New Pyranopyrazole Compounds Show Selective Cytotoxicity Against Cancer Cells in Vitro

Chemists have synthesized a new family of pyranopyrazole compounds and found that two of them, labeled AL-21 and AL-31, kill certain human cancer cells in laboratory dishes while leaving normal mouse fibroblast cells largely unharmed. The work, published in Results in Chemistry, describes a one-pot, solvent-free synthesis driven by a zinc Schiff base complex mounted on silica-coated copper ferrite nanoparticles, followed by a 48-hour MTT cytotoxicity screen against five human cancer cell lines and one normal cell line. The compounds are 6-amino-4-benzyloxyphenyl-dihydropyrano[2,3-c]pyrazole-5-carbonitriles, a class built around a pyran ring fused to a pyrazole core with a nitrile group and an amino group attached.

The synthesis itself is a four-component reaction. Ethyl acetoacetate, malononitrile, hydrazine, and an aromatic aldehyde were combined in equimolar amounts with just 3 milligrams of the nanomagnetic catalyst and stirred at 80 degrees Celsius for 20 minutes. Because the catalyst is magnetic, it was pulled out of the reaction mixture with an external magnet, and the crude product was recrystallized from 95 percent ethanol. The team, led by Ahmad Reza Moosavi-Zare with colleagues at Afyonkarahisar Health Sciences University in Türkiye, optimized the conditions using a model reaction of ethyl acetoacetate, benzaldehyde, malononitrile, and hydrazine. With 3 milligrams of catalyst at 80 degrees Celsius for 35 minutes, the model reaction reached a 95 percent isolated yield. Reducing the catalyst to 1.5 milligrams dropped the yield to 50 percent, while raising it to 6 milligrams lowered it to 60 percent. Temperature mattered as well: at 50 degrees Celsius the yield fell to 49 percent, and at 110 degrees it reached only 70 percent.

Solvent-free conditions proved superior to any solvent tested. When the same model reaction was run under reflux in ethanol, chloroform, ethyl acetate, dichloromethane, dimethylformamide, or n-hexane, yields ranged from 52 to 88 percent, all below the 95 percent achieved without solvent. The authors attribute the catalyst’s effectiveness partly to its nanoscale dimensions. Field emission scanning electron microscopy showed particles smaller than 100 nanometers, which increases the specific surface area available for contact with the starting materials. Zeta potential measurements at 25 degrees Celsius gave an average of 68.3 millivolts, a value above the 60-millivolt threshold the authors cite as indicating that particles resist aggregation and maintain a desirable active surface. The catalyst was recovered and reused three times in the model reaction without reported loss of performance.

The mechanism proposed for the reaction begins with ethyl acetoacetate and hydrazine coordinating to the zinc center in cis positions. Hydrazine attacks the carbonyl group of ethyl acetoacetate, releasing a molecule of water, and an intramolecular attack by the amine nitrogen on the second carbonyl closes the ring with loss of ethanol, forming 5-methyl-2,4-dihydro-3H-pyrazol-3-one. Meanwhile, malononitrile and the aldehyde also bind to zinc, raising its coordination number so the two reactants meet in activated form and condense into a cyanoolefin intermediate after losing water. The pyrazolone, in its enol form, then combines with this intermediate, and ring closure plus tautomerization delivers the final pyranopyrazole. Because no chiral catalyst is present, the C-4 stereocenter forms equally as R and S configurations, so each product is racemic and optically inactive.

Eight new compounds were characterized, designated AL-11 through AL-81, using melting point, infrared spectroscopy, proton and carbon NMR in DMSO-d6, and mass spectrometry. Isolated yields ranged from 60 percent for AL-71, an acetamide-bearing derivative, to 100 percent for AL-31 and AL-61. The benzyloxy substituents on the aromatic aldehydes varied widely: AL-11 carries a 4-methylbenzyl ether, AL-21 adds a methoxy group, AL-31 and AL-41 use 4-bromobenzyl and 4-chlorobenzyl ethers respectively, AL-51 and AL-61 combine those halogenated benzyl ethers with a methoxy group on the phenyl ring, and AL-81 incorporates a 2,4-dinitrophenyl group on the pyrazole nitrogen through the use of 2,4-dinitrophenylhydrazine. The cytotoxic screen focused on the six compounds AL-11 through AL-61.

For the biological testing, the researchers used MCF-7 breast adenocarcinoma, U-87 glioblastoma, HeLa cervical adenocarcinoma, HT-29 colon adenocarcinoma, and DU-145 prostate carcinoma cells from the American Type Culture Collection, plus L-929 normal mouse adipose fibroblasts from the European Collection of Authenticated Cell Cultures. Cells were seeded at 2 times 10 to the fourth per well in 96-well plates, treated for 48 hours with compound concentrations from 1 to 100 micromolar dissolved in DMSO and diluted with DMEM, and compared against a 1 percent DMSO negative control. Cisplatin and 5-fluorouracil served as positive controls. After MTT addition and formazan dissolution, absorbance at 570 nanometers was read on a plate reader, and viability was expressed as a percentage of the negative control. IC50 values came from nonlinear regression with a variable slope model in GraphPad Prism 8, and experiments were run in triplicate with one-way ANOVA and Dunnett’s post-hoc test, treating p below 0.05 as significant.

The results were cell-line specific. In U-87 glioblastoma cells, every compound except AL-41, which left 74 percent viability, pushed viability below 70 percent at 100 micromolar. AL-31 was the standout, reducing viability to 37 percent with an IC50 of 87.22 micromolar, while cisplatin reached 29 percent viability with an IC50 of 41.33 micromolar. In MCF-7 breast cancer cells, AL-21 was the most potent of the new compounds, cutting viability to 49 percent with an IC50 of 98.67 micromolar, compared with cisplatin’s 34 percent viability and IC50 of 67.45 micromolar; AL-11, AL-31, and AL-51 each beat 5-fluorouracil’s 73 percent viability. In DU-145 prostate cancer cells, AL-31 again led, dropping viability to 38 percent with an IC50 of 51.79 micromolar, better than cisplatin’s IC50 of 91.44 micromolar in that line. HeLa cells were more resistant: AL-11 matched 5-fluorouracil at roughly 79 to 80 percent viability, and no new compound came close to cisplatin’s 21 percent viability and 27.58 micromolar IC50. In HT-29 colon cells, AL-11 and AL-31 outperformed both reference drugs, at 61 and 60 percent viability respectively, though no IC50 below 100 micromolar was recorded for any new compound in that line.

Selectivity is where the new compounds distinguish themselves. In L-929 normal mouse fibroblasts, all six tested compounds preserved viability above 85 percent, with AL-41 and AL-51 at 96 percent, AL-61 at 94 percent, AL-21 at 91 percent, AL-31 at 89 percent, and AL-11 at 87 percent. Cisplatin, by contrast, cut fibroblast viability to 42 percent with an IC50 of 58.86 micromolar, and 5-fluorouracil left 73 percent viability. The selectivity index, defined as the IC50 in normal cells divided by the IC50 in cancer cells, exceeded 1.0 for AL-21 against MCF-7 cells and for AL-31 against U-87 cells, and reached approximately 2.0 for AL-31 against DU-145 cells. An index above 1.0 indicates greater toxicity toward tumor cells than toward normal cells, a profile the authors consider favorable for anticancer candidates.

The authors conclude that AL-21 and AL-31 are the most promising leads and propose follow-up work examining apoptotic pathways, cell cycle arrest, receptor targets, and eventual xenograft mouse models. Those are plans, not results; the current evidence consists of 48-hour MTT viability data in cell culture, and the compounds remain far from any clinical application. Pyranopyrazoles as a broader class have previously been reported to show antibacterial, antifungal, anti-inflammatory, and kinase-inhibitory activities, but this study tested only the six new derivatives in six cell lines. What the data do establish is a concrete synthetic achievement, a reusable magnetic catalyst that delivers high yields in 20 minutes without solvent, and early evidence that two of its products combine measurable cytotoxicity in specific cancer cell lines with minimal toxicity to normal fibroblasts in the same assay.

Subject of Research: Synthesis of new benzyloxy-substituted dihydropyrano[2,3-c]pyrazole compounds using a magnetically recoverable zinc catalyst and evaluation of their in vitro cytotoxicity against human cancer cell lines

Article Title: Synthesis and in vitro cytotoxic evaluation of some new 6-amino-4-benzyloxyphenyldihydropyrano[2,3- c ]pyrazole-5‑carbonitriles catalyzed by zinc (II) complex supported on magnetite copper ferrite nanoparticles

Article References: Moosavi-Zare, A. R., Najafi, R., Rezaeivala, M., Kılıçkaya, O., & Şahin, S. (2026). Synthesis and in vitro cytotoxic evaluation of some new 6-amino-4-benzyloxyphenyldihydropyrano[2,3-c]pyrazole-5‑carbonitriles catalyzed by zinc (II) complex supported on magnetite copper ferrite nanoparticles. Results in Chemistry, 32, Article 104011. https://doi.org/10.1016/j.rechem.2026.104011

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.104011

Keywords: pyranopyrazoles, cytotoxicity, MTT assay, nanocatalyst, copper ferrite nanoparticles, Schiff base zinc complex, solvent-free synthesis, selectivity index, MCF-7, U-87 glioblastoma, DU-145 prostate cancer, drug discovery