Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Preparation and biological evaluation of new antimicrotubule agents: Modification of the imidazolidin-2-one moiety of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates.

We prepared and biologically evaluated 32 novel molecules named phenyl 4-(dioxoimidazolidin-1-yl)benzenesulfonates (PID-SOs) and ethyl 2-(3-(4-(phenoxysulfonyl)phenyl)ureido)acetates (EPA-SOs). The antiproliferative activity of PID-SOs and EPA-SOs was assessed on four cancer cell lines (HT-1080, HT-29, M21, and MCF7). The most potent PID-SOs bearing an imidazolidin-2,4-dione group show antiproliferative activity in the nanomolar to low micromolar range (0.066 - 6 µM) while EPA-SOs and PID-SOs bearing an imidazolidin-2,5-dione moiety are mostly not active, exhibiting antiproliferative activity over 100 µM. The most potent PID-SOs (16-18) arrest the cell cycle progression in G2/M phase and interact with the colchicine-binding site leading to the microtubule and cytoskeleton disruption. Moreover, their antiproliferative activity is not impaired in vinblastine-, paclitaxel-, and multidrug-resistant cell lines. Finally, our study confirms that PID-SOs bearing the imidazolidin-2,4-dione moiety are a new family of promising antimitotics.

Branched alkyl of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates as unique cytochrome P450 1A1-activated antimitotic prodrugs: Biological evaluation and mechanism of bioactivation.

We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.

A facile synthesis of diaryl pyrroles led to the discovery of potent colchicine site antimitotic agents.

Three different series of cis-restricted analogues of combretastatin A-4 (CA-4), corresponding to thirty-nine molecules that contained a pyrrole nucleus interposed between the two aryl rings, were prepared by a palladium-mediated coupling approach and evaluated for their antiproliferative activity against six human cancer cell lines. In the two series of 1,2-diaryl pyrrole derivatives, results suggested that the presence of the 3',4',5'-trimethoxyphenyl moiety at the N-1 position of the pyrrole ring was more favorable for antiproliferative activity. In the series of 3,4-diarylpyrrole analogues, three compounds (11i-k) exhibited maximal antiproliferative activity, showing excellent antiproliferative activity against the CA-4 resistant HT-29 cells. Inhibition of tubulin polymerization of selected 1,2 pyrrole derivatives (9a, 9c, 9o and 10a) was similar to that observed with CA-4, while the isomeric 3,4-pyrrole analogues 11i-k were generally from 1.5- to 2-fold more active than CA-4. Compounds 11j and 11k were the only compounds that showed activity as inhibitors of colchicine binding comparable to that CA-4. Compound 11j had biological properties consistent with its intracellular target being tubulin. This compound was able to block the cell cycle in metaphase and to induce significant apoptosis at a concentration of 25 nM, following the mitochondrial pathway, with low toxicity for normal cells. More importantly, compound 11j exerted activity in vivo superior to that of CA-4P, being able to significantly reduce tumor growth in a syngeneic murine tumor model even at the lower dose tested (5.0 mg/kg).

A Pyranoxanthone as a Potent Antimitotic and Sensitizer of Cancer Cells to Low Doses of Paclitaxel.

Microtubule-targeting agents (MTAs) remain a gold standard for the treatment of several cancer types. By interfering with microtubules dynamic, MTAs induce a mitotic arrest followed by cell death. This antimitotic activity of MTAs is dependent on the spindle assembly checkpoint (SAC), which monitors the integrity of the mitotic spindle and proper chromosome attachments to microtubules in order to ensure accurate chromosome segregation and timely anaphase onset. However, the cytotoxic activity of MTAs is restrained by drug resistance and/or toxicities, and had motivated the search for new compounds and/or alternative therapeutic strategies. Here, we describe the synthesis and mechanism of action of the xanthone derivative pyranoxanthone 2 that exhibits a potent anti-growth activity against cancer cells. We found that cancer cells treated with the pyranoxanthone 2 exhibited persistent defects in chromosome congression during mitosis that were not corrected over time, which induced a prolonged SAC-dependent mitotic arrest followed by massive apoptosis. Importantly, pyranoxanthone 2 was able to potentiate apoptosis of cancer cells treated with nanomolar concentrations of paclitaxel. Our data identified the potential of the pyranoxanthone 2 as a new potent antimitotic with promising antitumor potential, either alone or in combination regimens.

Synthesis and biological evaluation of indolylglyoxylamide bisphosphonates, antimitotic microtubule-targeting derivatives of indibulin with improved aqueous solubility.

Indibulin (D-24851) derivatives with bisphosphonate fragment connected to the N1 atom of imidazole ring were synthesized by alkylation of (indolyl-3)methylglyoxylates with ethylenebisphosphonate. Biological evaluation of targeted compounds 4a-d using the phenotypic sea urchin embryo assay provided evidence that replacing of p-chlorobenzene ring in indibulin by bisphosphonate group did not eliminate antimitotic microtubule destabilizing activity. The most active molecule, tetraacid 5a, at physiological pH formed tetrasodium salt 6a with aqueous solubility value of at least 10 mg/mL. Molecule 5a was more potent in the sea urchin embryo assay than the parent indibulin. This compound also exhibited pronounced cytotoxicity against A549 lung carcinoma and A375 melanoma cell lines.

Pyrrolo[2',3':3,4]cyclohepta[1,2-d][1,2]oxazoles, a New Class of Antimitotic Agents Active against Multiple Malignant Cell Types.

A new class of pyrrolo[2',3':3,4]cyclohepta[1,2-d][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including neoplasms. The new compounds were screened in the 60 human cancer cell lines of the NCI drug screen and showed potent activity with GI50 values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 µM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC50 values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC50 values of 1.9-8.2 µM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.

Synthesis and Antiproliferative Screening Of Novel Analogs of Regioselectively Demethylated Colchicine and Thiocolchicine.

Colchicine, a pseudoalkaloid isolated from Colchicum autumnale, has been identified as a potent anticancer agent because of its strong antimitotic activity. It was shown that colchicine modifications by regioselective demethylation affected its biological properties. For demethylated colchicine analogs, 10-demethylcolchicine (colchiceine, 1) and 1-demethylthiocolchicine (3), a series of 12 colchicine derivatives including 5 novel esters (2b-c and 4b-d) and 4 carbonates (2e-f and 4e-f) were synthesized. The antiproliferative activity assay, together with in silico evaluation of physicochemical properties, confirmed attractive biological profiles for all obtained compounds. The substitutions of H-donor and H-acceptor sites at C1 in thiocolchicine position provide an efficient control of the hydration affinity and solubility, as demonstrated for anhydrate 3, hemihydrate 4e and monohydrate 4a.

Synthesis and biological evaluation of bergenin-1,2,3-triazole hybrids as novel class of anti-mitotic agents.

In continuation of our investigation of pharmacologically-motivated natural products, we have isolated bergenin (1) as a major compound from Mallotus philippensis, which is deployed in different Indian traditional systems of medicine. Here, a series of bergenin-1,2,3-triazole hybrids were synthesized and evaluated for their potentials against a panel of cancer cell lines. Several of the hybrid derivatives were found more potent in comparison to parent compound bergenin (1). Among them, 4j demonstrated potent activity against A-549 and HeLa cell lines with IC50 values of 1.86 µM and 1.33 µM, respectively, and was equipotent to doxorubicin. Cell cycle analysis showed that 4j arrested HeLa cells at G2/M phase and lead to accumulation of Cyclin B1 protein. Cell based tubulin polymerization assays and docking studies demonstrated that 4j disrupts tubulin assembly by occupying colchicine binding pocket of tubulin.

Synthesis and biological evaluation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides as antimitotic agents.

A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides (7a-al) have been designed, synthesized and screened for their anti-proliferative activity against some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable tubulin polymerization inhibition (IC50 value 2.04 µM) to the standard E7010 (IC50 value 2.15 µM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via cell cycle arrest at G2/M phase in A549 cells. Induction of apoptosis was further observed by examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that compound 7f binds to the colchicine binding site of the ß-tubulin. Thus, 7f exhibits anti-proliferative properties by inhibiting the tubulin polymerization through the binding at the colchicine active site and by induction of apoptosis.

Synthesis and biological evaluation of N-biphenyl-nicotinic based moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin Lymphoma.

We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas, they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis. PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted an antiangiogenic effect by suppressing microvascular formation.

A new class of diamide scaffold: Design, synthesis and biological evaluation as potent antimitotic agents, tubulin polymerization inhibition and apoptosis inducing activity studies.

A new series of diamide functional compounds has been designed, synthesized and confirmed by spectroscopic methods and elemental analyses. All the synthesized compounds were evaluated for their antiproliferative activity on HepG2 cell line. Compounds 3k and 3l were proved to have potent anticancer activity equipotent or more potent than reference compound Combretastatin A-4. The results of DNA flow cytometry analysis demonstrated cell cycle arrest at G2/M phase. The extent of apoptosis induced by 3k and 3l was also determined. Moreover, the compounds produced a significant reduction in cellular microtubules for microtubule loss and potently inhibited the binding of [3H]colchicine to tubulin. Compounds 3k and 3l were proved to upregulate expression of proteins triggering apoptosis, such as p53, Bax, and decreased Bcl-2 overexpression as well as increased the expression of effector caspase- 3/7.

Sea Urchin Embryo Model As a Reliable in Vivo Phenotypic Screen to Characterize Selective Antimitotic Molecules. Comparative evaluation of Combretapyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles as Tubulin-Binding Agents.

A series of both novel and reported combretastatin analogues, including diarylpyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles, were synthesized via improved protocols to evaluate their antimitotic antitubulin activity using in vivo sea urchin embryo assay and a panel of human cancer cells. A systematic comparative structure-activity relationship studies of these compounds were conducted. Pyrazoles 1i and 1p, isoxazole 3a, and triazole 7b were found to be the most potent antimitotics across all tested compounds causing cleavage alteration of the sea urchin embryo at 1, 0.25, 1, and 0.5 nM, respectively. These agents exhibited comparable cytotoxicity against human cancer cells. Structure-activity relationship studies revealed that compounds substituted with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B displayed the highest activity. 3-Hydroxy group in the ring B was essential for the antiproliferative activity in the diarylisoxazole series, whereas it was not required for potency of diarylpyrazoles. Isoxazoles 3 with 3,4,5-trimethoxy-substituted ring A and 3-hydroxy-4-methoxy-substituted ring B were more active than the respective pyrazoles 1. Of the azoles substituted with the same set of other aryl pharmacophores, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and 4,5-diaryl-1,2,3-triazoles 7 displayed similar strongest antimitotic antitubulin effect followed by 3,4-diarylisoxazoles 5, 1,5-diaryl-1,2,3-triazoles 8, and pyrroles 10 that showed the lowest activity. Introduction of the amino group into the heterocyclic core decreased the antimitotic antitubulin effect of pyrazoles, triazoles, and to a lesser degree of 4,5-diarylisoxazoles, whereas potency of the respective 3,4-diarylisoxazoles was increased.

Design and 22-step synthesis of highly potent D-ring modified and linker-equipped analogs of spongistatin 1.

Spongistatin 1 is among the most potent anti-proliferative agents ever discovered rendering it an attractive candidate for development as a payload for antibody-drug conjugates and other targeted delivery approaches. Unfortunately, it is unavailable from natural sources and its size and complex stereostructure render chemical synthesis highly time- and resource-intensive. As a result, the design and synthesis of more acid-stable and linker functional group-equipped analogs that retain the low picomolar potency of the parent natural product requires more efficient and step-economical synthetic access. Using uniquely enabling direct complex fragment coupling crotyl- and alkallylsilylation reactions, we report a 22-step synthesis of a rationally designed D-ring modified analog of spongistatin 1 that is characterized by GI50 values in the low picomolar range, and a proof-of-concept result that the C(15) acetate may be replaced with linker functional group-bearing esters with only minimal reductions in potency.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells.

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent, PAIB-SA 13, bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-expressing cells into potent antimitotics.

Synthesis and Biological Evaluation of Selenium-Containing 4-Anilinoquinazoline Derivatives as Novel Antimitotic Agents.

Twenty-eight novel selenium-containing 4-anilinoquinazoline derivatives were designed, synthesized, and evaluated as antiproliferative agents. Most of them had significant in vitro activities, particularly for compounds 23a, 25a, and 25d, which also exhibited the most potent antitumor activities against cisplatin-resistant cell lines and the doxorubicin-resistant cell lines, good selectivity toward normal cells, and obvious inhibitory effect on migration of A549 cell lines. Further mechanistic studies revealed that 23a, 25a, and 25d induce G2/M phase arrest and apoptosis in A549 cells, which was associated with a collapse of the mitochondrial membrane potential, alterations in the expression of some cell cycle-related and apoptosis-related proteins, and increasing the intracellular ROS level. Finally, compounds 23a, 25a, and 25d also effectively inhibited the tumor growth in the A549 xenograft model without obvious hints of toxicity. Taken together, these in vitro and in vivo results suggest that 23a, 25a, and 25d may be promising microtubule-stabilizing agents and can be used as a promising lead for the development of new antitumor agents.

Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-trisubstituted-2H-pyrazolo[4,3-c]pyridines.

An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent treatment of the coupling products with dry ammonia afforded a versatile library of 2H-pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI50 values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis, as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming apoptosis as the mechanism of cell death.

Recent Developments on Phenstatins as Potent Antimitotic Agents.

BACKGROUND: Phenstatin and their derivatives display remarkable antiproliferative activity toward a wide variety of preclinical tumor models. Structural simplicity and excellent stability of phenstatins offer a stimulating premise for developing various derivatives with profound antimitotic activity and excellent cytotoxicity. OBJECTIVE: To do analysis of literature that phenstatins derivatives inhibit tubulin polymerization through their interaction at the colchicine binding site of microtubules and arrest the G2/M phase of the cell cycle. In addition, phenstatin derivatives are undergoing clinical evaluation as vascular targeting/disrupting agents and also exhibit direct antiangiogenic properties. METHODS: An organised well designed and appropriately managed search of bibliographic databases for peer-reviewed research literature using a focused review question and inclusion/ exclusion criteria has been done for this article. CONCLUSION: In this review article, the synthesis and structure-activity relationships of phenstatin and a wide number of their reported analogues with modifications to ring A, ring B, and to the keto position are discussed in the perspective of medicinal chemistry with proper conclusion.

Novel antimitotic agents related to tubuloclustin: synthesis and biological evaluation.

Tubuloclustin [N-(7-adamant-2-yloxy-7-oxoheptanoyl)-N-deacetylcolchicine], a highly cytotoxic anti-tubulin compound is known for its ability to promote microtubule disassembly followed by the formation of tubulin clusters of unique morphology. Three series of antimitotic agents related to tubuloclustin were designed and synthesized in order to enhance the molecular diversity of "tubuloclustin-like" family of compounds. The series of compounds with modified adamantane moiety was highly potent in cytotoxic effect on human lung carcinoma A549 cells (EC50 = 6-400 nM) and was active in affecting the microtubule arrays and induction of strong tubulin clusterization. In two other sets of compounds, the colchicine moiety of tubuloclustin was replaced by podophyllotoxin or combretastatin A-4. All combretastatin A-4 derivatives displayed noticeable cytotoxic activity ([Formula: see text]) but their effect on microtubules depended on the position of the linker attachment. Podophyllotoxin derivatives were also toxic to A549 cells ([Formula: see text]) and caused both microtubule depolymerization and some tubulin clustering. The data obtained gave additional evidence that the whole panel of C7-colchicine, podophyllotoxin and combretastatin derivatives could manifest clustering effect, and the strength of this effect correlated with cytotoxic activity of the compounds.

Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents.

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI50 value of 3.8µM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays.