Bioorganic Chemistry 87 (2019) 810–820

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Bioorganic Chemistry

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Evaluation of angularly condensed diquinothiazines as potential anticancer T agents ⁎ Małgorzata Jeleńa, Krystian Plutaa, , Małgorzata Latochab, Beata Morak-Młodawskaa, Kinga Suwińskac,d, Dariusz Kuśmierzb a The Medical University of Silesia, School of Pharmacy with the Division of Laboratory Medicine, Department of Organic Chemistry, Jagiellońska 4, 41-200 Sosnowiec, Poland b The Medical University of Silesia, School of Pharmacy with the Division of Laboratory Medicine, Department of Cell Biology, Jedności 8, 41-200 Sosnowiec, Poland c Faculty of Mathematics and Natural Sciences, Cardinal Stefan Wyszyński University, K. Wóycickiego 1/3, 01-938 Warszawa, Poland d A.M. Butlerov Institute of Chemistry, Kazan Federal University, Kremlevskaya ul. 18, Kazan 420008, Russia

ARTICLE INFO ABSTRACT

Keywords: We present efficient synthesis of isomeric types of angularly fused diquinothiazines in the reactions of2,2′- Phenothiazines dichloro-3,3′-diquinolinyl disulfide and diquinodithiin with 3-, 5-, 6- and 8-aminoquinolines. The pentacyclic Azaphenothiazines 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 ring formation 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-dimen- Anti-proliferative activity sional 1H and 13C NMR techniques (COSY, ROESY, HSQC and HMBC) of N-methyl derivatives. The identification Gene expression analysis of pentacyclic ring system was confirmed by X-ray diffraction analysis of selected N-alkyl derivatives. The X-ray 2D NMR correlation X-ray analysis 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 sig- nificant 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).

1. Introduction novel scaffolds in medicinal chemistry [6,7]. One of the most bioactive heterocyclic rings is a 6-membered 1,4-thiazine ring, containing the Cancer is a major public health problem all over the world and one nitrogen and sulfur atoms [8]. When this ring is condensed with two of the leading causes of death worldwide affecting millions of people benzene rings on both sides, it forms a tricyclic dibenzothiazine ring every year. The main forms of a curative treatment for tumors are system called phenothiazine. Compounds with this system possessing surgery, radiation, chemotherapy, hormone therapy, immune therapy, the dialkylaminoalkyl groups at the nitrogen atom (and additional and targeted therapy. The goal of current chemotherapy is to cure the simple group at the carbon atom in position 2) are also called phe- tumor, to prolong survival and to reduce the tumor burden to alleviate nothiazines and are recognized for 5 decades as the neuroleptic, anti- symptoms. A lot of effort has been applied to the synthesis ofnew histaminic, antitussive and antiemetic drugs [9]. chemotherapeutic agents/potential anticancer drugs with better se- Antipsychotic phenothiazines are low toxic, inexpensive and rela- lectivity and minor side effects but the results still do not meet ex- tively easy to obtain. They have been used as good targets for drug pectations [1–5]. repurposing [5]. Thioridazine, one of the most known phenothiazines, Bioactive heterocyclic compounds of natural and synthetic origin exhibits significant properties for multidrug-resistant tuberculosis, contain various heterocyclic ring systems (differing in the ring size and methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-re- the kind of heteroatoms) play an important role for the development of sistant enterococci [5,10–12] and lung cancer therapy through

⁎ Corresponding author at: The Medical University of Silesia, Department of Organic Chemistry, Jagiellońska 4, 41-200 Sosnowiec, Poland. E-mail address: [email protected] (K. Pluta). https://doi.org/10.1016/j.bioorg.2019.04.005 Received 30 January 2019; Received in revised form 28 March 2019; Accepted 5 April 2019 Available online 06 April 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved. M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820 targeting lung cancer stem cells, and is both efficient and safe [13]. dialkylaminoalkyl substituents at the thiazine nitrogen atom, their The other way to explore the phenothiazine scaffold is a modifica- structural analysis, and the evaluation of their anti-proliferative activity tion of its structure towards novel phenothiazines with new bioactivity. against cancer cells. It can be achieved mainly by introduction of new substituents at the thiazine nitrogen atom (many intermediates are commercially available 2. Results and discussion as 10H-phenothiazines) and by replacement of one or two benzene rings with various azine rings (to form varied azaphenothiazines). 2.1. Synthesis Numerous original reports, reviews and chapters in monographs de- scribe new promising biological activity of modified phenothiazines For the synthesis of new diquinothiazines, 2,2′-dichloro-3,3′-diqui- such as anticancer, antiviral, anti-inflammatory, immunosupressive, nolinyl disulfide 3 and diquinodithiin 4 (Scheme 1) were used as antibacterial, and reversal of multi-drug resistance [14–19]. They are starting materials. Those compounds reacted with 3-, 5-, 6- and 8- promising candidates for further studies in the treatment of Creutzfeldt- aminoquinolines in boiling monomethyl ether of diethylene glycol (a Jakob’s, Alzheimer’s, Parkinson’s and Huntington’s diseases [20,21]. very effective solvent, high boiling and miscible with water [33]) or The quinoline scaffold is prevalent in a variety of pharmacologically with their hydrochlorides (without a solvent) at 200–205 °C. In the case active synthetic and natural compounds [22]. Quinoline compounds of diquinodithiin 4, the reaction runs as the thiazine ring opening at the have been known for almost 400 years as alkaloids of antimalarial ac- 2-quinolinyl carbon atom (in position 4a and 5a) which is susceptible tivity (quinine, quinidine) [23]. Synthetical quinoline derivatives based for nucleophilic substitution. Both substrates lead to diquinolinyl on the aminoquinoline moiety (chloroquine, amodiaquine, primaquine) amines 5, 8, 10 and 13 (not isolated) which cyclized to NH-diqui- are recognized antimalarial drugs [22,24]. Fluoroquinolones (cipro- nothiazines 6H or/and 7H, 9H, 11H or/and 12H and 14H depending floxacine and ofloxacine) are one of the most promising and widely on the direction of the thiazine ring closure. Only one product was used in contemporary anti-infective chemotherapy [25,26]. Other qui- observed in each case. The reactions of disulfide 3 were more effective noline compounds exhibit anticancer, antimycobacterial, antic- than those of dithiin 4. The microwave-assisted synthesis using dis- onvulsant, anti-inflammatory, cardiovascular and antileishmanial ac- ulfide 3 shortened the reaction time from 3 h (for classical synthesis) to tivities [24]. Recent studies have shown that quinoline compounds 0.5 h giving NH-diquinothiazines with better yield (from a few to over a derived from substitution of the quinoline ring and fusion this ring with dozen percent). other heterocyclic rings display potent anticancer activity targeting After the structure analysis, NH-diquinothiazines were further different sites like topoisomerase I, telomerase, farnesyl transferase, transformed into N-substituted derivatives (6a-i, 9a, 11a-h and 14a-g), tyrosine kinase, tubulin polymerization and protein kinase CK-II possessing the methyl, allyl, propynyl and varied dialkylaminoalkyl [24,27]. groups in the reactions with appropriate alkyl halides in various con- One type of modified phenothiazines are azaphenothiazines, where ditions (Scheme 2). In the case of 14H-diquinothiazine 9H an in- the thiazine ring is fused with an azine ring. The fusion with one or two troduction of other groups than the methyl was unsuccessful, probably quinoline moiety instead of the benzene rings leads to the formation of due to steric hindrance. The choice of those substituents was dictated quinobenzothiazines and diquinothiazines. Selected substituted quino- by spectroscopic structural analysis (the methyl group) and their benzothiazines 1a-b and diquinothiazines 2a-b (Fig. 1) exhibit sig- pharmacophoric activity found in other azaphenothiazines [19,28,32]. nificant anticancer activity against tens of cancer cells derived from leukemia, melanoma, non-small cell lung, colon, CNS, ovarian, renal, prostate, breast and skin cancers [28–32]. These compounds show also 2.2. Spectroscopic structural analysis promising antioxidant activity on rat hepatic microsomal membranes for protection of non-enzymatic lipid peroxidation [33], inhibitory ac- It is well known that the synthesis of phenothiazines from the tivity of mitogen-induced proliferation of human peripheral blood substrates possessing CeS bond and an amino group can proceed via the mononuclear cells, tumor necrosis factor alpha (TNFα) production in Smiles rearrangement of the SeN type, which can lead to other than human whole blood cultures [29,31,32], and against butyr- expected product, to its isomer [39,40]. The primary NH-diquinothia- ylcholinesterase [34]. They exerted suppressive activity in in vivo zines were methylated with methyl iodide to give appropriate N-methyl models of: delayed-type hypersensitivity to ovalbumin and cutaneous derivatives. The identification of the product structures was based on carrageenan reaction [35], contact sensitivity to oxazolone [36] and two-dimensional spectroscopic NMR (ROESY, COSY, HSQC and HMBC) experimental psoriasis in mice [37], and exhibited inhibitory activity of analysis [41] of these compounds. IFNβ expression and IFNβ-dependent downstream genes and proteins The ROESY experiment in CDCl3 with irradiation of the methyl involved in the pathogenesis of autoimmune diseases [38]. protons at 3.67–4.15 ppm showed spatial proximity of these protons Those diquinothiazines are structurally isomeric azaphenothiazines with the closest other proton (Fig. 2). with condensed ring systems where the quinoline ring is fused with the In the case of compound 14a, the irradiation of the methyl protons 1,4-thiazine ring along the quinoline bonds: C2-C3 (linearly condensed at 4.15 ppm resulted in a lack of the 1H–1H connectivity because the system) and C3-C4 (angularly condensed system). Both systems are nearest spatial atoms were two azine nitrogens. Such a negative result symmetrical with the plane of symmetry along the SeN atoms. confirmed the structure of the fused ring system as diquino[3,2-b;8′,7′- Theoretically, the quinoline ring can form various condensed ring sys- e][1,4]thiazine. In other cases the nearest atoms were one proton and tems with the thiazine ring depending on the fused places. The aim of one azine nitrogen. this paper is to present synthesis of new type of isomeric diquinothia- The irradiation of the methyl protons in compound 9a at 3.95 ppm zines of the unsymmetrical structure possessing alkyl and showed the 1H–1H connectivity with the proton doublet at 8.48 ppm. As the most deshielded proton in quinolone compounds α-quinoline

Fig. 1. Known quinobenzothiazines and diquinothiazines.

811 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

Scheme 1. Synthesis of NH-diqunothiazines.

Scheme 2. Synthesis of N-substituted diquinothiazines.

812 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

Fig. 2. The examples of 2D NMR experiments (ROESY, COSY, HSQC and HMBC) for diquinothiazines.

proton (H2quinoline [42]) (at 8.85 ppm in 9a), the proton signal at confirmed the structures concluded from NMR analysis as mono- 8.48 ppm has to be attributed to γ-quinoline proton (H4quinoline) and angularly fused in the L-shape and showed different spatial arrangement identified as H-1 proton of diquinothiazine moiety. The experiment of the pentacyclic ring system. The pentacyclic ring system in com- confirmed the product structure as 14-methyldiquino[3,2-b;5′,6′-e] pound 6d is almost planar with dihedral angles between the two halves [1,4]thiazine. of the thiazine ring equal to 170.54(6)° and between the quinoline rings The irradiation of the methyl protons at 3.78 pm in the product is equal to 172.63(2)°). The phenyl ring of the benzyl group is per- derived from the reaction of 3-aminoquinoline (6a or 7a) resulted in pendicular to the pentacyclic ring system. Compound 14a crystalized the connectivity with the proton singlet at 8.63 ppm, characteristic for with two symmetry independent molecules in the unit cell. The mole-

α-quinoline proton (H2quinoline). This signal was identified as derived cules are folded along the N···S axis with the dihedral angles of from H-6 proton in the diquinothiazine system. The experiment ex- 141.9(1)/146.7(1)° and 147.0(1)/154.7(1)° between the two halves of cluded linearly fused structure 6a and pointed at the structure as 7- the thiazine ring and between the quinoline rings, respectively. The methyldiquino[3,2-b;3′,4′-e][1,4]thiazine 7a. central thiazine ring in both diquinothiazines adopts the boat con- The irradiation of the methyl protons at 3.67 pm in the product formation with the substituent at N-atom in equatorial position with the derived from the reaction of 6-aminoquinoline (11a or 12a) showed the S···N–Cx (Cx = CH2 or CH3) angle of 177.3(1)° in compound 6d and connectivity with a doublet signal at 7.43 ppm with ortho-coupling of 151.9(2)/151.4(2)° in compound 14a. J = 9.0 Hz. This signal was found as derived from H-6 proton and The CeNeC and CeSeC bond angles in the thiazine ring are, re- therefore the linear structure 12a was excluded. The methylated pro- spectively, 123.6(1) and 101.88(7)° in compound 6d, and are larger duct was identified as 7-methyldiquino[3,2-b;6′,5′-e][1,4]thiazine 11a. than these found in compound 14a (120.4(2)/121.7(2)° and 99.7(1)/ The full proton signal assignments in those N-methyl derivatives 100.5(1)°, respectively), which allows the adoption of an almost flat (7a, 9a, 11a and 14a) were achieved by study other proton spatial conformation of the pentacyclic ring system. proximity in ROESY spectra and 1H–1H connectivities in COSY spectra. In both compounds only weak CeH···N and π···π intermolecular in- The confirmation of the proton assignments came from the 13C NMR teractions were found. spectra which were solved by the use of HSQC and HMBC spectra in- dicating the 13C–1H relationship. The HSQC spectra showed which 2.4. Anti-proliferative activity proton was bonded to the carbon atom (the CeH relationship through 1 one bond, JC,H connectivity) and the HMBC spectrum indicated the 3 Selected diquinothiazines (Table 1) were screened for their anti- CeH relationship through three (predominantly, JC,H, connectivity) 2 proliferative activity against in vitro using cultured glioblastoma SNB- and exceptionally two bonds ( JC,H connectivity) (see Fig. 3). 19, colorectal carcinoma Caco-2, breast cancer MDA-MB-231 and lung cancer A549 cell lines and cisplatin as a reference drug. Most of the 2.3. X-ray structural analysis compounds were very active at least against one cancer cell line with

the IC50 value < 3 µM being more active than cisplatin. Only diqui- X-ray analysis of compounds not only confirms the structure eluci- nothiazines 6b, 6c and 14H were weakly active or inactive. Even NH- dated from NMR analysis but also showed their spatial arrangement in a diquinothiazines 6H, 9H and 11H, which did not possess a substituent solid state. The X-ray structure of known diquinothiazines (linearly and at the thiazine nitrogen atom, exhibited good activity against some double angularly fused in the C-shape) revealed both planar and folded cancer cells. The most tested diquinothiazines showed their higher ac- pentacyclic ring system, and equatorial or axial positions of the sub- tivity against lung cancer A549 cell lines. Diquinothiazines with the stituents at the thiazine nitrogen atoms as a result of the steric inter- dialkylaminoalkyl groups were more active than those with the alkyl actions between the substituents and the hydrogen atoms and the groups with the exception of diquinothiazine 11c possessing the pro- nature of the substituents (electron-donating or electron-accepting) pynyl group. Compounds 6e-g, 6i, 11d-f, 11h, 14c, and 14f were very [43–47]. active against all cancer cells. The most active were dimethylamino- Not all of the obtained N-methyl derivatives provided good quality propyldiquinothiazine 6f against A549 cell line and pyrrolidiny- monocrystals for X-ray analysis but only compound 14a. Therefore, lethyldiquinothiazine 11f against SNB-19 cell line with the IC50 value of other alkyl derivatives were involved to grow crystals and only benzyl 0.3 µM (See Table 2). derivative 6d was found to give suitable crystals. Both compounds The location of the substituent in the pentacyclic ring system seems

813 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

Fig. 3. Molecular structures of 6d (left) and 14a (right) compounds. Top: views shoving the fused rings with atom labeling scheme; down: side views down the C···N direction of the thiazine ring showing the difference in folding the fused ring system. to be important as 7-substituted diquinothiazines (6 and 11) were more transcription, apoptosis, DNA repair, as well as cell motility. The ex- active than 14-substituted ones (9 and 14). In the former case the pression of the p21 protein was analyzed as a biomarker of the cell substituent is exo-orientated and in the last case is endo-orientated and response to different toxic stimuli [52,53]. less accessible for interaction with a biological target. Compound 11f induced meaningly a decrease in the expression of Cytotoxicity of diquinothiazines were tested against normal human CDKN1A in MDA-MB-231, A549 and SNB-19 cancer lines. The P53 dermal fibroblasts NHDF. Most active compounds were a few times less protein can also stimulate a cell to changes in gene expression of an- active to normal cells than to cancer cells. tiapoptotic BCL-2 and proapoptotic BAX involved in the mitochondrial pathway apoptosis [54–57]. Compound 11f reduced remarkably the expression of BCL-2 in A549 and SNB-19 cancer lines (in two other lines 2.5. Apoptosis assay to a lesser degree) and the expression of BAX in the A549 cancer cell lines (less in SNB-19 and MDA-MB 231 lines). 7-Dimethylaminopropyldiquinothiazine 11f was selected to study The ratio of antagonistic BAX/BCL-2 is associated with cell apop- the mechanism of anti-proliferative action using RT-QPCR method. This tosis or progression. In comparison with the control, the BAX/BCL-2 method measures the gene transcriptional activities of proliferation ratio was found to be greater for compound 11f inducing mitochondrial marker (H3), cell cycle regulator (TP53 and CDKNIA) and intracellular apoptosis in SNB-19 and Caco-2 cell lines. Two other cell lines, A549 apoptosis pathway (BACL-2 and BAX). and MDA-MB-231, were resistant for apoptosis and compound 11f in- Hundreds of genes working together control the growth, division duces the cell death in other way. and eventual death of the cells in the body. The gene encoding the histone H3, which is involved in the cell cycle progression, is con- sidered an indicator of proliferation in molecular studies. It plays a 3. Conclusion crucial role in regulation of the expression of the genetic information encoded in DNA [48,49]. Compound 11f decreased significantly the We report here efficient synthesis of new isomeric types of angularly number of mRNA copies in all cancer lines. Such a reduced action can fused 7H- and 14H-diquinothiazines in the reactions of 2,2′-dichloro- be the result of a modification of the chromatin structure. 3,3′-diquinolinyl disulfide and diquinodithiin with 3-, 5-, 6- and8- Cellular stresses such as the DNA damage, oncogene activation, aminoquinolines. The pentacyclic diquinothiazine ring system was spindle damage and hypoxia can change the expression of the TP53 identified as diquino[3,2-b;3′,4′-e][1,4]thiazine, diquino[3,2-b;5′,6′-e] gene encoding the p53 protein, which is considered the “guardian of the [,4]thiazine, diquino[3,2-b;6′,5′-e][1,4]thiazine and diquino[3,2- genome”. Activated p53 transactivates a number of target genes, many b;8′,7′-e][1,4]thiazine using advanced two-dimensional 1H and 13C of which are involved in the DNA repair, cell cycle arrest and apoptosis NMR (COSY, ROESY, HSQC and HMBC) spectra of N-methyl deriva- [50,51]. Compound 11f reduced considerably the number of mRNA tives. The identification of the way of pentacyclic ring fusion was copies in the A549, SNB-19 and MDA-MB-231 cancer lines. confirmed by X-ray diffraction analysis of selected N-alkyl derivatives. The p53 protein influences cell cycle arrest by changing theex- The X-ray analysis revealed different spatial structures of pentacyclic pression of CDKN1A gene encoding the p21 protein. The main function ring system (planar and folded). NH-diquinothiazines were further of the p21 protein is to arrest cell cycle progression by inhibiting the transformed into N-alkyl and N-dialkylaminoalkyl derivatives. Most of activity of cyclin-dependent kinases. This protein regulates also the the obtained diquinothiazines exhibited significant anti-proliferative

814 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

Table 1 method. The expression of H3, TP53, CDKN1A, BCL-2 and BAX genes The anti-proliferative activity of diquinothiazines against glioblastoma SNB-19, revealed that this compound inhibited the proliferation in all cells (H3) colorectal carcinoma Caco-2, breast cancer MDA-MB-231 and lung cancer A549 and activated mitochondrial events of apoptosis (BAX/BCL-2) in two cell lines, and normal fibroblasts NHDF. cancer cell lines (SNB-19 and Caco-2 cell lines).

Nr IC50 (µM) 4. Experimental SNB-19 Caco-2 MDA-MB- A549 NHDF 231 Melting points were determined in open capillary tubes on a Boetius 6H 21.4 64.0 93.6 19.9 331.8 melting point apparatus and were uncorrected. The NMR spectra were 6b 61.9 279.8 177.6 122.7 281.8 recorded on Bruker Fourier 300 and Bruker Avance spectrometers (1H 13 6c 90.6 176.4 87.6 83.6 194.3 at 300 and 600 MHz, C at 75 MHz) in CDCl3. Two-dimensional 6e 2.0 5.2 1.9 0.4 17.7 NOESY, HSQC and HMBC spectra of compounds 6a, 9a, 11a and 14a were recorded on a Bruker Avance spectrometer at 600 MHz. Fast Atom 6f 1.8 1.3 3.4 0.3 2.6 Bombardment (FAB MS) mass spectra were run on a Finnigan MAT 95 spectrometer at 70 eV, HR MS mass spectra were run on a Impact II 6g 1.2 1.9 1.6 1.1 13.8 spectrometer.

6h 3.6 12.7 2.3 1.5 14.3 4.1. Synthesis

6i 2.2 1.4 2.1 1.6 8.8 4.1.1. General procedure for the synthesis of 7H- and 14H-diquinothiazines (6H, 9H, 11H, 14H) from diquino-1,4-dithiin (4) Diquinodithiin 4 (0.16 g, 0.5 mmol) was finely powdered together with 3-, 5-, 6- or 8-aminoquinoline hydrochloride (0.46 g, 2.5 mmol) on an oil bath at 200–205 °C for 4 h. After cooling, the solution was poured 9H 22.5 16.5 24.0 24.2 118.3 into water (10 mL) and alkalized with 5% aqueous sodium hydroxide to pH 10. The resulting solid was filtered off, washed with water, and

purified by column chromatography (aluminum oxide,3 CHCl ) to give diquinothiazine.

11H 13.0 2.2 12.1 20.8 33.5 11b 105.6 73.1 87.8 95.5 215.7 4.1.1.1. 7H-diquino[3,2-b;3′,4′-e]thiazine (6H). Yield 0.17 g, 40%, mp 1 11c 1.7 2.6 25.5 2.7 25.0 240–241 °C. H NMR (DMSO‑d6) δ: 7.26 (t, J = 8.4 Hz, 1H, H-11), 7.49 11d 0.9 1.3 6.0 1.7 4.2 (d, J = 7.6 Hz, 2H, H-12, H-10), 7.61 (m, 4H, H-1, H-2, H-3, H-9), 7.87 (d, J = 7.8 Hz, 1H, H-4,), 7.92 (s, 1H, H-13), 8.45 (s, 1H, H-6). EI MS + 11e 1.2 2.0 2.6 1.7 7.7 m/z: 301 (M , 100), 269 (M−S, 25). HR MS m/z: 302.3728, calcd 302.3730. 11f 0.3 1.8 0.9 1.1 4.3 4.1.1.2. 14H-diquino[3,2-b;5′,6′-e]thiazine (9H). Yield 0.08 g, 27%, 11g 14.6 9.4 22.3 2.2 18.6 1 mp. 210–211ÂC° . H NMR (CDCl3) δ: 7.25 (d, J = 8.4 Hz, 1H, H-2), 7.36 (t, J = 7.3 Hz, 1H, H-10), 7.51 (d, J = 7.2 Hz, 1H, H-6), 7.52 (t, 11h 1.9 2.1 9.6 1.6 5.7 J = 7.2 Hz, 1H, H-11), 7.56 (m, 2H, H-9, H-12), 7.61 (s, 1H, H-8), 7.65 (d, J = 8.4 Hz, 1H, H-5), 7.76 (d, J = 8.6 Hz, 1H, H-1), 8.89 (d, J = 3.6 Hz, 1H, H-3). EI MS m/z: 301 (M+, 100), 269 (M−S, 30). HR MS m/z: 302.3726, calcd 302.3730.

4.1.1.3. 7H-diquino[3,2-b;6′,5′-e]thiazine (11H). Yield 0.10 g, 33%, mp 14H 201.0 216.3 331.8 249.2 331.8 14b 27.9 52.4 23.8 17.9 154.3 260–261 °C, ref [33] mp 260–261 °C. 14c 2.3 1.7 2.1 2.1 12.4 4.1.1.4. 14H-diquino[3,2-b;8′,7′-e]thiazine (14H). (0.10 g, 33%), mp. 1 14d 11.1 11.8 13.1 7.1 11.3 185–186 °C. H NMR (CDCl3) δ: 7.07 (d, J = 8.4 Hz, 1H, H-6), 7.7 (t, J = 7.5 Hz, 1H, H-10), 7.31 (d, J = 8.6 Hz, 1H, H-5), 7.38 (m, 1H, H-3), 14e 8.9 2.7 3.6 1.7 2.0 7.46 (d, J = 8.4 Hz, 1H, H-9), 7.50 (t, J = 8.6 Hz, 1H, H-11), 7.52 (s, 1H, H-8), 7.67 (d, J = 7.8 Hz, 1H, H-12), 8.03 (d, J = 7.6 Hz, 1H, H-4), + 14f 1.8 5.0 2.2 2.3 3.6 8.82 (d, J = 2.4 Hz, 1H, H-2). EI MS m/z: 301 (M , 100), 269 (M−S, 35). HR MS m/z: 302.3728, calcd 302.3734. 14g 14.1 13.0 10.8 13.5 16.5 4.1.2. General procedure for the synthesis of 7H- and 14H-diquinothiazines (6H, 9H, 11H, 14H) from from 2,2′-dichloro-3,3′-diquinolinyl disulfide cisplatin 29.2 17.9 2.7 9.3 225.4 (3) A solution of disulfide 3 (0.20 g, 0.5 mmol) with 3-, 5-, 6- or 8- aminoquinoline (0.22 g, 1.5 mmola) in monomethyl ether of diethylene glycol (MEDG) (5 mL) was refluxed for 3 h. After cooling, the solution activities against the human glioblastoma SNB-19, colorectal carcinoma was poured into water (20 mL) and alkalized with 5% aqueous sodium Caco-2, breast cancer MDA-MB-231 and lung cancer A549 cell lines hydroxide to pH 10. The resulting solid was filtered off, washed with with the IC50 values < 1 µg/mL and were more potent than cisplatin. water, and purified by column chromatography (aluminum oxide,

The most promising compound, 7-dimethylaminopropyldiquino[3,2- CHCl3) to give diquinothiazine. b;6′,5′-e]thiazine, was used for gene expression analysis by RT-QPCR a. 7H-diquino[3,2-b;3′,4′-e]thiazine (6H). Yield 0.17 g, 57%.

815 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

Table 2 The influence of compound 11f on the expression of genes encoding: H3, TP53, CDKN1A, BCL-2, BAX in the cancer cell lines.

Number of mRNA copies / µg total RNA

Gene A549 Caco-2 SNB-19 MDA-MB-231

H3 Control 119933 ± 12200 124653 ± 41211 424545 ± 69325 415337 ± 147036 11f 1032 ± 396 24120 ± 1056 35535 ± 5156 102568 ± 8572 P53 Control 165582 ± 62819 615738 ± 189545 673583 ± 186284 474781 ± 49655 11f 11921 ± 4551 512396 ± 64934 108577 ± 7947 126517 ± 23266 P21 Control 470585 ± 18029 353788 ± 97698 1233007 ± 129287 659408 ± 137062 11f 49884 ± 12351 326461 ± 64428 576439 ± 38045 71935 ± 38747 BCL-2 Control 99058 ± 20212 48297 ± 5165 170083 ± 22035 568878 ± 35743 11f 2660 ± 224 17819 ± 2978 13748 ± 3070 193931 ± 77547 BAX Control 39317 ± 12314 267782 ± 68738 320419 ± 96634 85989 ± 24400 11f 419 ± 90 263259 ± 5757 98830 ± 7832 41299 ± 20504 BAX/BCL-2 Control 0.40 5.54 1.88 0.15 11f 0.16 14.77 7.19 0.21

+ b. 14H-diquino[3,2-b;5′,6′-e]thiazine (9H). Yield 0.16 g, 53%. (C-8a), 152.73 (C-7a). EI MS m/z: 315 (M , 100), 300 (M−CH3, 15). c. 7H-diquino[3,2-b;6′,5′-e]thiazine (11H). Yield 0.20 g, 67%. HR MS m/z: 316.0908, calcd 316.0908. d. 14H-diquino[3,2-b;8′,7′-e]thiazine (14H). Yield 0.14 g, 47%. 4.1.4.2. 14-Methyldiquino[3,2-b;5′,6′-e]thiazine (9a). Yield 0.13 g, 1 4.1.3. General procedure for the microwave assisted synthesis of 7H- and 82%, mp 159–160ÂC° . H NMR (CDCl3) δ: 3.95 (s, 3H, CH3), 7.34 (t, 14H-diquinothiazines (6H, 9H, 11H, 14H) from from 2,2′-dichloro-3,3′- J = 7.5 Hz, 1H, H-10), 7.40 (m, 1H, H-2), 7.45 (d, J = 8.4 Hz, 1H, H-6), diquinolinyl disulfide (3) 7.58 (t, J = 7.8 Hz, 1H, H-11), 7.59 (d, J = 7.2 Hz, 1H, H-9), 7.72 (s, A sealed 10 mL glass tube containing a solution of disulfide 3 1H, H-8), 7.83 (d, J = 8.4 Hz, 1H, H-5), 7.84 (d, J = 8.4 Hz, 1H-12), (0.20 g, 0.5 mmol) and 3-, 5-, 6- or 8-aminoquinoline (0.22 g, 1.5 mmol) 8.48 (d, J = 8.4 Hz, 1H, H-1), 8.85 (dd, J = 4.2 Hz, J = 1.8 Hz, 1H, H- 13 in monomethyl ether of diethylene glycol (MEDG) (5 mL) was in- 3). C NMR (CDCl3) δ: 42.07 (CH3), 120.09 (C-2), 120.99 (C-7a), troduced in the cavity of a microwave reactor (CEM Co., Discover) and 122.26 (C-14b), 124.19 (C-6a), 124.73 (C-10), 125.18 (C-5), 126.55 (C- irradiated at 200 °C for 30 min under magnetic stirring. The reaction 9), 126.60 (C-8a), 127.40 (C-12), 127.97 (C-6), 129.53 (C-11), 132.08 mixture was poured into water (20 mL) and alkalized with 5% aqueous (C-8), 132.49 (C-1), 138.54 (C-14a), 145.72 (C-12a), 148.66 (C-4a), + sodium hydroxide to pH 10. The resulting solid was filtered off, washed 149.21 (C-3), 156.90 (C-13a). EI MS m/z: 315 (M , 100), 300 with water, and purified by column chromatography (aluminum oxide, (M−CH3, 35). HR MS m/z: 316.0902, calcd 316.0908.

CHCl3) to give diquinothiazine. a. 7H-diquino[3,2-b;3′,4′-e]thiazine (6H). Yield 0.20 g, 67%. 4.1.4.3. 7-Methyldiquino[3,2-b;6′,5′-e]thiazine (11a). Yield 0.14 g, 1 b. 14H-diquino[3,2-b;5′,6′-e]thiazine (9H). Yield 0.18 g, 60%. 88%, mp 189–190 °C. H NMR (CDCl3) δ: 3.67 (s, 3H, CH3), 7.23 (t, c. 7H-diquino[3,2-b;6′,5′-e]thiazine (11H). Yield 0.21 g, 70%. J = 8.7 Hz, 1H, H-11), 7.37 (t, J = 6.3 Hz, 1H, H-2), 7.43 (d, d. 14H-diquino[3,2-b;8′,7′-e]thiazine (14H). Yield 0.19 g, 63%. J = 8.6 Hz, 1H, H-6), 7.47 (t, J = 8.7 Hz, 1H, H-10), 7.49 (d, J = 8.4 Hz, 1H, H-12), 7.70 (s, 1H, H-13), 7.71 (d, J = 8.4 Hz, 1H, H- 4.1.4. General procedure for the synthesis of 7- and 14- 9), 7.95 (d, J = 8.6 Hz, 1H, H-5), 8.34 (d, J = 8.4 Hz, 1H, H-1), 8.74 (d, 13 alkyldiquinothiazines (6a-b, 6d, 9a, 11a-b and 14a-b) J = 4.2 Hz, 1H, H-3). C MNR (CDCl3) δ: 34.35 (CH3), 113.78 (C-14a), To a solution of diquino[1,4]thiazine 6H, 9H, 11H or 14H (0.15 g, 117.64 (C-13a), 119.65 (C-6), 121.56 (C-2), 124.47 (C-11), 125.81 (C- 0.5 mmol) in dry DMF (5 mL) NaH (0.12 g, 5 mmol, 60% NaH in mi- 14b), 126.21 (C-12a), 126.42 (C-12), 127.51 (C-9), 128.71 (C-5), neral oil was washed out with hexane) was added. The reaction mixture 129.49 (C-10), 131.72 (C-1), 132.73 (C-13), 141.22 (C-6a), 144.58 was stirred at room temperature for 1 h, alkyl halide (methyl iodide, (C-4a), 145.99 (C-8a), 148.41 (C-3), 154.10 (C-7a). EI MS m/z: 315 + allyl bromide, benzyl chloride 1.5 mmol) was added and the stirring (M , 100), 300 (M−CH3, 10). HR MS m/z: 316.0900, calcd 316.0908. was continued for 24 h. The reaction mixture was poured into water (25 mL). The resulting solid was filtered off, washed with water and 4.1.4.4. 14-Methyldiquino[3,2-b;8′,7′-e]thiazine (14a). Yield 0.11 g, 1 purified by column chromatography (aluminum oxide, CHCl3) to give: 70%, mp 262–263 °C. H NMR (CDCl3) δ: 4.15 (s, 3H, CH3), 7.28 (d, methyl-, allyl- and benzyldiquinothiazines. J = 8.4 Hz, 1H, H-6), 7.29 (t, J = 6.6 Hz, 1H, H-10), 7.31 (m, 1H, H-3), 7.44 (d, J = 8.4 Hz, 1H, H-5), 7.54 (t, J = 7.8 Hz, 1H, H-11), 7.55 (d, 4.1.4.1. 7-Methyldiquino[3,2-b;3′,4′-e]thiazine (6a). Yield 0.14 g, 88%, J = 6.9 Hz, 1H, H-9), 7.68 (s, 1H, H-8), 7.89 (d, J = 8.4 Hz, 1H, H-12), 1 mp 204–205 °C. H NMR (CDCl3) δ: 3.78 (s, 3H, CH3), 7.33 (t, 8.03 (d, J = 8.4 Hz, 1H, H-4), 8.85 (dd, J = 4.2 Hz, J = 1.8 Hz, 1H, H- 13 J = 7.2 Hz, 1H, H-11), 7.56 (d, J = 8.4 Hz, 1H, H-12), 7.57 (t, 2). C MNR (CDCl3) δ: 41.78 (CH3), 120.53 (C-7a), 120.66 (C-3), J = 7.2 Hz, 1H, H-10), 7.60 (t, J = 7.5 Hz, 1H, H-2), 7.64 (t, 123.12 (C-5), 124.15 (C-10), 124.89 (C-6a), 125.26 (C-6), 126.36 (C-9), J = 7.5 Hz, 1H, H-3), 7.73 (s, 1H, H-13), 7.79 (d, J = 8.4 Hz, 1H, H- 126.38 (C-8a), 127.52 (C-12), 129.16 (C-4a), 129.27 (C-11), 131.83 (C- 9), 7.94 (d, J = 8.4 Hz, 1H, H-1), 8.06 (d, J = 8.4 Hz, 1H, H-4), 8.63 (s, 8), 135.93 (C-4), 139.20 (C-14a), 141.65 (C-14b), 145.91 (C-12a), 13 + 1H, H-6). C NMR (CDCl3) δ: 33.57 (CH3), 116.09 (C-13a), 122.72 (C- 148.23 (C-2), 157.07 (C-13a). EI MS m/z: 301 (M , 100), 269 (M−S, 1), 124.67 (C-11), 124.78 (C-14b), 126,08 (C-14a, C-12a), 126.43 (C- 35). HR MS m/z: 316.0910, calcd 316.0908. 10), 127.49 (C-9), 127.58 (C-2), 128.15 (C-3), 129.25 (C-4), 129.76 (C- 12), 132.94 (C-13), 135.65 (C-6a), 138.40 (C-6), 143.63 (C-4a), 146.03 4.1.4.5. 7-Allyldiquino[3,2-b;3′,4′-e]thiazine (6b). Yield 0.10 g, 68%,

816 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

1 mp 190–192 °C. H NMR (CDCl3) δ: 5.05 (m, 2H, CH2), 5.39 (m, 2H, filtered off washed with water, air-dried and purified by column chro- CH2), 6.18 (m, 1H, CH), 7.29 (t, J = 8.1 Hz, 1H, H-11), 7.57 (m, 4H, H- matography (aluminium oxide, CHCl3) to give dialkylaminoalk- 12, H-10, H-2, H-9), 7.70 (s, 1H, H-13), 7.71 (t, J = 8.7 Hz, 1H, H-3), yldiquinothiazines. 7.89 (d, J = 7.8 Hz, 1H, H-13), 7,99 (d, J = 7.8 Hz, 1H, H-4), 8.66 (s, 1H, H-6). HR MS m/z: 342.1062, calcd 342.1065. 4.1.6.1. 7-(2′-Diethylaminoethyl)diquino[3,2-b;3′,4′-e]thiazine 1 (6e). Yield 0.12 g, 60%, an oil. H NMR (CDCl3) δ: 1.19 (t, J = 7.2 Hz, 4.1.4.6. 7-Allyldiquino[3,2-b;6′,5′-e]thiazine (11b). Yield 0.11 g, 75%, 6H, 2CH3), 2.77 (m, 4H, 2CH2), 3.04 (m, 2H, CH2), 4.53 (m, 2H, CH2), 1 mp 109–110 °C. H NMR (CDCl3) δ: 5.04 (m, 2H, CH2), 5.37 (m, 2H, 7.27 (t, J = 7.8 Hz, 1H, H-11), 7.55 (t, J = 7.2 Hz, 1H, H-2), 7.58 (d, CH2), 6.20 (m, 1H, CH), 7.29 (t, J = 7.4 Hz, 1H, H-11), 7.42 (m, 1H, H- J = 8.4 Hz, 1H, H-9), 7.65 (s,1H, H-13), 7.78 (t, J = 7.2 Hz,1H, H-3), 6), 7.53 (m, 3H, H-2, H-10, H-12), 7.74 (s, 1H, H-13), 7.75 (d, 7.85 (d, J = 8.4 Hz, 1H, H-1), 7.92 (m, 2H, H-12, H-10), 8.04 (d, J = 8.8 Hz, 1H, H-9), 7.92 (d, J = 8.6 Hz, 1H, H-5), 7.36 (d, J = 8.4 Hz, 1H,H-4), 8.75 (s, 1H, H-6). HR MS m/z: 401.1813, calcd J = 8.4 Hz, 1H, H-1), 8.81 (d, J = 4.4 Hz, 1H, H-3). HR MS m/z: 401.1800. 342.1066, calcd 342.1065. 4.1.6.2. 7-(2′-Diethylaminoethyl)diquino[3,2-b;6′,5′-e]thiazine 1 4.1.4.7. 14-Allyldiquino[3,2-b;8′,7′-e]thiazine (14b). Yield 0.11 g, 75%, (11d). Yield 0.13 g, 65%, an oil. H NMR (CDCl3) δ: 1.16 (t, 1 an oil. H NMR (CDCl3) δ: 4.95 (m, 1H,CH2), 5.37 (m, 1H, CH2), 5.74 J = 7.2 Hz, 6H, 2CH3), 2.72 (m, 4H, 2CH2), 2.98 (t, J = 7.5 Hz, 2H, (m, 2H, CH2), 5.93 (t, J = 4.5 Hz, 1H, CH2), 7.32 (m, 3H, H-3, H-6, H- CH2), 4.41 (m, 2H, CH2), 7.25 (t, J = 8.6 Hz, 1H, H-11), 7.35 (m, 1H, H- 10), 7.48 (d, J = 8.4 Hz, 1H, H-5), 7.54 (t, J = 7.2 Hz, 1H, H-11), 7.56 6), 7.49 (m, 2H, H-2, H-10), 7.55 (d, J = 8.4 Hz, 1H, H-12), 7.61 (s, 1H, (d, J = 7.0 Hz, 1H, H-9), 7.75 (s, 1H, H-8), 7.86 (d, J = 8.4 Hz, 1H, H- H-13), 7.69 (d, J = 8.4 Hz, 1H, H-9), 7.91 (d, J = 8.6 Hz, 1H, H-5), 8.26 12), 8.06 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H, H-4), 8.88 (dd, J = 6.0 Hz, (d, J = 8.4 Hz, 1H, H-1), 8.77 (d, J = 4.2 Hz, 1H, H-3). HR MS m/z: J = 1.8 Hz, 1H, H-2). HR MS m/z: 342.1064, calcd 342.1065. 401.1805, calcd 401.1800.

4.1.4.8. 7-Benzyldiquino[3,2-b;3′,4′-e]thiazine (6d). Yield 0.16 g, 82%, 4.1.6.3. 14-(2′-Diethylaminoethyl)diquino[3,2-b;8′,7′-e]thiazine 1 1 mp 208–209 °C. H NMR (CDCl3) δ: 5.72 (s, 2H, CH2), 7.32 (m, 4H, H- (14c). Yield 0.12 g, 60%, an oil. H NMR (CDCl3) δ: 0.93 (t, 6H, 2CH3, 11, C6H3), 7.43 (d, J = 7.2 Hz, 2H, C6H2), 7.52 (t, J = 7.6 Hz, 1H, H- J = 7.2 Hz), 2.54 (q, 4H, 2CH2), 2.75 (t, J = 7.4 Hz, 2H, CH2), 5.14 (t, 10), 7.58 (d, J = 8.6 Hz, 1H, H-12), 7.61 (m, 2H, H-2, H-3), 7.67 (d, J = 7.4 Hz, 2H, CH2), 7.27 (d, J = 8.4 Hz, 1H, H-6), 7.28 (t, J = 6.8 Hz, J = 8.4 Hz, 1H, H-9), 7.77 (s, 1H, H-13), 7.93 (d, J = 8.4 Hz, 1H-1), 1H, H-10), 7.31 (m, 1H, H-3), 7.44 (d, J = 8.4 Hz, 1H, H-5), 7.53 (t, 8.01 (d, J = 8.4 Hz, 1H, H-4), 8.85 (s, 1H, H-6). HR MS m/z: 392.1216, J = 7.2 Hz, 1H, H-11), 7.54 (d, J = 7.0 Hz, 1H, H-9), 7.70 (s, 1H, H-8), calcd 392.1221. 7.84 (d, J = 8.4 Hz, 1H, H-12), 8.03 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H, H- 4), 8.86 (dd, J = 4.8 Hz, J = 1.8 Hz, 1H, H-2). HR MS m/z: 401.1806, 4.1.5. General procedure for the synthesis of synthesis of calcd 401.1800. propargyldiquinothiazines (6c and 11c) To a solution of diquinobenzothiazine 6a or 11a (0.15 g, 0.5 mmol) 4.1.6.4. 7-(3′-Dimethylaminopropyl)diquino[3,2-b;3′,4′-e]thiazine 1 in dry DMF (5 mL), potassium tert-butoxide (0.08 g, 0.72 mmol) was (6f). Yield 0.12 g, 63%, mp 130–131 °C. H NMR (CDCl3) δ: 2,21 (m, added. The mixture was stirred at room temperature for 1 h. Then to the 2H, CH2), 2,41 (s, 6H, 2CH3), 2,66 (m, 2H, CH2), 4.43 (m, 2H, CH2), solution, a solution of propargyl bromide 0.08 g (0.64 mmol) in toluene 7.29 (t, J = 7.5 Hz, 1H, H-11), 7.54 (m, 2H, H-12, H-9), 7.56 (t, was added dropwise. The resulted solution was stirred at room tem- J = 7.2 Hz, 1H, H-2), 7.60 (t, J = 7.2 Hz, 1H, H-10), 7.66 (s, 1H, H-13), perature for 24 h and poured into water (20 mL), extracted with me- 7.72 (d, J = 8.4 Hz, 1H, H-1), 7.88 (t, J = 7.2 Hz, 1H, H-3), 7.99 (d, thylene chloride (20 mL), dried with Na2SO4, evaporated to a brown oil. J = 8.4 Hz, 1H, H-4), 8.65 (s, 1H, H-6). HR MS m/z: 387.1655, calcd The residue was purified by column chromatography (silica gel, CHCl3) 387.1643. to give propargyl derivative. 4.1.6.5. 7-(3′-Dimethylaminopropyl)diquino[3,2-b;6′,5′-e]thiazine 1 4.1.5.1. 7-Propargyldiquino[3,2-b;3′,4′-e]thiazine (6c). Yield 0.11 g, (11e). Yield 0.13 g, 67%, an oil. H NMR (CDCl3) δ: 2.14 (m, 2H, CH2), 1 75%, mp 264–265 °C. H NMR (CDCl3) δ: 2.41 (t, J = 2.4 Hz, 1H, 2.33 (s, 6H, 2CH3), 2.56 (m, 2H, CH2), 4.42 (m, 2H, CH2), 7.31 (t, CH), 5.12 (d, J = 2.4 Hz, 2H, CH2), 7.40 (t, J = 7.6 Hz, 1H, H-11), 7.63 J = 8.4 Hz, 1H, H-11), 7.42 (m, 1H, H-6), 7.55 (m, 3H, H-2, H-10, H- (m, 2H, H-12, H-10), 7.81 (m, 5H, H-1, H-2, H-3, H-9, H-13), 8.04 (d, 12), 7.72 (s, 1H, H-13), 7.75 (d, J = 8.4 Hz, 1H, H-9), 7.96 (d, J = 7.8 Hz, 1H, H-4), 8.71 (s, 1H, H-6). HR MS m/z: 340.0899, calcd J = 8.4 Hz, 1H, H-5), 8.35 (d, J = 8.2 Hz, 1H, H-1), 8.81 (d, 340.0908. J = 4.2 Hz, 1H, H-3). HR MS m/z: 387.1640, calcd 387.1643.

4.1.5.2. 7-Propargyldiquino[3,2-b;6′,5′-e]thiazine (11c). Yield 0.13 g, 4.1.6.6. 14-(3′-Dimethylaminopropyl)diquino[3,2-b;8′,7′-e]thiazine 1 1 77%), mp 228–229 °C. H NMR (CDCl3) δ: 2.36 (t, J = 4.5 Hz, 1H, (14d). Yield 0.13 g, 68%, an oil. H NMR (CDCl3) δ: 1.79 (m, 2H, CH2), CH), 5.09 (d, J = 4.8 Hz, 1H, CH2), 7.32 (t, J = 8.6 Hz, 1H, H-11), 7.44 2.11 (s, 6H, CH3), 2.41 (t, J = 7.2 Hz, 2H, CH2), 5.03 (t, J = 7.2 Hz, 2H, (m, 1H, H-6), 7.56 (m, 2H, H-2, H-12), 7.82 (m, 3H, H-9, H-10, H-13), CH3), 7.18 (m, 3H, H-3, H-6, H-10), 7.46 (d, J = 8.4 Hz, 1H, H-5), 7.52 8.04 (d, J = 8.6 Hz, 1H, H-5), 8.39 (d, J = 8.4 Hz, 1H, H-1), 8.84 (d, (t, J = 7.2 Hz, 1H, H-11), 7.55 (d, J = 7.2 Hz, 1H, H-9), 7.73 (s, 1H, H- J = 4.4 Hz, 1H, H-3). HR MS m/z: 340.0911, calcd 340.0908. 8), 7.85 (d, J = 8.4 Hz, 1H, H-12), 8.04 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H, H-4), 8.86 (dd, J = 6.0 Hz, J = 1.8 Hz, 1H, H-2). HR MS m/z: 387.1633, 4.1.6. General procedure for the synthesis of 7- and 14- calcd 787.1643. dialkylaminoalkyldiquinothiazines (6e-i, 11d-h and 14c-g) A mixture of diquinothiazine 6a, 11a or 14a (0.15 g, 0,5 mmol), 4.1.6.7. 7-(1′-Pyrrolidinylethyl)diquino[3,2-b;3′,4′-e]thiazine 1 sodium hydroxide (0.30 g, 7,5 mmol) and hydrochloride of dialkyla- (6 g). Yield 0.14 g, 70%, an oil. H NMR (CDCl3) δ: 1.94 (m, 4H, 2CH2), minoalkyl chloride (1,5 mmol, 2-diethylaminoethyl – 0.26 g, 3-di- 2.90 (m, 4H, 2CH2), 3.11 (m, 2H, CH2), 4.64 (m, 2H, CH2), 7.30 (t, methylaminopropyl – 0.24 g, 2-(1-pyrrolidinyl)ethyl – 0.26 g, 2-(1-pi- J = 8.4 Hz, 1H, H-11), 7.55 (m, 3H, H-2, H-10, H-12), 7.59 (t, peridinyl)ethyl 0.28 g, 2-(1′-methyl-2′-piperydinyl)ethyl – 0.30 g) in J = 7.2 Hz, 1H, H-3), 7.67 (s, 1H, H-13), 7.79 (d, J = 8.4 Hz, 1H, H- dry dioxane (5 mL) was refluxed for 3 h. After cooling the reaction 9), 7.87 (d, J = 8.4 Hz, 1H, H-1), 8.00 (d, J = 8.4 Hz, 1H, H-4), 8.77 (s, mixture was poured into water (50 mL) and the resulted solid was 1H, H-6). HR MS m/z: 399.1642, calcd 399.1643.

817 M. Jeleń, et al. Bioorganic Chemistry 87 (2019) 810–820

4.1.6.8. 7-(1′-Pyrrolidinylethyl)diquino[3,2-b;6′,5′-e]thiazine 1H, H-11), 7.56 (d, J = 7.2 Hz, 1H, H-9), 7.74 (s, 1H, H-8), 7.85 (d, 1 (11f). Yield 0.14 g, 70%, an oil. H NMR (CDCl3) δ: 1.88 (m, 4H, J = 8.4 Hz, 1H, H-12), 8.06 (dd, J = 8.2 Hz, J = 1.8 Hz, 1H, H-4), 8.87 2CH2), 2.77 (m, 4H, 2CH2), 3.07 (m, 2H, CH2), 4.58 (m, 2H, CH2), 7.29 (dd, J = 6.4 Hz, J = 1.8 Hz, 1H, H-2). HR MS m/z: 427.1958, calcd (t, J = 8.6 Hz, 1H, H-11), 7.41 (m, 1H, H-6), 7.53 (m, 2H, H-2, H-10), 427.1956. 7.60 (d, J = 8.4 Hz, 1H, H-12), 7.70 (s, 1H, H-13), 7.75 (d, J = 8.4 Hz, 1H, H-9), 7.96 (d, J = 8.4 Hz, 1H, H-5), 8.33 (d, J = 8.4 Hz, 1H, H-1), 4.2. Crystal structure determination 8.81 (d, J = 4.6 Hz, 1H, H-3). HR MS m/z: 399.1638, calcd 399.1643. The single-crystal X-ray diffraction study was carried out ona 4.1.6.9. 14-(1′-Pyrrolidinylethyl)diquino[3,2-b;8′,7′-e]thiazine Bruker D8 Venture Duo diffractometer at 150(2) K using CuKα radia- 1 (14e). Yield 0.13 g, 65%, an oil. H NMR (CDCl3) δ: 1.70 (m, 4H, tion with λ = 1.54184 Å. The supplementary crystallographic data have 2CH2), 2.56 (m, 4H, 2CH2), 2.81 (t, J = 7.2 Hz, 2H, CH2), 5.22 (t, been deposited at the Cambridge Crystallographic Data Centre (CCDC), J = 7.2 Hz, 2H, CH3), 7.64 (m, 2H H-6, H-10), 7.31 (m, 1H, H-3), 7.45 deposition CCDC 1,887,513 (compound 6d) and CCDC 1,887,514 (d, J = 8.2 Hz, 1H, H-5), 7.53 (t, J = 7.2 Hz, 1H, H-11), 7.54 (d, (compound 14a). These data can be obtained free of charge from CCDC J = 7.4 Hz, 1H, H-9), 7.71 (s, 1H, H-8), 7.84 (d, J = 8.2 Hz, 1H, H- via www.ccdc.cam.ac.uk/data_request/cif. 12), 8.03 (dd, J = 8.2 Hz, J = 1.2 Hz, 1H, H-4), 8.86 (dd, J = 6.4 Hz, J = 1.8 Hz, 1H, H-2). HR MS: m/z: 399.1639, calcd 399.1643. 4.2.1. Crystal structure determination of 7-benzyldiquino[3,2-b;3′,4′-e] thiazine (6d) 4.1.6.10. 7-(1′-Piperidinylethyl)diquino[3,2-b;3′,4′-e]thiazine C25H17N3S, M = 391.47, orange-brown block, 0.13 × 0.22 × 1 (6h). Yield 0.13 g, 63%, an oil. H NMR (CDCl3) δ: 1.27 (m, 2H, CH2), 0.35 mm, triclinic, space group P1, a = 9.729(1), b = 10.962(1), 1.70 (m, 4H, 2CH2), 2.69 (m, 4H, 2CH2), 2.97 (m, 2H, CH2), 4.56 (m, c = 10.9824(1) Å, α = 103.534(4), β = 114.195(4), γ = 108.515(3)°, 3 −3 2H, CH2), 7.29 (t, J = 8.4 Hz, 1H, H-11), 7.56 (m, 3H, H-2, H-10, H-12), V = 918.9(2) Å , Z = 2, Dc = 1.415 g·cm , F000 = 408, 2θmax = 7.58 (t, J = 7.4 Hz, 1H, H-3), 7.70 (s, 1H, H-13), 7.72 (d, J = 8.4 Hz, 68.41°, 22,741 reflections collected, 3347 unique (Rint = 0.034). Final 1H, H-9), 7.88 (d, J = 8.4 Hz, 1H, H-1), 8.00 (d, J = 8.4 Hz, 1H, H-4), GooF = 1.04, R = 0.035, wR = 0.096, R indices based on 3004 reflec- 2 8.78 (s, 1H, H-6). HR MS m/z: 413.1797, calcd 413.1800. tions with I > 2σ(I) (refinement on F ), 262 parameters, 0 restraints. Lp and absorption corrections applied, µ = 1.69 mm−1. 4.1.6.11. 7-(1′-Piperidinylethyl)diquino[3,2-b;6′,5′-e]thiazine 1 (11g). Yield 0.14 g, 68%, an oil. H NMR (CDCl3) δ: 1.49 (m, 2H, CH2), 4.2.2. Crystal structure determination of 14-methyldiquino[3,2-b;8′,7′-e] 1.67 (m, 4H, 2CH2), 2.60 (m, 4H, 2CH2), 2.88 (m, 2H, CH2), 4.50 (m, thiazine (14a) 2H, CH2), 7.26 (t, J = 8.4 Hz, 1H, H-11), 7.37 (m, 1H, H-6), 7.50 (m, C19H13N3S, M = 315.38, yellow needle, 0.02 × 0.08 × 0.33 mm, 2H, H-2, H-10), 7.60 (d, J = 8.4 Hz, 1H, H-12), 7.65 (s, 1H, H-13), 7.72 triclinic, space group P1, a = 3.8855(4), b = 20.376(2), c = 20.703(2) 3 (d, J = 8.4 Hz, 1H, H-9), 7.93 (d, J = 8.4 Hz, 1H, H-5), 8.29 (d, Å, α = 61.871(5), β = 85.691(6), γ = 89.738(6)°, V = 1440.6(3) Å , −3 J = 8.6 Hz, 1H, H-1), 8.79 (d, 1H, H-3). HR MS m/z: 413.1793, calcd Z = 4, Dc = 1.454 g·cm , F000 = 656, 2θmax = 63.86°, 35,404 reflec- 413.1800. tions collected, 4747 unique (Rint = 0.069). Final GooF = 1.046, R = 0.045, wR = 0.087, R indices based on 3556 reflections with 2 4.1.6.12. 14-(1′-Piperidinylethyl)diquino[3,2-b;8′,7′-e]thiazine I > 2σ(I) (refinement on F ), 417 parameters, 0 restraints. Lp and ab- 1 −1 (14f). Yield 0.14 g, 68%, an oil. H NMR (CDCl3) δ: 1.31 (m, 2H, CH2), sorption corrections applied, µ = 2.00 mm . 1.40 (m, 4H, 2CH2), 2.38 (m, 4H, 2CH2), 2.60 (t, J = 7.2 Hz, 2H, CH2), 5.19 (t, J = 7.2 Hz, 2H, CH2), 7.31 (m, 2H, H-6, H-11), 7.32 (m, 2H, H- 4.3. Biological evaluation 3), 7.45 (d, J = 8.2 Hz, 2H, H-5), 7.52 (d, J = 7.2 Hz, 1H, H-11), 7.54 (d, J = 7.4 Hz, 1H, H-9), 7.71 (s, 1H H-8), 7.83 (d, J = 8.2 Hz, 1H, H- 4.3.1. Cell culture 12), 8.04 (dd, J = 8.2 Hz, J = 1.2 Hz, 1H, H-4), 8.87 (dd, J = 6.4 Hz, Compounds were evaluated for their anti-proliferative activity using J = 1.8 Hz, 1H, H-2). HR MS m/z: 413.1803, calcd 413.1800. three cultured cell lines: SNB-19 (human glioblastoma, DSMZ - German Collection of Microorganisms and Cell Cultures, CaCo-2 (human colon 4.1.6.13. 7-(1′-Methyl-2′-piperydinylethyl)diquino[3,2-b;3′,4′-e]thiazine adenocarcinoma, DSMZ - German Collection of Microorganisms and 1 (6i). Yield 0.11 g, 54%, mp 144–145 °C. H NMR (CDCl3) δ: 1.70 (m, Cell Cultures, Braunschweig, Germany), A549 (human lung carcinoma, 4H, 2CH2), 1.86 (m, 2H, CH2), 2.19 (m, 4H, 2CH2), 2.54 (s, 3H, CH3), ATCC, Manassas, VA, USA), MDA-MB-231 (human adenocarcinoma 2.91 (m, 1H, CH), 4.51 (m, 2H, CH2), 7.28 (t, J = 8.4 Hz, 1H, H-11), mammary gland, ATCC, Manassas, VA, USA) and HFF-1 (human fi- 7.53 (m, 2H, H-2, H12), 7.60 (m, 2H, H-3, H-10), 7.66 (s, 1H, H-13), broblast cell line, ATCC, Manassas, VA, USA). The cultured cells were 4 7.69 (d, J = 8.4 Hz, 1H, H-9), 7.88 (d, J = 8.4 Hz, 1H, H-1), 7.99 (d, kept at 37 °C and 5% CO2. The cells were seeded (1 × 10 cells/well/ J = 7.8 Hz, 1H, H-4), 8.64 (s, 1H, H-6). HR MS m/z: 427.1956, calcd 100 μL DMEM supplemented with 10% FCS and streptomycin and pe- 427.1956. nicillin) using 96-well plates (Corning). The cells were counted in a hemocytometer (Burker’s chamber) using a phase contrast Olympus 4.1.6.14. 7-(1′-Methyl-2′-piperydinylethyl)diquino[3,2-b;6′,5′-e]thiazine IX50 microscope equipped with Sony SSC-DC58 AP camera and 1 (11h). Yield 0.12 g, 56%), an oil. H NMR (CDCl3) δ: 1.69 (m, 4H, Olympus DP10 digital camera. 2CH2), 1.88 (m, 2H, CH2), 2.23 (m, 4H, 2CH2), 2.49 (s, 6H, 2CH3), 2.98 (m, 1H, CH), 4.42 (m, 2H, CH2), 7.31 (t, J = 8.6 Hz, 1H, H-11), 7.44 (m, 4.3.2. Proliferation assay 1H, H-6), 7.54 (m, 3H, H-2, H-10, H-12), 7.73 (d, J = 8.4 Hz, 1H, H-9), The anti-proliferative effect of the compounds obtained from both 7.74 (s, 1H, H-13), 7.97 (d, J = 8.6 Hz, 1H, H-5), 8.37 (d, J = 8.4 Hz, the cancer and the normal cells was determined using the Cell 1H, H-1), 8.83 (d, J = 4.2 Hz, 1H, H-3). HR MS m/z: 427.1953, calcd Proliferation Reagent WST-1 assay (Roche Molecular Biochemicals 427.1956. Mannheim, Germany). This assay is based on a colorimetric method using the viable cell’s ability to cause the bright red-colored stable 4.1.6.15. 14-(1′-Methyl-2′-piperydinylethyl)diquino[3,2-b;8′,7′-e]thiazine tetrazolium monosodium salt (2-(4-iodophenyl)-3-(4-nitrophenyl)-5- 1 (14g). Yield 0.12 g, 56%, mp 144–145 °C. H NMR (CDCl3) δ: 1.66 (m, (2,4-disulfophenyl)–2H-tetrazolium) to cleave to the dark red soluble 4H, 2CH2), 1.69 (m, 2H, CH2), 2.05 (m, 4H, CH2), 2.10 (s, 3H, CH3), formazan by cellular enzymes. A greater number of viable cells results 2.75 (t, J = 7.2 Hz, 1H, CH2), 4.92 (t, J = 7.2 Hz, 2H, CH2), 7.31 (m, in an increase in the overall activity of mitochondrial dehydrogenases 3H, H-3, H-6, H-10), 7.47 (d, J = 8.2 Hz, 1H, H-5), 7.53 (t, J = 7.2 Hz, in the sample. An increase in the amount of formazan dye formed

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