Cytotoxic Products Research Articles

Total Enzyme Syntheses of Napyradiomycins A1 and B1.

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon-carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.

Enantioselective Stereodivergent Synthesis of Jaspine B and 4-epi-Jaspine B from Axially Chiral Allenols

A short enantioselective synthetic route to the cytotoxic marine natural product jaspine B has been developed. A chiral non-racemic primary α-allenol, obtained from pentadecanal, gave access to an enantioenriched 2-tetradecyl-2,5-dihydrofuran as key intermediate. A stereodivergent functionalization of this dihydrofuran allowed access in a few steps to jaspine B and its 4-epimer.

Association between Helicobacter pylori, Epstein-Barr virus, human papillomavirus and gastric adenocarcinomas.

AIMTo correlate Helicobacter pylori (H. pylori), Epstein-Barr virus (EBV) and human papillomavirus (HPV) with gastric cancer (GC) cases in Pará State, Brazil.METHODSTissue samples were obtained from 302 gastric adenocarcinomas. A rapid urease test was used to detect the presence of H. pylori, and the presence of the cagA gene in the HP-positive samples was confirmed by PCR. An RNA in situ hybridization test designed to complement Eber1 RNA was used to detect the presence of EBV in the samples, and the L1 region of HPV was detected using nested PCR. Positive HPV samples were genotyped and analyzed for E6 and E7 viral gene expression. Infections were also correlated with the clinical and pathological characteristics of the patients.RESULTSThe majority of the 302 samples analyzed were obtained from men (65%) aged 55 years or older (67%) and were classified as the intestinal subtype (55%). All three pathogens were found in the samples analyzed in the present study (H. pylori: 87%, EBV: 20%, HPV: 3%). Overall, 78% of the H. pylori-positive (H. pylori+) samples were cagA+ (H. pylori-cagA+), and there was an association between the cytotoxic product of this gene and EBV. Coinfections of H. pylori-cagA+ and EBV were correlated with the most advanced tumor stages. Although only 20% of the tumors were positive for EBV, infection with this virus was associated with distant metastasis. Only the HPV 16 and 18 strains were found in the samples, although no expression of the E6 and E7 oncoproteins was detected. The fundus of the stomach was the region least affected by the pathogens.CONCLUSIONHPV was not involved in gastric tumorigenesis. Prophylactic and therapeutic measures against H. pylori and EBV may prevent the development of GC, especially the more aggressive forms.

Macrocyclic Core of Salarin C: Synthesis and Oxidation.

The synthesis of the macrocyclic core of the cytotoxic natural product salarin C from the sponge Fascaplysinopsis sp. is described, with the two epoxides being replaced by alkene moieties. In the key step, ring-closing metathesis exclusively afforded the ( E)-product. NOESY-based conformational analysis of the macrolactone showed that the oxazole ring and its unsaturated side chains are located in a common plane. Mimicking the conversion of salarin C to salarin A, the trisubstituted oxazole unit embedded in the 17-membered ring underwent photooxidation on treatment with singlet oxygen, affording macrocyclic trisacylamines.

Eosinophilic inflammation in spontaneous coronary artery dissection: A potential therapeutic target?

Spontaneous coronary artery dissection (SCAD), defined as non-traumatic, non-iatrogenic dissociation of coronary vessel wall resulting from intimal disruption or intramural hemorrhage, represents an important cause of sudden death and myocardial infarction among young or middle-aged women without conventional risk factors for atherosclerosis. On histopathological examination, SCAD is featured by prominent eosinophilic infiltration of the adventitia or periadventitial layer of coronary artery. It has been estimated that approximately 15–30% of SCAD patients experience recurrent episodes of dissection despite medical therapy. Preliminary evidence suggests that injury to the vascular endothelium and myocytes in the arterial wall may be explained by cytotoxic products released from eosinophils in response to inflammatory mediators. In addition, neovascularization of vasa vasorum and dilatation of intimal capillaries may be stimulated by localized eosinophils. Newly formed fragile vasa vasorum may disrupt due to high intraluminal pressure from the interconnected capillary network, leading to the expansion of intramural hemorrhage. It is hypothesized that anti-inflammatory therapy targeting eosinophilic coronary periarteritis would be effective in preventing the recurrence of SCAD by promoting the healing of dissection. The article delineates the biological plausibility, empirical data, and future perspective regarding eosinophilic inflammation as a potential therapeutic target for SCAD.

Testosterone and Follicle Stimulating Hormone–Dependent Glyoxalase 1 Up-Regulation Sustains the Viability of Porcine Sertoli Cells through the Control of Hydroimidazolone– and Argpyrimidine-Mediated NF-κB Pathway

Because Sertoli cells (SCs) play a central role in germ cell survival, their death may result in marked germ cell loss and infertility. SCs are the only somatic cells within the seminiferous tubules and are essential for regulating spermatogenesis. Factors that enhance or diminish the viability of SCs may have profound effects on spermatogenesis. Yet the mechanisms underlying the maintenance of SC viability remain largely unknown. Glyoxalase 1 (Glo1) detoxifies methylglyoxal (MG), a highly reactive carbonyl species mainly formed during glycolysis, which is a potent precursor of cytotoxic advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are AGEs resulting from MG-mediated post-translational modification of arginine residues in various proteins. The role of Glo1 and MG-derived AGEs in regulating the fate of SCs has never been investigated. By using gene silencing and the specific MG scavenger, aminoguanidine, the authors demonstrate that Glo1, under testosterone and follicle-stimulating hormone control, sustains viability of porcine neonatal SCs through a mechanism involving the NF-κB pathway. Glo1 knockdown induces a mitochondrial apoptotic pathway driven by the intracellular accumulation of MG-H1 and ArgPyr that desensitizes NF-κB signaling by modifying the inhibitor of NF-κB kinase, IKKß. This is the first reportdescribing a role for Glo1 and MG-derived AGEs in SC biology, providing valuable new insights into the potential involvement of this metabolic axis into spermatogenesis.

A Catalytic Oxidative Quinone Heterofunctionalization Method: Synthesis of Strongylophorine-26.

The preparation of heteroatom-substituted p-quinones is ideally performed by direct addition of a nucleophile followed by in situ reoxidation. Albeit an appealing strategy, the reactivity of the p-quinone moiety is not easily tamed and no broadly applicable method for heteroatom functionalization exists. Shown herein is that Co(OAc)2 and Mn(OAc)3 ⋅2 H2 O act as powerful catalysts for oxidative p-quinone functionalization with a collection of O, N, and S nucleophiles, using oxygen as the terminal oxidant. Preliminary mechanistic observations and the first synthesis of the cytotoxic natural product strongylophorine-26 is presented.

Abstract LB-108: Generation of off-the-shelf TCR-less CAR-targeted cytotoxic T cells from renewable pluripotent cells for cancer immunotherapy

Abstract Genetically engineered T cells are at the vanguard of an emerging wave of scientific breakthrough focused on harnessing the power of the immune system to treat cancer and other immune disorders. Perhaps the most dramatic clinical outcome to date has been demonstrated in clinical trials evaluating autologous chimeric antigen receptor (CAR) therapy for the treatment of refractory B cell ALL where complete responses in the majority of the patients have been reported. While adoptive CAR-T cell treatments hold great promise, pressing challenges remain to ensure multi-parameter genetically engineered T-cell immunotherapies can be successfully derived, cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization.Human induced pluripotent stem cell (hiPSC)-derived T cells uniquely represent a practical and renewable supply of well-defined engineered CAR-T cells for various therapeutic applications. We have previously described a novel platform to facilitate multi-gene locus-specific engineering of hiPSCs at the single cell level to establish highly characterized master cell banks which can then be repeatedly applied to our stage-specific lymphocyte directed differentiation process to reproducibly and reliably generate engineered cytotoxic T cells.Here we present the first set of pre-clinical data for FT819, a first-of-kind off-the-shelf hiPSC-derived CAR-T cell product. To generate FT819, we successfully combined reprogramming of peripheral blood derived T cells with targeted insertion of a CD19 CAR into the T cell receptor α (TRAC) locus under the transcriptional control of its endogenous regulatory elements to generate a single cell-derived clonal TRAC-targeted CAR expressing master hiPSC line. The clone was characterized to be pluripotent (>95% SSEA4 / TRA181) and consisted of bi-allelic disruption of TRAC locus. During the stage-specific differentiation, the hiPSC line faithfully converted into CD34 positive cells which were then differentiated towards CD8 positive cells with uniform CAR expression (95 +/- 5%) in the absence of TCR expression, eliminating the likelihood of GvHD. In vitro functional studies demonstrated that FT819 elicits an efficient cytotoxic T lymphocyte response to CD19 antigen challenge with production of effector cytokines (IFNg, TNFa, IL2), degranulation (CD107a/b, Perforin, Granzyme B), proliferation (>85% entry into cell cycle) and upregulation of activation markers CD69 and CD25. Importantly, FT819 targets tumor in an antigen specific manner as demonstrated by lysis of CD19+, but not CD19-, Raji and Nalm6 tumor cell lines. In summary, FT819 holds the promise of a safe and efficacious off-the-shelf cytotoxic CAR-T cell product derived in a renewable and highly reproducible process analogous to biopharmaceutical drug products. Citation Format: Raedun L. Clarke, Sjoukje van der Stegen, Tom Lee, Jorge Mansilla-Soto, Chia-wei Chang, Jeffrey Sasaki, Mushtaq Husain, Eigen Peralta, Ian Hardy, Edwin Ortiz, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of off-the-shelf TCR-less CAR-targeted cytotoxic T cells from renewable pluripotent cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-108.

Abstract 2805: Combretastatins as highly cytotoxic natural compounds

Abstract Combretastatins originally isolated from the South African willow tree Combretum cafrum are highly cytotoxic natural products, but their use is limited because of the cis/trans isomerization of the cis-stilbene moiety under physiological conditions. Herein we report the preparation of cis-restrained carbocyclic analogs of CA-4. The compounds, which differ by the size and hybridization of the carbocyclic ring have been evaluated for cytotoxic activity in vitro against a panel of human cancer and two non-tumor fibroblast cell lines and for their ability to inhibit tubulin polymerization. Biological data, supported by molecular docking studies, identified a cyclobutenyl derivative of the natural product as a promising drug candidate with selective nanomolar cytotoxic activity towards cancer cell lines, showing high potency against multidrug-resistant cells and inhibiting tubulin polymerization. This work was supported by Ministry of Health of the Czech Republic (15-31984A), internal grant of Palacky University (IGA_LF_2017_026) and by the Czech Ministry of Education, Youth and Sports LO1304, CZ-OPENSCREEN, (LM2015063) and EATRIS (LM2015064). Citation Format: Petr Dzubak, Jiři Rehulka, Sona Gurska, Uwe Riener, Marian Hajduch. Combretastatins as highly cytotoxic natural compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2805.

Immune complex-induced neutrophil functions: A focus on cell death.

Neutrophils are amongst the first cells to be recruited to sites of infection or trauma. Neutrophil functional responsiveness is tightly regulated by many agents including immune complexes. These immune cells can generate reactive oxygen species and degranulate to release abundant cytotoxic products, making them efficient at killing invading microorganisms. If neutrophil function is dysregulated, however, these cells have the potential to cause unwanted host tissue damage as exemplified by pathological and chronic inflammatory conditions. In physiological inflammation, once the initial insult has efficiently been dealt with, neutrophils are thought to leave the tissues or undergo programmed cells death, especially apoptosis. Apoptotic neutrophils are then rapidly removed by other phagocytes, primarily macrophages, by mechanisms that do not elicit a pro-inflammatory response. In this review, we discuss the interesting observations and consequences that immune complexes have on neutrophil cell death processes such as apoptosis.

Drug Delivery System for Emodin Based on Mesoporous Silica SBA-15

In this study mesoporous silica SBA-15 was evaluated as a vehicle for the transport of cytotoxic natural product emodin (EO). SBA-15 was loaded with different quantities of EO (SBA-15|EO1–SBA-15|EO5: 8–36%) and characterized by traditional methods. Several parameters (stabilities) and the in vitro behavior on tumor cell lines (melanoma A375, B16 and B16F10) were investigated. SBA-15 suppresses EO release in extremely acidic milieu, pointing out that EO will not be discharged in the stomach. Furthermore, SBA-15 protects EO from photodecomposition. In vitro studies showed a dose dependent decrease of cellular viability which is directly correlated with an increasing amount of EO in SBA-15 for up to 27% of EO, while a constant activity for 32% and 36% of EO in SBA-15 was observed. Additionally, SBA-15 loaded with EO (SBA-15|EO3) does not disturb viability of peritoneal macrophages. SBA-15|EO3 causes inhibition of tumor cell proliferation and triggers apoptosis, connected with caspase activation, upregulation of Bax, as well as Bcl-2 and Bim downregulation along with amplification of poly-(ADP-ribose)-polymerase (PARP) cleavage fragment. Thus, the mesoporous SBA-15 is a promising carrier of the water-insoluble drug emodin.

A New Platform for Profiling Degradation-Related Impurities Via Exploiting the Opportunities Offered by Ion-Selective Electrodes: Determination of Both Diatrizoate Sodium and Its Cytotoxic Degradation Product.

Although the ultimate goal of administering active pharmaceutical ingredients (APIs) is to save countless lives, the presence of impurities and/or degradation products in APIs or formulations may cause harmful physiological effects. Today, impurity profiling (i.e., the identity as well as the quantity of impurity in a pharmaceutical) is receiving critical attention from regulatory authorities. Despite the predominant use of spectroscopic and chromatographic methods over electrochemical methods for impurity profiling of APIs, this work investigates the opportunities offered by electroanalytical methods, particularly, ion-selective electrodes (ISEs), for profiling degradation-related impurities (DRIs) compared with conventional spectroscopic and chromatographic methods. For a meaningful comparison, diatrizoate sodium (DTA) was chosen as the anionic X-ray contrast agent based on its susceptibility to deacetylation into its cytotoxic and mutagenic degradation product, 3,5-diamino-2,4,6 triiodobenzoic acid (DTB). This cationic diamino compound can be also detected as an impurity in the final product because it is used as a synthetic precursor for the synthesis of DTA. In this study, four novel sensitive and selective sensors for the determination of both DTA and its cytotoxic degradation products are presented. Sensors I and II were developed for the determination of the anionic drug, DTA, and sensors III and IV were developed for the determination of the cationic cytotoxic impurity. The use of these novel sensors not only provides a stability-indicating method for the selective determination of DTA in the presence of its degradation product, but also permits DRI profiling. Moreover, a great advantage of these proposed ISE systems is their higher sensitivity for the quantification of DTB relative to other spectroscopic and chromatographic methods, so it can measure trace amounts of DTB impurities in DTA bulk powder and pharmaceutical formulation without a need for preliminary separation.

P-48 - Redox-active quinones as regulators of free radical processes and potential antitumor agents

Redox regulation of cell activity is the basis for antitumor therapy. In cancer cells generation of ROS is reduced, thus such cells have resistance to apoptotic signals. Malignant cells reproduce under hypoxic conditions and this fact reduces the probability of oxidative processes. Therefore, the use of oxidizing agents, such as quinones and their derivatives, is significant, because of their capability to provide additional formation of ROS, cytotoxic oxidation products and oxidative destruction of biomolecules. We have shown that p-benzoquinone and its derivatives are effective regulators of free radical processes involving hydroxyl-containing C-centered radicals. Thymoquinone, a biologically active component of Nigella sativa, and other p-benzoquinones inhibit ROS-induced fragmentation of glycerophospholipids, significantly suppressing the formation of phosphatidic acid, which in cancer cells should reduce the effectiveness of antitumor therapy. It was found that p-benzoquinones inhibit the growth of HEp-2 cells in a dose-dependent manner. The tested quinones possess antitumor activity due to their ability to suppress the mitochondrial membrane potential. Possible relationship between radical-regulatory and antitumor properties of quinoid-type compounds is proposed.

Mechanism of Action of the Cytotoxic Asmarine Alkaloids.

The asmarines are a family of cytotoxic natural products whose mechanism of action is unknown. Here, we used chemical synthesis to reverse engineer the asmarines and understand the functions of their individual components. We found that the potent asmarine analog "delmarine" arrested the mammalian cell cycle in the G1 phase and that both cell cycle arrest and cytotoxicity were rescued by cotreatment with ferric and ferrous salts. Cellular iron deprivation was clearly indicated by changes in iron-responsive protein markers, and cytotoxicity occurred independently of radical oxygen species (ROS) production. Chemical synthesis allowed for annotation of the distinct structural motifs required for these effects, especially the unusual diazepine, which we found enforced an iron-binding tautomer without distortion of the NCNO dihedral angle out of plane. With this information and a correlation of cytotoxicity with logP, we could replace the diazepine by lipophilic group appendage to N9, which avoided steric clash with the N6-alkyl required to access the aminopyridine. This study transformed the asmarines, scarce marine metabolites, into easily synthesized, modular chemotypes that may complement or succeed iron-selective binders in clinical trials and use.

Photochemical degradation of trypan blue.

PurposeTo investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.MethodsDefined solution samples of TB and a mixture with lutein/zeaxanthin were exposed to blue light. Thermal degradation processes were ruled out using controls not subjected to irradiation. All samples were analyzed using optical microscopy, UV/Vis spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Degradation kinetics were determined based on changes in absorbance; intermediates were identified by analyzing mass differences of characteristic fragment ion peaks within the fragmentation patterns, and assignments were verified by NMR.ResultsTB demonstrated a photochemical degradation, which can be triggered by lutein/zeaxanthin. Intermediates vary depending on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of TB occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, within the presence of lutein/zeaxanthin the decomposition of TB indicates the formation of methoxyamine and sulfonyl arin. Thermal degradation processes were not observed.ConclusionsTB demonstrated a photodegradation that may be triggered by lutein/zeaxanthin and results in the formation of cytotoxic decomposition products. Our findings contribute to understand degradation mechanisms of TB and may elucidate previous clinical and experimental observations of cellular toxicity after TB application.

The potent pro-oxidant activity of rhododendrol-eumelanin is enhanced by ultraviolet A radiation.

RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol, RD), a skin-whitening agent, is known to induce leukoderma in some consumers. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products and RD-eumelanin exerts a potent pro-oxidant activity. Cellular antioxidants were oxidized by RD-eumelanin with a concomitant production of H2 O2 . In this study, we examined whether this pro-oxidant activity of RD-eumelanin is enhanced by ultraviolet A (UVA) radiation because most RD-induced leukoderma lesions are found in sun-exposed areas. Exposure to a physiological level of UVA (3.5mW/cm2 ) induced a two to fourfold increase in the rates of oxidation of GSH, cysteine, ascorbic acid, and NADH. This oxidation was oxygen-dependent and was accompanied by the production of H2 O2 . These results suggest that RD-eumelanin is cytotoxic to melanocytes through its potent pro-oxidant activity that is enhanced by UVA radiation.

ATG5 Promotes Death Signaling in Response to the Cyclic Depsipeptides Coibamide A and Apratoxin A.

Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5−/− cells relative to ATG5+/+ cells. Time-dependent changes in annexin V staining, membrane integrity, metabolic capacity and caspase activation indicated that MEFs with a functional autophagy pathway are more sensitive to coibamide A. This pattern could be distinguished from autophagy modulators that induce acute ER stress (thapsigargin, tunicamycin), ATP depletion (oligomycin A) or mTORC1 inhibition (rapamycin), but was shared with the Sec61 inhibitor apratoxin A. Coibamide- or apratoxin-induced cell stress was further distinguished from the action of thapsigargin by a pattern of early LC3-II accumulation in the absence of CHOP or BiP expression. Time-dependent changes in ATG5-ATG12, PARP1 and caspase-3 expression patterns were consistent with the conversion of ATG5 to a pro-death signal in response to both compounds.

Total Synthesis and Stereochemical Revision of Iriomoteolide‐2a

AbstractTotal syntheses of the proposed and correct structures of iriomoteolide‐2a, a cytotoxic marine macrolide natural product with an unusual 23‐membered macrolactone skeleton, have been accomplished for the first time. The synthesis of the correct structure involves an asymmetric epoxidation/diepoxide cyclization cascade for the construction of the bis(tetrahydrofuran) moiety, a Suzuki–Miyaura coupling for the fragment assembly, and a ring‐closing metathesis for the closure of the macrocyclic backbone. In addition, the original stereochemical assignment of iriomoteolide‐2a was revised.

Total Synthesis and Stereochemical Revision of Iriomoteolide-2a.

Total syntheses of the proposed and correct structures of iriomoteolide-2a, a cytotoxic marine macrolide natural product with an unusual 23-membered macrolactone skeleton, have been accomplished for the first time. The synthesis of the correct structure involves an asymmetric epoxidation/diepoxide cyclization cascade for the construction of the bis(tetrahydrofuran) moiety, a Suzuki-Miyaura coupling for the fragment assembly, and a ring-closing metathesis for the closure of the macrocyclic backbone. In addition, the original stereochemical assignment of iriomoteolide-2a was revised.

In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-One Derivatives via Apoptosis Induction

Systemic toxicity associated with drug resistance continues to be the major obstacle to curative therapy of cancer. Tumor cell resistance to chemotherapeutic drugs often results in coordinate resistance to other structurally and functionally unrelated drugs and the subsequent development of cross resistance phenotype. Therefore, it seems necessary to identify new molecules as anticancer agents. In this process, we synthesized a series of new pyridazin-3(2H)-one derivatives and evaluated their antitumor potential. These cyclic molecules were synthesized and designed as a combination of benzofuran with pyridazinones. All final compounds have been characterized by spectral and elemental analyses to confirm successful synthesis reactions. To evaluate their anticancer activity, all derivatives were assessed against the human breast adenocarcinoma cell line (MCF-7) and the murine mastocytoma cell line (P815) using the methyl tetrazolium Test (MTT assay). The cytotoxic activity was found to be dose-dependent and the IC50 values of the synthesized compounds ranged from 14.5 to 40 μM against MCF-7 and from 35 to 82.5 μM against P815. At the same time, no cytotoxic activity was observed against normal cells. In order to investigate the molecular mechanism of the most cytotoxic product (6f), apoptosis induction was measured against MCF-7 cells. Using the annexin-V FITC staining technique, we showed that the cytotoxic effect of this product is associated with apoptosis induction.