Removal of Gaseous Hydrogen Sulfide by a FeOCl/H2O2 Wet Oxidation System

The removal of gaseous hydrogen sulfide using FeOCl/H2O2 was studied. The effects of the FeOCl dosage, the H2O2 concentration, the reaction temperature, and the gas flow rate on the removal of H2S were investigated. The reaction products were analyzed, and the characterization of FeOCl was carried out by X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, and electron paramagnetic resonance spectroscopy. Furthermore, radical quenching experiments were carried out using butylated hydroxytoluene, isopropanol, and benzoquinone. It was found that the H2S removal rate for a H2S gas concentration of 160 ppm reached 85.6% when bubbling through 100 mL of an aqueous solution containing FeOCl (1 g/L) and H2O2 (0.33 mol/L) at 293 K with a flow rate of 135 mL/min. Although the dissolution of chlorine in FeOCl was found to result in reduced catalytic performance, the activity was restored after soaking the catalyst in concentrated hydrochloric acid (37%) and subsequent calcination. The mechanism of H2S removal was also discussed, and it was found that this process was controlled by H2S diffusion. FeOCl was found to activate H2O2 and produce radicals, such as •OH and •O2–, resulting in the formation of a water film rich in radicals on the FeOCl surface. Following the diffusion of H2S into the water film, it underwent oxidation by radicals to produce SO42–. Overall, the catalyst and the product can be effectively separated.

INTRODUCTION
Hydrogen sulfide (H 2 S) is a colorless, flammable, and toxic gas with the smell of rotten eggs, and it is a common impurity in natural gas and syngas. Upon its combustion, H 2 S produces sulfur dioxide, which is released into the atmosphere and leads to air pollution. 1 In addition, in industry, the acidity and corrosiveness of H 2 S can cause serious corrosion to equipment and pipelines, while in daily life, an atmospheric concentration of >500 ppm can lead to a partial loss of human neurological function and can even be fatal. 1−4 Thus, to reduce H 2 S emissions, a large number of H 2 S removal technologies have been developed to date, including adsorption/catalytic desulfurization, 5−17 biodesulfurization, 18 membrane separation, 19,20 and washing desulfurization. 21−31 More specifically, the adsorption desulfurization process is simple and the level of investment required is low; however, the regeneration energy consumption is high, and the adsorbent is easily deactivated. 3,4 In contrast, the biological desulfurization technology can operate at room temperature and pressure, its operating costs are low, and it does not tend to cause secondary pollution; however, in the presence of high concentrations of H 2 S, treatment is slow, and the process efficiency tends to be poor. 18 In addition, the membrane separation technology is easily operated and boasts both a high separation efficiency and a low level of pollution; however, the membranes exhibit poor corrosion resistances and short service lives, in addition to being particularly expensive. 19,20 In terms of the chemical washing desulfurization technology, the operation is also simple, and this process exhibits strong adaptability and a high desulfurization rate, resulting in it being the most widely used and mature technology at present. 3,21,23 Nonetheless, the most commonly used traditional absorbents for this process, including ethanolamine and carbonate, require high amounts of energy for their regeneration. 3 In contrast, the wet (solution) oxidation desulfurization technology does not suffer from issues related to absorbent regeneration, the process and equipment required are simple, and a high sulfur recovery tends to be achieved. As a result, this technology has attracted growing attention in recent years. 3,22,24−30 Based on these advantages, Liu et al. [26][27][28][29][30]32 reported the application of the wet oxidation desulfurization technology in an advanced oxidation system. More specifically, hydrogen peroxide, persulfate, and Oxone were used as oxidants, while Fe 3+ , Cu 2+ , Fe 2+ , and UV irradiation were used to promote the production of radicals. Hydrogen peroxide is a low-cost, widely used chemical that does not require complex safety measures. 3 Moreover, Fe 3+ , Cu 2+ , Fe 2+ , and hydrogen peroxide form a homogeneous Fenton or Fenton-like oxidation system, which can effectively remove gaseous H 2 S. 26,27 However, the recovery of Fe 3+ , Cu 2+ , and Fe 2+ is challenging, and during evaporation crystallization, wherein the final oxidation product (sulfuric acid) is recovered by the addition of ammonia to produce ammonium sulfate, 28,30 the product can become contaminated with these metal elements.
Compared with the homogeneous Fenton system, the heterogeneous Fenton system allows the more facile recovery of the catalyst. Among the various catalysts reported to date for this process, iron oxychloride (FeOCl) has been widely studied in terms of the degradation of organic pollutants present in wastewater due to its high catalytic activity, low raw material costs, and its simple preparation and regeneration. 31,33−39 However, to date, the removal of gaseous H 2 S by the FeOCl/ H 2 O 2 system has yet to be examined.
Thus, we, herein, report a novel desulfurization method wherein gaseous H 2 S is removed by bubbling in a FeOCl/ H 2 O 2 wet oxidation system. The technical feasibility of this new technology and the effects of various process parameters (e.g., the FeOCl dosage, the H 2 O 2 concentration, the reaction temperature, and the gas flow rate) on the removal of H 2 S are studied. In addition, the reaction products and the reaction mechanism of H 2 S removal are studied systematically. It is expected that the results of this study will provide the necessary theoretical basis and data for the development of new H 2 S removal technologies and will promote the practical application of the advanced oxidation technology in the field of gaseous H 2 S removal.

MATERIALS AND METHODS
All analytically pure reagents were provided by Aladdin Industrial Corporation (Shanghai, China), and were used directly as received without any additional treatment.

Catalyst Preparation.
FeOCl was prepared by thermal decomposition as described previously. 38,40 More specifically, ferric trichloride (FeCl 3 ·6H 2 O) was ground in an agate mortar, spread flat on the bottom of a crucible (thickness ≤ 3 mm), and heated to 220°C at a rate of 10°C/min in a muffle furnace. After maintaining this temperature for 2.5 h, the crucible was allowed to cool to room temperature, then washed with deionized water, and finally dried overnight at 60°C in a vacuum drying box to obtain the desired FeOCl as a dark fuchsia powder.
2.2. H 2 S Removal. As outlined in Figure 1, the experimental device employed herein consists of a raw material gas supply system, a reaction system, a gas absorption system, and a waste gas treatment system. The raw gas supply system is composed of 300 ppm H 2 S/N 2 (1), high-purity N 2 (2), and a gas mixing system (3−5). The reaction system is mainly composed of three flasks (7) and a heat-collecting magnetic agitator (10), while the gas absorption system to be tested and the waste gas treatment system were prepared using absorption bottles containing zinc acetate (12,14) and sodium hydroxide (13,15), respectively.
The H 2 S/N 2 gas mixture was prepared by mixing the two individual gases as raw materials. By adjusting the flow rates of H 2 S/N 2 and N 2 , the flow rate of the mixture was changed. A heat-collecting magnetic agitator was used to stir the system and to control the temperature. After mixing the gas evenly and adjusting the reaction temperature to the required value, a catalyst adsorption experiment was carried out using only the catalyst and water (100 mL) in a three-neck flask. Following saturation of the catalyst, the desired amount of H 2 O 2 was added to the three-port flask to allow the catalytic oxidation process to take place. The H 2 S concentrations of the inlet and outlet gases were determined by iodometry 41 after being subjected to absorption by zinc acetate. The tail gas was discharged after passing through the waste gas treatment system. The removal rate of H 2 S was calculated by eq 1. During the reaction time of 120 min, four samples were taken at each 30 min interval and the average value was calculated.  In eq 1, η is the removal rate of H 2 S, C in is the H 2 S inlet concentration, and C out is the H 2 S outlet concentration.
2.3. Characterization. The crystal structure of FeOCl was determined by X-ray diffractometry (XRD, DX-2700, China) with Cu Kα radiation. The scanning angle (2θ) was 5−80°and the step size was 0.02°. The morphology of the FeOCl sample was analyzed by scanning electron microscopy (SEM, FEI Inspect F50). X-ray photoelectron spectroscopy (XPS, Thermo Escalab 250Xi) was employed to determine the element valences and chemical information of the FeOCl surface. The binding energy data were calibrated with the C 1s signal at 284.6 eV. Paramagnetic signals were collected on an electron paramagnetic resonance spectrometer (EPR, Brooke A300, Germany) using 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO) to give the EPR spectra of the samples. The oxidation products of H 2 S were analyzed by ion chromatography 32 (IC, ICS-5000).

RESULTS AND DISCUSSION
3.1. Performance of H 2 S Removal by the FeOCl/H 2 O 2 System. The effects of the amount of an FeOCl catalyst, the H 2 O 2 concentration, the reaction temperature, the gas flow rate, and the reaction time on the removal of H 2 S by the FeOCl/H 2 O 2 system were investigated, and the results are presented in Figure 2.
As can be seen from Figure 2A, when the amount of FeOCl is increased from 0 to 1.0 g/L, η increased from 11.1 to 56.6%, indicating that FeOCl has a catalytic effect on the removal of H 2 S by H 2 O 2 , and so η increases with an increase in the FeOCl dosage. This can be accounted for by considering that increasing the amount of FeOCl will provide additional catalytic active centers, 42 which will produce a greater number of active species, and η increases. However, when the amount of FeOCl was increased further to 2 g/L, η increased only from 56.6 to 58.4%. This was likely due to the fact that an excess of the catalyst led to an agglomeration of catalyst particles, thereby hindering the catalytic action of the reaction active sites. 43 As outlined in Figure 2B, when the H 2 O 2 concentration was increased from 0 to 0.33 mol/L, η increased from 0 to 56.6%, although a further increase in the H 2 O 2 concentration to 1.65 mol/L gave only a slight increase in η, i.e., to 60.7%. This was considered that when the concentration of H 2 O 2 in the reaction system is low, the solid catalyst can fully interact with the available H 2 O 2 , and so the amount of an active material (and thus η) increases with an increase in the H 2 O 2 content. 44 However, on increasing hydrogen peroxide continuously, the amount of hydrogen peroxide was in excess to the catalyst relatively, and the reactive material did not increase with the increase in H 2 O 2 ; in addition, adding excess H 2 O 2 would result in the self-consumption of reactive substances. 43 Thus, for the purpose of subsequent experiments, an initial H 2 O 2 concentration of 0.33 mol/L was selected.
As shown in Figure 2C, upon increasing the temperature from 283 to 293 K, η increased from 43.6 to 56.6%, although a further increase in temperature to 313 K resulted in a slight decrease in η (i.e., from 56.6 to 55.4%). In this context, it should be noted that as the temperature increases, the reaction rate also increases initially. 32 However, the solubility of H 2 S in water is reduced at higher temperatures, and so the reduced concentration of reactants ultimately results in a lower reaction rate.
As can be seen from Figure 2D, when the gas flow rate was increased from 135 to 370 mL/min, η decreased from 85.6 to 20.5%. It should be noted that the initial pH of the reaction system is 3.16, and so H 2 S exists mainly in its molecular form in the aqueous environment (K a1 (H 2 S) = 1.3 × 10 −7 , [HS − ]/ [H 2 S] = 1 × 10 −3.56 ). For their effective removal, the H 2 S molecules must diffuse to the catalyst surface, and so as the gas flow rate is increased, the residence time of H 2 S in the reactor decreases, greater volumes of H 2 S are discharged from the reactor prior to diffusion to the catalyst surface, and η is reduced. These results clearly indicate that the removal of H 2 S is controlled by the diffusion of H 2 S.
As outlined in Figure 2E, when the H 2 S gas was continuously introduced into the reaction system over 540 min, η decreased gradually from 56.6 to 40.1%. This can be attributed to a reduced concentration of H 2 O 2 over time and/ or a loss in the catalytic performance of FeOCl. Generally, the oxidation products of H 2 S could be sulfur 32 and/or sulfur oxides (e.g., SO 3 2− and SO 4 2−4 ); in addition, the amount of ineffective H 2 O 2 decomposition is small under acidic conditions (i.e., pH ∼ 3). 45 Assuming that the consumption of H 2 O 2 is mainly attributed to the oxidation of H 2 S, the oxidation of H 2 S to S 0 consumes the least amount of H 2 O 2 (eq 2), 32 while the generation of SO 4 2− consumes the most amount of H 2 O 2 (eq 3). 32 3.2. Analysis of the Reduced Catalytic Performance of FeOCl. In general, the decreased performances of heterogeneous catalysts can be attributed to the reaction products covering or blocking the catalyst active centers or pores or changes in the composition and the structure of the catalyst. 46 As noted above, the products of H 2 S oxidation by H 2 O 2 are sulfur 32 and sulfur oxides. 4 If sulfur is produced, it can cover the active center of the catalyst or block the catalyst pores of the catalyst, whereas if SO 3 2− and SO 4 2− are produced, the pH of the reaction system may decrease. In addition, the interaction between FeOCl and H 2 S may produce FeS. Thus, after the completion of the reaction (i.e., 540 min), the reaction mixture was separated by centrifugation. In the case where SO 3 2− and SO 4 2− are formed, these components should be present in the supernatant, whereas if S 0 and FeS are formed, they would exist in the solid. The liquid phase (or supernatant) was analyzed by ion chromatography and its pH was determined (Table 1); the solid phase (i.e., "used FeOCl") was dried under vacuum at 333 K and characterized by XRD, SEM, and XPS. As listed in Table 1, after 540 min, the pH of the liquid phase was 2.88, and both SO 4 2− and Cl − were detected. Since only FeOCl, H 2 O 2 , H 2 O, and H 2 S/N 2 were added to the reaction system, Cl − present in the liquid phase must originate from FeOCl. The oxidation of H 2 S to SO 4 2− and the dissolution of chlorine from FeOCl to Cl − resulted in the reduced pH of the reaction system (i.e., from 3.16 to 2.88).
It was then considered whether a reduction in the pH of the reaction system would lead to a η decrease. Thus, dilute sulfuric acid was used to regulate the initial pH of the reaction system, and the effect of the initial pH on the removal of H 2 S was investigated ( Figure 3). As shown, when the initial pH values were 2.00, 2.88, and 3.16, the η values were 61.5, 57.2, and 56.6%, respectively, indicating that between values of 2.88 and 3.16, the pH had little effect on the removal of H 2 S. However, when the pH value was 2, η was slightly higher, and this was attributed to the strong acidity of the solution, leading to the dissolution of Fe from the FeOCl, and the amount of H 2 S removed by the homogeneous Fenton system cannot be ignored. 47 These results indicate that a reduction in the system pH during the reaction process does not result in a decrease in η. Figure 4 shows the XRD patterns and SEM images of FeOCl and used FeOCl. As can be seen from Figure 4A, the intensity of the XRD diffraction peaks of used FeOCl decreased significantly compared to FeOCl. In addition, the twodimensional flake morphology of FeOCl can be clearly seen in Figure 4B, 35 while in the case of used FeOCl, this structure was destroyed to a certain extent. Overall, these observations confirm that the crystal structure of FeOCl was partly destroyed during the reaction. Figure 5 shows the XPS spectra of FeOCl, which allowed the elemental composition and valence distribution of the elements on the catalyst surface to be calculated, as listed in Table 2. From Figure 5A, it is apparent that sulfur was not present in used FeOCl, indicating that little or no S 0 and FeS were produced during H 2 S removal. This observation, therefore, indicates that blockage of the catalyst pores or  covering of the catalyst active centers was not responsible for the decreased catalytic activity of FeOCl. As outlined in Table 2, the contents of chlorine in FeOCl and used FeOCl were 26.99 and 8.37% respectively, indicating the loss of chlorine during the reaction. It can be seen from Figure 5B that the O 1s peaks at 530. 35 49 resulting in a decrease in the chlorine content, an increase in the oxygen content, and a higher Fe−O/H 2 O ratio. It is, therefore, possible that the decreased catalytic activity may be caused by the dissolution of chlorine from FeOCl. It was, therefore, expected that the addition of chlorine to used FeOCl could restore its catalytic activity. 50 Thus, used FeOCl was supplemented with chlorine by soaking in a 37% HCl solution (used FeOCl/HCl = 1 g:10 mL) for 30 min, then calcined at 220°C for 1 h, and washed and dried to obtain the regenerated catalyst, i.e., regenerated FeOCl. 50 As shown in Figure 6A, the XRD peak intensity of the regenerated FeOCl is similar to that of FeOCl. Figure 6B shows that η reached 55.4% (average after 120 min) for regenerated FeOCl, which is similar to FeOCl. These results    43 and it is known that H 2 O 2 can approach this polarization plane more easily than water. 31 Thus, the H 2 O 2 molecules adsorbed on the catalyst active sites can be activated to form hydroxyl radicals ( • OH) and superoxide radicals ( • O 2 − ). 32,40,51−54 To confirm the presence of such radicals and their possible contribution to the reaction mechanism, EPR spectroscopy ( Figure 7) and radical quenching experiments (Figure 8) were carried out on the FeOCl/H 2 O 2 system. In the EPR experiment, the DMPO trap reacts with • OH and • O 2 − in solution to form adducts, resulting in the production of characteristic signals. 55 As can be seen from Figure 7 As can be seen from Figure 5C, two large peaks are visible for the Fe 2p orbitals at 711.05 and 724.46 eV, which correspond to the 2p 3/2 and 2p 1/2 binding energies of Fe III , respectively, 56 while two smaller peaks at 712.9 and 726.12 eV correspond to the 2p 3/2 and 2p 1/2 binding energies of Fe II , respectively, 42 To confirm the participation of radicals in the H 2 S removal process, radical quenching experiments were carried out using butylated hydroxytoluene (BHT). 57 As shown in Figure 8C, upon the addition of BHT, η was reduced from 56.6 to 7.8%, which is a similar value to that obtained in the absence of FeOCl (i.e., 10%). These results indicate that the H 2 S removal process did indeed involve participation from radicals. Subsequently, quenching experiments were carried out using isopropanol (IPA) and benzoquinone (BQ). 54 As shown in Figure 8D,E, upon the addition of IPA and BQ, η decreased from 56.6 to 35.4 and 38.5%, respectively, which further indicated that • OH and • O 2 − were involved in the process of H 2 S removal by the FeOCl/H 2 O 2 system. The chemical reactions involved in this process are given by eqs 8−10, 32 Upon the simultaneous addition of both IPA and BQ ( Figure 8F), η was 24.2%, which is higher than that obtained when BHT was added (i.e., 7.8% η), indicating that other radicals were also involved in the H 2 S removal process. As can be seen from eq 5, HO 2 • is also produced during the catalytic process, but this species is further decomposed to produce • O 2 − (eq 6), and the oxidizability of HO 2 • (oxidation potential 1.51) is significantly weaker than that of • O 2 − (oxidation potential 2.21) and • OH (oxidation potential 2.80). 59 In addition to • OH, • O 2 − , and HO 2

•
, there are other forms of radicals involved in the reaction.
Wang 60 found that the radicals produced by H 2 O 2 activation can be divided into two states, namely adsorbed and free states. IPA is a hydrophilic radical scavenger, which cannot effectively quench adsorbed radicals; 61 BQ is also hydrophilic and it was therefore considered that BQ can only quench free superoxide radicals. Radical quenching experiments show that free radicals are involved in the removal of H 2 S. According to the process of the catalytic reaction, eqs 4 and 13−15 can be given as follows In eqs 13−15, the subscript "ads" denotes radicals in the adsorbed state and "free" denotes radicals in the free state.
According to eqs 4 and 13−15, there are adsorbed radicals in the system. Qian 55 reported that the adsorbed radicals are easy to oxidize the pollutants adsorbed on the catalyst. If H 2 S in the solution can be adsorbed on FeOCl, it shows that the adsorbed radicals are likely to participate in the oxidation of H 2 S. Thus, the adsorption of H 2 S by FeOCl in the aqueous phase was investigated and the adsorption kinetics was analyzed, as shown in Figure 9 and Table 3.
It can be seen from Figure 9A that with an increase in temperature, the solubility of H 2 S in solution decreases, but the adsorption capacity of H 2 S increases. Obviously, the adsorption of H 2 S by FeOCl in solution accords with the characteristics of chemical adsorption. 62 As indicated in the fitting results shown in Figure 9B,C and Table 3, the R 2 of the  pseudo-second-order adsorption kinetics of H 2 S on FeOCl is larger than that of the pseudo-first-order adsorption kinetics at 283, 293, and 303 K, indicating that the pseudo-second-order model can better describe the adsorption process. 2,62 According to the results of kinetic analysis, enthalpy (ΔH) of H 2 S adsorption by FeOCl can be calculated by eqs 16−18 to be 21.5 kJ/mol. Generally, ΔH of the chemisorption is >40 kJ/mol, 63 which indicates that the interaction between FeOCl and H 2 S is relatively weak, and H 2 S could be activated by adsorbing. Therefore, we speculate that the adsorbed radicals were involved in the removal of H 2 S. This may also be the reason that FeS was not generated during the catalysis and sulfur was not detected in the XPS.
In eqs 16−18, K is the adsorption equilibrium constant and q e is the equilibrium adsorption capacity of H 2 S (saturated adsorption capacity, mg/g). According to the results of kinetic analysis, q e = q e2 and c e is the concentration of H 2 S in the aqueous solution at the point of adsorption equilibrium (mg/ g). Since the concentration of H 2 S in the gas phase remains constant (160 ppm) during the adsorption experiment, the value of c e can be obtained according to Henry's law. ΔG is the free energy (kJ/mol), ΔH is the enthalpy (kJ/mol), ΔS is the entropy (J/(mol·K)), R is the gas constant (8.314 J/(mol·K)), and T is the adsorption temperature (K).
3.4. Mechanistic Analysis. In general, the lifetimes of radicals are extremely short, and so they are mainly concentrated in the water film close to the surface of the solid catalyst. 34 In heterogeneous catalytic reactions, the reaction process is generally composed of several processes occurring in series, such as diffusion of the H 2 S to the surface of the catalyst, the adsorption of H 2 S onto the catalyst surface, and conversion of the adsorbed H 2 S. Due to the fact that a water film rich in radicals is present on the surface of FeOCl, and the free radicals can react directly with H 2 S, 32 it is possible that two reaction paths exist for H 2 S: (I) the adsorbed H 2 S is oxidized by adsorbed radicals and (II) the not adsorbed H 2 S is oxidized by free radicals. Based on the abovementioned analysis, a mechanism was established for H 2 S removal by the FeOCl/H 2 O 2 system, as shown in Figure 10. The oxidation product is mainly SO 4 2− , and the diffusion of H 2 S controls the rate of H 2 S removal.
The resulting SO 4 2− can be recovered by the addition of ammonia to produce ammonium sulfate, 32 thereby allowing the effective recovery of the reaction products to achieve zero discharge of liquid waste from our proposed system.

CONCLUSIONS
This paper reports the study on the performance of gaseous H 2 S removal by the FeOCl/H 2 O 2 system. It was found that upon increasing the amount of a catalyst, the H 2 O 2 concentration, and the reaction temperature, the removal efficiency of H 2 S initially increased prior to becoming stable. In contrast, upon increasing the gas flow rate, the removal efficiency of H 2 S decreased, and so it was apparent that the process of H 2 S removal by FeOCl/H 2 O 2 is a diffusioncontrolled process. The removal rate of 160 ppm H 2 S gas reached 85.6% when bubbling through 100 mL of an aqueous solution containing FeOCl (1 g/L) and H 2 O 2 (0.33 mol/L) at 293 K with a flow rate of 135 mL/min. Although the dissolution of chlorine in FeOCl caused a reduction in the catalytic performance, the catalytic activity could be restored by soaking in hydrochloric acid. The main active species involved in the removal of H 2 S were found to be radicals, such as • OH and • O 2 − , which were produced from H 2 O 2 upon catalysis by FeOCl. These radicals were likely present in the adsorbed and free states, and the free radicals were mainly distributed in the water film close to the FeOCl surface. In terms of the process mechanism, H 2 S initially diffuses toward the vicinity of FeOCl or adsorbs onto the catalyst surface. Subsequently, H 2 S is oxidized by free and adsorbed radicals, Figure 10. Schematic representation of the heterogeneous degradation of H 2 S by the FeOCl/H 2 O 2 system. Path I, the adsorbed H 2 S is oxidized by adsorbed radicals, and path II, the unadsorbed H 2 S is oxidized by free radicals. respectively, to give SO 4 2− . Importantly, the catalyst and the product of this system can be separated effectively. To enhance the removal rate of H 2 S further, it is necessary to increase the residence time of H 2 S in the reactor, such as replacing the three-neck flask with a tower reactor. Overall, this process was found to be a simple and efficient method for H 2 S purification, and following scale-up, could be suitable for industrial applications.


Antitumor Activity of Ethyl Acetate Extraction of Wikstroemia chamaedaphne: Cell Cycle Arrest and Apoptosis- Inducing Activity in Melanoma Cells

The ethyl acetate extraction (WCE) of Wikstroemia chamaedaphne Meisn on the viability of melanoma cell lines B16 and A375 were evaluated. The WCE exhibited a potent cytotoxic against B16 and A375 with IC50 values of 156.2 and 192.8 μg/mL, respectively. Cell migration was assessed using a wound healing assay. The WCE caused cell cycle arrest in the G0/G1 phase in B16 cells and the S phase in A375 cells, according to cell cycle analysis. Flow cytometric analysis revealed that WCE promoted apoptosis in B16 and A375 cells in a dosedependent manner. Chemical analysis of WCE resulted in the isolation of a new diterpenoid, wikstchalin A (6) with five known flavonoids (1−5) and two known diterpenoids (7 and 8). Spectroscopic analysis revealed their structures, and CD analysis revealed the absolute configurations of 6. The absolute structure of pimelotide A (7) was firstly confirmed by single-crystal X-ray diffraction. The cytotoxicities of all the compounds (1−8) were evaluated against B16 cell lines. Compound 8 was more active than the positive control, dacarbazine (IC50 300 μM), in terms of cytotoxicity against B16, with an IC50 value of 7.9 μM.

Introduction
Malignant melanoma is one of the most common malignancies. Around 232100 cases of all newly diagnosed primary malignant cancers are cases of cutaneous melanoma, and there are 55500 newly deaths worldwide every year [1]. Malignant melanoma is frequently resistant to chemotherapy and radiotherapy, and advanced melanoma patients have a dismal prognosis, with a median overall survival of 8-10 months and a 5-year survival rate of 6% [2]. Metastatic melanoma is one of the most difficult tumors to treat, imposing a financial burden on both individual patients and society. Despite major breakthroughs in chemotherapy regimens, there has been little change in malignant melanoma mortality during the last several decades. The finding of exact molecular targets for malignant melanoma chemoprevention is believed to be an accessible option for the development of new preventive medicines.
Wikstroemia chamaedaphne Meisn., a toxic shrub native to China, has been used in traditional Chinese medicine to treat edema, cough, hepatitis, schizophrenia, and infertility [3]. Previous chemical investigations on the W. chamaedaphne have resulted the isolation of daphnane diterpenoids [4][5][6], flavonoids [5], and phenolic constituents [7]. As part of our screening program to investigate antineoplastic compounds from this genus [8], ethyl acetate extraction (WCE) of W. chamaedaphne demonstrated cytotoxic activity. The WCE was tested for cytotoxicity against two melanoma cell lines, B16 and A375, respectively. The WCE was found to be moderate activity to the melanoma cell lines B16 and A375, with IC50 values of 156.2 and 192.8 μg/mL, respectively. Chemical investigation of WCE has led to the isolation of a new wikstchalin A (6) with the five known flavonoids (1−5) and two known diterpenoids (7 and 8). Spectroscopic analysis revealed their structures, and CD analysis revealed the absolute configurations of 6. The absolute structures of pimelotide A (7) were firstly confirmed by single-crystal X-ray diffraction. Herein, details of cytotoxic activities of WCE, the isolation, and structural elucidation are described.

General Methods
At 25°C, NMR spectra were collected using a Bruker AM-600 spectrometer. A semi-preparative HPLC Eclipse XDB-C18 column was used in conjunction with an Agilent 1200 series HPLC system. For column chromatography (CC), we used a silica gel (300-400 mesh, Qingdao Haiyang Chemical Co., Ltd.), a C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and a Sephadex LH-20 gel (Amersham Biosciences). All of the solvents were analytical or chromatographic grade (Shanghai J&K Scientific Ltd or Chemical Reagents Company, Ltd.).

Plant Material
W. chamaedaphne flower buds were purchased from Bozhou Jinwan Pharmaceutical Company (Bozhou, People's Republic of China) and collected in November 2019 from Taiyuan, Shanxi Province, People's Republic of China. One of the authors (L. Luo) identified the specimen. A standard sample (SWCZ-201911) was deposited at the Central Laboratory, Seventh People's Hospital of Shanghai University of TCM.

Cell Culture
B16 mouse malignant melanoma and A375 human malignant melanoma cells were provided by the cell bank of the Chinese Academy of Sciences (Shanghai, China). B16 cells were thawed and grown in RPMI-1640 and supplemented with 0.5% penicillin/streptomycin and 10% FBS, while A375 cells were thawed and expanded in DMEM. The cells were incubated at 37 °C with 5% CO2. For the following assays, all cells were harvested during the exponential growth phase.

Cell Viability Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability. Separately, B16 and A375 cells were seeded into 96-well plates at a density of 5×10 3 cells/well and incubated overnight. Following a 24-hour incubation with WCE extract at various concentrations, B16 and A375 cells were treated with MTT (5 mg/mL dissolved in PBS) for an additional 4-hour incubation at 37 °C. Following the dissolution of the formazan crystals in DMSO, the absorbance at 560 nm was measured with a spectrophotometer and the IC50 was calculated using GraphPad 8. All assays were carried out in triplicate.

Morphological Analysis
The AO/PI staining assay was used to investigate the morphological changes that occurred in the cells after WCE treatment. For 24 hours, cells were treated with 100, 200, 300, and 400 μg/mL of WCE, respectively. 300 μM of dacarbazine (DBZ) were used as positive controls. Acridine orange (AO) and propidium iodide were used to stain B16 and A375 cells (PI). The plate was incubated in the dark for 5 minutes before being washed with PBS to remove the unbound dye. An EVOS FL digital microscope (200 objective) was used to capture the cell morphology. All assays were carried out in triplicate.

Wound Healing Assay
B16 and A375 cells (1 × 10 6 cells/well) were seeded and incubated until the plates were full. To assess cell migration, wound lines in the shape of a cross were made in confluent cells by scraping with a plastic 200 μL pipette tip. After wounding, floating cells were washed with PBS and cultured for 48 hours in 2 percent FBS-containing DMEM or RPMI-1640 supplemented with or without WCE. Data were collected as previously described [9]. Specifically, under a microscope, the width of the wound healing was then observed and photographed. All assays were carried out in triplicate.

Cell Cycle Analysis
B16 and A375 cells were harvested and washed twice with cold PBS after 24 hours of WCE or DBZ treatment. The cells were fixed overnight in 75% frozen ethanol at 4°C before being resuspended in 1 mL cold PBS. After adding 500 μL of dye solution, the cells were incubated in the dark at room temperature for 30 minutes before being detected by flow cytometry. All assays were carried out in triplicate.

Cell Apoptosis Analysis
The effect of WCE on cell apoptosis was investigated using Annexin V-FITC apoptosis detection in conjunction with flow cytometry. For 24 hours, B16 and A375 cells were treated with varying doses of WCE or DBZ. After washing with PBS, cells at a concentration of 5 × 10 4 cells/mL were gently mixed with 5 μL Annexin V-FITC and 5 μL PI. Cells were then left at room temperature in the dark for 15 minutes. The cells were then placed in 400 μL binding buffer before being loaded onto the FACS Calibur. FLOW JO 10 was used for additional analysis. As controls, cells were left untreated or stained with Annexin V-FITC or PI alone. All assays were carried out in triplicate.

Cytotoxic Effects of WCE Against Melanoma Cells
An MTT assay was used to assess the WCE's in vitro cytotoxicity against B16 and A375 melanoma cell lines in vitro. WCE was found to have significant cytotoxic effects against B16 and A375, with IC50 values of 156.2 and 192.8 μg/mL after 24 hours of treatment, respectively ( Figure 1).

Morphological Changes in Melanoma Cells Treated with WCE
To confirm the apoptosis-inducing effects of WCE, melanoma cells were examined in fluorescence photomicrographs of B16 and A375 cells stained with acridine orange (AO) after treatment with various concentrations of WCE for 24 hours. After that, the inverted light or fluorescence microscope was used to examine the morphological features of apoptosis. The cells in the control group had normal morphology and were stained green in the nuclei. When the WCE concentration was between 100 and 200 µg/mL, the cell membrane shrank and the nuclear chromatin started to pyknosis, indicating the early manifestation of apoptosis. Most of the nuclei were yellow green and bead-like when the WCE concentration was 300 µg/mL, and the nuclear chromatin was orange red and pyknosis, indicating that many of the cells had entered the late stage of apoptosis. When the WCE concentration was raised to 400 µg/mL, the cell membrane ruptured and the nucleus turned red (Figure 2). Thus, WCE could cause apoptosis in B16 and A375 cells in a dose-dependent manner.

Antimigratory Activity of the WCE in Melanoma Cells
Wound-healing assays were used to investigate the effects of WCE on melanoma cell migration. B16 and A375 cells were treated with different concentrations of WCE for a total of 48 hours to see how it affected migration. The wound was measured in width after incubation and compared to the original ones. WCE's anti-migration activity was assessed using this method of analysis. In B16 and A375 cells, the WCE has equivalent activity to the positive control DBZ (Figure 3).

The WCE Induce Cell Cycle Arrest in B16 and A375 Melanoma Cells
The cell cycle profiles of B16 and A375 cells were analyzed using flow cytometry to further investigate the effects of WCE on the cell cycle. The cells were stained with PI after being treated with WCE at concentrations of 100, 200, 300, and 400 µg/mL for 48 hours. The cells were then flow cytometrically examined to determine their DNA content. When the concentration of WCE was increased, the percentage of cells in G0/G1 phase increased from 58.94% to 59.45%, 62.37%, 69.41 %, and 80.41 %, respectively, while the number of cells in S and G2/M phase decreased gradually ( Figure  4A), indicating that WCE could block the cell cycle in G0/G1 phase to inhibit B16 cell proliferation. The percentage of cells in the S phase increased significantly after WCE treatment, while the percentage of cells in the G0/G1 and G2/M phases decreased significantly ( Figure 4B). S phase values increased from 23.14% to 31.17%, 43.05%, 45.17%, and 45.63%, respectively, when compared to the control group, indicating that WCE could block the cell cycle in S phase to inhibit the proliferation of A375 cells.

The WCE Induce Apoptosis in B16 and A375 Melanoma Cells
The percentage of apoptotic cells was determined using flow cytometry and Annexin V-FITC/PI double staining. As shown in Figure 5, cells considered viable were annexin V-FITC and PI negative, cells in early apoptosis were annexin V-FITC positive and PI negative, and cells in late apoptosis or already dead were both annexin V-FITC and PI positive. The percentages of total apoptotic cells in B16 were 27.35 %, 45.38%, 69.18%, and 81.03% after 24 hours of treatment with 100, 200, 300, and 400 μg/mL, respectively, and the percentages of total apoptotic cells in A375 were 36.54%, 53.26%, 70.59%, and 74.81 %. These findings revealed that WCE induced apoptosis in B16 and A375 cells was dosedependent.

Cytotoxic Effects of the Isolates
The MTT assay was used to assess the in vitro cytotoxicity of all isolated compounds 1−8 against B16 cell lines. Compound 8 was discovered to be the most active compound, with an IC50 value of 7.9 μM, compared to the positive control DBZ against B16, which had an IC50 value of 300 μM.

Conclusion
The WCE was found to be highly cytotoxic to the melanoma cell lines B16 and A375, with IC50 values of 156.2 and 192.8 µg/mL, respectively. In an acridine orange and propidium iodide (AO/PI) staining assay, WCE-treated B16 and A375 cells showed typical morphological changes of cell apoptosis. To assess cell migration, a wound healing assay was used. WCE induced cell cycle arrest at the G0/G1 phase in B16 cells and the S phase in A375 cells, according to cell cycle analysis with propidium iodide staining. WCE induced apoptotic events in B16 and A375 cells in a dose-dependent manner, according to flow cytometric analysis using Annexin V-FITC and propidium iodide staining. These findings strongly suggested that WCE could be developed further as a potential antitumor agent. Chemical analysis of the WCE resulted in the isolation of a new wikstchalin A (6), in addition to the five known flavonoids (1−5) and two known diterpenoids (7 and 8). Single-crystal X-ray diffraction was used to confirm one of the diterpenoids, pimelotide A (7). All compounds were tested for cytotoxicity against the B16 cell line, and compound 8 inhibited B16 with an IC50 values of 7.9 μM, making it more active than the positive control, DBZ (IC50 = 300 μM). However, the mechanism of compound 8's inhibition of B16 cell line proliferation requires further investigation.


s2fieldsofstudy: Chemistry Biology

text:
New tricyclic systems as photosensitizers towards triple negative breast cancer cells

Nineteen pyrrolo[1,2-h][1,7]naphthyridinones and pyrido[2,3-c]pyrrolo[1,2-a]azepinones were synthesized as new tricyclic systems in which the pyridine ring is annelated to the 6,7-dihydroindolizin-8(5H)-one and 5,6,7,8-tetrahydro-9H-pyrrole[1,2-a]azepine-9-one moieties to obtain potential photosensitizing agents. They were tested for their photoantiproliferative activity on a triple-negative breast cancer cell line, MDA-MB-231, in the dark and under UVA light (2.0 J/cm2). We demonstrated that their toxicity, only when exposed to light, was primarily due to the generation of reactive oxygen species while their photodegradation products were not responsible for their activity. The most active compounds exhibited photocytotoxicity with IC50 values at low micromolar level inducing a decrease in the intracellular content of thiol, thus triggering cancer cell death through apoptosis. All the pyridone derivatives revealed to be pure photosensitizers with preferential photocytotoxic activity towards cancerous over healthy cells. Altogether, the results obtained confirm pyrrolo[1,2-h][1,7]naphthyridinones and pyrido[2,3-c]pyrrolo[1,2-a]azepinones as promising photosensitisers against triple-negative breast cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s12272-022-01414-1.

Introduction
Cancer represents one of the most challenging tasks in drug discovery, and despite several progress have been done, many efforts are still needed. Among the different kind of applications in the treatment of cancer and benign tumours, the use of light is considered a promising non-invasive modality with high selectivity towards malignant target versus normal cells. It can be selectively applied onto a region by illuminating the specific lesion using light with a proper wavelength to activate a tumour-targeted photosensitizer (PS). The latter, by in situ generation of highly cytotoxic reactive oxygen species (ROS), will be able to selectively destroy tumour tissue offering much lower toxic side effects. Over the years there has been a wide number of studies investigating different types of PSs, but the only approved one for clinical use have a tetrapyrrole backbone, such as porphyrins and their analogues. However, their large size is often related to some challenges such as limited tumour specificity and in some cases very important photosensitivity reactions caused by PS accumulation in off-target tissues. Small molecules show several advantages as they typically possess fast distribution throughout the body, rapid clearance, and target accumulation at the tumour site. During our studies aiming at the development of new bioactive heterocyclic compounds (Spanò et al. 2021a, b;Barreca et al. 2022b;Cilibrasi et al. 2022;Labbozzetta et al. 2022), we explored several classes of PSs, and some of them showed high potency, with outstanding selectivity index versus Abstract Nineteen pyrrolo [1,2-h] [1,7]naphthyridinones and pyrido [2,3-c]pyrrolo[1,2-a]azepinones were synthesized as new tricyclic systems in which the pyridine ring is annelated to the 6,7-dihydroindolizin-8(5H)-one and 5,6,7,8-tetrahydro-9H-pyrrole[1,2-a]azepine-9-one moieties to obtain potential photosensitizing agents. They were tested for their photoantiproliferative activity on a triple-negative breast cancer cell line, MDA-MB-231, in the dark and under UVA light (2.0 J/cm 2 ). We demonstrated that their toxicity, only when exposed to light, was primarily due to the generation of reactive oxygen species while their photodegradation products were not responsible for their activity. The most active compounds exhibited photocytotoxicity with IC 50 values at low micromolar level inducing a decrease in the intracellular content of thiol, thus triggering cancer cell death through apoptosis. All the pyridone derivatives revealed to be pure photosensitizers with preferential photocytotoxic activity towards cancerous over healthy cells. Altogether, the results obtained confirm pyrrolo [1,2-h] [1,7]naphthyridinones and pyrido [2,3-c]pyrrolo[1,2-a]azepinones as promising photosensitisers against triple-negative breast cancer. tumour cells inducing mitochondrial apoptotic death (Spanò et al. 2017). We recently reported our studies on two classes of small heterocyclic compounds, namely pyrimido [5,4-g] indolizines 1 and pyrimido [4,5-c]pyrrolo[1,2-a]azepines 2 ( Fig. 1) and their antiproliferative activity against the highly refractory triple negative human breast cancer (MDA-MB-231) and vesical human tumour (T24) cell lines, in the dark and under UVA light irradiation. Results indicated very promising antitumour effect, with IC 50 values up to nanomolar level and strong induction of reactive oxygen species (ROS) and a thiol redox stress responsible of apoptotic cell death (Barreca et al. 2022a). However, some compounds belonging to these series showed activity already in the dark, which is considered not optimal for their use as PSs. In order to extend our insight around their chemical space and inspired by our previous studies indicating the pyridine-2-one moiety as a valuable structural feature within our photoactivable compounds (Barraja et al. 2006(Barraja et al. , 2010(Barraja et al. , 2011Spanò et al. 2017), we decided to explore the effect of the replacement of the pyrimidine moiety generating new classes of tricyclic compounds containing the indolizine and azepine structures incorporating the pyridine-2-one moiety. The choice was supported by our previous evidences related to pyrroloquinolinones 3 and their positional isomers 4, 5 ( Fig. 1) which were able to produce phototoxicity in the submicromolar-micromolar range (IC 50 0.4-17.5 μM, 0.2-17.5 μM and 0.5-9.3 μM, respectively), high ROS level with the involvement of mitochondria and lysosomes (Barraja et al. 2006(Barraja et al. , 2010(Barraja et al. , 2011Spanò et al. 2017) without generating DNA strand breaks and DNA oxidative damages which represents a great disadvantage in this kind of applications. The enlargement of the central ring of the latter class of derivatives led to pyrrolo[3',2':6,7]cyclohepta[1,2b]pyridines 6 ( Fig. 1), which maintained phototoxic activity at submicromolar-micromolar level (EC 50 0.25-10.70 μM). It was demonstrated that they were able to trigger a rapid apoptotic process through the induction of ROS production with a clear involvement of mitochondrial disfunction and lysosomes.
In this paper we wish to report the synthesis and the evaluation of the photoantiproliferative activity of the new tricyclic systems pyrrolo[1,2-h][1,7]naphthyridinones 7 and pyrido[2,3-c]pyrrolo[1,2-a]azepinones 8 (Fig. 1) and their evaluation as PSs against a very difficult type of breast cancer (BC) to be treated, the triple-negative breast cancer (TNBC).
BC can be described as an etiologically and clinically heterogeneous group of tumours originating from mammary epithelial cells (Lüönd et al. 2021). They can be divided into five molecular subtypes: luminal A, luminal B, HER2enriched, basal-like (also known as triple-negative breast cancer) and normal-like breast cancer (Ali et al. 2020). These subtypes show differences in incidence, age at diagnosis, prognosis, therapeutic response, and disease progression (Liu et al. 2018). TNBC is an invasive breast carcinoma and represents around 15-20% of newly diagnosed breast   (Criscitiello et al. 2012). TNBC is associated with young age at diagnosis (< 40 years) and early metastases despite optimal adjuvant treatment. The metastatic behaviour is predominant in the lungs, central nervous system, and liver (Bergin and Loi 2019). Significant heterogeneity exists within the TNBC class, contrary to the basal-like subtype cancers that present a similar gene expression (Yao et al. 2017). Despite differences within this group, all TNBC subtypes are characterized by the absence of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression (Sporikova et al. 2018). Therefore, among all BC subtypes, TNBC has the most limited therapeutic options (Tong et al. 2018) as patients with TNBC do not benefit from therapies that are designed to target the above biomarkers (Tong et al. 2018). All the new synthesized derivatives were tested on TNBC cells, namely MDA-MB-231 human cancer cells, in the dark and upon UVA light. Furthermore, in order to assess their selectivity against tumour cells, all derivatives were additionally screened on a non-transformed human cell line (HEK293 cells). Finally, selected mechanistic investigations were also performed with the aim to deeply characterize their mechanism of action and to evaluate the cell death pathway triggered by the most representative compounds.

Chemistry
All melting points were taken on a Büchi melting point M-560 apparatus. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1 H and 13 C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d 6 or CDCl 3 solution using a Bruker Avance II series 200 MHz spectrometer. Column chromatography was performed with Merck silica gel (230 − 400 mesh ASTM) or a Büchi Sepacor chromatography module (prepacked cartridge system). Elemental analyses (C, H, N) were within ± 0.4% of theoretical values and were performed with a VARIO EL III elemental analyzer. The purity of all the tested compounds was > 95%, determined by HPLC (Agilent 1100 series).
Compounds 9a-e were prepared according to published procedure (Barreca et al. 2022a).

General procedure for the synthesis of N-methyl derivatives (7d-f and 8c, d) and O-methyl derivatives (7g-i and 8e, f)
To a solution of derivatives 7a-c and 8a, b (15 mmol) in anhydrous DMF (20 mL), NaH (Sigma-Aldrich, Schnelldorf, Germany) (0.64 g, 16 mmoli) was added at 0 °C and the reaction mixture was stirred at room temperature for 6 h. Then, iodomethane (Sigma-Aldrich, Schnelldorf, Germany) (16 mmol) was added at 0 °C and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was poured onto crushed ice. The precipitate was filtered off and dried, in absence the solution was extracted with ethyl acetate (3 × 30 mL). The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure. The crude product, containing N-methyl substituted derivatives 7d-f and 8c, d and O-methyl 7g-i and 8e, f, was purified by column chromatography (DCM/AcOEt 95:5).

Biology
All tested compounds were dissolved in the minimum DMSO amount prior to cell culture testing. A calculated amount of the stock DMSO solution was added to the cell culture media to reach a final maximum DMSO concentration of 0.5%, which had no discernable effects on cell viability.

Light exposure
The irradiation device used for the irradiation of cell cultures during the assays was a black box equipped with two Philips UVA lamps with an irradiance of 17 mW cm −2 and a centered emission peak at 365 nm; UVA irradiance was measured using a Waldmann Variocontrol radiometer, equipped with a UVA sensor. The box was maintained at room temperature inside with a fan and the multiwell plates were placed onto a water-refrigerated metal block.

MTT assay
Cells (5-8 × 10 3 cells/well, dependent upon the growth characteristics of the cell line) were seeded in 96-well microplates in growth medium (100 μL). After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. For photocytotoxicity studies, after 1 h of incubation the medium was replaced with PBS and cells were treated with 2.0 J cm −2 of UVA light. After irradiation, PBS was replaced with complete proliferation media. For pre-irradiation studies, instead, compounds in PBS were irradiated with 2.0 J cm −2 of UVA light and subsequently cells were treated with pre-irradiated solutions. Following 24 h, each well was treated with 10 μL of a 5 mg/mL MTT saline solution, and following 5 h of incubation, 100 μL of a sodium dodecyl sulfate (SDS) solution in HCl 0.01 M were added. After an overnight incubation, cell growth inhibition was detected by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted against drug concentration. IC 50 values, the drug concentrations that reduce the mean absorbance at 570 nm to 50% of those in the untreated control wells, were calculated by the four-parameter logistic (4-PL) model. Evaluation was based on means from at least four independent experiments.

Mitochondrial Membrane Potential (ΔΨ)
The ΔΨ was assayed using the Mito-ID® Membrane Potential Kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY, USA) as previously described. Briefly, MDA-MB-231 cells (8 × 10 3 per well) were seeded in 96-well plates; after 24 h, cells were washed with PBS and loaded with Mito-ID Detection Reagent for 30 min at 37 °C in the dark. Afterwards, cells were incubated with increasing concentrations of tested compounds for 90 min and after that exposed to UVA light (2.0 J/cm 2 ). CCCP (Carbonyl cyanide 3-chlorophenyl-hydrazone) was used as positive control. Fluorescence intensity was estimated using a VICTOR X3 (PerkinElmer, USA) plate reader at 490 (excitation) and 590 nm (emission).

ROS production
The production of ROS was measured in MDA-MB-231 cells (10 4 per well) grown for 24 h in a 96-well plate in RPMI medium without phenol red (Merk, Germany). Cells were then washed with PBS and loaded with 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H 2 DCFDA) (Molecular Probes-Invitrogen, Eugene, OR) for 45 min, in the dark. Afterwards, cells were washed with PBS and incubated with tested compounds (25 µM) for 90 min and after that exposed to UVA light (2.0 J/cm 2 ). Fluorescence increase was estimated utilizing the wavelengths of 485 nm (excitation) and 527 nm (emission) in a VICTOR X3 (PerkinElmer, USA) plate reader. Antimycin (3 μM, Merk), a potent inhibitor of Complex III in the electron transport chain, was used as positive control.

Quantification of thiols
The MDA-MB-231 cells (2 × 10 5 ) were seeded in a six-well plate in growth medium (4 mL). After 24 h, cells were incubated with tested compounds (25 µM) for 90 min, and after that exposed to UVA light (2.0 J/cm 2 ). Subsequently, after 24 h, the thiol content was measured as previously described (Rigobello et al. 2009).

Generation of active oxygen species
Singlet oxygen determination. Samples containing the compounds under examination (2.2 × 10 -5 M), p-nitrosodimethylaniline (4 × 10 -5 M) and imidazole (4 × 10 -5 M) in 0.02 M phosphate buffer (pH 7.3) were irradiated with increasing UVA doses and their absorbance at 440 nm was then measured (Kraljic and El Mohsni 1978). The data were expressed as percentage of RNO bleaching.

Cellular biodistribution studies
MDA-MB-231 cells were seeded into 4-well tissue-culture slides (BD Falcon, Bedford, MA, USA) at 2.5 × 10 4 cells/well (0.4 cm 2 ). After 24 h, after 1 h of incubation the medium was replaced with PBS and cells were treated with 2.0 J cm −2 of UVA light. After irradiation, PBS was replaced with complete proliferation media. Samples were examined at 20 × magnification in a Zeiss LSM 800 confocal microscope using the Zeiss ZEN 2.3 software system.

Statistical analysis
Data are expressed as mean ± the standard deviation (SD) of the mean. The difference between groups was evaluated using analysis of variance (ANOVA) with Graphpad Prism 9.0.
The latter compounds were reacted with an excess of N,Ndimethylformamide dimethylacetal (DMFDMA) in N,Ndimethylformamide (DMF) to yield enaminoketones 9a-e in good yields (56-87%) (Scheme 1). These kinds of intermediates have been widely used by us, as they are highly reactive and prone to react with dinucleophyles furnishing several opportunities of annelation. In particular, by reaction with cyanomethylene compounds, the pyridine-2-one ring closure can be achieved. We chose to react enaminoketones 9 with phenylsulfonylacetonitrile, as the phenylsulfonyl group demonstrated crucial for the activity in our previous studies, leading to the desired tricyclic derivatives 7a-c and 8a, b (54-71%) (Scheme 1, Table 1) which were further subjected to methylation at the pyridine ring using iodomethane and NaH as the base in DMF, allowing the isolation of the corresponding N-methyl 7d-f and 8c, d (51-57%) and O-methyl substituted 7g-i and 8e, f (35-46%) derivatives from the same reaction mixtures (Scheme 1, Table 1). Additionally, compounds 7a-c and 8a, b were subjected to bromination of the pyrrole ring with the aim of evaluating the effect of the presence of bromine atom, usually responsible of the so called "heavy atom effect", on cyto-and phototoxicity. Reactions were carried out using bromine as brominating agent, leading to pyrrolonaphthyridinones 7j, k and pyridopyrroloazepinones 8g, h (63-90%) (Scheme 1, Table 1). From the reaction of derivative 7a, it was not possible to isolate the desired bromo derivative as pure product for biological screenings.
Interestingly, all compounds were completely inactive in the dark and some of them behaved as pure PSs, being effective only upon irradiation. Within the pyrrolo[1,2-h][1,7] naphthyridinone series, derivatives 7b and 7h-k showed a detectable cytotoxicity and, in particular, compound 7h and 7i elicited IC 50 values in the low micromolar range (IC 50 4.4 and 7.9 μM, respectively). Considering the pyrido[2,3-c] pyrrolo[1,2-a]azepinone series, however, compounds 8c and 8e-g were less effective in inhibiting cancer cell viability upon irradiation, with IC 50 values in the micromolar range (IC 50 15.3-24.9 μM). It is of worth noting that the O-methyl COOEt substituted derivatives 7h and 8e were the most effective compounds among the two series.
In addition, as one of the main drawbacks of chemotherapeutics is the undesired cytotoxic effect toward noncancerous cells, we also evaluate the selectivity of the newly developed compounds against cancer cells with respect to non-cancer ones. To this attempt, compounds were screened against a human non-transformed, highly replicating cell line, the human embryonic kidney 293 (HEK293) cells.

Cell-free and in cells photogeneration of ROS
To corroborate the above reported hypothesis and to confirm the ability of all derivatives to form ROS when UVA irradiated, experiments in both cell-free and MDA-MB-231 human TNBC cells were performed. Concerning cell-free experiments, two in solution assays for singlet oxygen and superoxide anion have demonstrated to be highly predictable for many photosensitizing molecules (Kraljic and El Mohsni 1978;Pathak and Joshi 1984;Dalla Via et al. 2015;Zhang et al. 2021).

Cell-free ROS generation
With the two assays shown below, it is possible to distinguish the capacity of the test compounds of producing singlet oxygen over superoxide anion and therefore predict their behaviour as photosensitizers.
The percentage of RNO photobleaching of tested compounds under increasing light doses, which is a measure of singlet oxygen generation, is shown in Fig. 2. The majority of the compounds, with the exception of 7a and 7c, were able to generate singlet oxygen in a light dose-dependent manner (0-20 J/cm 2 ), thus attesting that a higher UVA dose increased the singlet oxygen concentration by these compounds and, as a consequence, led to stronger oxidation of RNO/higher percentage of photobleaching. Among the two series, derivatives 7j and 7k as well as 8g and 8h were the ones with a higher percentage of RNO photobleaching, thus indicating their greater ability of singlet oxygen generation.
Additionally, in Fig. 3 the changes in the absorbance values of NBT at 560 nm under increasing light doses are depicted. Some of the compounds only registered the production of superoxide anion under specific light dose(s) rather than under the whole light dose range. Compounds 7j, 7k, 8d, 8f, and 8h production was not reported since they did not produce superoxide anion at any light dose. Likely, superoxide anion is a free radical with a short halflife, making its measurement more difficult than singlet oxygen especially when produced in very low amount. Among all, the most effective in generating a high concentration of superoxide anion was compound 7a. Moreover, from a biological point of view, this radical species despite its high reduction potential of + 0.94 V, can oxidize very few biological compounds (D'Autréaux and Toledano 2007; Krumova et al. 2016), thus O 2˙− is less effective in destroying cell Cells (8 × 10 3 mL −1 ) were pre-treated for 1 h increasing concentrations of the tested compounds, irradiated with UVA light (2.0 J/cm 2 ) in the case of UVA irradiated samples, and recovered for total 24 h. components than singlet oxygen and therefore less powerful in provoking a general toxicity against treated cancer cells (see cell viability experiments).

Intracellular ROS generation
In order to understand if the intracellular action mechanism of our compounds was dependent on ROS production in intact cancer cells, the most representative compounds 7h and 8e were evaluated for their ability to increase intracellular basal ROS production in TNBC cells. With this kind of assay, all reactive oxygen species are detected without distinction among singlet oxygen, superoxide, hypochlorite, nitric oxide and hydrogen peroxide. ROS production under light (UVA, 2.0 J/cm 2 ) was evaluated in MDA-MB-231 cell samples treated with increasing concentrations of tested compounds or antimycin, a potent inhibitor of Complex III of mitochondria electron chain, used as a positive control under the same experimental conditions (Fig. 4).
Treatment of MDA-MB-231 cells with 7h and 8e determined the highest increase in cellular ROS production, in a time-dependent manner (Fig. 4A). Remarkably, cells treated with 7h showed a hydrogen peroxide content comparable to that induced by antimycin. Conversely, compounds 7e and 8d were hardly effective in inducing any ROS basal level production increase.
Coherently, intracellular thiols were significantly decreased by treatment with 7h and 8e in a time-dependent manner. Interestingly, 7h was much more effective than 8e, being able to decrease intracellular sulfhydryl content of about 45% after 24 h of exposure (Fig. 4B).

Mitochondrial membrane potential loss and apoptotic cell death
Induction of ROS production can in turn prompt the collapse of mitochondrial membrane potential as well as loss of mitochondrial integrity and functioning. We hence evaluated As summarized in Fig. 5A, a significant increase of cells with depolarized mitochondria was observed after treatment with compounds 7h and 8e which was like that exerted by the positive control CCCP (Carbonyl cyanide 3-chlorophenyl-hydrazone). On the contrary, treatment with 7e and 8d did not induce a substantial change in mitochondrial membrane potential of TNBC cells.
Loss of mitochondrial transmembrane potential is a crucial step in the activation of intrinsic apoptotic cell death. Hence, the ability to induce apoptosis by representative compounds 7h and 8e was assessed in human TNBC cells. MDA-MB-231 cells were treated with IC 50 doses of tested compounds and apoptotic cell death induction was confirmed through a Hoechst 33342 staining assay.
As reported in Fig. 5B, cells treated with 7h and 8e, as compared with control untreated cells, were positive to Hoechst 33342 staining, and displayed chromatin condensation and fragmentation as well as pyknotic nuclei, typical features of apoptosis. On the other hand, treated cells were poor stained with PI, thus indicating the absence of necrosis induction.

Photostability experiments
The most significant 7h and 8e pyridone derivatives were tested for their stability under UVA irradiation. These data might be useful to understand the mechanism of action of the compounds apart from the production of ROS. As shown by the changes of their UV-Vis spectra under increasing UVA doses (Fig. 6), compound taken in the dark = 0 J/cm 2 = red spectrum; compound under 20 J/cm 2 of UVA light = light green spectrum), the tested compounds were very unstable under light and behaved very similarly. Indeed, their main absorption peak gradually decreased under irradiation accompanied by a blue shift of the maximum and the change of its shape, suggesting the loss of the original structure. Moreover, isosbestic points were visible, indicating the formation of one or more new stable species in equilibrium with the intact dark compound.
In addition, to test the role of the photodegradation products on the photocytotoxicity profiles of the most representative derivatives 7h and 8e, the cytotoxic activity of the photodegraded mixture was evaluated by treating TNBC cells with the corresponding pre-irradiated (20 J/cm 2 ) PBS solutions. No cytotoxic activity was evidenced (IC 50 over 50 µM) clearly attesting that the photodegradation products did not frankly contribute to the cytotoxic activity of the test compounds; rather, these compounds lose their activity when photodegraded and suggest that they mainly act by inducing intracellular ROS.
By roughly calculating the decrease of absorbance of the main UV-Vis peaks (data non shown), the photodegradation was very weak under the dose of light used during the cytotoxic experiments (2 J/cm 2 , blue line in Fig. 5) and the yield of photoproducts formed able to participate to the activity of 7h and 8e was most likely not significant for their contribution in their general cytotoxicity. Anyway, even in very high amount (after 20 J/cm 2 of UVA, light green line in Fig. 5), they were not active at all as discussed above.

Cellular accumulation and biodistribution studies
With the aim of correlating cellular phototoxic activity with intracellular accumulation of tested compounds, we performed some preliminary in cell biodistribution studies. By exploiting their fluorescence characteristics, the most effective compounds 7h and 8e, as well as a completely ineffective derivative, namely 8h, were evaluated for their ability to enter MDA-MB-231 TNBC cells and localize into different cellular compartments by confocal microscopy analysis.
As evident form micrographs reported in Fig. 7, the most photocytotoxic compounds 7h and 8e were able to significantly enter cancer cell membrane and accumulate in cytoplasm. Compounds preferentially localize in the perinuclear space, with minimal accumulation in the periplasmatic site. Conversely, compound 8h was barely effective in accumulating into cancer cells, and only low levels of fluorescence were detected in the cytoplasm of treated cancer cells.
Overall, these cellular accumulation studies clearly support the hypothesis that the cell killing inactivity of compound 8h can be explained by taking into consideration its scarce efficacy to permeate TNBC cells. Despite behaving as PS in cell-free experiments, 8h was found completely ineffective in inhibiting cancer cell proliferation upon irradiation.

Discussion
Photochemotherapy broadly refers to a treatment method that uses a photosensitizer (PS) activated by a selected light irradiation. The combination of both produces a better effect than the use of one of these treatments alone (Ledo and Ledo 2000). This therapeutic technique comprises PUVA (psoralen-UV-A) therapy, in which a psoralen derivative damages DNA of proliferating cells under the exposure to ultraviolet A (UV-A) radiation (Schneider et al. 2008) and photodynamic therapy (PDT) that relies on the red-light activation of a porphyrin-like derivative that generates reactive oxygen species leading to the destruction of unwanted cells and diseased tissues (Benov 2015). The lack of toxicity of the above photosensitizers in the dark and the selective illumination of the targeted area avoids the general toxicity that is one of the main drawbacks of the conventional chemotherapy against cancer. Therefore, the ideal PS would be Fig. 6 UV-Vis Absorption spectra of 7h (left) and 8e (right) in PBS irradiated under 0, 2, 5, 10, 15 and 20 J/cm 2 with UVA light. 0 J/cm 2 , red spectrum; 20 J/cm 2 , light green spectrum a stable drug, with specific uptake by the targeted tissues, minimal dark cytotoxicity, strong absorption at relatively long wavelengths (600-800 nm) at which the light can penetrate efficiently into the tissues, rapid clearance to avoid phototoxic side effects, being amphiphilic, chemically pure and easy to access or synthesize (Dias et al. 2021). In the case of PS activated by less penetrating shorter wavelengths, such as psoralen derivatives activated by UVA light and, as demonstrated more recently, even by blue light (Sturaro et al. 2021), their application against proliferating cells contemplates the treatment of skin diseases, i.e. psoriasis and neurodermitis, or an extracorporeal treatment, the so called extracorporeal photopheresis (ECP) against some immunological disorders, i.e., cutaneous T cell lymphoma and graft versus host disease (Drexler et al. 2020). Although a big progress has been done towards the understanding of TNBC's biology and the development of more efficient treatment regimens, drug resistance, strong side effects and episodes of recurrence are still common among patients with this aggressive disease. Intensive research has been conducted to uncover a new therapeutical approach which maintains a patient's quality of life and has a more localized action.
Quinazoline derivatives arose from this diligent search as a promising direction in the treatment of this life-threatening cancer disease. Their enormous potential is the motivation for this study, whose aims consisted of testing the photocytotoxic activity of nineteen newly synthesised photosensitizing pyridone derivatives on a TNBC cancer cell line (MDA-MB-231) under UV-A light and study their ability to produce ROS as the main mechanism of their action.
The demonstrated antiproliferative activity in the micromolar range of some of these compounds in combination with a small non-toxic dose of UVA light (2.0 J/cm 2 ), their inactivity in the dark against both normal and cancer cells, their apoptotic mechanism of cell death make some of these compounds as a very promising alternative to the conventional and often not effective therapies against TNBC. Interestingly, all the compounds presenting a bromine as substituent at the pentatomic ring were able to generate high yields of singlet oxygen. This is in line with the effect of the heavy atom in the molecular structure which increases the spin orbit coupling between singlet and triplet states, leading to more efficient ISC with respect to the non-halogenated compounds (Lai et al. 2021). Unfortunately, these compounds Fig. 7 Intracellular accumulation studies. MDA-MB-231 TNBC cells were treated with 7h, 8e and 8h upon irradiation and live samples were examined at × 20 magnification by a confocal microscope bearing the heavy halogen bromine atom as inducer of singlet oxygen production could not enter the cells so efficiently and their potential higher activity with respect the other compounds was lost in the presence of cancer cells. Indeed, the localization of the compounds inside the cells confirmed this assumption.
It must be noted that, as in the case of PUVA, we evaluated the toxicity of these compounds under UVA light, where all absorbed significantly; but unfortunately, UVA light penetrates the tissues much less than the red light used in PDT. Theoretically, PUVA therapy could not be used to treat solid internal cancers or deep skin cancers as PDT. However, a direct irradiation of the diseased area in the presence of our compounds to get a clean tissue during and after the surgical removal of the tumour could be suggested. Moreover, for superficial breast cancers the use of a catheter for the instillation of the photoactive principle followed by irradiation through a probe, equipped with a tip emitting UVA light, could be another option for these potential phototherapeutic compounds.