Synthesis, optical properties and preliminary in vitro photodynamic effect of pyridyl and quinoxalyl substituted chlorins

Article abstract of DOI:10.1016/j.bmc.2015.03.021

A series of chlorophyll a-based chlorins conjugated with pyridyl or quinoxalyl group at different positions were synthesized, characterized and evaluated for their photodynamic effect in vitro. It was found that all the pyridyl and quinoxalyl chlorins showed promising photocytotoxicities but nontoxic without irradiation in HeLa cells, and the substituted types and positions had a significant influence on the photocytotoxicities of the chlorophyll a-based chlorins. All the chlorins with a pyridyl group at the C-D ring end exhibited relatively high photocytotoxicity as compared to those with 3²-pyridyl. Among them, compound 12 conjugated with a pyridyl group at its C12 position showed the best photodynamic effect in HeLa cells with an IC₅₀ value of 0.033 μM. These facts, associated with the relative high long wavelength absorptions of those chlorins may provide valuable ways to design and prepare promising photosensitizers for application in photodynamic therapy.

Full text of DOI:10.1016/j.bmc.2015.03.021

Bioorganic & Medicinal Chemistry 23 (2015) 1684–1690
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, optical properties and preliminary in vitro photodynamic
effect of pyridyl and quinoxalyl substituted chlorins
Jiazhu Li a, , Xin Zhang , Yang Liu , Il Yoon , Dong-Kyoo Kim , Jun-Gang Yin , Jin-Jun Wang ,
⇑
b
a
c
d
a
a
c,
⇑
Young Key Shim
a
College of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, China
b
c
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
PDT Research Institute, School of Nano System Engineering, Inje University, Gimhae 621-749, Republic of Korea
Department of Biomedicinal Chemistry and Institute of Basic Science, Inje University, Gimhae 621-749, Republic of Korea
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of chlorophyll a-based chlorins conjugated with pyridyl or quinoxalyl group at different positions
were synthesized, characterized and evaluated for their photodynamic effect in vitro. It was found that all
the pyridyl and quinoxalyl chlorins showed promising photocytotoxicities but nontoxic without irradia-
tion in HeLa cells, and the substituted types and positions had a significant influence on the
photocytotoxicities of the chlorophyll a-based chlorins. All the chlorins with a pyridyl group at the
C–D ring end exhibited relatively high photocytotoxicity as compared to those with 3 -pyridyl. Among
them, compound 12 conjugated with a pyridyl group at its C12 position showed the best photodynamic
effect in HeLa cells with an IC50 value of 0.033 lM. These facts, associated with the relative high long
wavelength absorptions of those chlorins may provide valuable ways to design and prepare promising
Received 6 February 2015
Revised 7 March 2015
Accepted 8 March 2015
Available online 14 March 2015
2
Keywords:
Chlorophyll a
Pyridyl chlorin
Quinoxalyl chlorin
Photosensitizer
Photodynamic therapy
photosensitizers for application in photodynamic therapy.
Ó 2015 Elsevier Ltd. All rights reserved.
5
,6
1
. Introduction
a-based photosensitizers such as purpurinimide, HPPH,7–9 and
1
0–12
NPe
6
have been developed and are studied at various stages
Photodynamic therapy (PDT) consists of the administration and
of preclinical or advanced clinical trials.
selective accumulation of a photosensitizer (PS) by target tumor
tissues, followed by generation of singlet oxygen and other cyto-
toxic reactive oxygen species (ROS) that result in cell membrane
Due to their applications in the construction of biomimetic
models of photosynthetic systems, pyridyl porphyrins or chlorins
and their metal complexes have recently attracted considerable
1
3–15
damage and subsequent cell death upon irradiation with light of
attention.
Their water-soluble quaternary salts have also been
1
2
appropriate wavelength in the presence of tissue oxygen (O ).
studied comprehensively, such as monocationic cycloimide deriva-
1
6
Photofrin is the first photosensitizer to receive regulatory approval
for the treatment of various cancers in more than 40 countries
6
tives of chlorin p (CICD), cationic water-soluble esters of chlorin
1
7,18
19,20
6
e ,
cationic meso-porphyrins
and cationic b-vinyl substi-
2
1
throughout the world, including the United States (by FDA in
tuted meso-tetraphenylporphyrins.
2
1
995). To overcome the drawbacks of the first generation PS
Based on these intriguing results, we have designed and synthe-
2
Photofrin, many new generation PSs have been developed and
introduced in clinical trials, most of whom are cyclic tetrapyrroles
comprising substituted derivatives of porphyrin, (bacterio)chlorin
and phthalocyanine.3 In recent years, PS which can easily be
prepared by partial synthesis starting from abundant natural
materials, such as heme, chlorophyll and bacteriochlorophyll,
attracted maximum interests in that they have both economical
sized a series of 3 -pyridyl or quinoxalyl substituted chlorins and
their quaternary salts via stereoselective aldol-like carbon–carbon
2
2,23
condensation (Fig. 1).
bond linked pyridyl or quinoxalyl groups which conjugated to the
chlorin macrocycle -system via a vinyl bridge. It has been proved
Such compounds possess carbon–carbon
p
that their quaternary salts showed promising photodynamic effect
2
in HeLa cells, but the photodynamic effect of 3 -pyridyl or
and environmental advantages.4
A
series of chlorophyll
quinoxalyl substituted chlorins is still an open question. In the
present work we proposed the synthesis of a new series of pyridyl
substituted chlorins which have a pyridyl group linked at the C–D
ring end of the macrocycle, and the comparative study of all these
⇑
Corresponding authors. Tel.: +86 535 6903490; fax: +86 535 6902078 (J.L.);
tel.: +82 55 320 3871; fax: +82 55 320 3872 (Y.K.S.).
E-mail addresses: jiazhu82@ytu.edu.cn (J. Li), ykshim@inje.ac.kr (Y.K. Shim).
http://dx.doi.org/10.1016/j.bmc.2015.03.021
0
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
1685
R
R
R
purified by silica gel chromatography to give a dark green solid
in 61% yield. While 12 -pyridyl chlorin 12 was prepared by the
2
similar method to that of chlorin 4 from 12-formyl-12-demethyl
pyropheophorbide a methyl ester 11, which has been reported by
NH
N
N
NH
N
N
NH
N
N
2
4
Wang et al. previously. The desired product 12 (m/z 638.5) was
obtained as a green solid in 51% yield after silica gel chro-
matography. As for the preparation of pyridyl purpurinimide 14,
purpurin 18 methyl ester 13, prepared from MPa 7 via air oxidation
HN
HN
HN
OCH
3
O
O
N
OCH
O
O
O
5
in alkaline solution, was refluxed with 4-(aminomethyl)pyridine
O
OCH3
O
O
3
in toluene under nitrogen atmosphere for 8 h to afford the desired
product 14 (m/z 699.4) in 85% yield as purple red solid.
OCH
3
OCH
3
OCH
3
1
or 4
2 or 5
3 or 6
The structures of all the new chlorins were characterized by the
H NMR spectra. As shown in Figure 2, the 3 -pyridyl proton
1, 2, 3: R =
N
4, 5, 6: R =
1
2
N
signals of chlorin 2 appeared as two doublets at d 8.80 and 7.72,
while its 3-vinyl proton signals showed at d 8.60 and 7.56 as two
doublets with the coupling constant (J) of 16.0 Hz, typical of an
E-geometry of the double bond. In contrast, the proton signals of
4-pyridylethylene at the C12 position of chlorin 12 appeared at d
8.77, 7.84 (two doublets, pyridyl) and d 8.98, 8.63 (two doublets,
Figure 1. Structures of the 32-pyridyl or quinoxalyl substituted chlorins 1–6
prepared via aldol-like condensation.
pyridyl or quinoxalyl chlorins about their optical properties and
photodynamic activities in HeLa cells.
1
2-vinyl), respectively. It is worth noting that the geometry of
the 12-ethylene linker is also an E-type based on the coupling
constant (16.3 Hz). The appearance of the typical ABX system of
2
2
. Results and discussion
3
-vinyl proton signals at d 7.91 (dd), 6.26 (d), 6.17 (d) and the
disappearance of the 12-CH proton signal further confirmed the
structure of chlorin 12. In the H NMR spectrum of chlorin 10,
.1. Synthesis and characterization
3
1
2
The synthesis of 3 -pyridyl or quinoxalyl substituted chlorins
2
3
2
2,23
the 13 -NH, 13 -CH and pyridyl proton signals appeared at d
1–3, 5 and 6 has been reported previously.
As shown in
2
2
6.70 as broad singlet, d 4.82, 4.42 as two doublet of doublets and
Scheme 1, Preparation of 3 -quinoxalyl substituted chlorin 4 was
started from the ring-opening reaction of methyl pheophorbide a
d 8.51 and 7.22 as two doublets, respectively. While for the chlorin
3
1
4, the proton signals of 13 -CH
2
was observed at d 5.68 as a sin-
(
MPa) 7 resulted in the production of chlorin e
followed by OsO /NaIO oxidation to give the 3-formyl-3-devinyl
chlorin e trimethyl ester 9, which was refluxed with quinaldine
in Ac O for 5 h to give the desired product 4 (m/z 766.4) as dark
6
trimethyl ester 8,
glet, and the pyridyl proton signals appeared at d 8.60 and 7.58
as two doublet of doublets.
4
4
6
2
2
.2. Optical property
brown solid in 55% yield. In order to compare the difference of
properties among chlorins with the pyridyl group at various substi-
tuted positions, a series of new pyridyl chlorins were prepared
using MPa 7 as the substrate. Chlorin 10 was prepared from MPa
The optical properties of the pyridyl or quinoxalyl chlorins are
shown in Figure 3 and summarized in Table 1. In the electronic
2
absorption spectra, it was observed that all the 3 -pyridyl or
7
via nucleophilic substitution with 4-(aminomethyl) pyridine at
2
1
quinoxalyl chlorins 1–6 and 12 -pyridyl chlorin 12 showed a
its C13 position, the desired product 10 (m/z 715.4) was readily
R
32
R
31
5
7
A
D
B
8
NH
N
N
2
NH
N
N
NH
N
N
NH
N
N
d
a
c
2
0
10
HN
HN
HN
HN
C
18
12
131
1
17
O
E
32
13
2
NH 13
OCH
3
OCH
3
O
O
1
O
O
O
O
O
O
OCH3 13
3
O
OCH3
O
OCH3
O
OCH3
OCH
3
N
OCH
3
OCH
3
OCH
3
10
7: MPa
8
: R = CH
1: R = pyridyl-4-yl
4: R = quinoxalyl-2-yl
2
b
g
9: R = O
e
NH
N
N
NH
N
N
NH
N
N
NH
N
N
f
h
HN
21
HN
HN
HN
1
CHO
1
22
N
O
O
13
O
O
N
1
O
O
O
33
O
12
O
11
O
O
14
OCH
3
OCH
3
OCH
3
OCH
3
N
2
Scheme 1. Synthetic routes of 3 -quinoxalyl substituted chlorin 4 and pyridyl chlorins 10, 12 and 14 which have a pyridyl group linked at the C–D ring end of the
macrocycles. Reaction conditions: (a) NaOMe, MeOH, 0 °C, 2 h; (b) OsO , NaIO , rt, 3 h; (c) quinaldine, Ac O, AcOH, reflux, 5 h; (d) 4-(aminomethyl) pyridine, rt, 12 h; e) LiOH/
O/MeOH/THF, rt, 2 h; (f) 4-picoline, Ac O, reflux, 5 h; (g) KOH/1-propanol/pyridine/Et O, O , 1 h; (h) 4-(aminomethyl) pyridine, toluene, reflux, 8 h.
4
4
2
H
2
2
2
2

1
686
J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
10 lM at 12 h incubation in dark or after photoirradiation (670-
710 nm light, total light dose 2 J/cm for 15 min). As shown in
2
CHCl
3
2
CH
2
Cl
2
Figure 4A and B, all the pyridyl or quinoxalyl chlorins showed very
low dark toxicity up to the highest concentration (10 lM) investi-
gated. However, upon exposure to a low light dose, all the com-
pounds showed significant reduction in HeLa cell viability in a
concentration-dependent manner. With an increase in the concen-
tration of the photosensitizer, the cell viability revealed decreasing
results. For example, chlorins 10 and 12 showed cell viabilities of
1
1
1
2
0
4
6
0
1
5.5% and 22.2% at 0.05
.1 M after PDT, their cell viabilities were changed to 28.1% and
.4%, respectively.
Table 2 summarized the IC50 values of the pyridyl or quinoxalyl
lM after PDT, respectively, while at
l
chlorins and the reference compound MPPa against HeLa cells after
PDT. It was observed that all the tested chlorins exhibited signifi-
cant photodynamic anticancer activity against HeLa cells at micro
molar concentration with IC50 values of <5
tuted chlorin e derivatives 1, 4, 10 and the 12 -pyridyl substituted
methyl pyropheophorbide 12 showed obviously increased
photodynamic activity against HeLa cells as compared to the refer-
ence compound MPPa (IC50 = 0.182 M). Among them, chlorin 12
(IC50 = 0.033 M) showed the best result, which increased the
lM. The pyridyl substi-
2
Figure 2. The comparative 1H NMR spectra (CDCl
d 4.0–10.0 ppm of pyridyl substituted chlorins 2, 10, 12 and 14.
500 MHz) in the region
3
,
6
a
bathochromic shift (10–53 nm) of their long wavelength absorption
l
bands (Q
y
bands) compared with methyl pyropheophorbide a
-extension effect
l
(
MPPa, 668 nm), which can be explained by the
p
photodynamic activity by almost an order of magnitude in com-
2
derived from the conjugated connection of the pyridyl or quinoxalyl
parison with MPPa. It is interesting to observe that 3 -pyridyl or
group surrounding the periphery of chlorin macrocycle. While
quinoxalyl substituents of different chlorin derivatives showed dif-
ferent effect on their phototoxicity.
chlorin 10 and 14 showed typical Q
chlorin e (663 nm) and purpurinimide (708 nm), respectively. In
comparison with 3 -pyridyl chlorins 1–3 (
y
absorptions of their precursors
2
As shown in Figure 4B and Table 2 and 3 -pyridyl substituted
6
2
D
Q
y
= 10, 10 and 48 nm,
respectively), the 3 -quinoxalyl chlorins 4–6 showed more batho-
chromic shift of their Q absorptions ( = 11, 12 and 53 nm,
chlorin e
6
derivative 1 (IC50 = 0.141
M) showed relative high phototoxicity in comparison
with 3 -quinoxalyl substituted derivatives 4 (IC50 = 0.170 M)
and 5 (IC50 = 0.631 M), respectively. While for the purpurin-
imide-based chlorins, the 3 -quinoxalyl substituted derivative 6
lM) and MPPa derivative 2
2
(IC50 = 0.581
l
2
D
Q
y
l
y
respectively). It was interesting to find that the conjugated position
of the pyridyl group surrounding the periphery of chlorin
macrocycle has a significant influence on their electronic absorp-
l
2
2
(IC50 = 0.992
lM) showed better phototoxicity than 3 -pyridyl
2
tion spectra. As shown in Figure 3A, the 12 -pyridyl chlorin 12
derivative 3 (IC50 = 3.118
lM). It was also observed that the differ-
showed not only relative high bathochromic shift of its Q
tion ( = 29 nm), but also the increased absorption intensities
of all the peaks including its Soret, Q and Q bands compared to
-pyridyl chlorin 2. Such properties are desired for a photosensiti-
zer in both photodynamic therapy and dye-sensitized solar cell
y
absorp-
ent substituted positions of pyridyl group have significant influence
on the phototoxicity of the chlorins. Generally speaking, chlorins
10, 12 and 14 which have a pyridyl group linked at the C–D ring
end of their macrocycles exhibited significant increased phototoxi-
DQ
y
x
y
2
3
2
city as compared to those have a 3 -pyridyl substituent (com-
2
5
study. In the fluorescence spectra, these chlorins showed distinct
pounds 1–3). For example, 13-pyridyl substituted chlorin e
derivative 10 and 12-pyridyl substituted MPPa 12 exhibited photo-
toxicity against HeLa cells at IC50 value of 0.071 and 0.033 M,
respectively, while the IC50 value of the corresponding 3 -pyridyl
derivatives 1 and 2 is only 0.141 and 0.581 M, respectively.
6
2
emission bands and intensities, among them, 3 -quinoxalyl chlorin
1
4 showed the largest Stokes shift (9 nm).
l
2
2
.3. In vitro photodynamic activity in HeLa cells
l
These results suggest that the types and positions of substituents
influenced significantly on the phototoxicity of chlorophyll a-based
chlorins, and those chlorins with a pyridyl group linked at the C–D
ring end are promising photosensitizers for application in photody-
namic therapy.
To investigate their potential in PDT, preliminary in vitro
photodynamic effects of these pyridyl or quinoxalyl chlorins 1–6,
0, 12 and 14 were evaluated by WST-8 assay against HeLa cells
exposed to increasing concentrations of each compound up to
1
Figure 3. Comparative electronic absorption spectra (10
CH Cl ) of pyridyl chlorins 2, 10, 12 and 14 (B).
2 2
lM in CH Cl ) of pyridyl or quinoxalyl substituted chlorins 2, 3, 5, 6, 12, 14 (A) and fluorescence spectra (1 lM in
2
2

J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
1687
Table 1
The absorption and emission properties of the novel chlorins in dichloromethane
4
ꢁ1
ꢁ1
*
Compound
Absorption kmax (nm) (
Soret (
)a
e
ꢀ 10
M
cm
)
Emission kmax (nm)
Excitation Emission
Stokes shift (nm)
y
y
DQ (De
)a
Soret
D
D
e
Q
1
2
3
4
5
6
1
1
1
412 (6.31)
421 (4.05)
420 (8.60)
412 (5.71)
424 (4.85)
422 (11.27)
403 (11.61)
427 (5.96)
419 (6.63)
ꢁ2 (ꢁ1.01)
7 (ꢁ3.27)
6 (1.28)
ꢁ2 (ꢁ1.61)
10 (ꢁ2.47)
8 (3.95)
ꢁ11 (4.29)
13 (ꢁ1.36)
5 (ꢁ0.69)
678 (2.26)
678 (1.86)
716 (2.60)
679 (2.30)
680 (2.33)
721 (4.88)
663 (5.04)
697 (3.56)
708 (2.42)
10 (ꢁ0.95)
10 (ꢁ1.35)
48 (ꢁ0.61)
11 (ꢁ0.91)
12 (ꢁ0.88)
53 (1.67)
412
421
420
412
424
422
403
427
419
685
684
723
685
685
728
670
705
717
7
6
7
6
5
7
7
8
9
0
2
4
ꢁ5 (1.83)
29 (0.35)
40 (ꢁ0.79)
a
band and absorbance intensity, respectively, between the novel chlorins and methyl pyropheophorbide a.23
D
Soret,
DQ and De represent the change of the Soret band, Q
y
y
*
Stokes shift a longest wavelength absorption.
by the 1O
DPBF) using methylene blue (MB) as the reference compound.
As shown in Figure 5, all the pyridyl or quinoxalyl substituted chlo-
-induced bleaching of 1,3-diphenylisobenzo-furan
2
26,27
(
1
rins produced
1
2
O efficiently after photoirradiation. Among them,
2
1
2 -pyridyl substituted MPPa 12 exhibited the best result of
O
2
generation, which is correlated well with the phototoxicity study,
1
while it should be noted that the O
2
generation order of these chlo-
rins is not correlated with their phototoxicities against HeLa cells.
2
.5. The hydrophobic property study
It is generally well established that the hydrophobic property of
the chlorophyll a-based photosensitizers play a significant role in
their photosensitizing efficacy and is closely related to their cellu-
2
8
lar uptake property. The hydrophobicity parameter (logP) of the
pyridyl or quinoxalyl substituted chlorins 1–6, 10, 12 and 14 were
calculated by a program module of the ACD/Labs software (version
1
4.02, Advanced Chemistry Development, Inc., Toronto, ON,
Canada). As shown in Table 3, all the pyridyl or quinoxalyl substi-
tuted chlorins have logP values in the range of 6.0–10.0. It is
Figure 4. Cell viability results in dark (A) and after PDT (B) in HeLa cells for pyridyl
or quinoxalyl substituted chlorins 1–6, 10, 12 and 14. The cell viabilities of these
photosensitizers after PDT were evaluated in the concentration ranges of 0–10 lM
after 12 h. Surviving cell population was measured by WST-8 assay. The data are
expressed as mean of three experiments.
2
.4. Singlet oxygen study
In most of the photosensitizers, the formation of singlet oxygen
Figure 5. Comparative absorbance decay (%) of 1,3-diphenylisobenzofuran (DPBF,
(1
O
2
) when the photosensitizer is exposed to light is believed to be
50
l
M in DMSO) at 418 nm after photoirradiation in the absence (control) and
methylene blue (MB), compounds 1–6, 10, 12 and 14,
one of the main causes of cytotoxicity. The relative difference of
singlet oxygen generation of the pyridyl or quinoxalyl substituted
chlorins 1–6, 10, 12 and 14 after photoirradiation were measured
presence of
1 lM
respectively. The data are expressed as mean of three experiments.
Table 3
Table 2
Hydrophobicity parameters (logP) of photosensitizers calculated by means of
IC50 values of photosensitizers in HeLa cells
ACD/Labs (version 14.02)
Compound
MPPa
0.182
1
2
3
4
Compound
LogP
Compound
LogP
a
IC50
(lM)
0.141
0.581
3.118
0.170
1
2
3
4
5
8.45 ± 1.64
8.31 ± 1.61
6.92 ± 1.65
9.81 ± 1.64
9.67 ± 1.61
6
8.27 ± 1.65
7.07 ± 1.65
8.33 ± 1.61
6.11 ± 1.65
Compound
5
6
10
0.071
12
0.033
14
0.189
10
12
14
a
IC50
(lM)
0.631
0.992
a
IC50 represents the half maximal (50%) inhibitory concentration of the
photosensitizers.

1
688
J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
interesting to find that the chlorins 10, 12 and 14 which have a
acid under nitrogen atmosphere for 5 h. The reaction mixture was
evaporated to remove the solvent, and the residue was dissolved in
dichloromethane (30 mL), washed with 1 M aqueous HCl
(3 ꢀ 50 mL), then with water (2 ꢀ 50 mL). The organic layer
pyridyl group linked at the C–D ring end of their macrocycles
2
showed relative low logP values as compared to 3 -pyridyl or
quinoxalyl substituted chlorins 1–6. While as shown in the
in vitro study, chlorins 10, 12 and 14 exhibited better phototoxicity
than compounds 1–6, which might be suggested that a lower
hydrophobicity of this kind of photosensitizers is desirable for
the photosensitizing efficacy against HeLa cells.
2 4
obtained was dried over anhydrous Na SO , concentrated, and pur-
ified on a silica gel column using acetone/dichloromethane (2%-5%)
as gradient eluent to yield 99 mg (0.129 mmol, 55%) of the title
4
product 4 as dark brown solid. UV–vis (CH
2
Cl
2
) kmax
(
e
ꢀ 10 ):
1
4
12 (5.71), 504 (0.82), 541 (0.72), 629 (0.52), 678 (2.30) nm.
H
3
NMR (500 MHz, CDCl ) d 9.74 (s, 1H, 10-H), 9.70 (s, 1H, 5-H),
3
3
. Experimental section
1
9
.14 (d, J = 16.3 Hz, 1H, 3 -H), 8.83 (s, 1H, 20-H), 8.32 (d,
0
0
J = 8.4 Hz, 1H, 3 -H), 8.28 (d, J = 8.3 Hz, 1H, 6 -H), 8.00 (d,
J = 16.5 Hz, 1H, 3 -H), 7.99 (d, J = 8.5 Hz, 1H, 9 -H), 7.91 (d,
J = 8.1 Hz, 1H, 4 -H), 7.82 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H, 7 -H), 7.60
.1. General information
2
0
0
0
All reactions were monitored by TLC using 0.20 mm silica gel
0
1
(
ddd, J = 8.0, 6.9, 1.1 Hz, 1H, 8 -H), 5.36 (d, J = 18.9 Hz, 1H, 15 -H),
plates with or without UV indicator (60F-254). Silica gel 60
230–400 mesh, Merck) was used for flash column chromatogra-
1
5
4
3
2
.25 (d, J = 19.0 Hz, 1H, 15 -H), 4.47 (q, J = 7.2 Hz, 1H, 18-H),
.44–4.39 (m, 1H, 17-H), 3.80 (q, J = 7.5 Hz, 2H, 8 -CH
.77, 3.65, 3.64, 3.58, 3.34 (each s, each 3H, CH + OCH
(
1
2
), 4.26,
), 2.63–
.53, 2.27–2.17, 2.07–1.99 (each m, total 4H, 17 + 17 -CH ), 1.76
), ꢁ1.24,
: C,
1
phy. H NMR spectra were measured at 500 MHz on a Bruker
Advance II spectrometer. Chemical shifts (d) are given in ppm rela-
tive to tetramethylsilane (TMS, 0 ppm) unless otherwise indicated.
Electronic absorption spectra were measured on a AOE A560 UV–
Vis spectrophotometer. The absorption maxima kmax are given in
3
3
1
2
2
2
(
d, J = 7.3 Hz, 3H, 18-CH
3
), 1.72 (t, J = 7.7 Hz, 3H, 8 -CH
3
ꢁ
47 5 6
1.49 (each br s, each 1H, NH). Anal. Calcd for C46H N O
7
2.14; H, 6.19; N, 9.14. Found: C, 72.15; H, 6.21; N, 9.17. MS
nm and molar absorbance coefficient (
e
) or relative intensity. The
+
(
(
48 5 6
FAB) m/z 766.4 (MH , 100). HRMS (FAB): Calcd for C46H N O
MH ) 766.3605; Found 766.3608.
fluorescence spectra were measured on a Varian Cary Eclipse fluo-
rescence spectrophotometer. FABMS and HRMS were obtained on a
Jeol JMS700 high resolution mass spectrometer at the Korea Basic
Science Institute (Daegu). Materials obtained from commercial
+
1
3
6
.2.3. Synthesis of chlorin e -13 -N-(pyridine-4-yl methylene)
2
3
amide-15 ,17 -dimethyl ester (10)
suppliers were used without further purification. Chlorins 1–3, 5
A solution of methyl pheophorbide a 7 (200 mg, 0.330 mmol) in
chloroform (10 mL) was stirred with 4-aminomethyl pyridine
and 6 have been reported by us previously.2
2,23
Methyl pheophor-
bide a 7 was prepared according to the procedure described in the
literature.
(
0.5 mL, excess) at room temperature under nitrogen atmosphere
2
9
until the disappearance of the starting material (TLC, 12 h). The
reaction mixture was diluted with dichloromethane (100 mL),
washed with water (3 ꢀ 100 mL), dried over anhydrous Na SO
3
3
.2. Synthesis and characterization of new compounds
.2.1. Synthesis of 3–formyl-3-devinyl chlorin e trimethyl ester
was prepared from methyl
2
4
and evaporated to dryness at 30–40 °C under reduced pressure.
After purification on a silica gel column using 20% MeOH in
CH Cl as eluent to yield 144 mg (0.201 mmol, 61%) of pure pro-
6
(
9)
2
2
4
Chlorin
e
6
trimethyl ester
8
max
duct 10 as dark green solid. UV–vis (CH Cl ) k (e
(11.61), 501 (1.22), 529 (0.33), 609 (0.41), 663 (5.04) nm.
ꢀ 10 ): 403
2
2
11
1
pheophorbide a 7 as previously reported, and its spectroscopic
characterization agreed fully with the published data. To a solution
H
3
NMR (500 MHz, CDCl ) d 9.58 (s, 1H, 10-H), 9.56 (s, 1H, 5-H),
0
0
of chlorin e
were added a solution of OsO
tion of NaIO (2.0 g, 9.35 mmol) in water (50 mL) successively. The
mixture was stirred violently at room temperature for 3 h. It was
then diluted with CH Cl (150 mL), washed with water
SO . The organic phase
6
trimethyl ester 8 (210 mg, 0.329 mmol) in 80 mL THF
8.79 (s, 1H, 20-H), 8.51 (dd, J = 4.5, 1.4 Hz, 2H, 2 ,6 -H), 7.89 (dd,
1
0
0
4
(10 mg) in CCl (20 mL) and a solu-
4
J = 17.8, 11.5 Hz, 1H, 3 -H), 7.22 (d, J = 5.4 Hz, 2H, 3 + 5 -H), 6.70
2
2
4
(s, 1H, 13 -NH), 6.17 (dd, J = 17.9, 1.1 Hz, 1H, 3 -H), 5.96 (dd,
J = 11.5, 1.1 Hz, 1H, 3 -H), 5.42 (d, J = 19.0 Hz, 1H, 15 -H), 5.19 (d,
J = 19.0 Hz, 1H, 15 -H), 4.82 (dd, J = 14.9, 6.2 Hz, 1H, 13 -H), 4.47
(q, 1H, 18-H), 4.42 (dd, J = 14.9, 6.2 Hz, 1H, 13 -H), 4.36 (dd,
2
1
1
3
2
2
3
(
3 ꢀ 150 mL), and dried over anhydrous Na
2
4
1
was evaporated and the residue was purified on a silica gel column
eluting with ethyl acetate/n-hexane (1:1). After the major dark
brown band was eluted from the column, the solvent was evapo-
J = 10.0, 2.1 Hz, 1H, 17-H), 3.77 (q, J = 7.7 Hz, 2H, 8 -CH ), 3.70,
2
3.61, 3.35, 3.32, 3.28 (each s, each 3H, OCH + CH ), 2.61–2.52,
3
3
1
2
2.25–2.13, 1.83–1.76 (each m, total 4H, 17 + 17 -CH ), 1.74 (d,
2
2
rated to yield 188 mg (0.293 mmol, 89%) of the title compound 9
as dark brown solid. UV–vis (CH
J = 7.3 Hz, 3H, 18-CH ), 1.71 (t, J = 7.7 Hz, 3H, 8 -CH ), ꢁ1.61,
3
3
4
2
Cl
2
) kmax
(e
ꢀ 10 ): 421 (7.42),
ꢁ1.84 (each br s, each 1H, NH). Anal. Calcd for C
42 46 6 5
H N O : C,
1
5
13 (0.54), 546 (0.60), 666 (0.93), 693 (1.91) nm. H NMR
70.57; H, 6.49; N, 11.76. Found: C, 70.60; H, 6.51; N, 11.85. MS
1
+
(
(
500 MHz, CDCl
s, 1H, 5-H), 9.37 (s, 1H, 20-H), 5.48 (d, J = 19.1 Hz, 1H, 15 -H),
3
) d 11.40 (s, 1H, 3 -H), 10.62 (s, 1H, 10-H), 9.99
(FAB) m/z 715.4 (MH , 100). HRMS (FAB): Calcd for C42H47N O
6
5
1
+
(MH ) 715.3563; Found 715.3605.
1
5
.43 (d, J = 19.4 Hz, 1H, 15 -H), 4.68 (q, J = 7.3 Hz, 1H, 18-H), 4.58
1
(
dd, J = 11.0, 2.2 Hz, 1H, 17-H), 3.90 (q, J = 7.5 Hz, 2H, 8 -CH
2
),
),
3.2.4. Synthesis of 12-formyl-12-demethyl pyropheophorbide a
methyl ester (11)
4
2
3
.30, 3.86, 3.84, 3.74, 3.64, 3.42 (each s, each 3H, CH + OCH
.78–2.68, 2.49–2.40, 2.22–2.12 (each m, total 4H, 17 + 17 -
3
2
1
Methyl pheophorbide a 7 was transformed to methyl pyro-
pheophorbide a (MPPa) by the method described in the litera-
2
CH
CH
C
2 3
), 1.90 (d, J = 7.3 Hz, 3H, 18-CH ), 1.66 (t, J = 7.7 Hz, 3H, 8 -
2
9
3
), ꢁ1.03, ꢁ1.70 (each br s, each 1H, NH). Anal. Calcd for
ture. To a solution of methyl pyropheophorbide a (550 mg,
1.00 mmol) in 50 mL THF was added 1.5 g Lithium hydroxide (dis-
solved in 10 mL water and 25 mL methanol). The mixture was stir-
red vigorously for 1 h in an open flask. The reaction mixture was
then diluted with water (100 mL) and acidified to pH 3 by adding
aqueous HCl (5%). It was then extracted with dichloromethane
(2 ꢀ 100 mL), the organic layer was separated and washed with
water (100 mL), dried over anhydrous Na SO and evaporated to
36
H
40
N
4
O
7
: C, 67.48; H, 6.29; N, 8.74. Found: C, 67.54; H, 6.31;
N, 8.75.
2
3
.2.2. Synthesis of (E)-3 -(quinoline-2-yl) chlorin e
6
trimethyl
ester (4)
Compound 9 (150 mg, 0.234 mmol) and quinaldine (0.1 mL)
were refluxed in acetic anhydride (10 mL) with one drop of acetic
2
4

J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
1689
dryness. The residue was dissolved in dichloromethane and treated
with diazomethane to convert the carboxylic acid back to methyl
ester. After chromatographic purification on a silica gel column
with 1–4% acetone/dichloromethane as gradient eluent, the brown
band and green band was collected. The tile product 11 (a green
solid) was obtained as minor product in 17% yield, and its spectro-
40 40 6 4
C H N O : C, 71.84; H, 6.03; N, 12.57. Found: C, 71.81; H, 6.07;
+
N, 12.62. MS (FAB) m/z 669.3 (MH , 100). HRMS (FAB): Calcd for
+
C
40 41 6 4
H N O (MH ) 669.3189; Found 669.3186.
3
.3. In vitro photosensitizing efficacy against HeLa cells
2
4
scopic characterization agreed fully with the published data.
The photodynamic effect of the selected photosensitizers on cell
viability was investigated in HeLa cell line. The HeLa cells obtained
from American Type Culture Collection (ATCC) were cultured in
3
.2.5. Synthesis of 12-demethyl-12-(pyridine-4-yl ethylene)pyr-
opheophorbide a methyl ester (12)
2
EMEM medium supplemented with 10% FBS at 37 °C (5% CO ) in
Compound 11 (150 mg, 0.267 mmol) and 4-picoline (0.1 mL)
were refluxed in acetic anhydride (10 mL) under nitrogen atmo-
sphere for 5 h. The reaction mixture was evaporated to remove
the solvent, and the residue was dissolved in dichloromethane
3
a humidified atmosphere. Cells were plated at 5 ꢀ 10 cells into
each well of a 96-well microplate. After 24 h of incubation, media
were substituted by fresh media containing photosensitizers at
various concentrations. Plates were returned to the incubator for
(
30 mL), washed with 1 M aqueous HCl (3 ꢀ 50 mL), then with
water (2 ꢀ 50 mL). The organic layer obtained was dried over
anhydrous Na SO , concentrated, and purified on a silica gel col-
2
4 h. And then the cells were replaced with fresh media and
2
exposed to light (BioSpec LED, 670–710 nm, 2.0 J/cm ) for 15 min
for the determination of the dark toxicity, to skip over this step
2
4
(
umn using acetone/dichloromethane (2–5%) as gradient eluent to
yield 87 mg (0.136 mmol, 51%) of the title product 12 as green
solid. UV–vis (CH
and incubate for 12 h directly). Following illumination, the plates
were incubated further for 12 h at 37 °C in the dark. CCK-8 reagent
4
2
Cl
2
) kmax
(e
ꢀ 10 ): 427 (5.96), 576 (1.23), 638
(
10 lL) was then added to each well followed by 2 h incubation.
1
(
1.41), 697 (3.56) nm. H NMR (500 MHz, CDCl
3
) d 9.41 (s, 1H,
The cell viability was assessed by WST-8 [2-(2-methoxy-4-nitro-
phenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium],
an indicator that is reduced by dehydrogenases in cells to give an
orange colored product (formazan), which is soluble in cell culture
medium. The optical density for living cells was read at 450 nm in a
1
1
2
7
0-H), 9.15 (s, 1H, 5-H), 8.98 (d, J = 16.3 Hz, 1H, 12 -H), 8.77 (m,
H, 2 + 6 -H), 8.63 (d, J = 16.3 Hz, 1H, 12 -H), 8.37 (s, 1H, 20-H),
.91 (dd, J = 18.1, 11.4 Hz, 1H, 3 -H), 7.84 (m, 2H, 3 + 5 -H), 6.26
0
0
2
1
0
0
2
2
(
d, J = 18.6 Hz, 1H, 3 -H), 6.17 (d, J = 10.9 Hz, 1H, 3 -H), 5.25 (d,
2
2
J = 20.2 Hz, 1H, 13 -H), 5.10 (d, J = 20.3 Hz, 1H, 13 -H), 4.45–4.33
m, 1H, 18-H), 4.25–4.17 (m, 1H, 17-H), 3.64 (q, J = 7.4 Hz, 2H,
Ò
multi-microplate reader (synergy HT, BIO-TEK ). Wells containing
(
cells and an appropriate volume of the vehicle (DMSO) served as
the control. Wells containing only culture medium and CCK-8
reagent served as the blank. Each experiment was repeated at least
three times.
1
8
2
3
-CH
2
), 3.63, 3.33, 3.18 (each s, each 3H, CH
3
+ OCH
), 1.79 (d, J = 7.1 Hz,
), 0.07, -0.72 (each br s,
: C, 75.33; H, 6.16; N,
0.98. Found: C, 75.41; H, 6.19; N, 10.94. MS (ESI) m/z 638.5
3
), 2.75–2.52,
1
2
.39–2.24 (each m, total 4H, 17 + 17 -CH
H, 18-CH ), 1.67 (t, J = 7.7 Hz, 3H, 8 -CH
3 3
each 1H, NH). Anal. Calcd for C40H N O
39 5 3
2
2
1
+
3.4. Measurement of singlet oxygen generation
(
MH , 100).
The relative difference of singlet oxygen generation of the pyr-
idyl or quinoxalyl substituted chlorins 1–6, 10, 12 and 14 after
3
.2.6. Synthesis of purpurin 18 methyl ester (13)
Purpurin 18 methyl ester 13 was prepared from methyl
photoirradiation were measured using 1,3-diphenylisobenzo furan
pheophorbide a 7 by air oxidation in alkaline solution as previously
published, and its spectroscopic characterization agreed with the
published data.
1
(
DPBF) as the selective
O
2
acceptor, and methylene blue (MB) as
M) in DMSO
was prepared as control sample, and sample solutions of DPBF
50 M) with 1 M methylene blue (reference compound), chlo-
5
,30
the reference compound. A solution of DPBF (50 l
(
l
l
3
.2.7. Synthesis of purpurin-18-N-(pyridine-4-yl methylene)imide
rins 1–6, 10, 12 and 14, respectively in DMSO were prepared in
dark. All the samples were placed in a 96-well plate and the con-
tainer was covered with aluminum foil. The samples were irradi-
methyl ester (14)
Purpurin 18 methyl ester 13 (200 mg, 0.346 mmol) and 4-ami-
nomethyl pyridine (0.2 mL, excess) were refluxed in anhydrous
toluene (10 mL) for 8 h under a nitrogen atmosphere. The reaction
was monitored periodically by UV–vis spectroscopy, the
disappearance of a peak at 698 nm (for the anhydride) and the
appearance of a new peak at 708 nm (for the purpurinimide) indi-
cated completion of the reaction. After evaporating the solvents
from the reaction mixture, the crude material was dissolved in
dichloromethane (50 mL) and washed with water (3 ꢀ 50 mL),
2
ated (2 J/cm ) for 15 min. After irradiation, visible spectra of the
sample solutions were measured spectrometrically. The normal-
ized absorbance of DPBF at 418 nm in these samples were
1
reported. The
2
O photogeneration activities of MB, chlorins 1–6,
1
0, 12 and 14 can be compared with the different absorbance
decay of each sample relative to the DPBF control sample.
4
. Conclusions
2 4
organic layer obtained was dried over anhydrous Na SO , concen-
trated, and purified over a silica gel column using ethyl acetate/n-
hexane (1:1) as eluent to yield 197 mg (0.295 mmol, 85%) of pure
In conclusion, three new chlorins 10, 12 and 14 with a pyridyl
4
group linked at the C-D ring end of their macrocycles have been
synthesized in good to excellent yields through simple synthetic
product 14 as purple red solid. UV–vis (CH
2
Cl
2
) kmax
(e
ꢀ 10 ):
4
19 (6.63), 481 (0.26), 511 (0.33), 551 (1.25), 649 (0.38), 708
3
) d 9.49 (s, 1H, 10-H), 9.25 (s,
H, 5-H), 8.60 (dd, J = 4.6, 1.6 Hz, 2H, 2 + 6 -H), 8.54 (s, 1H, 20-
H), 7.83 (dd, J = 17.8, 11.5 Hz, 1H, 3 -H), 7.58 (dd, J = 4.7, 1.6 Hz,
H, 3 + 5 -H), 6.25 (dd, J = 17.8, 1.2 Hz, 1H, 3 -H), 6.13 (dd,
2
1
approaches. These chlorins, as well as the 3 -substituted deriva-
(
2.42) nm. H NMR (500 MHz, CDCl
0
0
tives 1–6 were studied comparatively for their optical properties
and in vitro photosensitizing efficacies against HeLa cells. All of
the chlorins showed high photocytotoxic activity in HeLa cell line,
it was found that the types and positions of substituents influenced
significantly on the phototoxicity of chlorophyll a-based chlorins,
and those chlorins with a pyridyl group linked at the C–D ring
end generally showed better phototoxicity than others. Among
them, chlorin 12 had the highest efficacy at lower concentration,
which increased the photodynamic activity by almost an order of
1
1
0
0
2
2
2
3
J = 11.5, 1.2 Hz, 1H, 3 -H), 5.68 (s, 2H, 13 -CH
2
), 5.33 (dd, J = 9.1,
2
2
2
1
8
.2 Hz, 1H, 17-H), 4.34 (q, J = 7.4 Hz, 1H, 18-H), 3.54 (q, J = 7.7 Hz,
1
H, 8 -CH
2 3 3
), 3.75, 3.54, 3.32, 3.08 (each s, each 3H, OCH + CH ),
1
.73–2.64, 2.43–2.33, 2.00–1.93 (each m, total 4H, 17 +
2
7 -CH
2
), 1.76 (d, J = 7.4 Hz, 3H, 18-CH
3
), 1.61 (t, J = 7.7 Hz, 3H,
2
3
-CH ), 0.05, ꢁ0.08 (each br s, each 1H, NH). Anal. Calcd for

1
690
J. Li et al. / Bioorg. Med. Chem. 23 (2015) 1684–1690
10. Ferrario, A.; Kessel, D.; Gomer, C. J. Cancer Res. 1992, 52, 2890.
magnitude in comparison with MPPa. These facts, associated with
their relative high long wavelength absorptions may provide valu-
able ways to design and prepare promising photosensitizers for
application in photodynamic therapy. Further in vitro and in vivo
studies concerning the cellular uptake, intracellular localization
and the mechanism of cell death are presently in progress in our
laboratory.
11. Hargus, J. A.; Fronczek, F. R.; Graca, M.; Vicente, H.; Smith, K. M. Photochem.
Photobiol. 2007, 83, 1006.
12. Jinadasa, R. G. W.; Hu, X. K.; Vicente, M. G. H.; Smith, K. M. J. Med. Chem. 2011,
54, 7464.
1
1
1
3. Gryko, D. T.; Tasior, M. Tetrahedron Lett. 2003, 44, 3317.
4. Rui, X.; Zha, Q.-Z.; Wei, T.-T.; Xie, Y.-S. Inorg. Chem. Commun. 2014, 48, 111.
5. Rowland, G. B.; Barnett, K.; DuPont, J. I.; Akurathi, G.; Le, V. H.; Lewis, E. A.
Bioorg. Med. Chem. 2013, 21, 7515.
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6. Nazarova, A.; Ignatova, A.; Feofanov, A.; Karmakova, T.; Pljutinskaya, A.; Mass,
O.; Grin, M.; Yakubovskaya, R.; Mironov, A.; Maurizot, J. C. Photochem.
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Acknowledgments
1
1
1
7. Taima, H.; Okubo, A.; Yoshioka, N.; Inoue, H. Tetrahedron Lett. 2005, 46, 4161.
8. Taima, H.; Okubo, A.; Yoshioka, N.; Inoue, H. Chem. Eur. J. 2006, 12, 6331.
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This work was supported by the BK21 PLUS Project and the
Doctoral Science Foundation of Yantai University (HY13B11 to J.L.)
203.
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