J. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 293–297
297
BCPDs, 16 and 18 are more effective PDT photosensitiz-
ers based on the studies with BEL-7402 cells. MG-P
staining qualitative analysis also showed that PDT with
BCPD 16 can induced apoptosis in BEL-7402 cells. Fur-
ther developments such as partial hydrolysis and
improvements using BCPDs are in progress and more
extensive and deeper biological studies are ongoing.
Acknowledgments
This work was supported by the National Natural Sci-
ence Foundation of China (Grant No. 30371737) and
the special Foundation of Second Military Medical Uni-
versity (Grant No. 05JS08).
Supplementary data
Figure 2. The images of BEL-7402 cell apoptosis induced by BCPD
16-mediated PDT at a fluence of 10 J/cm2 (56 mW/cm2 for 3 min) with
a laser (670 nm) at concentration of 2.0 lg/mL.
Supplementary data associated with this article can be
value was 0.23 lg/mL (95% confidence limit was
0.13 ꢀ 0.46 lg/mL) for BPDMA. Among them, BCPDs
16 and 18 were determined to be more effective PDT
photosensitizers based on the studies with BEL7402 cells
and had nearly equal photodynamic efficacy to
BPDMA.
References and notes
1. (a) Brown, S. B.; Brown, E. A.; Walker, L. Lancet 2004, 5,
497; (b) Dougherty, T. J. J. Clin. Laser Med. Surg. 2002,
20, 3; (c) Ackroyd, R.; Kelty, C.; Brown, N.; Reed, M.
Photochem. Photobiol. 2001, 74, 656; (d) Moser, J. G.
Photodynamic Tumor Therapy; Harwood Academic:
Amsterdam B.V., 1998, pp 117–232.
2. DeRosa, M. C.; Crutchley, R. J. Coord. Chem. Rev. 2002,
233, 351.
3. Bonnett, R. J. Heterocycl. Chem. 2002, 39, 455.
4. Bonnett, R.; Martinez, G. Tetrahedron 2001, 57, 9513.
5. Castano, A. P.; Demidova, T. N.; Hamblin, M. R.
Photodiagn. Photodyn. Ther. 2004, 1, 279.
6. Detty, M. R.; Gibson, S. L.; Wagner, S. J. J. Med. Chem.
2004, 47, 3897.
7. Sternberg, E. D.; Dolphin, D.; Bruckner, C. Tetrahedron
1998, 54, 4151.
8. Bonnet, R. Chem. Rev. 1995, 24, 19.
9. Sharman, W. M.; Allen, C. M.; van Lier, J. E. Discovery
Today 1999, 4, 507.
10. Dennis, E. J.; Dolmans, G. C.; Fukumura, D.; Jain, R. K.
Nat. Rev. Cancer 2003, 3, 380.
11. Davis, W. M.; Vinson, M. C. Drug Topics 2001, 145, 89.
12. (a) Callot, H. L.; Johnson, A. W.; Sweeney, A. J. Chem.
Soc., Perkin Trans. 1973, 1, 1424; (b) Morgan, A. R.;
Pangka, V. S.; Dolphin, D. J. Chem. Soc., Chem.
Commun. 1984, 1047; (c) Dinello, R. K.; Dophin, D.
J. Org. Chem. 1980, 45, 5196; (d) Pandey, R. K.; Shiau,
F.-Y.; Ramachandran, K. R.; Dougherty, T. J.; Smith, K.
M. J. Chem. Soc., Perkin Trans. 1992, 1, 1377; (e) Yu, J.
X.; Xu, D. Y. Chin. J. Pharm. 1999, 30, 158 (in Chinese).
13. Richter, A. M.; Kelly, B.; Chow, J.;Liu, J. D.; Towers, G. H.
N.; Levy, J.; Dolphin, D. J. Natl. Cancer Inst. 1990, 52, 501.
14. (a) Yao, J. Z.; Shen, W. D.; Chen, W. H.; Liu, J. F.; Zhou,
Y. J. Chin. J. Pharm. 2000, 31, 215 (in Chinese); (b) Yao,
To further address the BEL-7402 cell death caused by
BCPD 16-mediated PDT, the apoptosis morphology,
which was captured by Olympus IX 41, was observed
through Methyl Green–Pyronin (MG-P) staining
(Fig. 2). Cells were pre-incubated with concentration
of BCPD 16 at 2.0 lg/mL in the dark for 24 h at 37 ꢁC
followed by irradiation with a laser (670 nm) at a fluence
of 10 J/cm2 (56 mW/cm2 for 3 min).Then, hepatoma
BEL-7402 cells were incubated for 24 h at 37 ꢁC. The
cells were fixed by ethanol, stained by MG-P, and subse-
quently photographed by Olympus IX 41.
In principle, Methyl Green–Pyronin staining is effective
in the identification of plasma cell and RNA in tissue
sections and cytological preparations. DNA is stained
green to blue by Methyl Green and RNA is colored
red by Pyronin. Apoptotic cells display positive response
to both Methyl Green and Pyronin. Necrotic cells show
positive response to Methyl Green, but negative re-
sponse to Pyronin. Normal cells exhibit negative re-
sponse to both Methyl Green and Pyronin. As shown
in Figure 2, hepatoma BEL-7402 cells upon treatment
with BCPD 16-mediated PDT showed typical apoptosis
morphology when stained by MG-P.
In summary, a series of novel benzochloroporphyrin
derivatives (BCPDs) have been designed, synthesized,
and characterized. The potentials of BCPDs as PDT-
sensitizers were evaluated by in vitro photocytotoxicity
measurement using MTT assay on human hepatoma
BEL-7402 cells. All title BCPD tested exhibit signifi-
cantly lower dark cytotoxicity than BPDMA. Notably,
BCPDs 16, 17, and 18 show significantly higher photo-
cytotoxicity on BEL-7402 than BCPD 15. Moreover,
J. Z.; Liu, J. F.; Zhang, W. N.; Zhou, Y. J.; Zhu, J.; Lu, J.
¨
G.; Wang, X. Y. Acta Pharm. Sin. 2001, 36, 188 (in
Chinese); (c) Yao, J. Z.; Liu, J. F.; Zhang, W. N.; Zhou,
Y. J.; Zhu, J.; Lu¨, J. G.; Wang, X. Y.; Li, K. . Chin. J. Org.
Chem. 2001, 21, 458 (in Chinese).
15. See Supplementary Information.