Novel Tetracyclic Azaphenothiazines with the Quinoline Ring as New Anticancer and Antibacterial Derivatives of Chlorpromazine

Phenothiazine derivatives are widely studied in various fields such as biology, chemistry, and medicine research because of their pharmaceutical effects. The first compound used successfully in the treatment of psychosis was a phenthiazine derivative, chlorpromazine. Apart from its activity in neurons, chlorpromazine has also been reported to display anticancer and antibacterial properties. In this study, we present the synthesis and research on the activity of A549, MDA, MiaPaCa, PC3, and HCT116 cancer cell lines and of S. aureus, S. epidermidis, E. coli, and P. aeruginosa bacterial strains against a series of new tetracyclic chlorpromazine analogues containing a quinoline scaffold in their structure instead of the benzene ring and various substituents at the thiazine nitrogen. The structure of these novel molecules has been determined by 1H NMR, 13C NMR, and HRMS spectral techniques. The seven most active of the twenty-four new chlorpromazine analogues tested were selected to study the mechanism of cytotoxic action. Their ability to induce apoptosis or necrosis in cancer cells was assessed by flow cytometry analysis. The results obtained confirmed the proapoptotic activity of selected compounds, especially in terms of inducing late apoptosis or necrosis in cancer cell lines A549, MiaPaCa-2, and HCT-116. Furthermore, studies on the induction of cell cycle arrest suggest that the new chlorpromazine analogues exert antiproliferative effects by inducing cell cycle arrest in the S phase and, consequently, apoptosis.


Introduction
Heterocyclic compounds containing sulfur and nitrogen atoms occupy a special place in medicinal chemistry due to their wide range of biological activities.Such heterocycles include a large group of β-lactam antibiotics and drugs containing a thiazole ring (including ritonavir, cobicistat, nizatidine, famotidine, meloxicam, mirabegron, and dasatinib) [1][2][3].This group includes also aromatic tricyclic compounds in which two benzene rings are linked by sulfur and nitrogen atoms-phenothiazines.These compounds were initially used in the dye and pigment industry and as insecticides, and quickly became the parent molecule of a multitude of drugs that have varied uses throughout medical and veterinary practice.For example, 10H-dibenzo-1,4-thiazine possesses insecticidal, antifungal, antibacterial, and anthelmintic properties [4].
One of the first important uses of phenothiazines, in addition to their use in the dye industry, was for their toxic effect on mosquito larvae and as anthelmintics (effective against swine roundworm) and antimalarials; however, they were not widely used for these purposes [5].
The history of these compounds goes back to the second half of the 19th century.Chlorpromazine, which belongs to this group and was initially used as an anesthetic, revolutionized the treatment of psychiatric disorders.It was originally synthesized by scientists at Rhone-Poulenc with the hopes that it would be an effective antimalarial [6,7].Chlorpromazine has been one of the most widely used antipsychotic medications for the treatment of schizophrenia and other psychiatric disorders.Although chlorpromazine is a first-generation antipsychotic medication, it is still widely used in psychiatry [8].Chlorpromazine demonstrates a high affinity for dopamine (D 1 -D 4 ) receptors and acts as a receptor antagonist by inhibiting adenylatecyclase activity.Chlorpromazine also inhibits other receptors, including receptors for 5-HT, H 1 histamine, α1 and α2 adrenaline, and M 1 and M 2 muscarinic acetylcholine receptors.N-Methyl-D-aspartate (NMDA) receptor inhibitory effects have also been described at high concentrations of chlorpromazine [9][10][11].
Several studies have reported on the potential anticancer activity of dopamine receptor antagonists.Among the drugs belonging to the neuroleptic phenothiazine, not only chlorpromazine, but also perphenazine, prochlorperazine, fluphenazine, and thioridazine have been tested for anticancer activity [12][13][14].
From the very beginning of the use of chlorpromazine in psychiatry, its impact on the course of cancer diseases has been observed.Among other things, studies conducted in Denmark in the second half of the twentieth century suggested a reduced risk of cancer in chlorpromazine-treated psychiatric patients.Significant inhibition of tumor growth has also been reported in a patient with laryngeal squamous cell carcinoma after direct injection of chlorpromazine into the tumor.Chlorpromazine was also shown to inhibit the growth of sarcoma tumors in mice.In line with the drug repurposing strategy, chlorpromazine was tested for its potential antitumorigenic effects, among others, against colorectal, breast, brain, lung, skin, pancreatic, and oral cancers, and also against leukemia, lymphoma, sarcoma, mastocytoma, and glioblastoma [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31].
The good results obtained in studies on cancer cell lines coupled with animal studies drew attention to chlorpromazine as a potential antitumor medication, leading to follow-up studies focused on chlorpromazine's anticancer mechanisms.Published research results have found that chlorpromazine inhibits cancer growth through multiple independent pathways, through various targets, ranging from histone deacetylases to ion channels.The overlap of molecular pathways between schizophrenia and cancer has been suspected for many years [32][33][34][35][36].
Therefore, in parallel with multidirectional research on neuroleptic phenothiazines, syntheses and research on the activity of new phenothiazine derivatives are carried out.New syntheses are realized in many ways, new substituents are introduced to the thiazine nitrogen atom and/or to the carbons of benzene rings.It is also increasingly common to replace one or both benzene rings with monocyclic or bicyclic azine systems (pyridine, pyrimidine, pyridazine, pyrazine, or quinoline) [37][38][39][40][41].Among the many activities determined for new phenothiazine analogues, such as antibacterial, antifungal, anti-tubercular, antiviral, anti-inflammatory, anti-filarial, antimalarial, anti-parasitic, and multidrug resistance reversal, the anticancer properties are of particular interest [42].
Phenothiazines containing one or two quinoline moieties instead of benzene rings are quinobenzothiazines and diquinothiazines.Selected compounds from substituted quinobenzothiazines I and II and diquinothiazines III-VIII (Figure 1) show significant anticancer activity against dozens of cancer cells derived from leukemia, melanoma, non-small cell lung, colon, CNS, ovary, prostate, breast, and skin cancers [37,40,41,43,44].These compounds also show promising antioxidant effects on rat liver microsomal membranes to protect non-enzymatic lipid peroxidation, inhibitory effects on mitogen-induced proliferation of human peripheral blood mononuclear cells, production of tumor necrosis factor-alpha (TNFα) in human whole blood cultures, against butyrylcholinesterase, and free radical scavenging, antiglycation, and alpha-glucosidase and alpha-amylase inhibition [37,40,44].They exerted a suppressive effect in in vivo models: delayed-type hypersensitivity to ovalbumin and cutaneous reaction to carrageenan, contact sensitivity to oxazolone, and experimental psoriasis in mice, and showed inhibitory effects on the expression of IFNβ and IFNβ-dependent further genes and proteins involved in the pathogenesis of autoimmune diseases [37,40].Some of the phenothiazines modified with the quinoline ring have also shown therapeutic effects in mouse experimental colitis, and prolongation of skin graft survival in mice [45].
hypersensitivity to ovalbumin and cutaneous reaction to carrageenan, contact sensitivity to oxazolone, and experimental psoriasis in mice, and showed inhibitory effects on the expression of IFNβ and IFNβ-dependent further genes and proteins involved in the pathogenesis of autoimmune diseases [37,40].Some of the phenothiazines modified with the quinoline ring have also shown therapeutic effects in mouse experimental colitis, and prolongation of skin graft survival in mice [45].In these studies, we developed an efficient synthesis of 6H-8chloroquinobenzothiazine as a substrate to obtain a series of new chlorpromazine analogues, which have a quinoline scaffold in their structure instead of the benzene ring and various substituents at the thiazine nitrogen atom (Figure 2).The starting point for planning such a modification was the significant and promising in vitro and in vivo activities of previously obtained 9-chloroquinobenzothiazine derivatives [37,38,40] and the great importance of chlorpromazine as a leading structure in medicinal chemistry.

Synthesis
For the synthesis of 6H-8-chloroquinobenzothiazine 5, 2-amino-4-chlorobenzenethiol 1 and 3-bromo-2-chloroquinoline 2 (Scheme 1) were used as starting materials.The reaction was carried out in boiling DMF for 1 h.Phenyl quinoline sulfide 3 is formed as an intermediate product in this type of phenothiazine ring synthesis reaction.Sulfide 3 can then undergo transformations in two directions.There is the possibility of direct cyclization (the Ullmann cyclization) toward quinobenzothiazine 4. The second variant leads through the Smiles rearrangement reaction (the S→N type, quinolinyl part migrates from the sulfur atom to the nitrogen atom, not isolated) to an amine 3′, which then undergoes cyclization to quinobenzothiazine 5.The literature data show that the course of this type of reaction for the synthesis of phenothiazine systems most often depends on the conditions used [37,[46][47][48][49]. Sometimes, it is impossible to state whether a reaction goes with or without the rearrangement because Ullmann's and Smiles's products are the same.The rearrangement proceeds under basic (most often) conditions, but also under acidic and neutral conditions.Using substituted o-aminobenzenethiol as a substrate, as in the reaction described, the possibility of creating two phenothiazines 4 and 5 should be considered.In these studies, we developed an efficient synthesis of 6H-8-chloroquinobenzothiazine as a substrate to obtain a series of new chlorpromazine analogues, which have a quinoline scaffold in their structure instead of the benzene ring and various substituents at the thiazine nitrogen atom (Figure 2).The starting point for planning such a modification was the significant and promising in vitro and in vivo activities of previously obtained 9-chloroquinobenzothiazine derivatives [37,38,40] and the great importance of chlorpromazine as a leading structure in medicinal chemistry.
hypersensitivity to ovalbumin and cutaneous reaction to carrageenan, contact sen to oxazolone, and experimental psoriasis in mice, and showed inhibitory effects expression of IFNβ and IFNβ-dependent further genes and proteins involved pathogenesis of autoimmune diseases [37,40].Some of the phenothiazines modifi the quinoline ring have also shown therapeutic effects in mouse experimental col prolongation of skin graft survival in mice [45].In these studies, we developed an efficient synthesis of chloroquinobenzothiazine as a substrate to obtain a series of new chlorpro analogues, which have a quinoline scaffold in their structure instead of the benze and various substituents at the thiazine nitrogen atom (Figure 2).The starting p planning such a modification was the significant and promising in vitro and activities of previously obtained 9-chloroquinobenzothiazine derivatives [37,38, the great importance of chlorpromazine as a leading structure in medicinal chemi

Synthesis
For the synthesis of 6H-8-chloroquinobenzothiazine 5, 2-amino-4-chlorobenze 1 and 3-bromo-2-chloroquinoline 2 (Scheme 1) were used as starting materia reaction was carried out in boiling DMF for 1 h.Phenyl quinoline sulfide 3 is for an intermediate product in this type of phenothiazine ring synthesis reaction.S can then undergo transformations in two directions.There is the possibility o cyclization (the Ullmann cyclization) toward quinobenzothiazine 4. The second leads through the Smiles rearrangement reaction (the S→N type, quinolinyl part m from the sulfur atom to the nitrogen atom, not isolated) to an amine 3′, whi undergoes cyclization to quinobenzothiazine 5.The literature data show that the of this type of reaction for the synthesis of phenothiazine systems most often dep the conditions used [37,[46][47][48][49]. Sometimes, it is impossible to state whether a reacti with or without the rearrangement because Ullmann's and Smiles's products same.The rearrangement proceeds under basic (most often) conditions, but als acidic and neutral conditions.Using substituted o-aminobenzenethiol as a substra the reaction described, the possibility of creating two phenothiazines 4 and 5 sh considered.

Synthesis
For the synthesis of 6H-8-chloroquinobenzothiazine 5, 2-amino-4-chlorobenzenethiol 1 and 3-bromo-2-chloroquinoline 2 (Scheme 1) were used as starting materials.The reaction was carried out in boiling DMF for 1 h.Phenyl quinoline sulfide 3 is formed as an intermediate product in this type of phenothiazine ring synthesis reaction.Sulfide 3 can then undergo transformations in two directions.There is the possibility of direct cyclization (the Ullmann cyclization) toward quinobenzothiazine 4. The second variant leads through the Smiles rearrangement reaction (the S→N type, quinolinyl part migrates from the sulfur atom to the nitrogen atom, not isolated) to an amine 3 ′ , which then undergoes cyclization to quinobenzothiazine 5.The literature data show that the course of this type of reaction for the synthesis of phenothiazine systems most often depends on the conditions used [37,[46][47][48][49]. Sometimes, it is impossible to state whether a reaction goes with or without the rearrangement because Ullmann's and Smiles's products are the same.The rearrangement proceeds under basic (most often) conditions, but also under acidic and neutral conditions.Using substituted o-aminobenzenethiol as a substrate, as in the reaction described, the possibility of creating two phenothiazines 4 and 5 should be considered.6H-8-Chloroquinobenzothiazine 5 was obtained in our previous studies by reacti 2,2'-dichloro-3,3'-diquinolinyl disulfide with 2,5-dichloroaniline [50].Comparison of t substances obtained in these reactions allowed the preliminary assumption that the re tion of 2-amino-4-chlrobenzenethiol 1 and 3-bromo-2-chloroquinoline 2 involves a Smi rearrangement.In order to identify unequivocally the structure of quinobenzothiazine we transformed it into 6-methyl derivative 6 and carried out 1 H NMR and two-dime sional NOESY and COSY spectra (Scheme 2, Table 1).6H-8-Chloroquinobenzothiazine 5 was obtained in our previous studies by reacting 2,2 ′ -dichloro-3,3 ′ -diquinolinyl disulfide with 2,5-dichloroaniline [50].Comparison of the substances obtained in these reactions allowed the preliminary assumption that the reaction of 2-amino-4-chlrobenzenethiol 1 and 3-bromo-2-chloroquinoline 2 involves a Smiles rearrangement.In order to identify unequivocally the structure of quinobenzothiazine 5, we transformed it into 6-methyl derivative 6 and carried out 1 H NMR and two-dimensional NOESY and COSY spectra (Scheme 2, Table 1).6H-8-Chloroquinobenzothiazine 5 was obtained in our previous studies by r 2,2'-dichloro-3,3'-diquinolinyl disulfide with 2,5-dichloroaniline [50].Compariso substances obtained in these reactions allowed the preliminary assumption that t tion of 2-amino-4-chlrobenzenethiol 1 and 3-bromo-2-chloroquinoline 2 involves a rearrangement.In order to identify unequivocally the structure of quinobenzothi we transformed it into 6-methyl derivative 6 and carried out 1 H NMR and twosional NOESY and COSY spectra (Scheme 2, Table 1).The 1 H NMR spectra were very useful for identification of the product as 8-substituted quinobenzothiazine 6.The proton signals of the quinoline part were found at low field (over 7.3 ppm) as a singlet (proton H12), doublet signal with one ortho-coupling (proton H4), triplet signal with two ortho-couplings (proton H2), doublet (proton H1), and triplet (proton H3).The proton signals of the benzene ring were found at a high field (below 7.1 ppm) and are observed as three doublets differing in shape and multiplicity depending on the proton to which they are assigned.The H10 and H9 (as double doublet signals) proton signals appear in the form of wide doublets with a coupling constant of 7, while the H7 proton signal appears in the form of a very narrow doublet with a coupling constant of 1.8.
In order to fully document the structure of derivative 6, a 13 C NMR spectrum and two-dimensional HSQC and HMBC spectra were also performed, which allowed for the assignment of appropriate C atoms to individual signals (Table 2).Therefore, the products were identified as 8-chloroquino [3,2-b]benzo [1,4]thiazines (8-chlorobenzo[b]-1azaphenothiazine).The next step in the modification of the phenothiazine system was the introduction of N,N-dialkylaminoalkyl, N-acylaminoalkyl, N-sulfonylaminoalkyl groups, and 1,2,3triazole substituents to the thiazine nitrogen atom in position 6.The N,N-dialkylaminoalkyl substituents were introduced in the N-alkylation reactions with hydrochlorides of selected acyclic and cyclic dialkylaminoalkyl chlorides in boiling dioxane in the presence of sodium hydroxide.As a result of such syntheses, the following was obtained five different 6-dialkylaminoalkyl derivatives 7-11 in 64-86% yield (Scheme 3).and triplet (proton H3).The proton signals of the benzene ring were found at a high field (below 7.1 ppm) and are observed as three doublets differing in shape and multiplicity depending on the proton to which they are assigned.The H10 and H9 (as double double signals) proton signals appear in the form of wide doublets with a coupling constant of 7 while the H7 proton signal appears in the form of a very narrow doublet with a coupling constant of 1.8.In order to fully document the structure of derivative 6, a 13 C NMR spectrum and two-dimensional HSQC and HMBC spectra were also performed, which allowed for the assignment of appropriate C atoms to individual signals (Table 2).Therefore, the products were identified as 8-chloroquino [3,2-b]benzo [1,4]thiazines (8-chlorobenzo[b]-1-azaphe nothiazine).The next step in the modification of the phenothiazine system was the introduction of N,N-dialkylaminoalkyl, N-acylaminoalkyl, N-sulfonylaminoalkyl groups, and 1,2,3-tri azole substituents to the thiazine nitrogen atom in position 6.The N,N-dialkylaminoalky substituents were introduced in the N-alkylation reactions with hydrochlorides of se lected acyclic and cyclic dialkylaminoalkyl chlorides in boiling dioxane in the presence o sodium hydroxide.As a result of such syntheses, the following was obtained five differen 6-dialkylaminoalkyl derivatives 7-11 in 64-86% yield (Scheme 3).Preparation of 6-substituted 8-chloroquinobenzothiazines with N-acylaminoalkyl and N-sulfonylaminoalkyl groups required a three-step synthesis.In the first stage, 6H-8-chloroquinobenzothiazine 5 was alkylated with phthalimidopropyl and phthalimidobutyl bromides in dry toluene in the presence of sodium hydride into the phthalimidoalkyl derivatives 12 and 13.Next, these compounds underwent reactions with hydrazine hydrate in aqueous ethanol to give aminopropyl derivative 14 and aminobutyl derivative 15 with yields of 85 and 84%, respectively (Scheme 4).Aminoalkylquinobenzothiazines 14 and 15 were transformed into the N-acyl derivatives.The reactions with acetic anhydride, ethyl chloroformate, and 2-chloroethyl isocyanate gave two 8-chloro-6-acetylaminoalkylquinobenzothiazines 16, 17, two 8-chloro-6ethoxycarbonylaminoalkylquinobenzothiazines 18 and 19, and two 8-chloro-6-chloroethylureidoalkylquinobenzothiazines 20 and 21 (possessing a half-mustard unit) in 65-86% yield.Aminoalkylquinobenzothiazines 14 and 15 were also transformed into the Nsulfonyl derivatives.The reactions with methanesulfonyl and p-toluenesulfonyl chlorides led to the sulfonamide derivatives: two 8-chloro-6-methanesulfonylaminoalkyl-and two 8-chloro-6-p-toluenesulfonylaminoalkylquinobenzothiazines 22-25 in 74-77% yield (Scheme 5).The synthesis of these derivatives was based on the interesting potential of phenothiazines containing such substituents.As previous studies have shown, phenothiazines with a half-mustard unit have great biological potential.For 10-chloroethylureidoalkylphenothiazines, cytotoxic effects have been found against various 54-60 human cancer cell lines: leukemia, melanoma, small cell lung, colon, central nervous system, kidneys, breast, ovary, and prostate cancer.Significant anticancer activity against nine types of human cancer cells (leukemia, melanoma, small cell lung, colon, central nervous system, kidney, breast, ovarian, and prostate cancers) was also found for 6-chloroethylureidoethyldquinothiazine [38,40].However, 9-chloro-and 9-methylthio-6-chloroethylureidoalkylquinobenzothiazines and 9-chloro-6-acetylaminopropylquinobenzothiazine showed very strong antiproliferative activity, no or low toxicity, and inhibited the production of TNF-α.They were also tested for anticancer activity against epidermoid carcinoma (A-431), lymphoma (L1210), and colorectal cell lines (SW-948).These compounds were as active as cisplatin [37,40].However, quinobenzothiazine with a methanesulfone fragment showed cytotoxic activity against cancer cell lines A-431, L1210, SW-948, and CX-1, comparable to the reference cisplatin [37,40].Aminoalkylquinobenzothiazines 14 and 15 were transformed into the N-acyl derivatives.The reactions with acetic anhydride, ethyl chloroformate, and 2-chloroethyl isocyanate gave two 8-chloro-6-acetylaminoalkylquinobenzothiazines 16, 17, two 8-chloro-6ethoxycarbonylaminoalkylquinobenzothiazines 18 and 19, and two 8-chloro-6-chloroethylu reidoalkylquinobenzothiazines 20 and 21 (possessing a half-mustard unit) in 65-86% yield.Aminoalkylquinobenzothiazines 14 and 15 were also transformed into the N-sulfonyl derivatives.The reactions with methanesulfonyl and p-toluenesulfonyl chlorides led to the sulfonamide derivatives: two 8-chloro-6-methanesulfonylaminoalkyl-and two 8-chloro-6p-toluenesulfonylaminoalkylquinobenzothiazines 22-25 in 74-77% yield (Scheme 5).The synthesis of these derivatives was based on the interesting potential of phenothiazines containing such substituents.As previous studies have shown, phenothiazines with a halfmustard unit have great biological potential.For 10-chloroethylureidoalkylphenothiazines, cytotoxic effects have been found against various 54-60 human cancer cell lines: leukemia, melanoma, small cell lung, colon, central nervous system, kidneys, breast, ovary, and prostate cancer.Significant anticancer activity against nine types of human cancer cells (leukemia, melanoma, small cell lung, colon, central nervous system, kidney, breast, ovarian, and prostate cancers) was also found for 6-chloroethylureidoethyldquinothiazine [38,40].However, 9-chloro-and 9-methylthio-6-chloroethylureidoalkylquinobenzothiazines and 9-chloro-6-acetylaminopropylquinobenzothiazine showed very strong antiproliferative activity, no or low toxicity, and inhibited the production of TNF-α.They were also tested for anticancer activity against epidermoid carcinoma (A-431), lymphoma (L1210), and colorectal cell lines (SW-948).These compounds were as active as cisplatin [37,40].However, quinobenzothiazine with a methanesulfone fragment showed cytotoxic activity against cancer cell lines A-431, L1210, SW-948, and CX-1, comparable to the reference cisplatin [37,40].

Cytotoxic Activity
Our biological study aimed to assess the cytotoxicity of newly synthesized derivatives of chlorpromazine (Schemes 3-6) and their potential utility in cancer treatment.Initially, all derivatives were tested against two human carcinoma cell lines (A549-lung cancer, and MDA-MB-231-breast cancer) and a normal cell line HaCaT (immortalized human keratinocytes) to determine their IC50 values using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) method [60].Comparisons to the IC50 values of the common chemotherapeutic agent, doxorubicin, are presented in Table 3.The selectivity index (SI) for these compounds ranged from 0.15 to 10.7, which was higher or equal to that of doxorubicin (0.14-0.15).Based on these findings, nine compounds were further evaluated on three additional cancer cell lines (human pancreas cancer-MiaPaca-2, human prostate cancer-PC3, and human colon cancer-HCT116) using the MTT method (Table 4).Apart from the A549 cell line, HCT116 cells exhibited the highest sensitivity to the tested substances, while PC3 cells showed lower sensitivity.It is important to note that while doxorubicin demonstrated high cytotoxicity against all cancer cell lines, it also affected normal cells.Compounds 8 and 23 displayed the highest selectivity index (39 and 143, respectively), with IC 50 values of 1.6 ± 0.8 µM and 0.7 ± 0.08 µM, respectively, against HCT116 cells, while showing no cytotoxic effects on HaCaT cells.

In Vitro Antibacterial Activity
The antibacterial efficacy of newly synthesized derivatives 5-33 was assessed by initially screening them for their minimal inhibitory concentrations (MICs) [61].
The results showed that investigated compounds (5-9, 20, 21) exhibited the potential to moderate antibacterial potency, mainly against standard staphylococcal strains (Table 5).In general, the most prominent activity against standard strains (except for P. aeruginosa) was observed for compound 21, with MIC (minimum inhibitory concentration) values ranging from 2 to 8 µg/mL.
The above results indicate that derivatives 8, 20, and 21 have both antibacterial and cytotoxic properties, which makes them promising compounds.

Mechanism of Cytotoxicity of Newly Synthesized Derivatives
• Apoptotic activity: Given the significant cytotoxic potential observed in the newly synthesized chlorpromazinederived compounds, namely 5, 8, 11, 20, 21, 23, and 25, against cancer cell lines such as A549 (lung cancer), MiaPaCa-2 (breast cancer), and HCT116 (colon cancer), with concentrations not exceeding 11 µM, these compounds were selected for further investigation to elucidate their mechanisms of biological action.Their ability to induce apoptosis or necrosis in cancer cells was assessed through flow cytometry analysis.As depicted in Figures 3 and 4, the tested derivatives, when administered at their IC 50 concentrations, exhibited proapoptotic properties in the selected cell lines compared to the untreated controls.
The obtained results indicated that the selected derivative 21 exhibited potent proapoptotic activity across all tested cancer cell lines (Figures 3 and 4).For derivative 8, a strong late apoptosis and necrosis-inducing effect was found in A549 (42% ± 0.76), whereas in HCT116 the same compound induced mainly early and late apoptosis/necrosis (9.09 ± 0.5 and 8.55 ± 0.33) (Figure 3).Furthermore, incubation with derivative 20 led to a significantly higher percentage of A549 and HCT116 cells in late apoptosis or necrosis (ranging from 15.2% to 17%) compared to the control.Additionally, a similar noticeable pro-apoptotic effect as compound 20 was observed with derivative 11 in HCT116 cells (15.64% in late apoptosis or necrosis).Compound 5 acted similarly, as it activated not only early but also late apoptosis/necrosis in MiaPaCa-2 cells, which accounted for 4.56% and 23%, respectively (Figures 3 and 4B).The strongest late apoptosis and necrotic activity (82% ± 2.89) was found for 25 towards A549 cancer cells, while compound 23 exhibited a high percentage of late apoptosis/necrosis (93% ± 3.29) in HCT116 cells (Figures 3 and 4C).
Our study confirmed the proapoptotic activity of selected compounds, especially in terms of inducing late apoptosis or necrosis in the A549, MiaPaCa-2, and HCT116 cancer cell lines (Figure 3).

• Induction of cell cycle arrest
The dysregulation of the cell cycle represents a hallmark of cancer cells, manifesting disruptions in various cellular pathways, particularly those governing the cell cycle and apoptosis.This dysregulation frequently enables cancer cells to evade crucial processe such as apoptosis or senescence, consequently leading to unchecked tumor proliferation and growth.Consequently, targeting this dysregulation has emerged as a promising ther apeutic strategy in cancer treatment [62][63][64].
Therefore, the effects of new derivatives on the cell cycle were investigated to eluci date the inhibition mechanisms.The cells were exposed to IC50 concentrations of com pounds 5, 8, 20, 21, 23, and 25 for 24 h.It was found that compounds 5, 8, 20, 21, and 25 induced G0/G1 phase arrest in both cell lines (the A549 and MiaPaca-2, respectively) (Fig ures 3 and 4A,B).A concomitant reduction in the number of cells in the S and G2/M phase was also observed (Figure 5).
This increase in the G0/G1-phase cell population was mostly at the expense of G2/M cells.These results clearly suggest that the studied derivatives exert an antiproliferative

• Induction of cell cycle arrest
The dysregulation of the cell cycle represents a hallmark of cancer cells, manifesting disruptions in various cellular pathways, particularly those governing the cell cycle and apoptosis.This dysregulation frequently enables cancer cells to evade crucial processes such as apoptosis or senescence, consequently leading to unchecked tumor proliferation and growth.Consequently, targeting this dysregulation has emerged as a promising therapeutic strategy in cancer treatment [62][63][64].
Therefore, the effects of new derivatives on the cell cycle were investigated to elucidate the inhibition mechanisms.The cells were exposed to IC 50 concentrations of compounds 5, 8, 20, 21, 23, and 25 for 24 h.It was found that compounds 5, 8, 20, 21, and 25 induced G0/G1 phase arrest in both cell lines (the A549 and MiaPaca-2, respectively) (Figures 3 and 4A,B).
A concomitant reduction in the number of cells in the S and G2/M phases was also observed (Figure 5).spectively) and statistically significant growth at the S phase (range 20.4 to 25.81 compounds 11 and 20 decreased HCT116 cells at their G0/G1 phase, and arrested population at the S phase (Figures 5 and 6C).Furthermore, cell cycle distribu HCT116 cells indicates proapoptotic action (Figure 3).This increase in the G0/G1-phase cell population was mostly at the expense of G2/M cells.These results clearly suggest that the studied derivatives exert an antiproliferative effect by inducing cell cycle arrest at the S phase and, consequently, apoptosis.The addition of derivative 21 significantly increased the percentage of Sub G1 phase A549 cells when tested at IC 50 (27.6%,p = 0.0001) and reduced the cell population in the G0/G1 and G2/M phases, compared to the cell cycle distribution monitored in untreated A459 cells (Figures 3 and 4A).Upon treatment of HCT116 cells with compounds 8, 21, and 23, a significant increase in the cell amount at the sub-G1 phase (5, 12, and 11-fold, respectively) was detected.Moreover, these three derivatives led to a significant increase in the number of cells accumulated in the S phase (approximately 2-fold) and in the G2/M phase (approximately 1.3-fold) as compared to the control.In addition, on exposure to compounds 11 and 20, there was a small increase in the sub-G1 population (range 2-and 3-fold, respectively) and statistically significant growth at the S phase (range 20.4 to 25.81%).The compounds 11 and 20 decreased HCT116 cells at their G0/G1 phase, and arrested the cell population at the S phase (Figures 5 and 6C).Furthermore, cell cycle distribution in HCT116 cells indicates proapoptotic action (Figure 3).

Methods and Materials
Melting points were determined in open capillary tubes on a Boetius melting point apparatus and were uncorrected.The standard NMR spectra were recorded on Bruker Avance spectrometers (Bruker, Billerica, MA, USA)( 1 H at 600 MHz, 13 C at 150 MHz) in

Methods and Materials
Melting points were determined in open capillary tubes on a Boetius melting point apparatus and were uncorrected.The standard NMR spectra were recorded on Bruker Avance spectrometers (Bruker, Billerica, MA, USA)( 1 H at 600 MHz, 13 C at 150 MHz) in CDCl 3 or DMSO-d 6 .Two-dimensional COSY, NOESY, HSQC, and HMBC spectra of selected compounds were recorded on a Bruker Avance spectrometer at 600 MHz, using COSYGPSW, NOESYGPPHSW, HSQCGPPH, and HMBCGP experiments.The HRMS spectra (EI-electroimpact ionization) were run on a Brucker Impact II (Bruker, Billerica, MA, USA). 1 H NMR, 13 C NMR and HRMS spectra are included in Supplementary Materials.Thin-layer chromatography was performed on aluminum oxide 60 F 254 neutral (type E) (Merck 1.05581) with CH 2 Cl 2 as eluents.
To a stirred solution of aminoalkyldiquinothiazines 14 or 15 (0.5 mmol) in a mixture of CH 2 Cl 2 (5 mL) and 10% Na 2 CO 3 solution (7 mL), a solution of methanesulfonyl chloride (0.06 mL, 0.75 mmol) was added.The mixture was stirred at rt for 24 h.The organic phase was separated and the aqueous phase was extracted with CH 2 Cl 2 (2 × 5 mL).The combined extracts were washed with water (2 × 10 mL) and dried over Na 2 SO 4 .The drying agent was filtered off and the filtrate was evaporated.

MTT Assay
To evaluate the cytotoxic effects of the newly synthesized compounds, a preliminary MTT (3-(4,5-dimethylthiadiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) test was performed on two different cancer cell lines, A549 and MDA-MB231, as well as on one healthy HaCaT cell line.After obtaining the results, additional MTT tests were performed on three additional cancer cell lines (MiaPaca-2, PC3, and HCT-116), only on a selected group of compounds showing cytotoxic activity (5, 8, 21, 23, and 25).The study derivatives were subjected to testing at various concentrations (ranging from 5 to 140 µM), alongside the reference drug doxorubicin.These compounds were added to 96-well plates containing study cells (1 × 10 4 cells per well) and incubated for 72 h.MTT analysis, as previously described [65], was employed.
Cell absorbance results were incorporated into the formula for calculating the relative MTT level (%), enabling the assessment of cell viability following exposure to the test compounds.The cell viability percentage represents the MTT reduction in cells treated with the test compounds compared to the control sample, where only the medium was added to the cells.The IC 50 values, representing the concentration at which 50% of cell viability is inhibited, were calculated using Prism 8.0.1, GraphPad software.

Apoptosis and Cell Cycle Analysis by Flow Cytometry (FCM)
To analyze the number of cells in early apoptosis, late apoptosis, or necrosis, A549, MiaPaca-2, and HCT-116 cell lines were cultured in 6-well plates with a seeding density of 1 × 10 5 cells per well.These cells were then treated with selected compounds (4, 5, and 8) at their respective IC 50 concentrations and incubated for 72 h.Subsequently, a commercially available kit, the FITC: Annexin V Apoptosis Detection Kit I from BD Biosciences Pharmingen in San Jose, CA, USA, was utilized to assess apoptosis.After 72 h the cells were harvested, washed, and labeled with Annexin V-FITC and propidium iodide (PI) following the manufacturer's protocol (Becton Dickinson), as previously described [65].The stained cells were analyzed by flow cytometry.The cells were identified as early apoptotic (Annexin V+/PI−) or late apoptotic/necrotic (Annexin V+/PI+).

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Cell cycle analysis: In brief, cells were seeded at a density of 1 × 10 5 cells/well on a six-well plate and allowed to adhere for 24 h.Subsequently, they were treated with selected compounds at their IC 50 concentrations for an additional 24 h.Both population cells (the attached and detached) were collected and centrifuged at 400× g for 5 min at 4 • C.After centrifugation, the cells were washed twice with 0.9% NaCl and subsequently fixed in 500 µL of 70 % cold ethanol overnight.
Before analysis, the fixed cells underwent another centrifugation step at 850× g for 5 min at 4 • C and were washed with PBS.Following the washes, the cells were incubated with 50 µL of RNase (100 µg/mL) and 200 µL of propidium iodide (PI) (50 µg/mL) at 37 • C for 30 min in the dark.Finally, 100 µL of PBS was added to each sample.Flow cytometry (Becton Dickinson FACS Verse, Franklin Lakes, NJ, USA) was then used to analyze the cell cycle distribution, identifying cells in various stages including sub-G1, G0/G1, S, and G2/M phases.Each assay was performed in quadruplicate.

In Vitro Antibacterial Studies
To evaluate the antibacterial effectiveness of chloroquinobenzothiazine derivatives, various bacterial strains from international microbe collections such as the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC) were examined, alongside a panel of clinical rods.This included two Gram-negative organisms, E. coli ATCC 25922 and P. aeruginosa ATCC 15442, and a series of six Gram-positive strains: S. aureus NCTC 4163; S. aureus ATCC 29213, 25923, and 6538; and S. epidermidis ATCC 12228 and 35984.
The antimicrobial activity was examined using the Minimal Inhibitory Concentration (MIC) method, following standard CLSI procedures with slight adjustments.The MIC was determined using the two-fold serial broth microdilution method in 96-well microtitration plates containing Mueller-Hinton II broth medium (Becton Dickinson, Franklin Lakes, NJ, USA).The final inoculum for all tested bacteria was adjusted to 10 −6 CFU/mL (colony-forming units per milliliter).The stock solution of the tested compounds was prepared in dimethyl sulfoxide (DMSO) and diluted to a maximum of 1% solvent content with a sterile medium.The MIC value recorded represents the lowest concentration of the tested antimicrobial agents (expressed in µg/mL) that inhibits visible growth of the microorganism after 19 h of incubation at 35 • C.
Additional details regarding the conducted biological studies, including cell culture, appropriate conditions, and methodology, were provided in our previous publication [66].

Statistical Analysis
The statistical analysis was performed using GraphPad Prism 9 software (Graph Pad Software, San Diego, CA, USA).The results were reported as mean ± SD from a minimum of three independent experiments.Statistical significance of differences between values was assessed using analysis of variance with Dunnett's multiple comparison post hoc test, and significance was defined as p < 0.05.

1 a
Data are expressed as mean SD, b IC 50 (µM)-the concentration of the compound that corresponds to a 50% growth inhibition of cell line (as compared to the control) after the cells are cultured for 72 h with the individual compound.cThe SI (selectivity index) was calculated using the formula: SI = IC 50 for normal cell line/IC 50 cancer cell line.d Human lung cancer (A549).e Human breast cancer (MDA-MB231).f Human immortal keratinocyte cell line from adult human skin (HaCaT).g The selected reference compound commonly used in cancer treatment (doxorubicin).

Figure 3 .
Figure 3.The effects of newly synthesized compounds 5, 8, 11, 20, 21, 23, and 25 on early and apoptosis or necrosis were assessed in A549, MiaPaca-2, and HaCaT cell lines.Cells were tre with the compounds at their IC50 concentrations for 72 h, followed by staining with annexin Vand PI, and analysis using flow cytometry.The results are presented as the percentage of cells i early stage of apoptosis and the percentage of cells in the late stage of apoptosis or necrosis.* 0.0001, ** p ≤ 0.001, * p ≤ 0.01, as compared to the control (C).

Figure 3 .Figure 3 .
Figure 3.The effects of newly synthesized compounds 5, 8, 11, 20, 21, 23, and 25 on early and late apoptosis or necrosis were assessed in A549, MiaPaca-2, and HaCaT cell lines.Cells were treated with the compounds at their IC 50 concentrations for 72 h, followed by staining with annexin V-FITC and PI, and analysis using flow cytometry.The results are presented as the percentage of cells in the early stage of apoptosis and the percentage of cells in the late stage of apoptosis or necrosis.*** p ≤ 0.0001, ** p ≤ 0.001, * p ≤ 0.01, as compared to the control (C).

Table 2 .
The proton-carbon correlation of compound 6.

Table 2 .
The proton-carbon correlation of compound 6.

Table 3 .
Cytotoxic activity (IC50, µM) of studied compounds estimated by the MTT assay a .

Table 3 .
Cytotoxic activity (IC 50 , µM) of studied compounds estimated by the MTT assay a .

Table 4 .
Cytotoxic activity (IC 50 , µM) of selected compounds estimated by the MTT assay a .

Cancer Cells Normal Cells MiaPaCa-2 d PC3 e HCT116 f HaCaT g IC 50 b SI c IC 50 SI IC 50 SI IC 50
Data are expressed as mean SD. b IC 50 (µM)-the concentration of the compound that corresponds to a 50% growth inhibition of cell line (as compared to the control) after culturing the cells for 72 h with the individual compound.
a c The SI (selectivity index) was calculated using formula: SI = IC 50 for normal cell line/IC 50 cancer cell line.d Human pancreas cancer (MiaPaCa-2).e Human prostate cancer (PC3).f Human colon cancer (HCT-116).g Human immortal keratinocyte cell line from adult human skin (HaCaT).h The selected reference compound commonly used in cancer treatment (doxorubicin).