Amino acid derivatives of pyropheophorbide-a ethers as photosensitizer: Synthesis and photodynamic activity

Article abstract of DOI:10.1016/j.cclet.2016.03.019

Ten new water-soluble amino acid conjugates of pyropheophorbide-a ethers 4a-4j were synthesized and investigated for their in vitro photodynamic antitumor activity. The results showed that all compounds exhibited higher phototoxicity and lower dark toxicity against three kinds of tumor cell lines than BPD-MA. In particular, the most phototoxic compound 4d and 4j individually showed IC₅₀ values of 41 nmol/L and 33 nmol/L against HCT116 cell, which represented 7.8- and 9.7-fold increase of antitumor potency compared to BPD-MA, respectively, suggesting that they were promising photosensitizers for PDT applications because of their strong absorption at long wavelength (λ_max > 650 nm), high phototoxicity, low dark cytotoxicity and good water-solubility.

Full text of DOI:10.1016/j.cclet.2016.03.019

G Model
CCLET 3638 1–4
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
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Amino acid derivatives of pyropheophorbide-a ethers
as photosensitizer: Synthesis and photodynamic activity
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Q1 Zhi Meng a,1, Bin Shan b,1, Ling Zhang a,1, Gui-Yan Han a, Ming-Hui Liu b, Ning-Yang Jia c,
a,
a,
Zhen-Yuan Miao a, Wan-Nian Zhang a, , Chun-Quan Sheng , Jiang-Zhong Yao
*
*
*
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a Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
c Department of Radiology, Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Ten new water-soluble amino acid conjugates of pyropheophorbide-a ethers 4a–4j were synthesized
and investigated for their in vitro photodynamic antitumor activity. The results showed that all
compounds exhibited higher phototoxicity and lower dark toxicity against three kinds of tumor cell lines
than BPD-MA. In particular, the most phototoxic compound 4d and 4j individually showed IC50 values of
41 nmol/L and 33 nmol/L against HCT116 cell, which represented 7.8- and 9.7-fold increase of antitumor
potency compared to BPD-MA, respectively, suggesting that they were promising photosensitizers for
Received 25 January 2016
Received in revised form 21 February 2016
Accepted 8 March 2016
Available online xxx
Keywords:
PDT applications because of their strong absorption at long wavelength (lmax > 650 nm), high
Photodynamic therapy (PDT)
Photosensitiser
Pyropheophorbide-a
Chlorin
phototoxicity, low dark cytotoxicity and good water-solubility.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Antitumor
10
11
1. Introduction
(
l
max > 650 nm) to take full advantage of greater tissue penetra- 29
tion [4–6]. Among them, talaporfin [7] and verteporfin (BPD-MA) 30
[8] were approved for PDT applications (Fig. 1). 31
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Photodynamic therapy (PDT) now is a well recognized approach
to cancer therapy for the selective destruction of tumors by visible
light in presence of a photosensitizer (PS) and cell oxygen [1]. It is
based on the principle that the interaction between light and PS in
tumor tissues generates cytotoxic reactive oxygen species (ROS)
created through either electron transfer (type I) or energy transfer
(type II) reactions to inactivate the tumor cells [2].
Porfimer sodium, the first clinically approved porphyrin-type
PS for the treatment of bladder cancer in the world, has suffered
some drawbacks such as its complex component, inefficient
Pyropheophorbid-a (2), the one of chlorophyll-a derivative as 32
chlorin-type PS, has poor water solubility to hamper its clinical 33
development. Introducing amino acid at 173-position and alkoxy at 34
31-position was reported to individually improve the water 35
solubility and the biological activity of chlorin-based derivatives 36
[9–12]. In this regard, a series of novel water-soluble amino acid 37
conjugates of pyropheophorbide-a ethers 4a–4j were synthesized 38
(Scheme 1) and investigated their in vitro photodynamic antitu- 39
mor activity against three kinds of tumor cell lines.
40
absorption
max = 630 nm), prolonged cutaneous phototoxicity up to 4–6
weeks due to its slow clearance in skin tissues [3].
(
e
= 1170 L molÀ1 cmÀ1
)
at
long
wavelength
(
l
2. Experimental
41
42
In the recent decade, the so-called second generation chlorin-
type PSs such as natural chlorophyll-a derivatives have generated
interest due to its low skin phototoxicity, rapid clearance from
tissues and strong absorption at long wavelengths
2.1. Chemicals and instruments
Melting points were measured on a XRD micro melting point 43
apparatus and uncorrected. 1H NMR spectra were recorded on 44
Bruker MSL-300 or MSL-600 using TMS as the internal reference. 45
Mass spectra were collected on an API-3000 LC-MS spectrometer. 46
UV absorption spectra were measured on an Agilent UV 47
8453 spectrophotometers. Elemental analysis was carried out 48
using a PE2400 II instrument. Column chromatography was 49
*
Corresponding author.
E-mail addresses: zhangwnk@hotmail.com (W.-N. Zhang),
shengcq@hotmail.com (C.-Q. Sheng), yaojz@sh163.net (J.-Z. Yao).
1
These authors contributed equally to this work.
performed on silica gel (size 10–40 mm, Qingdao Haiyang 50
http://dx.doi.org/10.1016/j.cclet.2016.03.019
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Z. Meng, et al., Amino acid derivatives of pyropheophorbide-a ethers as photosensitizer: Synthesis and
photodynamic activity, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.019

G Model
CCLET 3638 1–4
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Z. Meng et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
CH2Cl2 (100 mL) was individually added 1-ethyl-3-(3-dimethyla-
82
minopropyl)-carbodiimide hydrochlorate (EDCI) (0.48 mmol,
1.2 equiv.) and 1-hydroxybenzotriazole (HOBt) (0.48 mmol l,
1.2 equiv.) to stir until completely dissolved under in ice-salt
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bath. After 30 min,
L
-Asp(OBut)2ÁHCl or
L
-Glu(OBut)2ÁHCl
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98
99
(0.48 mmol 1.2 equiv.) and N,N-diisopropylethylamine (DIPEA)
(0.48 mmol l, 1.2 equiv.) were mixed in CH2Cl2 (30 mL) and poured
into above reaction mixture. The mixture was allowed to stir at
room temperature overnight under nitrogen. It was diluted with
CH2Cl2 (150 mL) and then washed with 5% aqueous citric acid,
brine and water, respectively. The organic layer was dried over
anhydrous Na2SO4 and evaporated. The residue was dissolved in
dry CH2Cl2/TFA (1:1, 30 mL) and stirred at room temperature for
2 h. The resulting mixture was diluted with CH2Cl2 and adjusted to
pH 5–6 with 10% NaHCO3 and purified on a silica gel column
(CH2Cl2:CH3COCH3:CH3OH:HCO2H = 80:1:1:0.1 as eluent) to give
the target compounds 4a–4j.
Fig. 1. Two clinical available semisynthetic chlorin-type photosensitizers.
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Chemical, China). All reagents and solvents purchased from
commercial vendors and were used without further purifications.
The key intermediate pheophorbide-a (1) was obtained via cond.
aqueous HCl degradation of chlorophyll-a in Et2O (Scheme 1) by
the methodology developed in our laboratory using crude
chlorophyll extracts in silkworm excrements [13].
N-(3-Devinyl-3-(1-methoxy)ethyl-pyropheophorbide-a-173-ac-
yl)-
L
-aspartic acid (4a): Yield 54.2%, mp 168–169 8C; 1H NMR
100
101
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(300 MHz, DMSO-d6):
d
12.10 (s, 2H, 2Â CO2H), 9.65 (s, 1H, 10-H),
9.38 (s, 1H, 5-H), 8.88 (s, 1H, 20-H), 8.20 (m, 1H, CONH), 6.02 (q, 1H,
J = 6.8 Hz, 31-H), 5.22 (d, 1H, J = 21.0 Hz, 132-Hb), 5.10 (d, 1H,
J = 21.0 Hz, 132-Ha), 4.56–4.59 (m, 1H, 17-H), 4.28–4.32 (m, 1H, 18-
H), 3.82 (m, 1H, CONHCHCO), 3.69 (q, 2H, J = 7.2 Hz, 81-CH2), 3.62 (s,
3H, 12-CH3), 3.42 (s, 3H, 31-OCH3), 3.35 (s, 3H, 7-CH3), 3.17 (s, 3H, 2-
CH3), 2.22–2.03 (m, 6H, 172-CH2 + 171-CH2 + CONHCHCH2), 2.02 (d,
3H, J = 6.8 Hz, 32-CH3), 1.76 (d, 3H, J = 7.2 Hz, 18-CH3), 1.59 (t, 3H,
J = 7.2 Hz, 82-CH3), À2.04 (s, 1H, NH); MS (ESI+) m/z: 682.57 [M+H]+
(100%); Anal. Calcd. for C38H43N5O7:C 66.94, H 6.36, N 10.27; Found:
C 67.14, H 6.31, N 10.33.
57
2.2. Chemical synthesis
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Pyropheophorbide-a (2): A suspension of 1 (2.0 g, 3.38 mmol)
in HOAc (100 mL) was refluxing under an atmosphere of N2 for 4 h.
The reaction mixture was poured into H2O (1 L) and then extracted
with CH2Cl2. The organic layer was washed with water, dried over
anhydrous Na2SO4, and evaporated. The residue was purified on a
silica gel column (CH2Cl2:CH3COCH3:CH3OH:HCO2H = 60:1:1:0.1
as eluent) to obtain 2 (1.5 g, 83.1%) as bright black solid. Physical
and spectroscopic characterization data of compound 2 was given
in Supporting information.
Pyropheophorbide-a ether derivatives (3a–3e): A suspension of
2 (0.5 g, 0.94 mmol) in 33% HBr–HOAc (50 mL) was stirred
overnight at room temperature and then evaporated. The residue
was added dissolved in CH2Cl2 (50 mL). K2CO3 (1.0 g) and the
appropriate alcohol donors (10 mL) were then added and allowed
to stir at room temperature for 2 h. The reaction mixture was
added H2O (300 mL) and then extracted with CH2Cl2. The organic
layer was washed with water, dried over anhydrous Na2SO4, and
evaporated. The residue was purified on a silica gel column
(CH2Cl2:CH3COCH3:CH3OH:HCO2H = 80:1:1:0.1 as eluent) to give
compounds 3a–3e as bright black solid in yield of 42.3–69.0%.
Physical and spectroscopic characterization data of compounds
3a–3e were given in Supporting Information.
N-(3-Devinyl-3-(1-propoxy)ethyl-pyropheophorbide-a-173-ac-
yl)-
L
-aspartic acid (4b): Yield 55.8%, mp > 300 8C; 1H NMR
(300 MHz, DMSO-d6):
d
9.80 (1H, splitted s, 10-H), 9.76 (s, 1H, 5-
H),8.83(s, 1H,20-H),7.95(m, 1H,CONH), 6.0(q, 1H, J = 6.8 Hz, 31-H),
5.23 (d, 1H, J = 21.0 Hz, 132-Hb), 5.09 (d, 1H, J = 21.0 Hz, 132-Ha),
4.51–4.58 (m, 1H, 17-H), 4.28 (m, 1H, 18-H), 3.74 (m, 1H,
CONHCHCO), 3.70 (q, 2H, J = 7.3 Hz, 81-CH2), 3.63 (s, 3H, 12-CH3),
3.49 (m, 2H, 31-OCH2), 3.39 (s, 3H, 7-CH3), 3.22 (s, 3H, 2-CH3), 2.12–
2.30 (m, 6H, 172-CH2 + 171-CH2 + CONHCHCH2), 2.02 (d, 3H,
J = 6.8 Hz, 32-CH3), 1.76 (d, 3H, J = 7.6 Hz, 18-CH3), 1.63 (t, 3H,
J = 7.3 Hz, 82-CH3), 0.82–0.93 (m, 5H, 31-OCH2CH2CH3), À1.98 (s, 1H,
NH); MS (ESI+) m/z: 710.56 [M+H]+ (100%); Anal. Calcd. for
C
40H47N5O7: C 67.68, H 6.67, N 9.87; Found: C 67.89, H 6.61, N 9.92.
N-(3-Devinyl-3-(1-pentyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-aspartic acid (4c): Yield 60.1%, mp > 300 8C; 1H NMR
(300 MHz, DMSO-d6):
d
9.80 (splitted s, 1H, 10-H), 9.75 (s, 1H, 5-H),
Amino acid conjugates of pyropheophorbide-a ethers (4a–4j):
To a solution of compounds 3a–3e (0.40 mmol) in anhydrous
8.83 (s, 1H, 20-H), 7.95 (m, 1H, CONH), 5.98 (q, 1H, J = 6.8 Hz, 31-H),
5.23 (d, 1H, J = 18.0 Hz, 132-Hb), 5.09 (d, 1H, J = 18.0 Hz, 132-Ha),
Scheme 1. Synthetic route for the titled compounds 4a–4j. Reagents and conditions: (a) cond. aqueous HCl–Et2O, 0–5 8C, 30 min; (b) HOAc, reflux, 4 h; (c) 33% HBr–HOAc,
24 h; (d) alcohol, CH2Cl2, K2CO3, 2 h; Alcohol donors: R = CH3 (3a), n-C3H7 (3b), n-C5H11 (3c), n-C6H13 (3d), n-C8H17 (3e); (e)
-Asp(OBut)2 or -Glu(OBut)2, EDCI, HOBt, DIPEA,
L
L
CH2Cl2, r.t., 12 h; (f) CH2Cl2–TFA (1:1), r.t., 2 h. Alcohol donors and amino acid residues: m = 1 and R = CH3 (4a), n-C3H7 (4b), n-C5H11 (4c), n-C6H13 (4d), n-C8H17 (4e); m = 2 and
R = CH3 (4f), n-C3H7 (4g), n-C5H11 (4h), n-C6H13 (4i), n-C8H17 (4j).
Please cite this article in press as: Z. Meng, et al., Amino acid derivatives of pyropheophorbide-a ethers as photosensitizer: Synthesis and
photodynamic activity, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.019

G Model
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4.59–4.56 (m, 1H, 17-H), 4.31–4.28 (m, 1H, 18-H), 3.74 (m, 1H,
CONHCHCO), 3.71 (q, 2H, J = 7.7 Hz, 81-CH2), 3.62 (s, 3H, 12-CH3),
3.46–3.51 (m, 2H, 31-OCH2), 3.38 (s, 3H, 7-CH3), 3.22 (s, 3H, 2-CH3),
2.33–2.12 (m, 6H, 172-CH2 + 171-CH2 + CONHCHCH2), 2.02 (d, 3H,
J = 6.8 Hz, 32-CH3), 1.76 (d, 3H, J = 6.6 Hz, 18-CH3), 1.63 (t, 3H,
J = 7.7 Hz, 82-CH3), 1.25 (m, 4H, 31-OCH2(CH2)2C2H5), 0.72–0.86
(m, 5H, O(CH2)3CH2CH3), À1.98 (s, 1H, NH); MS (ESI+) m/z:
738.51 [M+H]+ (100%); Anal. Calcd. for C42H51N5O7:C 68.36, H 6.97,
N 9.49; Found: C 68.58, H 6.92, N 9.56.
N-(3-Devinyl-3-(1-pentyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
-glutamic acid (4h): Yield 53.5%, mp 180–182 8C; 1H NMR 197
(300 MHz, DMSO-d6):
12.53 (s, 2H, 2Â CO2H), 9.81 (s, 1H, 10-H), 198
196
L
d
9.75 (s, 1H, 5-H), 8.84 (s, 1H, 20-H), 8.10 (m, 1H, CONH), 6.00 (q, 1H, 199
J = 6.0 Hz, 31-H), 5.22 (d, 1H, J = 18.0 Hz, 132-Hb), 5.09 (d, 1H, 200
J = 18.0 Hz, 132-Ha), 4.56 (m, 1H, 17-H), 4.32 (m, 1H, 18-H), 4.18– 201
4.20 (m, 1H, CONHCHCO), 3.71 (q, 2H, J = 8.3 Hz, 81-CH2), 3.62 (s, 202
3H, 12-CH3), 3.48–3.50 (m, 2H, 31-OCH2), 3.39 (s, 3H, 7-CH3), 3.21 203
(s, 3H, 2-CH3), 2.33–2.10 (m, 6H, 172-CH2 + 171-CH2 + CH2CO2H), 204
2.01 (d, 3H, J = 6.0 Hz, 32-CH3), 1.76 (d, 3H, J = 6.8 Hz, 18-CH3), 1.63 205
(t, 3H, J = 8.3 Hz, 82-CH3), 1.20 (m, 8H, CONHCH(CO2H)CH2CH2- 206
CO2H + 31-OCH2(CH2)3CH3), 0.90 (t, 3H, J = 6.8 Hz, O(CH2)4CH3), 207
À1.98 (s, 1H, NH); MS (ESI+) m/z: 752.99 [M+H]+ (100%). Anal. 208
Calcd. for C43H53N5O7:C 68.69, H 7.10, N 9.31; Found: C 69.81, H 209
N-(3-Devinyl-3-(1-hexyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-aspartic acid (4d): Yield 51.6%, mp > 300 8C; 1H NMR
(300 MHz, DMSO-d6):
d
9.80 (s, 1H, 10-H), 9.75 (s, 1H, 5-H), 8.81 (s,
1H, 20-H), 8.05 (m, 1H, CONH),6.21 (q, 1H, J = 6.8 Hz, 31-H), 5.20 (d,
1H, J = 18.0 Hz, 132-Hb), 5.05 (d, 1H, J = 18.0 Hz, 132-Ha), 4.55 (m,
1H, 17-H), 4.26 (m, 1H, 18-H), 4.12 (m, 1H, CONHCHCO), 3.71 (q,
2H, J = 7.5 Hz, 81-CH2), 3.62 (s, 3H, 12-CH3), 3.49 (m, 2H, 31-OCH2),
3.39 (s, 3H, 7-CH3), 3.22 (s, 3H, 2-CH3), 2.12–2.33 (m, 6H, 172-
CH2 + 171-CH2 + CONHCHCH2), 2.02 (d, 3H, J = 6.8 Hz, 32-CH3), 1.76
(d, 3H, J = 6.6 Hz 18-CH3), 1.63 (t, 3H, J = 7.5 Hz, 82-CH3), 1.27 (m,
6H, 31-OCH2(CH2)3C2H5), 0.80–0.90 (m, 5H, 31-O(CH2)4CH2CH3);
MS (ESI+) m/z: 752.53 [M+H]+ (100%). Anal. Calcd. for C43H53N5O7:C
68.69, H 7.10, N 9.31; Found: C 68.89, H 7.05, N 9.38.
7.05, N 9.37.
210
211
N-(3-Devinyl-3-(1-hexyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-glutamic acid (4i): Yield 69.3%, mp 184–185 8C; 1H NMR 212
(300 MHz, DMSO-d6):
d
12.29 (s, 2H, 2Â CO2H), 9.79 (s, 1H, 10-H), 213
9.71 (s, 1H, 5-H), 8.83 (splitted s, 1H, 20-H), 8.14 (m, 1H, CONH), 214
5.95 (q, 1H, J = 5.7 Hz, 31-H), 5.21 (d, 1H, J = 18.0 Hz, 132-Hb), 5.09 215
(d, 1H, J = 18.0 Hz, 132-Ha), 4.56 (m, 1H, 17-H), 4.32 (m, 1H, 18-H), 216
4.22 (m, 1H, CONHCHCO), 3.69 (q, 2H, J = 7.3 Hz, 81-CH2), 3.60 (s, 217
3H, 12-CH3), 3.42 (m, 2H, 31-OCH2), 3.35 (s, 3H, 7-CH3), 3.20 (s, 3H, 218
2-CH3), 2.23–2.11 (m, 6H, 172-CH2 + 171-CH2 + CH2CO2H), 2.02 (d, 219
3H, J = 5.7 Hz, 32-CH3), 1.76 (d, 3H, J = 6.5 Hz, 18-CH3), 1.61 (t, 3H, 220
J = 7.3 Hz, 82-CH3), 1.11–1.28 (m, 10H, CONHCH(CO2H)CH2CH2- 221
CO2H + 31-OCH2(CH2)4CH3), 0.81 (t, 3H, J = 5.4 Hz, 31-O(CH2)5CH3), 222
À1.97 (1H, s, NH); MS (ESI+) m/z: 766.26 [M+H]+ (100%). Anal. 223
Calcd. for C44H55N5O7:C 69.00, H 7.24, N 9.14; Found: C 69.23, H 224
N-(3-Devinyl-3-(1-octyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-aspartic acid (4e): Yield 53.0%, mp > 300 8C; 1H NMR
(300 MHz, (CD3)2CO):
d
9.87 (s, 1H, 10-H), 9.71 (s, 1H, 5-H), 8.78 (s,
1H, 20-H), 7.45 (m, 1H, CONH), 6.02 (q, 1H, J = 6.8 Hz, 31-H), 5.21 (d,
1H, J = 18.0 Hz, 132-Hb), 5.04 (d, 1H, J = 18.0 Hz, 132-Ha), 4.77 (m,
1H, 17-H), 4.60 (m, 1H, 18-H), 4.38 (m, 1H, CONHCHCO), 3.69 (q,
2H, J = 7.5 Hz 81-CH2), 3.58 (s, 3H, 12-CH3), 3.55 (m, 2H, 31-OCH2),
3.39 (s, 3H, 7-CH3), 3.22 (s, 3H, 2-CH3), 2.31–2.20 (m, 6H, 172-
CH2 + 171-CH2 + CONHCHCH2CO2H), 2.06 (d, 3H, J = 6.8 Hz, 32-
CH3), 1.78 (d, 3H, J = 6.6 Hz, 18-CH3), 1.64 (t, 3H, J = 7.5 Hz, 82-CH3),
1.20 (m, 4H, 31-OCH2(CH2)2C5H11), 0.81–0.82 (m, 8H, 31-
O(CH2)3(CH2)4CH3), 0.63 (t, 3H, J = 7.5 Hz, 31-O(CH2)7CH3), À1.84
(s, 2H, NH Â 2); MS (ESI+) m/z: 780.62 [M+H]+ (100%). Anal. Calcd.
for C45H57N5O7:C 69.30, H 7.37, N 8.98; Found: C 69.49, H 7.29,
N 9.05.
7.17, N 9.20.
225
226
N-(3-Devinyl-3-(1-octyloxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-glutamic acid (4j): Yield 79.3%, mp 180–182 8C; 1H NMR 227
(300 MHz, DMSO-d6):
d
12.58 (s, 2H, 2Â CO2H), 9.79 (s, 1H, 10-H), 228
9.72 (s, 1H, 5-H), 8.82 (s, 1H, 20-H), 7.90 (m, 1H, CONH), 5.95 (q, 1H, 229
J = 6.8 Hz, 31-H), 5.21 (d, 1H, J = 18.0 Hz, 132-Hb), 5.08 (d, 1H, 230
J = 18.0 Hz, 132-Ha), 4.56 (m, 1H, 17-H), 4.30 (m, 1H, 18-H), 4.16 (m, 231
1H, CONHCHCO), 3.69 (q, 2H, J = 7.8 Hz, 81-CH2), 3.60 (s, 3H, 12- 232
CH3), 3.45 (m, 2H, 31-OCH2), 3.35 (s, 3H, 7-CH3), 3.20 (s, 3H, 2-CH3), 233
2.20–2.13 (m, 6H, 172-CH2 + 171-CH2 + CH2CO2H), 2.02 (d, 3H, 234
J = 6.8 Hz, 32-CH3), 1.75 (d, 3H, J = 6.8 Hz, 18-CH3), 1.62 (t, 3H, 235
J = 7.8 Hz, 82-CH3), 0.82–1.37 (m, 17H, CONHCH(CO2H)CH2CH2- 236
CO2H + 31-OCH2(CH2)6CH3), À1.98 (s, 1H, NH); MS (ESI+) m/z: 237
792.63 [M+H]+ (100%). Anal. Calcd. for C46H59N5O7:C 69.58, H 7.49, 238
N-(3-Devinyl-3-(1-methoxy)ethyl-pyropheophorbide-a-173-ac-
yl)-
L
-glutamic acid (4f): Yield 73.3%, mp > 300 8C; 1H NMR
(300 MHz, DMSO-d6):
d
9.70 (s, 2H, 10- and 5-H), 8.85 (s, 1H, 20-
H), 8.15 (m, 1H, CONH), 5.95 (q, 1H, J = 6.6 Hz, 31-H), 5.20 (d, 1H,
J = 18.0 Hz, 132-Hb), 5.07 (d, 1H, J = 18.0 Hz, 132-Ha), 4.56 (m, 1H, 17-
H), 4.30 (m, 1H, 18-H), 4.10 (m, 1H, CONHCHCO), 3.68 (q, 2H,
J = 7.4 Hz,81-CH2), 3.58(s, 3H,12-CH3), 3.47(s, 3H,31-OCH3), 3.40(s,
3H, 7-CH3), 3.21 (s, 3H, 2-CH3), 2.25–2.03 (m, 6H, 172-CH2 + 171-
CH2 + CONHCH(CO2H)CH2CH2CO2H),2.02(d, 3H,J = 6.6 Hz, 32-CH3),
1.77 (d, 3H, J = 6.9 Hz, 18-CH3), 1.61 (t, 3H, J = 7.4 Hz, 82-CH3), 1.20–
1.40 (m, 2H, CONHCH(CO2H)CH2CH2CO2H), À1.99 (s, 1H, NH); MS
(ESI+) m/z: 696.55 [M+H]+ (100%). Anal. Calcd. for C39H45N5O7:C
67.32, H 6.52, N 10.07; Found: C 67.51, H 6.48, N 10.12.
N 8.82; Found: C 69.76, H 7.41, N 8.90.
239
The determination method of in vitro dark toxicity and 240
phototoxicity for target compounds 4a-4j was given in Supporting 241
Information.
242
243
244
3. Results and discussion
3.1. Synthesis
N-(3-Devinyl-3-(1-propoxy)ethyl-pyropheophorbide-a-173-
acyl)-
L
-glutamic acid (4g): Yield 59.5%, mp > 300 8C; 1H NMR
(300 MHz, DMSO-d6):
d
12.38 (s, 2H, 2Â CO2H), 9.80 (splitted s, 1H,
It was designed that pyropheophorbide-a ethers (3a–3e), which 245
was generated via the addition of pyropheophorbide-a (2) with 246
hydrobromic acid followed by substitution with alcohol donors 247
(ROH), coupled with carboxyl-protected amino acids in the 248
presence of condensation agent and catalyst followed by removal 249
of protective group with trifluoroacetic acid (TFA) to yield the 250
10-H), 9.74 (s, 1H, 5-H), 8.84 (s, 1H, 20-H), 8.16 (m, 1H, CONH), 6.0
(q, 1H, J = 6.3 Hz, 31-H), 5.21 (d, 1H, J = 18.0 Hz, 132-Hb), 5.09 (d, 1H,
J = 18.0 Hz, 132-Ha), 4.55 (m, 1H, 17-CH), 4.31 (m, 1H, 18-CH), 4.20
(m, 1H, CONHCHCO), 3.71 (q, 2H, J = 7.7 Hz, 81-CH2), 3.62 (s, 3H,
12-CH3), 3. 49 (m, 2H, 31-OCH2), 3.43 (s, 3H, 7-CH3), 3.21 (s, 3H, 2-
CH3), 2.25–2.07 (m, 6H, 172-CH2 + 171-CH2 + CONHCH(-
CO2H)CH2CH2CO2H), 2.02 (d, 3H, J = 6.3 Hz, 32-CH3), 1.76 (d, 3H,
J = 6.8 Hz, 18-CH3), 1.63 (t, 3H, J = 7.7 Hz, 82-CH3), 1.13–1.38 (m,
4H, CONHCH(CO2H)CH2CH2CO2H + 31-OCH2CH2CH3), 0.90 (t, 3H,
J = 7.5 Hz, 31-O(CH2)2CH3), À1.98 (s, 1H, NH); MS (ESI+) m/z:
724.54 [M+H]+ (40%); MS (ESIÀ) m/z: 722.55 [MÀH]+ (70%),
1445.46 [2MÀH]+ (100%). Anal. Calcd. for C41H49N5O7:C 68.03, H
6.82, N 9.68; Found: C 68.23, H 6.77, N 9.73.
target compounds (4a–4j).
251
Compound 2 was prepared by acid degradation of pheophor- 252
bide-a (1) in refluxing HOAc. Compound 1 was synthesized 253
according to our reported procedure [13]. All of the target amino 254
acid conjugates of pyropheophorbide-a ethers 4a–4j were new 255
compounds and their structures were confirmed by 1H NMR, MS 256
and elemental analysis. The UV–vis spectral data showed that they 257
possessed
more
efficient
absorption
(
e
= 1.7 Â 104
to 258
Please cite this article in press as: Z. Meng, et al., Amino acid derivatives of pyropheophorbide-a ethers as photosensitizer: Synthesis and
photodynamic activity, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.019

G Model
CCLET 3638 1–4
4
Z. Meng et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
Table 1
4. Conclusion
283
UV–vis data of the titled compounds 4a–4j.
In summary, 10 new water-soluble amino acid conjugates of
284
285
286
287
288
289
290
291
292
293
294
295
296
No.
lmax (CH2Cl2, nm) (e
 104 L molÀ1 cmÀ1
)
pyropheophorbide-a ethers (4a–4j) were synthesized and evalu-
ated for their in vitro photodynamic antitumor activity against
HCT116, MDA-MB-231 and MKN45 cells. All compounds exhibited
more potent phototoxicity and lower dark toxicity against all
tested tumor cell lines than BPD-MA. In particular, compounds 4d
and 4j were the two most effective PSs, which showed 7.8- and 9.7-
fold more potent antitumor activity against HCT116 cells
compared to BPD-MA. These results demonstrated that com-
pounds 4d and 4j could be promising PSs for PDT applications
because of their strong absorption at long wavelength, high
phototoxicity, low dark cytotoxicity and good water-solubility, and
worthy of further study.
Soret band
Visible bands
4a
4b
4c
4d
4e
4f
413 (4.8)
415 (6.3)
414 (7.1)
411 (4.1)
415 (11.0)
412 (8.6)
411 (16.0)
411 (34.0)
410 (11.0)
411 (3.8)
507 (0.49)
–
537 (0.43)
536 (0.41)
538 (0.65)
539 (0.36)
537 (0.71)
–
608(0.71)
602 (0.44)
605(0.69)
608 (0.44)
603(0.81)
614 (0.53)
605(1.7)
666 (1.7)
651 (1.7)
660 (2.4)
662 (1.7)
651 (3.1)
665 (1.9)
661 (7.5)
651 (9.8)
662(5.2)
661 (1.7)
507 (0.63)
507 (0.35)
–
507 (0.60)
507 (1.6)
–
4g
4h
4i
541 (1.7)
538 (2.1)
541 (1.2)
540 (0.43)
602 (2.4)
602(1.2)
507 (1.2)
507 (0.42)
4j
605 (0.44)
Table 2
Cytotoxicity (IC50, mmol/L) for the titled compounds against tumor cells.
Acknowledgments
297
Compd.
HCT116
DTa
MDA-MB-231
DTa PTa,b
36.11
MKN45
DTa
PTa,b
PTa,b
This work was supported by the National Natural Science Q2 298
Foundation of China (No. 81172950), the Project of Science and
Technology Commission of Shanghai (Nos. 11431920401 and
11430723201).
299
300
301
4a
10.08
11.82
35.19
15.06
18.01
62.30
11.41
11.99
10.48
11.80
1.95
0.183
0.076
0.061
0.041
0.050
0.126
0.093
0.092
0.063
0.033
0.32
0.297
0.108
0.11
0.09
0.12
0.21
0.17
0.13
0.13
0.10
0.56
32.70
35.35
>100
24.56
>100
>100
37.51
16.24
66.20
81.40
10.75
0.298
0.182
0.16
0.12
0.13
0.15
0.15
0.14
0.13
0.10
0.92
4b
33.06
61.48
38.23
42.03
4c
4d
4e
4f
100
Appendix A. Supplementary data
302
4g
23.69
26.74
21.84
39.73
9.5
4h
4i
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.
019.
303
304
305
4j
BPD-MA
a
Abbreviation: DT, dark toxicity; PT, phototoxicity.
Cells irradiated with the diode laser at 660 nm for a light dose of 10.8 J/cm2.
b
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9.8 Â 104 L molÀ1 cmÀ1) at longer maximum absorption wave-
length ranged from 651 nm to 666 nm than porfimer sodium
(Table 1), indicating their greater tissue penetration [4–6].
262
3.2. In vitro dark cytotoxicity and photodynamic efficacy
263
264
265
266
267
268
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The dark toxicity and phototoxicity of the titled compounds were
screened with the MTT assay against HCT116, MDA-MB-231 and
MKN45 cells using BPD-MA as control (Table 2). In order to realize
parallel control to eliminate the experimental error caused by the
solvent, the tested compounds 4a–4j and BPD-MA were both
prepared into the DMSO-mediated 10% PBS solution. The results
displayed that all compounds exhibited better phototoxicity and less
dark toxicity against three kinds of tumor cell lines than BPD-MA. A
structure-activity relationship analysis revealed that their PDT
antitumor activity, particularly against HCT116 cells, enhanced with
ether carbon chain growth, the one with 6–8 carbon atoms as the
best. In addition, the type of water-soluble amino acid such as
L-
asparticacidor -glutamicacid hadlittleeffect ontheactivity.Among
L
them, compound 4d and 4j exhibited the best in vitro PDT antitumor
efficacy and their IC50 values for HCT116 cell were individually
41 nmol/L and 33 nmol/L, which represented 7.8- and 9.7-fold
increase of antitumor potency compared to BPD-MA, respectively. In
the next in vivo animal antitumor trials, they could be made into
water-soluble sodium salt for intravenous administration.
282
Please cite this article in press as: Z. Meng, et al., Amino acid derivatives of pyropheophorbide-a ethers as photosensitizer: Synthesis and
photodynamic activity, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.019