Synthesis and cytotoxic activity of unsaturated macrolides and their hybrid molecules with a C60 fullerene.

Previously unreported macrodiolides containing a 1Z,5Z-diene fragment in the structure have been synthesized with high yields and stereoselectivity by our research group, using the intermolecular esterification of malonic acid with α,ω-diols containing bis-methylene-separated Z-double bonds, catalyzed by Hf(OTf)4 hafnium triflate. Under Bingel-Hirsch conditions, the synthesized macrodiolides were chemically bonded with a C60 fullerene to produce the corresponding methanofullerenes. The cytotoxic activity of macrodiolides and methanofullerenes in relation to Jurkat, K562, U937, HL60 tumor cell lines and normal fibroblasts was studied. Covalent binding of macrodiolides to the C60 fullerene molecule was found to significantly increase the cytotoxic effect (from 5 to 170 times) of the hybrid molecule as compared to the initial macrodiolide. Moreover, the synthesized hybrid molecules initiate apoptosis by uncoupling oxidation and phosphorylation of the mitochondrial membrane of tumor cells.

Direct Synthesis of Polyaromatic Cyclophanes Containing Bis-Methylene-Interrupted Z-Double Bonds and Study of Their Antitumor Activity In Vitro.

An original synthetic route was developed for the preparation of previously unknown unsaturated polyaromatic macrolactones containing a 1Z,5Z-diene moiety in 48-71% yields and with >98% stereoselectivity. The method is based on intermolecular cyclocondensation of aromatic dicarboxylic acids with α,ω-alka-nZ,(n+4)Z-dienediols (1,12-dodeca-4Z,8Z-dienediol, 1,14-tetradeca-5Z,9Z-dienediol, 1,18-octadeca-7Z,11Z-dienediol) mediated by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/4-dimethylaminopyridine (DMAP). The unsaturated diols were prepared by successive homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp2TiCl2 catalyst (10 mol.%) and subsequent treatment with 0.1 equiv. of para-toluenesulfonic acid of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The resulting cyclophanes exhibited high cytotoxic activity in vitro against Jurkat, K562, U937, and HL60 cancer lines. Additionally, the synthesized products were studied for their effect on mitochondria, ability to induce apoptosis, and influence on the cell cycle using modern flow cytometry methods.

Hybrid molecules based on fullerene C60 and 5Z,9Z-dienoic acids: Synthesis and cytotoxic activity.

The present research project details synthesis of new hybrid methanofullerenes based on acetylene and triazole esters of malonic acid containing 5Z,9Z-dienoic acids and fullerene C60 under Bingel-Hirsch conditions, including study of the cytotoxic activity with respect to Jurkat, K562, U937 and HL60 tumor cell lines. Hybrid methanofullerenes containing acetylenic fragments, unlike triazole substituents, were found to exhibit higher cytotoxicity, but are characterized by lower selectivity of action in relation to healthy cells.

Total Synthesis of Natural Lembehyne C and Investigation of Its Cytotoxic Properties.

The first Z-stereoselective method for the synthesis of the natural marine alkynol lembehyne C, containing a 1Z,5Z,9Z-triene moiety, in 41% yield was developed using the new Ti-catalyzed cross-coupling of oxygenated and aliphatic 1,2-dienes as the key step. It was found for the first time that lembehyne C exhibits moderate cytotoxicity against Jurkat, K562, U937, and HL60 cancer cells and also efficiently induces apoptosis in Jurkat cells, with the cell death mechanism being activated by the mitochondrial pathway. The lembehyne C inhibition of the cell cycle follows the mitotic catastrophe mechanism.

New 1Z,5Z-diene macrodiolides: Catalytic synthesis, anticancer activity, induction of mitochondrial apoptosis, and effect on the cell cycle.

An original scheme was developed for the synthesis of previously undescribed unsaturated macrodiolides containing a 1Z,5Z-diene moiety in 44-80% yields and with high stereoselectivity (>95%) based on the intermolecular esterification of α,ω-diols with α,ω-alka-nZ,(n + 4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-dienedicarboxylic acid, 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid, 1,18-octadeca-7Z,11Z-dienedicarboxylic acid) catalyzed by hafnium triflate [Hf(OTf)4]. The unsaturated dicarboxylic acids were prepared via homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp2TiCl2 catalyst (10 mol.%) and the subsequent Jones oxidation of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The thus prepared macrodiolides exhibit high cytotoxic activity in vitro against Jurkat, K562, U937, Hek293 and HeLa cancer cell lines. It was found that induction of the programmed cell death in Jurkat cells by macrodiolides corresponds to the mitochondrial apoptosis pathway. Also, it was shown that the prepared macrodiolides efficiently suppress phosphorylation of Akt and p38 kinases and CREB transcription factor in cancer cells.

New synthetic analogues of natural 5Z,9Z-dienoic acids: Stereoselective synthesis and study of the anticancer activity.

A stereoselective method was developed for the synthesis of synthetic analogues of natural 5Z,9Z-dienoic acids by esterification of aliphatic and aromatic alcohols and carboxylic acids with (5Z,9Z)-1,14-tetradeca-5,9-dienedioic acid and (5Z,9Z)-1,14-tetradeca-5,9-dienediol, synthesized by Ti-catalyzed homo-cyclomagnesiation of the tetrahydropyran ether of hepta-5,6-dien-1-ol with Grignard reagents. In order to establish the effect of molecular structure on the antitumor activity, the obtained 5Z,9Z-dienoic acids were tested for the inhibitory activity against human topoisomerase I, the cytotoxic activity in vitro against several cancer and normal cell lines (Jurkat, HL-60, K562, U937, fibroblasts), the effect on the cell cycle, and apoptosis-inducing ability using flow cytofluorometry. In addition, the effect of the synthesized acids on the cancer cell production of some phosphorylated and unphosphorylated proteins responsible for proliferation and apoptosis was studied by a new multiplex assay technology, MAGPIX.

The Synthesis of Bicyclo[4.2.1]nona-2,4,7-trienes by [6π + 2π]-Cycloaddition of 1-Substituted 1,3,5-Cycloheptatrienes Catalyzed by Titanium and Cobalt Complexes.

The [6π + 2π]-cycloaddition of alkynes to 1-methyl-, propyl-, benzyl-, and hydroxymethyl-substituted 1,3,5-cycloheptatrienes in the presence of catalytic systems Ti(acac)2Cl2-Et2AlCl and Co(acac)2(dppe)/Zn/ZnI2 was performed for the first time to give practically valuable bicyclo[4.2.1]nona-2,4,7-trienes in high yields (72-88%). The structures of the obtained bicyclic compounds were reliably proved by NMR methods and X-ray diffraction analysis. The newly synthesized bicycles have been investigated for their in vitro cytotoxic activity against Jurkat, K562, U937, and HL60 tumor and normal cell lines and induction of apoptosis.

Comparison of Predictive In Silico Tools on Missense Variants in GJB2, GJB6, and GJB3 Genes Associated with Autosomal Recessive Deafness 1A (DFNB1A).

In silico predictive software allows assessing the effect of amino acid substitutions on the structure or function of a protein without conducting functional studies. The accuracy of in silico pathogenicity prediction tools has not been previously assessed for variants associated with autosomal recessive deafness 1A (DFNB1A). Here, we identify in silico tools with the most accurate clinical significance predictions for missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes associated with DFNB1A. To evaluate accuracy of selected in silico tools (SIFT, FATHMM, MutationAssessor, PolyPhen-2, CONDEL, MutationTaster, MutPred, Align GVGD, and PROVEAN), we tested nine missense variants with previously confirmed clinical significance in a large cohort of deaf patients and control groups from the Sakha Republic (Eastern Siberia, Russia): Сх26: p.Val27Ile, p.Met34Thr, p.Val37Ile, p.Leu90Pro, p.Glu114Gly, p.Thr123Asn, and p.Val153Ile; Cx30: p.Glu101Lys; Cx31: p.Ala194Thr. We compared the performance of the in silico tools (accuracy, sensitivity, and specificity) by using the missense variants in GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) genes associated with DFNB1A. The correlation coefficient (r) and coefficient of the area under the Receiver Operating Characteristic (ROC) curve as alternative quality indicators of the tested programs were used. The resulting ROC curves demonstrated that the largest coefficient of the area under the curve was provided by three programs: SIFT (AUC = 0.833, p = 0.046), PROVEAN (AUC = 0.833, p = 0.046), and MutationAssessor (AUC = 0.833, p = 0.002). The most accurate predictions were given by two tested programs: SIFT and PROVEAN (Ac = 89%, Se = 67%, Sp = 100%, r = 0.75, AUC = 0.833). The results of this study may be applicable for analysis of novel missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes.

Diversity-oriented synthesis of spirothiazolidinediones and their biological evaluation.

We report a new synthetic approach to assemble spirothiazolidinediones via a [2 + 2 + 2] cyclotrimerization reaction and the derivatives were further functionalized through DA chemistry and click reaction. Using flow cytometry, it was shown for the first time that the new benzyl alcohol derivatives of thiazolidine-2,4-dione generated here are efficient apoptosis inducers in the HeLa, Hek293, U937, Jurkat, and K562 cell lines.

Cobalt-Catalyzed [6 + 2] Cycloaddition of Alkynes with 1,3,5,7-Cyclooctatetraene as a Key Element in the Direct Construction of Substituted Bicyclo[4.3.1]decanes.

A new, effective catalytic system based on Co(acac)2 has been developed for [6 + 2] cycloaddition of terminal alkynes to 1,3,5,7-cyclooctatetraene to give substituted bicyclo[4.2.2]deca-2,4,7,9-tetraenes in high yields (68-85%). The electrophilic activation of double bonds in the bicyclic products with m-CPBA is an efficient method for the synthesis of substituted bicyclo[4.3.1]deca-2,4,8-triene-7,10-diols, which form the key structural moieties of numerous natural biologically active compounds. The structures of the obtained compounds were reliably proven by modern spectral methods and X-ray diffraction. The mechanism of the discovered rearrangement was studied both using deuterium-labeled bicyclo[4.2.2]deca-2,4,7,9-tetraenes and utilizing quantum chemical calculations. The obtained substituted bicyclo[4.3.1]deca-2,4,8-triene-7,10-diols and their keto derivatives showed high antitumor activity in vitro against Hek293, Jurkat, K562, and A549 tumor cell lines.

The first total synthesis of the marine acetylenic alcohol, lembehyne B - a selective inducer of early apoptosis in leukemia cancer cells.

The communication reports a new stereoselective method for the synthesis of a natural acetylenic alcohol, lembehyne B. The key stage of the process uses new cross-cyclomagnesiation reaction of aliphatic and oxygenated 1,2-dienes with Grignard reagents in the presence of a catalytic amount of Cp2TiCl2. A study of the cytotoxic properties of lembehyne B on tumor cell lines using flow cytometry demonstrated that this is a selective inducer of early apoptosis of the Jurkat, HL-60 and K562 cell cultures and hypodiploid (sub-G1) sub-population inducer in cell cycle studies for all cell lines used.

Short Route to the Total Synthesis of Natural Muricadienin and Investigation of Its Cytotoxic Properties.

An original synthesis of the acetogenin muricadienin, the bioprecursor of solamin, has been developed. The key step in the five-step 41% overall yield synthesis is the catalytic cross-cyclomagnesiation reaction of functionally substituted 1,2-dienes with EtMgBr in the presence of Cp2TiCl2 and magnesium metal. It has been demonstrated for the first time that muricadienin exhibits a moderate in vitro inhibitory activity against topoisomerases I and IIα, key cell cycle enzymes. Using flow cytometry, muricadienin was shown to have high cytotoxicity toward the HEK293 kidney cancer cells (IC50 0.39 µM).

Stereoselective synthesis of 11-phenylundeca-5Z,9Z-dienoic acid and investigation of its human topoisomerase I and IIα inhibitory activity.

(5Z,9Z)-11-Phenylundeca-5,9-dienoic acid was stereoselectively synthesized, based on original cross-cyclomagnesiation of 2-(hepta-5,6-dien-1-yloxy)tetrahydro-2H-pyran and buta-2,3-dien-1-ylbenzene with EtMgBr in the presence of Cp2TiCl2 catalyst giving 2,5-dialkylidenemagnesacyclopentane in 86% yield. The acid hydrolysis of the product and the Jones oxidation of the resulting 2-{[(5Z,9Z)-11-phenylundeca-5,9-dien-1-yl]oxy}tetrahydro-2Н-pyran afforded (5Z,9Z)-11-phenylundeca-5,9-dienoic acid in an overall yield of 75%. A high inhibitory activity of the synthesized acid with respect to human topoisomerase I (hTop1) and II (hTop2α) was determined.

New Polyether Macrocycles as Promising Antitumor Agents-Targeted Synthesis and Induction of Mitochondrial Apoptosis.

A series of previously unknown aromatic polyether macrodiolides containing a cis,cis-1,5-diene moiety in the molecule were synthesized in 47-74% yields. Macrocycle compounds were first obtained by intermolecular esterification of aromatic polyether diols with α,ω-alka-nZ,(n+4)Z-dienedioic acids mediated by N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and 4-(dimethylamino)pyridine (DMAP). For the synthesized compounds, studies of cytotoxicity on tumor (Jurkat, K562, U937), conditionally normal (HEK293) cell lines, and normal fibroblasts were carried out. CC50 was determined, and the therapeutic selectivity index of cytotoxic action (SI) in comparison with normal fibroblasts was evaluated. With the involvement of modern methods of flow cytometry for the most promising macrocycles, their effect on mitochondria and the cell cycle was investigated. It was found that a new macrocycle exhibits pronounced apoptosis-inducing activity toward Jurkat cells and can retard cell division by blocking at the G1/S checkpoint. Also, it was shown that the synthesized macrodiolides influence mitochondria due to their high ability to penetrate the mitochondrial membrane.

Pentacyclic Triterpenoids-Based Ionic Compounds: Synthesis, Study of Structure-Antitumor Activity Relationship, Effects on Mitochondria and Activation of Signaling Pathways of Proliferation, Genome Reparation and Early Apoptosis.

The present research paper details the synthesis of novel ionic compounds based on triterpene acids (betulinic, oleanolic and ursolic), with these acids acting both as anions and connected through a spacer with various nitrogen-containing compounds (pyridine, piperidine, morpholine, pyrrolidine, triethylamine and dimethylethanolamine) and acting as a cation. Based on the latter, a large number of ionic compounds with various counterions (BF4-, SbF6-, PF6-, CH3COO-, C6H5SO3-, m-C6H4(OH)COO- and CH3CH(OH)COO-) have been synthesized. We studied the cytotoxicity of the synthesized compounds on the example of various tumor (Jurkat, K562, U937, HL60, A2780) and conditionally normal (HEK293) cell lines. IC50 was determined, and the influence of the structure and nature of the anion and cation on the antitumor activity was specified. Intracellular signaling, apoptosis induction and effects of the most active ionic compounds on the cell cycle and mitochondria have been discussed by applying modern methods of multiparametric enzyme immunoassay and flow cytometry.

Natural Acetogenins, Chatenaytrienins-1, -2, -3 and -4, Mitochondrial Potential Uncouplers and Autophagy Inducers-Promising Anticancer Agents.

The present paper details the complete stereoselective synthesis of four natural acetogenins, chatenaytrienins-1, -2, -3 and -4, previously isolated from the roots of fruit trees of the family Annonaceae (A. nutans and A. muricata), as an inseparable mixture. The novel organometallic reactions, developed by the authors, of Ti-catalyzed cross-cyclomagnesiation of O-containing and aliphatic allenes using available Grignard reagents were applied at the key stage of synthesis. We have studied the biological activity of the synthesized individual chatenaytrienins-1, -2, -3 and -4 in vitro, including their cytotoxicity in a panel of tumor lines and their ability to induce apoptosis, affect the cell cycle and mitochondria, and activate the main apoptotic signaling pathways in the cell, applying modern approaches of flow cytometry and multiplex analysis with Luminex xMAP technology. It has been shown that chatenaytrienins affect mitochondria by uncoupling the processes of mitochondrial respiration, causing the accumulation of ROS ions, followed by the initiation of apoptosis. The most likely mechanism for the death of cortical neurons from the consumption of tea from the seeds of Annona fruit is long-term chronic hypoxia, which leads to the development of an atypical form of Parkinson's disease that is characteristic of the indigenous inhabitants of Guam and New Caledonia.

Mono- and binuclear complexes of copper(II) with dimethylaminomethyl derivatives of 2-naphthol and 6-quinolinol: synthesis and in vitro study of antitumor properties.

1-(Dimethylamino)methyl-6-quinolinol scaffold, a structural moiety of the molecule of anticancer drug topotecan, was modified into copper-containing products to study cytotoxic properties. New mononuclear and binuclear Cu(II) complexes with 1-(N,N-dimethylamino)methyl-6-quinolinol were synthesized for the first time. The same way Cu(II) complexes with 1-(dimethylamino)methyl-2-naphtol ligand were synthesized. The structures of mono- and binuclear Cu(II) complexes with 1-aminomethyl-2-naphtol were confirmed by X-ray diffraction. The obtained compounds were examined for in vitro cytotoxic activity against Jurkat, K562, U937, MDA-MB-231, MCF7, T47D, and HEK293 cells. The induction of apoptosis and the effect of novel Cu complexes on the cell cycle were investigated. The cells showed a higher sensitivity to mononuclear Cu(II) complex with 1-(N,N-dimethylamino)methyl-6-quinolinolligand. All synthesized Cu(II) complexes had higher antitumor activity than the drugs topotecan, camptothecin, and platinum containing cisplatin.

Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect.

Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha Republic (Yakutia) located in Eastern Siberia (Russian Federation). The Yakut population exhibits high frequency of some Mendelian disorders, which are rare in other populations worldwide. Mutational analysis of GJB2 gene in 86 unrelated Yakut patients with congenital HI without other clinical features has been performed. In this study, we registered a large cohort of Yakut patients homozygous for the IVS1+1G>A mutation (70 unrelated deaf subjects in total). Detailed audiological analysis of 40 deaf subjects with genotype IVS1+1G>A/IVS1+1G>A revealed significant association of this genotype with mostly symmetrical bilateral severe to profound HI (85% severe-to-profound HI versus 15% mild-to-moderate HI, P<0.05). The highest among six investigated Eastern Siberian populations carrier frequency of the IVS1+1G>A mutation (11.7%) has been found in Yakut population. Reconstruction of 140 haplotypes with IVS1+1G>A mutation demonstrates the common origin of all mutant chromosomes found in Yakuts. The age of mutation was estimated to be approximately 800 years. These findings characterize Eastern Siberia as the region with the most extensive accumulation of the IVS1+1G>A mutation in the world as a result of founder effect.

Synthesis and Anticancer Activity of Hybrid Molecules Based on Lithocholic and (5Z,9Z)-Tetradeca-5,9-dienedioic Acids Linked via Mono(di,tri,tetra)ethylene Glycol and α,ω-Diaminoalkane Units.

For the first time, hybrid molecules were synthesized on the basis of lithocholic and (5Z,9Z)-1,14-tetradeca-5,9-dienedicarboxylic acids, obtained in two stages using the homo-cyclomagnesiation reaction of 2-(hepta-5,6-diene-1-yloxy)tetrahydro-2H-pyran at the key stage. The resulting hybrid molecules containing 5Z,9Z-dienoic acids are of interest as novel synthetic biologically active precursors to create modern drugs for the treatment of human oncological diseases. The synthesized hybrid molecules were found to exhibit extremely high in vitro inhibitory activity against human topoisomerase I, which is 2-4 times higher than that of camptothecin, a known topoisomerase I inhibitor. Using flow cytometry and fluorescence microscopy, it was first shown that these new molecules are efficient apoptosis inducers in HeLa, U937, Jurkat, K562, and Hek293 cell cultures. In addition, the results of investigations into the effect of the synthesized acids on mitochondria and studies of possible DNA damage in Jurkat tumor cells are also presented.

Synthesis, crystal structure, and in vitro evaluation of the anticancer activity of new Pt (Pd) complexes with 1-[(dimethylamino)methyl]-2-naphthol ligand.

The synthesis of new Pt(II) and Pd(II) complexes with 1-aminomethyl-2-naphtol ligands has been first performed. The adducts of [PtCl4]2- and [PdCl4]2- anions with the 1-aminomethyl-2-naphtol NH cation were synthesized. The structure for four Pt (Pd)-containing compounds was investigated using X-ray diffraction. The obtained compounds were examined for in vitro cytotoxic activity against Jurkat and K562 human leukemia cells, lymphoma U937cells, A2780 and the cisplatin-resistant A2780cis lines of human ovarian cancer, and normal fibroblasts. Study of induction of apoptosis and the effect of new palladium and platinum complexes on the cell cycle was carried out. The cells showed a higher sensitivity to Pt(II) compounds than to Pd(II) ones. All the synthesized metal complexes show much more antitumor activity compared with a platinum-containing cisplatin drug.