Synthesis and Antiproliferative Effect of New Alkyne-Tethered Vindoline Hybrids Containing Pharmacophoric Fragments.

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.

Unveiling the Oxazolidine Character of Pseudoproline Derivatives by Automated Flow Peptide Chemistry.

Pseudoproline derivatives such as Thr(ΨPro)-OH are commonly used in peptide synthesis to reduce the likelihood of peptide aggregation and to prevent aspartimide (Asi) formation during the synthesis process. In this study, we investigate notable by-products such as aspartimide formation and an imine derivative of the Thr(ΨPro) moiety observed in flow peptide chemistry synthesis. To gain insight into the formation of these unexpected by-products, we design a series of experiments. Furthermore, we demonstrate the oxazolidine character of the pseudoproline moiety and provide plausible mechanisms for the two-way ring opening of oxazolidine leading to these by-products. In addition, we present evidence that Asi formation appears to be catalyzed by the presence of the pseudoproline moiety. These observed side reactions are attributed to elevated temperature and pressure; therefore, caution is advised when using ΨPro derivatives under such harsh conditions. In addition, we propose a solution whereby thermodynamically controlled Asi formation can be kinetically prevented.

Stereoselective Synthesis and Catalytical Application of Perillaldehyde-Based 3-Amino-1,2-diol Regioisomers.

A library of regioisomeric monoterpene-based aminodiols was synthesised and applied as chiral catalysts in the addition of diethylzinc to benzaldehyde. The synthesis of the first type of aminodiols was achieved starting from (-)-8,9-dihydroperillaldehyde via reductive amination, followed by Boc protection and dihydroxylation with the OsO4/NMO system. Separation of formed stereoisomers resulted in a library of aminodiol diastereoisomers. The library of regioisomeric analogues was obtained starting from (-)-8,9-dihydroperillic alcohol, which was transformed into a mixture of allylic trichloroacetamides via Overman rearrangement. Changing the protecting group to a Boc function, the protected enamines were subjected to dihydroxylation with the OsO4/NMO system, leading to a 71:16:13 mixture of diastereoisomers, which were separated, affording the three isomers in isolated form. The obtained primary aminodiols were transformed into secondary derivatives. The regioselectivity of the ring closure of the N-benzyl-substituted aminodiols with formaldehyde was also investigated, resulting in 1,3-oxazines in an exclusive manner. To explain the stability difference between diastereoisomeric 1,3-oxazines, a series of comparative theoretical modelling studies was carried out. The obtained potential catalysts were applied in the reaction of aromatic aldehydes and diethylzinc with moderate to good enantioselectivities (up to 94% ee), whereas the opposite chiral selectivity was observed between secondary aminodiols and their ring-closed 1,3-oxazine analogues.

CuAAC-Based Synthesis, Copper-Catalyzed Aldehyde-Forming Hydrolytic Fission and Antiproliferative Evaluation of Novel Ferrocenoylamino-Substituted Triazole-Tethered Quinine-Chalcone Hybrids.

A series of novel triazole-tethered ferrocenoylamino-substituted cinchona-chalcone hybrids along with two representative benzoylamino-substituted reference compounds were prepared by three methods of CuAAC chemistry. In line with the limited success or complete failure of attempted conversions with low catalyst loadings, by means of DFT modeling studies, we demonstrated that a substantial part of the Cu(I) ions can be chelated and thus trapped in the aroylamino-substituted cinchona fragment and all of the accessible coordinating sites of the chalcone residues. Accordingly, increased amounts of catalysts were used to achieve acceptable yields; however, the cycloadditions with para-azidochalcones were accompanied by partial or complete aldehyde-forming hydrolytic fission of the enone C=C bond in a substituent-, solvent- and copper load-dependent manner. The experienced hydrolytic stability of the hybrids obtained by cycloadditions with ortho-azidochalcones was interpreted in terms of relative energetics, DFT reactivity indices and MO analysis of simplified models of two isomer copper-enone complexes. The novel hybrids were evaluated on HeLa, MDA-MB-231 and A2780 cell lines and showed substantial activity at low-to-submicromolar concentrations. An organometallic model carrying 3,4,5-trimethoxyphenyl residue in the enone part with a para-disubstituted benzene ring in the central skeletal region was identified as the most potent antiproliferative lead, characterized by submicromolar IC50 values measured on the three investigated cells. The biological assays also disclosed that this ferrocenoylamino-containing lead compound displays a ca. two- to five-fold more substantial antiproliferative effect than its benzoylamino-substituted counterpart.

Acetyl group for proper protection of ß-sugar-amino acids used in SPPS.

The synthesis of D-glucosamine-1-carboxylic acid based ß-sugar amino acids (ß-SAAs) is typically performed in nine consecutive steps via an inefficient OAc → Br → CN conversion protocol with low overall yield. Here, we present the improved and more efficient synthesis of both Fmoc-GlcAPC-OH and Fmoc-GlcAPC(Ac)-OH, ß-SAAs consisting of only 4-5 synthetic steps. Their active ester and amide bond formation with glycine methyl ester (H-Gly-OMe) was completed and monitored by 1H NMR. The stability of the pyranoid OHs protecting the acetyl groups was investigated under three different Fmoc cleavage conditions and was found to be satisfactory even at high piperidine concentration (e.g. 40%). We designed a SPPS protocol using Fmoc-GlcAPC(Ac)-OH to produce model peptides Gly-ß-SAA-Gly as well as Gly-ß-SAA-ß-SAA-Gly with high coupling efficiency. The products were deacetylated using the Zemplén method, which allows the hydrophilicity of a building block and/or chimera to be fine-tuned, even after the polypeptide chain has already been synthesized.

Novel Erlotinib-Chalcone Hybrids Diminish Resistance in Head and Neck Cancer by Inducing Multiple Cell Death Mechanisms.

In a search for novel therapeutic options for head and neck squamous cell carcinomas (HNSCCs) generally treated with limited therapeutic success, we synthesized a series of novel erlotinib-chalcone molecular hybrids with 1,2,3-triazole and alkyne linkers and evaluated them for their anticancer activity on Fadu, Detroit 562 and SCC-25 HNSCC cell lines. Time- and dose-dependent cell viability measurements disclosed a significantly increased efficiency of the hybrids compared to the 1:1 combination of erlotinib and a reference chalcone. The clonogenic assay demonstrated that hybrids eradicate HNSCC cells in low micromolar concentrations. Experiments focusing on potential molecular targets indicate that the hybrids trigger the anticancer effect by a complementary mechanism of action that is independent of the canonical targets of their molecular fragments. Confocal microscopic imaging and real-time apoptosis/necrosis detection assay pointed to slightly different cell death mechanisms induced by the most prominent triazole- and alkyne-tethered hybrids (6a and 13, respectively). While 6a featured the lowest IC50 values on each of the three HNSCC cell lines, in Detroit 562 cells, this hybrid induced necrosis more markedly compared to 13. The therapeutic potential indicated by the observed anticancer efficacy of our selected hybrid molecules validates the concept of development and justifies further investigation to reveal the underlying mechanism of action.

Synthesis and SAR Analysis of Novel 4-Hydroxytamoxifen Analogues Based on Their Cytotoxic Activity and Electron-Donor Character.

Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Fine-Tuned Reactivity of N-Containing Naphthol Analogues.

6-Hydroxyquinoline and 3-hydroxyisoquinoline as N-containing naphthol analogues were tested in modified Mannich reactions (mMr's). In the case of 6-hydroxyquinoline, the outcomes of the attempted Mannich reactions were strongly influenced by the amine components. Aminoalkylation of this substrate with reagents 1-naphthaldehyde and N-benzylmethylamine led to the isolation of a diol regarded as a stabilised water adduct of an ortho-quinone methide (o-QM), of which formation can be ascribed to the presence of a hydroxide ion in a relatively higher concentration generated by the bulky and basic amine component with decreased nucleophilicity. The classical Mannich base was isolated as a single product when the amine component was replaced for morpholine, featuring nucleophilicity rather than basic character under the applied reaction conditions. Starting from the isomer substrate 3-hydroxyisoquinoline, independently on the nucleophile (methanol or morpholine) besides the formation of the classical Mannich base, the nucleophilic attack at position one of the heterocyclic substrate was also observed. The DFT analysis of the acceptor molecular orbitals of the potential electrophilic components and the thermodynamics of the assumed-possible transformations demonstrated that this regioselective addition is a feasible process on the investigated heterocyclic skeleton. DFT modelling studies also suggest that besides the steric bulk, the orbital-controlled electronic properties of the aryl group, originating from the aldehyde components, have a strong influence on the ratios and the NMR-monitored interconversions of the C-1-substituted 3-hydroxyisoquinolines and the classical Mannich bases formed in multistep reaction sequences. On the basis of the DFT analysis of the thermodynamics of alternative pathways, a reaction mechanism was proposed for the rationalization of these characteristic substrate-controlled interconversions.

Synthesis and Antiproliferative Activity of Novel Imipridone-Ferrocene Hybrids with Triazole and Alkyne Linkers.

Imipridones, including ONC201, ONC206 and ONC212 (which are emblematic members of this class of compounds developed by Oncoceutics) constitute a novel class of anticancer agents, with promising results in clinical trials. With the aim of increasing the ROS (reactive oxygen species) responsivity of the synthesized molecules, a set of novel ferrocene-imipridone hybrids were designed and synthesized. Our strategy was motivated by the documented interplay between the imipridone-triggered activation of TRAIL (the tumor necrosis factor-related apoptosis-inducing ligand) and mitochondrial ClpP (Caseinolytic protease P) and the ROS-mediated effect of ferrocene-containing compounds. In order to obtain novel hybrids with multitarget characters, the ferrocene moiety was tethered to the imipridone scaffold through ethynylene and 1,2,3-triazolyl linkers by using Sonogashira coupling of Cu(I)- and Ru(II)-catalyzed azide-alkyne cycloadditions. The biological activities of the new hybrids were examined by using in vitro cell viability assays on four malignant cell lines (PANC-1, A2058, EBC-1 and Fadu), along with colony formation assays on the most resistant PANC-1 cell line. Several hybrids caused a significantly greater drop in the cell viability compared to ONC201, and two of them completely overcame the resistance, with IC50 values comparable to those produced by ONC201. The two most potent hybrids, but not ONC201, induced apoptosis/necrosis in PANC-1 and A2058 cells after 24 h of treatment.

Investigation of the Antitumor Effects of Tamoxifen and Its Ferrocene-Linked Derivatives on Pancreatic and Breast Cancer Cell Lines.

Tamoxifen is a long-known anti-tumor drug, which is the gold standard therapy in estrogen receptor (ER) positive breast cancer patients. According to previous studies, the conjugation of the original tamoxifen molecule with different functional groups can significantly improve its antitumor effect. The purpose of this research was to uncover the molecular mechanisms behind the cytotoxicity of different ferrocene-linked tamoxifen derivates. Tamoxifen and its ferrocene-linked derivatives, T5 and T15 were tested in PANC1, MCF7, and MDA-MB-231 cells, where the incorporation of the ferrocene group improved the cytotoxicity on all cell lines. PANC1, MCF7, and MDA-MB-231 express ERα and GPER1 (G-protein coupled ER 1). However, ERß is only expressed by MCF7 and MDA-MB-231 cells. Tamoxifen is a known agonist of GPER1, a receptor that can promote tumor progression. Analysis of the protein expression profile showed that while being cytotoxic, tamoxifen elevated the levels of different tumor growth-promoting factors (e.g., Bcl-XL, Survivin, EGFR, Cathepsins, chemokines). On the other hand, the ferrocene-linked derivates were able to lower these proteins. Further analysis showed that the ferrocene-linked derivatives significantly elevated the cellular oxidative stress compared to tamoxifen treatment. In conclusion, we were able to find two molecules possessing better cytotoxicity compared to their unmodified parent molecule while also being able to counter the negative effects of the presence of the GPER1 through the ER-independent mechanism of oxidative stress induction.

The Synergistic Activity of Bortezomib and TIC10 against A2058 Melanoma Cells.

Combination antitumor treatments are essential parts of modern tumor therapy as-compared to monotherapies-(i) they are more effective; (ii) the dose of the compounds can be reduced; and (iii) therefore the side effects are improved. Our research group previously demonstrated the antitumor character of bortezomib (BOZ) in A2058 melanoma cells. Unfortunately, dose-related side effects are common during BOZ therapy, which could be prevented by reducing the dose of BOZ. This study aimed to characterize synergistic combinations of BOZ with a TRAIL (TNF-related apoptosis-inducing ligand) -inducing compound (TIC10), where the doses can be cut down but the efficacy is preserved. Endpoint cell viability assays were performed on A2058 cells, and synergism of BOZ and TIC10 was observed after 72 h. Synergism was further validated in a real-time impedimetric assay, and our results showed that BOZ-treated melanoma cells survived the treatment, an effect not registered in the co-treatments. Treatment with the combinations resulted in increased apoptosis, which was not accompanied by enhanced LDH release. Nevertheless, the expression of death receptor 5 (DR5) was increased on the cell surface without transcriptional regulation. In summary, our findings support the theory that the application of BOZ and TIC10 in combination could provide higher efficacy in vitro.

2,3-Dihydroferroceno[3,4]pyrrolo[2,1-b]thiazol-5(8b)-ones: Synthesis, Structure and DFT Study on the Mechanism of Chemo- and Diastereoslective Annulations of (Sp)-2-Formylferrocenecarbonyl Fluoride and (Sp)-2-Formylferrocenecarboxylic Acid.

By means of the annulation of easy-to-handle yet sufficiently reactive (Sp)-2-formylferrocenecar- bonyl fluoride with the hydrochlorides of cysteamine and the methyl esters of enentiomer cysteines conducted in dichloromethane at room temperature in the presence of pyridine, the first members of 2,3-dihydroferroceno[3,4]pyrrolo[2,1-b]thiazol-5(8bH)-ones with the elements of planar- and central chirality were prepared as single enantiomers. An atom economic procedure was also elaborated for the synthesis of these organometallic heterocycles directly exploring (Sp)-2-formylferrocenecarboxylic acid in situ activated by CDI and TFA, sequentially added to the reaction mixture. The relative and consequently, the absolute, configuration of the isolated diastereomers was determined by NMR measurements supported by DFT structural optimization. On the basis of the results of synthetic control experiments and a series of further DFT modelling studies, including energetic and MO analysis of the iminium intermediates, we propose a mechanism for the thiazolidine-forming annulations that proceed via primary N-acylation followed by proton-mediated cyclocondensation and subsequent diastereoselective sulfhydryl-attack on the resulting iminium center.

Synthesis, structure and in vitro antiproliferative effects of alkyne-linked 1,2,4-thiadiazole hybrids including erlotinib- and ferrocene-containing derivatives.

Chemotherapy is an indispensable tool to treat cancer, therefore, the development of new drugs that can treat cancer with minimal side effects and lead to more favorable prognoses is of crucial importance. A series of eleven novel 1,2,4-thiadiazoles bearing erlotinib (a known anticancer agent), phenylethynyl, ferrocenyl, and/or ferrocenethynyl moieties were synthesized in this work and characterized by NMR, IR and mass spectroscopies. The solid-phase structures were determined by single-crystal X-ray diffraction. Partial isomerisation of bis(erlotinib)-1,2,4-thiadiazole into its 1,3,4-thiadiazole isomer, leading to the isolation of a 3 : 2 isomer mixture, was observed and a plausible mechanism for isomerisation is suggested. The in vitro cytostatic effect and the long-term cytotoxicity of these thiadiazole-hybrids, as well as that of erlotinib, 3,5-dichloro-1,2,4-thiadiazole and 3,5-diiodo-1,2,4-thiadiazole were investigated against A2058 human melanoma, HepG2 human hepatocellular carcinoma, U87 human glioma, A431 human epidermoid carcinoma, and PC-3 human prostatic adenocarcinoma cell lines. Interestingly, erlotinib did not exhibit a significant cytostatic effect against these cancer cell lines. 1,2,4-Thiadiazole hybrids bearing one erlotinib moiety or both an iodine and a ferrocenethynyl group, as well as 3,5-diiodo-1,2,4-thiadiazole demonstrated good to moderate cytostatic effects. Among the synthesized 1,2,4-thiadiazole hybrids, the isomer mixture of bis-erlotinib substituted 1,2,4- and 1,3,4-thiadiazoles showed the most potent activity. This isomer mixture was proven to be the most effective in long-term cytotoxicity, too. 3,5-Diiodo-1,2,4-thiadiazole and its hybrid with one erlotinib fragment were also highly active against A431 and PC-3 proliferation. These novel compounds may serve as new leads for further study of their antiproliferative properties.

Protoflavone-Chalcone Hybrids Exhibit Enhanced Antitumor Action through Modulating Redox Balance, Depolarizing the Mitochondrial Membrane, and Inhibiting ATR-Dependent Signaling.

Hybrid compounds combine fragments with complementary targets to achieve a common pharmacological goal. This approach represents an increasingly popular strategy for drug discovery. In this work, we aimed to design antitumor hybrid compounds based on an inhibitor of ataxia-telangiectasia and Rad3-related protein (ATR)-dependent signaling, protoapigenone, and a pro-oxidant ferrocene or chalcone fragment. Four new triazole-coupled hybrids were prepared. The compounds were cytotoxic against human breast cancer cell lines in vitro, showing IC50 values in the sub-micromolar range. The nature of interactions between relevant fragments of the hybrids was evaluated by the Chou-Talalay method. Experimental combination treatment with the fragments showed additive effects or slight/moderate synergism, while strong synergism was observed when the fragments were virtually combined into their hybrids, suggesting a relevant pharmacological benefit of the coupling. All hybrids were strong inhibitors of the ATR-mediated activation of Chk1, and they interfered with the redox balance of the cells leading to mitochondrial membrane depolarization. Additionally, they induced late apoptosis and primary necrosis in MDA-MB-231 and MCF-7 breast cancer cells, respectively. Our results demonstrate that coupling the ATR-dependent signaling inhibitor protoflavone with a pro-oxidant chalcone dramatically increases the antitumor activity compared with either fragment alone. Such compounds may offer an attractive novel strategy for the treatment of various cancers.

Novel Polycondensed Partly Saturated ß-Carbolines Including Ferrocene Derivatives: Synthesis, DFT-Supported Structural Analysis, Mechanism of Some Diastereoselective Transformations and a Preliminary Study of Their in vitro Antiproliferative Effects.

Use of a Pictet-Spengler reaction of tryptamine and l-tryptophan methyl ester and subsequent reduction of the nitro group followed by further cyclocondensation with aryl aldehydes and formyl-substituted carboxylic acids, including ferrocene-based components, furnished a series of diastereomeric 6-aryl-substituted 5,6,8,9,14,14b-hexahydroindolo[2',3':3,4]pyrido[1-c]-quinazolines and 5,5b,17,18-tetrahydroindolo[2',3':3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11-(15bH)-ones with the elements of central-, planar and conformational chirality. The relative configuration and the conformations of the novel polycyclic indole derivatives were determined by 1H- and 13C-NMR methods supplemented by comparative DFT analysis of the possible diastereomers. The structure of one of the pentacyclic methyl esters with defined absolute configuration "S" was also confirmed by single crystal X-ray diffraction measurement. Accounting for the characteristic substituent-dependent diastereoselective formation of the products multistep mechanisms were proposed on the basis of the results of DFT modeling. Preliminary in vitro cytotoxic assays of the products revealed moderate-to-significant antiproliferative effects against PANC-1-, COLO-205-, A-2058 and EBC-1 cell lines that proved to be highly dependent on the stereostructure and on the substitution pattern of the pending aryl substituent.

Synthesis of New C-3 Substituted Kynurenic Acid Derivatives.

The application of kynurenic acid (KYNA) as an electron-rich aromatic system in the modified Mannich reaction has been examined. The extension possibility of the reaction was tested by using amines occurring in a number of bioactive products, such as morpholine, piperidine, or N-methylpiperazine and aldehydes of markedly different reactivities, like formaldehyde and benzaldehyde. The influence of substituents attached to position 3 on the aminoalkylation was also investigated. Thus, reactions of 3-carbamoyl-substituted precursors with tertiary amine containing side-chains were also tested to afford new KYNA derivatives with two potential cationic centers. By means of NMR spectroscopic measurements, supported by DFT calculations, the dominant tautomer form of KYNA derivatives was also determined.

Synthetic- and DFT modelling studies on regioselective modified Mannich reactions of hydroxy-KYNA derivatives.

The syntheses of hydroxy-substituted kynurenic acid (KYNA) derivatives have been achieved by an optimised Conrad-Limpach procedure. The derivatives were then reacted with morpholine and paraformaldehyde, as a representative amine and aldehyde, in a modified Mannich reaction. The newly introduced substituents altered the preferred reaction centre of the KYNA skeleton. A systematic investigation of substitutions was carried out, using different reaction conditions, resulting in mono- or disubstituted derivatives. Product selectivity and regioselectivity were rationalised by DFT calculations disclosing HOMO distribution and NBO charges on the potential nucleophilic centres in the anion of the appropriate KYNA ester assumed to be active components towards the iminium ion intermediate.

Novel Crizotinib-GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems.

Several promising anti-cancer drug-GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]-GnRH-I targeting peptide. Our most prominent crizotinib-GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]-GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]-GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib-GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets-the ATP-binding site of RTKs- and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib-GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug-GnRH conjugates.

Synthesis, Structure and In Vitro Cytotoxic Activity of Novel Cinchona-Chalcone Hybrids with 1,4-Disubstituted- and 1,5-Disubstituted 1,2,3-Triazole Linkers.

By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine- and quinidine-derived alkynes with azide-substituted chalcones a systematic series of novel cinchona-chalcone hybrid compounds, containing 1,4-disubstituted- and 1,5-disubstituted 1,2,3-triazole linkers, were synthesized and evaluated for their cytotoxic activity on four human malignant cell lines (PANC-1, COLO-205, A2058 and EBC-1). In most cases, the cyclization reactions were accompanied by the transition-metal-catalyzed epimerization of the C9-stereogenic centre in the cinchona fragment. The results of the in vitro assays disclosed that all the prepared hybrids exhibit marked cytotoxicity in concentrations of low micromolar range, while the C9-epimerized model comprising quinidine- and (E)-1-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl) fragments, connected by 1,5-disubstituted 1,2,3-triazole linker, and can be regarded as the most potent lead of which activity is probably associated with a limited conformational space allowing for the adoption of a relatively rigid well-defined conformation identified by DFT modelling. The mechanism of action of this hybrid along with that of a model with markedly decreased activity were approached by comparative cell-cycle analyses in PANC-1 cells. These studies disclosed that the hybrid of enhanced antiproliferative activity exerts significantly more extensive inhibitory effects in subG1, S and G2/M phases than does the less cytotoxic counterpart.

Ferrocene-Containing Impiridone (ONC201) Hybrids: Synthesis, DFT Modelling, In Vitro Evaluation, and Structure⁻Activity Relationships.

Inspired by the well-established clinical evidence about the interplay between apoptotic TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) mechanism and reactive oxygen species (ROS)-mediated oxidative stress, a set of novel ONC201 hybrids containing the impiridone core and one or two differently positioned ferrocenylalkyl groups were synthesised in our present work. These two types of residues have been implicated in the aforementioned mechanisms associated with cytotoxic activity. A straightforward, primary amine-based synthetic approach was used allowing the introduction of a variety of N-substituents into the two opposite regions of the heterocyclic skeleton. Reference model compounds with benzyl and halogenated benzyl groups were also synthesised and tested. The in vitro assays of the novel impiridones on five malignant cell lines disclosed characteristic structure-activity relationship (SAR) featuring significant substituent-dependent activity and cell-selectivity. A possible contribution of ROS-mechanism to the cytotoxicity of the novel metallocenes was suggested by density functional theory (DFT)studies on simplified models. Accordingly, unlike the mono-ferrocenylalkyl-substituted products, the compounds containing two ferrocenylalkyl substituents in the opposite regions of the impiridone core display a much more pronounced long-term cytotoxic effect against A-2058 cell line than do the organic impiridones including ONC201 and ONC212. Furthermore, the prepared bis-metallocene derivatives also present substantial activity against COLO-205- and EBC-1 cell lines.