Synthesis and evaluation of new 5-aminolevulinic acid derivatives as prodrugs of protoporphyrin for photodynamic therapy

Article abstract of DOI:10.1039/c7pp00203c

Protoporphyrin IX (PpIX) is used as a photosensitizer in the photodynamic diagnosis (PDD) and photodynamic therapy (PDT) of cancer and is synthesized intracellularly from 5-aminolevulinic acid (5-ALA) precursors. Thirteen novel 5-ALA derivatives were designed and synthesized appropriately with tailored hydrophilicity and lipophilicity. The generation of PpIX was detected and their antitumor activity in vitro and in vivo was also investigated. It was shown that compounds 9b-c, 11b-c and 13a displayed a characteristic long wavelength absorption peak at 593 nm after 5 h incubation in mice fibrosarcoma S180 cells. After being exposed to 600 nm laser light irradiation, these compounds can inhibit cell proliferation in S180 cells in vitro. The growth of S180 cell tumors in Kunming mice was significantly inhibited by these compounds in vivo. Among these compounds, 13a has low dark toxicity and high phototoxicity, which makes it an effective and promising prodrug for PDT.

Full text of DOI:10.1039/c7pp00203c

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Synthesis and evaluation of new 5-aminolevulinic
acid derivatives as prodrugs of protoporphyrin for
photodynamic therapy†
Cite this: DOI: 10.1039/c7pp00203c
Wei Zhu, a Ying-Hua Gao,a Chun-Hong Song,a Zhi-Bin Lu,a Tabbisa Namulinda,a
a
Yi-Ping Han,b Yi-Jia Yan,c Lai-Xing Wang*b and Zhi-Long Chen
*
Protoporphyrin IX (PpIX) is used as a photosensitizer in the photodynamic diagnosis (PDD) and photo-
dynamic therapy (PDT) of cancer and is synthesized intracellularly from 5-aminolevulinic acid (5-ALA) pre-
cursors. Thirteen novel 5-ALA derivatives were designed and synthesized appropriately with tailored
hydrophilicity and lipophilicity. The generation of PpIX was detected and their antitumor activity in vitro
and in vivo was also investigated. It was shown that compounds 9b–c, 11b–c and 13a displayed a charac-
teristic long wavelength absorption peak at 593 nm after 5 h incubation in mice fibrosarcoma S180 cells.
After being exposed to 600 nm laser light irradiation, these compounds can inhibit cell proliferation in
S180 cells in vitro. The growth of S180 cell tumors in Kunming mice was significantly inhibited by these
compounds in vivo. Among these compounds, 13a has low dark toxicity and high phototoxicity, which
makes it an effective and promising prodrug for PDT.
Received 2nd June 2017,
Accepted 22nd August 2017
DOI: 10.1039/c7pp00203c
rsc.li/pps
Introduction
The photodynamic therapy (PDT) of cancer is based on the
administration of a photosensitizer with tumor-localizing pro-
perties, and subsequent irradiation with light of an appropri-
ate wavelength leading to selective damage to the treated
tissue.1–3 5-Aminolevulinic acid (5-ALA) is a natural amino acid
biosynthesized not only in animals, but also in plant mito-
chondria. Together with light or laser irradiation, it is used as
a photosensitizer precursor as part of fluorescence diagnosis
(FD) and photodynamic therapy (PDT) to identify/kill tumor
cells, resulting in a new strategy for cancer diagnosis and
therapy.4,5 In mammalian cells, 5-ALA is converted to proto-
porphyrin IX (PpIX), which is the precursor of hemin and a
potent photosensitizer (Fig. 1).6,7 Meanwhile, in most clinical
and preclinical studies, it has been proved that systemic or
topical application of 5-ALA is capable to temporarily increase
the concentration of PpIX in the target tissues.8
Fig. 1 General principle of 5-aminolevulinic acid metabolism.
cellular membranes.9,10 To overcome this obstacle, a lot of
compounds have been synthesized to improve the uptake of
5-ALA and its selectivity.11,12 One approach has been adopted,
involving the use of more lipophilic 5-ALA derivatives, such as
alkyl or ethyleneglycol esters, which are potential substrates
for cellular esterases.13–15 As prodrugs 5-aminolevulinic acid
ester derivatives have been widely studied. Methylamino levuli-
nate (MAL) was approved for the treatment of actinic keratosis
(AK) and basal cell carcinoma (BCC) in Europe and Australia
and hexylaminolevulinate (HAL) has been used to improve the
detection of superficial bladder cancer.16 The use of alkyl
esters of 5-ALA results in a nonspecific distribution of 5-ALA in
all cell types, but with an increased PpIX production in
tumour cells.17
A significant drawback of 5-ALA-PDT is the fact that 5-ALA
is a zwitterion at physiological pH resulting in low lipid solubi-
lity and limiting passage through biological barriers such as
aDepartment of Pharmaceutical Science & Technology, College of Chemistry and
Biology, Donghua University, Shanghai 201620, China. E-mail: zlchen1967@qq.com
bShanghai Changhai Hospital, 200433, China. E-mail: wlx920@163.com
cShanghai Xianhui Pharmaceutical Co. Ltd., Shanghai 200433, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7pp00203c
An attractive way to obtain 5-ALA prodrugs that have both
improved physicochemical properties and can selectively
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release ALA in specific cell lines is to incorporate 5-ALA into a
short peptide derivative.18,19 Following this approach, upon
cellular uptake, 5-ALA release is mediated by the action of cyto-
plasmic esterases and/or proteases, and it may be possible to
design 5-ALA prodrugs that target the disease-dependent levels
of a given enzymatic activity. A previous investigation showed
that the general structure Ac-Xaa-ALA-OR (Xaa is L-Phe, L-Leu)
was the most effective structure to deliver 5-ALA to cells among
a series of derivatives, with an enhancement of PpIX and PDT
effectiveness.20–22
Scheme 3 Reagents and reaction conditions: (i) LiOH, THF, rt, 1 h.
In this context, the syntheses of thirteen new 5-ALA deriva-
tives are described. Their chemical characterization, photo-
physical properties, and photodynamic activities in vitro and
in vivo are reported.
Scheme 4 Reagents and reaction conditions: (i) Succinic acid, HOBt,
EDCI, DIPEA, rt, 6 h.
inducing the production of high levels of PpIX. Following the
same synthetic method, the two carboxyls of succinic acid
were linked with two molecular benzyl 5-ALA to obtain 7a
(Fig. S6†) (Scheme 4).
Results and discussion
Synthesis and characterization
According to the previous literature,29 Cbz-protected amino
acids and Boc-protected amino acids were used for the con-
densation reaction to prepare 9a–c (Fig. S7–S9†) (Scheme 5). It
was found that the deprotection of the Cbz-group was more
difficult than that of the Boc-group. So Boc-protected amino
acids (11a–c) were synthesized (Scheme 6) (Fig. S10–S12†), and
then 11a was selected to give the mono-deprotected peptide
12a as trifluoroacetate in good yield. Without purification, the
acetylation of 12a directly provides product 13a as white
powder (Fig. S13†) (Scheme 7).
Synthesis of the ALA esters. The desired 5-ALA esters were
obtained according to the method previously described.23 The
esters (3a–d) derived from 5-aminolevulinic acid (1), and
methanol, 1-propanol, 1-hexanol, and benzyl alcohol (2a–d)
were obtained in excellent yields (Scheme 1). In the process of
preparation, the major problem was the removal of the corres-
ponding alcohol used in excess, especially high boiling point
and water-insoluble alcohol.24 The characterization of com-
pounds was entirely in accordance with the previous
literature.25
Synthesis of the amide derivatives of N-terminal modified
5-ALA. The condensation of 5-aminolevulinate esters (3a–d),
and phenylpropionic acid, propionic acid, and benzoic acid
(4a–c) to obtain the analogues (5a–d) was straightforward
(Scheme 2).26 At room temperature, 5a was hydrolyzed with
LiOH solution to give 6a (Scheme 3). Compounds 5a–d and 6a
were both purified using column chromatography and charac-
terized (Fig. S1–S5†). Previous literature studies27,28 reported
that the 5-ALA derivatives formed from three molecular 5-ALA
esterification and 5-ALA-dendrimers were both capable of
Scheme 5 Reagents and reaction conditions: (i) HOBt, EDCI, DIPEA, rt,
5 h.
Scheme 1 Reagents and reaction conditions: (i) ROH, SOCl2, 30 °C–
70 °C, 2 h–5 h, 75%–96%.
Scheme 6 Reagents and reaction conditions: (i) HOBt, EDCI, DIPEA, rt,
6 h.
Scheme 2 Reagents and reaction conditions: (i) Acids (4a–c), HOBt,
Scheme 7 Reagents and reaction conditions: (i) TFA, rt, 1 h; (ii) acetic
EDCI, DIPEA, rt, 5 h.
anhydride, TEA, 20 h.
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UV-visible absorption and fluorescence spectra
5-ALA and its derivatives are frequently used as photosensiti-
zers for PDT, although they are not sensitizers themselves.30
However, after they are transformed and metabolized into
PpIX in vivo, the UV-visible absorption and fluorescence
spectra can be obtained.31 The production of PpIX after incu-
bation with ALA and its derivatives was evaluated in mice
fibrosarcoma S180 cells, and the results of these experiments
are summarized in Table 1. After 5 h of incubation, five com-
pounds (9b, 9c, 11b, 11c, and 13a) displayed photosensitivity
characteristics compared to equimolar ALA, which had the
characteristic Soret and Q-band absorption at 405 nm (Soret)
and 593 nm (Q-band), respectively (Fig. 2a). Meanwhile, when
excited at 405 nm, these compounds showed the strongest
emission peaks at 635 nm similar to equimolar ALA (Fig. 2b).
However, UV-visible absorption and fluorescence were not
detected in a parallel series when S180 cells were exposed to
the dipeptides 5a, 5b, 5c, 5d, 6a, 7a, 9a, and 11a (data not
shown). So it can be speculated that in molecules 5a, 5b, 5c,
5d, 6a, 7a, 9a, and 11a, the 5-aminolevulinic ester groups are
Fig. 3 Dark toxicity and phototoxicity of ALA and its derivatives. (a)
Dark toxicity of different concentrations assayed. (b) Light dose depen-
dence effect on the cell viability after incubation with 0.02 mM of ALA
or its derivatives for 19 h. Cell survival is expressed as a percentage of
the control nonirradiated and exposed to ALA or its derivatives. Data
correspond to mean values SD from three different experiments.
much easier to be metabolized and decomposed by enzymes
in vivo before 5-aminolevulinic acid was released to form PpIX
compared with molecules 9b, 9c, 11b, 11c, and 13a.
Cytotoxicity on mice fibrosarcoma S180 cells
The effect of ALA and its derivatives on the viability of cultured
S180 cells was evaluated by using the MTT assay. As shown in
Fig. 3a, the dark cytotoxicities of 13a, 11c, 9c, 11b, and 9b
were visibly higher than that of ALA. When the concentration
was low (0.01 mM and 0.02 mM), the cell viabilities of 13a,
11c, 9c, 11b, and 9b were all more than 80%. However, with
the concentration of prodrugs increased to 0.05 mM, 0.1 mM
and 0.2 mM, the cell viability decreased gradually to about
20% (Fig. 3a). Among these five compounds, 13a showed the
lowest dark-toxicity. After irradiation with laser light, a signifi-
cant cytotoxicity was detected. At low concentration (0.02 mM),
the cell viability of 13a was lowest, upon varying the light dose
(Fig. 3b). With the increase of light dose, the cell viability of
13a significantly decreased, but ALA, 11b, 11c, 9c and 9b had
no obvious change.
Table 1 Detectability of PpIX from N-modified 5-ALA derivatives
Detectability
Code
R1
R
of PpIXa
5a
5b
5c
5d
6a
7a
9a
9b
9c
11a
11b
11c
13a
C6H5CH2CH2–
CH3CH2–
C6H5–
CH3CH2–
–CH3
−
−
−
−
−
−
−
+
+
−
+
+
+
–CH2(CH2)4CH3
–CH2CH2CH3
–CH2CH2CH3
–H
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
C6H5CH2CH2–
Bn-ALA-succinyl–
C6H5-CH2-OCONH-Gly–
C6H5-CH2-OCONH-Leu–
C6H5-CH2-OCONH-Phe–
(CH3)3C-OCONH-Gly–
(CH3)3C-OCONH-Leu–
(CH3)3C-OCONH-Phe–
CH3CO-Phe–
In vivo photodynamic antitumor potency
The PDT antitumor efficacy of 5-ALA derivatives (9b–9c, 11b–c,
13a) was evaluated in S180 tumor bearing Kunming mice. By
comparing the tumor weight in different groups, the inhi-
bition rates of tumor growth could be calculated. When tumor
sizes had reached 5–7 mm in diameter, the mice were given
a “+” means PpIX is detectable. “−” means PpIX is undetectable.
intravenous injection via the tail vein at a dose of 15 mg kg−1
.
After 3 h incubation, the tumor site was irradiated with laser
light (600 nm, 180 J cm−2, 250 mW cm−2). After five days of
administration, treatment effects became significant. Although
compounds 11b, 11c, 9b, and 9c can induce the production of
PpIX in vivo, their dark toxicity resulted in tumor fester and
mice death (Fig. S14–17†). Among these compounds, 13a had
the most medicinal properties with low dark toxicity and high
phototoxicity. The volume of tumors in three sets of parallel
control groups was larger than that in the 13a-PDT group. The
volume growth curves of tumors are provided in Fig. 4a, the
tumor volume increased about 10-fold for 14 days in the
control group. Tumor images are shown in Fig. 4b, and the
tumors began to swell after 1 day post PDT. The tumors
Fig. 2 (a) UV-vis absorption spectrum registered after exposure of S180
cells to 2 mM ALA and its derivatives. The results obtained after 5 h of
incubation are shown. (b) Fluorescence spectrum obtained after incu-
bation of S180 cells to 2 mM ALA and its derivatives for 5 h, excitation
wavelength is 405 nm.
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75 mL of 1 M citric acid, 10% NaHCO3, and NaCl saturated
water. The organic layer was dried over MgSO4, and the solvent
was removed under reduced pressure. The residue was purified
by flash chromatography (CH2Cl2 : MeOH = 50 : 1) to afford the
pure product as a white solid (5a–d).
Methyl 4-oxo-5-(3-phenylpropanamido)pentanoate (5a).
66.2% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.21
(t, J = 5.7 Hz, 1H), 7.30–7.15 (m, 5H), 3.93 (d, J = 5.7 Hz, 2H),
3.58 (s, 3H), 2.82 (dd, J = 8.8, 6.8 Hz, 2H), 2.64 (t, J = 6.5 Hz,
2H), 2.47 (td, J = 6.9, 4.2 Hz, 4H). 13C NMR (100 MHz, DMSO-
d6) δ ppm: 206.06, 173.09, 172.21, 141.70, 128.72, 128.67,
126.33, 51.84, 48.81, 37.08, 34.28, 31.44, 27.61. HRMS
(MALDI-TOF) calcd for C15H20O4N [M + H]+ 278.1387, found
278.1388.
Fig. 4 (a) Tumor volume at different times after 13a-PDT. The tumors
in control groups continued to grow and were significantly larger than
in the PDT-treated group after 14 days post-treatment. (b) Images of
mice bearing S180 tumors. Before and following PDT for 13a and ALA at
different times and mice with saline were set as the control.
Hexyl 4-oxo-5-propionamidopentanoate (5b). 65.3% yield.
1H NMR (400 MHz, CDCl3) δ ppm: 6.24 (s, 1H), 4.24 (t, J =
3.1 Hz, 2H), 4.08 (td, J = 6.8, 2.0 Hz, 2H), 2.80–2.73 (m, 2H),
2.69 (d, J = 6.4 Hz, 2H), 2.35–2.24 (m, 2H), 1.62 (q, J = 6.8 Hz,
2H), 1.33 (d, J = 9.7 Hz, 6H), 1.19 (td, J = 7.7, 2.1 Hz, 3H),
0.94–0.81 (m, 3H). 13C NMR (100 MHz, CDCl3) δ ppm: 204.11,
173.83, 172.46, 65.09, 49.24, 34.62, 31.40, 29.36, 28.51, 27.84,
25.53, 22.53, 14.00, 9.69. HRMS (MALDI-TOF) calcd for
C14H26O4N [M + H]+ 272.1856, found 272.1857.
became dark, necrotic and a scar formed at 3 days post PDT.
Then the scar of tumors fell off and normal healthy skin was
reconstructed 14 days post PDT.
Experimental
Materials and instrumentation
Propyl 5-benzamido-4-oxopentanoate (5c). 45.7% yield.
1H NMR (400 MHz, CDCl3) δ ppm: 7.87–7.81 (m, 2H),
7.57–7.51 (m, 1H), 7.47 (dd, J = 8.2, 6.7 Hz, 2H), 6.95 (s, 1H),
4.45 (d, J = 4.5 Hz, 2H), 4.07 (t, J = 6.7 Hz, 2H), 2.84 (dd, J = 7.4,
5.1 Hz, 2H), 2.74 (dd, J = 7.2, 5.0 Hz, 2H), 1.71–1.64 (m, 2H),
0.96 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ ppm:
204.08, 172.48, 167.28, 133.72, 131.78, 128.61, 127.08, 66.53,
49.70, 34.71, 27.88, 21.93, 10.36. HRMS (MALDI-TOF) calcd for
C15H20O4N [M + H]+ 278.1387, found 278.1387.
Propyl 4-oxo-5-propionamidopentanoate (5d). 67.5% yield.
1H NMR (400 MHz, CDCl3) δ ppm: 6.22 (s, 1H), 4.25 (d, J =
4.6 Hz, 2H), 4.06 (t, J = 6.7 Hz, 2H), 2.77 (dd, J = 7.4, 5.0 Hz,
2H), 2.70 (dd, J = 7.2, 4.8 Hz, 2H), 2.30 (q, J = 7.6 Hz, 2H),
1.66 (s, 2H), 1.20 (t, J = 7.6 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ ppm: 204.06, 173.77, 172.45,
66.51, 49.26, 34.65, 29.39, 27.83, 21.93, 10.36, 9.71. HRMS
(MALDI-TOF) calcd for C11H20O4N [M + H]+ 230.1387, found
230.1388.
All solvents and reagents were obtained from commercial
suppliers and used without further purification unless other-
wise stated. Melting points were determined on a Reichert-
Jung Thermo Galen Kofler block and are uncorrected. 1H NMR
and 13C NMR spectra were recorded on a Bruker 400 MHz
spectrometer. Chemical shifts were reported as δ ppm values
relative to the internal standard tetramethylsilane. ESI-MS
spectra were recorded on a Micromass triple quadrupole mass
spectrometer. HRMS spectra were recorded on a Bruker
Daltonics APEXIII 7.0 tesla FT mass spectrometer (Bruker
Daltonik GmbH, Bremen, Germany), which allowed the deter-
mination of the molecular formula of the [M + H]+, the
[M + Na]+ or the [M + K]+ peak. The solvent was removed by rotary
evaporation under reduced pressure, and silica gel chromato-
graphy was performed using silica gel H (300–400 mesh). The
UV-vis absorption spectrum was recorded on an ultraviolet
visible spectrophotometer (Model V-530, Japan). Fluorescence
spectra were recorded on a fluorescence spectrophotometer
(FluoroMax-4, France). The synthesis of compounds (3a–d) was
performed according to previous reports.
4-Oxo-5-(3-phenylpropanamido)pentanoic acid (6a). To a
solution of 5a (100 mg, 0.36 mmol) in THF (100 ml) was
added 1 M LiOH (1.8 ml, 1.8 mmol) at room temperature
under N2, and the solution was stirred at room temperature for
2 h. After evaporation of the solvent, the residue was dissolved
in water. Then the pH of the mixture was adjusted to 3 with
1 M HCl, and the product was precipitated. Product 6a was
General procedures for the preparation of the peptide
analogues 5a–d via coupling of 5-aminolevulinic acid
esters 3a–d with acids 4a–c
A total of acids (4a–c) (6.1 mmol) in dry DCM (10 mL) were obtained by filtration (73.3 mg, 77.2%). 1H NMR (400 MHz,
activated over 1 h by the addition of HOBt (0.82 g, 6.1 mmol) CDCl3) δ ppm: 7.29 (d, J = 7.7 Hz, 2H), 7.20 (dd, J = 7.6, 5.0 Hz,
and EDCI (1.16 g, 6.1 mmol) at room temperature under N2. 3H), 6.31 (s, 1H), 4.18 (d, J = 4.7 Hz, 2H), 2.96 (t, J = 7.9 Hz,
A solution of 5-ALA esters (3a–d) (5.5 mmol) in DCM (5 mL) 2H), 2.69 (s, 4H), 2.55 (dd, J = 8.9, 6.7 Hz, 2H). 13C NMR
was added, and then DIPEA (2.87 mL, 16.5 mmol) was added. (100 MHz, CDCl3) δ ppm: 204.02, 176.11, 172.60, 140.56,
After 5 h stirring at room temperature, the solvent was evapo- 128.56, 128.31, 126.31, 77.24, 49.23, 37.95, 34.41, 31.48, 27.55.
rated under reduced pressure. The crude material was extracted HRMS (MALDI-TOF) calcd for C14H18O4N [M + H]+ 264.1231,
with 3 × 100 mL of ethyl acetate and washed sequentially with found 264.1230.
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Dibenzyl 5,5′-(succinylbis(azanediyl))bis(4-oxopentanoate) 67.13, 66.72, 56.11, 49.09, 38.47, 34.44, 27.82. HRMS
(7a). To a solution of succinic acid (0.72 g, 6.1 mmol) in dry (MALDI-TOF) calcd for C26H33O6N2 [M + H]+ 469.2334, found
DCM (10 ml) were added HOBt (1.64 g, 12.2 mmol) and EDCI 469.2333.
(2.32 g, 6.1 mmol) and the solution was stirred at room tem-
Benzyl 5-(2-(((benzyloxy)carbonyl)amino)-3-phenyl-
perature for 1 h under N2. A solution of benzyl 5-aminolevulinic propanamido)-4-oxopentanoate (9c). 85.3% yield, 1H NMR
acid hydrochloride (3d) (3.2 g, 12.5 mmol) in DCM (6 mL) was (400 MHz, CDCl3) δ ppm: 7.36 (tt, J = 12.7, 6.3 Hz, 10H),
added and then DIPEA (2.87 mL, 16.5 mmol) was added. After 7.29–7.12 (m, 5H), 6.51 (s, 1H), 5.30 (s, 1H), 5.12 (d, J = 8.6 Hz,
stirring for 6 h, the solvent was evaporated under reduced 4H), 4.50 (d, J = 8.0 Hz, 1H), 4.25–4.03 (m, 2H), 3.11 (d, J =
pressure. The crude material was dissolved with ethyl acetate 6.7 Hz, 2H), 2.71 (s, 4H), 1.65 (s, 2H). 13C NMR (100 MHz,
and washed sequentially with 1 M citric acid (3 × 75 mL), 10% CDCl3) δ ppm: 203.13, 172.16, 170.95, 136.21, 129.25, 128.75,
NaHCO3, and NaCl saturated water. The organic layer was 128.63, 128.56, 128.39, 128.26, 128.22, 128.06, 127.12, 77.26,
dried over MgSO4, and the solvent was evaporated under 67.13, 66.72, 56.11, 49.09, 38.47, 34.44, 27.82. HRMS
reduced pressure. Purification by flash chromatography (MALDI-TOF) calcd for C29H31O6N2 [M + H]+ 503.2176, found
(CH2Cl2 : MeOH = 50 : 1) gave the pure product as a white solid 503.2177.
1
(2.0 g, 63.1%). H NMR (400 MHz, CDCl3) δ ppm: 7.37 (q, J =
General procedures for the preparation of the peptide
analogues 11a–c via coupling of 5-aminolevulinic acid
esters with Boc-protected amino acids 10a–c
6.2, 4.7 Hz, 10H), 6.59 (s, 2H), 5.13 (d, J = 2.2 Hz, 4H), 4.19 (d,
J = 3.9 Hz, 4H), 2.74 (q, J = 4.5 Hz, 8H), 2.62 (s, 4H). 13C NMR
(100 MHz, CDCl3) δ ppm: 203.92, 172.25, 172.14, 135.64,
128.62, 128.36, 128.23, 66.68, 49.30, 34.50, 31.29, 27.82. HRMS A total of Boc-amino acids (2.0 mmol) dissolved in dry DCM
(MALDI-TOF) calcd for C28H33O8N2 [M + H]+ 525.2231, found (10 mL) was activated over 1 h by the addition of HOBt (0.28 g,
525.2231.
2.1 mmol) and EDCI (0.39 g, 2.1 mmol) at room temperature
under N2. A solution of benzyl aminolevulinic acid hydro-
chloride (3d) (0.54 g, 2.1 mmol) in DCM (6 mL) was added and
then DIPEA (1.1 mL, 6.3 mmol) was added. After 6 h stirring at
room temperature, the solvent was removed under reduced
General procedures for the preparation of the peptide
analogues 9a–c via coupling of 5-aminolevulinic acid
esters with Cbz-protected amino acids 8a–c
A total of Cbz-amino acids (2.0 mmol) dissolved in dry DCM pressure. The crude material was dissolved with ethyl acetate
(10 mL) was activated over 1 h by the addition of HOBt (0.28 g, and washed sequentially with 1 M citric acid (3 × 75 mL) and
2.1 mmol) and EDCI (0.39 g, 2.1 mmol) at room temperature 10% NaHCO3 (3 × 50 mL). The organic layer was dried over
under N2. A solution of benzyl 5-aminolevulinic acid hydro- MgSO4, and the solvent was evaporated in vacuo. Purification
chloride (3d) (0.54 g, 2.1 mmol) in DCM (6 mL) was added and by flash chromatography (CH2Cl2 : methanol = 100 : 1) gave the
then DIPEA (1.1 mL, 6.3 mmol) was added. After 5 h stirring at product as a white solid (11a–c).
room temperature, the solvent was removed under reduced
Benzyl 5-(2-((tert-butoxycarbonyl)amino)acetamido)-4-oxo-
pressure. The crude material was extracted with 3 × 100 mL of pentanoate (11a). 80.3% yield, 1H NMR (400 MHz, CDCl3)
ethyl acetate and washed sequentially with 75 mL of 1 M citric δ ppm: 7.43–7.31 (m, 5H), 6.77 (s, 1H), 5.18 (s, 1H), 5.13 (s,
acid, 10% NaHCO3, and NaCl saturated water. The organic 2H), 4.22 (d, J = 4.6 Hz, 2H), 3.86 (d, J = 5.6 Hz, 2H), 2.84–2.66
layer was dried over MgSO4, and the solvent was removed (m, 4H), 1.48 (s, 9H). 13C NMR (100 MHz, CDCl3) δ ppm:
under reduced pressure. Purification by flash chromatography 203.39, 172.10, 169.52, 155.91, 135.64, 128.58, 128.32, 128.19,
(CH2Cl2 : MeOH = 50 : 1) gave the product as a white solid 80.39, 77.21, 66.68, 49.02, 44.22, 34.53, 28.29, 27.87. HRMS
(9a–c).
Benzyl 5-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-oxo- 379.1864.
pentanoate (9a). 75.4% yield, 1H NMR (400 MHz, CDCl3)
Benzyl
(MALDI-TOF) calcd for C19H27O6N2 [M + H]+ 379.1864, found
5-(2-((tert-butoxycarbonyl)amino)-4-methyl-
δ ppm: 7.37 (d, J = 7.2 Hz, 10H), 6.70 (s, 1H), 5.45 (s, 1H), 5.14 pentanamido)-4-oxopentanoate (11b). 67.8% yield, 1H NMR
(d, J = 12.6 Hz, 4H), 4.22 (d, J = 4.3 Hz, 2H), 3.94 (d, J = 5.5 Hz, (400 MHz, CDCl3) δ ppm: 7.37 (q, J = 7.1, 6.5 Hz, 5H), 6.82
2H), 2.75 (s, 4H). 13C NMR (100 MHz, CDCl3) δ ppm: 203.42, (s, 1H), 5.13 (s, 2H), 5.05–4.80 (m, 1H), 4.20 (d, J = 4.2 Hz, 3H),
172.12, 169.03, 156.55, 136.15, 128.59, 128.54, 128.33, 128.22, 2.76 (dt, J = 12.2, 5.3 Hz, 4H), 1.78–1.61 (m, 2H), 1.47 (s, 9H),
128.20, 128.09, 77.21, 67.26, 66.70, 49.04, 44.43, 34.52, 27.88. 0.97 (dd, J = 5.9, 2.8 Hz, 6H). 13C NMR (101 MHz, CDCl3)
HRMS (MALDI-TOF) calcd for C22H25O6N2 [M + H]+ 413.1708, δ ppm: 203.79, 173.05, 172.25, 155.70, 135.66, 128.58, 128.31,
found 413.1707.
Benzyl 5-(2-(((benzyloxy)carbonyl)amino)-4-methyl- 24.72, 23.03, 21.84. HRMS (MALDI-TOF) calcd for C23H35O6N2
pentanamido)-4-oxopentanoate (9b). 77.8% yield, 1H NMR [M + H]+ 435.2490, found 435.2490.
128.19, 79.95, 66.61, 52.97, 49.05, 41.44, 34.45, 28.31, 27.77,
(400 MHz, CDCl3) δ ppm: 7.42–7.33 (m, 10H), 6.72 (s, 1H),
Benzyl 5-(2-((tert-butoxycarbonyl)amino)-3-phenyl-
5.23–5.17 (m, 1H), 5.13 (d, J = 2.2 Hz, 4H), 4.27 (s, 1H), propanamido)-4-oxopentanoate (11c). 85.7% yield, 1H NMR
4.23–4.14 (m, 2H), 2.75 (s, 4H), 1.68–1.66 (m, 2H), 1.55 (d, J = (400 MHz, DMSO-d6) δ ppm: 8.31 (d, J = 5.7 Hz, 1H), 7.46–7.14
9.0 Hz, 1H), 0.96 (d, J = 5.4 Hz, 6H). 13C NMR (100 MHz, (m, 10H), 7.04 (d, J = 8.6 Hz, 1H), 5.08 (s, 2H), 4.20 (s, 1H),
CDCl3) δ ppm: 203.13, 172.16, 170.95, 136.21, 129.25, 128.75, 3.97 (dd, J = 12.9, 5.4 Hz, 2H), 3.04–2.95 (m, 1H), 2.76 (d, J =
128.63, 128.56, 128.39, 128.26, 128.22, 128.06, 127.12, 77.26, 10.8 Hz, 1H), 2.72 (d, J = 7.9 Hz, 2H), 2.56 (d, J = 6.7 Hz, 2H),
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1.29 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ ppm: 206.03, harvested and seeded in 96-well plates at 2 × 104 cells per well.
172.63, 172.54, 155.75, 138.72, 129.65, 128.89, 128.48, 128.44, The cells were allowed to attach to the bottom of the wells for
128.32, 126.63, 78.47, 65.97, 56.16, 48.93, 37.86, 34.30, 28.59, 24 hours before starting the experiment in 5% CO2 at 37 °C.
27.79. HRMS (MALDI-TOF) calcd for C26H33O6N2 [M + H]+ The medium containing ALA and its derivatives (13a, 11c, 11b,
469.2335, found 469.2333.
9c, and 9b) at different concentrations (0.01, 0.02, 0.05, 0.1,
Benzyl 5-(2-acetamido-3-phenylpropanamido)-4-oxopentanoate and 0.2 mM) was administered to cells and uptake was allowed
(13a). The protected amino acid-ALA derivative (11c) (234 mg, for 24 hours to test the dark toxicity. RPMI-1640 medium con-
0.5 mmol) was dissolved in a mixture of TFA (1 mL, 13 mmol) taining drugs was removed and the cells were washed with
and DCM (3 mL) under N2. After the solution was stirred for fresh PBS before irradiation with different light doses (range
1 h at room temperature, TFA was evaporated under high from 1 to 16 J cm−2) using an Nd:YAG laser at 600 nm when
vacuum. The products (12a) were obtained in good purity the phototoxicity was tested at 0.02 mM. The cell viability was
without further purification. A suspension of the crude tri- evaluated by using the MTT colorimetric assay 24 hours after
fluoroacetic acid salt thus prepared in dry DCM (10 mL) was treatment.
cooled to 0 °C under an argon atmosphere and DIPEA
(0.18 mL, 1.0 mmol) was added, followed by acetic anhydride
In vivo experiments
(0.1 mL, 0.98 mmol). The mixture was allowed to attain room
temperature with stirring overnight and then the solvent was
evaporated. The crude product was purified by flash chromato-
graphy (CH2Cl2 : MeOH = 50 : 1). Recrystallization from ethanol
gave a white solid (93.1 mg, 45.4%). 1H NMR (400 MHz,
CDCl3) δ ppm: 7.37 (dq, J = 7.0, 4.0, 2.9 Hz, 7H), 7.31 (d, J =
1.6 Hz, 2H), 7.27–7.19 (m, 4H), 6.54 (s, 1H), 6.17 (d, J = 7.8 Hz,
1H), 5.13 (s, 2H), 4.74 (q, J = 7.2 Hz, 1H), 4.21–3.98 (m, 2H), 3.09
(d, J = 6.9 Hz, 2H), 2.70 (s, 4H), 2.20 (s, 2H), 2.00 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ ppm: 203.24, 172.17, 171.20, 170.20,
136.41, 129.21, 128.63, 128.59, 128.33, 128.19, 127.01, 66.67,
54.30, 49.05, 38.33, 34.39, 27.80, 23.06. HRMS (MALDI-TOF)
calcd for C23H27O5N2 [M + H]+ 411.1915, found 411.1914.
In vivo PDT efficacy. Five-week-old Kunming male mice were
purchased from Shanghai Slack Laboratory Animal Co., Ltd.
The mouse tumor models were set up by the subcutaneous
injection of 5 × 106 S180 cells in the flank. When growing to
an approximate diameter of 10 mm, tumors were excised and
small pieces of the tumor (approximately 1 mm square pieces)
were then implanted subcutaneously into the right dorsal area
of Kunming male mice. When tumor sizes had reached
5–7 mm in diameter after implantation (14–21 days), 18 mice
were randomly divided into three groups: laser radiation
group, ALA (15 mg kg−1) + laser radiation group, and 13a
(15 mg kg−1) + laser radiation group followed 5 h later by laser
radiation. The mice were restrained in rat holders and exposed
to the laser (λ = 600 nm) with a light dose of 180 J cm−2. After
PDT, the mice were observed daily for tumor regrowth or
tumor cure. Visible tumors were measured using two ortho-
gonal measurements L and W (perpendicular to L), and the
volumes were calculated using the formula V = LW2/2 and
recorded.
Photophysical and photochemical measurements
Absorption and emission spectra. The UV-visible absorption
spectrum was recorded on an ultraviolet visible spectro-
photometer (Model V-530, Japan). Fluorescence spectra were
recorded on a fluorescence spectrophotometer (FluoroMax-4,
France). Slits were kept narrow to 1 nm in excitation and 2 nm
in emission. All the measurements were carried out at room
temperature in quartz cuvettes with a path length of 1 cm. All
compounds were dissolved in DMSO, and then wrapped in
castor oil to obtain 2 mM of solution after evaporation. After
5 h incubation in the culture, the sample was obtained
through the added PBS buffer solution.
Human & animal welfare
All experiments were performed in triplicate and the data were
expressed as mean plus and minus the standard error of the
mean. Analysis of variance (ANOVA) and Student’s t-test were
used to determine the statistically significant difference
among different groups when appropriate.
The experiments were carried out in accordance with the
guidelines issued by the Ethical Committee of Donghua
University.
In vitro experiments
Cell line and culture conditions. Mice fibrosarcoma S180
cells were obtained from the Type Culture Collection of the
Chinese Academy of Sciences. All cell culture related reagents
were purchased from Shanghai Ming Rong Bio-Science
Technology Co., Ltd. Cells were cultured in normal RPMI-1640
Conclusions
culture medium. All media were supplemented with 10% In summary, thirteen novel ALA derivatives were synthesized
fetal bovine serum (FBS), 100 units per mL penicillin G and and their photodynamic activities were evaluated in vitro and
100 μg mL−1 streptomycin. All cells were incubated at 37 °C in in vivo. Among these compounds, 5a–5d, 6a, 7a, 9a, and 11a
5% CO2 in a humidified incubator.
are unable to show photosensitivity characteristics, and 13a,
11c, 11b, 9c, and 9b are able to metabolize and induce PpIX.
After 5 h incubation in S180 cells, compounds 9b–c, 11b–c and
MTT cell viability assay
S180 cells were cultured in RPMI-1640 medium with 10% (v/v) 13a display a characteristic long wavelength absorption peak at
FBS, collected by centrifugation for 5 min. The cells were 593 nm, in accordance with PpIX induced by the control group
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Paper
5-ALA. Among them, 13a has the most medicinal properties
which possess suitable dark toxicity and high phototoxicity.
In vivo therapeutic efficacy of PDT using 13a, after being
exposed to 600 nm laser light irradiation, the growth of S180
cell tumors in Kunming mice was significantly inhibited.
Therefore, 13a is a promising antitumor photosensitizer for
photodynamic therapy.
8 P.
Uehlinger,
M.
Zellweger,
G.
Wagnières,
L. Juilleratjeanneret, d. B. H. Van and N. Lange,
5-Aminolevulinic acid and its derivatives: physical chemical
properties and protoporphyrin IX formation in cultured
cells, J. Photochem. Photobiol., B, 2000, 54, 72–80.
9 L. Bourré, F. Giuntini, I. M. Eggleston, M. Wilson and
A. J. Macrobert, Protoporphyrin IX enhancement by
5-aminolaevulinic acid peptide derivatives and the effect of
RNA silencing on intracellular metabolism, Br. J. Cancer,
2009, 100, 723–731.
10 M. J. Dixon, L. Bourre, A. J. MacRobert and I. M. Eggleston,
Novel prodrug approach to photodynamic therapy:
Fmoc solid-phase synthesis of a cell permeable peptide
incorporating 5-aminolaevulinic acid, Bioorg. Med. Chem.
Lett., 2007, 17, 4518–4522.
Conflicts of interest
The authors have no other relevant affiliations or financial
involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript apart from those
disclosed.
11 T. Feuerstein, G. Berkovitch-Luria, A. Nudelman,
A. Rephaeli and Z. Malik, Modulating ALA-PDT efficacy
of mutlidrug resistant MCF-7 breast cancer cells using
ALA prodrug, Photochem. Photobiol. Sci., 2011, 10, 1926–
1933.
Acknowledgements
This work was supported by the National Natural Science 12 G. Berkovitch-Luria, S. Yakobovitch, M. Weitman,
Foundation of China (no. 21372042, 21402236), the
Foundation of Shanghai Government (no. 14431906200,
15XD1523400, 15431904100, 14ZR1439900, 14ZR1439800,
15ZR1439900, 16ZR1400600, 15411960400), the Foundation of
A. Nudelman, G. Rozic, A. Rephaeli and Z. Malik,
A multifunctional 5-aminolevulinic acid derivative induces
erythroid differentiation of K562 human erythroleukemic
cells, Eur. J. Pharm. Sci., 2012, 47, 206–214.
Donghua University (17D110513), and the Foundation of 13 J.-M. Gaullier, K. Berg, Q. Peng, H. Anholt, P. K. Selbo,
Songjiang District (no. 15SJGG45, 16SJGG20).
L.-W. Ma and J. Moan, Use of 5-aminolevulinic acid esters
to improve photodynamic therapy on cells in culture,
Cancer Res., 1997, 57, 1481–1486.
14 G. Berkovitch, D. Doron, A. Nudelman, Z. Malik and
A. Rephaeli, Novel multifunctional acyloxyalkyl ester pro-
drugs of 5-aminolevulinic acid display improved anticancer
activity independent and dependent on photoactivation,
J. Med. Chem., 2008, 51, 7356–7369.
Notes and references
1 D. Kessel, Hematoporphyrin and HPD: photophysics,
photochemistry and phototherapy, Photochem. Photobiol.,
1984, 39, 851–859.
2 P. Agostinis, K. Berg and K. A. Cengel, Photodynamic 15 G. Berkovitch-Luria, M. Weitman, A. Nudelman,
therapy of cancer: an update, CA-Cancer J. Clin., 2011, 61,
250–281.
A. Rephaeli and Z. Malik, Multifunctional 5-aminolevulinic
acid prodrugs activating diverse cell-death pathways, Invest.
New Drugs, 2012, 30, 1028–1038.
3 D. E. Dolmans, D. Fukumura and R. K. Jain, Photodynamic
therapy for cancer, Nat. Rev. Cancer, 2003, 3, 380–387.
4 J. C. Kennedy and R. H. Pottier, New trends in photo-
biology: endogenous protoporphyrin IX, a clinically useful
photosensitizer for photodynamic therapy, J. Photochem.
Photobiol., B, 1992, 14, 275–292.
16 N. Fotinos, M. A. Campo, F. Popowycz, R. Gurny and
N. Lange, 5-aminolevulinic acid derivatives in photomedi-
cine: characteristics, application and perspectives,
Photochem. Photobiol., 2006, 82, 994–1015.
17 J. Webber, D. Kessel and D. Fromm, Side effects and photo-
sensitization of human tissues after aminolevulinic acid,
J. Surg. Res., 1997, 68, 31–37.
5 C. Perotti, H. Fukuda, G. Divenosa, A. J. Macrobert,
A. Batlle and A. Casas, Porphyrin synthesis from ALA
derivatives for photodynamic therapy. In vitro and in vivo 18 A. Casas, A. del C. Batlle, A. Butler, D. Robertson, E. Brown,
studies, Br. J. Cancer, 2004, 90, 1660–1665.
6 Q. Peng, K. Berg, J. Moan, M. Kongshaug and
J. M. Nesland, 5-Aminolevulinic acid-based photodynamic
A. MacRobert and P. Riley, Comparative effect of ALA
derivatives on protoporphyrin IX production in human and
rat skin organ cultures, Br. J. Cancer, 1999, 80, 1525–1532.
therapy: principles and experimental research, Photochem. 19 R. Schneider, L. Tirand, C. Frochot, R. Vanderesse,
Photobiol., 1997, 65, 235–251.
N. Thomas, J. Gravier, F. Guillemin and M. Barberi-Heyob,
Recent improvements in the use of synthetic peptides for a
selective photodynamic therapy, Anti-Cancer Agents Med.
Chem., 2006, 6, 469–488.
7 E. Endlicher, P. Rümmele, F. Hausmann, R. Krieg and
R. Knüchel, Protoporphyrin IX distribution following local
application of 5-aminolevulinic acid and its esterified
derivatives in the tissue layers of the normal rat colon, 20 Y. Berger, L. Ingrassia, R. Neier and L. Juillerat-Jeanneret,
Br. J. Cancer, 2001, 85, 1572–1576.
Evaluation of dipeptide-derivatives of 5-aminolevulinic acid
This journal is © The Royal Society of Chemistry and Owner Societies 2017
Photochem. Photobiol. Sci.

View Article Online
Paper
Photochemical & Photobiological Sciences
as precursors for photosensitizers in photodynamic 26 L. Pare, E. Marcuello, A. Altes, E. del Rio, L. Sedano,
therapy, Bioorg. Med. Chem., 2003, 11, 1343–1351.
21 Y. Berger, A. Greppi, O. Siri, R. Neier and L. Juillerat-
Jeanneret, Ethylene glycol and amino acid derivatives of
5-aminolevulinic acid as new photosensitizing precursors
of protoporphyrin IX in cells, J. Med. Chem., 2000, 43,
4738–4746.
22 F. Giuntini, L. Bourre, A. J. MacRobert, M. Wilson and
I. M. Eggleston, Improved Peptide Prodrugs of 5-ALA for
PDT: Rationalization of Cellular Accumulation and
Protoporphyrin IX Production by Direct Determination of
J. Salazar, A. Cortes, A. Barnadas and M. Baiget,
Pharmacogenetic prediction of clinical outcome in
advanced colorectal cancer patients receiving oxaliplatin/
5-fluorouracil as first-line chemotherapy, Br. J. Cancer,
2009, 100, 1368.
27 C. Martucci, S. Franchi, E. Giannini, H. Tian, P. Melchiorri,
L. Negri and P. Sacerdote, Bv8, the amphibian homologue
of the mammalian prokineticins, induces a proinflamma-
tory phenotype of mouse macrophages, Br. J. Pharmacol.,
2006, 147, 225–234.
Cellular Prodrug Uptake and Prodrug Metabolization, 28 A. François, S. Battah, A. J. Macrobert, L. Bezdetnaya,
J. Med. Chem., 2009, 52, 4026–4037.
23 H. Brunner, F. Hausmann and R. Knuechel, New
5-Aminolevulinic Acid Esters—Efficient Protoporphyrin
F. Guillemin and M. A. D’Hallewin, Fluorescence diagnosis of
bladder cancer: a novel in vivo approach using 5-aminolevuli-
nic acid (ALA) dendrimers, BJU Int., 2012, 110, E1155–E1162.
Precursors for Photodetection and Photodynamic Therapy, 29 L. Bourre, F. Giuntini, I. M. Eggleston, M. Wilson and
Photochem. Photobiol., 2003, 78, 481–486.
24 A. Godal, N. O. Nilsen, J. Klaveness, J. E. Braenden,
J. M. Nesland and Q. Peng, New derivatives of 5-aminolevu-
A. J. MacRobert, 5-Aminolaevulinic acid peptide prodrugs
enhance photosensitization for photodynamic therapy,
Mol. Cancer Ther., 2008, 7, 1720–1729.
linic acid for photodynamic therapy: chemical synthesis 30 J. Kloek and G. M. J. Beijersbergen van Henegouwen,
and porphyrin production in in vitro and in vivo biological
Prodrugs of 5-Aminolevullinic Acid for Photodynamic
systems, J. Environ. Pathol., Toxicol. Oncol., 2006, 25, 109–126.
Therapy, Photochem. Photobiol., 1997, 64, 994–1000.
25 J. Kloek, W. Akkermans and G. van Henegouwen, 31 Y. Ninomiya, Y. Itoh, S. Tajima and A. Ishibashi, In vitro
Derivatives of 5-Aminolevulinic Acid for Photodynamic
Therapy: Enzymatic Conversion into Protoporphyrin,
Photochem. Photobiol., 1998, 67, 150–154.
and in vivo expression of protoporphyrin IX induced by
lipophilic 5-aminolevulinic acid derivatives, J. Dermatol.
Sci., 2001, 27, 114–120.
Photochem. Photobiol. Sci.
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