2016 Volume 64 Issue 9 Pages 1310-1314
A novel fluoroporphyrin–anthraquinone hybrid with dipeptide link and its metal complexes were synthesized and evaluated for anti-proliferation activity in human cancer cell line HeLa. The preliminary results demonstrated that all the compounds showed moderate to excellent antitumor activities. Among the active compounds, compound 3 which contains fluorinated porphyrin–anthraquinone and zinc ion exhibited the highest potency with IC50 value of 8.83 µM, indicating that it was a promising antitumor candidate.
Cancer is the second fatal cause worldwide after cardiovascular disease. Billions of dollars are spent annually on research in order to cure or improve the life-standards of the cancer patients.1–3) The traditional cancer treatments, including surgery, radiation therapy and chemotherapy, result in serious adverse effects caused by the loss of the functions of normal organs.1) Photodynamic therapy (PDT) is a form of phototherapy using nontoxic light-sensitive compounds that are exposed selectively to light, whereupon they become toxic to targeted malignant and other diseased cells.4,5) PDT is a promising method for physical treatment of cancer and other diseases, including bacteria, fungi and viruses.6) It is recognized as a treatment strategy which is both minimally invasive and minimally toxic.7,8)
Porphyrins are an important class of naturally occurring macrocyclic compounds found in biological compounds that play a very important role in PDT.9,10) They bind to DNA or RNA and accumulate in tumor cells, which are enabled to have an easy access in accepting the light radiation because of the strong absorption and luminescence properties of porphyrins.11) Moreover, the anthraquinone derivatives can be used as the light of DNA cleavage agents mainly due to three reasons12): 1) the aromatic ring skeleton of anthraquinones can be embedded into the base of DNA to bridge DNA bond together; 2) the excited state of anthraquinones is a good oxidant and the embedded anthraquinone can oxidize poly(sugar-phosphate skeleton) and initiate the DNA breakage; 3) anthraquinones are very stable in the redox process.13)
In this study, we have designed and incorporated anthraquinone scaffold into the porphyrin to increase the binding of porphyrin to cancer cells, and further synthesized a novel fluoroporphyrin–anthraquinone hybrid with dipeptide link and its metal complex. We also found that introduction of fluorine to porphyrin can enhance the binding interactions, metabolic stability and physicochemical properties.14) The preliminary study of antitumor activities of the porphyrin derivatives against HeLa cell line was evaluated.
Infrared spectra were measured using a BX fourier transform infrared spectroscopy (FT-IR) spectrometer (Perkin-Elmer Inc., U.S.A.) using smear KBr crystal or KBr plate. 1H-NMR spectroscopy were recorded on a JEOL EX-300 (300 MHz) spectrometer; J values are in Hz. UV absorption spectra were recorded on a 751 UV/Vis spectrophotometer. TLC was self-made silica gel (GF254) sheets. Flash chromatography was performed using 200–300 mesh silica gel. The yields were calculated by the last step reaction.
Preparation of 5-(4-Hydroxyphenyl)-10,15,20-O-trifluorophenylporphyrin (1)A solution of p-hydroxybenzaldehyde (0.3053 g, 2.5 mmol) and 2-fluorobenzaldehyde (0.8 mL, 7.5 mmol) in propionic acid (20 mL) was stirred at 100°C in microwave oven under the power of 195 W for 3 min. Then the mixture of acetic anhydride (2 mL) and pyrrole (0.7 mL, 10.0 mmol) was slowly added to the flask. Upon the completion of addition, the reaction mixture was heated under the power of 195 W for 9 min. After cooling to room temperature, the mixture was kept under static conditions overnight. The precipitate separated by suction filtration and washed four times on the funnel with hot water. The residue was purified by silica gel column chromatography to afford 5-(4-hydroxyphenyl)-10,15,20-O-trifluorophenylporphyrin (1, 62 mg, 65%). UV-Vis in CHCl3: Soret (nm) 418, Q (nm) 512, 544, 586, 646. IR (infra-red spectrum) (KBr, cm−1): 3447 (O–H), 1544, 1465 (benzene skeleton vibration), 1152 (O–H bending vibration), 1406 (CH3 bending vibration, 1030 (porphine frame vibration), 975 (porphine ring N–H bending vibration). 1H-NMR (600 MHz, CDCl3, δ ppm): 8.94 (d, J=28 Hz, 8H, Hβ), 8.67 (s, 3H, Ho), 8.49 (d, J=40 Hz, 2H, Ho′), 8.25 (d, J=40 Hz, 2H, Hm), 8.04 (s, 3H, Hm2), 7.88 (s, 3H, Hm), 7.62 (m, 3H, Hp), 2.69 (s, 2H, PyNH).
Preparation of 5,10,15-O-Trifluorophenyl-20-[4-(glycyl-1-aminoanthraquinoneimide)] Sub Methoxy Phenyl Porphyrin (2)A mixture of porphyrin (0.060 g, 0.1 mmol) and anhydrous K2CO3 (0.6910 g, 5.0 mmol) in N,N-dimethylformamide (DMF) 10 mL was stirred at 76°C for 1 h. 2-Chloro-N-(2-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino)-2-oxoethyl)acetamide (0.0714 g, 0.2 mmol) in DMF (10 mL) was slowly added to the mixture. When finished dropping, the reaction mixture was stirred under reflux condition for 20 h. The solvent was removed under vacuum to give the crude product, which was washed by water to remove K2CO3. The residue was further purified by silica gel column chromatography to afford compound 5,10,15-O-trifluorophenyl-20-[4-(glycyl-1-aminoanthraquinoneimide)] sub methoxy phenyl porphyrin (2, purple, 45 mg, 45%). UV-Vis (CHCl3): Soret (nm) 418, Q (nm) 514, 546, 588, 644. IR (KBr, cm−1): 3396 (stretching vibration of chlorin N–H), 2923, 2852 (CH2 stretching vibration), 1720, 1703, 1686, 1655 (C=O stretching vibration), 1560, 1544, 1465 (benzene skeleton vibration), 1459 (CH2 bending vibration), 1119 (C–O stretching vibration), 1074 (porphine skeleton stretching vibration), 968 (porphine ring N–H bending vibration). 1H-NMR (600 MHz, CDCl3, δ ppm): 8.94 (d, J=30 Hz, 8H, Hβ), 8.70 (d, J=20 Hz, 3H, Ho), 8.52 (s, 2H, Ho′), 8.61(m, 2H), 8.36 (s, 2H), 8.21 (s, 2H, Hm′), 8.04 (s, 3H, Hm2), 7.88 (s, 3H, 3Hm), 7.62 (m, 3H, Hp), 8.14 (s, 1H), 7.78 (s, 1H), 7.23 (s, 1H, anthraquinone hydrogen), 6.58 (s, 2H, OCH2CO), 5.22 (s, 2H, NHCH2CO), 2.77 (s, 2H, PyNH).
Preparation of Compounds 3–7General Procedure for Synthesis of Compounds 3–7A solution of compound 2 (10.4 mg, 0.1 mmol) in CHCl3 and CH3OH (30 mL, 3 : 2, v/v) was added to various metal acetates (0.2 mmol). The mixture was stirred under 10% (65 W) microwave heating until the raw point disappeared under the detection of TLC. After cooling to ambient temperature, the solvent was removed under vacuum to yield compounds 3–7. The analysis data of compounds 3–7 are listed as following.
Compound 3, purple solid (9.2 mg); yield, 71%; UV-Vis (CHCl3): Soret (nm) 418, Q (nm) 546. IR (KBr, cm−1): 3447 (stretching vibration of amide N–H), 2924, 2853 (CH2 stretching vibration), 1716, 1703, 1654 (C=O stretching vibration), 1561, 1540, 1465 (benzene skeleton vibration), 1459 (CH2 bending vibration), 1127 (C–O stretching vibration), 997 (porphine skeleton stretching vibration).
Compound 4, yield, 86%; UV-Vis (CHCl3): Soret (nm) 418, 426 Q (nm) 516, 548, 615. IR (KBr, cm−1): 3456 (stretching vibration of amide N–H), 2927, 2850 (CH2 stretching vibration), 1712, 1701, 1655 (C=O stretching vibration), 1560, 1539, 1470 (benzene skeleton vibration), 1464 (CH2 bending vibration), 1123 (C–O stretching vibration), 1010 (porphine skeleton stretching vibration).
Compound 5, yield, 87%; UV-Vis (CHCl3): Soret (nm) 417, Q (nm) 542. IR (KBr, cm−1): 3470 (stretching vibration of amide N–H), 2930, 2857 (CH2 stretching vibration), 1723, 1708, 1656 (C=O stretching vibration), 1559, 1532, 1455 (benzene skeleton vibration), 1468 (CH2 bending vibration), 1132 (C–O stretching vibration), 999 (porphine skeleton stretching vibration).
Compound 6, yield, 71%; UV-Vis (CHCl3): Soret (nm) 417, Q (nm) 550. IR (KBr, cm−1): 3436 (stretching vibration of amide N–H), 2920, 2848 (CH2 stretching vibration), 1720, 1705, 1656 (C=O stretching vibration), 1564, 1539, 1462 (benzene skeleton vibration), 1460 (CH2 bending vibration), 1128 (C–O stretching vibration), 1002 (porphrine skeleton stretching vibration).
Compound 7, yield, 71%; UV-Vis (CHCl3): Soret (nm) 426, Q (nm) 562. IR (KBr, cm−1): 3449 (stretching vibration of amide N–H), 2924, 2855 (CH2 stretching vibration), 1714, 1701, 1655 (C=O stretching vibration), 1560, 1541, 1466 (benzene skeleton vibration), 1462 (CH2 bending vibration), 1128 (C–O stretching vibration), 996 (porphine skeleton stretching vibration).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) AssayCell viability was measured by MTT assay to assess the chemosensitivity of tumor cells.15,16) The growth inhibitory activity of target compounds was determined on cell lines HeLa using a modified version of the microculture tetrazolium assay. Once the cells reached 90% confluence, a cell suspension was prepared by trypsinization of monolayer cultures. Cell counts was performed, and the suspensions were diluted accordingly to give 4–5×104 cells/mL with the appropriate medium. Aliquots (10 µL) of the cell suspension were added to each well in a 96-well microtitre plate. The cells were incubated for 24 h (37°C, 5% CO2). Stock solutions of the test compound were prepared in dimethyl sulfoxide (DMSO). And serial dilutions were made with medium to over a 100 fold concentration range. Not more than 1% DMSO (final concentration) was present in each well. The test sample was incubated with the wells for 72 h. Cultured for 6 h in the ultraviolet light irradiation conditions. Wells without cells and those with cells in culture medium/DMSO were examined in parallel. At the end of the incubation period, the medium was decanted and replaced with 20 µL MTT solution (5 mg/mL). The cells were incubated for another 4 h, after which the medium was removed from each well by pipetting. DMSO (100 µL) was added to each well to lyse the cells. The optical density (OD) values were measured within 30 min at 570 nm on an ELISA reader (WellscanMK2). The cell inhibitory rate was calculated with the following equation:
 |
HeLa cells were routinely grown on 22 mm square coverslips placed into 35 mm culture dishes. Cells were stimulated to undergo apoptosis with 25 µM compound 3 for 6 h. After induction of apoptosis, cells were stained with the AO dye mix for 5 min. The dye mix was 100 µg/mL acridine orange in phosphate buffered saline (PBS). Acridine orange permeates all cells, making the nuclei appear green. Live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented chromatin and membrane blebbing.17)
After AO staining, culture dishes were inverted and fixed with formaldehyde vapor for 1 min to prevent the photo-damaging effects of continuous excitation on living cells due to the photosensitizing effects of most fluorescent dyes. A total of 600 cells per cell line per group were counted from each independent experiment to determine the percentage of apoptotic cells.17)
Transferase-Mediated Deoxyuridine Triphosphate-Biotin Nick End Labeling (TUNEL) AssayThe cells apoptosis were assessed by fluorescent staining and the TUNEL method. The cryostat sections were washed in PBS (3×5 min), mounted in glycerol–PBS (1 : 1) and coverslipped. Photographs were taken with an Olympus camera attached to an Olympus fluorescence microscope (Olympus Optical Co., Ltd., Japan). The TUNEL assay, used to investigate biochemical feature of apoptosis, was carried out according to the kit manufacturer’s protocol (Chemicon, U.S.A.). The sensory neurons were then photographed under a light microscope.
The synthesis of compounds 1–7 was following literature procedure and it was a relatively straightforward microwave procedure. Compound 1, 5-(4-hydroxyphenyl)-10,15,20-O-trifluorophenylporphyrin, was prepared as shown in Chart 1 by mixing four equivalent of pyrrole, three equivalent of 2-fluorobenzaldehyde and one equivalent of p-hydroxybenzaldehyde in propionic acid under microwave conditions. The preparation of 5,10,15-O-trifluorophenyl-20-[4-(glycyl-1-aminoanthraquinoneimide)] sub methoxy phenyl porphyrin (compound 2) started with the reaction of compound 1 above with anthraquinone chloride under potassium carbonate and yielded compound 2 after purification. Compounds 3–7 were synthesized by chelating compound 2 with five metal acetates, respectively. All the compounds were structurally confirmed by UV-Vis, IR and 1H-NMR spectrascopy.
Compounds 1–7 were dissolved in CHCl3 (5×10−6 mol/L). The fluorescence emission spectrum (FS) data (Table 1) was received by fluorescence emission scanning in fluorescence spectroscopy using UV-Vis absorption spectra (420 nm) of maximum absorption band (Soret band) as the excitation wavelength. Under the same conditions, the fluorescent emission intensity order was: 1>2>4>7>6>5>3, and the fluorescence quenching order was: 3>5>6>7>4>2>1. The spectral wavelength and peak shape of the compound 2 were similar with porphyrin 1, while the absorption intensity of the compound 2 was lower than that of porphyrin 1 (Table 1). Due to the linker was flexible, resulting in face-to-face overlapping between porphyrin and anthraquinone ring. The excited state electrons can directly transfer from the porphyrin through the surface-to-surface channel to anthraquinone, namely the intramolecular electron transfer, leading to fluorescence quench. The fluorescence intensity of compounds 3–7 was weaker than that of compounds 1 and 2.
| Compounds | The maximum emission wavelength (nm) | Fluorescence intensity |
|---|---|---|
| 1 | 653.04 | 223.04 |
| 2 | 648.98 | 153.10 |
| 4 | 646.37 | 97.58 |
| 7 | 646.29 | 97.32 |
| 6 | 645.03 | 96.98 |
| 5 | 643.78 | 96.01 |
| 3 | 643.04 | 95.70 |
Fluorescence emission wavelength of the metal compounds shifted to short wavelength. This may be because the positive charged metal ion was an electron withdrawing group, and the electron density of porphine ring became lower after it coordinated with metal ion, leading to violet shift of the fluorescence emission wavelength.
Antitumor ActivityThe compounds 1–7 were evaluated by MTT assay for their inhibitory activities against human cancer cell line HeLa cells. The results, expressed as IC50, are summarized in Table 2. The antitumor activity order was: 3>5>6>7>4>2>1. Compound 1, only containing porphyrin ring, showed no inhibitory activity up to 100 µM, while compound 2 by introducing an anthraquinone scaffold, showed an increasing antitumor activity, with an IC50 value of 13.96±1.12 µM. Compound 3, chelated with a zinc ion, was proved to be the most potent compound with an IC50 of 8.83±0.75 µM, which can be used for further development novel antitumor agents. The better bioactivity of compound 3, a zinc complex, can be explained from its property of less stereo-hindrance from DNA binding and stronger binding affinity between Zn–porphyrin and DNA. Because of the smaller size of zinc ion comparing to other four metals, stereo hindrance of the zinc–porphyrins is less than other metalloporphyrins, inferring that zinc–porphyrin complex is more approachable than other four complexes. On the other aspect, the coordination bonds between zinc and porphine are the weakest among the five metal complexes because zinc has the most tendency to form a covalent bond alike with a porphine among the five metals, increasing the stability of metal–porphine complex. Thus we speculate that the zinc–porphyrin complex has stronger binding affinity toward to DNA than that of other metalloporpyrins and free porphyrins.18)
| Compounds | Unlighted IC50 (µM) | Lighted IC50 (µM) |
|---|---|---|
| 1 | >100 | >100 |
| 2 | >100 | 13.96±1.12 |
| 4 | >100 | 11.38±0.95 |
| 7 | >100 | 11.24±0.83 |
| 6 | >100 | 10.82±1.02 |
| 5 | >100 | 9.35±0.86 |
| 3 | >100 | 8.83±0.75 |
AO is a nucleic acid selective metachromatic stain useful for cell cycle determination. AO interacts with DNA and RNA by intercalation or electrostatic attraction respectively. DNA intercalated AO fluoresce green (525 nm). In this study, AO staining was chosen for locating the zinc–porphyrin complex, compound 3. First, HeLa cells were treated with compound 3, red fluorescence was observed (Fig. 1A). Then, the cells were stained with AO, and yellow fluorescence (green+red) was observed inside of red fluorescence (Fig. 1B), indicating that compound 3 acted on nucleus.
(A) HeLa cells were treated with compound 3, (B) HeLa cells were first treated with compound 3, then treated with AO staining.
We used TUNEL method for detecting DNA fragmentation that results from apoptotic signaling cascades.19–22) The cancer cells (negative control) showed a large cell body, large nucleus and the expected distribution of nuclear material with no apoptotic signs (Fig. 2A). In contrast, a considerable number of TUNEL positive nuclei were detected after the treatment with compound 3 (Fig. 2B), which suggested that the cells were in the last phase of apoptosis. The self-accumulation of metal ion porphyrin–anthraquinone compounds on DNA effectively improved the yield of singlet oxygen, and elevated the levels of DNA breakage.
(A) Negative control, (B) HeLa cells were treated with compound 3.
In this study, we have synthesized a novel fluoroporphyrin–anthraquinone hybrid with dipeptide link and its metal complexes, which were structurally confirmed by UV-Vis, IR and 1H-NMR spectra. MTT assay demonstrated that the entire series of compounds exhibited moderate to excellent antitumor activities. Particularly, the zinc–porphyrin complex (compound 3) showed the highest activity against HeLa cells (IC50=8.83 µM). AO staining revealed that compound 3 targeted the nucleus, which provided a good lead compound for designing novel antitumor agents. Further modifications of this novel family of potential antitumor agents are ongoing in our laboratories and will be reported in due course.
This work was funded by General Research Fund of Huanggang Normal University (No. 2014022203), Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains (2015TD07), The Natural Science Foundation of Hubei Province of China (2006ABA186) and the Natural Science Foundation of Hubei Province of Youth Project (Q200727004).
The authors declare no conflict of interest.
