Małgorzata Jeleń

Anticancer activity of newly synthesized azaphenothiazines from NCI’s anticancer screening bank#

The activity of the newly synthesized azaphenothiazines: tricyclic 10-substituted dipyridothiazines 1-9, pentacyclic 6-substituted diquinothiazines 10-22 and hexacyclic diquinothiazinium salt 23 was tested on 55-60 in vitro cell lines. The cell lines included nine types of cancer: leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer (National Cancer Institute, Bethesda, MD, USA). The features of the chemical substituent at the thiazine nitrogen atom confer the anticancer activity of diquinothiazines 10-23. Unexpectedly, the most active of the dipyridothiazines 1-9 was the unsubstituted compound 1 (the substituent is a hydrogen atom). The most cytotoxic compound was the half-mustard derivative 18. The GI(50) value of this compound was -7.06 (corresponding to 40 ng/ml) when tested on the melanoma cell line SK-MEL-5 and -6.0 - -6.62 using cell lines from various cancers including: leukemia (CCRF-CEM), the MOLT-4 cell line, colon cancer (HCT-116), central nervous system cancer (SNB-75 and SF-295), prostate cancer (PC-3), non-small cell lung cancer (NCI-H460 and HOP-92), ovarian cancer (IGROV1 and OVCAR-4) and breast cancer (MDA-MB-460). The ethylene group in the aminoalkylazaphenothiazines is as a good linker and is similar to the propylene and butylene linkers in aminoalkylphenothiazines. To our knowledge, this is the first demonstration of significant azaphenothiazine anticancer activity.

The immunosuppressive activities of newly synthesized azaphenothiazines in human and mouse models

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 μg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.

Synthesis and selected immunological properties of 10-substituted 1,8-diazaphenothiazines

A new type of tricyclic azaphenothiazines—1,8-diazaphenothiazines—was obtained in the reaction of 2,3- and 3,4-disubstituted pyridines. The reaction ran as the Smiles rearrangement. The 1,8-diazaphenothiazine system was determined using NOE experiment and 2D NMR spectra (COSY, HSQC, HMBC). 10H-1,8-diazaphenothiazine was transformed into 10-derivatives with alkyl, aminoalkyl, amidoalkyl, sulfonamidoalkyl, and nitrogen half-mustard groups. The compounds were tested for their effects on phytohemagglutinin A-induced proliferative response of human peripheral blood mononuclear cells (PBMC) and lipopolysaccharide-induced tumor necrosis factor alpha production by human whole blood cultures. The compounds exhibited differential, dose-dependent inhibitory activities in these tests. All the compounds were low toxic against PBMC. The compounds showing the highest antiproliferative activity strongly inhibited the growth of leukemia L-1210 and colon cancer SW-948 cell lines, similarly as cisplatin, a reference drug.

6-Substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines display strong antiproliferative and antitumor properties

6-Substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines - a new type of tetracyclic azaphenothiazines-were obtained from of 6H-9-fluoroquinobenzothiazine by the introduction of appropriate substituents to the thiazine nitrogen atom (alkyl, aminoalkyl, amidoalkyl, sulfonamidoalkyl and nitrogen half-mustard groups). The compounds displayed differential cytotoxic as well as antiproliferative actions against human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin A (PHA). In addition, they suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production by whole blood human cell cultures. Two compounds (4 and 15, with the propargyl and methanesulfonamidopropyl groups) were selected for further experiments because of lack of cytotoxicity and strong antiproliferative actions. Compound 4 showed strong suppressive actions on growth of L1210, SW948, A-431 and CX-1 tumor cell lines which were close to those of cisplatin, the reference drug (e.g. GI50 of 2.28 μg/mL vs. 1.86 μg/mL for L1210 cells). Further, the compound appeared to be equally effective as cyclosporine A (CsA) in the inhibition of human two-way mixed lymphocyte reaction (MLR). The compound did not significantly inhibit interleukin 2 (IL-2)-induced growth of CTLL-2 cell line. In addition, inhibition of prostaglandin (PG) synthesis by indomethacin or block of PG receptors did not interfere with the inhibitory effect of the compound on PHA-induced cell proliferation. Therefore, it is likely that the compound acts by inhibiting cell cycle as proposed for other phenothiazines. Further studies are required for the elucidation of the mechanism of action and therapeutic utility of these compounds in more advanced in vivo models.

DETERMINATION OF THE LIPOPHILICITY PARAMETERS LOG P CALCD, RM0 AND LOG P TLC OF NEW ANTICANCER ACYLAMINOALKYL- AND SULFONYLAMINOALKYLAZAPHENOTHIAZINES BY COMPUTATIONAL METHODS AND REVERSED-PHASE THIN-LAYER CHROMATOGRAPHY

The lipophilicity parameters of twenty-two new acylaminoalkyl- and sulfonylaminoalkyl-azaphenothiazines of two types (dipyridothiazines A1-A11 and diquinothiazines B1-B11 ) were determined theoretically using 11 computational methods and experimentally by reversed-phase thin-layer chromatography. The theoretical log P calcd values differed dramatically depending on the calculating programs. The experimental R M values were linearly dependent on the concentration of acetone in the mobile phase and extrapolated to 0% of acetone gave the lipophilicity parameter R M0, which was further transformed into the parameter log P TLC by use of a calibration curve. The parameter R M0 and specific hydrophobic surface area b were significantly intercorrelated showing congeneric classes of azaphenothiazines. The parameter R M0 was correlated with molecular descriptors (molar mass, molar volume and molar refractivity). Diquinothiazines B1-B11 were much more lipophilic than dipyridothiazines A1-A11 by 2.75–4.02 in logarithmic units. The theoretical log P calcd values were compared with experimental log P TLC values showing low prediction power of calculated programs. The determined parameters were discussed in the terms of the structure-lipophilicity relationships.

Thin-layer chromatographic detection of new azaphenothiazines

Many new phenothiazines exhibit promising anticancer and antibacterial activities, reversal of multidrug resistance and potential treatment in Alzheimers, Creutzfeldt-Jakob and AIDS diseases. Their synthesis may proceed through a stage of the Smiles rearrangement and may lead to different products: cyclic phenothiazines (rearranged or not), cyclic side-products of isosteric structures and non-cyclic products when the ring-closure processes did not occur. The TLC method was found suitable for detection of new modified phenothiazines (being tri-, tetra- and pentacyclic azaphenothiazines with hydrogen, alkyl, dialkylaminoalkyl, aryl and heteroaryl substituents at the thiazine nitrogen atom and in a few cases additional substituents in the benzene ring) during their synthesis. The natural fluorescence of phenothiazines and azaphenothiazines under irradiation of UV light of 365nm is very characteristic and becomes useful additional analytical information of these compounds. The spots of azaphenothiazines were also distinguished from the spots of substrates and side-products by giving color reactions with the visualizing reagents: sulfuric acid in ethanol, concentrated nitric acid and citric acid in acetic anhydride. The combination of the separation (the R(F) values) and the spot color detection (as the native fluorescence and the results of the usage of visualizing reagents) facilitated the identifications of new azaphenothiazines in the reaction mixtures containing also other compounds. This paper is the first attempt of the determination of new azaphenothiazines by TLC method preceding the identification by spectroscopic methods. It facilitates the separation of the proper fraction in column chromatography and preparative TLC.

Synthesis and anticancer and lipophilic properties of 10-dialkylaminobutynyl derivatives of 1,8- and 2,7-diazaphenothiazines

Abstract New derivatives of two isomeric types of azaphenothiazines, 1,8- and 2,7-diazaphenothiazine, containing the triple bond substituents and additionally tertiary cyclic and acyclic amine groups, were synthesized and tested for their anticancer activity. The compounds exhibited differential inhibitory activities. Better results were obtained when the acetylenic group was transformed via the Mannich reaction to the dialkylaminobutynyl groups. The most active was 2,7-diazaphenothiazine with the N-methylpiperazine-2-butynyl substituent against the human ductal breast epithelial tumor cell line T47D, more potent than cisplatin. The 2,7-diazaphenothiazine system turned out to be more active than isomeric 1,8-diaza one. For the most active compound, the expression of TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The gene expression ratio BACL-2/BAX suggests the mitochondrial apoptosis in T47D cells. The synthesis makes possible to obtain many new bioactive phenothiazines with the dialkylaminoalkynyl substituents inserting various tertiary cyclic and acyclic amine moieties to the substituents.

DETERMINATION OF THE LIPOPHILICITY PARAMETERS OF NEW ANTIPROLIFERATIVE 8-10-SUBSTITUTED QUINOBENZOTHIAZINES BY COMPUTATIONAL METHODS AND RP TLC

The lipophilicity parameters of fourteen new antiproliferative 8-10-substituted 6H-quinobenzothiazines were determined theoretically using 9 computational methods and experimentally by reversed-phase thin-layer chromatography on the RP-18 silica plates with acetone-aqueous TRIS buffer as the mobile phase. The experimental R M values were linearly dependent on the concentration of acetone in the mobile phase and extrapolated to 0% of acetone that produced the lipophilicity parameter R M0. The parameter R M0 and specific hydrophobic surface area b were significantly intercorrelated showing congeneric classes of quinobenzothiazines. The parameter R M0 was correlated with molecular descriptors (molar mass, molar volume, and molar refractivity), predicted and tested biological activities, and was further transformed into parameter log PTLC by use of the calibration curve. The theoretical log P calcd values differed depending on the calculating programs and were compared with experimental log PTLC values showing different prediction powers of calculated programs. Tetracyclic 6H-quinobenzothiazine was as lipophilic as tricyclic 10H-phenothiazine. The determined parameters are discussed in the terms of the structure-lipophilicity relationships.

Synthesis, spectroscopic characterization, and anticancer activity of new 10-substituted 1,6-diazaphenothiazines.

New phenothiazine derivatives as 10-substituted dipyridothiazines of the 1,6-diazaphenothiazine structure were obtained in the cyclization reaction of 3-amino-3′-nitro-2,2′-dipyridinyl sulfide and 3,3′-dinitro-2,2′-dipyridinyl disulfide, and in the reaction of 2-chloro-3-ntropyridine with sodium 3-amino-2-pyridinethiolate followed by various alkylation and arylation reactions. The reaction of the thiazine ring formation ran via the Smiles rearrangement of the S-N type. As the alkylation reactions could proceed at the thiazine, azine or both nitrogen atoms, the product structure elucidation was based on the 2D NMR (Rotating-frame Overhauser Effect Spectroscopy, Correlated Spectroscopy, Heteronuclear Single Quantum Coherence, and Heteronuclear Multiple Bond Correlation) spectra of the N-methylated product. Some 10-substituted 1,6-diazaphenothizines (5, 10, 12, 13) were at least anticancer active against melanoma C-32 and breast cancer MCF-7 cell lines as a reference drug – cisplatin. The monoazaphenothiazine drug, prothipendyl, turned out to be less active than least 6 derivatives of the 1,6-diazaphenothiazine structure.

Discovery of butyrylcholinesterase inhibitors among derivatives of azaphenothiazines

Abstract The study presents the discovery of novel butyrylcholinesterase (BuChE) inhibitors among derivatives of azaphenothiazines by application of in silico and in vitro screening methods. From an in-house library of compounds, 143 heterocyclic molecules derived from the azaphenothiazine scaffold were chosen for virtual screening. Based on results of the docking procedure, 15 compounds were identified as exhibiting the best fit for the two screening complexes (ligand – AChE and ligand – BuChE). Five compounds displayed moderate AChE and good BuChE inhibitory activity at screening concentrations of 10 µM. The IC50 values for active BuChE inhibitors were in the 11.8–122.2 nM range. Three of the most active inhibitors are tetra- or pentacyclic derivatives of azaphenothiazines with the same N-methyl-2-piperidinethyl substituent.