounds with selectivity ratios below this cutoff are regarded as nonselective, whilst those with ratios above 2 may suggest a preference for specific tumor types. With selectivity ratios ranging from 0.54 to 2.46, the drugs in this investigation showed broad-spectrum anticancer efficacy across the nine tumor subpanels examined. With an average MID of 1.33 μM, compound 8c demonstrated the highest potency overall and the most effective and selective treatment of leukemia (MID = 0.54 μM, selectivity = 2.46). Likewise, compound 8e had significant potency and selectivity against leukemia, attaining a MID of 0.97 μM and a selectivity ratio of 1.98, with an average MID of 1.93 μM.

2.4. Antiproliferative Activity Against Leukemia Cells

Following the NCI 60 screening, the synthesized derivatives (8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f) were further evaluated for their antiproliferative activity against leukemia cancer cell lines, including chronic myeloid leukemia (K562) and acute monocytic leukemia (MV4-11), as shown in Table 4.
With the exception of compound 8e, the chosen pyrazolo [3,4-b]pyridine derivatives efficiently suppressed the proliferation of MV4-11 cells, with GI50 values ranging from 0.72 to 9.03 µM. With a GI50 value of 0.72 µM, pyrazolo [3,4-b]pyridine 8c exhibited the strongest sub-micromolar cytotoxic effect. Furthermore, with GI50 values of 3.55 and 3.70 µM, respectively, compounds 8b and 8f demonstrated low single-digit micromolar cytotoxicity against the MV4-11 cell line. The remaining compounds (10b, 10c, 10e, and 10f), on the other hand, demonstrated substantial single-digit micromolar cytotoxic activity, as indicated by Table 4, with GI50 values ranging from 8.03 to 9.03 µM.
The pyrazolo [3,4-b]pyridine derivatives showed GI50 values ranging from 0.72 to 6.52 µM for the K562 leukemia cell line. Compound 8c once more exhibited exceptional sub-micromolar cytotoxicity (GI50 = 0.72 µM). With GI50 values ranging from 2.50 to 6.52 µM, compounds 8b, 8f, 10b, 10c, 10e, and 10f demonstrated single-digit micromolar cytotoxic action. However, as Table 4 summarizes, compound 8e exhibited noticeably less activity, with a GI50 value exceeding 10 µM.
Overall, SAR analysis indicates that subtle structural modifications have a marked influence on biological activity, notably enhancing potency against leukemia cancer cell lines. Among the tested compounds, 8c exhibited the highest potency and was therefore selected for further experiments to elucidate the molecular mechanism of action of the presented derivatives.

2.5. Immunodetection and Cell Cycle Analysis of MV4-11

Based on the promising antiproliferative activity observed in the initial screening and SAR analysis, further experiments were conducted to characterize the molecular mechanisms underlying the cytotoxic effects of the most potent compound 8c. Asynchronously growing MV4-11 cells were incubated with increasing concentrations of 8c (1.25, 2.5, 5.0, and 10 µM) for 24 h and subsequently analyzed by immunodetection (Figure 5) and cell cycle flow cytometry (Figure 6).
Treatment with 8c efficiently induced the cleavage of initiator caspase-9, which was accompanied by cleavage of executioner caspases −3 and −7. Once activated, they cleave a variety of cellular substrates, including poly(ADP-ribose) polymerase 1 (PARP-1), thereby promoting commitment to cell death [66]. The presence of the cleaved 89 kDa fragment of PARP-1 clearly indicates a concentration-dependent progression of cell death in MV4-11 cells following 8c treatment. This was further associated with a slight increase in the proapoptotic protein Bax and, conversely, a decrease in the antiapoptotic protein XIAP. In addition, 8c induced phosphorylation of histone H2AX at serine 139 (γ-H2AX), indicative of DNA damage, which is likely the trigger responsible for the initial activation of caspases [67,68] (Figure 5).
To further support these findings, flow cytometry analysis of cell cycle distribution was performed in MV4-11 cells treated with 8c for 24 h, using propidium iodide staining. Treatment with 8c resulted in a dose-dependent decrease in the S-phase cell population, accompanied by a reduction in the number of cells progressing to the G2/M phase and a significant increase in the sub-G1 population. These changes are indicative of replication stress during the S-phase and corroborate the strong induction of cell death previously observed by immunoblotting (Figure 6).

2.6. Topoisomerase Relaxation Assay

Finally, we sought to investigate the underlying mechanism responsible for the replication stress induced by compound 8c, leading to cell death. Replication stress is often associated with inhibiting TOPs, enzymes essential for maintaining DNA topology during replication. Notably, several established TOP inhibitors, such as camptothecin (a TOPI inhibitor) and etoposide (a TOPII inhibitor), possess planar aromatic structures that facilitate DNA intercalation [44]. Given the planar aromatic character of the synthesized compound series, we hypothesized that 8c might interfere with the activity of TOPI or TOPII.
To test this hypothesis, we performed topoisomerase relaxation assays to evaluate the inhibitory potential of 8c on the ability of TOPI and TOPIIα to relax supercoiled plasmid DNA. Reaction products were separated by agarose gel electrophoresis and visualized using GelRed nucleic acid stain. Camptothecin (CPT) and etoposide were employed as positive controls for TOPI and TOPIIα inhibition, respectively. 8c did not inhibit TOPI-mediated DNA relaxation; however, it impaired the relaxation activity of TOPIIα in a concentration-dependent manner (Figure 7).

2.7. Kinase Assays

The newly prepared pyrazolo [3,4-b]pyridines (8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f) underwent further evaluation against a focused kinase panel (Abl, FLT3-ITD, and PDGFR) to assess off-target effects and confirm TOPIIα selectivity. At concentrations up to 10 µM, these molecules exhibited minimal inhibitory activity (Table S1) across the tested kinases, demonstrating no clinically relevant off-target interactions. Furthermore, we intend to extend the profiling to include a broader kinase panel in our future work, which will allow for a thorough assessment of kinome-wide selectivity.

2.8. Docking

The structure of 8c was subjected to molecular docking modelling in order to assess the potential mode of binding inside TOPII. As anticipated from the best docking pose, 8c fits well into the major groove region of the DNA double helix (Figure 8). The intercalation was observed for the cocrystal with the DNA bases DC8 and DC13, while 8c was observed with DC8, DC9, DC12, and DC13. The π-interactions were observed between these bases and the aromatic systems of 8c, including the main scaffold, one of the phenyl rings, the indole ring, and the phenolic ring. Additionally, the cyano group formed a hydrogen bond with Gln778, and a similar interaction occurred with one of the oxygen atoms of the cocrystal ligand. In addition, lipophilic interactions with Met782 and Arg503 were also observed with 8c. Furthermore, the OH group in the para position enabled sulfur-x interaction with Met782 and improved the π-stacking interaction with DT9. The docking score of 8c (−10.4 kcal/mol) was lower than that of the cocrystal ligand (−14.7 kcal/mol) while maintaining the important interaction patterns.

3. Materials and Methods

3.1. Chemistry

3.1.1. General

Commercial suppliers provided all of the materials, which were used exactly as supplied. Silica gel plates (Merck KGaA, Darmstadt, Germany) were subjected to TLC in order to track the purity and development of the reaction. Uncorrected melting points were measured using a Stuart SMP30 apparatus (Cole-Parmer Ltd., Stone, Staffordshire, UK), and standard equipment was used for elemental analysis using a Vario EL III elemental analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany), FTIR using a PerkinElmer Spectrum Two FTIR spectrometer (Waltham, MA, USA), and NMR (1H, 13C, and DEPT-135) were obtained on a Bruker Avance III HD 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). A Bruker MicroTOF spectrometer was used to record high-resolution mass spectra (Bruker Daltonics, Billerica, MA, USA). As stated in the literature, starting ingredients 2 [54], 5 [55], and 9 [55] were synthesized. A ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA) was used for HPLC analysis, which verified that the produced chemicals were more than 95% pure.

3.1.2. General Procedure for Synthesis of Pyrazolo [3,4-b]Pyridines (8a–g, 10a–g, and 12)

TLC was used to track the reaction progress of a combination of different aldehydes (3a–g and 11) (0.0015 mole), equimolar quantities of 3-oxo-3-arylpropanenitriles (2 and 4) (0.0015 mole), and 3-substituted-1-phenyl-1H-pyrazol-5-amines (5 and 9) (0.0015 mole) that were refluxed in 20 mL of absolute ethanol for 12 h. The reaction mixture was finished and then left to cool to room temperature. Filtration, air drying, and recrystallization of the resultant precipitate from ethanol produced the required very pure pyrazolo [3,4-b]pyridine derivatives (8a–g, 10a–g, and 12).

3.1.3. 6-(1H-Indol-3-yl)-1,3,4-Triphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8a)

Yield (85%) as a white powder, with Mp: 245 °C. HPLC: RT 7.098 min (purity: 96.00%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.68 (d, J = 2.9 Hz, 1H, NH), 7.81–7.78 (m, 3H, Arom-H), 7.63–7.57 (m, 4H, Arom-H), 7.49–7.39 (m, 3H, Arom-H), 7.33–7.29 (m, 6H, Arom-H), 7.20–7.16 (m, 2H, Arom-H), 7.13 (tt, J = 7.2, 2.3 Hz, 2H, Arom-H). 13C NMR (101 MHz, DMSOd6) δ (ppm): 149.7, 145.6, 145.3, 139.1, 138.2, 136.3, 134.5, 130.7, 129.8, 129.3, 129.1, 128.9, 128.3, 127.4, 127.2, 125.7, 125.2, 123.7, 122.4, 122.2, 121.4, 120.0, 111.8, 108.1, 99.4, 85.7. HRMS (ESI): m/z: [M + H]+ calcd. 488.1870 and found 488.1862. Anal. Calcd. (Found) For C33H21N5: C, 81.29 (81.15); H, 4.34 (4.35); N, 14.36 (14.29)%.

3.1.4. 4-(3-Hydroxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8b)

Yield (83%) as a white powder, with Mp: 232 °C. HPLC: RT 5.770 min (purity: 99.40%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.68 (d, J = 2.9 Hz, 1H, NH), 9.80 (s, 1H, OH), 7.81–7.78 (m, 3H, Arom-H), 7.72–7.54 (m, 7H, Arom-H), 7.21–7.07 (m, 5H, Arom-H), 6.83–6.74 (m, 2H, Arom-H), 6.71 (t, J = 2.1 Hz, 1H, Arom-H), 6.59 (dd, J = 8.1, 2.4 Hz, 1H, Arom-H). 13C NMR (101 MHz, DMSOd6) δ (ppm): 158.5, 150.5, 144.8, 143.8, 139.3, 136.0, 134.1, 130.8, 129.7, 128.6, 128.1, 128.0, 127.3, 124.0, 121.5, 119.0, 115.2, 114.6, 112.9, 102.1. HRMS (ESI): m/z: [M + H]+ calcd. 504.1819 and found 504.1810. Anal. Calcd. (Found) For C33H21N5O: C, 78.71 (78.87); H, 4.20 (4.18); N, 13.91 (13.86)%.

3.1.5. 4-(4-Hydroxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8c)

Yield (89%) as a white powder, with Mp: 295 °C. HPLC: RT 5.165 min (purity: 98.53%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.92 (s, 1H, NH), 9.85 (s, 1H, OH), 8.53–8.44 (m, 2H, Arom-H), 8.39–8.29 (m, 2H, Arom-H), 7.69–7.61 (m, 2H, Arom-H), 7.57 (dd, J = 8.3, 3.3 Hz, 1H, Arom-H), 7.49–7.43 (m, 1H, Arom-H), 7.31–7.25 (m, 2H, Arom-H), 7.23–7.11 (m, 7H, Arom-H), 6.71–6.56 (m, 2H, Arom-H). 13C NMR (126 MHz, DMSOd6) δ (ppm): 159.2, 156.3, 153.7, 151.0, 147.5, 138.9, 136.9, 132.1, 131.5, 130.1, 129.7, 129.4, 128.5, 128.0, 127.4, 126.4, 124.8, 123.1, 122.3, 122.0, 121.4, 119.7, 115.2, 113.3, 112.7, 110.8, 100.3. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 131.5 (↑), 130.1 (↑), 129.7 (↑), 129.4 (↑), 128.5 (↑), 128.0 (↑), 127.4 (↑), 123.1 (↑), 122.3 (↑), 122.0 (↑), 121.4 (↑), 115.2 (↑), 112.7 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 504.1819 and found 504.1809. Anal. Calcd. (Found) For C33H21N5O: C, 78.71 (78.59); H, 4.20 (4.23); N, 13.91 (13.97)%.

3.1.6. 4-(2-Hydroxy-3-Methoxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8d)

Yield (85%) as a white powder, with Mp: 271 °C. HPLC: RT 7.888 min (purity: 99.41%). 1H NMR (400 MHz, DMSOd6) δ (ppm): 11.33 (s, 1H, NH), 9.41 (s, 1H, OH), 8.45–6.76 (m, 18H, Arom-H), 3.88 (s, 3H, OCH3). 13C NMR (101 MHz, DMSOd6) δ (ppm): 160.3, 153.2, 150.0, 149.7, 147.1, 143.0, 139.1, 138.3, 136.2, 130.5, 129.9, 129.8, 128.9, 127.8, 127.5, 126.9, 126.2, 122.4, 121.7, 120.5, 118.3, 116.9, 112.5, 112.0, 106.4, 102.5, 56.2. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.38); H, 4.34 (4.30); N, 13.13 (13.09)%.

3.1.7. 4-(3-Hydroxy-4-Methoxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8e)

Yield (91%) as a white powder, with Mp: 266 °C. HPLC: RT 7.413 min (purity: 96.44%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.92 (s, 1H, NH), 9.13 (s, 1H, OH), 8.48 (d, J = 7.9 Hz, 2H, Arom-H), 8.36–8.33 (m, 2H, Arom-H), 7.67–7.63 (m, 2H, Arom-H), 7.57 (d, J = 8.0 Hz, 1H, Arom-H), 7.48–7.45 (m, 1H, Arom-H), 7.28 (ddt, J = 8.1, 5.6, 1.9 Hz, 2H, Arom-H), 7.22 (td, J = 7.5, 1.2 Hz, 1H, Arom-H), 7.18–7.11 (m, 4H, Arom-H), 6.82 (d, J = 2.1 Hz, 1H, Arom-H), 6.78 (d, J = 8.3 Hz, 1H, Arom-H), 6.73 (dd, J = 8.2, 2.1 Hz, 1H, Arom-H), 3.77 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 156.2, 153.4, 150.9, 149.3, 147.4, 146.8, 138.9, 136.9, 132.1, 130.1, 129.7, 129.2, 128.6, 127.9, 127.4, 126.9, 126.4, 123.1, 122.4, 122.0, 121.4, 119.5, 117.0, 113.3, 112.7, 112.2, 110.8, 100.3, 56.3. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.1 (↑), 129.7 (↑), 129.2 (↑), 128.6 (↑), 127.9 (↑), 127.4 (↑), 123.1 (↑), 122.4 (↑), 122.0 (↑), 121.4 (↑), 117.0 (↑), 112.7 (↑), 112.2 (↑), 56.3 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 534.1925 and found 534.1922. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.65); H, 4.34 (4.32); N, 13.13 (13.16)%.

3.1.8. 4-(4-Hydroxy-3-Methoxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8f)

Yield (87%) as a white powder, with Mp: 239 °C. HPLC: RT 7.355 min (purity: 98.55%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.92 (d, J = 2.9 Hz, 1H, NH), 9.40 (s, 1H, OH), 8.49 (d, J = 2.7 Hz, 1H, Arom-H), 8.47 (d, J = 7.9 Hz, 1H, Arom-H), 8.36–8.31 (m, 2H, Arom-H), 7.68–7.63 (m, 2H, Arom-H), 7.57 (d, J = 8.0 Hz, 1H, Arom-H), 7.49–7.45 (m, 1H, Arom-H), 7.31–7.25 (m, 2H, Arom-H), 7.22 (td, J = 7.4, 1.2 Hz, 1H, Arom-H), 7.17 (d, J = 4.4 Hz, 4H, Arom-H), 7.05 (dd, J = 8.1, 2.1 Hz, 1H, Arom-H), 6.82 (d, J = 8.1 Hz, 1H, Arom-H), 6.74 (d, J = 2.0 Hz, 1H, Arom-H), 3.34 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 156.4, 153.5, 151.0, 148.6, 147.5, 147.4, 138.9, 136.9, 132.4, 130.2, 129.7, 129.4, 128.6, 127.9, 127.4, 126.4, 125.0, 123.1, 123.0, 122.4, 122.0, 121.4, 119.8, 115.5, 114.9, 113.3, 112.7, 110.7, 100.2, 55.7. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.2 (↑), 129.7 (↑), 129.4 (↑), 128.6 (↑), 127.9 (↑), 127.4 (↑), 123.1 (↑), 123.0 (↑), 122.4 (↑), 122.0 (↑), 121.4 (↑), 115.5 (↑), 114.9 (↑), 112.7 (↑), 55.7 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 534.1925 and found 534.1912. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.67); H, 4.34 (4.38); N, 13.13 (13.18)%.

3.1.9. 4-(3,4-Dimethoxyphenyl)-6-(1H-Indol-3-yl)-1,3-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (8g)

Yield (88%) as a white powder, with Mp: 228 °C. HPLC: RT 10.632 min (purity: 99.55%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 12.26 (s, 1H, NH), 8.44 (s, 2H, Arom-H), 8.22–8.18 (m, 4H, Arom-H), 7.81 (d, J = 2.2 Hz, 2H, Arom-H), 7.73 (dd, J = 8.5, 2.1 Hz, 2H, Arom-H), 7.56 (d, J = 7.1 Hz, 2H), 7.28 (td, J = 7.6, 1.5 Hz, 4H, Arom-H), 7.19 (d, J = 8.5 Hz, 2H, Arom-H), 3.88 (s, 3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 181.9, 153.1, 152.9, 149.1, 137.1, 135.8, 126.7, 126.3, 125.4, 123.9, 122.8, 121.9, 119.1, 114.2, 113.3, 112.9, 112.3, 108.4, 56.3, 56.0. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 152.9 (↑), 135.8 (↑), 126.3 (↑), 123.9 (↑), 122.8 (↑), 121.9 (↑), 113.3 (↑), 112.9 (↑), 112.3 (↑), 56.3 (↑), 56.0 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 548.2081 and found 548.2080. Anal. Calcd. (Found) For C35H25N5O2: C, 76.77 (76.91); H, 4.60 (4.57); N, 12.79 (12.89)%.

3.1.10. 3-(1H-Indol-3-yl)-1,4,6-Triphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10a)

Yield (86%) as a white powder, with Mp: 280 °C. HPLC: RT 7.001 min (purity: 98.27%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.22–11.19 (m, 1H, NH), 8.38 (d, J = 8.0 Hz, 2H, Arom-H), 8.20 (d, J = 7.7 Hz, 1H, Arom-H), 8.02 (dd, J = 6.6, 2.9 Hz, 2H, Arom-H), 7.68–7.61 (m, 6H, Arom-H), 7.59 (s, 1H, Arom-H), 7.56–7.53 (m, 2H, Arom-H), 7.48 (t, J = 7.6 Hz, 2H, Arom-H), 7.42 (t, J = 7.4 Hz, 1H, Arom-H), 7.35 (d, J = 7.8 Hz, 1H, Arom-H), 7.17–7.12 (m, 2H, Arom-H). 13C NMR (126 MHz, DMSOd6) δ (ppm): 160.4, 153.1, 150.0, 142.8, 139.0, 138.2, 136.1, 135.0, 130.6, 130.5, 129.9, 129.9, 129.9, 129.0, 128.9, 127.3, 127.1, 126.2, 122.4, 121.8, 121.4, 120.5, 118.1, 112.4, 112.0, 106.3, 102.4. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.6 (↑), 130.5 (↑), 129.9 (↑), 129.9 (↑), 129.9 (↑), 129.0 (↑), 128.9 (↑), 127.3 (↑), 127.1 (↑), 122.4 (↑), 121.8 (↑), 121.4 (↑), 120.5 (↑), 112.0 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 488.1870 and found 488.1868. Anal. Calcd. (Found) For C33H21N5: C, 81.29 (81.42); H, 4.34 (4.37); N, 14.36 (14.41)%.

3.1.11. 4-(3-Hydroxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10b)

Yield (83%) as a white powder, with Mp: 247 °C. HPLC: RT 5.736 min (purity: 99.79%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.32 (d, J = 2.9 Hz, 1H, NH), 9.88 (s, 1H, OH), 8.40 (d, J = 8.0 Hz, 2H, Arom-H), 8.03–8.01 (m, 2H, Arom-H), 7.68–7.62 (m, 6H, Arom-H), 7.39–7.32 (m, 3H, Arom-H), 7.20–7.13 (m, 3H, Arom-H), 7.04–7.02 (m, 1H, Arom-H), 6.98 (d, J = 7.6 Hz, 1H, Arom-H), 6.96 (d, J = 2.1 Hz, 1H, Arom-H). HRMS (ESI): m/z: [M+H]+ calcd. 504.1819 and found 504.1824. Anal. Calcd. (Found) For C33H21N5O: C, 78.71 (78.90); H, 4.20 (4.19); N, 13.91 (13.94)%.

3.1.12. 4-(4-Hydroxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10c)

Yield (85%) as a white powder, with Mp: 259 °C. HPLC: RT 5.054 min (purity: 99.32%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.33 (d, J = 2.8 Hz, 1H, NH), 10.07 (s, 1H, OH), 8.38 (d, J = 8.1 Hz, 2H, Arom-H), 8.24–8.22 (m, 1H, Arom-H), 8.02–8.00 (m, 2H, Arom-H), 7.65–7.61 (m, 5H, Arom-H), 7.42 (s, 1H, Arom-H), 7.37 (dd, J = 8.6, 6.9 Hz, 3H, Arom-H), 7.20–7.09 (m, 3H, Arom-H), 6.86–6.83 (m, 2H, Arom-H). HRMS (ESI): m/z: [M + H]+ calcd. 504.1819 and found 504.1817. Anal. Calcd. (Found) For C33H21N5O: C, 78.71 (78.86); H, 4.20 (4.16); N, 13.91 (13.95)%.

3.1.13. 4-(2-Hydroxy-3-Methoxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10d)

Yield (90%) as a white powder, with Mp: 277 °C. HPLC: RT 7.845 min (purity: 99.81%). 1H NMR (400 MHz, DMSOd6) δ (ppm): 11.67 (s, 1H, NH), 9.21 (s, 1H, OH), 8.27 (d, J = 7.6 Hz, 1H, Arom-H), 7.88 (d, J = 2.5 Hz, 1H, Arom-H), 7.81–7.71 (m, 3H, Arom-H), 7.60 (t, J = 7.9 Hz, 3H, Arom-H), 7.51–7.42 (m, 3H, Arom-H), 7.32–7.30 (m, 1H, Arom-H), 7.21–7.14 (m, 5H, Arom-H), 6.95 (s, 1H, Arom-H), 3.82 (s, 3H, OCH3). 13C NMR (101 MHz, DMSOd6) δ (ppm): 163.3, 150.1, 148.9, 148.8, 148.4, 139.4, 137.0, 129.6, 127.6, 125.4, 124.6, 124.5, 123.4, 122.1, 121.4, 120.2, 120.1, 119.7, 116.3, 112.2, 109.3, 91.9, 56.3. HRMS (ESI): m/z: [M + H]+ calcd. 534.1925 and found 534.1918. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.66); H, 4.34 (4.32); N, 13.13 (13.16)%.

3.1.14. 4-(3-Hydroxy-4-Methoxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10e)

Yield (93%) as a white powder, with Mp: 249 °C. HPLC: RT 7.380 min (purity: 99.95%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.35 (d, J = 2.9 Hz, 1H, NH), 9.45 (s, 1H, OH), 8.39 (d, J = 8.0 Hz, 2H, Arom-H), 8.33–8.30 (m, 1H, Arom-H), 8.03–7.99 (m, 2H, Arom-H), 7.67–7.62 (m, 5H, Arom-H), 7.55–7.35 (m, 3H, Arom-H), 7.17 (tt, J = 7.1, 5.4 Hz, 2H, Arom-H), 7.05 (d, J = 8.3 Hz, 1H, Arom-H), 6.98 (d, J = 2.2 Hz, 1H, Arom-H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H, Arom-H), 3.89 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 160.4, 153.3, 150.0, 149.7, 147.1, 143.0, 139.1, 138.3, 136.2, 130.6, 129.9, 129.9, 129.6, 129.0, 128.6, 127.8, 127.5, 127.0, 126.2, 122.4, 121.8, 121.7, 121.2, 120.6, 118.3, 116.9, 112.5, 112.4, 112.0, 106.4, 102.6, 56.2. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.6 (↑), 129.9 (↑), 129.9 (↑), 129.0 (↑), 127.8 (↑), 127.0 (↑), 122.4 (↑), 121.8 (↑), 121.7 (↑), 121.2 (↑), 120.6 (↑), 116.9 (↑), 112.5 (↑), 112.0 (↑), 56.2 (OCH3 ↑). HRMS (ESI): m/z: [M + H]+ calcd. 534.1925 and found 534.1929. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.38); H, 4.34 (4.28); N, 13.13 (13.08)%.

3.1.15. 4-(4-Hydroxy-3-Methoxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10f)

Yield (88%) as a white powder, with Mp: 276 °C. HPLC: RT 7.283 min (purity: 99.42%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.34 (d, J = 2.8 Hz, 1H, NH), 9.61 (s, 1H, OH), 8.38 (d, J = 8.3 Hz, 2H, Arom-H), 8.17 (d, J = 7.8 Hz, 1H, Arom-H), 8.04–7.99 (m, 2H, Arom-H), 7.67–7.59 (m, 5H, Arom-H), 7.45–7.36 (m, 2H, Arom-H), 7.15 (dt, J = 21.7, 7.2 Hz, 2H, Arom-H), 7.05 (dt, J = 8.1, 1.8 Hz, 1H, Arom-H), 7.01 (d, J = 1.7 Hz, 1H, Arom-H), 6.88 (dd, J = 8.0, 1.4 Hz, 1H, Arom-H), 6.16 (t, J = 2.0 Hz, 1H, Arom-H), 3.38 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 160.6, 153.4, 150.1, 148.9, 147.7, 142.9, 139.0, 138.3, 136.1, 130.6, 130.0, 129.8, 128.9, 127.7, 127.0, 126.2, 125.1, 123.2, 122.3, 121.8, 121.2, 120.5, 118.6, 115.7, 114.9, 112.4, 111.9, 106.5, 102.5, 55.9. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.6 (↑), 130.0 (↑), 129.8 (↑), 128.9 (↑), 127.7 (↑), 127.0 (↑), 123.3 (↑), 122.3 (↑), 121.8 (↑), 121.2 (↑), 120.5 (↑), 115.7 (↑), 114.9 (↑), 111.9 (↑), 55.9 (OCH3 ↑). HRMS (ESI): m/z: [M + H]+ calcd. 534.1925 and found 534.1920. Anal. Calcd. (Found) For C34H23N5O2: C, 76.53 (76.37); H, 4.34 (4.33); N, 13.13 (13.11)%.

3.1.16. 4-(3,4-Dimethoxyphenyl)-3-(1H-Indol-3-yl)-1,6-Diphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (10g)

Yield (91%) as a white powder, with Mp: 284 °C. HPLC: RT 9.795 min (purity: 99.32%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.30 (d, J = 2.8 Hz, 1H, NH), 8.38 (d, J = 8.0 Hz, 2H, Arom-H), 8.12 (d, J = 7.9 Hz, 1H, Arom-H), 8.05–8.00 (m, 2H, Arom-H), 7.69–7.61 (m, 5H, Arom-H), 7.41 (dd, J = 20.8, 7.7 Hz, 2H, Arom-H), 7.19–7.01 (m, 5H, Arom-H), 6.12 (d, J = 2.7 Hz, 1H, Arom-H), 3.85 (s, 3H, OCH3), 3.36 (s, 3H, OCH3). 13C NMR (126 MHz, DMSOd6) δ (ppm): 160.6, 153.1, 150.7, 150.1, 148.7, 142.9, 139.0, 138.3, 136.1, 130.6, 130.0, 129.9, 129.0, 127.6, 127.1, 126.6, 126.2, 122.9, 122.3, 121.8, 121.1, 120.4, 118.5, 114.3, 112.5, 112.0, 111.9, 106.4, 102.4, 56.2, 55.7. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.6 (↑), 130.0 (↑), 129.9 (↑), 129.8 (↑), 129.0 (↑), 127.6 (↑), 127.1 (↑), 122.9 (↑), 122.3 (↑), 121.8 (↑), 121.1 (↑), 120.4 (↑), 114.3 (↑), 112.0 (↑), 111.9 (↑), 56.2 (OCH3 ↑), 55.7 (OCH3 ↑). HRMS (ESI): m/z: [M + H]+ calcd. 548.2081 and found 548.2074 Anal. Calcd. (Found) For C35H25N5O2: C, 76.77 (76.59); H, 4.60 (4.62); N, 12.79 (12.85)%.

3.1.17. 4-(1H-Indol-3-yl)-1,3,6-Triphenyl-1H-Pyrazolo [3,4-b]Pyridine-5-Carbonitrile (12)

Yield (89%) as a white powder, with Mp: 257 °C. HPLC: RT 7.166 min (purity: 99.25%). 1H NMR (500 MHz, DMSOd6) δ (ppm): 11.62 (d, J = 2.8 Hz, 1H, NH), 8.35–8.29 (m, 2H, Arom-H), 8.09–8.03 (m, 2H, Arom-H), 7.66–7.61 (m, 5H, Arom-H), 7.46–7.39 (m, 4H, Arom-H), 7.18–7.09 (m, 4H, Arom-H), 7.02–6.94 (m, 3H, Arom-H). 13C NMR (126 MHz, DMSOd6) δ (ppm): 161.4, 150.7, 147.7, 147.6, 138.8, 138.4, 136.2, 132.2, 130.6, 130.0, 129.8, 129.0, 128.9, 128.7, 127.7, 127.4, 126.2, 122.4, 122.3, 120.4, 120.0, 118.9, 112.7, 112.2, 109.0, 102.9. 13C NMR-DEPT-135 (126 MHz, DMSOd6) δ: 130.6 (↑), 130.0 (↑), 129.8 (↑), 129.0 (↑), 128.9 (↑), 128.9 (↑), 128.7 (↑), 127.7 (↑), 127.4 (↑), 122.4 (↑), 122.3 (↑), 120.4 (↑), 120.0 (↑), 112.2 (↑). HRMS (ESI): m/z: [M + H]+ calcd. 488.18697 and found 488.18698. Anal. Calcd. (Found) For C33H21N5: C, 81.29 (81.44); H, 4.34 (4.32); N, 14.36 (14.41)%.

3.2. Biological Evaluation

3.2.1. In Vitro Antitumor Screening Against 60 Cancer Cell Lines

In accordance with the NCI’s usual technique, the synthetic compounds’ in vitro anticancer activity was assessed against a panel of 60 human tumor cell lines from nine different tissue types (Figures S67–S105). To evaluate each compound’s potency and cytotoxic profile, key dose–response parameters, including GI50, TGI, and LC50, were computed [69,70,71,72,73,74,75,76,77], and detailed data are provided in the Supplementary Information.

3.2.2. Cell Lines

Recognized cell banks provided the MV4-11 and K562 cancer cell lines, which were then cultivated at 37 °C in a humidified CO2 incubator under standard conditions in RPMI-1640 or DMEM medium supplemented with fetal bovine serum and antibiotics, and detailed data are provided in the Supplementary Materials Information.

3.2.3. Cell Viability Assay

In order to calculate GI50 values from dose–response curves using fluorescence measurements, cells were seeded in 96-well plates and treated with different doses of the test substances for 72 h. This was followed by a viability assessment based on resazurin, and detailed data are provided in the Supplementary Materials Information.

3.2.4. Flow Cytometry

The test substance was applied at different concentrations to asynchronously developing cells, which were then harvested after 24 h, fixed in ice-cold 70% ethanol, and incubated on ice for 30 min. Cell cycle distribution was examined by flow cytometry with a 488 nm laser after PBS washing and propidium iodide staining, and ModFit LT software (version 5.0.9) was used to quantify the results, and detailed data are provided in the Supplementary Materials Information.

3.2.5. Immunoblotting

RIPA buffer was used to generate cell lysates, and proteins were separated using SDS-PAGE before being transferred to nitrocellulose membranes. Following blocking, membranes were incubated with certain primary antibodies overnight at 4 °C. Peroxidase-conjugated secondary antibodies were then added, and SuperSignal West Pico reagents and a LAS-4000 CCD camera were used for detection, and detailed data are provided in the Supplementary Materials Information.

3.2.6. Topoisomerase Relaxation Assay

Supercoiled pBR322 plasmid and the corresponding assay buffers were used for the Topoisomerase I and IIα assays. The assays were then incubated for 30 min at 37 °C at 350 rpm. Complete experimental details are included in the Supplementary Materials Information. Reactions were halted, products were separated using 5% agarose gel electrophoresis, and they were then seen using GelRed staining and a FLA-7000 digital image analyzer.

3.2.7. Molecular Docking

The RCSB-PDB site was used to obtain the coordinates of TOPII (PDB: 3QX3 [78]). The three-dimensional structures of 8c and the cocrystal ligand were prepared and optimized using Marvin Sketch [79] as part of the docking investigation. AutoDock Vina [80] was used to carry out docking operations. The active site’s coordinates (x, y, z) were 32.9/95.4/50.8 with a size 24.5/20.2/17.5. The 2D schematic presentation and 3D visualization were created using the Discovery Studio 2021 client [81].

4. Conclusions

The synthesized pyrazolo [3,4-b]pyridine derivatives demonstrated antiproliferative activity, particularly against leukemia-derived cancer cell lines, with 8c emerging as the most potent candidate. Detailed mechanistic studies revealed that 8c induces DNA damage leading to cell death in MV4-11 cells, as evidenced by caspase activation, PARP-1 cleavage, and modulation of pro- and antiapoptotic proteins. Additionally, 8c caused S-phase cell cycle perturbations indicative of replication stress and confirmed strong induction of cell death. Topoisomerase relaxation assays identified TOPIIα as a potential molecular target, with 8c inhibiting its activity in a concentration-dependent manner. These findings highlight 8c as a promising lead compound for the further development of anticancer agents targeting TOPIIα-associated pathways.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph18111770/s1, Figures S1–S37. NMR spectra of compounds 8a-g, 10a-g and 12, Figures S38–S51. HRMS spectra of compounds 8a-c, 8e-g, 10a-g and 12, Figures S52–S66. HPLC spectrum of compounds 8a-g, 10a-g and 12: title, Figures S67–S105. NCI charts of compounds 8a-g, 10a-g and 12; Table S1. Kinases assays.

Author Contributions

Conceptualization, W.M.E., V.K. and H.A.A.-A.; Formal analysis, W.M.E., H.O.T., A.T.N., T.A.M., M.M.E., P.K. and V.K.; Investigation, D.V., V.V., A.T.N., Z.M.E., M.A.S., P.K. and G.H.A.-A.; Resources, W.M.E. and V.K.; Writing—original draft preparation, W.M.E., H.O.T., Z.M.E., D.V. and V.K.; Writing—review and editing, H.O.T., D.V., A.T.N., M.A.S. and V.K.; Visualization, D.V., M.M.E., G.H.A.-A., V.K. and H.A.A.-A.; Supervision, W.M.E., and V.K.; Project administration, W.M.E., and V.K.; Funding acquisition, W.M.E., V.K. and H.A.A.-A. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number (RGP2/500/46). The study was also supported by The European Union—Next Generation EU (The project National Institute for Cancer Research, Programme EXCELES, ID No. LX22NPO5102) and Palacky University (IGA_PrF_2025_011).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structure of some reported pyrazolo [3,4-b]pyridine (I–XI) derivatives alongside their biological activity.
Figure 1. Structure of some reported pyrazolo [3,4-b]pyridine (I–XI) derivatives alongside their biological activity.
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Figure 2. Structure of some reported indole (XII–XVI) derivatives alongside their biological activity.
Figure 2. Structure of some reported indole (XII–XVI) derivatives alongside their biological activity.
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Figure 3. The adopted hybridization strategy for the design of small molecules 8a–g, 10a–g, and 12.
Figure 3. The adopted hybridization strategy for the design of small molecules 8a–g, 10a–g, and 12.
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Scheme 1. The synthetic route of target pyrazolo [3,4-b]pyridine compounds (8a–g). Reagents and conditions: i. Cyanoacetic acid, acetic anhydride, 85 °C 30 min; ii. Abs. EtOH, C6H5NHNH2, reflux 1 h; iii. Abs. EtOH, reflux 12 h.
Scheme 1. The synthetic route of target pyrazolo [3,4-b]pyridine compounds (8a–g). Reagents and conditions: i. Cyanoacetic acid, acetic anhydride, 85 °C 30 min; ii. Abs. EtOH, C6H5NHNH2, reflux 1 h; iii. Abs. EtOH, reflux 12 h.
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Scheme 2. The synthetic route of target pyrazolo [3,4-b]pyridine compounds (10a–g and 12). Reagents and conditions: i. Abs. EtOH, C6H5NHNH2, reflux 1 h; ii. Abs. EtOH, reflux 12 h.
Scheme 2. The synthetic route of target pyrazolo [3,4-b]pyridine compounds (10a–g and 12). Reagents and conditions: i. Abs. EtOH, C6H5NHNH2, reflux 1 h; ii. Abs. EtOH, reflux 12 h.
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Figure 4. The target compounds’ structure–activity relationship (SAR) as in vitro anticancer agents based on their mean GI% values across 60 human cancer cell lines.
Figure 4. The target compounds’ structure–activity relationship (SAR) as in vitro anticancer agents based on their mean GI% values across 60 human cancer cell lines.
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Figure 5. Effect of 8c on the induction of cell death and DNA damage in MV4-11 cells treated for 24 h with indicated concentrations (µM). Representative results from three independent experiments are presented; quantitative data are provided in the Supplementary Materials Information.
Figure 5. Effect of 8c on the induction of cell death and DNA damage in MV4-11 cells treated for 24 h with indicated concentrations (µM). Representative results from three independent experiments are presented; quantitative data are provided in the Supplementary Materials Information.
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Figure 6. The cell cycle phases (%) in MV4-11 cells treated with the indicated concentrations (µM) of compound 8c for 24 h.
Figure 6. The cell cycle phases (%) in MV4-11 cells treated with the indicated concentrations (µM) of compound 8c for 24 h.
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Figure 7. Effect of 8c on the ability of (A) TOPI and (B) TOPIIα to relax supercoiled plasmid DNA at the indicated concentrations using camptothecin (CPT) and etoposide as references.
Figure 7. Effect of 8c on the ability of (A) TOPI and (B) TOPIIα to relax supercoiled plasmid DNA at the indicated concentrations using camptothecin (CPT) and etoposide as references.
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Figure 8. Molecular docking inside the active site of TOPII (PDB: 3QX3); (A) overlay of the docked and cocrystal ligand “green” with RMSD 0.3503 and (B) docking of 8c.
Figure 8. Molecular docking inside the active site of TOPII (PDB: 3QX3); (A) overlay of the docked and cocrystal ligand “green” with RMSD 0.3503 and (B) docking of 8c.
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Table 1. Percentage cell growth inhibition (GI%) of an in vitro panel of 60 human tumor cell lines at 10 µM concentration of compounds 8a–g, 10a–g, and 12.
Table 1. Percentage cell growth inhibition (GI%) of an in vitro panel of 60 human tumor cell lines at 10 µM concentration of compounds 8a–g, 10a–g, and 12.
Cell LinesGrowth Inhibition Percentage (GI%)
8a8b8c8d8e8f8g10a10b10c10d10e10f10g12
LeukemiaCCRF-CEM308083֍8399֍63L9280L894856
HL-60(TB)129993֍9593֍18LL61L93֍25
K-562699596֍9999117397951599956460
MOLT-4468284֍8994֍67L9942L904859
RPMI-822629L96159698֍44L9232L912135
SRNTNTNTNTNTNTNT69LNT38L9931NT
Lung
Cancer (NSC)		A5492296L֍L91֍398893֍LL3934
EKVX27LL15LL2970LL28LL5027
HOP-6229LL22LL1222LL֍LL1816
HOP-92֍LL֍8270֍NTNT89NTNTNTNT֍
NCI-H22636LL48LL2042LL13LL2330
NCI-H2315LL֍LL2052LL15LL2339
NCI-H322M11LL֍L90֍1973982090822613
NCI-H4606193L֍LL֍75LL֍LL4247
NCI-H522֍90L֍L98֍LLL21LL4419
Colon
Cancer		COLO 20513LL֍LL֍37LL֍LL1613
HCC-299823LL֍LL֍70LL֍LL3425
HCT-11660LL֍LL֍4498L֍96L4352
HCT-1580LL14LL֍56LL23LL3556
HT2936LL֍LL֍41LL֍LL2946
KM 1240LL֍LL֍69LL֍LL3934
SW-62027LL֍LL֍37LL֍LL1113
CNS
Cancer		SF-268176592158172֍41885629L933115
SF-29545LL22LL֍35LL19LL2623
SF-53953LL14LL1226LL11LL1428
SNB-1932LL24L991140L8722L903316
SNB-75186591139983֍25704728857226֍
U25152LL13LL֍38LL17LL3035
MelanomaLOX IMVI66LL13LL1149LL17LL3340
MALME-3M25LL13LL֍58LL13LL6617
M14֍LL֍LL֍3198L֍L922718
MDA-MB-43516LL֍L97֍44LL֍LL3213
SK-MEL-2֍67L֍9844֍40L2916LL21֍
SK-MEL-2826LL֍LL֍57L93֍LL60֍
SK-MEL-525LL26LL143087L22L53֍42
UACC-257֍5982֍L55֍246051֍909615֍
UACC-6216LL12LL֍19666413L5523֍
Ovarian CancerIGROV119LL֍L97֍27L98֍LL1715
OVCAR-3֍LL֍LL֍֍LL֍LL֍֍
OVCAR-4֍9599229387֍2582701996912912
OVCAR-5֍LL֍LL֍15L98֍L92֍֍
OVCAR-836LL18L96֍28L9928L992819
ADR-RES40LL21LL֍29LL24LL2817
SK-OV-3֍5783֍6633֍257633֍8278֍֍
Renal
Cancer		786-06493L֍LL֍4189L1499812819
A498֍LL֍LL֍54LL֍LL֍֍
ACHN44LL֍9798֍36LL֍L862226
CAKI-11599L1792851754LL27LL4931
RXF 393LLL36LL3171LL21LL7249
SN 12C17LL14LL֍48LL14LL2926
TK-10֍6882֍6357֍֍5045֍6158֍֍
UO-3142LL12LL293346L40LL3547
Prostate CancerPC-337LL12L92֍NTNT98NTNTNTNT58
DU-14521LL֍LL֍42LL֍LL3215
Breast
Cancer		MCF746LL16LL1656LL21LL4157
MDA-MB-23131LL֍LL2239LL30LL4227
HS 578T32LL18L97֍52LL֍LL6319
BT-549֍LL֍LL114183L֍LL28֍
T-47D֍8374147260֍66L743196993948
MDA-MB-468֍LL֍LL֍35LL֍L981941
GI Mean241201548138125343126118151381222923
Compounds that were not tested are indicated by “NT”. A growth inhibition percentage (GI%) of less than 10% denotes low cytotoxic activity, as shown by the symbol “֍”. Moderate anticancer activity is indicated by a GI% between 10 and 60, and substantial growth inhibition is indicated by values between 60 and 100. The letter “L” stands for GI% values higher than 100%, which suggests strong cytotoxicity and a fatal impact on the cancer cells. The effectiveness of each chemical throughout the NCI-60 cancer cell line panel can be interpreted with the use of these activity classifications.
Table 2. Five dose in vitro anticancer activity results expressed as GI50 (μM) for compounds 8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f against all sixty cancer cell lines.
Table 2. Five dose in vitro anticancer activity results expressed as GI50 (μM) for compounds 8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f against all sixty cancer cell lines.
Subpanel
Cell Lines		GI50 (μM)
8b8c8e8f10b10c10e10f
LeukemiaCCRF-CEM2.670.320.742.444.282.852.922.95
HL-60(TB)3.470.650.994.773.563.042.922.47
K-5622.670.451.623.683.212.742.852.07
MOLT-42.820.481.233.773.312.002.362.12
RPMI-82262.900.880.573.744.27֍2.522.39
SR2.870.480.712.763.572.602.532.21
Lung
Cancer (NSC)		A5492.841.572.343.323.434.072.662.80
EKVX2.371.271.462.923.212.362.752.37
HOP-622.321.163.522.302.113.191.732.02
HOP-921.392.311.182.652.923.102.872.65
NCI-H2262.191.871.861.84֍֍֍4.03
NCI-H232.251.441.522.681.862.021.881.86
NCI-H322M2.121.892.083.834.00֍3.242.96
NCI-H4602.361.272.172.102.673.091.751.90
NCI-H5222.401.682.382.462.071.831.802.03
Colon
Cancer		COLO 2051.632.052.612.021.922.791.651.85
HCC-29982.221.591.501.811.782.091.851.90
HCT-1162.321.371.621.781.912.331.831.75
HCT-151.971.161.261.701.743.412.391.90
HT292.541.242.282.153.053.141.841.82
KM 121.591.341.542.041.983.091.911.73
SW-620֍2.26֍֍2.893.141.503.12
CNS
Cancer		SF-2682.793.643.654.672.85֍2.982.61
SF-2952.080.230.372.022.61֍2.432.14
SF-5391.690.310.621.803.72֍2.151.82
SNB-19NTNTNTNT֍NT4.263.31
SNB-751.840.230.442.132.043.432.172.81
U2511.790.620.852.282.193.181.851.67
MelanomaLOX IMVI1.531.231.361.691.661.901.701.65
MALME-3M2.122.542.313.052.723.141.671.68
M142.371.572.032.362.95֍2.412.20
MDA-MB-4352.311.302.712.032.413.122.192.16
SK-MEL-22.311.492.564.08֍֍3.994.93
SK-MEL-283.211.361.944.02֍֍2.742.91
SK-MEL-52.131.661.523.87֍֍4.703.13
UACC-2574.124.43֍֍֍֍4.70֍
UACC-621.721.372.282.86֍֍֍֍
Ovarian
Cancer		IGROV11.822.221.934.713.69֍2.812.75
OVCAR-32.091.611.351.851.583.141.881.96
OVCAR-43.082.84֍4.493.67֍2.942.69
OVCAR-52.471.542.782.78֍֍3.053.53
OVCAR-82.582.132.583.323.654.673.082.73
NCI/ADR-RES2.141.871.822.152.73֍3.122.41
SK-OV-33.672.414.00֍3.561.302.253.75
Renal
Cancer		786-03.220.360.841.922.154.371.872.00
A4984.901.202.012.034.88֍2.812.08
ACHN2.391.091.333.163.473.593.172.41
CAKI-12.481.181.793.382.883.322.602.50
RXF 3932.170.150.231.893.344.572.712.33
SN 12C1.601.672.211.772.202.461.941.58
TK-10NTNTNTNT1.58NT4.144.33
UO-311.830.931.652.082.47֍2.101.81
Prostate
Cancer		PC-31.721.101.483.252.763.232.762.56
DU-1452.201.401.762.242.192.712.111.75
Breast
Cancer		MCF71.920.591.211.842.982.872.642.22
MDA-MB-2311.961.461.782.052.212.251.921.48
HS 578T1.750.851.211.933.012.011.763.19
BT-5492.961.273.952.533.234.783.762.19
T-47D2.612.804.683.572.103.662.503.85
MDA-MB-4682.431.841.683.033.14֍2.792.26
“NT” Not tested. “֍” indicates GI50 equal to or more than 5 μM. Red bolded numbers indicate activity less than 1 μM.
Table 3. Selectivity ratio of the compounds 8b, 8c, 8e, 8f, 10b, 10c, 10e and 10f towards NCI nine tumor subpanels.
Table 3. Selectivity ratio of the compounds 8b, 8c, 8e, 8f, 10b, 10c, 10e and 10f towards NCI nine tumor subpanels.
CodeParameterPanelGI50-MIDMID a
LeukemiaLung Cancer (NSC)Colon CancerCNS CancerMelanomaOvarian CancerRenal CancerProstate CancerBreast Cancer
8bMID b2.902.242.932.032.422.552.651.962.2721.952.43
Selectivity c0.831.080.821.191.000.950.911.231.07
8cMID b0.541.601.251.001.882.080.941.251.4612.001.33
Selectivity c2.460.831.061.330.700.631.331.060.91
8eMID b0.972.052.421.182.492.801.431.622.4117.371.93
Selectivity c1.980.940.791.630.770.681.341.190.80
8fMID b3.522.673.672.583.823.632.312.742.4927.433.04
Selectivity c0.861.130.821.170.790.831.311.101.22
10bMID b3.703.422.183.256.163.572.872.472.7730.393.37
Selectivity c0.910.981.541.030.540.941.171.361.21
10cMID b3.193.542.855.126.604.595.812.973.4338.104.23
Selectivity c1.321.191.480.820.640.920.721.421.23
10eMID b2.682.721.852.643.602.732.662.432.5623.872.65
Selectivity c0.980.971.431.000.730.970.991.091.03
10fMID b2.362.512.012.393.322.832.382.152.5322.482.49
Selectivity c1.050.991.231.040.750.871.041.150.98
a MID is the average sensitivity of all cell lines in µM; b MID is the average sensitivity of all cell lines in a particular subpanel in µM; c Selectivity is the ratio of average sensitivity of all cell lines to the average sensitivity of all cell lines in a particular subpanel.
Table 4. Antiproliferative activity of pyrazolo [3,4-b]pyridines 8b, 8c, 8e, 8f, 10b, 10c, 10e and 10f against leukemia MV4-11 and K562 cell lines.
Table 4. Antiproliferative activity of pyrazolo [3,4-b]pyridines 8b, 8c, 8e, 8f, 10b, 10c, 10e and 10f against leukemia MV4-11 and K562 cell lines.
Pharmaceuticals 18 01770 i001Pharmaceuticals 18 01770 i002
CodeArGI50 (µM) a ± SDCodeArGI50 (µM) a ± SD
MV4-11K562MV4-11K562
8b3-OH-Ph3.55 ± 0.376.52 ± 2.2410b3-OH-Ph8.88 ± 0.043.32 ± 0.11
8c4-OH-Ph0.72 ± 0.270.73 ± 0.0110c4-OH-Ph8.03 ± 1.412.50 ± 0.30
8e3-OH-4-OCH3-Ph>10>1010e3-OH-4-OCH3-Ph9.03 ± 0.205.13 ± 0.12
8f4-OH-3-OCH3-Ph3.70 ± 0.043.17 ± 0.0310f4-OH-3-OCH3-Ph8.73 ± 0.404.00 ± 0.21
Cisplatin0.75 ± 0.214.57 ± 1.40Quizartinib0.003 ± 2.24>10
a Assayed in triplicate.
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Eldehna, W.M.; Tawfik, H.O.; Veselá, D.; Vojáčková, V.; Negmeldin, A.T.; Elsayed, Z.M.; Majrashi, T.A.; Krňávková, P.; Elbadawi, M.M.; Shaldam, M.A.; et al. Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity. Pharmaceuticals 2025, 18, 1770. https://doi.org/10.3390/ph18111770

AMA Style

Eldehna WM, Tawfik HO, Veselá D, Vojáčková V, Negmeldin AT, Elsayed ZM, Majrashi TA, Krňávková P, Elbadawi MM, Shaldam MA, et al. Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity. Pharmaceuticals. 2025; 18(11):1770. https://doi.org/10.3390/ph18111770

Chicago/Turabian Style

Eldehna, Wagdy M., Haytham O. Tawfik, Denisa Veselá, Veronika Vojáčková, Ahmed T. Negmeldin, Zainab M. Elsayed, Taghreed A. Majrashi, Petra Krňávková, Mostafa M. Elbadawi, Moataz A. Shaldam, and et al. 2025. "Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity" Pharmaceuticals 18, no. 11: 1770. https://doi.org/10.3390/ph18111770

APA Style

Eldehna, W. M., Tawfik, H. O., Veselá, D., Vojáčková, V., Negmeldin, A. T., Elsayed, Z. M., Majrashi, T. A., Krňávková, P., Elbadawi, M. M., Shaldam, M. A., Al-Ansary, G. H., Kryštof, V., & Abdel-Aziz, H. A. (2025). Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity. Pharmaceuticals, 18(11), 1770. https://doi.org/10.3390/ph18111770

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