Tetracyclic Thioxanthene Derivatives: Studies on Fluorescence and Antitumor Activity

Thioxanthones are bioisosteres of the naturally occurring xanthones. They have been described for multiple activities, including antitumor. As such, the synthesis of a library of thioxanthones was pursued, but unexpectedly, four tetracyclic thioxanthenes with a quinazoline–chromene scaffold were obtained. These compounds were studied for their human tumor cell growth inhibition activity, in the cell lines A375-C5, MCF-7 and NCI-H460. Photophysical studies were also performed. Two of the compounds displayed GI50 values below 10 µM for the three tested cell lines, and structure–activity relationship studies were established. Three compounds presented similar wavelengths of absorption and emission, characteristic of dyes with a push-pull character. The structures of two compounds were elucidated by X-ray crystallography. Two tetracyclic thioxanthenes emerged as hit compounds. One of the two compounds accumulated intracellularly as a bright fluorescent dye in the green channel, as analyzed by both fluorescence microscopy and flow cytometry, making it a promising theranostic cancer drug candidate.


Introduction
Thioxanthones have sparked interest in terms of medicinal chemistry, due to the fact that they are bioisosteres of xanthones, a privileged structure with occurrence in natural products [1,2]. They are S-heterocycles, with three rings and a dibenzo-γ-pyrone scaffold (1, Figure 1). This class of heterocycles has gained increasing importance in medicinal chemistry, due to its versatility and presence in bioactive compounds in a wide range of biological targets. Therefore, the substitution of a natural scaffold with a sulfur opens the  (1), the anticancer thioxanthones hycanthone (2) and lucanthone (3), the dual tumor cell line growth/P-glycoprotein inhibitor (4) and the general structure of the compounds studied in this work (5).
Recent studies performed by our group have proven the usefulness of thioxanthones in a multitude of diverse activities. A library of thioxanthones was tested for their antimicrobial and synergistic activity, proving the potential of these compounds to inhibit the growth of pathogenic bacteria and to revert antimicrobial resistance [5]. They have also proven successful in reducing lipids, with no associated toxicity in the tested model, uncovering potential anti-obesity activity [6]. Moreover, thioxanthones have been investigated in the last decades for their antitumor activity [7][8][9][10] and have also been studied for their photoinitiator features [11][12][13][14]. Hycanthone (2, Figure 1) and its prodrug, lucanthone (3, Figure 1), were the first thioxanthones introduced in therapy as antischistosomal agents [1,15,16]. However, these drugs led to mutagenicity, likely associated with their C-4 methylene moiety, which resulted in their removal from therapeutics [17]. This effect led to the discovery of their ability to sensitize cancer cells, suggesting new and promising applications in chemotherapy. Previous results by our group found that aminated thioxanthone 4 (Figure 1), structurally similar to 2 and 3, is a potent cell growth inhibitor in the myeloid leukemia K562 cell line, presenting GI50 values of 1.9 µM for both the doxorubicin-sensitive and resistant cell line. Regarding solid tumors, compound 4 also emerged as a hit compound for the three tested cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma), displaying the best results out of the library of thioxanthones tested [18]. Deeper insights into the mechanism revealed compound 4 as capable of modulating autophagy and inducing apoptosis, thus becoming a lead compound for tumor treatment, with proven efficiency in mice xenografts [10,19]. Furthermore, 4 also displayed inhibition of P-glycoprotein, a known efflux pump, working as a dual antitumor/P-glycoprotein inhibitor agent with improved efficacy in sensitizing a resistant P-gp overexpressing cell line to doxorubicin [7,20].
Theranostic, the combination of therapy and diagnostic, has been gathering importance, particularly in cancer, through the application of molecular imaging towards  (1), the anticancer thioxanthones hycanthone (2) and lucanthone (3), the dual tumor cell line growth/P-glycoprotein inhibitor (4) and the general structure of the compounds studied in this work (5).
Recent studies performed by our group have proven the usefulness of thioxanthones in a multitude of diverse activities. A library of thioxanthones was tested for their antimicrobial and synergistic activity, proving the potential of these compounds to inhibit the growth of pathogenic bacteria and to revert antimicrobial resistance [5]. They have also proven successful in reducing lipids, with no associated toxicity in the tested model, uncovering potential anti-obesity activity [6]. Moreover, thioxanthones have been investigated in the last decades for their antitumor activity [7][8][9][10] and have also been studied for their photoinitiator features [11][12][13][14]. Hycanthone (2, Figure 1) and its prodrug, lucanthone (3, Figure 1), were the first thioxanthones introduced in therapy as antischistosomal agents [1,15,16]. However, these drugs led to mutagenicity, likely associated with their C-4 methylene moiety, which resulted in their removal from therapeutics [17]. This effect led to the discovery of their ability to sensitize cancer cells, suggesting new and promising applications in chemotherapy. Previous results by our group found that aminated thioxanthone 4 (Figure 1), structurally similar to 2 and 3, is a potent cell growth inhibitor in the myeloid leukemia K562 cell line, presenting GI 50 values of 1.9 µM for both the doxorubicinsensitive and resistant cell line. Regarding solid tumors, compound 4 also emerged as a hit compound for the three tested cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma), displaying the best results out of the library of thioxanthones tested [18]. Deeper insights into the mechanism revealed compound 4 as capable of modulating autophagy and inducing apoptosis, thus becoming a lead compound for tumor treatment, with proven efficiency in mice xenografts [10,19]. Furthermore, 4 also displayed inhibition of P-glycoprotein, a known efflux pump, working as a dual antitumor/P-glycoprotein inhibitor agent with improved efficacy in sensitizing a resistant P-gp overexpressing cell line to doxorubicin [7,20].
Theranostic, the combination of therapy and diagnostic, has been gathering importance, particularly in cancer, through the application of molecular imaging towards the identification of cancer targets and the optimization of the therapy. Specifically, these agents concomitantly foster the transport of both therapeutic and diagnostic imaging agents, therefore reducing the biodistribution and therapeutic efficacy differences between them [21]. The focus of this work is the synthesis and biological evaluation of novel aminated thioxanthones based on the lead compound 4 as tumor cell line inhibitors and preliminary studies to understand their potential for theranostics. For that purpose, the amines chosen were guanidine and urea derivatives to allow a tandem reaction with further C-9 ketone functionalization which yielded tetracyclic derivatives. This tetracyclic scaffold is based on the D-π-A structure ("push-pull" system), architected by an electron donor group (C-4 propoxy chain), an electron acceptor group (C-1 guanidine/urea) and bridged by a conjugated π system (5, Figure 1). Most of the near-infrared fluorescence (NIR) probes are based on this structure, combining a rigid planarity capability and beneficially shifting the emission wavelength into the NIR region. Indeed, during the development of this work, new tetracyclic thioxanthenes, with a quinazoline-chromene scaffold, were synthesized and described as potential antitumor agent kinase inhibitors [8,22], while three other new thioxanthenes were revealed to be promising as theranostic agents [23][24][25][26], highlighting the potential of the rationale herein followed.

Synthesis of Aminated Tetracyclic Thioxanthenes
Thioxanthenes 11-14 were synthesized from 1-chloro-4-propoxy-9H-thioxanthen-9one (6) by nucleophilic aromatic substitution (Ullmann type C-N coupling) of the chlorine in position C-1 by a secondary amine (7)(8)(9)(10). A dehydrative cyclization happens in a concerted manner, leading to the formation of a tetracyclic structure [27]. Scheme 1 shows the general reaction for the synthesis of aminated thioxanthenes, and Table 1 shows the amines and thioxanthenes obtained. the identification of cancer targets and the optimization of the therapy. Specifically, these agents concomitantly foster the transport of both therapeutic and diagnostic imaging agents, therefore reducing the biodistribution and therapeutic efficacy differences between them [21]. The focus of this work is the synthesis and biological evaluation of novel aminated thioxanthones based on the lead compound 4 as tumor cell line inhibitors and preliminary studies to understand their potential for theranostics. For that purpose, the amines chosen were guanidine and urea derivatives to allow a tandem reaction with further C-9 ketone functionalization which yielded tetracyclic derivatives. This tetracyclic scaffold is based on the D-π-A structure ("push-pull" system), architected by an electron donor group (C-4 propoxy chain), an electron acceptor group (C-1 guanidine/urea) and bridged by a conjugated π system (5, Figure 1). Most of the near-infrared fluorescence (NIR) probes are based on this structure, combining a rigid planarity capability and beneficially shifting the emission wavelength into the NIR region. Indeed, during the development of this work, new tetracyclic thioxanthenes, with a quinazoline-chromene scaffold, were synthesized and described as potential antitumor agent kinase inhibitors [8,22], while three other new thioxanthenes were revealed to be promising as theranostic agents [23][24][25][26], highlighting the potential of the rationale herein followed.

Amine Thioxanthene Yield
Molecules 2021, 26, 3315 3 of 13 the identification of cancer targets and the optimization of the therapy. Specifically, these agents concomitantly foster the transport of both therapeutic and diagnostic imaging agents, therefore reducing the biodistribution and therapeutic efficacy differences between them [21]. The focus of this work is the synthesis and biological evaluation of novel aminated thioxanthones based on the lead compound 4 as tumor cell line inhibitors and preliminary studies to understand their potential for theranostics. For that purpose, the amines chosen were guanidine and urea derivatives to allow a tandem reaction with further C-9 ketone functionalization which yielded tetracyclic derivatives. This tetracyclic scaffold is based on the D-π-A structure ("push-pull" system), architected by an electron donor group (C-4 propoxy chain), an electron acceptor group (C-1 guanidine/urea) and bridged by a conjugated π system (5, Figure 1). Most of the near-infrared fluorescence (NIR) probes are based on this structure, combining a rigid planarity capability and beneficially shifting the emission wavelength into the NIR region. Indeed, during the development of this work, new tetracyclic thioxanthenes, with a quinazoline-chromene scaffold, were synthesized and described as potential antitumor agent kinase inhibitors [8,22], while three other new thioxanthenes were revealed to be promising as theranostic agents [23][24][25][26], highlighting the potential of the rationale herein followed.

Amine
Thioxanthene Yield Molecules 2021, 26, 3315 3 of 13 the identification of cancer targets and the optimization of the therapy. Specifically, these agents concomitantly foster the transport of both therapeutic and diagnostic imaging agents, therefore reducing the biodistribution and therapeutic efficacy differences between them [21]. The focus of this work is the synthesis and biological evaluation of novel aminated thioxanthones based on the lead compound 4 as tumor cell line inhibitors and preliminary studies to understand their potential for theranostics. For that purpose, the amines chosen were guanidine and urea derivatives to allow a tandem reaction with further C-9 ketone functionalization which yielded tetracyclic derivatives. This tetracyclic scaffold is based on the D-π-A structure ("push-pull" system), architected by an electron donor group (C-4 propoxy chain), an electron acceptor group (C-1 guanidine/urea) and bridged by a conjugated π system (5, Figure 1). Most of the near-infrared fluorescence (NIR) probes are based on this structure, combining a rigid planarity capability and beneficially shifting the emission wavelength into the NIR region. Indeed, during the development of this work, new tetracyclic thioxanthenes, with a quinazoline-chromene scaffold, were synthesized and described as potential antitumor agent kinase inhibitors [8,22], while three other new thioxanthenes were revealed to be promising as theranostic agents [23][24][25][26], highlighting the potential of the rationale herein followed.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2. Scheme 2. Proposed reaction mechanism for the cyclization of compound 12.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2. Scheme 2. Proposed reaction mechanism for the cyclization of compound 12.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2. Scheme 2. Proposed reaction mechanism for the cyclization of compound 12.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2.

13%
Noteworthy is also the fact that, in the case of thioxanthene 12, a reduction occurs, probably mediated by copper iodide, yielding the pyrimidine-like fourth ring, instead of the expected aromatic ring substituted with a carbonyl [28]. The proposed reaction mechanism for the cyclization and reduction, leading to compound 12, is presented in Scheme 2.

Scheme 2.
Proposed reaction mechanism for the cyclization of compound 12. Scheme 2. Proposed reaction mechanism for the cyclization of compound 12.

Biological Activity Antitumor Activity
The synthesized compounds were tested for their capability of inhibiting the growth of human tumor cell lines. The tetracyclic thioxanthenes herein described show potential as antitumor agents for the tested cell lines, particularly 11 and 14, which have emerged as hit compounds, presenting concentrations required to reduce growth rates to 50% (GI 50 ) in A375-C5 (IL-1 insensitive malignant melanoma), MCF-7 (breast adenocarcinoma) and NCI-H460 (non-small cell lung cancer) of 5-7 µM and 8-11 µM, respectively. Compound 12 presented concentrations between 31 and 39 µM for the tested cell lines and 13 displayed GI 50 between 16 and 23 µM. The results are presented in Table 2. The antitumor activity is presented as the concentrations that cause 50% cell growth inhibition (GI 50 ) after a continuous exposure for 48 h and represents mean ± SD from at least three independent experiments. Doxorubicin was used as positive control for the SRB assay.
We performed cell toxicity study of the compounds using non-tumor cell lines. We found that the GI 50 of the four compounds in the non-tumor Human Pulmonary Alveolar Epithelial Cells (HPAEpiC) was higher than that in cancer cell lines under study ( Table 3). The compounds exhibited a high degree of selectivity, as determined by selectivity index calculation, suggesting that they are not toxic to healthy cells, at least at concentrations that are toxic to cancer cells. Interestingly, compounds 11 and 13 exhibited a better degree of selectivity than doxorubicin. Overall, compound 11 showed the best GI 50 values for all the tested cell lines, meaning this could be a starting point for the development of new antitumor compounds. This compound is the only one bearing a free amino group, which makes it easier for it to pass negatively charged cell membranes. This is evidenced by the much different GI 50 between this compound and the structurally related 12, which is substituted with a hydrogen and presents much lower activity. Substitution of the free amino group with a 2-chlorobenzyl moiety (compound 13) also leads to lower activity in the tested cell lines. The imine derivative 14 presented activity comparable to 11, but had slightly higher GI 50 . This can suggest that substitution of the free amino group does not necessarily lead to lack of activity, but synthesis of different derivatives will lead to clearer conclusions over structureactivity relationships.

Fluorescence
The absorption and emission spectra of all compounds were recorded in diluted DMSO solutions (Figure 2), and their main characteristics are summarized in Table 4.

Fluorescence
The absorption and emission spectra of all compounds were recorded in diluted DMSO solutions (Figure 2), and their main characteristics are summarized in Table 4.   [29].
Compounds 11, 13 and 14 present similar wavelengths of absorption and emission, with a large Stokes' shift characteristic of dyes with a push-pull character. When the nitrogen is absent from the position 1′, as in compound 12, both absorption and emission are blue shifted. This nitrogen therefore seems to have an important contribution to the push-pull character of the dyes. On the other hand, the best quantum yield is obtained for compound 12, lacking this nitrogen atom, which therefore seems detrimental to the emission intensity. Moreover, compound 14, in which the aromaticity is disrupted because the nitrogen bears an extra group, is barely emissive in solution. Both the aromaticity of the heterocycle and the electron donating nature of the substituent seem important to obtain a high quantum yield and a large Stokes' shift.

Fluorescence Microscopy and Flow Cytometry Analysis
A fluorescence microscopy analysis was undertaken to check the brightness of the four compounds in culture cells. The analysis revealed that compound 11 emitted a bright intracellular fluorescence in the green channel, when cells were excited with 470 nm light, in the cancer cells tested (Figure 3a).  Compounds 11, 13 and 14 present similar wavelengths of absorption and emission, with a large Stokes' shift characteristic of dyes with a push-pull character. When the nitrogen is absent from the position 1 , as in compound 12, both absorption and emission are blue shifted. This nitrogen therefore seems to have an important contribution to the push-pull character of the dyes. On the other hand, the best quantum yield is obtained for compound 12, lacking this nitrogen atom, which therefore seems detrimental to the emission intensity. Moreover, compound 14, in which the aromaticity is disrupted because the nitrogen bears an extra group, is barely emissive in solution. Both the aromaticity of the heterocycle and the electron donating nature of the substituent seem important to obtain a high quantum yield and a large Stokes' shift.

Fluorescence Microscopy and Flow Cytometry Analysis
A fluorescence microscopy analysis was undertaken to check the brightness of the four compounds in culture cells. The analysis revealed that compound 11 emitted a bright intracellular fluorescence in the green channel, when cells were excited with 470 nm light, in the cancer cells tested (Figure 3a). The fluorescence staining accumulated within intracellular round-shaped structures that closely resemble endosome/lysosome structures, which led us to suggest that the component may accumulate in those structures. However, this was not confirmed by the colocalization study using the lysosome marker LAMP-1 antibody (data not shown). The fluorescence staining remained associated with the cells until cell death. The fluorescence property of the compound 11 was confirmed when treated cells were analyzed by flow cytometry (Figure 3b). A peak of fluorescent cells was obtained in the green channel, near the peak of Rhodamine 123, used as positive control. Moreover, the fluorescence was stable after keeping the cell preparations in the refrigerator for more than two weeks. Additional studies are needed to elucidate the exact subcellular localization of compound 11 and its significance in terms of therapeutic application.

X-ray Crystallography
The structures of compounds 11 and 12 were determined by single crystal X-ray diffraction (Figure 4). We have previously shown that while the most stable structure of tetrahydrothio-γ-pyrone is a chair conformation, thioxanthone molecules, like xanthones in general [30], are essentially planar [31]. The four-ring systems of 11 and 12 are also almost planar as expected. The central pyranoid rings have partial aromatic character with C-S bond lengths (from 1.741(4) to 1.752(4) Å) shorter than the corresponding bonds in the tetrahydrothio-γ-pyrone (1.806(4) and 1.814(4) Å) [31]. The fluorescence staining accumulated within intracellular round-shaped structures that closely resemble endosome/lysosome structures, which led us to suggest that the component may accumulate in those structures. However, this was not confirmed by the colocalization study using the lysosome marker LAMP-1 antibody (data not shown). The fluorescence staining remained associated with the cells until cell death. The fluorescence property of the compound 11 was confirmed when treated cells were analyzed by flow cytometry (Figure 3b). A peak of fluorescent cells was obtained in the green channel, near the peak of Rhodamine 123, used as positive control. Moreover, the fluorescence was stable after keeping the cell preparations in the refrigerator for more than two weeks. Additional studies are needed to elucidate the exact subcellular localization of compound 11 and its significance in terms of therapeutic application.

X-ray Crystallography
The structures of compounds 11 and 12 were determined by single crystal X-ray diffraction (Figure 4). We have previously shown that while the most stable structure of tetrahydrothio-γ-pyrone is a chair conformation, thioxanthone molecules, like xanthones in general [30], are essentially planar [31]. The four-ring systems of 11 and 12 are also almost planar as expected. The central pyranoid rings have partial aromatic character with C-S bond lengths (from 1.741(4) to 1.752(4) Å) shorter than the corresponding bonds in the tetrahydrothio-γ-pyrone (1.806(4) and 1.814(4) Å) [31].

Chemistry
All reagents and solvents were purchased from Sigma Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan, UK) and no further purification process was implemented. Solvents were evaporated using a rotary evaporator under reduced pressure, Buchi Waterchath B-480. All reactions were monitored by thin-layer chromatography (TLC) carried out on precoated plates with 0.2 mm of thickness using Merck silica gel 60 (GF254) with appropriate mobile phases. Compounds were easily detectable at 254 nm or 365 nm.
Flash column chromatography using silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany) was used in the purification of the synthesized compounds. Melting points (m.p.) were measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncorrected. 1 H-and 13 C-nuclear magnetic resonance (NMR) spectra were recorded at the University of Aveiro, Department of Chemistry in CDCl3 or DMSO-d6 (Deutero GmbH, Ely, UK) at room temperature on a Bruker Avance 300 spectrometer (300.13 MHz for 1 H and 75.47 MHz for 13 C, Bruker Biosciences Corporation, Billerica, MA, USA). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. Coupling constants are reported in hertz (Hz). 13 C-NMR assignments were made by bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR experiments (longrange C, H coupling constants were optimized to 7 Hz) or by comparison with the assignments of similar molecules. High-resolution mass spectroscopy (HRMS) spectra were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) and recorded as electrospray ionization (ESI) mode in Centro de Apoio Cientifico e Tecnolóxico á Investigación (CACTI, University of Vigo, Pontevedra, Spain) or on an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) at CEMUP, University of Porto, Portugal. The following compounds were synthesized and purified by the described procedures.

Chemistry
All reagents and solvents were purchased from Sigma Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan, UK) and no further purification process was implemented. Solvents were evaporated using a rotary evaporator under reduced pressure, Buchi Waterchath B-480. All reactions were monitored by thin-layer chromatography (TLC) carried out on precoated plates with 0.2 mm of thickness using Merck silica gel 60 (GF 254 ) with appropriate mobile phases. Compounds were easily detectable at 254 nm or 365 nm.
Flash column chromatography using silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany) was used in the purification of the synthesized compounds. Melting points (m.p.) were measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncorrected. 1 H-and 13 C-nuclear magnetic resonance (NMR) spectra were recorded at the University of Aveiro, Department of Chemistry in CDCl 3 or DMSO-d 6 (Deutero GmbH, Ely, UK) at room temperature on a Bruker Avance 300 spectrometer (300.13 MHz for 1 H and 75.47 MHz for 13 C, Bruker Biosciences Corporation, Billerica, MA, USA). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. Coupling constants are reported in hertz (Hz). 13 C-NMR assignments were made by bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR experiments (long-range C, H coupling constants were optimized to 7 Hz) or by comparison with the assignments of similar molecules. Highresolution mass spectroscopy (HRMS) spectra were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) and recorded as electrospray ionization (ESI) mode in Centro de Apoio Cientifico e Tecnolóxico á Investigación (CACTI, University of Vigo, Pontevedra, Spain) or on an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) at CEMUP, University of Porto, Portugal. The following compounds were synthesized and purified by the described procedures.

Antitumor Activity
The antitumor activity of compounds was accessed through the determination of the GI 50 by the sulforhodamine B assay (SRB, Sigma-Aldrich). Tumor cells were seeded in 96-well plates (0.05 × 10 6 cells/well) in complete culture medium and incubated at 37 • C for 24 h, allowing cell attachment. Cells were then incubated with two-fold serial dilutions of the test compounds, with concentrations ranging from 0 to 75 µM. Dimethyl sulfoxide-treated cells (DMSO), up to 0.25% concentration, were included as compounds solvent control. Forty-eight hours later, cells were fixed with 50% (m/v) trichloroacetic acid (Merck Millipore, Darmstadt, Germany), washed with distilled water and stained with SRB for 30 min at room temperature. The SRB-stained cells were washed 5 times with 1% (v/v) acetic acid (Merck Millipore) and afterwards dried at RT; SRB complexes were solubilized by adding 10 mM Tris-Base buffer (Sigma-Aldrich) for 30 min. Absorbance was measured at 515 nm in a microplate reader (BioTek Synergy 2, BioTek Instruments Inc., Winooski, VT, USA).The concentration that caused cell growth inhibition of 50% (GI 50 ) was determined based on a dose-response curve obtained for each cell line with each test compound.
The non-tumor Human Pulmonary Alveolar Epithelial Cells (HPAEpiC) were used to assess the degree of selectivity of the compounds, as the ratio of GI 50 in non-tumor cells over G I50 in cancer cells. A selectivity index value less than 2.0 indicates the general toxicity of the compound [32].

Photophysical Studies
The absorption spectra in dimethyl sulfoxide solutions were obtained on a Shimadzu UV-2501 PC spectrophotometer (1 cm path length quartz cell) and the emission spectra were recorded on a JASCO spectrofluorometer. Molar absorption coefficients were determined using 4 measurements in the concentration range 10 −3 -10 −6 mol.L −1 . Fluorescence quantum yields were determined by comparison with fluorescein in 0.1 M NaOH water solution as a fluorescence standard (ϕf = 90%) [29].
For flow cytometry analysis, cells exposed to compounds for 48 h were harvested, washed twice in PBS and the mean fluorescence intensity (MFI) of FITC-A was recorded in BD Accuri™ C6 Plus Flow cytometer (BD Biosciences, Qume Drive, San Jose, CA, USA). Cells treated with 1 µM of Rhodamine-123 were used as positive control. A total of 20.000 events per sample were collected. Data were analyzed with BD Accuri TM C6 Plus software, version 1.0.27.1 (www.bdbiosciences.com, accessed in 20 March 2021).

X-ray Crystallography
Diffraction data were collected at 291 K with a Gemini PX Ultra (Rigaku/Oxford, Neu-Isenburg, Germany) equipped with CuKα radiation (λ = 1.54184 Å). The structures were solved by direct methods using SHELXS-97 [33] and refined with SHELXL-97 [33]. Carbon, oxygen, nitrogen and sulfur atoms were refined anisotropically. Hydrogen atoms were either placed at their idealized positions using appropriate HFIX instructions in SHELXL, and included in subsequent refinement cycles, or were directly found from difference Fourier maps and were refined freely with isotropic displacement parameters. Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles and torsion angles have been deposited with the Cambridge Crystallographic Data Centre (CCDC).

Conclusions
In this study, we report the synthesis of an unexpected tetracyclic thioxanthene scaffold, with very sparse descriptions in the literature. Different substitutions at the C-1 position yielded four compounds, whose antitumor activity was studied for three different cell lines. Compounds 11 and 14 have been shown to be the best compounds in the three tested cell lines. The tetracyclic thioxanthenes were also studied for their fluorescence and it has been shown that, the higher the conjugation level, the more emissive and absorbent these compounds are in solution. In order to conjugate these two results, they were studied for their theranostic potential, and it was shown that compound 11 exhibits both high cytotoxic activity and a bright fluorescence property, making it a promising theranostic cancer drug candidate. Its easy intracellular uptake and accumulation at endosome-like structures could be used to target the respective subcellular structure, as a plausible therapeutic intervention to potentiate cancer cell death. Future work is needed to unveil the subcellular localization of the compound and its application to cancer therapy.