Mn(III) porphyrins as photosensitizers: structural, photophysical and anticancer studies

Abstract

Herein, we synthesized, characterized and explored the photo-triggered anticancer activity of five Mn(III) porphyrins Mn1–Mn5, viz. (diaqua)meso-(tetraphenylporphyrinato)manganese(III) propionate, [Mn(III)TPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn1; (diaqua)meso-tetrakis(4-methylphenylporphyrinato)manganese(III) propionate, [Mn(III)TMeP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn2; (diaqua)meso-tetrakis(4-methoxyphenylporphyrinato)manganese(III) propionate, [Mn(III)TMP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn3; (diaqua)meso-tetrakis(4-fluorophenylporphyrinato)manganese(III) propionate, [Mn(III)FTPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn4 and (diaqua)meso-tetrakis(4-chlorophenylporphyrinato)manganese(III) propionate, [Mn(III)ClTPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn5, which remain virtually unexplored as photodynamic therapy (PDT) agents like other paramagnetic metalloporphyrins. These Mn(III) porphyrins, bearing different para-substituents on their meso-phenyl rings and two water molecules as axial ligands, were characterized using spectroscopic techniques and structurally through single-crystal X-ray diffraction, revealing an octahedral MnN₄O₂ geometry. Binding studies demonstrated a strong affinity of the metalloporphyrins for human serum albumin (HSA), indicating their potential for biological applications. The visible light-assisted generation of reactive oxygen species (ROS) by these Mn(III) porphyrins was confirmed via 1,3-diphenylisobenzofuran (DPBF) titration, identifying singlet oxygen (¹O₂) as one of the primary ROS. Photoredox activity under visible light, displayed by the Mn(III) porphyrins in the presence of ascorbic acid involving +3 and +2 oxidation states of manganese, further underscores the photochemotherapeutic potential of Mn1–Mn5. The ROS generation ability was further validated intracellularly in HeLa cells using Mn4 with the help of 2′,7′-dichlorofluorescein diacetate (DCFDA) assay under visible light irradiation (λ = 400–700 nm). Furthermore, among the five Mn(III) porphyrin complexes (Mn1–Mn5) evaluated for photo-triggered anticancer activity using MTT assays, Mn4 exhibited superior photocytotoxicity, with a half-maximal inhibitory concentration (IC₅₀) of 4.93 ± 0.7 μM against HeLa cancer cells under visible light irradiation and negligible dark toxicity (IC₅₀ > 50 μM). These results suggest that both type-I and type-II ROS generation pathways contribute to the observed photocytotoxicity. This study highlights the potential of paramagnetic metalloporphyrins, particularly Mn(III) porphyrins, in anticancer application by demonstrating their effectiveness as photosensitizers for photodynamic cancer therapy.

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Introduction

Porphyrins belong to the family of tetrapyrrolic macrocycles that have gained massive research interest due to their many biological relevance and potential applications in diverse fields such as catalysis, sensing, and medicinal chemistry. Their unique optical, electronic, redox and catalytic properties make them ligands par excellence in both biology and chemistry.^1–4 The use of porphyrins as photosensitizers (PSs) in photodynamic therapy (PDT) has grown considerably over the past few decades as a promising approach to combat cancer. PDT is a recently developed technique that relies on the ability of the PSs to generate cell-damaging reactive oxygen species (ROS) in the presence of light of an appropriate wavelength and molecular oxygen, thereby eliminating malignant cells.^5–7 This therapy offers several advantages over traditional cancer treatments due to its non-invasiveness, spatiotemporal controllability and minimal systemic toxicity.^8–10 Porphyrinic PSs are reported to preferentially accumulate in tumour cells over normal cells and exhibit photocytotoxicity via the type-II PDT pathway, i.e. through the production of singlet oxygen (¹O₂).^8,9,11–13 However, some literature reports suggest that paramagnetic metalloporphyrins are not considered good photosensitizers as they negatively affect the ¹O₂ generation process.^14,15 Nonetheless, in 2014, Antoni et al. reported the photocytotoxicity of a paramagnetic Cu(II) porphyrin in A2780 cancer cell lines, which was attributed to its ability to generate cytotoxic hydroxyl radicals (˙OH) upon exposure to red light.¹⁶ This study prompted us to explore the photocytotoxicity of other paramagnetic metalloporphyrins and their mechanism of action.

Manganese plays an enormous roles in various biological activities, such as crucial roles in different metabolic functions, including cofactor for different type of enzymes, which calls for it use in pharmaceuticals.¹⁷ Mn-porphyrins bearing high positive charges were initially developed as SOD mimics and redox-active antioxidants.¹⁸ Their anticancer activity is thought to result from redox-induced cytotoxicity, which can affect the redox balance of cancer cells.^18–20 However, there are only a few reports on the photoinduced anticancer activity of Mn-porphyrins.^21–23 In 2003, Banfi et al. reported that positively charged Mn(III) porphyrins could oxidatively damage Plasmid Bluescript DNA in the presence of either an artificial oxygen donor or light,²¹ demonstrating the potential of Mn(III) porphyrins in PDT. In 2017, Lei Shi and co-workers demonstrated considerable photocytotoxicity of an anionic Mn(III) porphyrin in HepG2 cells.²² More recently, Nemeth and colleagues reported the PDT activity of a cationic Mn(III) pyridyl porphyrin in HeLa cells, and their EPR studies indicated that ¹O₂ was the primary cytotoxic species.²³ In another study, Kim et al. showed that Mn(III) porphyrins undergo photoreduction in the presence of polynucleotides and can oxidize DNA via a photo-redox pathway.²⁴

With these facts in mind, in this work, we synthesized five Mn(III) porphyrins bearing simple substituents at the para position of the meso-phenyl rings and two axial aqua ligands, viz. (diaqua)meso-(tetraphenylporphyrinato)manganese(III) propionate, [Mn(III)TPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn1; (diaqua)meso-tetrakis(4-methylphenyl porphyrinato)manganese(III) propionate, [Mn(III)TMeP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn2; (diaqua)meso-tetrakis(4-methoxyphenylporphyrinato)manganese(III) propionate, [Mn(III)TMP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn3; (diaqua)meso-tetrakis(4-fluorophenyl porphyrinato)manganese(III) propionate, [Mn(III)FTPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn4; and (diaqua)meso-tetrakis(4-chlorophenylporphyrinato)manganese(III) propionate, [Mn(III)ClTPP(H₂O)₂]⁺(C₃H₅O₂⁻) or Mn5, in order to examine their photoinduced anticancer activity (Chart 1). The compounds were fully characterized by various spectroscopic and analytical techniques and structurally characterized by single crystal X-ray diffraction. They showed significant binding affinity towards human serum albumin (HSA) protein. UV-visible titration experiments with DPBF confirmed the generation of ¹O₂ by the compounds. The Mn(III) porphyrins also displayed redox shuttling between Mn(III) and Mn(II) in air in the presence of ascorbic acid and visible light, which is believed to induce additional oxidative stress in cells. Furthermore, 2′,7′-dichlorofluorescein diacetate (DCFDA) assay demonstrated the generation of ROS by the metalloporphyrins in the cellular environment. MTT assays showed significant photocytotoxicity of the Mn(III) porphyrins in HeLa cells under visible light.

Chart 1 Schematic representations of Mn(III) porphyrins (Mn1–Mn5).

Experimental section

Materials and measurements

Chemicals and solvents used in the work were obtained from commercial sources such as SD Fine Chemicals, India; Spectrochem, India; Loba Chemie, India; and Sigma-Aldrich, USA, and were used as received unless otherwise specified. Freshly purified pyrrole, prepared by passing it through a short column of basic alumina, was used for the synthesis of the free bases. Human serum albumin (HSA), 1,3-diphenylisobenzofuran (DPBF), L-ascorbic acid, Dulbecco's modified Eagle's medium (DMEM), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 2′,7′-dichlorofluorescein diacetate (DCFDA), Dulbecco's phosphate-buffered saline (DPBS) and fetal bovine serum (FBS) were all purchased from Sigma-Aldrich (USA). Tris-(hydroxylmethyl)aminomethane-HCl (Tris-HCl) buffer solution was prepared by using deionised and sonicated double-distilled water.

Elemental CHN analyses were performed using a Thermo Finnigan Flash EA 1112 CHN analyzer. FT-IR spectra were recorded on a Bruker Alpha-II spectrophotometer, with samples prepared as KBr pellets. Electronic absorption and emission spectra were recorded on Shimadzu UV-1800 and Hitachi F-7000 spectrophotometers, respectively. ¹H NMR spectra were recorded at room temperature on Bruker 300 MHz and 400 MHz NMR spectrometers. Room-temperature magnetic susceptibility measurements of the Mn(III) porphyrins were obtained using a Sherwood Scientific instrument (Cambridge, England), with Hg[Co(NCS)₄] as the standard. The experimental susceptibility data were corrected for diamagnetic contributions.²⁵ Electrochemical measurements were carried out at 25 °C using a CHI660D electrochemical workstation (CH Instruments, Inc., USA) with a conventional three-electrode configuration consisting of a glassy carbon working electrode, platinum wire auxiliary electrode and an Ag/AgCl reference electrode. Tetrabutylammonium perchlorate (TBAP, 0.1 M) in DMF was used as the supporting electrolyte. High-resolution electrospray ionization mass spectral measurements were recorded using a Waters Xevo G2-XS QTof mass spectrometer. Photoirradiation for cellular experiments was performed using a Luzchem Photoreactor (model LZC-1, Ontario, Canada) equipped with eight Sylvania white fluorescent tubes (λ = 400–700 nm). A TECAN microplate reader provided the formazan absorbance values in the MTT assay, and the resulting data were analyzed using GraphPad Prism 8 software. A fluorescence-activated cell sorting (FACS) Verse device (BD Biosciences) was used to conduct the flow cytometric analysis. The instrument is equipped with a MoFLo XDP cell sorter and analyzer featuring 10-color detection capability, and three lasers (λ = 488, 365, and 640 nm).

Synthesis

Synthesis of free-base porphyrins (L1–L5). The free-base porphyrins (L1–L5) were synthesized following the methodology of Adler et al., in which the precursor aldehydes (40 mmol) were mixed with freshly purified pyrrole (40 mmol) in a 100 mL round-bottom flask containing 50 mL of propionic acid.²⁶ The solution was refluxed for 30 minutes, then cooled to room temperature and filtered. The filter cake was thoroughly washed with methanol followed by hot water. The crude solid obtained was dried in a vacuum desiccator. The solid was further purified by column chromatography [DCM : petroleum ether (1 : 1)] to obtain the pure ligands as purple crystalline solids [yield: 1.125 g, 19.23% for L1; 1.310 g, 19.87% for L2; 1.287 g, 19.8% for L3; 0.980 g, 14.30% for L4; 1.240 g, 16.45% for L5] (Scheme S1, ESI†).

Anal. calcd for C₄₄H₃₀N₄ (L1): C, 85.97; H, 4.92; N, 9.11. Found: C, 85.71; H, 4.87; N, 9.23. Selected IR data (KBr, cm⁻¹): 3314w (N–H), 3052w and 3021w (C–H), 2920w, 1678s, 1592m (C=C), 1553m, 1470s (C=C), 1437s, 1402m, 1347s (C–N), 1219w, 1174s, 1071m, 963vs, 799vs (C–H_bending), 700vs (vs, very strong; s, strong; m, medium; w, weak; br, broad). UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 416 (S, 1.87 × 10⁵), 513 (Q, 8.42 × 10³), 548 (Q, 3.8 × 10³), 590 (Q, 2.60 × 10³), 647 (Q, 2.56 × 10³). ¹H NMR (300 MHz, CDCl₃): δ ppm, 8.88 (s, 8H, β PyH), 8.25–8.23 (d, J = 6.0 Hz, 8H, Ar o-H), 7.79–7.77 (d, J = 6.0 Hz, 12H, Ar m & p H), −2.76 (s, 2H, NH). ESI-MS in MeOH (m/z): 615.2551 for [M + H]⁺ (calcd: 615.2549).

Anal. calcd for C₄₈H₃₈N₄ (L2): C, 85.94; H, 5.71; N, 8.35. Found: C, 85.73; H, 5.77; N, 8.27. Selected IR data (KBr, cm⁻¹): 3326w (N–H), 3021w (C–H), 2902w, 1699w, 1557m (C=C), 1509m, 1468s (C=C), 1347s (C–N), 1217w, 1180s, 1108w, 1021w, 966vs, 846m, 799vs (C–H_bending), 733s, 615m. UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 418 (S, 2.8 × 10⁵), 515 (Q, 1.2 × 10⁴), 550 (Q, 6.2 × 10³), 593 (Q, 3.5 × 10³), 649 (Q, 3.9 × 10³). ¹H NMR (400 MHz, CDCl₃): δ ppm, 8.86 (s, 8H, β PyH), 8.11–8.09 (d, J = 8.0 Hz, 8H, Ar o-H), 7.57–7.55 (d, J = 8.0 Hz, 8H, Ar m-H), 2.71 (s, 12H, CH₃), −2.78 (s, 2H, NH). ESI-MS in MeOH (m/z): 671.3177 for [M + H]⁺ (calcd: 671.3175).

Anal. calcd for C₄₈H₃₈N₄O₄ (L3): C, 78.45; H, 5.21; N, 7.62. Found: C, 78.21; H, 5.29; N, 7.55. Selected IR data (KBr, cm⁻¹): 3314w (N–H), 2928w (C–H), 2832w (C–H of –OCH₃), 1744m, 1604s (C=C), 1505s, 1464s (C=C), 1347m (C–N), 1289s, 1248vs (C–O–C), 1174vs, 1108w, 1033s, 966s, 803s (C–H_bending), 737s, 640m. UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 421 (S, 1.77 × 10⁵), 518 (Q, 7.06 × 10³), 555 (Q, 5.48 × 10³), 595 (Q, 2.40 × 10³), 651 (Q, 3.9 × 10³). ¹H NMR (300 MHz, CDCl₃): δ ppm, 8.86 (s, 8H, β PyH), 8.14–8.11 (d, J = 9.0 Hz, 8H, Ar o-H), 7.30–7.27 (d, J = 9.0 Hz, 8H, Ar m-H), 4.10 (s, 12H, OCH₃), −2.78 (s, 2H, NH). ESI-MS in MeOH (m/z): 735.2908 for [M + H]⁺ (calcd: 735.2971).

Anal. calcd for C₄₄H₂₆N₄F₄ (L4): C, 76.96; H, 3.82; N, 8.16. Found: C, 76.84; H, 3.79; N, 8.22. Selected IR data (KBr, cm⁻¹): 3309m (N–H), 3042w (C–H), 1699w, 1600m (C=C), 1555w, 1503vs, 1470s (C=C), 1400w, 1351m (C–N), 1225vs, 1157vs (Ar–F), 1091w, 968s, 846m, 807vs (C–H_bending), 729s. UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 414 (S, 4.2 × 10⁴), 511 (Q, 2.7 × 10³), 545 (Q, 1.2 × 10³), 586 (Q, 7.8 × 10²), 645 (Q, 5.7 × 10²). ¹H NMR (300 MHz, DMSO-d₆ with NaOH): δ ppm, 8.53 (s, 8H, β PyH), 8.14–8.12 (d, J = 6.0 Hz, 8H, Ar o-H), 7.57–7.51 (t, J = 9 Hz, 8H, Ar m-H). ESI-MS in MeOH (m/z): 687.4095 for [M + H]⁺ (calcd: 687.2172).

Anal. calcd for C₄₄H₂₆Cl₄N₄ (L5): C, 70.23; H, 3.48; N, 7.45. Found: C, 70.11; H, 3.42; N, 7.49. Selected IR data (KBr, cm⁻¹): 3312w (N–H), 2920m (C–H), 2850w, 2310w, 1656w, 1553m (C=C), 1470s (C=C), 1394m, 1344m (C–N), 1174m, 1089vs (Ar–Cl), 1015s, 966s, 844m, 795vs (C–H_bending), 729s. UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 417 (S, 1.87 × 10⁵), 514 (Q, 8.72 × 10³), 549 (Q, 3.67 × 10³), 590 (Q, 2.58 × 10³), 647 (Q, 2.14 × 10³). ¹H NMR (300 MHz, CDCl₃): δ 8.84 (s, 8H, β PyH), 8.15–8.12 (d, J = 9.0 Hz, 8H, Ar o-H), 7.76–7.74 (d J = 6.0 Hz, 8H, Ar m-H), −2.88 (s, 2H, NH). ESI-MS in MeOH (m/z): 753.0969 for [M + H]⁺ (calcd: 753.0960).

Synthesis of the Mn(III) porphyrins (Mn1–Mn5). The metalloporphyrins (Mn1–Mn5) were synthesized using a general procedure, in which the respective free bases (0.25 mmol; 153 mg for L1, 167 mg for L2, 183 mg for L3, 171 mg for L4, 188 mg for L5) were dissolved in a 20 mL of a chloroform/propionic acid mixture (3 : 1). This was followed by the addition of 1.25 mmol (306 mg) Mn(OAc)₂·4H₂O and the reaction mixture was refluxed for 1 h.²⁷ The organic layer was separated using a separating funnel, collected in chloroform, and evaporated to dryness in a rotary evaporator. The resulting solid was loaded onto a silica gel column and eluted with 1% ethanol in chloroform to remove any unreacted free base. The pure product, isolated as a greenish-black solid, was dried in vacuo over anhydrous calcium chloride, and the yield was calculated [yield: 0.162 g, 83.93% for Mn1; 0.170 g, 82.12% for Mn2; 0.178 g, 79.82% for Mn3, 0.142 g, 67.07% for Mn4; 0.197 g, 86.40% for Mn5] (Scheme S1, ESI†).

Anal. calcd for C₄₇H₃₇N₄O₄Mn (Mn1): C, 72.67; H, 4.80; N, 7.21. Found: C, 72.49; H, 4.88; N, 7.13. Selected IR data (KBr, cm⁻¹): 3442br (O–H), 3052w & 2924w (C–H), 1631w, 1598m (C=C), 1439w (C=C), 1384vs, 1350m (C–N), 1176w, 1072m, 1005vs, 834w, 793s (C–H_bending), 742s, 697s, 526w. UV-Visible in DMSO, [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 466 (S, 1.47 × 10⁵), 568 (Q, 1.44 × 10⁴), 603 (Q, 1.10 × 10⁴). ESI-MS in MeCN (m/z): 667.1685 for [M-(2H₂O + C₃H₅O₂⁻)]⁺ (calcd: 667.1694). μ_eff = 4.5μ_B at 298 K.

Anal. calcd for C₅₁H₄₅N₄O₄Mn (Mn2): C, 73.55; H, 5.45; N, 6.73. Found: C, 73.32; H, 5.38; N, 6.79. Selected IR data (KBr, cm⁻¹): 3438br (O–H), 2930w (C–H), 1631w, 1564m (C=C), 1413w (C=C), 1384m, 1352m (C–N), 1209w, 1180w, 1107w, 1074w, 1001s, 799s (C–H_bending), 717m, 603w, 524w. UV-Visible in DMSO [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 465 (S, 1.29 × 10⁵), 569 (Q, 1.42 × 10⁴), 607 (Q, 1.36 × 10⁴). ESI-MS in MeCN (m/z): 723.2330 for [M-(2H₂O + C₃H₅O₂⁻)]⁺ (calcd: 723.2320). μ_eff = 4.7μ_B at 298 K.

Anal. calcd for C₅₁H₄₅N₄O₈Mn (Mn3): C, 68.30; H, 5.06; N, 6.25. Found: C, 68.11; H, 5.12; N, 6.17. Selected IR data (KBr, cm⁻¹): 3444br (O–H), 2924w (C–H), 2832w (C–H of –OCH₃), 1606s (C=C), 1507s, 1460m (C=C), 1384w, 1352s (C=C), 1286m, 1248vs (C–O–C), 1174s, 1105w, 1074w, 1027w, 1001s, 803s (C–H_bending), 717m, 607m, 540w. UV-Visible in DMSO [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 467 (S, 1.38 × 10⁵), 572 (Q, 1.46 × 10⁴), 609 (Q, 1.66 × 10⁴). ESI-MS in MeCN (m/z): 787.2111 for [M-(2H₂O + C₃H₅O₂⁻)]⁺ (calcd: 787.2117). μ_eff = 4.9μ_B at 298 K.

Anal. calcd for C₄₇H₃₃N₄O₄F₄Mn (Mn4): C, 66.51; H, 3.92; N, 6.60. Found: C, 66.39; H, 3.84; N, 6.69. Selected IR data (KBr, cm⁻¹): 3442br (O–H), 2975w & 2922w (C–H), 1602m (C=C), 1564s, 1498s (C=C), 1415m, 1341m (C–N), 1296w, 1225s, 1158s (Ar–F), 1072m, 1011s, 854w, 803s (C–H_bending), 719w, 597m, 532m. UV-Visible in DMSO [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 465 (S, 1.22 × 10⁵), 567 (Q, 1.24 × 10⁴), 603 (Q, 8.64 × 10³). ESI-MS in MeCN (m/z): 739.1324 for [M-(2H₂O + C₃H₅O₂⁻)]⁺ (calcd: 739.1318). μ_eff = 4.4μ_B at 298 K.

Anal. calcd for C₄₇H₃₃N₄O₄Cl₄Mn (Mn5): C, 61.73; H, 3.64; N, 6.13. Found: C, 61.73; H, 3.61; N, 6.21. Selected IR data (KBr phase, cm⁻¹): 3421br (O–H), 2977w & 2938w (C–H), 1715m, 1641w, 1566m (C=C), 1484s (C=C), 1394m, 1339w (C–N), 1282w, 1205m, 1091vs (Ar–Cl), 1009vs, 883w, 850w, 803vs (C–H_bending), 717m, 632w, 562w, 501m. UV-Visible in DMSO [λ_max/nm (ε/M⁻¹ cm⁻¹)]: 463 (S, 1.51 × 10⁵), 567 (Q, 1.68 × 10⁴), 605 (Q, 1.34 × 10⁴). ESI-MS in MeCN (m/z): 805.0097 for [M-(2H₂O + C₃H₅O₂⁻)]⁺ (calcd: 805.0106). μ_eff = 4.6μ_B at 298 K.

X-ray crystallographic procedure

The structures of the Mn(III) porphyrins Mn1, Mn2, Mn4 and Mn5 were determined by single-crystal X-ray diffraction. Diffraction-quality crystals were isolated by slow evaporation of aqueous propionic acid solutions of the metalloporphyrins over 2–3 days. Crystals were mounted on glass fibres using epoxy cement. All geometric and intensity data were collected at room temperature on a Bruker SMART APEX II CCD diffractometer equipped with a Mo-K_α fine-focus sealed-tube X-ray source (λ = 0.71073 Å), using increasing ω (width of 0.3° per frame) at a scan speed of 5 s per frame. Intensity data, collected in ω–2θ scan mode, were corrected for Lorentz-polarisation effects and for absorption using SADABS.²⁸ Structure solution and refinement were performed using a combination of Patterson and Fourier methods, followed by full-matrix least-squares refinement using Bruker SHELXTL suite.²⁹ For Mn5, data were collected using a dual-source (Cu/Mo) Rigaku Saturn 724+ CCD and a Synergy-S kappa diffractometer at room temperature. All hydrogen atoms belonging to the complex were refined isotropically, whereas all the non-hydrogen atoms were refined anisotropically. Perspective molecular views were generated using ORTEP.³⁰

HSA binding experiment

The binding affinity of the compounds Mn1–Mn5 for human serum albumin (HSA) were determined by tryptophan fluorescence quenching experiments using a solution of HSA (2 μM) in Tris-HCl buffer (pH 7.2). Quenching of the emission intensity of the single tryptophan residue (Trp-214) of HSA at 340 nm (excitation wavelength: 280 nm) by increasing concentrations of Mn1–Mn5 as quenchers was monitored using a fluorescence spectrophotometer. Plots of I₀/I vs. [compound] were constructed, and the data were linearly fitted using the Stern–Volmer-like equation I₀/I = 1 + K[Q], where I₀ and I represent the emission intensities of HSA in the absence and presence of the quencher, respectively, and [Q] denotes the quencher concentration. The resulting slopes provided the binding constant K_HSA values.³¹

Partition coefficient of the metalloporphyrins between n-octanol and water

The partition coefficients of the Mn(III) porphyrins Mn1–Mn5 between n-octanol and water were determined using a modified shake-flask method reported in the literature.³² In summary, 5 ml solution of the compound in n-octanol-saturated water (OSW) was vortexed with 5 ml of water-saturated n-octanol (WSO) in a Falcon tube for ∼15–20 min. The biphasic mixture was then centrifuged at ∼3000 rpm for ∼5 min to separate the layers. The concentrations of Mn1–Mn5 in each layer were quantified by UV-visible spectroscopy. The partition coefficients of the compounds were determined by repeating the experiment three times at varying concentration of Mn1–Mn5 and averaging the results using the formula log P = log{[Mn1–Mn5]_oct/[Mn1–Mn5]_aq}.

UV-visible titration using DPBF to ascertain generation of ¹O₂

1,3-Diphenylisobenzofuran (DPBF) was used as a chemical probe to detect the generation of any singlet oxygen (¹O₂) by the Mn(III) porphyrins (Mn1–Mn5) under visible light irradiation. DPBF reacts specifically with ¹O₂ to form an endoperoxide, and a decrease in the absorbance intensity of DPBF at ∼415 nm indicates its conversion.³³ The experiment was conducted in two different solvents, i.e. DMF and DMSO. A 50 : 1 molar ratio of DPBF to compound was prepared in the respective solvent, and the absorption maxima of DPBF were monitored before and after irradiation with visible light (irradiation provided by a 12 W LED bulb fitted inside a rectangular box of area 9 cm × 4.5 cm, λ = 400–700 nm) at various time intervals. The absorbance data recorded at different time interval of light exposure were fitted in a pseudo first-order rate equation: ln([A]_t/[A]₀) = −kt, where [A]₀ and [A]_t represent the absorbance of DPBF before and after light irradiation for a duration of “t”, and k is the DPBF decay constant, indicating the rate of ¹O₂ generation. The value of k was obtained from the slope of the plot.³⁴ Various control experiments were also performed to confirm the formation of ¹O₂, including (a) photoirradiation of untreated DPBF for similar time intervals, (b) photoirradiation of compound-treated DPBF in the presence of a ¹O₂ quencher (NaN₃) and (c) compound-treated DPBF kept in dark. Relative quantum yields of the compounds for ¹O₂ generation were calculated from the k values using TPP as the reference standard (Φ^R_Δ = 0.64 in DMF and 0.52 in DMSO), based on the following equation:^35,36

Here, the superscripts S and R denote sample and reference, respectively, and F is the absorption correction factor given by 1–10^−OD, where OD is the absorbance of the compounds.

Photoreduction study in the presence of L-ascorbic acid

Ability of the Mn(III) porphyrins (Mn1–Mn5) to undergo photoreduction in the presence of a reducing agent such as L-ascorbic acid was monitored by UV-visible spectroscopy.³⁷ A 10 μM solution of each Mn(III) porphyrin was treated with ascorbic acid (1.4 mM in case of Mn1, Mn4 and Mn5; 2 mM for Mn2 and Mn3) in 1 : 1 DMSO : Tris-HCl buffer medium. The varying amounts of ascorbic acid required for reduction likely reflect differences in the reduction potentials of the compounds. The resulting solutions were then exposed to visible light (λ = 400–700 nm) for 60 min in air, and UV-visible spectra were recorded at 15 min intervals during photoirradiation. A dark control experiment was also performed to validate the photoreduction behavior.

DCFDA assay for detection of ROS

The DCFDA assay was used to detect the generation of cellular reactive oxygen species (ROS), using the most active Mn(III) porphyrin, i.e.Mn4, as a representative compound for the series. The experiment was conducted via flow cytometry following a reported protocol.³⁸ Approximately 1 × 10⁶ HeLa cells were incubated with Mn4 (10 μM) for 24 h. The cells were then treated with 10 μM DCFDA solution (in DMSO) in dark for 5 min at room temperature, followed by photoirradiation (400–700 nm) in PBS for varying durations (15, 30, 45 and 60 min). After irradiation, the cells were incubated for an additional 23 h in fresh medium. Corresponding dark control experiments were also conducted. Cells were harvested by trypsinization and resuspended as a single-cell suspension in PBS. Flow cytometry was used to assess the intracellular accumulation of H₂DCF in HeLa cells via the FL-1 channel.

Cell viability assay

The cytotoxicity of the compounds Mn1–Mn5 under visible light was evaluated in HeLa (human cervical carcinoma) cells using the MTT assay. This assay is based on the intracellular reduction of MTT dye by mitochondrial dehydrogenases in viable cells, resulting in the formation of dark blue, membrane-impermeable formazan crystals. The extent of absorbance at 540 nm provides a quantitative estimate of cell viability.³⁹ HeLa cells were cultured in DMEM with 10% FBS, 100 IU ml⁻¹ penicillin, 100 mg ml⁻¹ streptomycin and 2 mM glutamax at 37 °C in a humidified incubator with 5% CO₂. Monolayer adherent cultures were passaged every 4–5 days by trypsinization with 0.25% trypsin–EDTA. For the assay, ∼1 × 10⁴ HeLa cells were seeded per well in a 96-well culture plate containing DMEM with 10% fetal bovine serum and incubated overnight. Different concentrations of Mn1–Mn5 were prepared by serial dilution in 1% DMSO–DMEM medium and added to the cells, followed by a 24 h incubation in the dark. Following incubation, the medium was replaced with 50 mM phosphate-buffered saline (PBS; pH 7.4, containing 150 mM NaCl), and one set of cells was photoirradiated for 1 h with visible light (λ = 400–700 nm) at a dose of 10 J cm⁻². PBS was then replaced with 10% DMEM, and incubation was continued for an additional 23 h in the dark for light-exposed cells, and for 24 h in the dark for the unexposed cells. Subsequently, 25 μL of MTT solution (4 mg mL⁻¹) was added to each well and incubated for an additional 3 h. After discarding the culture medium, formazan crystals were dissolved in DMSO, and absorbance was measured at 540 nm using a TECAN microplate reader (BioRad, Hercules, CA, USA). The cytotoxicity of the compounds was expressed in terms of their half-maximal inhibitory concentration (IC₅₀) values, calculated using GraphPad Prism 8 software.

Results and discussion

Synthesis and general aspects

Five Mn(III) meso-tetraaryalporphyrins (Mn1–Mn5) bearing different substituents at the para positions of the meso-aryl groups along with two water molecules as axial ligands were synthesized in good yields by reacting manganese(II) acetate tetrahydrate with the corresponding free bases (L1–L5) in a chloroform/propionic acid mixture. The formation of the free bases (L1–L5) was confirmed by ¹H NMR and mass spectral data (Fig. S1–S10, ESI†). The corresponding metalloporphyrins (Mn1–Mn5) were characterised using various spectroscopic and analytical techniques, which supported the proposed structures. Selected physicochemical data are presented in Table 1. Characteristic N–H stretching vibrations at ∼3310–3325 cm⁻¹ were observed in the IR spectra of the free bases (Fig. S11–S15, ESI†), which disappeared in case of their Mn(III) derivatives (Fig. S16–S20, ESI†). The absorption spectra of free-base porphyrins (L1–L5) in DMSO displayed a typical Soret (or B) band at ∼414–421 nm and four Q-bands spanning ∼511–651 nm. On metallation with manganese, the electronic spectra exhibited dramatic changes resembling those of hyperporphyrins.⁴⁰ The Soret band was significantly red shifted (∼50 nm) to ∼460 nm, and the two Q-bands extended well into the red region, up to ∼605–610 nm (Fig. 1 and Fig. S21, ESI†). More specifically, the absorption intensities of the Q-bands in this region were markedly enhanced compared to their free-base counterparts, a feature highly desirable for PDT.^9,14 These metalloporphyrins also exhibited additional absorption bands in the ∼370–400 nm range, which were attributed to LMCT transitions.⁴⁰ Unlike the free-base porphyrins, the Mn(III) derivatives were found to be non-emissive in nature, as revealed by their emission spectra (Fig. 1 and Fig. S22, ESI†). The presence of a high-spin 3d⁴-Mn(III) center with four unpaired electrons was confirmed by the observed magnetic moment values of ∼4.5μ_B at 25 °C. The solution-phase stability of compounds Mn1–Mn5 was evidenced by their high-resolution ESI-MS spectra, which showed the base peak as [M-(2H₂O + C₃H₅O₂⁻)]⁺, corresponding to the loss of two weakly bound axial water molecules and a propionate counterion in acetonitrile (Fig. S23–S27, ESI†). Cyclic voltammetric measurements displayed a Mn(III)/Mn(II) redox couple at ∼−0.3 to −0.4 V vs. Ag/AgCl electrode, using TBAP (0.1 M) as the supporting electrolyte. All compounds exhibited quasi-reversible behavior with ΔE_p > 60 mV for the Mn(III)/Mn(II) redox couple (Fig. 2 and Fig. S28, ESI†). The observed large ΔE_p value is most likely associated with a change in the coordination number of the manganese center from six to five upon reduction of the Mn(III) porphyrins from d⁴ to d⁵.⁴¹ The negative potential required for one-electron reduction of Mn(III) porphyrins is consistent with the electrochemical stabilization of the reduced species. Mn4 and Mn5 were comparatively more readily reduced, likely due to the electron-withdrawing nature of the F and Cl atoms, respectively.

Fig. 1 (a) UV-visible absorption spectra of L4 and Mn4 (5 μM) (inset: enlarged view of the Q-bands) and (b) emission spectra of L4 and Mn4 (5 μM) in DMSO with λ_ex set at their Soret band maxima.

Fig. 2 Cyclic voltammetric responses of Mn4 (a) and Mn5 (b) in DMF using 0.1 M TBAP as the supporting electrolyte at a scan rate of 100 mV s⁻¹vs. Ag/AgCl.

Table 1 Selected physicochemical data for the Mn(III) porphyrins Mn1–Mn5

Compound	λ abs ^a/nm (ε/10^3 M^−1 cm^−1)	E f/V (ΔEp/mV)^b Mn(III)–Mn(II)	μ eff ^c/μB	log Po/w	Φ Δ ^d^,^e
a 5 μM solution in DMSO. b Mn(III)–Mn(II) couple in DMF with 0.1 M TBAP, Ef = 0.5(Epa + Epc), ΔEp = (Epa − Epc), where Epa and Epc are the anodic and cathodic peak potentials, respectively. The potentials are referenced against the Ag/AgCl electrode. Scan rate = 100 mV s^−1. c Magnetic moment measured at 298 K. d In DMF. e In DMSO.
Mn1	466 (B, 147), 568 (Qy 14), 603 (Qx 11)	−0.373 (110)	4.5	0.38	0.09, 0.04
Mn2	465 (B, 129), 569 (Qy 14), 607 (Qx 13)	−0.400 (198)	4.7	1.45	0.11, 0.06
Mn3	467 (B, 138), 572 (Qy 14), 609 (Qx 16)	−0.402 (157)	4.9	0.36	0.13, 0.08
Mn4	465 (B, 122), 567 (Qy 12), 603 (Qx 8.6)	−0.351 (161)	4.6	0.88	0.17, 0.09
Mn5	463 (B, 151), 567 (Qy 16), 605 (Qx 13)	−0.336 (173)	4.4	1.71	0.14, 0.08

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Solubility, stability and aggregation properties

The Mn(III) porphyrins Mn1–Mn5 exhibited good solubility in MeOH, EtOH, MeCN, CH₂Cl₂, CHCl₃, THF, DMF and DMSO, but were insoluble in hydrocarbon solvents. The compounds were found to be quite stable in DMSO–DMEM (1 : 1, v/v) solution phase for up to 24 h, as evidenced by their electronic absorption spectra in the ∼300–700 nm range (Fig. S29, ESI†). Photostability of the compounds was also evaluated in the same medium by irradiating the solutions with visible light for 60 min. The compounds remained stable under photoirradiation, suggesting their suitability for PDT without any significant degradation into undesirable oxidative products (Fig. S30, ESI†).⁴² Moreover, the Mn(III) porphyrins are expected to exhibit reduced aggregation in biological media due to the presence of two axial aqua ligands, which prevent individual porphyrin units from approaching each other closely.⁴³ In the support of this, Mn1–Mn5 displayed sharp and intense absorption bands in DMSO–DMEM (1 : 1, v/v) media compared to the broad and less intense bands observed for the free-base porphyrins at similar concentrations, indicating significantly lower aggregation properties for the Mn(III) porphyrins (Fig. S31, ESI†). This finding suggests that Mn(III) porphyrins possess superior aqueous solubility compared to their free-base counterparts, thereby facilitating their evaluation for in vitro anticancer activities.

Crystal structure

Structural characterization of the Mn(III) porphyrins Mn1, Mn2, Mn4 and Mn5 was successfully achieved by single-crystal X-ray diffraction analyses in the form of six coordinated cationic species [Mn(III)Por(H₂O)₂]⁺C₃H₅O₂⁻. The crystal structure of Mn4 is entirely new, whereas those of other Mn(III) porphyrins, i.e.Mn1, Mn2 and Mn5, have been previously reported, albeit with different counterions crystallized from different solvents.^44–49 To the best of our knowledge, the crystal structure of an Mn(III) porphyrin with propionate as the counterion has not been reported before. The ORTEP views of Mn4, Mn5 and the remaining compounds are shown in Fig. 3 (Fig. S32 and S33, ESI†). Mn4 crystallizes in the P1̄ space group of the triclinic crystal system with 2 molecules per unit cell (Fig. S34, ESI†). Mn1 also crystallizes in the P1̄ space group (triclinic) but with 1 molecule per unit cell (Fig. S35, ESI†). Mn2 and Mn5 crystallize in the P2₁/c space group of the monoclinic crystal system, with 2 molecules per unit cell (Fig. S36 and S37, ESI†). Selected crystallographic data are summarized in Table 2 (Table S1, ESI†). Selected bond distances and angle data are provided in Tables S2–S5 (ESI†). All the Mn(III) porphyrins exhibit an octahedral geometry around Mn(III) center, with four coordination sites occupied by the core nitrogen atoms of the flat planar porphyrin ring and two water molecules occupying the fifth and sixth coordination sites, i.e. the axial positions, with respect to the plan of the porphyrin ring. The Mn–N bond distances lie in the range of 2.0054–2.0207 Å. The Mn–O bond distances for the coordinated water molecules range from 2.2200–2.314 Å, indicating significant distortion in the octahedral structures imparted by tetragonal elongation anticipated in high-spin d⁴ Mn(III) complexes. The Mn–O and Mn–N bond distances are comparable to those reported structures that contain water, methanol, DMF, acrylamide etc. as axial ligands along with [Ag(CN)₂]⁻, Br⁻, SbF₆⁻, ClO₄⁻etc. as counterions.^44–49 The plane of the phenyl rings at the meso positions is not same with the plane of the porphyrin ring, suggesting a lack of conjugation between the two aromatic systems.

Fig. 3 ORTEP views of (diaqua)meso-tetrakis(4-fluorophenylporphyrinato)manganese(III) propionate (Mn4) (a) and (diaqua)meso-tetrakis(4-chlorophenylporphyrinato)manganese(III) propionate (Mn5), (b) showing 30% probability thermal ellipsoids and the atom-numbering scheme for the metal and heteroatoms. The counterion and hydrogen atoms are omitted for clarity.

Table 2 Selected crystallographic data for the compounds (diaqua)meso-tetrakis(4-fluoro phenylporphyrinato)manganese(III) propionate (Mn4) and (diaqua)meso-tetrakis(4-chloro phenylporphyrinato)manganese(III) propionate (Mn5)

Empirical formula	C44H28F4MnN4O2(C6H11O4)·3(C3H6O2) (Mn4)	C44H28Cl4MnN4O2(C6H11O4)·4(C3H6O2) (Mn5)
a R = ∑||Fo| − |Fc||/∑|Fo|. b R w = {∑[w(Fo^2 − Fc^2)^2]/∑[w(Fo)^2]}^½; w = [σ^2(Fo)^2 + (AP)^2 + BP]^−1, where P = (Fo^2 + 2Fc^2)/3, A = 0.0921; B = 0.8341 (for Mn4) and A = 0.0871; B = 1.3753 (for Mn5).
F w, g M^−1	1145.02	1284.90
Crystal system	Triclinic	Monoclinic
Space group	P1̄	P21/c
a, Å	9.2702(12)	12.7394(2)
b, Å	14.4396(18)	9.48030(10)
c, Å	22.878(3)	26.7201(4)
α, °	72.099(4)	90
β, °	89.438(5)	98.5660(10)
γ, °	72.965(5)	90
V, Å^3	2775.4(6)	3191.08(8)
Z	2	2
T, K	296(2)	294(2)
ρ calcd, g cm^−3	1.370	1.337
λ, Å	0.71073	1.54184
μ, cm^−1	0.318	3.768
Data/restraints/parameters	12 091/6/755	6028/1/378
F(000)	1192	1336
Goodness-of-fit	1.039	1.076
R(Fo)^a, I > 2σ(I) [Rw(Fo)^b]	0.0707 [0.1664]	0.0536 [0.1531]
R (all data) [Rw (all data)]	0.1261 [0.1932]	0.0594 [0.1582]
Largest diff. peak and hole (e Å^−3)	0.512, −0.475	0.644, −0.468

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Binding affinity for HSA

Neutral and anionic porphyrins are known for their ability to form complexes with HSA.⁵⁰ The affinity of the synthesized metalloporphyrins Mn1–Mn5 for human serum albumin (HSA) was evaluated by determining their binding constant values (K_HSA) with HSA using a fluorescence quenching experiment. The emission intensity of HSA at 334 nm was gradually quenched, accompanied by a significant blue shift, in Tris-HCl buffer (pH 7.2) on increasing the concentration of Mn1–Mn5 (Fig. S38, ESI†). The observed decrease in emission intensity and the blue shift are attributed to structural changes in HSA on binding to the Mn(III) porphyrins, leading to perturbations in the microenvironment around the Trp-214 residue responsible for fluorescence. The blue shift in the emission maximum indicates a more hydrophobic environment surrounding the Trp-214 residue upon binding of the Mn(III) porphyrins to HSA.^51,52 A linear plot of I₀/I vs. [compound] yielded K_HSA values in the following order: Mn5 > Mn4 > Mn2 > Mn3 ≥ Mn1 (Table S6, ESI†). Although this order is not completely parallel to their lipophilicity, it can be concluded that an increase in lipophilic character generally favors interaction with HSA. The K_HSA values ranged from 9.08 (±0.01) × 10⁴ to 4.03 (±0.02) × 10⁵ M⁻¹. Compared to these, the manganese derivative of protoporphyrin IX, Mn(III)PPIX, exhibited a relatively higher binding affinity towards HSA, with a K_HSA value of 2.13 (±0.07) × 10⁶ M⁻¹.⁵³ The moderate binding affinities of Mn1–Mn5 for HSA suggest that they can probably bind reversibly to HSA, potentially allowing them to be transported through blood plasma and released at the target tissue.

Measurement of lipophilicity

An essential property for a PDT drug is its ability to incorporate and localize within vital cellular compartments. This is particularly crucial for hydrophobic compounds, for which passive diffusion across the cytoplasmic membrane is often a pre-requisite for cellular uptake.⁵⁴ A partition coefficient between n-octanol and water serves as an indicator of a drug candidate's potential to diffuse through the lipid bilayer of cell membranes. The n-octanol/water partition coefficients (expressed as log P_O/W) were determined using the shake-flask method and were found to follow the order: Mn5 > Mn2 > Mn4 > Mn1 ≥ Mn3 (Table 1). The Mn(III) porphyrins bearing –Cl and –CH₃ substituents at their periphery were found to be highly lipophilic, with log P_O/W > 1.

Detection of ¹O₂ generation using DPBF as a chemical probe

Paramagnetic metalloporphyrins such as those of Co(II) and Cu(II) are reported to be unsuitable candidates for ¹O₂ generation process.^16,55 To evaluate whether the synthesized Mn(III) porphyrins are capable of generating ¹O₂ on electronic excitation, both extracellular and intracellular experiments were conducted using UV-visible titration with DPBF and a DCFDA assay as probes (Fig. 4). It is observed that DPBF, in the presence of Mn(III) porphyrins Mn1–Mn5, exhibited significant decrease in its absorption maximum under visible light irradiation (Fig. 4a and Fig. S39, ESI†). In contrast, the same experiment performed in the dark showed no significant changes in the DPBF absorption spectrum (Fig. 4a and Fig. S40, ESI†). Control experiments confirmed that DPBF did not undergo self-photodegradation under similar experimental conditions (Fig. S40, ESI†). To further verify whether the observed photodegradation of DPBF in the presence of Mn(III) porphyrins was due to ¹O₂ generation, a control experiment was performed using NaN₃ as a ¹O₂ quencher (Fig. 4a and Fig. S41, ESI†). This results of this experiment closely resembled the photodegradation profile of untreated DPBF. This indicates that the significant decrease in absorption maximum of DPBF in the presence of Mn(III) porphyrins upon photoirradiation under visible light is due to the generation of ¹O₂. The rate constant values obtained from the linear plot of −ln([A]_t/[A]₀) vs. kt were 5.82 × 10⁻³ s⁻¹, 8.20 × 10⁻³ s⁻¹, 8.90 × 10⁻³ s⁻¹, 1.14 × 10⁻² s⁻¹ and 9.10 × 10⁻³ s⁻¹ for Mn1, Mn2, Mn3, Mn4 and Mn5, respectively, following the order: Mn4 > Mn5 ≥ Mn3 > Mn2 > Mn1 (Fig. S42, ESI†). The ¹O₂ quantum yields calculated from these rate constants were found to be 0.09, 0.11, 0.13, 0.17 and 0.14 for Mn1, Mn2, Mn3, Mn4 and Mn5, respectively (Table 1). The whole sets of these experiments were further repeated in DMSO, where a similar trend of ¹O₂ quantum yields was observed (Fig. S43–S46, ESI†). However, ¹O₂ quantum yield values obtained in DMSO were in the range of 0.04 to 0.09, which were lower compared to those in DMF (Table 1). This difference is likely due to the variable lifetime of ¹O₂ in different solvents.⁵⁶ Although paramagnetic Mn(III) metal centre in porphyrin-type macrocycles promotes non-radiative decay pathways that limit ¹O₂ generation process, it does not completely eliminate the process. Thus, we observed some residual generation of ¹O₂ in the Mn(III) porphyrins. The unpaired electrons on Mn(III) in these compounds interact with the ring electrons and hence the (π,π*) states are not the typical singlets and triplets. The ground state becomes a quintet (⁵S₀), and the quintet excited states (⁵S₂ and ⁵S₁) are derived from the excited ring (π,π*) singlet state, while a “tripmultiplet” manifold (³T₁, ⁵T₁, ⁷T₁) arises from the lowest ring (π,π*) triplet state. Paramagnetism in Mn(III) porphyrin increases spin–orbit coupling, which in turn significantly enhances the rate of intersystem crossing compared to other metalloporphyrins (e.g. for Mn^IIITPP(Cl) τ_ISC = 2.8 ps).⁵⁷ Consequently, singlet excited states are rapidly converted to triplet manifolds. Although strong spin–orbit coupling also influences non-radiative decay of excited triplet states, some of these triplet excited states are still relatively long-lived compared to their excited singlet states. A portion of ⁵T₁ state decays back to the ground state, while another portion decays to comparatively long-lived ⁷T₁ state (Fig. S47, ESI†). This state can likely participate in energy transfer with molecular oxygen to generate ¹O₂. According to Harriman, for Mn^IIITPP(Cl), the energies of the ⁵T₁ and ⁷T₁ states are 1.8 eV and 1.6 eV, respectively, while the energy required for ¹O₂ generation is 0.98 eV.^57–60 Although, the Φ_Δ values of the Mn(III) porphyrins are lower than those of typical type-II PDT agents, they may still contribute to their photocytotoxicity due to their relatively better solubility and stability profiles.

Fig. 4 (a) Absorption spectral traces of DPBF (50 μM) treated with Mn4 (1 μM) in DMF, monitored at 415 nm: (i) upon photo-irradiation, (ii) kept in the dark and (iii) upon photo-irradiation in the presence of NaN₃ (1 μM). (b) Determination of ROS generation in HeLa cells treated with Mn4 (10 μM) using the DCFDA assay under various conditions: cells alone; cells with DCFDA and Mn4 in the dark (D); and cells with DCFDA and Mn4 exposed to visible light (L) for different time intervals, as represented in the bar diagram.

Mn(III)/Mn(II) redox shuttle in visible light

Mn(III) porphyrins can reversibly switch between +2 and +3 oxidation states. In general, Mn(III) porphyrins that bearing high positive charges on their periphery, such as water-soluble cationic Mn(III) porphyrins, are considered good SOD mimics. These compounds exhibit an optimum positive E_1/2 value for the Mn(III)/Mn(II) redox cycle, making them readily reducible by common reducing agents.⁶¹ However, the E_1/2 values of the Mn(III)/Mn(II) redox cycle for these Mn(III) porphyrins (Mn1–Mn5) fall within the negative range, rendering them difficult to reduce, especially under oxygenated conditions. As a result, reduction of such porphyrins is typically carried out under deaerated conditions.⁶² However, when we carried out the reduction experiment of these Mn(III) porphyrins with ascorbic acid, which is a cellular reductant, in 1 : 1 DMSO : Tris-HCl buffer in air with light irradiation, it was observed that with gradual increase in light irradiation, the UV-visible spectra of the porphyrins show some distinct changes that correlate to their reduction to Mn(II) porphyrin. The absorption intensity of the Soret band at ∼460 nm gradually decreases and a new band at approximately 434–440 nm appears, which is typical for Mn(II) porphyrins. The other two less intense bands observed at UV region (∼370–400 nm) also exhibited decrease in their absorption intensity, and the two Q-bands shifted somewhat towards the red region (Fig. 5 and Fig. S48, ESI†).^37,62 When the light irradiation was stopped and the system was left exposed to air, it exhibited reversibility as shown by their UV-visible spectra (Fig. 5 and Fig. S49, ESI†). These findings suggest that light irradiation can catalyse the reduction of these Mn(III) porphyrins even in aerated conditions. Mn(III) porphyrins are generally stable against photodecomposition, and our photostability data confirmed this behavior (Fig. S30, ESI†). When the same experiment was conducted in the absence of light, the UV-visible spectra of these Mn(III) porphyrins did not show significant changes, suggesting that they are stable against the reduction process even at a higher concentration of ascorbic acid (5 mM), except Mn5 (Fig. 5 and Fig. S50, ESI†). This photo-redox activity of Mn(III) porphyrins in turn can imbalance the overall redox status of cancer cell. Some literature reports suggest that Mn(II) porphyrins are oxidized to Mn(III) porphyrin by molecular oxygen and, generating H₂O₂ during the process. The hydrogen peroxide produced may then lead to the formation of ROS via the Fenton reaction, which can induce oxidative stress that in turn can kill cancer cells.^63,64 However, under our experimental conditions, we were unable to detect H₂O₂, likely due to its very low concentration. No reduction of Mn(III) to Mn(II) porphyrin was observed in the presence of glutathione, either in the dark or under visible light (Fig. S51, ESI†). This is likely due to coordination of the glutathione carboxylate group to the axial position of Mn(III) porphyrin, as previously in the literature.⁶² Furthermore, we were also unable to detect any hydroxyl radical formation by the Mn(III) porphyrins at physiological pH, either in the dark or under visible light irradiation, using terephthalic acid as a probe (Fig. S52, ESI†). Nonetheless, literature reports indicate that at alkaline pH (10–11), Mn(III) porphyrins can generate hydroxyl radicals through electron transfer reactions with hydroxide ions.³⁷

Fig. 5 UV-visible spectra of Mn4 (10 μM) in 1 : 1 DMSO : Tris-HCl buffer in the presence of ascorbic acid (AA): (a) under light irradiation (1.4 mM AA), (b) post-irradiation reversibility and (c) in the dark (5 mM AA).

Intracellular ROS generation

DCFDA assay was employed to evaluate the ability of Mn(III) porphyrins to generate reactive oxygen species in cancer cells, using the most active Mn(III) porphyrin, i.e.Mn4 (10 μM), as a representative compound. H₂DCF, formed intracellularly from DCFDA via enzymatic conversion, is oxidized by ROS to yield DCF.⁶⁵ The emissive property of DCF in green light (λ_em = 528 nm) was used to monitor the ability of Mn4 to generate ROS in HeLa cells through flow cytometry. A significant increase in DCF emission, resulting from intracellular oxidation of H₂DCF by ROS generated upon photoexcitation of Mn4, was observed at various time points, viz. 15, 30, 45 and 60 min, in a time-dependent manner, as represented in the bar diagram (Fig. 4b). This was evident from the significant increase in emission count of DCF at different time in the light-exposed HeLa cells treated with Mn4. Control experiments using only cells in the dark, cells exposed to light alone and Mn4-treated cells kept in the dark did not show any significant ROS generation. The ROS generated by Mn4 on light exposure is believed to be primarily responsible of the observed cell death.

Photocytotoxicity study

The toxicities of Mn(III) porphyrins Mn1–Mn5 against HeLa (human cervical carcinoma) cancer cell line under dark and visible light conditions (400–700 nm) were evaluated using the MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Fig. 6). The compounds Mn1–Mn5 exhibited remarkable dose-dependent photocytotoxicity in visible light (400–700 nm) in HeLa cell, while showing minimal toxicity in the dark at concentrations up to 50 μM. As the solubility of the compounds were not very high in the permissible composition of DMSO–DMEM mixture, the maximum concentration of the compounds employed was restricted to 50 μM. A >5–10-fold increase in cytotoxicity was observed for Mn1–Mn5 in HeLa cells following 1 h of light exposure, compared to compound-treated cells kept in the dark under similar experimental conditions. Among the tested compounds, metalloporphyrin Mn4 was found to be the most phototoxic to HeLa cells, with an IC₅₀ value of ∼4.9 μM under visible light, while remaining non-toxic in the dark up to a concentration of 50 μM. The phototoxicity index (PI), defined as the ratio of the IC₅₀ value in the dark to that in the light, was >10 for Mn4. Mn5, which contains a chloro substituent in the para position of the meso-phenyl rings, exhibited significant photocytotoxicity, with an IC₅₀ value of ∼5.5 μM in visible light and no apparent toxicity in the dark. This implies that the toxicity of the compound is enhanced by more than 9-fold (PI) under visible light. Mn(III) porphyrins bearing –CH₃ and –OCH₃ substituents in the para positions of the meso-phenyl rings of the porphyrins, i.e.Mn2 and Mn3, exhibited slightly higher IC₅₀ values of ∼6.25 μM and ∼6.70 μM, respectively, under light exposure. Both compounds showed no significant toxicity toward HeLa cells in the dark at concentrations up to 50 μM. The PI values were found to be greater than 8.0 and 7.5 for Mn2 and Mn3, respectively. (Diaqua)meso(tetraphenylporphyrinato)manganese(III) propionate, i.e.Mn1, showed the lowest phototoxicity among the series, with an IC₅₀ value of ∼9.54 μM in visible light. It is reported that porphyrins containing paramagnetic metal ions in the core such as Co(II), Cu(II), Mn(III) generally exhibit very poor singlet oxygen quantum yields and are therefore considered unsuitable for PDT.^14,15 However, some studies have demonstrated that Mn(III) porphyrins can exert PDT effects via the generation of reactive oxygen species.^22,23,66 Mn(III) porphyrin containing carboxyl groups at the meso positions showed a significant enhancement in cytotoxicity in HepG2 cells, with an IC₅₀ value of ∼19.0 μM when irradiated with 625 nm LED light for 60 min. Photoinduced production of superoxide and hydroxide radicals was reported to be responsible for the photocytotoxicity, as revealed by EPR studies.²² A Mn(III) porphyrin, viz. Pt-MnPor-PPh₃, bearing triphenylphosphonium cation and Pt(II) moieties at its meso-peripheral positions, exhibited significant PDT effect against HeLa cells upon light irradiation for 10 min using yellow light (4 J cm⁻²) generated from a 400 W tungsten lamp. The IC₅₀ value was reported to be 30.74 μM under light irradiation, whereas no significant cytotoxicity was observed in the dark up to a concentration of 50 μM.⁶⁶ Recently, a water-soluble Mn(III) porphyrin bearing cationic pyridyl side groups has been reported to show significant photocytotoxicity in HeLa cells when irradiated with a green LED of 527 nm.²³ The IC₅₀ values were found to be 178 and 4 μM in the dark and under light irradiation, respectively, giving a PI value of 44.5. The IC₅₀ values of the Mn(III) porphyrins tested, including the Mn(III) porphyrin bearing cationic pyridyl groups, Photofrin®, protoporphyrin IX (PpIX) and cisplatin, are listed in Table 3.^23,67–69 The observed photocytotoxicity is comparable to that of the clinically used PDT drug Photofrin®.⁶⁷ PpIX, another established anticancer PDT drug, is found to be slightly more toxic than these metalloporphyrins, with IC₅₀ values of 2.8 ± 0.5 μM and 2.1 ± 0.3 μM when irradiated at 510 nm (20 min, 5.0 J cm⁻²) at 540 nm (40 min, 9.5 J cm⁻²), respectively.⁶⁸ The widely used chemotherapeutic agent cisplatin does not show any significant photocytotoxicity in HeLa cells under similar conditions.⁶⁹ Overall, it can be concluded that the Mn(III) porphyrins exhibit significant photodynamic activity in HeLa cells under visible light irradiation through the generation of cytotoxic ROS. The MTT assay for the free-base porphyrins was not performed due to their poor solubility in the acceptable composition of DMSO-DMEM for photochemotherapeutic studies in cancer cells. In general, singlet oxygen quantum yields (Φ_Δ) of free bases (e.g. Φ_Δ for TPP 0.64 in DMF) are higher than those of the corresponding Mn(III) porphyrins. However, given their better solubility and lower aggregation properties in aqueous solutions, which normally limit the potential of hydrophobic free-base porphyrins as PS, the use of Mn(III) porphyrins for photodynamic therapeutic application cannot be precluded.^42,70

Fig. 6 Cell viability plots showing the cytotoxic effects of Mn1–Mn5 (a–e) on HeLa cells in the dark (black) and under visible light (400–700 nm, 10 J cm⁻², 1 h) (red).

Table 3 IC₅₀ values of the Mn(III) porphyrins Mn1–Mn5 and Photofrin® in HeLa cells

Compound	HeLa
	IC50 (μM) dark^a	IC50 (μM) light^b	PI^c
a The IC50 values correspond to 24 h treatment followed by 24 h incubation in the dark. b The IC50 values correspond to 24 h treatment in the dark followed by photo-exposure to visible light (400–700 nm, 10 J cm^−2) for 1 h. Subsequently, cells were incubated for 23 h in the dark. c PI (phototoxic index) is the ratio of the IC50 in the dark to that under visible light exposure. d The IC50 values are taken from ref. 23 and correspond to incubation of cells with the compound for 24 h in the dark, followed by illumination with a green LED (527 nm) for 15 min at a power of 45 mW. e The Photofrin® IC50 values are taken from ref. 67. Light source reported: red light at 633 nm. f The IC50 values are taken from ref. 68. g It corresponds to irradiation at 510 nm (20 min, 5.0 J cm^−2). h It corresponds to irradiation at 540 nm (40 min, 9.5 J cm^−2). i The IC50 value for 4 h treatment is taken from ref. 69.
Mn1	>50	9.54 ± 1.2	>5.2
Mn2	>50	6.25 ± 1.1	>8.0
Mn3	>50	6.70 ± 0.9	>7.5
Mn4	>50	4.93 ± 0.7	>10.1
Mn5	>50	5.50 ± 0.7	>9.1
MnT(HeOH)PyP^5+	178^d	4^d	44.5^d
Photofrin®	>41^e	4.3 ± 0.2^e	—
PpIX	>100^f^,^g	2.8 ± 0.5^f^,^g	>35.7^f^,^g
	>100^f^,^h	2.1 ± 0.3^f^,^h	>47.6^f^,^h
Cisplatin	71.3 ± 2.9^i	68.7 ± 3.4^i	—

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Conclusion

In summary, five meso-tetraaryl Mn(III) porphyrins bearing various para substituents on the meso-phenyl ring were synthesized, comprehensively characterized, and evaluated for their visible light-induced anticancer activity. The Mn(III) porphyrins exhibited strong binding affinity toward human serum albumin. Electrochemical studies via cyclic voltammetry provided insights into their redox behaviour, while lipophilicity studies highlighted the influence of substituents on their aqueous solubility and membrane permeability. Upon visible light irradiation, these complexes induced significant cytotoxicity in HeLa cells through the generation of ROS, as confirmed by DCFDA assay, while remaining essentially non-toxic in the dark. Photocytotoxicity data demonstrated that fluorine- and chlorine-substituted Mn(III) porphyrins, i.e., Mn4 and Mn5, respectively, displayed enhanced photocytotoxicity toward HeLa cells compared to the other compounds. In addition, these findings highlight the importance of substituent effects on the photophysical and biological properties of Mn(III) porphyrins, offering valuable insights for the rational design of next-generation photosensitizers for targeted cancer therapy. This study opens up the unexplored area of 3d metalloporphyrins for photodynamic therapy.

Conflicts of interest

There are no conflicts to declare.

Data availability

All relevant data supporting the findings of this study are included within the article and its ESI.†

Acknowledgements

The authors are grateful to Department of Science and Technology (DST)-SERB, Government of India, Core Research Grant (CRG) (CRG/2023/002246) for financial assistance. We also acknowledge financial support from the DST-PURSE 2022 program (TPN-84912). The authors are thankful to DST and Department of SAIF, Gauhati University, for single crystal X-ray diffractometer facility. We also thank Dr Ranjit Thakuria, Dr Tejender S. Thakur, Dr S. Brahma and Dr Debajit Sarma for their kind assistance in X-ray data collection and structure solution. We sincerely thank Prof. A. R. Chakravarty, Indian Institute of Science, Bangalore, for allowing us to use the cell culture facility in his laboratory. JPS thanks DST-SERB for research fellowship.

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Footnote

† Electronic supplementary information (ESI) available: Characterization, selected X-ray crystallographic data, HSA binding data (Tables S1–S6, Scheme S1, Fig. S1–S52). CCDC 2408932, 2408982, 2408984 and 2408985 for compounds Mn1, Mn2, Mn4 and Mn5, respectively. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4dt03432e

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