Design and synthesis of novel water-soluble amino acid derivatives of chlorin p6 ethers as photosensitizer

Article abstract of DOI:10.1016/j.cclet.2018.04.029

Eight new water-soluble amino acid conjugates 6a-h of chlorin p₆ ethers (5a-d) were synthesized and preliminarily investigated for their in vitro PDT antitumor activity and structure-activity relationship (SAR). The results showed that all compounds exhibited much higher phototoxicity against tumor cells than talaporfin. SAR analysis indicated that PDT antitumor effect enhanced with the increase of carbon chain length of alkoxyl ether bonds at 3¹-position, and L-aspartic acid was superior to L-glutamic acid. In particular, the IC₅₀ values of most phototoxic compound 6d were 0.20 μmol/L against A549 cell and 0.41 μmol/L against B16-F10 cell, which individually represented 31- and 24-fold increase of antitumor potency compared to talaporfin, suggesting that it was a promising candidate photosensitizer (PS) for PDT applications due to its strong absorption at long wavelength, high phototoxicity, low dark cytotoxicity and good water-solubility.

Full text of DOI:10.1016/j.cclet.2018.04.029

Accepted Manuscript
Title: Design and synthesis of novel water-soluble amino acid
derivatives of chlorin p6 ethers as photosensitizer
Authors: Xingjie Zhang, Zhi Meng, Zhiqiang Ma, Junhong
Liu, Guiyan Han, Fujia Ma, Ningyang Jia, Zhenyuan Miao,
Wannian Zhang, Chunquan Sheng, Jianzhong Yao
PII:
DOI:
Reference:
S1001-8417(18)30180-3
https://doi.org/10.1016/j.cclet.2018.04.029
CCLET 4525
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
27-3-2018
24-4-2018
24-4-2018
Please cite this article as: Xingjie Zhang, Zhi Meng, Zhiqiang Ma, Junhong
Liu, Guiyan Han, Fujia Ma, Ningyang Jia, Zhenyuan Miao, Wannian Zhang,
Chunquan Sheng, Jianzhong Yao, Design and synthesis of novel water-soluble
amino acid derivatives of chlorin p6 ethers as photosensitizer, Chinese Chemical
Letters https://doi.org/10.1016/j.cclet.2018.04.029
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Communication
Design and synthesis of novel water-soluble amino acid derivatives of chlorin p
6
ethers as
photosensitizer
a,1
a,1
a,1
a
a
b
c
a
Xingjie Zhang , Zhi Meng , Zhiqiang Ma , Junhong Liu , Guiyan Han , Fujia Ma , Ningyang Jia , Zhenyuan Miao , Wannian
a,*
a,*
a,*
Zhang , Chunquan Sheng , Jianzhong Yao
a
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
Department of Pharmacy, 455th Hospital of Chinese People’s Liberation Army, Shanghai 200052, China
b
c
Department of Radiology, Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
Graphical Abstract
6
Eight new water-soluble amino acid derivatives of chlorin p ethers 6a-h were designed and synthesized using purpurin-18 (2) as key intermediate. All target
compounds exhibited better phototoxicity than talaporfin and the most phototoxic compound 6d showed IC50 values of 0.20 μmol/L against A549 cell and 0.41
μmol/L against B16-F10 cell, which represented 31- and 24-fold increase of PDT antitumor efficacy compared to talaporfin
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
6
Eight new water-soluble amino acid conjugates 6a-h of chlorin p ethers (5a-d) were
synthesized and preliminarily investigated for their in vitro PDT antitumor activity and
structure-activity relationship (SAR). The results showed that all compounds exhibited much
higher phototoxicity against tumor cells than talaporfin. SAR analysis indicated that PDT
antitumor effect enhanced with the increase of carbon chain length of alkoxyl ether bonds at 31-
position, and L-aspartic acid was superior to L-glutamic acid. In particular, the IC50 values of
most phototoxic compound 6d were 0.20 mol/L against A549 cell and 0.41 mol/L against
B16-F10 cell, which individually represented 31- and 24-fold increase of antitumor potency
compared to talaporfin, suggesting that it was a promising candidate photosensitizer (PS) for
PDT applications due to its strong absorption at long wavelength, high phototoxicity, low dark
cytotoxicity and good water-solubility.
Available online
Keywords:
Photodynamic therapy (PDT)
Photosensitizer
Silkworm excrement
Chlorin p
Antitumor
6
Photodynamic therapy (PDT) now is an attractive approach to innovative cancer therapy involving combined use of visible light and
a photosensitizer (PS) [1]. PDT relies on the interaction between light and PS in tumor tissues to generate superoxide anions and
radicals (type I reaction) or highly cytotoxic singlet oxygen (type II reaction) with the ultimate formation of reactive oxygen species
(ROS) to inactivate the tumor cells [2].
Porfimer sodium, the first generation of porphyrin-type PS, has achieved enormous clinical efficacy for the treatment of bladder
cancer in the world. It also has suffered from some serious drawbacks such as complex component, poor tissue penetration due to its
-
1
-1
limited maximum absorption wavelength of 630 nm, inefficient absorption ( = 1170 L mol cm ) at 630 nm, and prolonged cutaneous
phototoxicity up to 4-6 weeks after treatment caused by its slow elimination in skin tissues [3].
A great number of so-called the second generation of PSs especially related to chlorins such as chlorophyll-a derivatives etc., are
becoming more and more concerned owing to rapid clearance from tissues and intense absorption in near-infrared region ( 650 nm,
also called “phototherapeutic window”), which are relatively harmless and penetrate deeply in biological tissues [4-6]. Among them,
talaporfin [7], verteporfin (BPD-MA) [8] and temoporfin (m-THPC) [9] were clinically approved for PDT applications (Fig. 1).
—
——
*
Corresponding authors.
E-mail addresses: zhangwnk@hotmail.com (W. Zhang), shengcq@hotmail.com (C. Sheng), yaojz6601@sina.com (J. Yao).
1
These three authors contributed equally to this work.

Fig. 1. Three clinical available chlorin-type photosensitizers.
Chlorin p
it is easily converted into stable purpurin-18 (2) with poor water solubility by its automatically intramolecular dehydration in the
neutral condition. Chlorin p trimethylester (4), which is formed by methylation of 3, has good stability but poor water solubility.
Because introducing amino acid was reported to be an effective strategy to improve the water-solubility and the biological activity of
chlorin- and porphyrin-based derivatives [10-12], we previously synthesized some chlorin p - and pyropheophorbide-a-based water-
6
(3), the one of chlorophyll-a derivatives as chlorin-type PS, has poor stability to hamper its clinical development because
6
6
soluble amino acid derivatives and obtained a candidate PS with a better efficacy than verteporfin [13,14]. Considering that alkoxyl
1
ether derivatives of chlorin at 3 -position exhibited stronger photosensitive activity than parent compound [15], a series of novel water-
6
soluble amino acid conjugates 6a-h of chlorine p ethers (5a-d) were further designed, synthesized and preliminarily investigated their
photodynamic antitumor activity against melanoma B16-F10 and mammary carcinoma cells (Scheme 1).
o
Scheme 1. Synthetic route for the titled compounds 6a-h. Reagents and conditions: (a) cond. aqueous HCl-Et
4.4%; (c) THF-CH
with Et O, dried by Na
(5a), n-C (5b), n-C
Cl , r.t., 12 h; (i) CH Cl -TFA (3:1), r.t., 6 h. Alcohol donors and amino acid residues: n = 1 and R = CH
(6e), n-C (6f), n-C (6g), n-C 11 (6h).
2
O, 0-5 C, 30 min; (b) KOH, i-PrOH-Et
2
O, 12 h,
O, extraction
, r.t., 2.5 h. Alcohol donors: R =
•HCl (n = 2), EDC•HCl, HOBt, DIPEA,
(6a), n-C (6b), n-C (6c), n-C 11 (6d); n =
3
3
OH- aqueous NaOH (0.5 mol/L) (1:4:5, v/v/v), r.t., 1 h; (d) 0.5 mol/L aqueous HCl to adjust pH value to 5-6, dilution with H
SO for 1 h; (e) CH , 83.6% (from c to e); (f) 33% HBr-HOAc, r.t., 36 h; (g) alcohol, CH Cl , K CO
(5c), n-C
2
2
2
4
N
2 2
2
2
2
3
t
t
CH
CH
2
3
H
3 7
H
4 9
5 2 2
H11 (5d); (h) L-(S)-(+)-Asp(OBu ) •HCl (n = 1) or L-(S)-(+)-Glu(OBu )
2
2
2
2
3
H
3 7
4
H
9
5
H
and R = CH
3
3
H
7
H
4 9
5
H
6
As shown in Scheme 1, all intermediates pheophorbide-a (1), purpurin-18 (2) and chlorin p trimethylester (4) were obtained via
acid and base degradation of chlorophyll-a followed by carboxyl methylation according to our previous methodology developed in our
laboratory using crude chlorophyll extracts in Chinese traditional herb named silkworm excrements [16,17]. Briefly, intermediate 1
was got via cond. aqueous HCl degradation of chlorophyll-a in Et
of O gave 2 in 34.4% yield. 13,15-Anhydride ring of 2 was hydrolyzed in the presence of tetrahydrofuran (THF) and CH
NaOH as the base to form unstable chlorin 3, which was rapidly methylated in Et O with CH to give 4 in 83.6% yield from 2.
2
O. Treatment of 1 in Et
2
O with KOH-i-PrOH under an atmosphere
2
3
OH using
2
2 2
N
In this paper, the details of the synthesis of key intermediate 5a-d and target compounds 6a-h from initial intermediate 4 were also
given in Supporting information. Briefly, addition of 4 with 33% HBr in HOAc followed by substitution with excessive alcohol donors
2 3 6 6
(ROH) in the presence of K CO produced chlorin p ether derivatives 3-devinyl-3-(1-(R/S)-alkoxy)ethyl-chlorin p -13,15-
dimethylester (5a-d) in modest yields ranged from 26.7% to 32.7%. Obviously, intermediate 5 consisted of two epimerides of R- and
1
S-configuration at 3 -alkoxyl. High performance liquid chromatography (HPLC) analysis with chiral column showed that the general
peak area ratio of the two epimerides was individually 22.5% vs. 77.5% for 5a, 35.9% vs. 64.1% for 5b, 31.3% vs. 68.7% for 5c, 33.3%
vs. 65.7% for 5d (Figs. S1-S4 in Supporting information). Then, key intermediate 5a-d was each coupled with L-(S)-()-aspartic acid
hydrochlorate or L-(S)-()-glutamic acid hydrochlorate whose carboxyl protected by tert-butyl in the presence of 1-ethyl-3-(3-

dimethylaminopropyl)-carbodiimide hydrochlorate (EDC•HCl), 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine
DIPEA) followed by removal of tert-butyl with trifluoroacetic acid (TFA) to generate the target compounds N-(3-devinyl-3-(1-(R/S)-
(
3
alkoxy)ethyl-chlorin p
p
6
-13,15-dimethylester-17 -acyl)-L-(S)-()-aspartic acid (6a-e) and N-(3-devinyl-3-(1-(R/S)-alkoxy)ethyl-chlorin
3
6
-13,15-dimethyl ester-17 -acyl)- L-(S)-()-glutamic acid (6f-h) in receivable yields ranged from 50.0% to 77.1%. Similarly, target
1
compound 6 also was composed of two anisometric epimerides of R- and S-configuration at 3 -alkoxyl, which possessed characteristic
of dextral rotation (Supporting information), and its general peak area ratio of two epimerides analyzed by HPLC with chiral column
was 35.2% vs. 64.8% for 6c, 35.3% vs. 64.7% for 6d, 41.8% vs. 58.2% for 6g, 48.2% vs. 51.8% for 6h, respectively (Figs.S5-S8 in
Supporting information). It is limited to our technical conditions that a pair of epimers in both 5 and 6 was even failure to complete
effective separation and preparation. In general, the change of minor group configuration has generally little effect on the PDT
antitumor activity as PS belongs to structural nonspecific drugs without specific drug target such as enzymes, receptors and proteins etc.
1
13
The structures of key intermediate 5a-d and target compounds 6a-h were identified by H NMR, C NMR, ESIMS and Elemental
analysis data (Supporting information). In addition, the UV-visible spectral data showed that they possessed more efficient absorption
5
5
-1
-1
(
 = 1.67×10 −2.42×10 L mol cm ) at longer maximum absorption wavelength of 660-662 nm than porfimer sodium ( = 1170 L
-
1
-1
mol cm ) at 630 nm (Table 1), suggesting their greater tissue penetration [4-6].
Table 1
UV-vis data of the synthetic titled compounds.
5
-1
-1
Compd.
λmax (THF, nm) (ε, ×10 L mol cm )
Soret band
397 (6.9)
399 (9.1)
400 (11.8)
400 (13.6)
396 (6.7)
398 (8.8)
399 (11.2)
399 (12.8)
Visible bands
497 (0.52)
499 (0.69)
499 (0.88)
499 (1.18)
498 (0.37)
499 (0.54)
499 (0.73)
499 (1.02)
6
6
6
6
6
6
6
6
a
b
c
d
e
f
525 (0.23)
543 (0.21)
544 (0.50)
546 (0.75)
527 (0.21)
542 (0.19)
545 (0.42)
544 (0.67)
608(0.23)
578 (0.27)
577 (0.38)
576 (0.54)
606 (0.21)
576 (0.25)
576 (0.33)
578 (0.48)
662 (1.91)
661 (2.08)
661 (2.23)
662 (2.42)
661 (1.67)
660 (1.85)
661 (2.02)
660 (2.17)
g
h
Table 2
Cytotoxicity (IC50, μmol/L) for the titled compounds against tumor cells
Compd.
A549
DT
B16-F10
DT
a
b
a
b
PT
PT
6
6
6
6
6
6
6
6
a
b
c
d
e
f
165.91
151.60
148.10
142.30
138.43
131.39
130.84
127.64
181.20
0.78
0.33
0.27
0.20
0.93
0.52
0.39
0.36
6.21
> 300
> 300
249.50
225.10
> 300
116.30
67.13
1.69
0.58
0.47
0.41
1.98
0.83
0.55
0.54
9.95
g
h
61.69
215.40
Talaporfin
a
Abbreviation: DT, dark toxicity; PT, phototoxicity.
Cells received irradiation with the diode laser at 660 nm for a light dose of 10 J/cm .
b
2
3
1
In order to evaluate the effect of the introduced different amino acid moiety at 17 -carboxyl and alkoxyl at 3 -alkoxyl on the growth
inhibitory activity of the target compounds against human cancer cell lines and to clarify the SAR, the dark toxicity and phototoxicity
of all the target compounds 6a-h were measured by the CCK-8 assay against human mammary carcinoma and murine melanoma B16-
F10 cells using talaporfin as positive control (Supporting information). To eliminate the experimental error caused by solvent, all
tested compounds and talaporfin were both made into water-soluble sodium salt. As shown in Table 2, all compounds exhibited better
phototoxicity and considerable dark toxicity against two tested tumor cell lines compared to talaporfin. SAR analysis showed that their
1
PDT antitumor activity enhanced with the increase of carbon chain length of alkoxyl ether bonds at 3 -position, and L-aspartic acid was
superior to L-glutamic acid. Among them, compound 6d exhibited the best in vitro PDT antitumor efficacy and its IC50 values against
A549 and B16-F10 cells were individually 0.20 μmol/L and 0.41 μmol/L, which represented 31- and 24-fold increase of antitumor
potency compared to talaporfin, respectively.
In summary, 8 new water-soluble amino acid conjugates 6a-h of 3 -alkoxyl ethers (5a-d) for chlorin p
1
6
trimethylester (4) were
synthesized and evaluated for their preliminary in vitro photodynamic antitumor activity against A549 and B16-F10 cells. All target
compounds exhibited much stronger phototoxicity against tested tumor cell lines than talaporfin. In particular, compounds 6d was the
most effective, which individually showed 31- and 24-fold antitumor potency on A549 and B16-F10 cells compared to talaporfin. As a
result, compound 6d represents a promising PS for PDT applications owing to its strong absorption at long wavelength, high
phototoxicity, low dark cytotoxicity and good water-solubility. Moreover, its more extensive and deeper physical and biological study
including singlet oxygen quantum yield, in vivo PDT antitumor efficacy, subcellular localization and tumor cell apoptosis detection etc.
are ongoing.
Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (Nos. 81172950 and 81671739), and the Project
of Science and Technology Commission of Shanghai (No. 11431920401) and the College Students’ Innovation Ability Training
Project of Second Military Medical University (No. MS2017040).
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