Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2026 Sep 25;17(10):2219–2229. doi: 10.1021/acsmedchemlett.6c00344

Fluorinated 4‑Methylideneisoxazolidin-5-ones: Synthesis and Apoptosis-Inducing Antiproliferative Activity

Zi-Han Li †, Ya Han †, Yi-Chao Li †, Xue-Qiong Zhang †,*, Subarna Jyoti Kalita ‡,*, Yi-Yong Huang †,*
PMCID: PMC13656280  PMID: 42866442

Abstract

Sesquiterpene lactones, featuring an α-methylene-γ-lactone moiety, serve as lead compounds for anticancer drug development. Their nitrogen-containing analogues, namely, 4-methylideneisoxazolidin-5-ones, have attracted special attention for their promising anticancer properties. Installing a fluorine atom at the exocyclic double bond of these analogues is highly desirable for improving metabolic stability, bioavailability, and lipophilicity, yet this has not been achieved to date due to the lack of an efficient synthetic method, representing a major obstacle in drug development. Herein, we report a Ni­(II)-catalyzed [3 + 2] cycloaddition/rearrangement cascade reaction of gem-difluorocyclopropenyl imides and N-Bn nitrones, providing direct access to (Z)-4-(monofluoroalkylidene)­isoxazolidin-5-ones in good to high yields. The compounds 3aw and 3as show promising in vitro antiproliferative activity against mouse mammary carcinoma 4T1 cells (IC50 = 6.85 ± 0.43 μM) and human lung carcinoma A549 cells (IC50 = 10.34 ± 0.07 μM), respectively. Mechanistic studies reveal that the activity occurs via a mitochondria-mediated intrinsic apoptotic pathway.

Keywords: gem-difluorocyclopropene, isoxazolidin-5-one, skeletal rearrangement, antiproliferative activity, fluoroalkene, sesquiterpene lactone

graphic file with name ml6c00344_0010.webp

graphic file with name ml6c00344_0009.webp

Introduction

Breast cancer is estimated as the most commonly diagnosed malignancy and the leading cause of cancer-related death among women worldwide. In 2023, 2.3 million new cases and 764,000 deaths were recorded globally, and annual incidence is projected to exceed 3.5 million by 2050. Despite advances in early detection and treatment, conventional chemotherapy is limited by severe systemic toxicities, including cardiotoxicity, myelosuppression, and neuropathy. , Furthermore, long-term efficacy is substantially compromised by multidrug resistance, which is driven by enhanced drug efflux, activated pro-survival signaling, and metabolic reprogramming. , These challenges highlight the pressing demand for novel anticancer agents with improved selectivity and minimized off-target toxicity.

Sesquiterpene lactones, featuring an α-methylene-γ-lactone moiety, are a group of natural products found in plants of the Compositae family. They are well-known for their cytotoxicity and antitumor activities, , which are attributed to the Michael-type addition of sulfhydryl-containing enzymes to the exocyclic double bond of the α-methylene-γ-lactone moiety, thereby interfering with key biological processes. Even though their selectivity is not sufficient to consider them for clinical applications, they serve as lead compounds. Significant efforts have been devoted to synthesize their analogues for drug development. , Particularly promising results have been obtained with the nitrogen-containing analogues 4-methylideneisoxazolidin-5-ones. , Given the central role of fluorine in drug design, − the introduction of a fluorine atom to the exocyclic double bond of such analogues is valuable, yet has not been achieved to date due to the lack of an efficient synthetic method (Figure A). Such modifications are known to enhance metabolic stability, bioavailability, and lipophilicity. −

1.

1

Open in a new tab

Background and synthetic developments of (Z)-4-(monofluoroalkylidene)­isoxazolidin-5-ones.

Isoxazolidin-5-one skeletons are prevalent in various natural products and bioactive molecules (Figure B) − and also find applications as key building blocks − or intermediates in the synthesis of β-amino acids, − nucleosides, , and aminosugars. , Consequently, they remain highly sought-after synthetic targets, prompting the development of various synthetic strategies. − Among these, the 1,3-dipolar cycloaddition of nitrones stands out as one of the most straightforward and efficient approaches. Meldrum’s acids (acting as ketene equivalents), oxazol-5-(4H)-ones, and ynolates were explored as reaction partners. Over the past decade, gem-difluorocyclopropenes have emerged as versatile fluorine-containing C3 building blocks. , The highly strained structures , enable them to serve as dipolarophiles in cycloadditions − and to undergo hydroalkylation, hydroalkenylation, hydroborylation, hydrosilylation, and reduction reactions , to give the corresponding gem-difluorocyclopropyl derivatives. Notably, in the past few years, researchers have leveraged these building blocks for the endocyclic incorporation of a β-monofluorinated C3 synthon into various heterocycles. − By contrast, the exocyclic incorporation of this synthon remains underexplored. , This pathway is especially attractive as it provides direct access to fluoroalkene motifs, which are also regarded as valuable bioisosteres of peptide bonds (Figure C). Building on this underexplored reactivity, we sought to address the challenge of installing a fluorine atom at the exocyclic double bond of 4-methylideneisoxazolidin-5-ones.

The [3 + 2] cycloaddition/rearrangement cascade reaction of nitrones has emerged as a transformative strategy that expands chemical space by providing modular access to diverse heterocycles. , We reasoned that a strained three-membered carbocyclic dipolarophile would readily undergo [3 + 2] cycloaddition with a nitrone, and that the resulting cycloadduct, bearing both the strained three-membered ring and a labile N–O bond, would be poised for subsequent rearrangement. Accordingly, to achieve our goal of installing a fluorine atom at the exocyclic double bond of 4-methylideneisoxazolidin-5-ones, we describe herein the use of gem-difluorocyclopropenyl imides bearing an easily removable oxazolidinyl group as dipolarophiles in the presence of a Ni­(II) catalyst. As outlined in Figure D, the cycloadducts resulting from the initial [3 + 2] cycloaddition with N-Bn nitrones undergo skeletal rearrangement − via water incorporation, N–O bond cleavage, and HF elimination, providing direct access to (Z)-4-(monofluoroalkylidene)­isoxazolidin-5-ones, featuring a fluorine atom on the exocyclic double bond. These derivatives were evaluated against mouse mammary carcinoma 4T1 cells. Several of them exhibited promising in vitro antiproliferative activity, with compound 3aw displaying the most potent effect (IC50 = 6.85 ± 0.43 μM). The antitumor mechanism was identified as mitochondria-mediated intrinsic apoptosis, where the active derivative 3aw modulated Bcl-2 family proteins, increased the Bax/Bcl-2 ratio, induced mitochondrial dysfunction and oxidative stress, and activated the caspase-3 cascade, ultimately leading to tumor cell apoptosis. Furthermore, among the derivatives evaluated against human lung carcinoma A549 cells, compound 3as was the most potent (IC50 = 10.34 ± 0.07 μM).

Results and Discussion

Synthetic Method Development for (Z)-4-(Monofluoroalkylidene)­isoxazolidin-5-ones

At the outset, we investigated the cyclorearrangement cascade reaction of gem-difluorocyclopropenyl imide 1a and nitrone 2a to establish the optimized reaction conditions. The optimal outcome was achieved using Ni­(ClO4)2·6H2O and bis-oxazoline ligand L1 catalyst system in DCM at 30 °C, with Na2CO3 (1.0 equiv) as an additive, furnishing the [3 + 2] cycloadduct I. Upon consumption of the starting materials (indicated by TLC), the crude reaction mixture was loaded on a preparative silica gel TLC plate. When exposed to silica gel, the cycloadduct underwent rearrangement to form the product 3aa in 82% yield as a single Z-isomer − (Scheme ).

1. Optimized Reaction Conditions for (Z)-4-(Monofluoroalkylidene)­isoxazolidin-5-one 3aa .

1

Open in a new tab

The substrate scope of the reaction was then examined under the optimized reaction conditions (Scheme ), and the results are summarized in Scheme . A range of N-Bn C-aryl nitrones having substituents with varied electronic and steric properties at para, meta, and ortho positions of the aryl moiety (R2) reacted smoothly with gem-difluorocyclopropenyl imide 1a to give the corresponding products 3aa–3ak in 40–82% yields as a single Z-isomer. For para-substituted substrates, the electron-donating 4-OMe group (3ab: 43%) and the 4-F halogen group (3ad: 45%) gave a slightly higher yield than the electron-withdrawing 4-CF3 group (3ac: 40%). Conversely, for meta-substituted substrates, the electron-donating 3-Me group (3ae: 50%) afforded a lower yield compared to the electron-withdrawing 3-NO2 (3af: 61%). The product 3ag containing a 3-Cl halogen group was formed in 76% yield. The reactivity trend of ortho-substituted substrates was similar to that of para-substituted counterparts, with the electron-donating 2-Me group (3ah: 65%) and the 2-Cl halogen group (3aj: 62%) delivering a higher yield than the electron-withdrawing 2-CF3 group (3ai: 47%). The nitrone bearing a 2-naphthyl group was converted to the product 3ak in 48% yield. The N-Bn C-alkyl nitrones with 1°, 2°, and 3° alkyl groups also performed well in the reaction, and the corresponding products 3al–3aw were obtained as a single Z-isomer in 51–87% yields. Substrates with smaller alkyl groups like methyl (3al: 62%) and ethyl (3am: 66%) provided higher yields than those with longer chains of butyl (3an: 51%) and pentyl (3ao: 52%). However, the yield increased significantly to 75% for the product 3ap when a much longer dodecyl chain was introduced. The one with a phenethyl group was converted into the product 3aq in 60% yield. The isopropyl group afforded a higher yield (3ar: 80%) than the tert-butyl group (3as: 60%). Cycloalkyl groups of different sizes, including cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl were well tolerated to deliver the corresponding products 3at–aw in 51–73% yields. A clear correlation between ring size and yield was observed. As the ring expanded from three to five members (cyclopropyl to cyclopentyl), the yield increased progressively (3at: 61%; 3au: 64%; 3av: 73%). However, a further increase to a six-membered cyclohexyl ring resulted in a pronounced decrease in yield (3aw: 51%). The products 3ax and 3ay were obtained in 57% and 40% yields from the nitrones bearing 4’-tetrahydropyranyl and N-Boc-piperidinyl groups, respectively. The reaction also proceeded well with a ketonitrone bearing both methyl and propyl substituents, affording the Z-isomeric product 3az in 42% yield. Next, the feasibility of diverse gem-difluorocyclopropenyl imides was investigated with N-Bn C-isopropyl nitrone 2r, and the corresponding products 3br–jr were obtained as a single Z-isomer in 43–83% yields. For para-substituted substrates, those with the electron-donating 4-OMe (3br: 75%) and 4-Me (3cr: 70%) groups on the aryl moiety (R1) delivered the corresponding products in higher yields than the variants with electron-withdrawing 4-CF3 (3dr: 44%) and 4-Cl halogen (3er: 43%) groups. A similar trend was observed with meta and ortho-substituted analogues. The substrate with the electron-donating 3-Me group afforded the product 3fr in 85% yield, significantly higher than the 55% yield of the product 3gr obtained from the 3-Cl case. Likewise, the 2-Me-substituted product 3ah was given in 82% yield, whereas a lower yield of 63% was observed for 3ir containing the 2-F substituent. The nitrone with the 1-naphthyl group (R1) was also compatible, providing the product 3jr in 70% yield. A plausible reaction mechanism is presented in the Supporting Information (Figure S1).

2. Substrate Scope for the Synthesis of (Z)-4-(Monofluoroalkylidene)­isoxazolidin-5-ones .

2

Open in a new tab

a Unless otherwise noted, the reactions were carried out using 1 (0.10 mmol), 2 (0.12 mmol), Ni­(ClO4)2·6H2O (10 mol%), L1 (12 mol%), 4 Å MS (25 mg), Na2CO3 (0.10 mmol) in anhydrous DCM (0.5 mL) under N2 atmosphere at 30 oC. Isolated Yields. (b) Without base. (c) Anhydrous DCE was used as a solvent at 60 °C.

To demonstrate the practical application of the method, a scale-up reaction of gem-difluorocyclopropenyl imide 1a (5.0 mmol) and nitrone 2r (6.0 mmol) was carried out under the standard reaction conditions. After completing the [3 + 2] cycloaddition, the crude reaction mixture was subjected to silica gel column chromatography, where the cycloadduct underwent rearrangement to give the desired product 3ar in 51% yield (Scheme ).

3. Scale-Up Synthesis of 3ar .

3

Open in a new tab

In Vitro Evaluation of Antiproliferative Activity

The Cell Counting Kit-8 (CCK-8) assay was employed to evaluate the in vitro antiproliferative activity of each final compound against 4T1 cells. Additionally, the seven most active compounds were selected for evaluation of their in vitro antiproliferative activity against A549 cells. The half-maximal inhibitory concentration (IC50) values of the tested compounds against 4T1 and A549 cells are presented in Figure . Among all synthesized derivatives, compound 3aw demonstrated the strongest in vitro cell proliferation inhibitory activity against 4T1 cells, with an IC50 value of 6.85 ± 0.43 μM, which was significantly lower than those of the other derivatives. Consequently, 3aw was selected as the lead compound for subsequent in vitro antibreast cancer phenotype screening. Compound 3as exhibited the most active antiproliferative activity against A549 cells with an IC50 value of 10.34 ± 0.07 μM. A profound structure–activity relationship (SAR) was observed between the nature of the substituents at R1 and R2 of the compounds and antiproliferative activity against 4T1 cells. Compounds 3aa–3ak bearing a phenyl group (R1) and various aryl groups (R2) with substituents at the para, meta, and ortho positions were generally inactive, except for 3aa (IC50 = 139.37 ± 0.75 μM) and 3ak (IC50 = 58.63 ± 0.36 μM), exhibiting moderate activity. With primary alkyl groups (R2), compounds 3al–3ap showed moderate activities (IC50 = 67.41 ± 2.16 μM to 159.03 ± 0.06 μM), while 3aq was inactive. A 4-fold increase in activity was observed when the isopropyl group (3ar: IC50 = 66.89 ± 1.82 μM) at R2 was replaced by the tert-butyl group (3as: IC50 = 17.42 ± 1.53 μM). The compounds 3at and 3au bearing cyclopropyl and cyclobutyl groups, respectively, at R2 were inactive, whereas the compound 3av (IC50 = 56.50 ± 0.25 μM) with a cyclopentyl group at R2 exhibited moderate activity. The cyclohexyl-substituted analogue 3aw (IC50 = 6.85 ± 0.43 μM) displayed the best activity. The activity of compound 3ay (IC50 = 20.39 ± 0.11 μM) bearing a 4-N-Boc-piperidinyl group increased nearly 6-fold compared to compound 3ax (IC50 = 118.00 ± 0.85 μM) bearing a 4-tetrahydropyranyl group. The tertiary stereogenic center-containing compound 3az was inactive. Except for 3br and 3gr, all compounds possessing an isopropyl group (R2) and different aryl groups (R1) having substituents at the para, meta, and ortho positions were moderately active (IC50 = 42.71 ± 0.09 μM to 78.45 ± 1.46 μM). When a 1-naphthyl group was present at R,2 the activity was moderate (3jr: IC50 = 38.32 ± 1.65 μM). Against A549 cells, compound 3aw (IC50 = 25.22 ± 0.47 μM) and 3av (IC50 = 71.36 ± 1.02 μM) were moderately active. A significant improvement in activity was observed with compound 3as (IC50 = 10.34 ± 0.07 μM). The remaining compounds 3ay, 3cr, 3ir, and 3jr were moderately active (IC50 = 16.02 ± 0.33 μM to 120.57 ± 1.48 μM).

2.

2

Open in a new tab

Screening of the tested compounds in 4T1 mouse mammary carcinoma cells and A549 human lung carcinoma cells after 24 h of treatment. IC50 values represent the concentration of compound required to inhibit 50% of cell proliferation. Data are representative of three independent experiments, and error bars indicate mean ± SD.

To provide a contextual benchmark, literature IC50 values of reference anticancer agents in the same cell lines were incorporated. Specifically, docetaxel, a standard chemotherapeutic for breast cancer, exhibited an IC50 of 31.45 μM against 4T1 cells, while cisplatin, a first-line agent for lung cancer, showed an IC50 of 16.48 μM against A549 cells. Notably, compound 3aw (IC50 = 6.85 ± 0.43 μM) displayed approximately 4.6-fold greater potency than docetaxel in the same 4T1 cell line, and compound 3as (IC50 = 10.34 ± 0.07 μM) was about 1.6-fold more active than cisplatin against A549 cells. These comparisons suggest that the newly synthesized fluorinated isoxazolidinone scaffold possesses antiproliferative efficacy comparable to or even superior to clinically used drugs, despite being at an early discovery stage. Moreover, compound 3aw exhibited an IC50 of 39.49 ± 0.15 μM against normal L929 cells after 24 h of treatment, leading to a selectivity index (SI = IC50(L929)/IC50(4T1)) of 5.76. This result indicates that 3aw exhibits favorable selectivity for breast cancer cells over normal cells, supporting its potential as a safe lead candidate for further development.

Induction of Apoptosis in 4T1 Cells by Compound 3aw

Previous studies have demonstrated that parthenolide (PTL) induces apoptosis in various human tumor cells, with its core mechanism of action attributed to the α-methylene-γ-lactone pharmacophore present in its structure.

To determine whether the antiproliferative effect of compound 3aw is associated with the induction of apoptosis, Annexin V FITC/PI double-staining and flow cytometric analysis were performed. As shown in Figure A, compared with the untreated control group, the population of apoptotic 4T1 cells increased significantly in a dose-dependent manner following 24 h of treatment with 2, 4, or 6 μM of 3aw. Quantitative analysis revealed that the total apoptosis rate increased markedly with rising concentrations of 3aw, rising from 5.09 ± 0.11% in the control group to 8.96 ± 0.14%, 12.41 ± 0.18%, and 18.74 ± 0.25%, respectively. Dose-dependent increases were also observed in both early and late apoptosis rates. These results confirm that compound 3aw effectively induces apoptosis in 4T1 cells in a dose-dependent manner.

3.

3

Open in a new tab

Pro-apoptotic effects of 3aw on 4T1 breast cancer cells. Representative flow cytometry plots of Annexin V/PI staining (A) and statistical analysis of the proportion of apoptotic cells (B) in 4T1 cells after treatment with different concentrations of compound 3aw for 24 h. Data are representative of three independent experiments, and error bars indicate mean ± SD ***P < 0.001 vs control.

Compound 3aw Effectively Induces Mitochondrial Damage in 4T1 Cells

Depolarization of the mitochondrial membrane potential (ΔΨm) is recognized as a hallmark and irreversible early event in the initiation of the intrinsic apoptosis pathway. The extent of membrane potential decline directly reflects the degree of mitochondrial dysfunction. , Concurrently, mitochondrial dysfunction triggers excessive production of reactive oxygen species (ROS) within the cell. The abnormal accumulation of ROS further exacerbates the disruption of mitochondrial membrane structure and function, thereby creating a cascading, self-amplifying vicious cycle that ultimately and irreversibly activates the apoptotic program. As a result, ROS are considered both a core product of mitochondrial damage and a key upstream regulator driving the activation of the mitochondrial apoptosis pathway. ,

To determine whether 3aw has mitochondrial damage potential, the JC-1 fluorescent probe was first employed to detect changes in ΔΨm in 4T1 cells following treatment with different concentrations of 3aw, as shown in Figure A. In the control group, JC-1 aggregated within the mitochondrial matrix to form clusters, emitting intense red fluorescence, which indicated a high ΔΨm. Following treatment with 2, 4, and 6 μM of 3aw for 24 h, the intensity of red fluorescence was significantly reduced in a dose-dependent manner, whereas the green fluorescence signal was markedly enhanced. The fluorescence phenotype exhibited a gradual shift from red to green, reflecting the conversion of JC-1 to its monomeric form in the cytoplasm due to the collapse of ΔΨm. Quantitative analysis revealed a significant decrease in the red to green fluorescence ratio (Figure B), indicating that 3aw induced a dose-dependent reduction in mitochondrial membrane potential. To further validate the 3aw mediated mitochondrial dysfunction, the DCFH DA fluorescence staining method was then used to detect intracellular ROS levels (Figure C and D). Following the treatment with 3aw, the intensity of green fluorescence increased in a dose-dependent manner, indicating a significant elevation in intracellular ROS levels. The mean fluorescence intensity (MFI) exhibited a significant dose-dependent increase. In summary, these data indicate that 3aw is capable of inducing severe mitochondrial dysfunction and oxidative stress in 4T1 cells.

4.

4

Open in a new tab

Mitochondrial damage induction effects of 3aw on 4T1 breast cancer cells. (A) Representative fluorescence imaging of mitochondrial membrane potential changes in 4T1 cells detected by the JC-1 probe after treatment with different concentrations of 3aw for 24 h (scale bar: 100 μm). (B) Quantitative analysis of the JC-1 red/green fluorescence intensity ratio (n = 3). (C) Representative fluorescence imaging of intracellular ROS in 4T1 cells detected by the ROS assay kit with different concentrations of 3aw for 24 h (scale bar: 200 μm). (D) Quantitative analysis of the relative mean fluorescence intensity (MFI) of ROS. Data are representative of three independent experiments, and error bars indicate mean ± SD *P < 0.05 and **P < 0.01 vs control.

Compound 3aw Regulates Mitochondrial Apoptotic Pathway-Related Protein and Gene Expression

The mitochondria-mediated intrinsic apoptosis pathway represents a central mechanism regulating apoptosis in tumor cells, with Bcl-2 family proteins serving as the key molecular switches in this pathway. , The expression ratio of the antiapoptotic protein Bcl-2 to the pro-apoptotic protein Bax directly determines the occurrence of mitochondrial outer membrane permeabilization (MOMP), an irreversible key step in the initiation of the mitochondrial apoptosis pathway. Caspase-3, as the core effector protease of apoptosis, serves as a hallmark marker of the final execution of the mitochondrial apoptotic cascade through the cleavage and activation of its precursor protein (pro-caspase-3). ,

To elucidate the downstream molecular regulatory mechanism of 3aw in 4T1 cells, Western blot analysis was employed to detect the expression levels of key regulatory proteins in the mitochondrial apoptosis pathway, while real-time quantitative PCR (qRT-PCR) was used to assess the transcriptional changes of these markers. The Western blot results are presented in Figure A and B. Following treatment of 4T1 cells with 3aw for 24 h, the expression of the antiapoptotic protein Bcl-2 was downregulated in a dose-dependent manner, whereas the expression of the pro-apoptotic protein Bax was significantly upregulated. Consequently, the Bax/Bcl-2 ratio increased markedly, suggesting the occurrence of MOMP. Meanwhile, the protein level of the apoptotic executor pro-caspase-3 decreased in a dose-dependent manner, whereas the expression of its activated form, cleaved caspase-3 (C-Caspase-3), showed the opposite trend. These findings confirm the robust activation of the caspase-mediated apoptotic cascade.

5.

5

Open in a new tab

Regulatory effects of 3aw on mitochondrial apoptotic markers. (A) Protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 (C-Caspase-3) in 4T1 cells after treatment with different concentrations of compound 3aw for 24 h. (B) Quantitative analysis of the corresponding protein bands, normalized against GAPDH. (C) Relative mRNA expression levels of Bcl-2, Bax, and caspase-3 in 4T1 cells after treatment with different concentrations of compound 3aw for 24 h. Data are representative of three independent experiments, and error bars indicate mean ± SD, ns = not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 vs control.

Consistent with the protein expression data, the qRT-PCR results (Figure C) showed that the 3aw treatment, in a dose-dependent manner, significantly upregulated the mRNA levels of Bax and caspase-3 as well as downregulated Bcl-2 mRNA. These results indicate that 3aw promotes the activation of the mitochondrial apoptotic cascade by transcriptionally regulating apoptosis-related genes.

Conclusion

In conclusion, we have developed a Ni­(II)-catalyzed [3 + 2] cycloaddition/rearrangement cascade reaction of gem-difluorocyclopropenyl imides and N-Bn nitrones, affording (Z)-4-(monofluoroalkylidene)­isoxazolidin-5-ones in up to 85% yield, featuring a fluorine atom at the exocyclic double bond. In this transformation, the [3 + 2] cycloadducts, bearing a strained three-membered ring and a labile N–O bond, underwent skeletal rearrangement during silica gel chromatography through a cascade water incorporation, N–O bond cleavage, and HF elimination. The product 3aw emerged as the most potent antiproliferative agent against mouse breast cancer 4T1 cells in vitro (IC50 = 6.85 ± 0.43 μM). Further mechanistic investigations revealed that 3aw induces apoptosis in a concentration-dependent manner and causes severe structural and functional mitochondrial damage by triggering mitochondrial membrane potential depolarization and excessive intracellular accumulation of reactive oxygen species (ROS). At the molecular regulatory level, 3aw significantly down-regulates the antiapoptotic protein Bcl-2 and up-regulates the pro-apoptotic protein Bax, thereby elevating the Bax/Bcl-2 ratio and initiating mitochondrial outer membrane permeabilization (MOMP). Concurrently, 3aw promotes the cleavage and activation of pro-caspase-3, leading to the activation of the caspase cascade apoptotic response. These regulatory effects were confirmed at both protein and transcriptional levels. In brief, compound 3aw exerts its antibreast cancer activity by activating the mitochondria-mediated intrinsic apoptotic pathway and can be further developed as a mitochondria-targeted lead compound for antibreast cancer drug development. Against the human lung carcinoma A549 cells, compound 3as (IC50 = 10.34 ± 0.07 μM) displayed promising activity. Overall, this work opens new avenues in the field of cyclorearrangement cascade reactions and holds considerable potential in pharmaceutical research.

Supplementary Material

ml6c00344_si_001.pdf (8.6MB, pdf)

Acknowledgments

Y.H. gratefully acknowledges the financial support from the National Natural Science Foundation of China (22372128), and the Innovative Research Group Project of National Natural Science Foundation of Hubei (2025AFA049).

Glossary

ABBREVIATIONS

TLC

thin layer chromatography

DCM

dichloromethane

THF

tetrahydrofuran

MS

molecular sieves

ND

not detected

SLs

sesquiterpene lactones

CCK-8

cell counting kit-8;

PTL

parthenolide

ROS

reactive oxygen species

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

MFI

mean fluorescence intensity

MOMP

mitochondrial outer membrane permeabilization

qRT-PCR

quantitative reverse transcription polymerase chain reaction

mRNA

messenger ribonucleic acid.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00344.

  • Experimental details, characterization and crystallographic data, spectra of all unknown compounds, and computational details (PDF)

¶.

Z.-H.L. Y.H. and Y.-C.L. contributed equally.

The authors declare no competing financial interest.

References

  1. Breast Cancer; World Health Organization, 2024. [Google Scholar]
  2. GBD 2023 Breast Cancer Collaborators. Global, Regional, and National Burden of Breast Cancer among Females, 1990–2023, with Forecasts to 2050: A Systematic Analysis for the Global Burden of Disease Study 2023. Lancet Oncol. 2026;27:302–326. doi: 10.1016/S1470-2045(25)00730-2. [DOI] [PubMed] [Google Scholar]
  3. Liu C., Chen H., Guo S., Liu Q., Chen Z., Huang H., Zhao Q., Li L., Cen H., Jiang Z., Luo Q., Chen X., Zhao J., Chen W., Yang P. C., Wang L.. Anti-breast cancer-induced cardiomyopathy: Mechanisms and future directions. Biomed Pharmacother. 2023;166:115373. doi: 10.1016/j.biopha.2023.115373. [DOI] [PubMed] [Google Scholar]
  4. You Z.. et al. Assessing the Optimal Regimen: A Systematic Review and Network Meta-Analysis of the Efficacy and Safety of Long-Acting Granulocyte Colony-Stimulating Factors in Patients with Breast Cancer. Cancers. 2023;15:3675. doi: 10.3390/cancers15143675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kinnel B., Singh S. K., Oprea-Ilies G., Singh R.. Targeted Therapy and Mechanisms of Drug Resistance in Breast Cancer. Cancers. 2023;15:1320. doi: 10.3390/cancers15041320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen R., Yu Y., Liu R., Chen Q.. Targeting Breast Cancer Resistance Protein (BCRP/ABCG2) in Cancer. Transl. Cancer Res. 2024;13:6550–6564. doi: 10.21037/tcr-24-1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Himaja A., Routholla G., Patel T., Banerjee S., Begum D., Regula S., Pulya S., Biswas S., Adhikari N., Ghosh B.. Design and Synthesis of Pyridine-Based Benzamides as Potent HDAC3 Inhibitors as an Armament against Breast Cancer with In Vivo Validation. Eur. J. Med. Chem. 2025;291:117636. doi: 10.1016/j.ejmech.2025.117636. [DOI] [PubMed] [Google Scholar]
  8. Dirsch V. M., Stuppner H., Ellmerer-Müller E. P., Vollmar A. M.. Structural Requirements of Sesquiterpene Lactones to Inhibit LPS-Induced Nitric Oxide Synthesis in RAW 264.7 Macrophages. Bioorg. Med. Chem. 2000;8:2747–2753. doi: 10.1016/S0968-0896(00)00202-9. [DOI] [PubMed] [Google Scholar]
  9. Kupchan S. M., Eakin M. A., Thomas A. M.. Tumor Inhibitors. 69. Structure-Cytotoxicity Relations among the Sesquiterpene Lactones. J. Med. Chem. 1971;14:1147–1152. doi: 10.1021/jm00294a001. [DOI] [PubMed] [Google Scholar]
  10. Janecka A., Wyrębska A., Gach K., Fichna J., Janecki T.. Natural and Synthetic α-Methylenelactones and α-Methylenelactams with Anticancer Potential. Drug Discovery Today. 2012;17:561–572. doi: 10.1016/j.drudis.2012.01.013. [DOI] [PubMed] [Google Scholar]
  11. Kupchan S. M., Fessler C. D., Eakin M., Giacobbe T. J.. Reactions of Alpha Methylene Lactone Tumor Inhibitor with Model Biological Nucleophiles. Science. 1970;168:376–377. doi: 10.1126/science.168.3929.376. [DOI] [PubMed] [Google Scholar]
  12. González A. G., Silva M. H., Padrón J. I., León F., Reyes E., Álvarez-Mon M., Pivel J. P., Quintana J., Estévez F., Bermejo J.. Synthesis and Antiproliferative Activity of a New Compound Containing an α-Methylene-γ-Lactone Group. J. Med. Chem. 2002;45:2358–2361. doi: 10.1021/jm025518n. [DOI] [PubMed] [Google Scholar]
  13. Janecki T., Błaszczyk E., Studzian K., Janecka A., Krajewska U., Różalski M.. Novel Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationship Studies of a Series of r-Alkylidene-γ-lactones and Lactams. J. Med. Chem. 2005;48:3516–3521. doi: 10.1021/jm048970a. [DOI] [PubMed] [Google Scholar]
  14. Rózalski M., Krajewska U., Panczyk M., Mirowski M., Rózalska B., Wąsek T., Janecki T.. Synthesis and Biological Evaluation of 4-Methylideneisoxazolidin-5-ones-A New Class of Highly Cytotoxic α-Methylidene-γ-Lactones. Eur. J. Med. Chem. 2007;42:248–255. doi: 10.1016/j.ejmech.2006.09.005. [DOI] [PubMed] [Google Scholar]
  15. Janecki T., Wąsek T., Różalski M., Krajewska U., Studzian K., Janecka A.. 4-Methylideneisoxazolidin-5-Ones–A New Class of α-Methylidene-γ-Lactones with High Cytostatic Activity. Bioorg. Med. Chem. Lett. 2006;16:1430–1433. doi: 10.1016/j.bmcl.2005.11.032. [DOI] [PubMed] [Google Scholar]
  16. Purser S., Moore P. R., Swallow S., Gouverneur V.. Fluorine in Medicinal Chemistry. Chem. Soc. Rev. 2008;37:320–330. doi: 10.1039/B610213C. [DOI] [PubMed] [Google Scholar]
  17. Wang J., Sánchez-Roselló M., Aceña J. L., del Pozo C., Sorochinsky A. E., Fustero S., Soloshonok V. A., Liu H.. Fluorine in Pharmaceutical Industry: Fluorine-Containing Drugs Introduced to the Market in the Last Decade (2001–2011) Chem. Rev. 2014;114:2432–2506. doi: 10.1021/cr4002879. [DOI] [PubMed] [Google Scholar]
  18. Zhou Y., Wang J., Gu Z., Wang S., Zhu W., Aceña J. L., Soloshonok V. A., Izawa K., Liu H.. Next Generation of Fluorine-Containing Pharmaceuticals, Compounds Currently in Phase II–III Clinical Trials of Major Pharmaceutical Companies: New Structural Trends and Therapeutic Areas. Chem. Rev. 2016;116:422–518. doi: 10.1021/acs.chemrev.5b00392. [DOI] [PubMed] [Google Scholar]
  19. Furuya T., Kamlet A. S., Ritter T.. Catalysis for Fluorination and Trifluoromethylation. Nature. 2011;473:470–477. doi: 10.1038/nature10108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zhu Y., Han J., Wang J., Shibata N., Sodeoka M., Soloshonok V. A., Coelho J. A. S., Toste F. D.. Modern Approaches for Asymmetric Construction of Carbon–Fluorine Quaternary Stereogenic Centers: Synthetic Challenges and Pharmaceutical Needs. Chem. Rev. 2018;118:3887–3964. doi: 10.1021/acs.chemrev.7b00778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kalita S. J., Qi J., Xiao L., Saha D., Huang Y.-Y., Shibata N.. Recent Advances on Catalytic Asymmetric Synthesis of Molecules Bearing a Fluorine-Containing Stereogenic Carbon Center (2015–2024) Chem. Rev. 2025;125:8477–8654. doi: 10.1021/acs.chemrev.5c00177. [DOI] [PubMed] [Google Scholar]
  22. Li Y.-C., Wang Y.-H., Kalita S. J., Huang Y.-Y.. Trivalent Phosphine-Catalyzed Reactions Using Fluorinated Building Blocks. Adv. Synth. Catal. 2023;365:2487–2510. doi: 10.1002/adsc.202300381. [DOI] [Google Scholar]
  23. Li G. Q., Zhang Y. B., Guan H. S.. A new isoxazol from Glehnia littoralis. Fitoterapia. 2008;79:238–239. doi: 10.1016/j.fitote.2008.01.002. [DOI] [PubMed] [Google Scholar]
  24. Dietrich, H. ; Döller, U. ; Gatzweiler, E. ; Helmke, H. ; Lehr, S. ; Machettira, A. ; Mcleod, M. ; Müller, T. ; Peters, O. ; Rosinger, C. ; Schmutzler, D. . Substituted Isoxazoline-5-ones and Salts Thereof and Use Thereof as Herbicidal Agents. World Patent WO2018178008A1 2018.
  25. Ferrazzano L., Viola A., Lonati E., Bulbarelli A., Musumeci R., Cocuzza C., Lombardo M., Tolomelli A.. New Isoxazolidinone and 3,4-Dehydro-β-Proline Derivatives as Antibacterial Agents and MAO-Inhibitors: A Complex Balance Between Two Activities. Eur. J. Med. Chem. 2016;124:906–919. doi: 10.1016/j.ejmech.2016.09.007. [DOI] [PubMed] [Google Scholar]
  26. Prakash M., Venneti N. M., Westrick J., Kodanko J. J.. A Highly Convergent Synthesis Route for Adda: A Non-Proteinogenic Amino Acid Unit in Cyanobacteria. J. Org. Chem. 2025;90:11753–11756. doi: 10.1021/acs.joc.5c01357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Eitzinger A., Winter M., Schörgenhumer J., Waser M.. Quaternary β2,2-Amino Acid Derivatives by Asymmetric Addition of Isoxazolidin-5-Ones to para-Quinone Methides. Chem. Commun. 2020;56:579–582. doi: 10.1039/C9CC09239K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wang T.-T., Jin H.-S., Cao M.-M., Wang R.-B., Zhao L.-M.. Rh­(III)-Catalyzed Regioselective Annulations of 3-Arylisoxazolones and 3-Aryl-1,4,2-Dioxazol-5-Ones with Propargyl Alcohols: Access to 4-Arylisoquinolines and 4-Arylisoquinolones. Org. Lett. 2021;23:5952–5957. doi: 10.1021/acs.orglett.1c02049. [DOI] [PubMed] [Google Scholar]
  29. Luisi R., Capriati V., Florio S., Vista T.. New Synthesis of Optically Active 5-Isoxazolidinones and β-Amino Acids. J. Org. Chem. 2003;68:9861–9864. doi: 10.1021/jo035356i. [DOI] [PubMed] [Google Scholar]
  30. Bentley S. A., Davies S. G., Lee J. A., Roberts P. M., Russell A. J., Thomson J. E., Toms S. M.. Conjugate Addition of Lithium N-tert-Butyldimethylsilyloxy-N-(α-Methylbenzyl)-Amide: Asymmetric Synthesis of β2,2,3-Trisubstituted Amino Acids. Tetrahedron. 2010;66:4604–4620. doi: 10.1016/j.tet.2010.04.027. [DOI] [Google Scholar]
  31. Pearson C., Rinehart K. L., Sugano M., Costerison J. R.. Enantiospecific Synthesis of N-Boc-Adda: A Linear Approach. Org. Lett. 2000;2:2901–2903. doi: 10.1021/ol006347o. [DOI] [PubMed] [Google Scholar]
  32. Tak R. K., Noda H., Shibasaki M.. Ligand-Enabled, Copper-Catalyzed Electrophilic Amination for the Asymmetric Synthesis of β Amino Acids. Org. Lett. 2021;23:8617–8621. doi: 10.1021/acs.orglett.1c03328. [DOI] [PubMed] [Google Scholar]
  33. Merino P., del Alamo E. M., Bona M., Franco S., Merchan F. L., Tejero T., Vieceli O.. Lewis Acid Stereocontrolled Additions of a Silyl Ketene Acetal to 2,3-Di-O-Isopropylidene-D-glyceraldehyde Nitrones. Synthesis of L-Isoxazolidinyl Nucleosides. Tetrahedron Lett. 2000;41:9239–9243. doi: 10.1016/S0040-4039(00)01674-9. [DOI] [Google Scholar]
  34. Merino P., Franco S., Garces N., Merchan F. L., Tejero T.. Modified Nucleosides from Nitrones: A New and Efficient Stereoselective Approach to Isoxazolidinyl Thymidine Derivatives. Chem. Commun. 1998;34:493. doi: 10.1039/a705673g. [DOI] [Google Scholar]
  35. Panfil I., Urbańczyk-Lipkowska Z., Chmielewski M.. Isoxazolidin-5-One Analogs of β-Lactam Antibiotics. Carbohydr. Res. 1998;306:505–515. doi: 10.1016/S0008-6215(97)10107-0. [DOI] [Google Scholar]
  36. Socha D., Jurczak M., Chmielewski M.. Synthesis of Acosamine and Daunosamine from Sugar σ-Enelactones. Tetrahedron. 1997;53:739–746. doi: 10.1016/S0040-4020(96)01016-2. [DOI] [Google Scholar]
  37. Annibaletto J., Oudeyer S., Levacher V., Brière J.-F.. Catalytic Enantioselective Syntheses of Isoxazolidin-5-Ones. Synthesis. 2017;49:2117–2128. doi: 10.1055/s-0036-1588765. [DOI] [Google Scholar]
  38. Macchia A., Eitzinger A., Brière J.-F., Waser M., Massa A.. Asymmetric Synthesis of Isoxazol-5-Ones and Isoxazolidin-5-Ones. Synthesis. 2021;53:107–122. doi: 10.1055/s-0040-1706483. [DOI] [Google Scholar]
  39. Annibaletto J., Martzel T., Levacher V., Oudeyer S., Brière J.-F.. Multicomponent Catalytic Enantioselective Synthesis of Isoxazolidin-5-Ones. Adv. Synth. Catal. 2021;363:4447–4451. doi: 10.1002/adsc.202100719. [DOI] [Google Scholar]
  40. Postikova S., Tite T., Levacher V., Brière J.-F.. Organocatalyzed Synthesis of Isoxazolidin-5-Ones: The Meldrum’s Acid Approach. Adv. Synth. Catal. 2013;355:2513–2517. doi: 10.1002/adsc.201300465. [DOI] [Google Scholar]
  41. Zhao H.-W., Liu Y.-Y., Zhao Y.-D., Pang H.-L., Chen X.-Q., Song X.-Q., Tian T., Li B., Yang Z., Du J., Feng N.-N.. [3 + 2] Cycloaddition of Oxazol-5-(4H)-Ones with Nitrones for Diastereoselective Synthesis of Isoxazolidin-5-Ones. Org. Lett. 2017;19:26–29. doi: 10.1021/acs.orglett.6b03206. [DOI] [PubMed] [Google Scholar]
  42. Shindo M., Itoh K., Tsuchiya C., Shishido K.. Anionic Inverse Electron-Demand 1,3-Dipolar Cycloaddition of Nitrones with Ynolates. Facile Stereoselective Synthesis of 5-Isoxazolidinones Leading to β-Amino Acids. Org. Lett. 2002;4:3119–3121. doi: 10.1021/ol0264455. [DOI] [PubMed] [Google Scholar]
  43. Jiapian H., Fei L., Jie W.. Recent Advances in the Transformation of Difluorocyclopropenes. Acta Chim. Sinica. 2023;81:520–532. doi: 10.6023/A23030088. [DOI] [Google Scholar]
  44. Dolbier W. R. Jr., Battiste M. A.. Structure, Synthesis, and Chemical Reactions of Fluorinated Cyclopropanes and Cyclopropenes. Chem. Rev. 2003;103:1071–1098. doi: 10.1021/cr010023b. [DOI] [PubMed] [Google Scholar]
  45. Wiberg K. B., Marquez M.. Effect of Fluorine Substitution on the Energies of Small Ring Compounds. J. Am. Chem. Soc. 1998;120:2932–2938. doi: 10.1021/ja971413b. [DOI] [Google Scholar]
  46. Nosik P. S., Pashko M. O., Poturai A. S., Kvasha D. A., Pashenko A. E., Rozhenko A. B., Suikov S., Volochnyuk D. M., Ryabukhin S. V., Yagupolskii Y. L.. Monosubstituted 3,3-Difluorocyclopropenes as Bench-Stable Reagents: Scope and Limitations. Eur. J. Org. Chem. 2021;2021:6604–6615. doi: 10.1002/ejoc.202100921. [DOI] [Google Scholar]
  47. Law D. C. F., Tobey S. W.. Diels-Alder Reactions of Tetrahalocyclopropenes. J. Am. Chem. Soc. 1968;90:2376–2386. doi: 10.1021/ja01011a030. [DOI] [Google Scholar]
  48. Muriel B., Gagnebin A., Waser J.. Synthesis of Bicyclo[3.1.0]­hexanes by (3 + 2) Annulation of Cyclopropenes with Aminocyclopropanes. Chem. Sci. 2019;10:10716–10722. doi: 10.1039/C9SC03790J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Donnelly K., Singh A., Tuttle T., Baumann M.. [3 + 2]-Cycloaddition Reactions of gem-Difluorocyclopropenes with Azomethine Ylides–Access to Novel Fluorinated Scaffolds. Chem.Eur. J. 2023;29:e202301861. doi: 10.1002/chem.202301861. [DOI] [PubMed] [Google Scholar]
  50. Pan D., Liu F.-X., Zeng Z., Ye J., Cai Y., Wang S., Zhou Z., Yi W.. Practical Conversion of gem-Difluorocyclopropenes for the Chemodivergent Assembly of Fluorinated Heterocyclic Frameworks. Green Chem. 2023;25:10630–10637. doi: 10.1039/D3GC03203E. [DOI] [Google Scholar]
  51. Liu B., Liu D., Niu C., Xia Y., Lu X., Fu Y., Liu Q.. Cobalt-Catalyzed Stereoselective Synthesis of Chiral gem-Difluorocyclopropanes with Vicinal Stereocenters. Org. Chem. Front. 2024;11:6617–6626. doi: 10.1039/D4QO01585A. [DOI] [Google Scholar]
  52. Zhang Z., Gevorgyan V.. Palladium Hydride-Enabled Hydroalkenylation of Strained Molecules. J. Am. Chem. Soc. 2022;144:20875–20883. doi: 10.1021/jacs.2c09045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhao X., Xu S., He J., Zhou Y., Cao S.. Highly Regio- and Diastereoselective Cu-Catalyzed Hydroborylation and Hydrosilylation of Difluorocyclopropenes with B2pin2 and PhMe2Si-Bpin. Org. Chem. Front. 2019;6:2539–2543. doi: 10.1039/C9QO00580C. [DOI] [Google Scholar]
  54. Sekine K., Akaishi D., Konagaya K., Ito S.. Copper-Catalyzed Enantioselective Hydrosilylation of gem-Difluorocyclopropenes Leading to a Stereochemical Study of the Silylated gem-Difluorocyclopropanes. Chem.Eur. J. 2022;28:e202200657. doi: 10.1002/chem.202200657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yamani K., Pierre H., Archambeau A., Meyer C., Cossy J.. Asymmetric Transfer Hydrogenation of gem-Difluorocyclopropenyl Esters: Access to Enantioenriched gem-Difluorocyclopropanes. Angew. Chem., Int. Ed. 2020;59:18505–18509. doi: 10.1002/anie.202008572. [DOI] [PubMed] [Google Scholar]
  56. Sekine K., Ushiyama A., Endo Y., Mikami K.. Enantioselective Functionalization of Difluorocyclopropenes Catalyzed by Chiral Copper Complexes: Proposal for Chiral gem-Dimethyl and tert-Butyl Analogues. J. Org. Chem. 2020;85:7916–7924. doi: 10.1021/acs.joc.0c00622. [DOI] [PubMed] [Google Scholar]
  57. Xu H., Chen W., Bian M., Xu H., Gao H., Wang T., Zhou Z., Yi W.. gem-Difluorocyclopropenes as Versatile β-Monofluorinated Three sp2 Carbon Sources for Cp*Rh­(III)-Catalyzed [4 + 3] Annulation: Experimental Development and Mechanistic Insight. ACS Catal. 2021;11:14694–14701. doi: 10.1021/acscatal.1c04508. [DOI] [Google Scholar]
  58. He Y., Tian L., Chang X., Qu Z., Huang Y., Huang C., Sun Q., Wang H.. Rhodium-Catalyzed Formal [4 + 3] Annulation Reaction of N-Methoxybenzamides with gem-Difluorocyclopropenes: A Combination of Experimental and Theoretical Studies. Chin. Chem. Lett. 2022;33:2987–2992. doi: 10.1016/j.cclet.2022.01.068. [DOI] [Google Scholar]
  59. Dong J., Feng W., Wang L., Li M., Chen Z., Xu X.. Cu/Base Co-Catalyzed [3 + 3] Cycloaddition for the Synthesis of Highly Functionalized 4-Fluoropyridines. Chem. Commun. 2021;57:12635–12638. doi: 10.1039/D1CC05412K. [DOI] [PubMed] [Google Scholar]
  60. Tang X., Liu K., Qu Z., Zhan J., Zhu R., Teng F., Meng L., Huang Y., Huang C., He Y., Zhu Q.. Convenient and Efficient Access to Tri- and Tetra-Substituted 4-Fluoropyridines via a [3 + 2]/[2 + 1] Cyclization Reaction. Chin. Chem. Lett. 2022;33:2982–2986. doi: 10.1016/j.cclet.2022.02.031. [DOI] [Google Scholar]
  61. Tran G., Gomez Pardo D., Tsuchiya T., Hillebrand S., Vors J.-P., Cossy J.. Modular, Concise, and Efficient Synthesis of Highly Functionalized 5-Fluoropyridazines by a [2 + 1]/[3 + 2]-Cycloaddition Sequence. Org. Lett. 2015;17:3414–3417. doi: 10.1021/acs.orglett.5b01370. [DOI] [PubMed] [Google Scholar]
  62. Feraldi-Xypolia A., Fredj G., Tran G., Tsuchiya T., Vors J.-P., Mykhailiuk P., Gomez Pardo D., Cossy J.. Synthesis of Functionalized 4-Fluoropyridazines. Asian J. Org. Chem. 2017;6:927–935. doi: 10.1002/ajoc.201700216. [DOI] [Google Scholar]
  63. Shen M., Li H., Zhang X., Fan X.. Rh­(III)-Catalyzed Simultaneous [3 + 3]/[5 + 1] Annulation of 1-Arylpyrazolidinones with gem-Difluorocyclopropenes Leading to Fluorinated Pyridopyrimidinone Derivatives. Org. Chem. Front. 2022;9:5976–5982. doi: 10.1039/D2QO01230H. [DOI] [Google Scholar]
  64. Li Q., Zhu Y., Wang Y., Qi G., Hao W.-J., Yan K., Jiang B.. Catalytic C–H Activation-Initiated Transannulation: An Oxygen Transfer Route to Ring-Fluorinated Tricyclic γ-Lactones. Chin. Chem. Lett. 2024;35:109494. doi: 10.1016/j.cclet.2024.109494. [DOI] [Google Scholar]
  65. Nihei T., Hoshino T., Konno T.. An Efficient Approach to gem-Difluorocyclopropylstannanes via Highly Regio- and Stereoselective Hydrostannylation of gem-Difluorocyclopropenes and Their Unique Ring-Opening Reaction to Afford β-Fluoroallylic Alcohols. Org. Biomol. Chem. 2015;13:3721–3731. doi: 10.1039/C5OB00046G. [DOI] [PubMed] [Google Scholar]
  66. He Y., Yuan J., Lv W.-X., Liu P., Teng F., Mo Q., Wu Z., Huang C., Liu Q., Wang H.. Simple Nucleophile/H2O Promoted Defluorinative Ring-Opening of gem-Difluorocyclopropenes. Green Synth. Catal. 2024;5:14–19. doi: 10.1016/j.gresc.2023.01.003. [DOI] [Google Scholar]
  67. Landelle G., Bergeron M., Turcotte-Savard M.-O., Paquin J.-F.. Synthetic Approaches to Monofluoroalkenes. Chem. Soc. Rev. 2011;40:2867–2908. doi: 10.1039/C0CS00201A. [DOI] [PubMed] [Google Scholar]
  68. Murahashi S.-I., Imada Y.. Synthesis and Transformations of Nitrones for Organic Synthesis. Chem. Rev. 2019;119:4684–4716. doi: 10.1021/acs.chemrev.8b00476. [DOI] [PubMed] [Google Scholar]
  69. Anderson L. L.. Diverse Applications of Nitrones for the Synthesis of Heterocyclic Compounds. Asian J. Org. Chem. 2016;5:9–30. doi: 10.1002/ajoc.201500211. [DOI] [Google Scholar]
  70. Wang Y., Gui J.. Bioinspired Skeletal Reorganization Approach for the Synthesis of Steroid Natural Products. Acc. Chem. Res. 2024;57:568–579. doi: 10.1021/acs.accounts.3c00716. [DOI] [PubMed] [Google Scholar]
  71. Al-Ahmad R., Dai M.. Advancing Total Synthesis Through Skeletal Editing. Acc. Chem. Res. 2025;58:1392–1406. doi: 10.1021/acs.accounts.5c00030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Xie J., Dong G.. Cyclopropylcarbinyl Cation Chemistry in Synthetic Method Development and Natural Product Synthesis: Cyclopropane Formation and Skeletal Rearrangement. Org. Chem. Front. 2023;10:2346–2358. doi: 10.1039/D3QO00282A. [DOI] [Google Scholar]
  73. Zhang R., Dong G.. Skeletal Rearrangements of Amides via Breaking Inert Bonds. Chem.Eur. J. 2025;31:e202500595. doi: 10.1002/chem.202500595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Vala D. P., Dunne Miller A., Atmasidha A., Parmar M. P., Patel C. D., Upadhyay D. B., Bhalodiya S. S., González-Bakker A., Khan A. N., Nogales J., Padrón J. M., Banerjee S., Patel H. M.. Click-Chemistry Mediated Synthesis of OTBN-1,2,3-Triazole Derivatives Exhibiting STK33 Inhibition with Diverse Anti-Cancer Activities. Bioorg. Chem. 2024;149:107485. doi: 10.1016/j.bioorg.2024.107485. [DOI] [PubMed] [Google Scholar]
  75. Upadhyay D. B., Mokariya J. A., Patel P. J., Patel S. G., Parmar M. P., Vala D. P., Ferro F., Rajani D. P., Narayan M., Kumar J., Banerjee S., Patel H. M.. Antimicrobial Efficacy of 1,2,3-Triazole-Incorporated Indole Pyrazolone against Drug-Resistant ESKAPE Pathogens: Design and Synthesis. ACS Bio Med. Chem. Au. 2025;5:66–77. doi: 10.1021/acsbiomedchemau.4c00060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Parmar M. P., Kashyap S., Vala D. P., Bhalodiya S. S., Patel C. D., Nogales J., Nandi A., Banerjee S., Patel H. M.. Ethanol-Mediated One-Pot Synthesis of 3-Phenyl-3,4,5,12-tetrahydrobenzo­[4,5]­imidazo­[2,1-b]­quinazolin-1­(2H)-ones as PDGFRA Inhibitors. Eur. J. Med. Chem. 2025;297:117914. doi: 10.1016/j.ejmech.2025.117914. [DOI] [PubMed] [Google Scholar]
  77. Vala D. P., Parmar M. P., Patel H. M.. Electrochemically Driven Azide–Alkyne Cycloaddition (E-CuAAC) via Anodic Oxidation Using Dual Copper Electrodes. Green Chem. 2026;28:2724–2735. doi: 10.1039/D5GC04792G. [DOI] [Google Scholar]
  78. Yescas-Galicia D., Restrepo-Osorio R. A., García-González A. N., Hernández-Benítez R. I., Espinoza-Hicks J. C., Escalante C. H., Barrera E., Santoyo B. M., Delgado F., Tamariz J.. Divergent Pd-Catalyzed Functionalization of 4-Oxazolin-2-ones and 4-Methylene-2-oxazolidinones and Synthesis of Heterocyclic-Fused Indoles. J. Org. Chem. 2022;87:13034–13052. doi: 10.1021/acs.joc.2c01563. [DOI] [PubMed] [Google Scholar]
  79. Parmar M. P., Vala D. P., Baraiya K. A., Bhalodiya S. S., Patel C. D., Konda P., Lakshmanan V. K., Sutariya P. G., Patel H. M.. Room-Temperature Accessible Stereospecific Synthesis of Methyl-(Z)-2-(7-methyl-3-oxo-5H-thiazolo­[3,2-a]­pyrimidin-2­(3H)-ylidene)­acetates:Cd2+ Fluorescence Probe and DU145 Cell Study. ChemPlusChem. 2025;90:e202500266. doi: 10.1002/cplu.202500266. [DOI] [PubMed] [Google Scholar]
  80. Burus A., Ozcan M., Canpinar H., Bozdemir O., Zeybek N. D., Bayazit Y.. The Effect of the Combination Therapy with Chlorophyllin, a Glutathione Transferase P1–1 Inhibitor, and Docetaxel on Triple-Negative Breast Cancer Invasion and Metastasis in Vivo/in Vitro. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2025;398:10569–10580. doi: 10.1007/s00210-025-03929-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Liu M., Cui L., Li X., Xia C., Li Y., Wang R., Ren F., Liu H., Chen J.. PD-0332991 Combined with Cisplatin Inhibits Nonsmall Cell Lung Cancer and Reversal of Cisplatin Resistance. Thorac. Cancer. 2021;12:924–931. doi: 10.1111/1759-7714.13866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wyrębska A., Gach K., Szemraj J., Szewczyk K., Hrabec E., Koszuk J., Janecki T., Janecka A.. Comparison of Anti-Invasive Activity of Parthenolide and 3-Isopropyl-2-Methyl-4-Methyleneisoxazolidin-5-One (MZ-6) – A New Compound with α-Methylene-γ-Lactone Motif – on Two Breast Cancer Cell Lines. Chem. Biol. Drug Des. 2012;79:112–120. doi: 10.1111/j.1747-0285.2011.01257.x. [DOI] [PubMed] [Google Scholar]
  83. Agarwal A., Kasinathan A., Ganesan R., Balasubramanian A., Bhaskaran J., Suresh S., Srinivasan R., Aravind K. B., Sivalingam N.. Curcumin Induces Apoptosis and Cell Cycle Arrest via the Activation of Reactive Oxygen Species–Independent Mitochondrial Apoptotic Pathway in Smad4 and p53 Mutated Colon Adenocarcinoma HT29 Cells. Nutr. Res. (N.Y.) 2018;51:67–81. doi: 10.1016/j.nutres.2017.12.011. [DOI] [PubMed] [Google Scholar]
  84. Patel P. S., Castelow C., Patel D. S., Bhattacharya S. K., Kuscu C., Kuscu C., Makowski L., Eason J. D., Bajwa A.. Mitochondrial Role in Oncogenesis and Potential Chemotherapeutic Strategy of Mitochondrial Infusion in Breast Cancer. Int. J. Mol. Sci. 2022;23:12993. doi: 10.3390/ijms232112993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Liou G.-Y., Storz P.. Reactive Oxygen Species in Cancer. Free Radical. Res. 2010;44:479–496. doi: 10.3109/10715761003667554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Circu M. L., Aw T. Y.. Reactive Oxygen Species, Cellular Redox Systems, and Apoptosis. Free Radical Biol. Med. 2010;48:749–762. doi: 10.1016/j.freeradbiomed.2009.12.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Martinou J.-C., Youle R. J.. Mitochondria in Apoptosis: Bcl-2 Family Members and Mitochondrial Dynamics. Dev. Cell. 2011;21:92–101. doi: 10.1016/j.devcel.2011.06.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Dong M., Zhou J.-P., Zhang H., Guo K.-J., Tian Y.-L., Dong Y.-T.. Clinicopathological Significance of Bcl-2 and Bax Protein Expression in Human Pancreatic Cancer. World J. Gastroenterol. 2005;11:2744–2747. doi: 10.3748/wjg.v11.i18.2744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Croce C. M., Tait S. W. G., Garcia-Sáez A. J., Villunger A., Letai A., Walter H. S., Dyer M. J. S., Green D. R., Shi Y., Melino G.. What Does BCL-2 Do? From New Molecular Insights to the Clinical Implications. Cell Death Differ. 2026;33:673–693. doi: 10.1038/s41418-025-01607-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Czabotar P. E., Garcia-Saez A. J.. Mechanisms of BCL-2 Family Proteins in Mitochondrial Apoptosis. Nat. Rev. Mol. Cell Biol. 2023;24:732–748. doi: 10.1038/s41580-023-00629-4. [DOI] [PubMed] [Google Scholar]
  91. Dho S. H., Cho M., Woo W., Jeong S., Kim L. K.. Caspases as Master Regulators of Programmed Cell Death: Apoptosis, Pyroptosis and Beyond. Exp. Mol. Med. 2025;57:1121–1132. doi: 10.1038/s12276-025-01470-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml6c00344_si_001.pdf (8.6MB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES