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Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.

Steroids with a nitrogen-containing heterocycle in the side chain are known as effective inhibitors of androgen signaling and/or testosterone biosynthesis, thus showing beneficial effects for the treatment of prostate cancer. In this work, a series of 3β-hydroxy-5-ene steroids containing an isoxazole fragment in the side chain was synthesized. The key steps included the preparation of Weinreb amide, its conversion to acetylenic ketones, and the 1,2- or 1,4-addition of hydroxylamine, depending on the solvent used. The biological activity of the obtained compounds was studied in a number of tests, including their effects on 17α-hydroxylase and 17,20-lyase activity of human CYP17A1 and the ability of selected compounds to affect the downstream androgen receptor signaling. Three derivatives diminished the transcriptional activity of androgen receptor and displayed reasonable antiproliferative activity. The candidate compound24j, (17R)-17-((3-(2-hydroxy-propan-2-yl)isoxazol-5-yl)methyl)-androst-5-en-3β-ol, suppressed the androgen receptor signaling and decreased its protein level in two prostate cancer cell lines, LNCaP and LAPC-4. Interaction of compounds with CYP17A1 and androgen receptor was confirmed and described by molecular docking.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 μM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki = 8.1 ± 1.2 μM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.

Haptoglobin (Hp) is a blood plasma glycoprotein that plays a critical role in tissue protection and the prevention of oxidative damage. Haptoglobin is an acute-phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein and is the subject of research as a potential biomarker of many diseases, including malignant neoplasms. The Human Hp gene is polymorphic and controls the synthesis of three major phenotypes—homozygous Hp1-1 and Hp2-2, and heterozygous Hp2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual’s predisposition to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). Glioblastoma multiform (GBM) is the most malignant primary brain tumor. In our study, we have analyzed the state of Hp proteoforms in plasma and cells using 1D (SDS-PAGE) and 2D electrophoresis (2DE) with the following mass spectrometry (LC ES-MS/MS) or Western blotting. We found that the levels of α2- and β-chain proteoforms are up-regulated in the plasma of GBM patients. An unprocessed form of Hp2-2 (PreHp2-2, zonulin) with unusual biophysical parameters (pI/Mw) was also detected in the plasma of GBM patients and glioblastoma cells. Altogether, this data shows the possibility to use proteoforms of haptoglobin as a potential GBM-specific plasma biomarker.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Conjugates of 17α-substituted testosterone (1 and 2) and 17β-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17β-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.

A general scheme for the synthesis of oxazoline and benzoxazole derivatives of [17(20)E]-21-norpregnene differing in the structure of the steroid moiety as well as amides of 17β-hydroxy-3-oxopregn-4-en-21-oic and 17α-hydroxy-3-oxopregn-4-en-21-oic acids was developed. The scheme involved synthesis of the steroid building blocks (appropriately protected derivatives of pregn-17(20)-en-21-oic, 17β-hydroxypregnan-21-oic, and 17α-hydroxypregnan-21-oic acids) and subsequent transformation of these building blocks into the target compounds. Following the developed scheme, synthesis of new nitrogen-containing steroid derivatives exhibiting antiandrogenic activity was enabled.

Myelin basic protein is a potential biomarker for the central nervous system diseases in which the myelin sheath is destroyed. Using pseudo-selected reaction monitoring and the method of standard additions, we have measured the myelin basic protein level in the cerebrospinal fluid of patients with neurotrauma (n = 6), chronic neurodegenerative diseases (n = 2) and brain cancer (n = 5). Myelin basic protein was detected only in four out of five cerebrospinal fluid samples of patients with brain cancer. The cerebrospinal fluid myelin basic protein level ranged from 3.7 to 8.8 ng ml-1. We suggest that monitoring of myelin basic protein in cerebrospinal fluid can serve as a diagnostic test for the brain cancer.

During recent decades significant progress in studies of the molecular basis of socially significant diseases has been achieved due to introduction of high-throughput methods of genomics and proteomics. Numerous studies, performed within the global program “Human Proteome,” were aimed at identifying all possible proteins in various (including cancer) cell cultures and tissues. One of the aims was to identify socalled biomarkers—the proteins, specific for certain pathologies. However, many studies have shown that the development of the disease is not associated with appearance of new proteins, but it depends on the expression level of certain genes or specific proteoforms representing splice variants, single amino acid polymorphism (SAP) and post-translational modifications (PTM) of proteins. PTMs can play a key role in the development of pathology, because they activate various regulatory or structural proteins in most cellular processes. Among such modifications, phosphorylation appears to be the most significant PTM. This review considers methods of analysis of protein phosphorylation used in studies of the molecular basis of oncological diseases; it contains examples illustrating contribution of modified proteins directly involved in their development as well as examples of screening of such crucial PTMs in diagnostics and selection of methods for treatment.

To date lung adenocarcinoma (LAC) is the most common type of lung cancer. Numerous studies on LAC biology resulted in identification of crucial mutations in protooncogenes and activating neoplastic transformation pathways. Therapeutic approaches that significantly increase the survival rate of patients with LAC of different etiology have been developed and introduced into clinical practice. However, the main problem in the treatment of LAC is early diagnosis, taking into account both factors and mechanisms responsible in tumor initiation and progression. Identification of a wide biomarker repertoire with high specificity and reliability of detection appears to be a solution to this problem. In this context, proteins with differential expression in normal and pathological condition, suitable for detection in biological fluids are the most promising biomarkers. In this review we have analyzed literature data on studies aimed at search of LAC biomarkers. The major attention has been paid to protein biomarkers as the most promising and convenient subject of clinical diagnosis. The review also summarizes existing knowledge on posttranslational modifications, splice variants, isoforms, as well as model systems and transcriptome changes in LAC.Na segodniashniĭ den' naibolee rasprostranennym tipom raka legkikh iavliaetsia adenokartsinoma (AKL). V rezul'tate mnogochislennykh issledovaniĭ prirody dannogo zabolevaniia byli ustanovleny osnovnye mutatsii v onkogenakh, aktiviruiushchie signal'nye puti opukholevoĭ transformatsii kletok. Razrabotany i vvedeny v praktiku terapevticheskie podkhody v lechenii patsientov s AKL razlichnoĭ étiologii, sushchestvenno povysivshie ikh vyzhivaemost'. Odnako glavnoĭ problemoĭ v lechenii AKL iavliaetsia ranniaia diagnostika, uchityvaiushchaia vse mnogoobrazie mekhanizmov vozniknoveniia i razvitiia opukholi. Reshenie dannoĭ zadachi mozhet byt' sviazano s vyiavleniem shirokogo kruga biomarkerov, kharakterizuiushchikhsia vysokoĭ spetsifichnost'iu i nadezhnost'iu detektsii. Naibolee perspektivnymi markerami, ochevidno, iavliaiutsia belki, differentsial'no ékspressiruemye v norme i patologii i detektiruemye v biologicheskikh zhidkostiakh. Poslednee osobenno vazhno dlia maloinvazivnykh metodov diagnostiki, primeniaemykh na rannikh étapakh zabolevaniia. V dannoĭ rabote proveden analiz literaturnykh dannykh, posviashchennykh poisku biomarkerov AKL s ispol'zovaniem shirokogo spektra podkhodov i metodov issledovaniia. Osnovnoĭ aktsent sdelan na markerakh belkovogo proiskhozhdeniia, poskol'ku imenno oni iavliaiutsia perspektivnymi i udobnymi ob"ektami v klinicheskoĭ diagnostike. Privedeny rezul'taty issledovaniĭ post-transliatsionnykh modifikatsiĭ, splaĭs-variantov, izoform, a takzhe model'nykh sistem i izmeneniĭ transkriptoma pri AKL.

Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).

We report the first direct electrochemistry of CYP109C1, CYP109C2 and CYP109D1 from the myxobacterium Sorangium cellulosum So ce56 immobilized on the screen-printed graphite electrode (SPE) modified by a didodecyldimethylammonium bromide (DDAB) film. Electrochemical response was investigated by cyclic voltammetry. Cyclic voltammograms in a deoxygenated argon saturated 0.1 M potassium phosphate buffer, pH 7.4, demonstrated a reversible redox process with E ° ’ of −0.305 ± 0.005 V, −0.313 ± 0.005 V, −0.309 ± 0.005 V ( vs. Ag/AgCl) for CYP109C1, CYP109C2 and СYP109D1, respectively. The apparent surface coverages were (2.5 ± 0.2) × 10 −11 , (2.5 ± 0.2) × 10 −11 , (3.1 ± 0.2) × 10 −11 mol cm −2 , which corresponded to 5-6% of the total amount of protein that was consumed by the immobilization process. The rate constant k s of heterogeneous electron transfer between the hemoproteins and the SPE modified by DDAB film (SPE/DDAB) were determined as 0.39 ± 0.01 s −1 , 0.35 ± 0.01 s −1 , and 0.43 ± 0.02 s −1 for CYP109C1, CYP109C2 and СYP109D1, respectively. The investigated CYPs (CYP109C1, CYP109C2 and CYP109D1) demonstrated electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen. Upon addition of the substrates (myristic acid and the norisoprenoid α-ionone) in the air-saturated solution, the reduction peak current of dissolved oxygen increased, which manifested the catalytic behavior of CYP109C2 and CYP109D1 towards the substrates. CYP109D1-depended electrocatalytic hydroxylation of the myristic acid was analyzed by mass-spectrometry after electrolysis at controlled working electrode potential.

Phosphorylation is one of the most common and the most important posttranslational modifications (PTM) of proteins. The study of protein phosphoforms is complicated by their low abundance in analyzed biological samples compared with the abundance of corresponding unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective PTM analysis with high specificity and sensitivity. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining phosphoproteins in a reconstituted kinase system and following usage of tryptic fragments of the phosphorylated protein as a template for the development of the SRM method. The developed method has been successfully applied for detection of phosphopeptides of myelin basic protein in human brain glioma samples.

Study of the proteomic composition of exhaled breath condensate (EBC), is a promising non-invasive method for the diagnosis of the respiratory tract diseases in patients. In this study the EBC proteomic composition of the 79 donors, including patients with different pathologies of the respiratory system has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins the type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of protein have been recognized: found in all pathologies (Dermcidin, Alpha-1-microglobulin, SHROOM3), found in several pathologies (CSTA, LCN1, JUP, PIP, TXN), and specific for a single pathology (PRDX1, Annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used to identify potential protein markers characteristic for pathologies such as chronic obstructive pulmonary disease (PRDX1) and pneumonia (Annexin A1/A2).Issledovanie proteomnogo sostava kondensata vydykhaemogo vozdukha (KVV), predstavliaetsia perspektivnym neinvazivnym metodom diagnostiki bolezneĭ respiratornogo trakta cheloveka. V ramkakh dannogo issledovaniia proanalizirovan proteomnyĭ sostav KVV 53 donorov, vkliuchaia patsientov s razlichnymi zabolevaniiami dykhatel'noĭ sistemy. Invariantnymi dlia vsekh prob iavlialis' tsitoskeletnye keratiny II tipa (1, 2, 3, 4, 5, 6) i tsitoskeletnye keratiny I tipa (9, 10, 14, 15, 16). Analiziruia chastotu vstrechaemosti belkov v raznykh gruppakh mozhno vydelit' neskol'ko kategoriĭ belkov: obnaruzhennye u vsekh (dermtsidin, al'fa-1-mikroglobulina, SHROOM3), v dvukh gruppakh odnovremenno (CSTA, LCN1, JUP, PIP, TXN) i spetsifichnye dlia odnoĭ gruppy (PRDX1, Anneksin A1/A2). S ispol'zovaniem VÉZhKh-MS/MS mozhno identifitsirovat' potentsial'nye belki-markery, kharakternye dlia vospaleniia legkikh infektsionnogo (pnevmoniia) i neinfektsionnogo geneza (khronicheskaia obstruktivnaia bolezn' legkikh, KhOBL).

In recent decades, studies in the molecular origins of socially significant diseases have made a big step forward with the development and using of high-performance methods in genomics and proteomics. Numerous studies in the framework of the global program "Human Proteome" were aimed at the identification of all possible proteins in various cell cultures and tissues, including cancer. One of the objectives was to identify biomarkers - proteins with high specificity to certain pathologies. However, in many cases, it is shown that the development of the disease is not associated with the appearance of new proteins, but depends on the level of gene expression or forming of proteoforms - splice variants, single amino acid substitutions (SAP variants), and post-translational modifications (PTM) of proteins. PTM may play a key role in the development of pathology because they activate a variety of regulatory or structural proteins in the majority of cell physiological processes. Phosphorylation is among the most significant of these protein modifications.This review will describe methods for analysis of protein phosphorylation used in the studies of such diseases as cancer and neurodegenerative diseases, as well as examples of cases when the modified proteins are involved directly to their development, and screening such significant PTM is used for the diagnosis and choice of treatment.V poslednie desiatiletiia issledovaniia molekuliarnykh osnov razvitiia sotsial'no znachimykh zabolevaniĭ sdelali bol'shoĭ shag vpered blagodaria razvitiiu vysokoproizvoditel'nykh metodov genomiki i proteomiki. Mnogochislennye issledovaniia v ramkakh global'noĭ programmy “Proteom cheloveka” byli napravleny na identifikatsiiu vsekh vozmozhnykh belkov v razlichnykh kletochnykh kul'turakh i tkaniakh, v tom chisle i rakovykh. Odnoĭ iz tseleĭ bylo vyiavlenie tak nazyvaemykh biomarkerov – belkov, spetsificheskikh dlia opredelennykh patologiĭ. Odnako vo mnogikh sluchaiakh pokazano, chto razvitie zabolevaniia sviazano ne s poiavleniem novykh belkov, a zavisit ot urovnia ékspressii genov ili spetsificheskikh proteoform belkov – splaĭs-variantov, odinochnykh aminokislotnykh zamen (SAP) i post-transliatsionnykh modifikatsiĭ (PTM) belkov. PTM mogut igrat' kliuchevuiu rol' v razvitii patologii, potomu chto oni aktiviruiut razlichnye reguliatornye ili strukturnye belki v bol'shinstve kletochnykh fiziologicheskikh protsessov. Sredi takikh modifikatsiĭ naibolee znachimoĭ iavliaetsia fosforilirovanie. V dannom obzore rassmotreny metody analiza fosforilirovaniia, primeniaemye v issledovaniiakh molekuliarnykh osnov onkologicheskikh zabolevaniĭ, a takzhe privedeny primery, kogda modifitsirovannye belki vnosiat neposredstvennyĭ vklad v ikh razvitie. Skrining takikh znachimykh PTM primeniaetsia dlia diagnostiki i vybora metodov lecheniia.

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20–47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10–6 M (transthyretin, P02766) to 10–11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in Proteom...

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E0′ of −299 ± 16 mV and −297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

The facile synthesis of six [17(20) Z ]- and [17(20) E ]-isomeric 3β-hydroxy-pregna-5,17(20)-dien-21-oyl amides and three [17(20) E ]-3β-hydroxy-2-[prergna-5,17(20)-dien-20-yl]-oxazolines from pregnenolone is presented. The synthetic scheme consists of transformation of pregnenolone into the known 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one followed by reaction with ethanolamine, 2-methyl-2-aminopropanol, and (1-aminocyclohexyl)methanol resulted in mixture of [17(20) E ]- and [17(20) Z ]-pregna-5,17(20)-dien-21-(2-hydroxy)-oyl amides; separation of [17(20) E ]- and [17(20) Z ]-isomers; their cyclization into [17(20) E ]-oxazolines under action of POCl 3 in pyridine, and removal of acetate protecting groups. Significantly different orientation of nitrogen containing substituents in [17(20) Z ]- and [17(20) E ]-isomers regarding to steroid backbone enables their configuration to be easily identified by NMR spectroscopy. All synthesized compounds did not exhibit marked toxic effects in three cell lines (MCF-7, Hep G2, and LNCaP). In androgen-sensitive LNCaP cells all testing compounds at concentrations of 50 nM potently stimulated proliferation.

Reaction of 17alpha-bromo-21-iodo-3beta-acetoxypregn-5-en-20-one with ammonia, primary, and secondary amines is simple and convenient method for preparation of [17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-oylamides. Synthesis and characteristics of 12 related amides are presented. Primary testing on cells proliferation indicated differing effects of synthesized compounds on androgen insensitive MCF-7 cells and androgen sensitive LNCaP cells.

The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3β-yloxyethyl-) bound to 13(1)-, 15(2)-, 17(3)-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy.