Design, synthesis, and in vitro photodynamic activities of benzochloroporphyrin derivatives as tumor photosensitizers

Article abstract of DOI:10.1016/j.bmcl.2007.10.086

Novel benzochloroporphyrin derivatives (BCPDs) were designed, synthesized, and characterized. In vitro dark cytotoxicity and photodynamic efficacy of BCPDs were evaluated by MTT assay on human hepatoma BEL-7402 cells. The experimental results showed that BCPDs 15, 16, 17, and 18 have strong long wavelength absorptions around 670 nm and exhibit significantly lower dark cytotoxicity than BPDMA and possess potent photocytotoxicity, IC₅₀ values 1.32 μg/mL for 15, 0.26 μg/mL for 16, 0.47 μg/mL for 17 of 0.27 μg/mL for 18, and 0.23 μg/mL for BPDMA. Among them, BCPDs 16 and 18 are more effective and promising PDT photosensitizers based on the studies with BEL-7402 cells and show nearly the same photodynamic efficacy as BPDMA. MG-P staining qualitative analysis also indicated that PDT with BCPDs 16 can induce apoptosis in BEL7402 cells.

Full text of DOI:10.1016/j.bmcl.2007.10.086

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 293–297
Design, synthesis, and in vitro photodynamic activities of
benzochloroporphyrin derivatives as tumor photosensitizers
Jianzhong Yao, Wannian Zhang,* Chunquan Sheng, Zhenyuan Miao, Feng Yang,
Jianxin Yu, Ling Zhang, Yunlong Song, Ting Zhou and Youjun Zhou
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
Received 25 May 2007; revised 1 October 2007; accepted 25 October 2007
Available online 5 November 2007
Abstract—Novel benzochloroporphyrin derivatives (BCPDs) were designed, synthesized, and characterized. In vitro dark cytotox-
icity and photodynamic efficacy of BCPDs were evaluated by MTT assay on human hepatoma BEL-7402 cells. The experimental
results showed that BCPDs 15, 16, 17, and 18 have strong long wavelength absorptions around 670 nm and exhibit significantly
lower dark cytotoxicity than BPDMA and possess potent photocytotoxicity, IC50 values 1.32 lg/mL for 15, 0.26 lg/mL for 16,
0.47 lg/mL for 17 of 0.27 lg/mL for 18, and 0.23 lg/mL for BPDMA. Among them, BCPDs 16 and 18 are more effective and prom-
ising PDT photosensitizers based on the studies with BEL-7402 cells and show nearly the same photodynamic efficacy as BPDMA.
MG-P staining qualitative analysis also indicated that PDT with BCPDs 16 can induce apoptosis in BEL7402 cells.
ꢀ 2007 Published by Elsevier Ltd.
Photodynamic therapy (PDT) is an attractive modality
for the treatment of cancer and other diseases.1 It in-
volves the illumination of a photosensitizer with light
of an appropriate wavelength, that is, in the range of
630–800 nm for which tissue penetration is optimal.
The light-activated photosensitizer can form lethal cyto-
toxic active singlet oxygen (1O2) and reactive oxygen
species (ROS).2–5 By directing the light beam to the tar-
get area, PDT can be used to selectively destroy diseased
tissue without harming surrounding cells.
ment, due to its accumulation and retention in skin tis-
sues.7 Therefore, much research has been devoted to
developing new photosensitive compounds with im-
proved photophysical efficiency and fewer side effects.
In the recent decade, a number of so-called second gen-
eration photosensitizers especially related to chlorins
have been reported in the literature.8 Among them, a
benzoporphyrin derivative (BPD) obtained from
Diels–Alder reaction of protoporphyrin IX dimethyl es-
ter 1 with dimethyl acetylenedicarboxylate (DMAD) as
its most effective analog—ring A modified cis-isomer
monocarboxylic acid form, so-called BPDMA 2, has
generated interest due to its low skin phototoxicity, ra-
pid clearance from tissues, and strong absorption at
Porphyrin-related photosensitizer such as porfimer so-
dium (Photofrin II), a complex mixture of hematopor-
phyrin derivatives, is the first clinically approved
photosensitizer for the treatment of bladder cancer by
PDT in 1993.1,6 Although encouraging results have been
obtained, it has some serious limitations. The molecular
absorption coefficient of Photofrin II at the clinically
used wavelength of 630 nm is low (e = 1170 MÀ1 cmÀ1).6
More importantly, it causes skin photosensitivity for a
prolonged period of time, up to 30–90 days after treat-
long wavelengths (kmax = 690 nm, e = 35,000 MÀ1 cmÀ1
)
to take full advantage of greater tissue penetration.6,9 It
is undergoing clinical trials for the treatment of basal
cell carcinoma10 and has had clinical success in the treat-
ment for age-related macular degeneration (ARMD) by
PDT.11
BPD-MA 2 is synthesized by first treatment of proto-
porphyrin IX dimethyl ester 1 with DMAD followed
by rearrangement with base and subsequent partial
hydrolysis (Scheme 1).12 In principle, there is a synthetic
key problem in efficiently obtaining the pure BPDMA 2.
Treatment of protoporphyrin IX dimethyl ester 1 with
Keywords: Photodynamic therapy (PDT); Photosensitizer; Ben-
zochloroporphyrin derivatives (BCPDs); Silkworm excrement;
Synthesis.
*
Corresponding author. Tel.: +86 021 25074460; e-mail:
zhangwnk@hotmail.com
0960-894X/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.10.086

294
J. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 293–297
Scheme 1.
DMAD yields a mixture of ring A and ring B Diels–Al-
der adducts which is followed to be rearranged to a mix-
ture of ring A and ring B cis-isomers by base. BPD-MA
2, the more effective ring A modified cis-isomer,13 is then
separated from the isomeric mixture by column
chromatography.
sis and preliminary photocytotoxicity in vitro on human
hepatoma BEL-7402 cells of BCPDs.
For our study, starting materials chlorin e6 3, chlorin e4
4, and chlorin p6 5b (or purpurin-18 5a) were obtained
through base-degradation of pheophorbide-a which is
prepared via 36% HCl degradation of chlorophyll a in
Et2O by the methodology developed in our laboratory
using crude chlorophyll extracts in Chinese traditional
herb named Silkworm excrement (Scheme 2).14
In order to solve the synthetic problem associated with
preparation of biologically active BPDMA 2 and obtain
novel highly potent, low toxic photosensitizers with
strong absorption at long wavelength (>630 nm), we
would like to use chlorin e6 3, chlorin e4 4, and chlorin
p6 5b (or purpurin-18 5a which is actually steady exis-
tence state of chlorin p6 5b) as substrates in the Diels–
Alder reaction to synthesize the novel BPDMA ana-
logs—benzochloroporphyrin derivatives (BCPDs). Be-
cause all substrates contain only one vinyl group
which is situated in the more efficacious ring A site
and thus only one Diels–Alder adduct with DMAD in
principle is possible. In this paper, we report the synthe-
The synthesis pathway of a series of novel BPDMA ana-
logs BCPDs are outlined in Scheme 3. Originally, we
planned to use chlorin e6 3, chlorin e4 4, chlorin p6 5b
(or purpurin-18 5a) or their methyl ester to react with
DMAD. However, the Diels–Alder reaction did not
take place, and only chloroporphyrin e6 trimethyl ester6,
chloroporphyrin e4 dimethyl ester7, and chloroporphy-
rin p6 trimethyl ester8, which were prepared through
methylation of chlorin e6 3, chlorin e4 4, and chlorin
Scheme 2. Reagents and conditions: (a) Concd HCl/Et2O; (b) NaOH–EtOH/N2, reflux; (c) pyridine/N2, reflux; (d) NaOH–i-PrOH/O2; (e) i—NaOH,
ii—adjust to pH 6–7; (f) standing.

J. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 293–297
295
Scheme 3.
p6 5b individually with CH2N2 followed by oxidation
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),
respectively, could undergo the Diels–Alder reaction
with DMAD.
6.74 (s, 1H) ppm and the shortage of one methoxy pro-
ton, which suggested the formation of an additional exo-
cyclic ring system. Compared to the resonances for
various protons in the DBU rearranged major product
1
15, the H NMR spectrum of 16 showed no significant
Treatment of 6, 7, and 8 individually with DMAD in
refluxing toluene under an atmosphere of nitrogen gave
the desired Diels–Alder adducts 9, 10, and 11 in 40.9%,
36.1%, and 43.4% yield, respectively. Rearrangement of
9, 10 and 11 individually with triethylamine (TEA) gave
the conjugated BCPDs trans-isomers 12, 13, and 14 in
quantitative yield, respectively. Reaction of 13 and 14
individually with 1,8-diazabicyclo[5,4,0]undec-7-ene
(DBU) produced the conjugated BCPDs cis-isomers 17
and 18 in 87.5% and 100% yield, respectively. The
BCPDs cis-isomers 17 and 18 can also be obtained by di-
rect treatment of the Diels–Alder adducts 10 and 11
individually with DBU in 81.3% and 94.7% yield,
respectively.
shifts in the resonances of the protons at 31-, 32-, 21-,
2-, 171-, and 172-positions, which appeared at d 7.81
(d, 1H, J = 5.7 Hz), 7.46 (d, 1H, J = 5.7 Hz), 5.04 (s,
1H), 1.81 (s, 3H), 4.08 (m, 2H), and 2.90 (m, 2H)
ppm, respectively. These results supported the forma-
tion of a rearranged cis-isomer with an additional five-
membered exocyclic ring between 131- and 151-posi-
tions. The mechanism for the formation of the exocyclic
ring is a base-induced Dieckmann condensation. De-
spite extensive studies of Diels–Alder reactions in por-
phyrin systems by us and others, this is the first
example which illustrates the formation of such a novel
ring system.
All new compounds, the Diels–Alder adducts 9, 10, 11
and the BCPDs trans-isomers 12, 13, 14 and BCPDs
cis-isomers 15, 16, 17, 18, essentially belong to side-
chain modified ‘Chlorin’ derivatives from naturally
occurring sources and their structures were character-
ized by 1H NMR, ESI-MS, and elemental analysis stud-
ies.15 The UV–visible spectral data of compounds 9–18
in CH2Cl2 are listed in Table 1.
However, rearrangement of 9 and 12 individually with
DBU both gave the same mixture of two compounds.
Separated individually by silica gel column chromatog-
raphy (CH3COCH3/CH3OH/CH2Cl2, 7:7:100), the de-
sired DBU rearrangement conjugated BCPD cis-
isomer 15, the slower moving band, was obtained as a
major product in 70% and 80% yield, respectively; the
undesired DBU rearrangement conjugated BCPD cis-
isomer 16, the faster moving band, was obtained as a
minor product in 20% yield.
As shown in Table 1, the UV–visible spectra of the
Diels–Alder adducts 9, 10, and 11 showed the intense
Soret bands (416 nm, 416 nm and 413 nm, respectively),
which were all slightly red-shifted than that of the inter-
mediate chloroporphyrins 6, 7, and 8 (409 nm, 410 nm,
and 406 nm, respectively), and the long wavelength
Compared to the resonances for the Diels–Alder ad-
ducts 9 and 12, the H NMR spectrum of 16 showed
1
the presence of only one proton at 151-position at d

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J. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 293–297
Table 1. UV–vis data of Diels–Alder adducts 9–11, BCPDs trans-isomers 12–14 and BCPDs cis-isomers 15–18
Compound
kmax (nm) (e · 10À4, MÀ1 cmÀ1
Visible bands
)
Soret band
9
416 (6.70)
416 (24.05)
413 (14.68)
440 (7.07)
440 (3.72)
434 (8.05)
443 (7.74)
444 (7.46)
451 (16.21)
439 (11.30)
521 (0.39)
521 (1.27)
519 (0.90)
552 (0.42)
554 (1.30)
550 (1.09)
597 (0.24)
597 (0.76)
600 (0.46)
581 (1.45)
583 (0.79)
578 (1.89)
596 (2.24)
620 (2.21)
591 (3.76)
586 (3.09)
653 (0.72)
653 (2.20)
655 (1.99)
668 (0.90)
670 (0.45)
670 (1.20)
675 (1.06)
666 (0.92)
677 (2.71)
677 (2.26)
10
11
12
13
14
15
16
17
18
—
—
—
—
—
—
—
—
—
—
—
—
—
—
absorption maxima in visible bands at 653 nm, 653 nm,
and 655 nm, respectively, which were absent in the inter-
mediate chloroporphyrins 6, 7, and 8. Compared to the
Diels–Alder adducts 9, 10, and 11, the absorption bands
of the TEA rearranged BCPDs trans-isomers 12, 13, 14,
and the DBU rearranged BCPDs cis-isomers 15, 16, 17,
and 18 were obviously red-shifted and broader due to
the extended conjugation. They showed the intense
broad Soret bands around 440 nm and the long wave-
length absorption maxima in visible bands around
670 nm.
tion-dependent cell survival curves for BCPDs 15–18
and BPDMA.
As shown in Figure 1, the cell survival rates of BCPDs
15–18 were all higher than 92% in the absence of light
for concentrations up to 2.5 lg/mL, which revealed the
minimal cytotoxicity in darkness (so-called ‘dark cyto-
toxicity’). However, the cell survival rates of positive
control BPDMA ranged from 97% to 5% in darkness
under the wide concentrations ranging from
0.03125 lg/mL to 2.5 lg/mL and its IC50 value (calcu-
lated by SPSS 10.0 for windows) was 1.35 lg/mL (95%
confidence limit was 1.19 ꢀ 1.54 lg/mL, which showed
significantly higher dark cytotoxicity on BEL-7402 cells
than that of BCPDs 15–18).
The preliminary in vitro dark cytotoxicity and photody-
namic efficacy of BCPDs cis-isomers 15–18 were evalu-
ated by MTT assay on human hepatoma BEL-7402
cells (Fig. 1).15
In addition, BCPDs 16, 17, and 18 exhibited signifi-
cantly higher photocytotoxicity on BEL-7402 cells than
BCPD 15. Calculated by SPSS 10.0 for windows, IC50
values were 0.26 lg/mL (95% confidence limit was
0.16 ꢀ 0.47 lg/mL) for BCPD 16, 0.47 lg/mL (95% con-
fidence limit was 0.22 ꢀ 1.05 lg/mL) for BCPD 17,
0.27 lg/mL (95% confidence limit was 0.24 ꢀ 0.32 lg/
mL) for BCPD 18, and 1.32 lg/mL (95% confidence
limit was 1.15 ꢀ 1.52 lg/mL) for BCPD 15, while IC50
Cells were pre-incubated with various concentrations of
BCPDs 15–18 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 cell survival frac-
tion was measured for photo-induced growth inhibition
by photosensitizers using MTT assay. Experiments were
carried out in triplicates. Figure 1 shows the concentra-
120
100
80
60
40
20
0
15
16
17
18
BPDMA
15+light
16+light
17+light
18+light
BPDMA+light
0
0.0313 0.0625 0.125
0.25
0.5
1
1.5
2
2.5
Concentration (µg/mL)
Figure 1. Cytotoxic effect of BCPD 15–18 on BEL-7402 cells in the absence of light and by irradiation for 3 min with a laser at a power density of
56 mW/cm2 and wavelength 670 nm.

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
found, in the online version, at doi:10.1016/j.bmcl.
2007.10.086.
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.
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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.
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15. See Supplementary Information.