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. 2026 Jul 13;16(37):39987–39998. doi: 10.1039/d6ra03196j

Design, synthesis, anticancer activity and molecular modeling of new pyrazoline derivatives as telomerase inhibitors

Mai M Elghonemy a, Eman Y Ahmed a,✉, Mohamed K El-Ashrey b,c, Manar E A Elasasy d, Hanem M Awad e, Rasha Z Batran a,✉, Nehad A Abdel Latif a,✉
PMCID: PMC13359362  PMID: 42444702

Abstract

Telomerase is an enzyme that rebuilds chromosomal ends called telomeres, allowing cells to keep dividing. Blocking telomerase is an important strategy in cancer treatment, as tumor cells are then forced to continue dividing without repairing their telomeres, leading to progressive telomere shortening, genomic stress, and ultimately the cessation of cell division or death, thereby slowing tumor growth and enhancing the effect of chemotherapies or targeted therapies. Twelve new pyrazoline derivatives were synthesized and evaluated for their cytotoxicity against MCF-7 and MDA-MB-231 breast adenocarcinoma cells along with BJ-1 normal cells. Compounds 2c and 2g showed promising selectivity indices against both cell lines, inhibited telomerase enzymatic activity and gene expression compared to the reference standards and triggered cell cycle arrest and apoptosis. The docking study revealed that the synthesized compounds exhibited scores close to those of the co-crystallized ligand, indicating comparable binding affinities.

Telomerase is an enzyme that rebuilds chromosomal ends called telomeres, allowing cells to keep dividing.graphic file with name d6ra03196j-ga.jpg

1. Introduction

Breast cancer is the most commonly diagnosed cancer in females worldwide.1 Cancer formation and development is a genetic phenomenon characterized by invasive cell growth and uncontrolled proliferation.2 Telomerase activity determines cellular proliferation,3 a telomere is a protective cap found at the end of a chromosome, each time a cell replicates, its telomere shortens. Telomerase helps maintain telomere length till it becomes too short that a cellular senescence or apoptosis is triggered.4–6 The amount of telomerase in our cells, limits how many times a cell can divide and researchers think it may be a way of protection against cancer. Unfortunately, up to 80% of human cancers and more than 90% of breast carcinomas express abnormally high levels of telomerase that allows for unlimited proliferation.7 Telomerase inhibition has been a target for cancer research, whereas treatment with telomerase inhibitors may not exhibit the toxicity associated with other chemotherapeutic agents since telomerase is inactive or undetectable in normal tissues and unlike growth factor receptors, the gene nonredundancy of telomerase makes tumors less likely to become resistant to treatments.8–11

Telomerase can be inhibited either indirectly or directly. Indirect telomerase inhibitors are G-quadruplex stabilizers that can stabilize telomeric G-quadruplexes and render them unavailable for the action of telomerase, an example is 6-thio-dG (6-thio-2′-deoxyguanosine) which is in phase II clinical trials, but toxicity is always expected with these inhibitors due to their binding to nontelomeric G-quadruplexes. Direct telomerase inhibitors can act on human telomerase RNA component (hTERC) as oligonucleotides or on human telomerase reverse transcriptase (hTERT) as small molecules. Imetelstat (GRN163L), that complements the hTERC template region, is a thiophosphoramidate oligonucleotide and a palmitoyl lipid group. However, phase II clinical trials of GRN163L did not indicate any improvement in the overall survival among advanced non-small cell lung cancer patients. On the other hand, BIBR1532 is a selective non-nucleoside telomerase inhibitor, these kinds of inhibitors have the advantage of acting by direct noncompetitive inhibition on the hTERT allosteric site. Unfortunately, BIBR1532 demonstrated poor pharmacokinetics characterized by inadequate cellular absorption, hindering its advancement from preclinical to clinical trials.12–16 As evident, efforts to design telomerase inhibitors have not been satisfactory to date, highlighting the urgent need for the development of new inhibitors, Fig. 1.

Fig. 1. Chemical structures of telomerase inhibitors.

Fig. 1

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Pyrazoline and related pyrazole scaffolds represent an attractive starting point for designing hTERT inhibitors as they possess a compact, flexible five-membered heterocycles with substitutable positions which permits structural diversity and optimization of protein-binding interactions. Several medicinal-chemistry reports have synthesized a series of aryl-2H-pyrazole and N-substituted dihydropyrazole telomerase inhibitors, for example, compound I displayed a potent inhibitory activity, confirming that pyrazoline frameworks can represent suitable pharmacophores capable of interfering with telomerase function.17 Structure–activity relationship and molecular modeling studies in previous literature showed that introduction of aryl substituents (e.g. compound II) resulted in improving telomerase inhibition in a model where pyrazoline derivatives either interacted with the hTERT protein or otherwise interfere with telomerase activity.18 Moreover, some reviews of pyrazoline hybrids as anticancer agents mentioned a number of examples where pyrazoline derivatives exhibited telomerase inhibition activities, an example of which is compound III, Fig. 2.19 The pyrazoline core offers a rigid yet partially flexible template that can project pharmacophoric groups at the C-3, C-5, and N-1 positions into distinct telomerase active site pockets. The adjacent nitrogen atoms within the ring can provide essential hydrogen-bonding interactions, while the attached aromatic substituents facilitate hydrophobic and pi–pi stacking interactions with the active site residues.20,21

Fig. 2. Reported telomerase inhibitors and design strategy of the new compounds.

Fig. 2

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On the other hand, the thiazole moiety is a well-established scaffold in anticancer drug discovery.22–24 Thiazole-containing derivatives possess favorable electronic characteristics, hydrogen-bonding capability and aromaticity that can offer desired interactions with biological targets.25,26 In addition, sulfur-containing heterocycles such as some reported isothiazolone derivatives (e.g. TMPI, TFPI, EOIB) have previously demonstrated direct telomerase inhibitory activity in cancer cells.27

The previously mentioned biological and chemical aspects justify the suitability of sulfur/nitrogen-containing heteroaromatic systems for telomerase-targeted drug design. Guided by the reported SAR analyses and pharmacophoric feature studies, the newly synthesized derivatives constituted a pyrazoline core incorporated with a thiazole moiety and two aromatic rings, one of which carries different substituents. The design preserves the main pharmacophoric features of hTERT inhibitors: the pyrazoline, thiazole and phenyl moieties provide the needed interactions with the protein, while the halogen and the methyl substitutions allow for hydrophobic interactions, and the polar substituents (–OCH3 and NO2) enhance hydrogen bonding with the active site, Fig. 2.28–30

2. Results and discussion

2.1. Chemistry

The target compounds were synthesized following the procedures outlined in Scheme 1. The key intermediate carbothioamide derivative 1 was subjected to cyclo-condensation reaction with the appropriate hydrazonyl chlorides in an alkaline medium using few drops of triethylamine to afford the target thiazolyl pyrazoline derivatives 2a–l. The structures of the newly synthesized compounds were assigned by different spectral and analytical methods as depicted in the experimental part.

Scheme 1. (a) NH2CSNHNH2, ethanol, AcOH, reflux, (b) Created by potrace 1.16, written by Peter Selinger 2001-2019 CH3COCH(Cl)N=N–Ar, dioxane, N(C2H5)3, reflux.

Scheme 1

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2.2. Biology

2.2.1. In vitro antiproliferative activity

The new compounds were designated for their cytotoxicity on the MCF-7 (hormone-dependent) and MDA-MB-231 (hormone-independent) human breast adenocarcinomas, as well as the BJ-1 human healthy cell line through the employment of the MTT assay. The percentages of the viable cells and their IC50 values were measured and were subsequently assessed with those of the positive control, doxorubicin, Table 1. In assessment with the positive control doxorubicin, six compounds (2a, 2c, 2f, 2g, 2h and 2i) recorded superior activities on MCF-7 and two compounds (2c and 2g) exhibited better activities on MDA-MB-231 compared to the other derivatives. 4-Fluoro 2c and 3-chloro derivatives 2g showed the most potent effects on both types of breast cancer cell lines (IC50 = 2.8 µM for MCF-7 and 5.5 and 4.2 µM for MDA-MB-231). Regarding the BJ-1 healthy human skin cell line, compounds 2c and 2g showed remarkable selectivity indices, implying their safety as anticancer agents.

Table 1. The cytotoxic IC50 values of the compounds according to the MTT assay on cancer and normal cells.
Compound IC50 (µM) ± SD
MCF-7 MDA-MB-231 BJ-1
2a 2.7 ± 0.2 7.8 ± 0.6 16.1 ± 1.1
2b 5.9 ± 0.4 8.2 ± 0.7 17.3 ± 1.2
2c 2.8 ± 0.3 5.5 ± 0.5 15.6 ± 1.2
2d 5.4 ± 0.3 7.3 ± 0.5 16.6 ± 1.2
2e 4.4 ± 0.2 26.0 ± 2.1 16.1 ± 1.3
2f 3.1 ± 0.3 8.6 ± 0.6 17.1 ± 1.3
2g 2.8 ± 0.2 4.2 ± 0.4 16.2 ± 1.3
2h 2.5 ± 0.2 7.9 ± 0.5 8.8 ± 0.7
2i 3.5 ± 0.2 7.4 ± 0.5 17.5 ± 1.4
2j 5.3 ± 0.3 8.2 ± 0.6 16.1 ± 1.1
2k 5.0 ± 0.4 28.6 ± 2.2 17.6 ± 1.5
2l 4.7 ± 0.3 25.1 ± 2.1 16.6 ± 1.2
Doxorubicin 4.0 ± 0.4 6.6 ± 0.6 16.0 ± 1.3
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Generally, the meta substituted derivatives 2f–2i showed more effectiveness than the para substituted ones 2b–2e against the tested cell lines MCF-7 (IC50; 2f–2i = 2.5–3.5 µM, 2b–2e = 2.8–5.9 µM) and MDA-MB-231 (IC50; 2f–2i = 4.2–8.6 µM, 2b–2e = 5.5–26.0 µM). The un-substituted derivative 2a showed significant effect on MCF-7 cells with IC50 value of 2.7 µM and comparable effect to doxorubicin on MDA-MB-231 cells (IC50 = 7.8 µM). The meta bromo derivative 2h showed outstanding potency against MCF-7 cells (IC50 = 2.5 µM) being 1.6-fold more active than doxorubicin and its 4-bromo congener 2e (IC50 = 4.4 µM) while exerting considerable effect against MDA-MB-231 (IC50 = 7.9 µM). The 3-methyl 2f and 3-nitro 2i derivatives showed remarkable equipotent activity towards MCF-7 cells (IC50 = 3.1 and 3.5 µM) with noticeable effect against MDA-MB-231 cells (IC50 = 8.6 and 7.4 µM).

Mono substituted 3-nitro derivative 2i was more active than di-substituted 2,4-dinitro compound 2k when tested against MCF-7 cells (IC50 = 3.5 µM and 5.0 µM) while a dramatic decrease in the activity was observed towards MDA-MB-231 (IC50 = 7.4 µM and 28.7 µM). Within the disubstituted derivatives, the 2,4-dichloro derivative 2j was the most active against both cancer cells; MCF-7 (IC50 = 5.3 µM) and MDA-MB-231 (IC50 = 8.2 µM), Fig. 3.

Fig. 3. Structure-dependent activity trends among the synthesized compounds.

Fig. 3

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2.2.2. Telomerase enzymatic activity assay

A cell-based ELISA kit was used to perform the enzymatic assay. The promising compounds 2c and 2g were selected for further screening as hTERT inhibitors. The assay results revealed that compound 2c achieved 78.3% ± 4.1% inhibition, while compound 2g exhibited a higher inhibition of 92.1% ± 5.1%, compared to the reference inhibitor BIBR1532 which showed 88.0% ± 5.5% inhibition.

2.2.3. Telomerase gene expression

The effects of compounds 2c and 2g on telomerase gene expression were investigated compared to the reference standard BIBR1532. Compound 2c exhibited a significant reduction in telomerase gene expression, achieving 71.9% ± 3.9% inhibition, while compound 2g revealed a stronger effect with 93.3% ± 5.3% inhibition, compared to that of BIBR1532 (93.7% ± 4.7%). The results point out that both compounds not only suppressed telomerase enzymatic activity but also downregulated its gene expression.

2.2.4. Cell cycle inhibition

MCF-7 cells were treated with the promising compounds and analyzed using the flow cytometry technique. The results revealed that compounds 2c and 2g inhibited MCF-7 cells proliferation by inducing 33.8% ± 3.2% and 37.6% ± 3.5% cell growth arrest at G2/M phase, respectively as compared to 13.1% ± 1.5% in the control cells, indicating a higher degree of cell growth arrest for compound 2g, Fig. 4.

Fig. 4. Effect of compounds 2c and 2g on cell cycle dynamics in MCF-7 cells. (A) Untreated control cells, (B) cells treated with compound 2c and (C) cells treated with compound 2g.

Fig. 4

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2.2.5. Cell apoptosis

MCF-7 cells apoptosis was detected by annexin V-FITC/PI flow cytometry. MCF-7 cells treated with compounds 2c and 2g revealed an increase in apoptosis, with 15.6% ± 2.1% and 22.1% ± 2.3% in early apoptosis and 9.8% ± 0.9% and 13.6% ± 1.5% in late apoptosis, respectively, as compared to the control cells, Fig. 5.

Fig. 5. Flow cytometric analysis of apoptosis in MCF-7 cells using annexin V-FITC/PI dual staining. Cells were either left (A) untreated, (B) treated with 2c or (C) treated with 2g for 48 h.

Fig. 5

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2.3. Molecular docking

In the 5CQG structure, telomerase's druggable pocket is an allosteric site (not the catalytic nucleotide site) on the thumb domain called the FVYL pocket. Here the inhibitor BIBR1532 is accommodated into a small, hydrophobic cavity, where it is held mainly by tight packing against phenylalanine/valine/tyrosine/leucine side chains (the FVYL motif) and a polar contact near the pocket rim; no metal or nucleotide contacts are required. Occupying this pocket disturbs telomerase activation by interfering with interactions between the thumb and RNA elements (CR4/5), which explains the inhibitory effect seen for BIBR1532 in 5CQG.31

Prior to carrying out the docking simulation, we validated the process by redocking of the protein co-crystallized ligand BIBR1532 into the same active site and calculating the root mean square deviation (RMSD) value which should not exceed 2. The RMSD value was 0.44 which ensures this validity, Fig. 6.

Fig. 6. Validation of the docking procedure to telomerase, the co-crystallized ligand in cyan and the re-docked ligand in purple.

Fig. 6

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Docking into the BIBR1532 allosteric (FVYL) pocket of the target enzyme showed that compound 2c (−8.5 kcal mol−1) and compound 2g (−8.7 kcal mol−1) exhibited docking scores close to that of the co-crystallized ligand (−9.0 kcal mol−1), indicating comparable binding affinity toward the allosteric binding site. Detailed interaction analysis revealed that both compounds adopt compact planar conformations deeply embedded within the hydrophobic FVYL cleft.

For compound 2c, the aromatic scaffold established π–π stacking and π–π T-shaped interactions with Tyr551 and Phe494, respectively, which play important roles in stabilizing the ligand orientation inside the aromatic-rich pocket. Additional π–sigma and hydrophobic π–alkyl interactions were observed with Ile550, Ile497, and Leu554, reinforcing hydrophobic complementarity within the binding site. Moreover, π–cation interactions involving Arg486 and Arg547, together with a π–sulfur interaction with Met483, contributed to additional stabilization of the docked complex. van der Waals contacts with Gly495, Gly553, Trp498, and Arg557 further supported favorable ligand accommodation within the pocket.

Similarly, compound 2g exhibited a closely related interaction profile, including π–π interactions with Tyr551 and Phe494, π–sigma interaction with Leu554, and extensive hydrophobic alkyl/π–alkyl contacts with Ile497, Ile550, and Met482. The fluorinated aromatic ring additionally formed favorable halogen-associated interactions near Arg557, while π–cation and π–sulfur interactions involving Arg486 and Met483 contributed to stabilization of the ligand within the allosteric site. Multiple van der Waals interactions with surrounding residues including Gly495, Gly553, Trp498, and Glu549 further enhanced the overall binding complementarity.

The observed interaction pattern closely resembles that of the co-crystallized inhibitor BIBR1532, where binding is predominantly driven by aromatic stacking and hydrophobic packing within the FVYL pocket. These findings, together with the close docking energies, provide structural support for the potent telomerase inhibitory activity observed for compounds 2c and 2g, Fig. 7 and 8. In addition, overlay analysis demonstrated high spatial similarity between compounds 2c, 2g, and the reference ligand within the allosteric pocket, Fig. 9.

Fig. 7. Interactions of compound 2c into the active site of telomerase enzyme (PDB: 5CQG).

Fig. 7

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Fig. 8. Interactions of compound 2g into the active site of telomerase enzyme (PDB: 5CQG).

Fig. 8

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Fig. 9. Overlay of compounds 2c (cyan) and 2g (purple) with the co-crystallized ligand (yellow) within the telomerase active site (PDB ID: 5CQG), illustrating a comparable binding orientation and interaction pattern.

Fig. 9

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2.4. Pharmacokinetics (ADME) prediction

The in silico ADME profiling of compounds 2c and 2g provides valuable insight into their developability as potential anticancer telomerase inhibitors. Both compounds exhibit comparable physicochemical characteristics, with molecular weights around 380–396 g mol−1 and TPSA values of 81.45 Å2, which fall within the acceptable range for orally active small molecules. The moderate fraction of sp3 carbons (0.20) and limited number of rotatable bonds (four) suggest a favorable balance between molecular rigidity and flexibility, which may support target binding while maintaining acceptable pharmacokinetic behavior.

Lipophilicity analysis revealed relatively high consensus log P values (5.03 for 2c and 5.18 for 2g), consistent with the aromatic-rich scaffolds required for effective telomerase interaction. Although elevated lipophilicity is often associated with reduced aqueous solubility, both compounds showed predicted high gastrointestinal absorption, indicating that permeability is unlikely to be a limiting factor for oral administration. Water solubility predictions classified both compounds as moderately to poorly soluble depending on the applied model, which is a common feature among anticancer agents with extended aromatic systems. This limitation may be addressed in future optimization through formulation strategies rather than structural compromise of telomerase inhibitory activity.

From a pharmacokinetic standpoint, both compounds are predicted to be non-permeant to the BBB, which is advantageous for anticancer agents targeting peripheral tumors, as it may reduce central nervous system-related adverse effects. Additionally, neither compound is predicted to be a P-glycoprotein substrate, suggesting a lower susceptibility to efflux-mediated resistance mechanisms.

Drug-likeness assessment demonstrated full compliance with Lipinski's rule of five for both compounds. Minor deviations were observed in Ghose and Muegge filters due to elevated lipophilicity, a known and often tolerated characteristic in anticancer drug discovery. Both compounds achieved a bioavailability score of 0.55, supporting their potential as orally bioavailable leads.

The bioavailability radar plots show that compounds 2c and 2g largely occupy the optimal physicochemical space, with lipophilicity and unsaturation being the primary parameter approaching the upper boundary. Importantly, polarity, size and flexibility, remain within the recommended ranges, reinforcing the overall balance of their ADME profiles, Fig. 10.

Fig. 10. SwissADME bioavailability radar plots of compounds 2c and 2g showing key physicochemical parameters affecting oral bioavailability. The pink area represents the optimal range, while the black polygon indicates the calculated properties of each compound.

Fig. 10

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The BOILED-Egg model further supports these findings, placing both compounds within the region predictive of high human intestinal absorption (HIA) while remaining outside the BBB-permeable zone. This distribution is particularly suitable for anticancer telomerase inhibitors, where systemic exposure without CNS penetration is often preferred, Fig. 11.

Fig. 11. BOILED-Egg model depicting predicted gastrointestinal absorption and blood–brain barrier permeability of compounds 2c and 2g based on log P and TPSA values.

Fig. 11

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Overall, the in silico ADME evaluation suggests that compounds 2c and 2g possess a favorable pharmacokinetic and drug-likeness profile that complements their telomerase inhibitory activity. These results support their classification as promising lead compounds for further optimization and experimental pharmacokinetic validation.

3. Conclusion

Based on the rational design, a series of pyrazoline derivatives was designed and synthesized. Most of the new compounds showed significant inhibitory activity against the tested MCF-7 and MDA-MB-231 breast adenocarcinoma cells. Among the synthesized compounds, derivatives 2c and 2g had remarkable selectivity indices, with compound 2g showing telomerase protein inhibition (92.06%) compared to the reference inhibitor BIBR1532 (88.00%), gene expression suppression, induction of cell cycle arrest, promotion of early and late apoptosis and a significant docking score. The compound showed protein/gene inhibition percentages and a docking score of 92.06, 93.28 and −8.7 kcal mol−1, respectively, outperforming or matching those of the standard reference BIBR1532

4. Materials and methods

4.1. Chemistry

All melting points were uncorrected and measured using Electrothermal IA 9000 apparatus. The nuclear magnetic resonance spectra 1H NMR (400 MHz) and 13C NMR spectra (100 MHz), were recorded on a Bruker spectrometer using TMS as the internal standard. The reactions were followed by TLC (silica gel, aluminum sheets 60 F254, Merck) using toluene–ethylacetate (8 : 2, v/v) as eluent and sprayed with iodine–potassium iodide reagent. The purity of the newly synthesized compounds was assessed by TLC and elemental analysis and was found to be higher than 95%. Compound 1 was previously prepared.32

4.1.1. Synthesis of 5-((aryl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2a–l)

A mixture of the carbothioamide compound 1 (2.5 mmol, 0.55 g) and the appropriate hydrazonoyl chloride (2.5 mmol) in dioxane (20 mL) and triethylamine (0.35 mL, 2.5 mmol) was heated under reflux for 8–10 h. After cooling, the solution was poured onto crushed ice and concentrated hydrochloric acid. The separated solid was collected by filtration and crystallized from ethanol to give compounds 2a–l.

4.1.1.1. 4-Methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5-(phenyldiazenyl)thiazole (2a)

Yield (87%); m.p. 170–171 °C. Anal. calcd for C20H19N5S (361.13): C, 66.46; H, 5.30; N, 19.38; S, 8.87. Found: C, 66.54; H, 5.37; N, 19.44; S, 8.95. 1H NMR (DMSO-d6, δ, ppm): 2.26 (3H, s, CH3), 2.50 (3H, s, CH3), 2.59–2.65 (1H, dd, J = 3.2 Hz, 18.32 Hz, CH2-pyrazoline), 3.52–3.59 (1H, dd, J = 11.52 Hz, 18.44 Hz, CH2-pyrazoline), 5.72–5.76 (1H, dd, J = 3.36 Hz, 11.28 Hz, CH-pyrazoline), 7.02–7.47 (10H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.21, 25.82, 46.87, 62.74, 115.32, 123.26, 123.29, 125.75, 127.24, 128.86, 129.77, 142.99, 143.61, 158.82, 166.13.

4.1.1.2. 5-((4-Methoxyphenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2b)

Yield (86%); m.p. 115–116 °C. Anal. calcd for C21H21N5OS (391.49): C, 64.43; H, 5.41; N, 17.89; S, 8.19. Found: C, 64.52; H, 5.55; N, 17.98; S, 8.30. 1H NMR (DMSO-d6, δ, ppm): 2.12 (3H, s, CH3), 2.43 (3H, s, CH3), 2.87–2.93 (1H, dd, J = 4.88 Hz, 18.20 Hz, CH2-pyrazoline), 3.68–3.76 (1H, dd, CH2-pyrazoline), 3.82 (3H, s, OCH3), 5.62–5.66 (1H, dd, J = 4.56 Hz, 11.40 Hz, CH-pyrazoline), 7.01–7.67 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.06, 16.30, 47.98, 55.94, 62.81, 114.97, 123.45, 123.59, 126.23, 128.03, 129.24, 129.76, 131.86, 140.40, 141.77, 146.67, 160.56, 164.50.

4.1.1.3. 5-((4-Fluorophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2c)

Yield (88%); m.p. 157–158 °C. Anal. calcd for C20H18FN5S (379.46): C, 63.31; H, 4.78; N, 18.46; S, 8.45. Found: C, 63.39; H, 4.86; N, 18.55; S, 8.55. 1H NMR (DMSO-d6, δ, ppm): 2.13 (3H, s, CH3), 2.46 (3H, s, CH3), 2.87–2.93 (1H, dd, J = 4.20 Hz, 18.32 Hz, CH2-pyrazoline), 3.69–3.76 (1H, dd, J = 11.20 Hz, 18.80 Hz, CH2-pyrazoline), 5.64–5.68 (1H, dd, J = 4.44 Hz, 11.36 Hz, CH-pyrazoline), 7.24–7.69 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.20, 25.80, 47.97, 62.78, 116.84, 123.89, 125.75, 126.22, 127.23, 128.85, 129.24, 140.22, 141.72, 149.45, 160.64, 165.13. HR-MS (m/z): calcd 379.1267; found 379.1266 (M+) (18.7%).

4.1.1.4. 5-((4-Chlorophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2d)

Yield (90%); m.p. 150–151 °C. Anal. calcd for C20H18ClN5S (395.90): C, 60.68; H, 4.58; N, 17.69; S, 8.10. Found: C, 60.77; H, 4.64; N, 17.77; S, 8.18. 1H NMR (DMSO-d6, δ, ppm): 2.13 (3H, s, CH3), 2.46 (3H, s, CH3), 2.88–2.94 (1H, dd, J = 4.16 Hz, 18.32 Hz, CH2-pyrazoline), 3.69–3.77 (1H, dd, J = 11.16 Hz, 18.36 Hz, CH2-pyrazoline), 5.66–5.70 (1H, dd, J = 4.36 Hz, 11.32 Hz, CH-pyrazoline), 7.23–7.67 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.12, 25.84, 47.99, 62.82, 119.89, 123.97, 125.75, 126.22, 126.87, 128.08, 128.85, 129.26, 129.64, 133.27, 140.29, 142.01, 159.99, 165.41.

4.1.1.5. 5-((4-Bromophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2e)

Yield (88%); m.p. 128–129 °C. Anal. calcd for C20H18BrN5S (440.36): C, 54.55; H, 4.12; N, 15.90; S, 7.28. Found: C, 54.66; H, 4.23; N, 15.99; S, 7.34. 1H NMR (DMSO-d6, δ, ppm): 2.13 (3H, s, CH3), 2.46 (3H, s, CH3), 2.88–2.94 (1H, dd, J = 4.2 Hz, 18.36 Hz, CH2-pyrazoline), 3.70–3.77 (1H, dd, J = 11.48 Hz, 17.64 Hz, CH2-pyrazoline), 5.66–5.70 (1H, dd, J = 3.68 Hz, 11.56 Hz, CH-pyrazoline), 7.23–7.66 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.11, 25.83, 47.99, 62.82, 117.31, 122.06, 123.77, 124.08, 126.21, 129.27, 132.51, 132.68, 141.73, 142.40, 161.24.

4.1.1.6. 4-Methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5-(m-tolyldiazenyl)thiazole (2f)

Yield (89%); m.p. 138–139 °C. Anal. calcd for C21H21N5S (375.49): C, 67.17; H, 5.64; N, 18.65; S, 8.54. Found: C, 67.27; H, 5.80; N, 18.79; S, 8.66. 1H NMR (DMSO-d6, δ, ppm): 2.13 (3H, s, CH3), 2.32 (3H, s, CH3), 2.47 (3H, s, CH3), 2.87–2.93 (1H, dd, J = 4.96 Hz, 18.20 Hz, CH2-pyrazoline), 3.68–3.76 (1H, dd, J = 10.96 Hz, 19.52 Hz, CH2-pyrazoline), 5.64–5.68 (1H, dd, J = 4.36 Hz, 11.08 Hz, CH-pyrazoline), 6.85–7.50 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.21, 21.71, 25.82, 47.93, 66.81, 112.63, 115.71, 123.17, 124.07, 126.23, 128.03, 129.24, 129.62, 139.14, 142.93, 146.67, 156.48, 160.56.

4.1.1.7. 5-((3-Chlorophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2g)

Yield (87%); m.p. 170–171 °C. Anal. calcd for C20H18ClN5S (395.90): C, 60.68; H, 4.58; N, 17.69; S, 8.10. Found: C, 60.78; H, 4.66; N, 17.76; S, 8.21. 1H NMR (DMSO-d6, δ, ppm): 2.12 (3H, s, CH3), 2.46 (3H, s, CH3), 2.89–2.95 (1H, dd, J = 3.64 Hz, 17.20 Hz, CH2-pyrazoline), 3.70–3.78 (1H, dd, J = 10.64 Hz, 18.20 Hz, CH2-pyrazoline), 5.67–5.71 (1H, dd, J = 4.20 Hz, 11.60 Hz, CH-pyrazoline), 7.06–7.48 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.12, 25.84, 47.97, 66.81, 124.48, 125.74, 126.22, 126.90, 128.13, 128.85, 129.14, 129.64, 131.50, 134.29, 144.49, 159.99, 165.44. HR-MS (m/z): calcd 395.0971; found 395.0976 (M+) (21.50%).

4.1.1.8. 5-((3-Bromophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2h)

Yield (88%); m.p. 143–144 °C. Anal. calcd for C20H18BrN5S (440.36): C, 54.55; H, 4.12; N, 15.90; S, 7.28. Found: C, 54.69; H, 4.22; N, 15.98; S, 7.36. 1H NMR (DMSO-d6, δ, ppm): 2.12 (3H, s, CH3), 2.46 (3H, s, CH3), 2.89–2.95 (1H, dd, J = 4.08 Hz, 18.28 Hz, CH2-pyrazoline), 3.70–3.76 (1H, dd, J = 11.18 Hz, 18.52 Hz, CH2-pyrazoline), 5.67–5.71 (1H, dd, J = 4.12 Hz, 11.36 Hz, CH-pyrazoline), 7.20–7.62 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.13, 25.89, 47.94, 66.78, 117.74, 122.73, 124.53, 125.64, 126.22, 129.27, 131.78, 134.68, 144.60, 159.27, 165.64.

4.1.1.9. 4-Methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5-((3-nitrophenyl)diazenyl)thiazole (2i)

Yield (89%); m.p. 120–121 °C. Anal. calcd for C20H18N6O2S (406.46): C, 59.10; H, 4.46; N, 20.68; S, 7.89. Found: C, 59.22; H, 4.55; N, 20.76; S, 7.97. 1H NMR (DMSO-d6, δ, ppm): 2.16 (3H, s, CH3), 2.55 (3H, s, CH3), 2.91–2.97 (1H, dd, J = 3.96 Hz, 18.32 Hz, CH2-pyrazoline), 3.72–3.80 (1H, dd, J = 10.08 Hz, 18.56 Hz, CH2-pyrazoline), 5.70–5.74 (1H, dd, J = 3.96 Hz, 11.16 Hz, CH-pyrazoline), 7.24–8.27 (9H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.19, 25.90, 46.88, 62.72, 117.74, 121.23, 124.53, 125.37, 126.22, 129.27, 131.32, 133.24, 138.71, 144.28, 149.05, 159.27, 166.12.

4.1.1.10. 5-((2,4-Dichlorophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2j)

Yield (80%); m.p. 122–123 °C. Anal. calcd for C20H17Cl2N5S (430.35): C, 55.82; H, 3.98; N, 16.27; S, 7.45. Found: C, 55.97; H, 4.08; N, 16.35; S, 7.54. 1H NMR (DMSO-d6, δ, ppm): 2.14 (3H, s, CH3), 2.47 (3H, s, CH3), 2.89–2.95 (1H, dd, J = 4.08 Hz, 18.28 Hz, CH2-pyrazoline), 3.72–3.79 (1H, dd, J = 10.52 Hz, 19.08 Hz, CH2-pyrazoline), 5.69–5.73 (1H, dd, J = 4.16 Hz, 11.44 Hz, CH-pyrazoline), 7.23–7.73 (8H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.13, 25.82, 48.04, 62.87, 126.19, 127.41, 127.88, 129.04, 129.29, 129.68, 130.23, 132.53, 133.24, 137.71, 141.46, 147.56, 166.12.

4.1.1.11. 5-((2,4-Dinitrophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2k)

Yield (85%); m.p. 150–152 °C. Anal. calcd for C20H17N7O4S (451.46): C, 53.21; H, 3.80; N, 21.72; S, 7.10. Found: C, 53.31; H, 3.91; N, 21.88; S, 7.22. 1H NMR (DMSO-d6, δ, ppm): 2.02 (3H, s, CH3), 2.16 (3H, s, CH3), 2.59–2.65 (1H, dd, J = 3.12 Hz, 18.20 Hz, CH2-pyrazoline), 3.52–3.59 (1H, dd, J = 10.52 Hz, 19.08 Hz, CH2-pyrazoline), 5.73–5.76 (1H, dd, J = 3.40 Hz, 11.32 Hz, CH-pyrazoline), 7.08–8.81 (8H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.19, 25.82, 46.88, 62.73, 120.25, 123.89, 125.74, 127.26, 128.87, 129.16, 129.80, 135.61, 143.58, 147.56, 150.28, 158.92, 166.12.

4.1.1.12. 5-((2-Chloro-4-nitrophenyl)diazenyl)-4-methyl-2-(3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (2l)

Yield (82%); m.p. 80–82 °C. Anal. calcd for C20H17ClN6O2S (440.90): C, 54.48; H, 3.89; N, 19.06; S, 7.27. Found: C, 54.58; H, 3.97; N, 19.15; S, 7.38. 1H NMR (DMSO-d6, δ, ppm): 2.19 (3H, s, CH3), 2.58 (3H, s, CH3), 2.96–3.01 (1H, dd, J = 4.04 Hz, 18.44 Hz, CH2-pyrazoline), 3.75–3.83 (1H, dd, J = 10.48 Hz, 18.80 Hz, CH2-pyrazoline), 5.77–5.80 (1H, dd, J = 4.12 Hz, 10.76 Hz, CH-pyrazoline), 7.24–8.43 (8H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 16.14, 25.93, 46.93, 62.75, 119.84, 124.85, 125.14, 125.81, 126.11, 126.15, 130.26, 136.35, 142.65, 143.82, 151.84, 166.26.

4.2. Biology

4.2.1. Antiproliferative activity

The cells of MCF-7 (hormone-dependent human breast adenocarcinoma), MDA-MB-231 (hormone-independent human breast adenocarcinoma), and the normal human skin fibroblast (BJ-1) were purchased from the American Type Culture Collection (Rockville, MD). All cells were maintained in a DMEM medium, which was supplemented with 10% of heat-inactivated fetal bovine serum (FBS), 100 U mL−1 of each penicillin and streptomycin. The cells were grown at 37 °C in a humidified atmosphere of 5% CO2.

The cytotoxic activities on the MCF-7 and MDA-MB-231 human cancer cell lines in addition to BJ-1 human healthy cell line 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 the mitochondrial dehydrogenases in viable cells.33–36 The cells were dispensed in a 96 well sterile microplate (3 × 104 cells per 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, 40 µL of MTT (2.5 mg mL−1) were added to each well, and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 570 nm applying 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 the values were represented as mean ± SD. The IC50 values were determined by the SPSS probit analysis software program (SPSS Inc., Chicago, IL).

4.2.2. Telomerase enzymatic activity assay

The assay was performed according to the manufacturer's using TERT (Human) ELISA Kit (Biovision, Catalog #K4187-100).

4.2.3. Telomerase gene expression

The real-time PCR assay was done per the manufacturer's instructions using Quantitative Telomerase Detection Kit (QTD Kit), (US Biomax, catalog no. MT3010, MT3011, MT3012).

4.2.4. Cell cycle inhibition and apoptosis assay

MCF-7 cells were seeded at 8 × 104 cells per well, incubated overnight at 37 °C. Cells were then treated with compounds 2c and 2g and incubated for 48 h of treatment. At the end of incubation time, cell pellets were collected and centrifuged at 300g for 5 min. For cell cycle analysis, cell pellets were fixed in 70% ethanol on ice for 15 min. The collected pellets were incubated with propidium iodide (PI) staining solution (50 µg mL−1 PI, 0.1 mg mL−1 RNaseA, and 0.05% Triton X-100) at room temperature for 1 h. Apoptosis detection was performed using FITC Annexin-V/PI kit (Becton Dickinson, Franklin Lakes, NJ, USA) following a previous protocol. The sample was analyzed by flow cytometry on a Beckman Coulter Gallios (Beckman Coulter, Brea, CA, USA) within 1 h of the staining. Data were analyzed using Kaluza v1.2 (Beckman Coulter).37,38

4.3. Molecular docking

The molecular docking simulation was functioned with the aid of the free trustable software; AutoDock Vina 1.1.2.39 The target protein; telomerase enzyme; was downloaded as pdb file with PDB ID: 5CQG. MGL Tools 1.5.7 were used to prepare and convert the protein and ligand files to pdbqt format. The results were pictured with Discovery Studio Visualizer v21.1.0.20298.40 The docking grid was centered on the FVYL allosteric pocket defined by the crystallographic ligand, a grid box size of [20 × 20 × 20 Å], sufficient to fully encompass the binding cavity and allow conformational freedom of the ligands.

4.4. Pharmacokinetics (ADME) prediction

In silico absorption, distribution, metabolism, and excretion (ADME) properties of the most active telomerase inhibitors, compounds 2c and 2g, were predicted using the SwissADME online platform (https://www.swissadme.ch). The chemical structures were input in SMILES format, and physicochemical descriptors including molecular weight, lipophilicity, topological polar surface area (TPSA), hydrogen bond donors and acceptors, and molecular flexibility were calculated.

Pharmacokinetic parameters such as gastrointestinal (GI) absorption, blood–brain barrier (BBB) permeation, P-glycoprotein (P-gp) substrate liability, and cytochrome P450 (CYP) inhibition profiles were predicted. Water solubility was estimated using different computational models (ESOL, Ali, and SILICOS-IT). Drug-likeness was evaluated according to Lipinski, Veber, Ghose, Egan, and Muegge rules, in addition to bioavailability score estimation.

Furthermore, the bioavailability radar was generated to visualize the overall oral drug-likeness profile, while the BOILED-Egg model was employed to predict passive GI absorption and BBB permeation characteristics.

Conflicts of interest

The authors declared that there are no actual or potential conflicts of interest and have approved the article.

Supplementary Material

RA-016-D6RA03196J-s001

Acknowledgments

This research article was funded by NRC, Cairo, Egypt (2022–2024), through project no. 13010137, ethical approval number (13010137-5), entitled “Synthesis and computational studies of new naturally derived anticancer heterocyclic compounds”.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03196j.

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Associated Data

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Supplementary Materials

RA-016-D6RA03196J-s001
RA-016-D6RA03196J-s001.pdf (1.7MB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03196j.

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