14-Substituted Diquinothiazines as a New Group of Anticancer Agents.

A series of novel double-angularly condensed diquinothiazines with aminoalkyl, amidoalkyl, sulfonamidoalkyl, and substituted phenyl groups was designed, synthesized, and evaluated for their anticancer activity against four selected human tumor cell lines (HTC116, SH-SY5Y, A549, and H1299). The cytotoxicity of the novel diquinothiazines was investigated against BEAS-2B cells. The activities of the compounds were compared to etoposide. Among them, compounds with aminoalkyl and phenyl groups showed excellent broad-spectrum anticancer activity. The most active 14-(methylthiophenyl)diquinothiazine, 3c, showed low cytotoxicity against BEAS-2B cells and high activity against tumor cell lines HTC116, SH-SY5Y, A549, and H1299, with IC50 values of 2.3 µM, 2.7 µM, 17.2 µM, and 2.7 µM, respectively (etopiside 8.6 µM, 3.9 µM, 44.8 µM, and 0.6, respectively). Live long-term microscopic observations of cell survival using the starting molecule M0 were also performed. Flow cytometry showed the proapoptotic effects of the studied diquinothiazines. Inhibition of the cell cycle in the S phase was observed, which is associated with damage to nucleic acids and connected to DNA replication arrest.

Interaction of new tri-, tetra-, and pentacyclic azaphenothiazine derivatives with calf thymus DNA: Spectroscopic and molecular docking studies.

Azaphenothiazines (AZA), modified phenothiazine derivatives, have been reported to exhibit a wide spectrum of biological activities, including anticancer activities, but the mechanisms of their interactions with biomolecules are not fully recognized. In this work, the mode of interaction of selected AZA with calf thymus DNA was investigated using UV-Vis absorption, fluorescence spectroscopy (competition experiment with ethidium bromide, quenching of fluorescence) and molecular docking. The investigated AZA represent dipyrido[3,4-b;3'4'-e][1,4]thiazine, quino[3,2-b]benzo[1,4]thiazine and diquino[3,2-b;2',3'-e][1,4]thiazine possessing tricyclic, tetracyclic and pentacyclic ring system with the additional N,N-dimethylaminopropyl group at the nitrogen atom in the 1,4 thiazine ring. The results obtained from spectroscopic studies showed that AZA bind to DNA by insertion of a fragment of the fused rings system between the base pair stack in the double helix of DNA. In addition, the number of rings in the AZA structures seemed to be related to the strength of the interaction, because pentacyclic AZA (binding constant Kb = 6.31 × 106 L/mol) demonstrated 10-fold higher affinity towards DNA than the tetracyclic AZA and about 100-fold higher affinity than that of tricyclic AZA. The molecular docking results showed that the binding mode of AZA to DNA helix was an intercalation mode with the partial insertion of one planar part of the AZA structure (the pyridine or quinoline ring) into the neighboring bases of one of the DNA chains with additional hydrogen bonding with the minor groove through the positively charged N,N-dimethylaminopropyl group. Chemical potential (µ), chemical hardness (ƞ), electronegativity (χ) and the value of electrons transferred from one system to another (ΔN) calculated from the HOMO and LUMO energies by the density functional theory method indicated that AZA acted as the electron acceptors to the DNA bases.

Phenothiazines Modified with the Pyridine Ring as Promising Anticancer Agents.

Azaphenothiazines are the largest and most perspective group of modified phenothiazines, and they exhibit variety of biological activities. The review sums up the current knowledge on the anticancer activity of isomeric pyridobenzothiazines and dipyridothiazines, which are modified azaphenothiazines with one and two pyridine rings, respectively, against 10 types of cancer cell lines. Some 10-substituted dipyridothiazines and even 10-unsubstituted parent compounds, such as 10H-1,9-diazaphenothiazine and 10H-3,6-diazaphenothiazine, exhibited very potent action with the IC50 values less than 1 µg/mL and 1 µM against selected cancer cell lines. The strength of the anticancer action depends both on the tricyclic ring scaffolds and the substituents at the thiazine nitrogen atom. The review discusses the kind of the substituents, nature of tricyclic ring scaffolds with the location of the azine nitrogen atoms, the types of the cancer cell lines, and the mechanism of action.

Dual Action of Dipyridothiazine and Quinobenzothiazine Derivatives-Anticancer and Cholinesterase-Inhibiting Activity.

The inverse correlation observed between Alzheimer's disease (AD) and cancer has prompted us to look for cholinesterase-inhibiting activity in phenothiazine derivatives that possess anticancer properties. With the use of in silico and in vitro screening methods, our study found a new biological activity in anticancer polycyclic, tricyclic, and tetracyclic compounds. The virtual screening of a library of 120 ligands, which are the derivatives of azaphenothiazine, led to the identification of 25 compounds that can act as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Biological assays revealed the presence of selective inhibitors of eeAChE (electric eel AChE) or eqBuChE (equine serum BuChE) and nonselective inhibitors of both enzymes among the tested compounds. Their potencies against eeAChE were in a submicromolar-to-micromolar range with IC50 values from 0.78 to 19.32 µM, while their IC50 values against eqBuChE ranged from 0.46 to 10.38 µM. The most potent among the compounds tested was the tetracyclic derivative, 6-(4-diethylaminobut-2-ynyl)-9-methylthioquinobenzothiazine 24, which was capable of inhibiting both enzymes. 9-Fluoro-6-(1-piperidylethyl)quinobenzothiazine 23 was found to act as a selective inhibitor of eqBuChE with an IC50 value of 0.46 µM. Compounds with such a dual antitumor and cholinesterase-inhibitory activity can be considered as a valuable combination for the treatment of both cancer and AD prevention. The results presented in this study might open new directions of research on the group of tricyclic phenothiazine derivatives.

Therapeutic effects of an azaphenothiazine derivative in mouse experimental colitis.

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed.

Design, Synthesis, and Structural Characterization of Novel Diazaphenothiazines with 1,2,3-Triazole Substituents as Promising Antiproliferative Agents.

A series of novel 1,2,3-triazole-diazphenothiazine hybrids was designed, synthesized, and evaluated for anticancer activity against four selected human tumor cell lines (SNB-19, Caco-2, A549, and MDA-MB231). The majority of the synthesized compounds exhibited significant potent activity against the investigated cell lines. Among them, compounds 1d and 4c showed excellent broad spectrum anticancer activity, with IC50 values ranging from 0.25 to 4.66 µM and 0.25 to 6.25 µM, respectively. The most promising compound 1d, possessing low cytotoxicity against normal human fibroblasts NHFF, was used for gene expression analysis using reverse transcription-quantitative real-time PCR (RT-qPCR). The expression of H3, TP53, CDKN1A, BCL-2, and BAX genes revealed that these compounds inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BAX/BCL-2).

10H-1,9-diazaphenothiazine and its 10-derivatives: synthesis, characterisation and biological evaluation as potential anticancer agents.

10H-1,9-diazaphenothiazine was obtained in the sulphurisation reaction of diphenylamine with elemental sulphur and transformed into new 10-substituted derivatives, containing alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenothiazine ring system was identified with advanced 1H and 13C NMR techniques (COSY, NOESY, HSQC and HMBC) and confirmed by X-ray diffraction analysis of the methyl derivative. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB-231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza- and 3,6-diazaphenothiazine).

Evaluation of angularly condensed diquinothiazines as potential anticancer agents.

We present efficient synthesis of isomeric types of angularly fused diquinothiazines in the reactions of 2,2'-dichloro-3,3'-diquinolinyl disulfide and diquinodithiin with 3-, 5-, 6- and 8-aminoquinolines. The pentacyclic diquinothiazine ring systems were identified as diquino[3,2-b;3',4'-e][1,4]thiazine, diquino[3,2-b;5',6'-e][1,4]thiazine, diquino[3,2-b;6',5'-e][1,4]thiazine and diquino[3,2-b;8',7'-e][1,4]thiazine with advanced two-dimensional 1H and 13C NMR techniques (COSY, ROESY, HSQC and HMBC) of N-methyl derivatives. The identification of pentacyclic ring system was confirmed by X-ray diffraction analysis of selected N-alkyl derivatives. The X-ray analysis revealed different spatial structures of the ring system (planar and folded). NH-diquinothiazines were further transformed into N-alkyl and N-dialkylaminoalkyl derivatives. Most of diquinothiazines exhibited significant cancer cell growth inhibition against the human glioblastoma SNB-19, colorectal carcinoma Caco-2, breast cancer MDA-MB-231 and lung cancer A549 cell lines with the IC50 values < 3 µM. This anti-proliferative activity was found to be more than for cisplatin. The most promising compound, 7-dimethylaminopropyldiquino[3,2-b;6',5'-e]thiazine, was used for gene expression analysis by reverse transcription-quantitative real-time PCR (RT-QPCR) method. The expression of H3, TP53, CDKN1A, BCL-2 and BAX genes revealed that this compound inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BAX/BCL-2) in two cancer cell lines (SNB-19 and Caco-2).

Prolongation of skin graft survival in mice by an azaphenothiazine derivative.

Azaphenothiazines are predominantly immunosuppressive compounds. We evaluated the efficacy of an azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT) in prolongation of survival of skin allografts between BALB/c and C57Bl/6 mice. The mice were treated intraperitoneally (i.p.) with 100 µg of DQT on alternate days, on days 1-13 of the experiment (7 doses). The effect of DQT on a two-way mixed lymphocyte reaction (MLR) in the human model, as well as its effect on production of TNF α and IL-10 in a whole blood cell culture, stimulated by lipopolysaccharide (LPS), were evaluated. In addition, DQT effects were investigated regarding the proportion of T cell subsets in human peripheral blood lymphocytes (PBMC) by flow cytometry. Lastly, the effect of DQT on expression of signaling molecules involved in pro apoptotic pathways was determined by RT PCR. The results showed that DQT significantly extended skin graft survival. The compound also strongly suppressed two-way MLR in the human model at a concentration range of 2.5-5.0 µM. In addition, DQT inhibited LPS-inducible TNF α, but not IL-10 production. The compound preferentially caused a loss of the CD3-CD8+CD11b + PBMC cell subset, and transformed CD3+CD8+high into CD3+CD8+low cells. Lastly, we demonstrated significant increases in expression of caspases (in particular caspase 8) and of p53 in a culture of Jurkat T cells. We conclude that the immunosuppressive actions of the compound in allograft rejection may be predominantly associated with induction of cell apoptosis and inhibition of TNF α production. The apoptosis could be predominantly selective for the CD3-CD8+CD11b + cell phenotype.

Synthesis, Anticancer Activity, and Apoptosis Induction of Novel 3,6-Diazaphenothiazines.

New 10-substituted derivatives of 3,6-diazaphenothiazine, containing the triple bond linker terminated with tertiary cyclic and acyclic amine groups, were synthesized and screened for their anticancer action. The compounds exhibited varied anticancer activities against human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cell lines, depending on the nature of the substituents. The most active 3,6-diazaphenothiazine, 4, was the derivative with the N,N-diethylamino-2-butynyl substituent against glioblastoma SNB-19, and was ten times more potent than cisplatin. For this compound, the expression of H3, TP53, CDKN1A, BCL-2, and BAX genes was detected by the RT-qPCR method. The gene expression ratio BAX/BCL-2 indicated the induction of mitochondrial apoptosis in cancer cell lines. The transformation of the propynyl substituent into amino-2-butynyl can be a method applicable to the search for more anticancer-active azaphenothiazines.

An Anti-Inflammatory Azaphenothiazine Inhibits Interferon ß Expression and CXCL10 Production in KERTr Cells.

An azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT), has recently been shown to exhibit immunosuppressive activities in mouse models. It also inhibited the expression of CXCL10 at the protein level, at non-toxic concentrations, in the culture of KERTr cells treated with double-stranded RNA, poly(I:C). In this report, we demonstrated that DQT inhibits the transcription of the CXCL10 gene. Although CXCL10 is an IFNγ-inducible protein, we found that the CXCL10 protein was induced without the detectable release of IFNγ or IκB degradation. Hence, we concluded that IFNγ or NFκB was not involved in the regulation of the CXCL10 gene in KERTr cells transfected with poly(I:C), nor in the inhibitory activity of DQT. On the other hand, we found that IFNß was induced under the same conditions and that its expression was inhibited by DQT. Kinetic analysis showed that an increase in IFNß concentrations occurred 4⁻8 h after poly(I:C) treatment, while the concentration of CXCL10 was undetectable at that time and started to increase later, when IFNß reached high levels. Therefore, DQT may be regarded as a new promising inhibitor of IFNß expression and IFNß-dependent downstream genes and proteins, e.g., CXCL10 chemokine, which is implicated in the pathogenesis of autoimmune diseases.

Synthesis and anticancer activity of multisubstituted purines and xanthines with one or two propynylthio and aminobutynylthio groups.

A synthesis of new 2,6-disubstituted and 2,6,8-trisubstituted 7-methylpurines as well as 8-substituted 3,7-dimethylxanthines containing a triple bond chain have been worked out. Purinethiones and xanthinethiones were converted into propynylthio derivatives, which were then further transformed via a Mannich reaction into aminobutynylthio derivatives (amine = pyrrolidine, piperidine, morpholine, and diethylamine). The products thus obtained represent various types of the purine and xanthine structure: 8-mono-, 2,6- and 6,8-dipropynylthio, 6- and 8-monoaminobutynylthio, 2,6- and 6,8-diaminobutynylthio derivatives. All of these compounds were tested for their anticancer activity against human glioblastoma SNB-19, human adenocarcinoma MDA-MB-231, and melanoma C-32 cell lines. The anticancer activity depends on the nature of the substituent and its localization in the purine and xanthine framework. Generally, compounds possessing two alkynylthio groups (propynylthio or aminobutynylthio) were more active than those possessing only one group. Some compounds exhibited stronger or similar anticancer activity to cisplatin. All compounds were also tested for their cytotoxic activity against normal human fibroblasts (HFF-1). The most promising anticancer compounds were found to be 2,6-dipropynylthio-7-methylpurine 4, 2-chloro-6,8-dipropynylthio-7-methylpurine 14, and 2-chloro-6,8-di(N-morpholinylbutynylthio)-7-methylpurine 15c acting selectively on glioblastoma SNB-19, melanoma C-32, and adenocarcinoma MDA-MB-231 with the IC50 = 0.07-4.08 µg/mL.

Topically applied azaphenothiazines inhibit experimental psoriasis in mice.

The therapeutic efficacy of topically applied azaphenothiazine derivatives: 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5) in the amelioration of inflammatory symptoms of imiquimod-induced psoriasis in mice was investigated. Clobederm®, containing clobetasol propioniate, served as a reference drug. The application of the compounds led to thinning of the epidermis and reduction of the cell layers. The suppressive actions of the compounds were even stronger with regard to pathological changes of the dermis. The compounds also exerted generalized, anti-inflammatory effects by decreasing the number of circulating leukocytes, lowering subiliac lymph node weight and partially normalizing an altered blood cell composition. The changes in the composition of main cell types in the epidermis and dermis were less affected by the compounds. In addition, both compounds inhibited to a similar degree production of tumor necrosis factor α (TNF α) in human whole blood cell culture. Whereas compound 5 strongly inhibited IL-8 and CXCL10 chemokines in human keratinocytes - KERTr cell line, transfected with poly(I:C), the suppressive action of compound 4 in this model was weak. In addition, compound 5, but not compound 4, exhibited at low doses proapoptotic properties with regard to colonic cell lines. In summary, we demonstrated the therapeutic potential of two selected azaphenotiazines in the amelioration of the skin pathology elicited in a mouse experimental model of psoriasis.

Lipophilicity of New Anticancer 1,6- and 3,6-diazaphenothiazines by of Use RP TLC and Different Computational Methods.

The lipophilicity of new two series of anticancer active 10-substituted 1,6- and 3,6-diazaphenothiazines has been investigated using reversed-phase thin-layer chromatography. Their lipophilicity (RM0 and log PTLC) was determined with mixtures of acetone and Tris buffer as mobile phases. The relative lipophilicity parameter RM0 and specific hydrophobic surface area b were significantly intercorrelated showing congeneric classes of diazaphenothiazines. The parameter RM0 was transformed into parameter log PTLC by use of the calibration curve. The parameter log PTLC was compared with computationally calculated lipophilic parameters log Pcalcd. The lipophilicity was discussed with the structure elements and was correlated with molecular descriptors, ADME properties and in vitro anticancer activities.

Topically applied azaphenothiazines inhibit contact sensitivity to oxazolone in mice.

In this work we investigated the efficacy of two topically applied azaphenothiazine derivatives, 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5), in the amelioration of inflammatory symptoms of contact sensitivity (CS) to oxazolone in mice, in relation to the commercial ointment Protopic® (tacrolimus), the reference drug. The compounds were administered 24 h following elicitation of CS and, 24 h later, the parameters of inflammation, such as ear edema, blood composition, leukocyte level, numbers of cells in the draining lymph nodes, histological picture of the inflamed tissue, and the morphometric analysis, were analyzed. The study showed that the effectiveness of the studied azaphenothiazines applied as a 0.1% ointment was comparable to the reference drug regarding suppression of the inflammatory process, when all the investigated histological parameters are taken into account.

10H-3,6-Diazaphenothiazine induces G2/M phase cell cycle arrest and caspase-dependent apoptosis and inhibits cell invasion of A2780 ovarian carcinoma cells through the regulation of NF-κB and (BIRC6-XIAP) complexes.

The asymptomatic properties and high treatment resistance of ovarian cancer result in poor treatment outcomes and high mortality rates. Although the fundamental chemotherapy provides promising anticancer activities, it is associated with severe side effects. The derivative of phenothiazine, namely, 10H-3,6-diazaphenothiazine (PTZ), was synthesized and reported with ideal anticancer effects in a previous paper. In this study, detailed anticancer properties of PTZ was examined on A2780 ovarian cancer cells by investigating the cytotoxicity profiles, mechanism of apoptosis, and cell invasion. Research outcomes revealed PTZ-induced dose-dependent inhibition on A2780 cancer cells (IC50 =0.62 µM), with significant less cytotoxicity toward HEK293 normal kidney cells and H9C2 normal heart cells. Generation of reactive oxygen species (ROS) and polarization of mitochondrial membrane potential (ΔΨm) suggests PTZ-induced cell death through oxidative damage. The RT2 Profiler PCR Array on apoptosis pathway demonstrated PTZ-induced apoptosis via intrinsic (mitochondria-dependent) and extrinsic (cell death receptor-dependent) pathway. Inhibition of NF-κB and subsequent inhibition of (BIRC6-XIAP) complex activities reduced the invasion rate of A2780 cancer cells penetrating through the Matrigel™ Invasion Chamber. Lastly, the cell cycle analysis hypothesizes that the compound is cytostatic and significantly arrests cell proliferation at G2/M phase. Hence, the exploration of the underlying anticancer mechanism of PTZ suggested its usage as promising chemotherapeutic agent.

Azaphenothiazines - promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties.

For the last two decades, classical phenothiazines have attracted attention of researchers, as the hitherto investigations have revealed many significant biological activities within this class of compounds, other than originally discovered neuroleptic ones. Important, new pharmaceutical results on phenothiazines, as 10-substituted dibenzothiazines, were recently highlighted in several reviews. Azaphenothiazines are structurally modified phenothiazines by substitution of one or both benzene rings in the phenothiazine ring system with the azine rings, such as: pyridine, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, quinoline, quinoxaline, benzoxazine and benzothiazine. They form over 50 different heterocyclic systems, of tri-, tetra-, penta- and hexacyclic structures, and contain from one to even four azine nitrogen atoms. This review summarizes the methodical knowledge on azaphenothiazines, referring to their nomenclature, synthesis, structure analysis and above all significant varied biological activities, examined in vitro and in vivo. It describes, in addition, current trends in the synthesis of azaphenothiazines. The influence of the azaphenothiazine ring system, the nature of the substituents, predominantly at the thiazine nitrogen atom, as well as at the azine nitrogen atom and carbon atom, on the biological activities, were also discussed.

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.

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

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.

Selected azaphenothiazines inhibit delayed type hypersensitivity and carrageenan reaction in mice.

Several, previously selected azaphenothiazines, as strongly antiproliferative agents in in vitro models, were subjected to evaluation for their potential immunosuppressive effects in the model of delayed type hypersensitivity (DTH) to ovalbumin (OVA) in BALB/c mice and in foot pad inflammation induced by carrageenan in CBA mice. In the DTH model the compounds were given to mice intraperitoneally (i.p.) in 50µg or 250µg doses, 1h before the elicitation of the response. In the carrageenan-induced foot pad inflammation the compounds were given i.p. in 50µg or 250µg doses, 24h and 2h before administration of carrageenan. Among the compounds, the significantly suppressive activities in both models were exhibited only by compound 5 (6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine) and compound 4 (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine). Structure-activity relationship, plausible mechanism of action and potential application in therapy of the compounds are discussed.