Pheophorbide a

Amphiphilic chlorins 15-(4-carboxyphenyl)-17,18-dihydro-18,18-dimethyl-5-p-tolylporphyrin (11) and Zn(ii) 15-(4-carboxyphenyl)-17,18-dihydro-18,18-dimethyl-3-(phenylethynyl)porphyrin (12) were synthesized with a de novo method that enables introduction of substituents at desired sites about the perimeter of the macrocycle.³⁶ Acid-promoted condensation of the Eastern and Western halves is followed by metal-mediated oxidative cyclization. The selective introduction of substituents relies on (i) the use of substituted precursors (Eastern and Western halves); or (ii) bromination of the chlorin macrocycle followed by Pd-mediated coupling reaction. Both strategies were used in the synthesis of target chlorins. Pheophorbide a is purchased from Frontier scientific and Zn is inserted by refluxing 5 molar equivalents of ZnCl₂ in methanol as previously described.³⁷ Bacteriochlorophyll was extracted from Rb. sphaeroides, then demetalated, phytyl chain cleaved before Zn insertion to form Zn bacteriochlorophyllide.³⁷

In order to investigate the activity of BCRP, a pheophorbide A (Frontier Scientific Inc., Logan, UT, USA) accumulation assay was performed as described [4 (link)]. Cells were cultured and detached by using EDTA and suspended in Krebs–HEPES buffer (10 mM HEPES, 118.6 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 4.2 mM NaHCO₃, 11.7 mM glucose). A total of 40,000 cells were added per well of a 96-well plate. To selectively inhibit BCRP, the inhibitor Ko143 (IC₅₀ 0.276 μM, Tocris Bioscience, Bristol, UK) was used at a concentration of 3 μM, and pheophorbide A was used at a concentration of 0.5 μM. After 2 h incubation, fluorescence (λ_ex 405 nm, λ_em 695 ± 50 nm) was measured by flow cytometry (Guava^©, Merck). The KHB values were normalized to each inhibited sample to calculate the functional transporter activity as a ratio to maximal inhibition.

In order to investigate the activity of ABC transporters, dye accumulation assays were performed. Cells were cultured 72 h in the presence or absence of COL1 up to 60–70% and/or 24 h in the presence or absence of ERK1/2-inhibitor SCH772984 (1 µM), detached using trypsin/EDTA (Pan Biotech) and suspended in Krebs-HEPES-Buffer (KHB). 40,000 cells were added per well of a 96-well plate. For BCRP transporter investigation, its substrate dye pheophorbide A (Frontier Scientific Inc., Logan, UT, USA) was used at a concentration of 0.5 µM. To obtain a total BCRP-inhibition, the inhibitor Ko143 (IC₅₀ 0.276 µM [70 (link)], Tocris Bioscience, Bristol, UK) was used at a concentration of 3 µM. After 2 h incubation, fluorescence (λex = 405 nm, λem = 695 ± 50 nm) was measured by flow cytometry (Guava^®, Merck). The values of KHB were normalized to each inhibited sample to get the functional transporter activity as a ratio to maximal inhibition. To analyze the structural effect of the inhibitors used, BCRP overexpressing MDCK II BCRP were used [70 (link)]. In these terms, inhibitors were added directly without any incubation.