Chlorin p6-Based Water-Soluble Amino Acid Derivatives as Potent Photosensitizers for Photodynamic Therapy

Article abstract of DOI:10.1021/acs.jmedchem.6b00352

The development of novel photosensitizer with high phototoxicity, low dark toxicity, and good water solubility is a challenging task for photodynamic therapy (PDT). A series of chlorin p₆-based water-soluble amino acid conjugates were synthesized and investigated for antitumor activity. Among them, aspartylchlorin p₆ dimethylester (7b) showed highest phototoxicity against melanoma cells with weakest dark toxicity, which was more phototoxic than verteporfin while with less dark toxicity. It also exhibited better in vivo PDT antitumor efficacy on mice bearing B16-F10 tumor than verteporfin. The biological assays revealed that 7b was localized in multiple subcellular organelles and could cause both cell necrosis and apoptosis after PDT in a dose-dependent manner, resulting in more effective cell destruction. As a result, 7b represents a promising photosensitizer for PDT applications because of its strong absorption in the phototherapeutic window, relatively high singlet oxygen quantum yield, highest dark toxicity/phototoxicity ratio, good water solubility, and excellent in vivo PDT antitumor efficacy.

Full text of DOI:10.1021/acs.jmedchem.6b00352

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Article
Chlorin p6-based Water-soluble Amino Acid Derivatives
as Potent Photosensitizers for Photodynamic Therapy
Zhi Meng, Bin Yu, Guiyan Han, Minghui Liu, Bin Shan, Guoqiang Dong, Zhenyuan Miao, Ningyang
Jia, Zou Tan, Buhong Li, Wannian Zhang, Haiying Zhu, Chunquan Sheng, and Jianzhong Yao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00352 • Publication Date (Web): 02 May 2016
Downloaded from http://pubs.acs.org on May 3, 2016
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Chlorin p -based Water-soluble Amino Acid Derivatives as Potent
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Photosensitizers for Photodynamic Therapy
†,1
†,2
†,1
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1
Zhi Meng , Bin Yu , Guiyan Han , Minghui Liu , Bin Shan , Guoqiang Dong , Zhenyuan
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*,2
Miao , Ningyang Jia , Zou Tan , Buhong Li , Wannian Zhang , Haiying Zhu , Chunquan
*
,1
*,1
Sheng , Jianzhong Yao
1
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
Department of Cell Biology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China
Department of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Qiuyang Road, Fuzhou, 350122,
2
3
China
4
Department of Radiology, Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University,
2
25 Changhai Road, Shanghai 200438, China
5
Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian
Normal University, 32 Shangshan Road, Fujian 350007, China
ABSTRACT: The development of novel photosensitizer with high phototoxicity, low darkꢀtoxicity and good
6
water solubility is a challenging task for photodynamic therapy (PDT). A series of chlorin p ꢀbased waterꢀsoluble
amino acid conjugates were synthesized and investigated for antitumor activity. Among them, aspartylchlorin p
6
dimethylester (7b) showed highest phototoxicity against melanoma cells with weakest darkꢀtoxicity, which was
more phototoxic than verteporfin while with less darkꢀtoxicity. It also exhibited better in vivo PDT antitumor
efficacy on mice bearing B16ꢀF10 tumor than verteporfin. The biological assays revealed that 7b was localized in
multiple subcellular organelles, and could cause both cell necrosis and apoptosis after PDT in a dose dependent
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manner, resulting in more effective cell destruction. As a result, 7b represents a promising photosensitizer for PDT
applications because of its strong absorption in the phototherapeutic window, relatively high singlet oxygen
quantum yield, highest darkꢀtoxicity/phototoxicity ratio, good waterꢀsolubility and excellent in vivo PDT
antitumor efficacy.
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ꢀ
INTRODUCTION
PDT is clinically approved, minimally invasive protocol for the treatment that can be curative of early
1
ꢀ4
disease and palliative in advanced disease.
It is a photochemistryꢀbased treatment procedure in which the
photosensitizer (PS) is typically administered intravenously or topically, and after a time named the drugꢀtoꢀlight
interval (DLI), PS is activated by light of an appropriate wavelength (e.g. phototherapeutic window 650~850 nm)
to form an excited triplet state. Excited PS molecular transfers an electron to molecular oxygen or other electron
acceptors generating superoxide anions and radicals (type I reaction), or transfers its energy to the triplet ground
state of molecular oxygen to produce the highly cytotoxic singlet oxygen (type II reaction) with the ultimate
formation of reactive oxygen species (ROS). ROS mainly initiates three biological mechanisms that make PDT an
effective anticancer procedure: 1) direct tumor killing induced by the ROS; 2) tumorꢀassociated vascular
5
ꢀ13
shutdown and massive ischemice death; 3) activation of antitumor immune memory and systemic response.
Photofrin, the first generation of porphyrinꢀtype PS, has achieved some clinical efficacy, but it also suffered
from several drawbacks such as chemical heterogeneity, poor tissue penetration due to its limited maximum
absorption wavelength, weak absorption at therapeutic wave length (630 nm), and prolonged cutaneous
1
4
photosensitivity caused by its slow elimination in normal tissue. Compared to porphyrins, chlorinꢀtype PSs are
receiving considerable attention owing to their intense absorption in near infrared region (≥ 650 nm, also called
"
phototherapeutic window"), which are relatively harmless and penetrate deeply in biological tissues. In particular,
chlorophyll a derivatives are inherently amphiphilic macrocycles that have been extensively investigated. As
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compared to commercial photofrin, chlorophyll a derivatives generally showed low dark toxicities, rapid
clearance from normal tissue and low cutaneous phototoxicity. However, poor water solubility is the main
limitation of the chlorophyll a derivatives, which has hampered their PDT clinical application. Thus, chlorophyll a
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analogues of the chlorin e
6
series have been derivatized in multiple ways to produce novel waterꢀsoluble PSs for
PDT investigations. For this, chlorophyll a derivatives have been conjugated to amino acids, peptides and sugars
1
5, 16
17, 18
have been reported. Among them, talaporfin
and verteporfin
were approved for PDT treatment. In
addition, bacteriochlorinꢀtype PSs received intensive attention mainly due to their advantage of intense absorption
in near infrared region (740~780 nm), which penetrate more deeply in biological tissues than chlorins, and
5
,6,19ꢀ25
Pdꢀbacteriopheophorbide, padeliporfin and redaporfin have entered clinical trials.
In our previous studies, a series of novel chlorinꢀtype PSs such as benzochloroporphyrin derivatives (BCPDs)
were designed and synthesized using crude chlorophyll extracts from Chinese traditional herb named silkworm
2
6
excrement as the starting material. Among them, benzochloroporphyrin p6 dimethylester cisꢀisomer
(BCPDꢀ18MA, 11) was a potent antitumor candidate for PDT due to its good photobiological properties, but its
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water solubility remained to be further improved. Introducing amino acids was reported to be an effective
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strategy to improve the water solubility and the biological effects of chlorinꢀ and porphyrinꢀbased derivatives.
Inspired by these encouraging results, herein we synthesized a series of new waterꢀsoluble amino acid conjugates
of 11 and chlorin p dimethylester (5) and investigated their phototoxicity against melanoma B16ꢀF10 and A375
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cells because melanoma is a kind of malignant tumor with increasing incidence and high mortality and therapeutic
33ꢀ35
effects of clinically available drugs (e.g. dacarbazine and carmustine) are far from ideal.
In vitro and in vivo
photodynamic antitumor assays highlighted 7b as a promising PS for PDT treatment of melanoma. Moreover, the
subcellular location as well as the mode of cell death induction of 7b was clarified by biological assays.
ꢀ
RESULTS AND DISCUSSION
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. Synthesis. The synthetic route of the amino acid conjugates (7) is shown in Scheme 1. Pheophorbide a (1)
was prepared via 36% aqueous HCl degradation of chlorophyll a in Et O by using our previously established
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protocol. Treatment of 1 in Et
2
O with KOH and iꢀPrOH under an atmosphere of O
2
gave purpurinꢀ18 (2) in
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34.4% yield. 15ꢀAnhydride ring of 2 was hydrolyzed in the presence of tetrahydrofuran (THF) and CH
3
OH
using NaOH as the base. The resulting chlorin p (3) was rapidly methylated in Et O with CH to gave chlorin
6
2
2 2
N
3
7
p
6
trimethylester (4) in 83.6% yield. Selective hydrolysis of 4 in THF with 25% aqueous HCl produced 5 in
3
8
1.8% yield. The 17 ꢀcarboxylic acid of 5 was activated using 1ꢀhydroxybenzotriazole (HOBt) and
1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)ꢀcarbodiimide hydrochlorate (EDCI) and coupled individually with Lꢀglutamic
acid diꢀtertꢀbutyl ester hydrochlorate and Lꢀaspartic acid diꢀtertꢀbutyl ester hydrochlorate in
N,Nꢀdiisopropylethylamine (DIPEA) to form its tertꢀbutylꢀprotected glutamyl (6a) and aspartyl (6b) derivatives,
which were followed by deprotection with trifluoroactic acid (TFA) to afford the target compounds
glutamylchlorin p dimethyl ester (7a) or 7b in 61.9% and 58.0% yields, respectively.
6
a
Scheme 1
a
Reagents and Conditions: (a) 36% HCl, Et
2
O, 5~10 °C, 30 min; (b) KOH, iꢀPrOH, O
2
, 12 h, 34.4%; (c) THFꢀCH
3
OH (1:4), 0.5 M
t
NaOH, rt, 3 h (d) CH
2
N
2
, rt, 5 min, two steps (c→d) 83.6%; (e) THFꢀ25% aqueous HCl (1:1), rt, 4 h, 81.8%; (f) LꢀGlu(OBu )
2
•HCl
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t
or LꢀAsp(OBu )
2
•HCl, EDCI, HOBt, DIPEA, CH
2
Cl
2
, rt, 12 h; (g) CH
2
2
Cl ꢀTFA (3:1), rt, 6 h; two steps (f→g) 61.9% (7a) and 58.0%
(7b).
a
Scheme 2
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a
2 2
Reagents and Conditions: (a) DDQ, CH Cl ꢀtoluene (6:1), rt, 30 min, 53.5%; (b) DMAD, toluene, microwave 3 h; (c) DMAD,
2 2
toluene, reflux, 24 h, two steps (b→c) 42.3%; (d) DBU, CH Cl , rt, 30 min, 94.9%; (e) THFꢀ25% aqueous HCl (1:1), rt, 2.5 h, 96.8%;
t
t
(f) EDCI, HOBt, LꢀGlu(OBu ) •HCl or LꢀAsp(OBu ) •HCl, DIPEA, CH Cl , rt, 12 h; (g) CH Cl ꢀTFA (3:1), 6 h, two steps (f→g)
2 2 2 2 2 2
7
5.1% (13a) and 62.7% (13b).
The synthesis of the amino acid conjugates (13) of 11 is outlined in Scheme 2. Oxidation of 4 by 2,
ꢀdichloroꢀ5, 6ꢀdicyanoꢀ1, 4ꢀbenzoquine (DDQ) in toluene produced chloroporphyrin p trimethylester (8) in
3.5% yield. Microwave reaction of 8 with dimethyl acetylenedicarboxylate (DMAD) in toluene for 3 h
3
5
6
27
followed by refluxing for 24 h afforded the DielsꢀAlder adduct (9) in 42.3% yield. Compared to the procedure of
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Zhang et al., the reaction time was greatly shortened from 6 d to 27 h under the microwave condition.
Rearrangement of 9 with 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU) formed benzochloroporphyrin p
6
3
trimethylester cisꢀisomer (BCPDꢀ18, 10) in 94.9% yield and selective 17 ꢀester hydrolyzation of 10 by 25%
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aqueous HCl provided 11 in 96.8% yield. The 17ꢀcarboxylic acid of 11 was activated using HOBt and EDCI and
coupled individually with Lꢀglutamic acid diꢀtertꢀbutyl ester hydrochlorate and Lꢀaspartic acid diꢀtertꢀbutyl ester
hydrochlorate in DIPEA to obtain its tertꢀbutylꢀprotected glutamyl (12a) or aspartyl (12b) derivatives, which were
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followed by deprotection in CH
2 2
Cl with TFA to afford the target compounds glutamyl BCPDꢀ18MA (13a) and
aspartyl BCPDꢀ18MA (13b) in 75.1% and 62.7% yields, respectively.
2
. Photophysical properties and water solubility. As shown in Table 1, UVꢀvisible spectra of the amino
acid conjugates 7a, 7b, 13a and 13b showed the long wavelength absorption maxima in visible bands at 666, 667,
75 and 675 nm, respectively. They exhibited stronger absorption at long wavelength than corresponding lead
compounds 5 and 11. Among them, 7b possessed the largest molar absorption coefficient at long wavelength of
67 nm.
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Moreover, the UV absorption spectra (Figure 1A), the timeꢀresolved fluorescence (Figure 1B) and O
2
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luminescence spectra (Figure 1C) of 5, 7a and 7b were determined. The lifetimes of fluorescence and O
2
could
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be derived by fitting the corresponding spectra (Figure 1D) and the results indicated that the O
2
luminescence
intensities of 5, 7a, 7b and Al(III)phthalocyanine chloride tetrasulfonic acid (AlPcS) showed a linear dependence
on their absorbance. The data gave an excellent fit to a straight line with zero intercept, and the slope of the line
1
was predicted by the computational fit. The determination of O
2
quantum yields was performed for three
independent measurements with a maximum standard deviation of 5%. As summarized in Table 2, no significant
1
differences were observed for the lifetimes of fluorescence, O
2
and tripletꢀstate among 5, 7a and 7b, while the
1
O quantum yield of 7b was 2ꢀfold of the standard control AlPcS. As compared to 5 and 7a, 7b could be a
2
1
1
potential PS for PDT with higher O
2 2
quantum yield. The O lifetimes (10.4 ± 0.4 ꢁs) of 5, 7a and 7b in methanol
38
were agreed well with the value obtained by Young et al. In addition, the water solubility of 5 and 7b was
39
determined by our reported protocol. As expected, the aqueous solubility of 7b (solubility: 41.7 ꢀM) was
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significantly better than its lead compound 5 (water solubility: < 1.6 ꢀM).
Table 1. UV-vis Data for Lead Compounds (5, 11) and Their Corresponding Amino Acid Conjugates
4
ꢀ1
ꢀ1
λmax (CH
3
OH, nm) (ε×10 , M cm )
Compounds
Soret band
397 (34.7)
399 (48.1)
398 (61.5)
431 (7.91)
437 (9.41)
435 (8.97)
Visible bands
5
497 (6.22)
529 (2.31)
528 (5.39)
529 (6.14)
612 (2.23)
667 (17.3)
666 (27.2)
667 (37.3)
674 (1.50)
675 (1.86)
675 (2.81)
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a
497 (8.92)
497 (10.1)
7b
612 (5.46)
582 (1.93)
583 (2.44)
581 (3.49)
11
1
3a
3b
1
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Table 2. Fluorescence Lifetime, O
2
Lifetime, Triplet-state Lifetime and O
2
Quantum Yield for Lead
Compound (5) and its Amino Acid Conjugates
Compounds
τfluorescence (ns)
3.52
τSinglet Oxygen (ꢁs)
10.98
τtriplet (ꢁs)
0.19
ΦSinglet Oxygen
0.43
5
7a
3.45
10.36
0.17
0.30
7b
3.48
10.21
0.18
0.59
Figure 1. Photophysical properties of the target compounds. (A) UV absorption spectra of 5, 7a, 7b and AlPcS in methanol; (B) The
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timeꢀresolved fluorescence of 5, 7a, 7b; (C) O luminescence spectra (λ = 532 nm) of 5, 7a, 7b; (D) Plot of O luminescence
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ex
2
counts against the absorbance for 5, 7a, 7b and AlPcS in methanol. The error bars represent the standard deviation of the mean of
three independent measurements.
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. Cytotoxicity. An effective PS needs to possess high phototoxicity and low darkꢀtoxicity, which is
measured as the darkꢀtoxicity/phototoxicity ratio. The darkꢀtoxicity and phototoxicity of the amino acid
conjugates 7a, 7b, 13a, 13b as well as their corresponding lead compounds 5 and 11 were evaluated in melanoma
B16ꢀF10 and A375 cells exposed to various concentrations of each compound using the cellꢀcounting kitꢀ8
(
CCKꢀ8) assay.
As shown in Table 3, compared to lead compounds 5 and 11, target conjugates 7a, 7b, 13a and 13b exhibited
higher darkꢀtoxicity/phototoxicity ratio although both darkꢀtoxicity and phototoxicity was decreased. Among them,
7
b showed the lowest darkꢀtoxicity against both B16ꢀF10 and A375 cells with IC50 larger than 300 ꢀM, which was
2
more than 8.3ꢀfold lower than verteporfin. Interestingly, after exposure to light (10 J/cm ), all the conjugates were
found to be highly toxic to B16ꢀF10 and A375 cells, and the phototoxicity of aspartic acid conjugates 7b and 13b
was better than that of the corresponding glutamic acid conjugates 7a and 13a, which was consistent well with the
1
result that 7b had higher O
2
quantum yield than 7a. Moreover, 7b was the most phototoxic compound, which was
more potent than verteporfin.
Table 3. Cytotoxicity for Lead Compounds (5, 11) and Their Corresponding Amino Acid Conjugates against
B16-F10 and A375 cells Using CCK-8 Assay
B16ꢀF10
phototoxicity
(IC50, ꢀM)
0.30
A375
phototoxicity
(IC50, ꢀM)
0.52
Compounds
darkꢀtoxicity
(IC50, ꢀM)
15.67
dark toxicity
(IC50, ꢀM)
31.33
ratio
ratio
5
52.23
232.95
> 441.18
16.66
60.25
> 194.81
> 306.12
15.49
7
7
a
284.20
1.22
> 300
1.54
b
> 300
0.68
> 300
0.98
11
3a
15.33
0.92
22.77
1.47
1
224.40
4.06
55.27
286.20
7.10
40.31
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3b
271.20
24.92
2.29
1.20
118.43
20.77
> 300
36.33
3.02
2.06
> 99.34
17.64
verteporfin
In order to further confirm the reliability of the obtained results, the survival fraction of B16ꢀF10 and A375
2
cells treated by 5, 7a, 7b and verteporfin under dark or light irradiation at dosage of 10 J/cm were measured at
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various concentrations. As shown in Figure 2, the conjugates 7a and 7b had far lower darkꢀtoxicity than lead
compound 5, and their phototoxicities were in a good drugꢀdose dependent manner. In addition, the phototoxicity
of the most phototoxic compound 7b with various light dosages at a concentration of 5 ꢀM was also determined in
order to validate the rationality of light dosage used in all above phototoxicity assays. As shown in Figure 3, the
phototoxicity of compound 7b correlated well with light dose within a certain range, and the light dose of 10
2
J/cm was really appropriate for the above phototoxicity measurement. Moreover, the darkꢀtoxicity and
phototoxicity of 7b and verteporfin against B16ꢀF10 cells was further validated using the MTT assay (Figure 4
and Table 4). As expected, the phototoxicity of 7b was also in a good drugꢀdose dependent manner. As shown in
Table 3 and Table 4, similar darkꢀtoxicity and phototoxicity of 7b and verteporfin were obtained in both CCKꢀ8
assay and MTT assay. Taken together, all the results suggested that 7b might be a promising PS for the PDT
applications because it has the highest darkꢀtoxicity/phototoxicity ratio (> 400 for B16ꢀF10 cells and > 306 for
A375 cells). Thus, in vivo PDT antitumor efficacy and mechanism for 7b were further investigated.
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Figure 2. Survival fraction of B16ꢀF10 and A375 cells treated by 5, 7a,7b and verteporfin (“ve”) at the concentrations of 300 ꢁM and
2
50 ꢁM under dark or at the concentrations of 2 ꢁM and 0.2 ꢁM with a light dose of 10 J/cm using the CCKꢀ8 assay.
Figure 3. Survival fraction of B16ꢀF10 cells treated by 7b with various light dosages at the concentration of 5 ꢁM using the
CCKꢀ8 assay.
Table 4. Cytotoxicity for Compound 7b and Verteporfin against B16-F10 Cells Using the MTT Assay
darkꢀtoxicity
phototoxicity
(IC50, ꢁM)
0.75
Compounds
ratio
(
IC50, ꢁM)
> 300
7
b
> 400.0
23.39
verteporfin
25.03
1.07
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Figure 4. In vitro phototoxicity of verteporfin (A), 7b (C) and darkꢀtoxicty of verteporfin (B), 7b (D) toward B16ꢀF10 cells using the
MTT assay.
4
. Subcellular localization. The subcellular localization of the PS is of special significance, since it
determines the site of primary photodamages and the type of cellular response to the therapy. While the ROS,
1
especially O
2
, is shortꢀlived, its intracellular targets are close to the sites where the PS is located. Therefore,
13, 40, 41
cellular structures containing PS would be preferentially damaged upon illumination.
The preferential sites of subcellular localization of conjugate 7b were evaluated by laser confocal microscopy
upon exposure of B16ꢀF10 cells to 7b (10 ꢀM) for 24 h. Figure 5 showed the fluorescent pattern observed for 7b
and its overlay with the organelle specific fluorescent probes Lyso Tracker Green (lysososomes), Mito Tracker
Green FM (mitochondria), Golgi Tracker Green (golgi apparatus), and ER Tracker Green (endoplasmic reticulum).
The images were obtained by merging the fluorescence of Lyso Tracker Green DNDꢀ26 (green signal, Figure 5G),
Mito Tracker Green FM (green signal, Figure 5C), Golgi Tracker Green (green signal, Figure 5K), and ER
Tracker Green (green signal, Figure 5O) with that of 7b (red signal, Figures 5B, 5F, 5J and 5N). After entry into
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cells, 7b was not observed inside the nucleus of B16ꢀF10 cells.
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Figure 5. Subcellular location of 7b in B16ꢀF10 cells at 10 ꢀM for 24 h: (A, E, I, M) phase contrast; (B, F, J, N) red fluorescence
corresponds to 7b; (C) Mito Tracker Green fluorescence; (G) Lyso Tracker Green fluorescence; (K) Golgi Tracker Green
fluorescence; (O) ER Tracker Green fluorescence; (D, H, L, P) overlays of organelle trackers with 7b fluorescence. Scale bar: 10
ꢀ
m.
The merged stained images revealed the overlaps of 7b with Mito Tracker Green FM (yellow signal, Figure
D), Lyso Tracker Green DNDꢀ26 (yellow signal, Figure 5H), Golgi Tracker Green (yellow signal, Figure 5L),
5
and ER Tracker Green (yellow signal, Figure 5P), suggesting that mitochondria, lysosomes, Golgi, and ER were
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all the sites of its intracellular distribution. Mitochondria were the most important cell organs related to apoptosis
and also exhibited peripheral benzodiazepine receptor (PBR) binding, which might be an important target for
42ꢀ44
PDT.
Photodamage to lysosomes could inhibit autophagy, a potentially cytoꢀprotective effect. Therefore, the
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loss of autophagy could improve the overall photodynamic effect in cells capable of initiating an apoptotic
6, 45
program in response to photodamage.
ER is one of the most critical sites for PDT and also could induce
apoptosis. Thus, localization in ER could lead to activation of apoptotic pathways. In addition, the localization in
6
, 46
ER could also cause photodamage to Bclꢀ2 or release of cytochrome C with proꢀapoptotic consequences.
Presumably, the photodamage effect to multiple organelles caused by 7b could trigger various apoptotic pathways,
4
7, 48
leading to effective cell destruction.
5
. Determination of the apoptotic cell death in vitro. Many excited PS generate ROS that lead to induction
2
7,49ꢀ51
of apoptosis in malignant cells.
Nuclear fragmentation and phosphatidylserine exposure were studied to
evaluate the effects of 7b on inducing cell apoptosis. The redistribution of plasma membrane phosphatidylserine is
an early marker of apoptosis. Since Annexin VꢀFITC can also be used to detect necrotic cells as a result of the loss
of membrane integrity, apoptotic cells are differentiated from necrotic cells by an increase in PI (propidium iodide)
positivity, which selectively labels necrotic but not apoptotic cells. To monitor the concentration gradient of
phosphatidylserine exposure, Annexin VꢀFITC binding and PI uptake were measured by flow cytometry. As
shown in Figure 6, the significant increase in both Annexin VꢀFITC binding and PI uptake was observed at 24 h
postꢀirradiation at a dose of 2 ꢀM (Figure 6B) or 5 ꢀM (Figure 6C). The results indicated that 7b could mainly
cause both cell necrosis and cell late period apoptosis in a dose dependent manner. The result was consistent with
the fact that 7b was localized in multiple subcellular organelles such as mitochondria, lysosomes, golgi apparatus
and ER (Figure 5).
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Figure 6. Flow cytometry analysis of B16ꢀF10 cells with Annexin V/PI double staining after PDT: (A) Blank; (B) Treated with 2.0
2
2
ꢁ
M of 7b at a light dose of 10 J/cm ; (C) Treated with 5.0 ꢁM of 7b at a light dose of 10 J/cm . UL (upper left quadrant), Annexin V
(ꢀ) PI (+), cell fragment; UR (upper right quadrant), Annexin V (+) PI (+), necrosis or the late period apoptosis; LL (lower left
quadrant), Annexin V (ꢀ) PI (ꢀ), survival cell; LR (lower right quadrant), Annexin V (+) PI (ꢀ), early period apoptosis
6. In vivo photodynamic antitumor potency. PDT antitumor efficacy of 7b on C57BL/6 mice bearing
B16ꢀF10 tumor was evaluated. As illustrated in Figures 7 and 8, the growth of the implanted tumors in 7bꢀPDT
group (1 mg/kg), and verteporfinꢀPDT group (2 mg/kg, positive control) was significantly inhibited compared to
negative control group (P < 0.05), and no significant difference was found among 7bꢀPDT group (1 mg/kg) and
verteporfinꢀPDT group (2 mg/kg) (P > 0.05). Furthermore, KaplanꢀMeier analysis also showed that compare to
negative control group (median survival = 13 d), the survival times of 7bꢀPDT group (median survival = 21 d) and
verteporfinꢀPDT group (median survival = 18 d) were both significantly prolonged (P < 0.05), while 7bꢀPDT
group and verteporfinꢀPDT group also showed no significant difference (P > 0.05) on the survival time. Overall,
these results demonstrated that the in vivo antitumor potency of 7b at dose of 1 mg/kg was comparable to that of
verteporfin at dose of 2 mg/kg, indicating that 7b might possess more potent PDT antitumor efficacy at equal
doses. It should be noted that verteporfin was in liposome form and could selectively accumulate at tumor site by
52
EPR (enhanced permeability and retention) effect, and 7b was in form of waterꢀsoluble sodium salt. The
preparation of 7b in a better formulation such also as liposome might result in better in vivo antitumor potency.
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Figure 7. PDT efficacy on C57BL/6 mice bearing B16ꢀF10 tumor. The mice were divided into 3 groups (5 mice in each group):
negative control (black line), 7b-PDT (1 mg/kg, green line), verteporfinꢀPDT (2 mg/kg, red line). The tumors were irradiated with
2
laser light (678 nm, 150 J/cm ) at 2 h after intravenous administration.
Figure 8. KaplanꢀMeier survival curve of C57BL/6 mice bearing B16ꢀF10 tumor. P < 0.05 for verteporfinꢀPDT group and 7bꢀPDT
group, compared with negative control by logꢀrank test (n = 5).
ꢀ
CONCLUSIONS
6
In summary, we designed and synthesized a series of novel chlorin p ꢀbased waterꢀsoluble amino acid
conjugates containing glutamic acid and aspartic acid residues using Chinese traditional herb silkworm excrement
as the starting material. The target conjugates 7a, 7b, 13a and 13b exhibited higher in vitro
darkꢀtoxicity/phototoxicity ratio against melanoma B16ꢀF10 and A375 cells compared to corresponding lead
compounds 5 and 11. Among them, 7b showed the highest phototoxicity and the lowest darkꢀtoxicity, which was
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even more phototoxic and less darkꢀtoxic than verteporfin. It also exhibited better in vivo photodynamic antitumor
efficacy on C57BL/6 mice bearing B16ꢀF10 tumor than verteporfin(in liposome formulation). In addition, 7b was
found to localize in multiple subcellular organelles including lysosomes, mitochondria, golgi apparatus and
endoplasmic reticulum (ER), and could cause both cell necrosis and apoptosis after PDT in a dose dependent
manner, leading to more effective cell destruction. Taken together, our data support the fact that 7b is a promising
PS for PDT applications due to its strong absorption in the phototherapeutic window (λmax = 667 nm, ε = 3.73
0
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5
ꢀ1
ꢀ1
1
×
10 M cm ), relatively high O quantum yield, hightest darkꢀtoxicity/phototoxicity ratio (> 400 for B16ꢀF10
2
cells and > 306 for A375 cells), good waterꢀsolubility and excellent in vivo PDT antitumor efficacy. Further
evaluation of PS candidate 7b for clinical trial was in progress.
ꢀ
EXPERIMENTAL SECTION
1
. Chemistry. All chemicals were of regent grade. All air and moisture sensitive reactions were performed in
dried solvents under a nitrogen atmosphere. All solvents and reagents were purchased from commercial sources,
unless otherwise stated. Thinꢀlayer chromatography (TLC) analysis was carried out on silica gel plates GF254
(QindaoHaiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60G
1
13
(
QindaoHaiyang Chemical, China). H and C spectra were recorded on Bruker MSLꢀ300 or MSLꢀ600 or Varian
3 3 3 2 6
Unity Inovaꢀ500 spectrometers, with TMS as the internal standard and CDCl or CD OD or (CD ) SO (DMSOꢀd )
as the solvents. Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively. ESI mass
spectrometric data were collected on an APIꢀ3000 LCꢀMS spectrometer or a Micromass QtofꢀMicro LCꢀMSꢀMS.
UV absorption spectra were measured on an Agilent UV 8453 or Techcomp UV 1102 spectrophotometers. Purity
of the target compounds were analyzed by high performance liquid chromatography (HPLC) (Agilent
Technologies 1260 Infinity) using MeOH / THF / H
2
O / HOAc (30:25:44.5:0.5, volume ratio) of 1 mL / min on a
C18 column (Aglilent 20RBA × SBꢀC18, 5 ꢀm, 4.6 mm × 150 mm). All compounds exhibited more than 95%
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purity. All solvents and regents were analytically pure, and no further purification was needed. All starting
materials were commercially available.
36,37
Chlorin p
6
dimethylester (5). 4 (375 mg, 0.601 mmol), which was synthesized as previously reported.
,
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60
was dissolved in THF (100 mL) with addition of 25% HCl aqueous solution (100 mL). The mixture was stirred at
room temperature for 4 h. The reaction mixture was diluted with water and adjusted to pH 5~6 with aqueous
2 2
sodium hydroxide and then extracted with CH Cl . The organic layer was washed with water, dried over
anhydrous Na SO , and evaporated. The residue was dissolved in CH Cl and purified on a silica gel column (3%
2
4
2
2
+
2 2 38 4 6
methanol in CH Cl as eluent) to afford 300 mg, 0.492 mmol, 81.8% yield of 5 (C35H N O ). MS (ESI ) m/z:
+
ꢀ1
ꢀ1
5
4
4
6
11.39 [M+H] (100%). UVꢀvis λmax (CH
3
OH, nm) (ε / M cm ): 667 (1.73×10 ), 610 (2.23×10 ), 529 (2.31×10 ),
4
5
1
497 (6.22×10 ), 397 (3.47×10 ). H NMR [CDCl
3
, 600 MHz]: δ 9.69 (s, 1H), 9.47 (s, 1H), 8.66 (s, 1H), 7.95 (dd,
J = 18.0, 11.4 Hz, 1H), 6.29 (d, J = 18.0 Hz, 1H), 6.12 (d, J = 11.4 Hz, 1H), 5.16 (m, 1H), 4.39 (m, 1H), 4.20 (s,
3H), 4.14 (s, 3H), 3.71 (m, 2H), 3.64 (s, 3H), 3.38 (s, 3H), 3.23 (s ,3H), 2.43ꢀ2.10 (m, 4H), 1.85 (d, J = 6.0 Hz,
3H), 1.69 (t, J = 7.8 Hz, 3H), ꢀ1.0 (br s, 2H). HPLC purity: 98.8%.
Glutamylchlorin p dimethylester (7a). 5 (110 mg, 0.180 mmol) was dissolved in anhydrous CH Cl (30 mL).
6
2 2
EDCI (41.5 mg, 0.216 mmol, 1.2 equiv.) and HOBt (29.2 mg, 0.216 mmol l, 1.2 equiv.) were then added and
t
allowed to stir until completely dissolved under argon in iceꢀsalt bath. After 30 min, LꢀGlu(OBu )
2
•HCl (60.9 mg,
0
.216 mmol l, 1.2 equiv.) and DIPEA (0.038 mL, 0.216 mmol l, 1.2 equiv.) were mixed in CH Cl (10 mL) and
2 2
added to the reaction mixture. The mixture was allowed to stir at room temperature overnight under nitrogen. It
was diluted with CH Cl (200 mL) and then washed with 5% aqueous citric acid, brine and water, respectively.
2
2
2 4
The organic layer was dried over anhydrous Na SO and evaporated. The residue was dissolved in dry
2 2 2 2
CH Cl /TFA (3:1, 15 mL) and stirred at room temperature for 6 h. The resulting mixture was diluted with CH Cl
and adjusted to pH 3~4 with 10% NaHCO and purified on a silica gel column (5% CH OH in CH Cl as eluent)
3
3
2
2
+
+
45 5 9
to give 82.5 mg, 0.112 mmol, 61.9% yield of 7a (C40H N O ). MS (ESI ) m/z: 740.42 [M+H] (100%). UVꢀvis
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ꢀ1
ꢀ1
5
4
4
5
1
λ
max (CH
3
OH, nm) (ε / M cm ): 666 (2.72×10 ), 528 (5.39×10 ), 497 (8.92×10 ), 397 (4.81×10 ). H NMR
[ (CD ) SO, 600 MHz]: δ 12.33 (br s, 2H)), 9.67 (s, 1H), 9.45 (s, 1H), 8.98 (s, 1H), 8.10 (dd, J = 18, 12 Hz, 1H),
3
2
8
.03 (d, J = 12 Hz, 1H), 6.34 (d, J = 18 Hz, 1H), 6.10 (d, J = 12 Hz, 1H), 4.99 (m, 1H), 4.58 (m, 1H), 4.15 (s, 3H),
0
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4
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0
1
2
3
4
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7
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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7
8
9
0
1
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3
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8
9
0
4.12 (s, 3H), 3.98 (m, 2H),3.56 (s, 3H), 3.40 (s ,3H), 3.38 (m, 1H), 3.09 (s, 3H), 2.28 (m, 2H), 2.18 (m, 2H), 2.01
13
(
m, 2H), 1.88 (m, 2H), 1.83 (d, J = 6.0 Hz, 3H), 1.65 (t, J = 6.0 Hz, 3H), ꢀ1.14 (s, 1H), ꢀ1.29 (s, 1H). C NMR
[ (CD
37.28, 136.03, 135.11, 134.20, 131.19, 128.76, 128.66, 122.63, 122.53, 104.22, 103.13, 99.79, 94.13, 52.79,
52.62, 52.29, 51.10, 48.16, 32.43, 31.53, 30.01, 28.89, 26.22, 23.49, 18.54, 17.51, 12.00, 11.85, 10.64. HRMS
3 2
) SO, 600 MHz]: δ 173.59, 173.42, 173.30, 171.74, 169.75, 167.27, 166.09, 154.16, 148.17, 144.95, 140.56,
1
+
+
(
46 5 9
ESI ) m/z: 740.3290 [M + H] , calcd for C40H N O 740.3217. HPLC purity: 97.6%.
t
Aspartylchlorin p
6 2
dimethylester (7b). 5 (120 mg, 0.197 mmol) was reacted with LꢀAsp(OBu ) •HCl (66.4
mg, 0.237 mmol, 1.2 equiv.) by following the procedure for the synthesis of 7a to obtain 82.7 mg, 0.114 mmol,
+
+
ꢀ1
ꢀ1
5
8.0% yield of 7b (C H N O ). MS (ESI ) m/z: 726.43 [M+H] (100%). UVꢀvis λ (CH OH, nm) (ε / M cm ):
39
43
5
9
max
3
5
4
4
5
5
1
6
3 2
67 (3.73×10 ), 612 (5.46×10 ), 529 (6.14×10 ), 497 (1.01×10 ), 398 (6.15×10 ). H NMR [(CD ) SO, 600 MHz]:
δ 9.48 (s, 1H), 9.19 (s, 1H), 8.95 (s, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.89 (dd, J = 18, 12 Hz, 1H), 6.17 (d, J = 18 Hz,
1
H), 5.96 (d, J = 12 Hz, 1H), 5.01 (m, 1H), 4.61 (m, 1H), 4.45 (m, 1H), 4.17 (s, 3H), 4.16 (s, 3H), 3.51 (s, 3H),
.36 (m, 1H), 3.31 (s, 3H), 2.88 (s, 3H), 2.61 (m, 1H), 2.49 (s, 2H), 2.35 (m, 1H), 2.09 (m, 3H), 1.84 (d, J = 6.6
3
13
Hz, 3H), 1.44 (t, J = 7.2 Hz, 3H), ꢀ1.25 (s, 1H), ꢀ1.33 (s, 1H). C NMR [(CD ) SO, 600 MHz]: δ 173.37, 172.84,
3
2
1
1
2
7
72.03, 171.24, 169.79, 167.31, 166.13, 154.03, 148.05, 144.73, 140.50, 137.17, 135.81, 134.95, 134.21, 131.05,
28.62, 122.48, 122.39, 104.05, 103.13, 99.61, 94.10, 52.82, 52.69, 52.28, 48.62, 48.18, 37.31, 32.73, 31.64,
ꢀ
+
42 5 9
8.83, 23.51, 18.41, 17.42, 11.97, 11.77, 10.47. HRMS (ESI ) m/z: 724.2987 [M ꢀ H] , calcd for C39H N O
24.3059. HPLC purity: 96.3%.
BCPDꢁ18MA (11). 4 (300 mg, 0.481 mmol) was dissolved in dry CH Cl (90 mL) and added DDQ (70 mg)
2
2
which was dissolved in methylbenzene (15 mL). The mixture was stirred for 30 min at room temperature and then
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washed with 0.1 M aqueous hydrochloric acid and water, respectively. Organic layer was dried over anhydrous
Na SO and evaporated. The residue was purified on a silica column to obtain 160 mg, 0.257 mmol, 53.5% yield
2
4
ꢀ1
ꢀ1
3
3
5
1
of 8 (C36
H N
38 4
O
6
). UVꢀvis λmax (CH
2 2
H , nm) (ε / M cm ): 549 (7.3×10 ), 511 (6.0×10 ), 406 (1.0×10 ). H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[CDCl , 500 MHz]: δ 10.01 (s, 1H), 9.98 (s, 1H), 9.91 (s, 1H), 8.08 (dd, J = 17.9, 11.4 Hz, 1H), 6.24 (d, J = 17.9
3
Hz, 1H), 6.10 (d, J = 11.4 Hz, 1H), 4.45 (s, 3H), 4.32 (s, 3H), 4.07 (t, J = 8.5 Hz, 2H), 3.99 (q, J = 7.7 Hz, 2H),
3.80 (s, 3H), 3.67 (s, 3H), 3.60 (s ,3H), 3.54 (s, 3H), 3.52 (s ,3H), 3.08 (t, J = 8.5 Hz, 2H), 1.80 (t, J = 7.7 Hz, 3H),
ꢀ3.78 (s, 1H), ꢀ3.82 (s, 1H). 8 (228.4 mg, 0.367 mmol) and DMAD (1 mL) were dissolved in methylbenzene (30
mL) and reacted by microwave under 145℃ for 3 h, followed by refluxing under inert N atmosphere for 24 h.
2
2 2
The mixture was evaporated and then purified via gel column chromatography (1% methanol in CH Cl as eluent)
+
+
to afford 118 mg, 0.154 mmol, 42.3% yield of 9 (C42
H
44
N
4
O
10). MS (ESI ) m/z: 765.18 [M+H] (100%). UVꢀvis
ꢀ1
ꢀ1
4
3
4
4
5
1
λ
max (CH
2
H
2
, nm) (ε / M cm ): 655 (2.0×10 ), 600 (4.6×10 ), 550 (1.1×10 ), 519 (0.9×10 ), 413 (1.5×10 ).
H
NMR [CDCl , 300 MHz]: δ 9.74 (s, 1H), 9.26 (s, 1H), 9.16 (s, 1H), 7.39 (m, 1H), 4.37 (s, 3H), 4.23 (s, 3H), 3.96
3
(
s, 3H), 3.90 (s, 3H), 4.05 (m, 2H), 3.94 (q, J = 7.6 Hz, 2H), 3.81 (s, 3H), 3.52 (s, 3H), 3.60 (m, 2H), 3.45 (s ,3H),
.42 (s, 3H), 3.02 (m, 2H), 2.13 (s, 3H), 1.76 (t, J = 7.6 Hz, 3H), ꢀ2.15 (s, 1H), ꢀ2.60 (s, 1H). The DielsꢀAlder
adduct 9 (118 mg, 0.154 mmol) was dissolved it in dry CH Cl (100 mL). DBU (1 mL) was added, and stirred for
0 min at room temperature. The reaction mixture was washed with 3% HCl, saturated brine, water, respectively.
Organic layer was dried over anhydrous Na SO and then evaporated. The residue was purified via gel column
3
2
2
3
2
4
+
chromatography (CH
Cl
2 2
as eluent) to afford 112 mg, 0.154 mmol, 94.9% yield of 10 (C42
44 4
H N O
10). MS (ESI )
+
ꢀ1
ꢀ1
4
4
5
m/z: 765.28 [M+H] (100%). UVꢀvis λmax (CH
H
2 2
, nm) (ε / M cm ): 677 (2.3×10 ), 586 (3.1×10 ), 439 (1.1×10 ).
1
3
H NMR [CDCl , 300 MHz]: δ 9.74 (s, 1H), 9.30 (s, 1H), 9.02 (s, 1H), 7.82 (d, J = 5.7 Hz, 1H), 7.44 (d, J = 5.7
Hz, 1H), 5.07 (s, 1H), 4.36 (s, 3H), 4.23 (s, 3H), 3.99 (s, 3H), 3.80 (s, 3H), 3.99~3.91 (m, 4H), 3.52 (s, 3H), 3.44
s ,3H), 3.39 (s, 3H), 3.04 (m, 2H), 3.0 (s, 3H), 1.79 (s, 3H), 1.75 (t, J = 7.6 Hz, 3H), ꢀ1.92 (s, 1H), ꢀ2.23 (s, 1H).
0 (100 mg, 0.131 mmol) was dissolved in THF (30 mL). 25% HCl aqueous solution (30 mL) was added and
(
1
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2
2
2
2
2
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3
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stirred at room temperature for 4 h. The mixture was diluted with CH
2 2
Cl and washed with water. Organic layer
was then dried over anhydrous Na SO and evaporated. The residue was purified via silica gel column
2
4
2 2
chromatography (2.5% methanol in CH Cl as eluent) to afford 95 mg, 0.116 mmol, 96.8% yield of 11
ꢀ1
ꢀ1
4
4
4
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(C41H
42
N
4
O
10). UVꢀvis λmax (CH
OH, nm) (ε / M cm ): 674 (1.50×10 ), 582 (1.93×10 ), 431 (7.91×10 ). H
3
NMR (DMSOꢀd , 500 Mz): δ 12.43 (s, 1H), 9.88 (s, 1H), 9.68 (s, 1H), 9.41 (s, 1H), 7.83 (d, J = 5.6 Hz, 1H), 7.78
6
(d, J = 5.6 Hz, 1H), 5.34 (s, 1H), 4.25 (s, 3H), 4.15 (s, 3H), 3.97 (q, J = 7.5 Hz, 2H), 3.93 (s, 3H), 3.81 (t, J = 8.4
Hz, 2H), 3.49 (s, 3H), 3.47 (s, 3H), 3.45 (s ,3H), 2.96 (s, 3H), 2.90 (t, J = 8.4 Hz, 2H), 1.79 (s, 3H), 1.71 (t, J = 7.5
+
+
43 4
Hz, 3H), ꢀ2.08 (s, 1H), ꢀ2.39 (s, 1H). HRMS (ESI ) m/z: 751.2967 [M] , calcd for C41H N O10 751.2894. HPLC
purity: 98.9%.
2 2
Glutamyl BCPDꢁ18MA (13a). 11 (30 mg, 0.04 mmol) was dissolved in dry CH Cl (50 mL). EDCI (9.2 mg,
0
.048 mmol, 1.2 equiv) and HOBt (6.6 mg, 0.048 mmol, 1.2 equiv.) were then added and allowed to stir until
t
completely dissolved under argon in iceꢀsalt bath. After 30 min, LꢀGlu(OBu ) •HCl (14.3 mg, 0.048 mmol, 1.2
2
2 2
equiv.) and DIPEA (0.010 mL) were dissolved in CH Cl (5 mL) and added to the reaction mixture. The mixture
2 2
was allowed to stir at room temperature overnight under nitrogen. It was then diluted with CH Cl and washed
2 4
with 5% aqueous citric acid, brine and water, respectively. The organic layer was dried over anhydrous Na SO
2 2
and evaporated. The residue was dissolved in dry CH Cl /TFA (3:1, 20 mL) and stirred at room temperature for 4
h. The resulting mixture was diluted with CH Cl and adjusted to pH 3~4 with 10% NaHCO and purified on a
2
2
3
3 2 2
silica gel column (5% CH OH in CH Cl as eluent) to afford 26.4 mg, 0.03 mmol, 75.1% yield of 13a
ꢀ
+
ꢀ1
ꢀ1
4
(
C
H
46 49
N
5
O
13). MS (ESI ) m/z: 878.33 [MꢀH] (100%). UVꢀvis λmax (CH
3
OH, nm) (ε / M cm ): 675 (1.86×10 ),
OD, 600 MHz): δ 9.39 (s, 1H), 9.27 (s, 1H), 9.23 (s, 1H), 8.16 (s,
H), 7.84 (d, J = 6.0 Hz, 1H), 7.65 (d, J = 6.0 Hz, 1H), 5.18 (s, 1H), 4.38 (s, 3H), 4.24 (s, 3H), 4.14 (m, 1H), 4.02
4
4
1
5
83 (2.44×10 ), 437 (9.41×10 ). H NMR (CD
3
1
(q, J = 6.0 Hz, 2H), 3.99 (s, 3H), 3.97 (m, 2H), 3.54 (s, 3H), 3.39 (s, 3H), 3.35 (m, 2H), 3.04 (s, 3H), 2.94 (m, 2H),
ꢀ
+
2
.89 (s, 3H), 2.25 (m, 2H), 1.82 (t, J = 6.0 Hz, 3H), 1.77 (s, 3H). HRMS (ESI ) m/z: 878.3260 [M ꢀ H] , calcd for
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C
46
H
48
N
5
O
13 878.3331. HPLC purity: 95.8%.
t
Aspartyl BCPDꢁ18MA (13b). 11 (26 mg, 0.035 mmol) was reacted with LꢀAsp(OBu ) •HCl (11.8 mg, 0.042
2
mmol, 1.2 equiv) by following method for the synthesis of 13a to give 18.8 mg, 0.022 mmol, 62.7% yield of 13b
+
+
ꢀ1
ꢀ1
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
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36
37
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47
48
49
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53
54
55
56
57
58
59
60
(C45
H
N
5
O
13). MS (ESI ) m/z: 866.51 [M+H] (100%). UVꢀvis λmax (CH
OH, nm) (ε / M cm ): 675 (2.81×10 ),
OD, 300 MHz): δ 9.27 (s, 1H), 9.07 (s, 1H), 9.00 (s, 1H), 8.35 (s,
1H), 7.87 (d, J = 6.0 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 5.23 (s, 1H), 4.44 (s, 3H), 4.30 (s, 3H), 4.09 (m, 1H), 4.07
s, 3H), 4.01 (q, J = 9.0 Hz, 2H), 3.59 (s, 3H), 3.50 (m, 2H), 3.38 (s, 3H), 3.35 (m, 2H), 3.16 (s, 3H), 2.97 (m, 2H),
47
3
4
4
1
581 (3.49×10 ), 435 (8.97×10 ). H NMR (CD
3
(
1
3
2.53 (s, 3H), 2.05 (t, J = 6.0 Hz, 3H), 1.72 (s, 3H). C NMR (CD
3
OD, 600 MHz): δ 173.99, 173.05, 171.37,
171.11, 169.60, 167.59, 166.49, 154.87, 154.14, 147.18, 144.26, 143.57, 140.47, 138.51, 137.68, 136.78, 136.02,
134.38, 132.73, 131.96, 131.08, 129.73, 129.39, 124.53, 122.69, 118.59, 114.01, 109.23, 99.12, 93.59, 92.38,
+
53.16, 52.60, 51.73, 51.44, 50.79, 37.04, 31.60, 30.37, 29.56, 22.77, 22.26, 15.37, 12.98, 11.11. HRMS (ESI ) m/z:
+
866.3246 [M + H] , calcd for C H N O 866.3174. HPLC purity: 96.6%.
45
48
5
13
1
1
2 2
Photophysical study. The determination of O quantum yield is based on the comparison of O
luminescence counts (λmax = 1270 nm) generated from the photosensitization of Al (III) phthalocyanine chloride
tetrasulfonic acid (AlPcS, Φꢂ= 0.27) inmethanol and the samples were tested under the same conditions by using
5
3
1
1
the previously established method. The O
2 2
lifetimes can be indirectly derived by fitting the O luminescence
spectra.
Cell lines and culture. B16ꢀF10 cells and A375 cells were originally obtained from the Chinese Academy of
Sciences Shanghai Institute of Cell Bank (Shanghai, China). B16ꢀF10 cells were cultured in RPMI 1640 medium
(Hyclone, Logan, UT, USA) and A375 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA),
supplemented with 2 mg/mL sodium bicarbonate, 4.5 mg/mL glucose, 100 ꢁg/mL streptomycin sulfate, 40 mg/mL
gentamicin, 100 U/mL penicillin and 10% (v/v) heatꢀinactivated fetal bovine serum (FBS) in 5% CO , 95% air in a
2
humidified incubator at 37℃.
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Mouse bearing tumor model. C57BL/6 male mice aged 6ꢀ8 weeks with a mean body weight of 22 ± 2 g
4
were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). B16ꢀF10 cells (2×10 ) were
subcutaneously inoculated into the right thigh of mice. Measurement of the tumor size was started on day 8 after
inoculation. Only those mice with tumor diameter between 6 to 8 mm were used. The animals were given free
access to food and water during the experiment.
0
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8
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5
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8
9
0
Two parameters (length and width) of the tumor were measured using a micrometer digital caliper
2
(
500ꢀ157ꢀ20, Mitutoyo, Japan). The tumor volume was calculated using the following formula: V = (W × L)/2,
according to the US National Cancer Institute protocol, where W is the width and L is the length of the tumor.
3
Dark cytotoxicity. 5×10 cells were seeded on a 96ꢀwell culture transwell apparatus (Costar, Cambridge,
MA, USA) and cultured in RPMI 1640 medium or DMEM medium with 10% (v/v) fetal bovine serum (FBS),
respectively. The tested compounds were diluted to different final concentration and the cells were incubated for
4
8 h in a CO incubator at 37 ℃.
2
CCKꢁ8 assay. Above loading media were removed, and the cells were fed new medium, followed by 20 ꢀL
of Cell Counting Kitꢀ8 (CCKꢀ8) per 200 ꢀL of medium and incubated for 1.5 h. The cell viability was then
assessed by CellꢀCounting Kitꢀ8 assay (Dojindo Laboratories, Japan) following the manufacturers’ protocol. The
5
4
absorbance of each well was monitored by a spectrophotometer (Tecan, Switzerland) at 450 nm.
MTT assay. Above loading media were removed, and the cells were fed new medium, followed by 20 ꢀL of
ꢀ(4,5ꢀdimethylthiazolꢀ2 yl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT) per 150 ꢀL of medium and incubated for 4
3
h. Dimethyl sulfoxide (DMSO) was then used to dissolve the formazan crystals and the absorbance was measured
5
5
at 490 nm using anspectrophotometer (Tecan, Switzerland).
Phototoxicity. The B16ꢀF10 cells and A375 cells were prepared as described above with the tested
compounds of different concentrations and incubated for 24 h in a 5% CO incubator at 37℃. After receiving
2
2
irradiation with the diode laser at 678 nm for a light dose of 10 J/cm , cells were incubated for another 24 h. Cell
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viability was then measured as described above using CCKꢀ8 assay or MTT assay.
Subcellular localization. The B16ꢀF10 cells were cultured in RPMI 1640 medium and then the cells were
4
seeded onto thin glassꢀbottomed 35 mm petri dishes (NEST Biotechnology, Hangzhou, China) at 1×10 cells per
10
11
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dish and incubated for 24 h at 37℃ under 5% CO
2
atmosphere to reattach. The medium was removed and new
medium containing 10 ꢁM aspartylchlorin p dimethylester (7b) was added, and the cells were incubated in dark
6
for 24 h. The medium was removed and washed twice with PBS, following incubation with MitoꢀTracker Green
FM (200 nM, for 45 min, Molecular Probes, Life Technologies, USA), LysoꢀTracker Green DNDꢀ26 (200 nM, for
45 min, Molecular Probes, Life Technologies, USA), GolgiꢀTraker Green (5 ꢁM, for 30 min, Molecular Probes,
Yeasen, China), and ERꢀTraker Green (1 ꢁM, for 30 min, Molecular Probes, Yeasen, China) at 37℃ in a 5% CO
2
incubator. Then both the medium and the trackers were removed and washed 3 times with PBS and the cells were
examined using a Leica TCS SP5 spectral confocal microscope equipped with ArgonꢀHeliꢀumNenon (ArꢀHeNe)
laser. MitoꢀTracker, LysoꢀTracker, GolgiꢀTraker, ERꢀTraker and compound 7b were respectively excited at the
wavelength of 488, 488, 488, 488, 633 nm and the signals from different probes were acquired in a sequential scan
mode.
Apoptosis measurement by Annexin V/PI staining. B16ꢀF10 cells in the longꢀphase were seeded into
sixꢀwell culture plate and incubated with compound 7b (2.0, 5.0 ꢁM). After 24 h, the cells were exposed to the
2
laser at 678 nm for a light dose of 10 J/cm , then the cells were incubated in a 5% CO incubator at 37℃ for 24 h.
2
After that, apoptotic cells and necrotic cells were analyzed by double staining with Alexa Fluor 488 annexin V and
propidium iodide (PI) (BD, USA) following the manufacturer’s instructions. Cells were coꢀstained with 5 ꢀL PI
and immediately analyzed using Beckman Cyanflow Cytometer (Beckman,USA). The percentage of apoptotic
5
5
(
annexinþ/PI) and necrotic (annexinþ/PI) cells was determined using software.
In vivo phototoxicity. The potential PDT efficacy of aspartylchlorin p dimethylester (7b) on B16ꢀF10
6
bearing mice was investigated by irradiation with the diode laser at 678 nm. The B16ꢀF10 bearing C57BL/6 mice
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1
2
2
2
2
2
2
2
2
2
2
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5
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5
5
6
were randomly divided into 3 groups, including negative control, 7b (1 mg/kg), and verteporfin (2 mg/kg), each
group contained 5 mice. Two hours after the intravenous administration, compound 7b and verteporfin group were
2
anesthetized and their tumor sites were irradiated with laser light (250 mW/cm ) for 10 min, and the total light
2
0
1
2
3
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9
0
dose was 150 J/cm . After the treatment, the tumor was measured with a caliper, and recorded the survival of mice
daily. The statistical differences between groups with respect to the mean tumor volume were evaluated by using
SPSS 19.0 for windows software. The significance was accepted at P < 0.05.
ꢀ
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XXXXXX/acs.jmedchem.XXXXXXX.
1
13
H and C NMR spectra (Figure S1ꢀS9), high resolution mass spectra (Figure S10ꢀS17) and HPLC purity
spectra (Figure S18ꢀS23).
SMILES data (CSV)
ꢀ
AUTHOR INFORMATION
Corresponding Authors
*
*
*
(H.Z.) Phone / Fax: +86 21 81870944. Eꢀmail: zinnia69@163.com.
(C. S.) Phone / Fax: +86 21 81871239. Eꢀmail: shengcq@hotmail.com.
(J.Y.) Phone / Fax: +86 21 81871243. Eꢀmail: yaojz@sh163.net.
Author Contributions
†
Zhi Meng, Bin Yu and Guiyan Han contributed equally to this work.
The manuscript was written through contributions of all authors. All authors have given approval to the final
version of the manuscript.
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Notes
The authors declare no competing financial interest.
ꢀ
ACKNOWLEDGMENT
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This work was supported by the Natural Science Foundation of China (Grant Nos. 81172950 and 61275216),
the Key Project of Science and Technology Commission of Shanghai (Grant No. 11431920400 and 11431920401)
and the Project of Science and Technology Commission of Shanghai (Grant No. 11430723200 and 11430723201).
ꢀ
ABBREVIATIONS LIST
PS, photosensitizer; PDT, photodynamic therapy; BCPDs, benzochloroporphyrin derivatives; BCPDꢀ18MA,
6
benzochloroporphyrin p dimethylester cisꢀisomer; THF, tetrahydrofuran; DDQ, 2, 3ꢀdichloroꢀ5, 6ꢀdicyanoꢀ1,
4ꢀbenzoquine; DMAD, dimethyl acetylenedicarboxylate; EDCI, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)ꢀcarbodiimide
hydrochlorate; DMF, dimethylformamide; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; DBU,
1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene; TFA, trifluoroactic acid; DIPEA, N,Nꢀdiisopropylethylamine; HOBt,
1
ꢀhydroxybenzotriazole; HPLC, high performance liquid chromatography; PBS, phosphate buffered saline; PI,
propidium iodide; FBS, fetal bovine serum; EPR, enhanced permeability and retention.
ꢀ
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