Abstract
Photodynamic therapy (PDT) is a modern cancer therapy. But it is still difficult to obtain ideal photosensitizers. We synthesized six new peri-xanthenoxanthene derivatives rapidly and efficiently using solid-phase carbon-bath microwave irradiation technology, and investigated their in vitro photodynamic antitumor activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results showed that all compounds exhibited extremely low dark cytotoxicity and good phototoxicity against four human cancer cell lines. In particular, compound 3c showed the best in vitro PDT activity against Hela cells and Bel-7402 cells with IC50 values of 91 and 74 nmol/L, respectively. Its value of 1-octanol/water partition coefficient (log Kow) was 0.5309, suggesting that it is a promising photosensitizer for PDT due to its low dark cytotoxicity, high phototoxicity, and potential water solubility.
Introduction
Photodynamic therapy (PDT) is an oxygen-dependent photoactive physiotherapy that can be used to treat tumors and benign diseases. It combines photosensitizers (PS) with light to produce reactive oxygen species (ROS) which act on the target and produces photodynamic damage to biological tissues and cells.1–5)
The first generation of photosensitizer, porfimer sodium, has a poor therapeutic effect on large lesions. And it will produce long-lasting skin photosensitivity after being absorbed by the skin, which has a long retention time (4–6 weeks). It is not only has little selective effect for tumor cells, but also has a low molar absorption coefficient.6)
Newer PS, such as talaporfin and 3-(4-hydroxyphenyl)propionic acid hydrazide (HPPH), have higher absorption bands at longer wavelengths. They are showing increased efficacy. But these second-generation PS still have certain limitations, such as low phototoxicity, difficulty in synthesis, and poor water-solubility. They may cause hemoptysis, itching, erythema, dysphagia, etc. Therefore, researchers continue to search for more suitable photosensitizers.7,8)
Peri-xanthenoxanthene (PXX) derivatives are polycyclic aromatic compounds with strong fluorescence and good optical properties. With good charge transfer properties, thermal stability, and chemical stability, these compounds are often used as compositions of pigments, charge transfer complexes, and organic semiconductors in electronic devices.9–15) To the best of our knowledge, PXX derivatives have never been used in anti-tumor applications.
In order to develop new potential PDT photosensitizers, we synthesized six new PXX derivatives in this research. These compounds were investigated for their in vitro phototoxicity against human tumor cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), their absorption spectra, and 1-octanol/water partition coefficients.
Results and Discussion
The synthetic route is outlined in Chart 1. Substituted 2-naphthol underwent self-coupling or cross-coupling reactions to obtain the target products via solid-phase carbon-bath microwave method15) in the presence of copper acetate. The reaction was fast and took only 6 to 12 min. High purity products could be obtained and the post-treatment was simple. Due to competitive reaction, the yield of monosubstituted PXXs was lower than the disubstituted ones. The results are shown in Table 1. All of the targets 3a–3f were new compounds, and their structures were confirmed by 1H-NMR, 13C-NMR, and Fourier transform ion cyclotron resonance （FT-ICR) MS.
Chart 1. Synthesis of PXX Derivatives
Table 1. Synthesis Results of PXX Derivatives in Microwave Carbon Bath
| Compound | R1 | R2 | Time (min) | Yield (%) |
|---|
| 3a | H | CN | 8 | 57.8 |
| 3b | H | COOCH3 | 6 | 61.2 |
| 3c | H | CONH(CH2)2OH | 9 | 55.3 |
| 3d | CN | CN | 10 | 89.6 |
| 3e | COOCH3 | COOCH3 | 12 | 85.3 |
| 3f | CONH(CH2)2OH | CONH(CH2)2OH | 12 | 84.2 |
The in vitro dark toxicity and phototoxicity of the synthesized PXX derivatives were measured with the MTT assay against HeLa, A549, SGC-7901, and Bel-7402 cell lines. According to the results (listed in Table 2), the dark IC50 values of 3a–3f against tumor cells were all >100 µmol/L, which showed that they had extremely low dark toxicity. All compounds exhibited high phototoxicity against four kinds of human tumor cell lines. Compounds 3b, 3c, 3e and 3f showed stronger phototoxicity than 3a and 3d, which indicated that the presence of moderate electron-withdrawing groups, CONH(CH2)2OH and COOCH3, can significantly improve the phototoxicity of PXX derivatives, while strong electron-withdrawing group, CN, would reduce the phototoxicity. The introduction of strong electron-withdrawing group is not beneficial to improve the phototoxicity of PXX molecules. In general, the phototoxicity of monosubstituted PXXs, 3a–3c, were better than the disubstituted ones, 3d–3f. In particular, compound 3c exhibited the strongest phototoxicity, with its IC50 values of 91 and 74 nmol/L against HeLa cells and Bel-7402 cells respectively. The IC50 value of compound 3a for SGC-7901 was 0.36 µmol/L, which was far lower than the IC50 values on the other three kinds of tumor cells, and showing strong selectivity. All these results indicate that the synthetic PXX derivatives, especially 3a and 3c, are potential PDT antitumor agents.
Table 2. Cytotoxicity (IC
50, µmol/L) for Compounds
3a–
3f against Tumor Cells
| Compound | HeLa | A549 | SGC-7901 | Bel-7402 |
|---|
| Dark | Light | Dark | Light | Dark | Light | Dark | Light |
|---|
| 3a | >100 | 12.31 ± 0.91 | >100 | 34.56 ± 1.25 | >100 | 0.36 ± 0.12 | >100 | 16.88 ± 1.98 |
| 3b | >100 | 0.28 ± 0.03 | >100 | 9.30 ± 1.53 | >100 | 0.52 ± 0.32 | >100 | 0.26 ± 0.02 |
| 3c | >100 | 0.091 ± 0.01 | >100 | 0.359 ± 0.11 | >100 | 0.108 ± 0.01 | >100 | 0.074 ± 0.02 |
| 3d | >100 | 8.47 ± 3.90 | >100 | 40.29 ± 0.86 | >100 | 12.16 ± 0.05 | >100 | 41.24 ± 1.21 |
| 3e | >100 | 0.61 ± 0.05 | >100 | 10.21 ± 1.25 | >100 | 0.55 ± 0.15 | >100 | 3.70 ± 0.26 |
| 3f | >100 | 5.39 ± 0.19 | >100 | 2.479 ± 0.56 | >100 | 10.83 ± 1.05 | >100 | 30.50 ± 2.00 |
The ROS produced by PS exist for a very short time, so the distribution and accumulation of PS in tumor cells is of great significance for the therapeutic effect of PDT. PXX derivatives have strong planarity, small molecular space, and strong lipid-solubility. Hence, they may be easier to enter cancer cells through biofilm and aggregate, showing strong phototoxicity. At the same time, the functional groups substituted onto the PXX structure may mainly affect the localization of PXX derivatives to different cancer cells. Thus the phototoxicity of PXX derivatives with different substituents may vary greatly.
The electronic absorption and basic photophysical properties for 3a–3f were characterized in dimethyl sulfoxide (DMSO) by UV-vis spectroscopy (Table 3). You can see these compounds possessed high molar extinction coefficient (ε = 0.37 × 104 to 1.21 × 104 L·mol−1·cm−1) at maximum absorption wavelength ranged from 409 to 442 nm. The fluorescence quantum yields of 3a–3f were from 0.80 till 0.95. The high fluorescence quantum yields may be mainly attributed to PXX’s high conjugate system.
Table 3. The Absorption Spectra and Φ
f of Compounds
3a–
3f| Compound | λmax (DMSO, nm) (ε × 104 L·mol−1·cm−1) | Φf |
|---|
| 3a | 412 (0.88) | 440 (1.04) | 0.88 |
| 3b | 413 (0.98) | 441 (1.21) | 0.80 |
| 3c | 415 (0.54) | 442 (0.67) | 0.81 |
| 3d | 409 (0.37) | 437 (0.39) | 0.95 |
| 3e | 412 (0.62) | 440 (0.75) | 0.93 |
| 3f | 414 (0.80) | 442 (1.06) | 0.80 |
From PDT point of view, absorption at 409–442 nm is not suitable for PDT applications compared to other available PSs (excited >600 nm). But the maximum absorption wavelength of PXX can reach more than 600 nm after structural modification.16) So PXX still has application prospects.
To provide information about the physicochemical properties of the PXX derivatives, we used a shake-flask method to detect the 1-octanol/water partition coefficient of compounds 3a and 3c. The partition coefficients were calculated according to the ratio Coct/Cwater and the values of log Kow were 1.2496 and 0.5309, which suggested that compounds 3a and 3c may potentially soluble in water.
In summary, six new PXX derivatives 3a–3f were synthesized by microwave irradiation efficiently. Although their ultraviolet absorption wavelength is relatively short, compounds 3a–3f are promising photosensitizers for PDT applications. Especially compound 3c has the advantages of high phototoxicity, low dark toxicity, high efficient absorption, and potential water solubility. In future work, we will modify the structure of these compounds to further improve their absorption wavelength and optimize their activity.
Acknowledgments
This work was supported by Guangzhou Innovation Environment Construction Project (2018, No. 110).
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
The online version of this article contains supplementary materials.
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