Design and synthesis of 5-aminolaevulinic acid/3-hydroxypyridinone conjugates for photodynamic therapy: Enhancement of protoporphyrin IX production and photo-toxicity in tumor cells

Article abstract of DOI:10.1039/c6md00040a

5-Aminolaevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy (PDT) as precursors of the photosensitizer, protoporphyrin IX (PpIX) in dermatology and urology. However, ALA-PDT is limited by the low bioavailability of ALA due to the fact that ALA is poorly absorbed by cells by virtue of its zwitterionic nature at physiological pH. In order to improve the therapeutic effect and induce higher levels of PpIX, a series of ALA prodrugs were synthesized by the conjugation of ALA to 3-hydroxypyridin-4-one (HPO) iron chelator using an amino acid linkage via amide bonds. Pharmacokinetic studies indicated that one ALA-HPO conjugate significantly enhanced PpIX production in a range of tumor cell lines over ALA alone or the co-administration of ALA and CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The intracellular porphyrin fluorescence levels showed good correlation with cellular photo-toxicity following light exposure, suggesting the potential application of the ALA-HPO conjugates in photodynamic therapy.

Full text of DOI:10.1039/c6md00040a

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Design and synthesis of 5-aminolaevulinic Acid/3-hydroxypyridinone
conjugates for photodynamic therapy: enhancement of
protoporphyrin IX production and photo-toxicity in tumor cells†
Received 00th January 20xx,
Accepted 00th January 20xx
Tao Zhoua*, Le-Le Shaoa, Sinan Battahb*, Chun-Feng Zhua, Robert C. Hiderc, Brandon J. Reederb,
Asma Jabeenb, Sandy J. MacRobertd, Gerui Rena, Xinle Lianga
DOI: 10.1039/x0xx00000x
www.rsc.org/
5-Aminolaevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy (PDT) as precursors of the
photosensitizer, protoporphyrin IX (PpIX) in dermatology and urology. However, ALA–PDT is limited by the low
bioavailability of ALA due to the fact that ALA is poorly absorbed by cells by virtue of its zwitterionic nature at
physiological pH. In order to improve the therapeutical effect and induce higher levels of PpIX, a series of ALA prodrugs
were synthesized by the conjugation ALA to 3-hydroxypyridin-4-ones (HPO) iron chelator using an amino acid linkage via
amide bonds. Pharmacokinetic studies indicated that one ALA–HPO conjugate significantly enhanced PpIX production in a
range of tumor cell lines over above that caused by ALA alone or the co-administration of ALA and CP94 (1,2-diethyl-3-
hydroxypyridin-4-one). The intracellular porphyrin fluorescence levels showed good correlation with cellular photo-toxicity
following light exposure, suggesting the potential application of the ALA–HPO conjugates in photodynamic therapy.
solubility, such as esters,12,13 peptide derivatives.14,15 According
to the action mechanism, ALA is metabolized via the heme
1. Introduction
Photodynamic therapy (PDT), based on the activation of
exogenously applied or endogenously formed photosensitizers
by visible light in the presence of molecular oxygen, is a
promising treatment strategy for malignant and non-malignant
lesions. Upon exposure to light, a photosensitizer generates
singlet oxygen and/or free radicals, which oxidize cellular
macromolecules, leading to the damage of a variety of
subcellular substrates (such as phospholipid membranes,
nucleic acids, and proteins), resulting in cell death.1-3ALA has
been widely used in PDT for the treatment of actinic keratosis,
squamous cell carcinoma, and Bowen’s disease, as well as
cutaneous microbial infections such as acne, onychomycosis,
and verrucae.4-8 ALA-induced PDT can also be used as a
diagnostic tool for the visualization of precancerous changes in
the mucosae by fluorescence spectroscopy.9,10 The main
disadvantage of this therapy is that ALA is poorly absorbed by
cells due to its high hydrophilicity, resulting in low
bioavailability.11 In order to overcome this drawback,
numerous effforts have been made, which are mainly centred
on the development of ALA prodrugs with more favorable lipid
biosynthesis pathway to produce the fluorescent
photosensitizer protoporphyrin IX (PpIX).16,17 Ferrochelatase
can catalyze the conversion of PpIX to light inactive heme,
leading to a decrease in the therapeutical effect.18 Thus, the
activity of ferrochelatase is a key factor that influences the
accumulation of PpIX in the cell.19,20 Iron chelators can inhibit
the activity of ferrochelatase by scavenging the intracellular
labile iron pool. It has been demonstrated that ALA–PDT can
be modulated in the presence of iron chelators such as EDTA,21
and desferioxamine.22-24 Several studies have also
demonstrated that a combination of ALA and the membrane
permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-
one) in PDT is an effective technique to increase the efficacy of
ALA-PDT within cells under both in vitro and in vivo
conditions.25–28 Previously, we reported that ALA–HPO
conjugates, in which the ALA moiety was linked to a HPO
moiety via an ester bond, significantly enhanced PpIX
formation in cells of the human breast adenocarcinoma (MDA-
MB-468), in comparison with ALA alone or with a combination
of ALA with deferiprone (1,2-dimethyl-3-hydroxypyridin-4-
one).29 In the present study, a range of novel ALA–HPO
conjugates with reasonable lipophilicity were synthesized. In
these molecules, ALA methyl ester was linked to phenylalanine
or leucine via a peptide bond, while the HPO moiety was
coupled to amino group on phenylalanine or leucine. The ALA–
HPO conjugates are anticipated to be hydrolyzed by
peptidases and esterases in the cytosol of tumor cells,
liberating free ALA. The PpIX fluorescence induced by these
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MedChemComm
O
O
O
ALA–HPO conjugates in a range of human tumor cell lines and
their photo-toxicity have been investigated.
OBn
H
N
OBn
OBn
H
N
a
b
c
R'
R'
N
R
N
R
COOH
N
R
COOH
COOEt
O
O
2. Results and discussion
5
6
7
2.1. Chemistry
O
The synthetic route of benzyl protected 3-hydroxypyridinones
containing a carboxyl group (5) using maltol (1) as a starting
O
OH
OBn
H
N
R'
R'
O
O
d
H
H
H
N
N
O
material is presented in Scheme 1. The benzylation of maltol
with benzyl chloride provided
O
N
N
R
N
R
2 in 86% yield. Condensation of
with primary amines under basic conditions provided the 3-
3 in good yield (84–90%), which
O
O
O
O
O
O
2
hydroxypyridinone derivatives
Z01-10
8
R' =PhCH2
R' =(CH3)2CHCH2
underwent selective oxidation of the methyl group at the 2-
position with selenium dioxide in acetic anhydride to give
Z01: R=CH3
Z06: R=CH3
Z02: R=CH2CH3
Z03: R=n-C4H9
Z04: R=n-C6H13
Z07: R=CH2CH3
Z08: R=n-C4H9
Z09: R=n-C6H13
aldehyde
presence of sodium hypochlorite and sulfonic acid provided
the carboxylic acids in reasonable yield (54-62%). Coupling
with L-amino acid esters was carried out in the
of 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-
(HCTU), giving
in moderate to good yield (68-81%) (Scheme 2).
Compounds were then hydrolyzed with lithium hydroxide to
the corresponding carboxylic acids in excellent yield (91-
94%). Conjugation of and methyl 5-aminolaevulinate was
achieved in the presence of HCTU to give (75-81% yield),
4 in 77-80% yield. Further oxidation of 4 in the
Z05: R=CH2CH2OCH3
Z10: R=CH2CH2OCH3
Scheme 2.Reagents and conditions
：
(a) HCTU, Et3N, DMF,
5
overnight; (b) i) LiOH, ii) Amberlite IR-120 (H form) ion
exchange resin; (c) HCTU, Et3N, DMF, overnight; (d) CH3OH,
Pd/C, 3h.
reaction of
presence
5
tetramethylaminiumhexafluorophosphate
product
6
6
7
Firstly, the efficacy of Z01–Z10 to produce PpIX in A549, KB,
LNCap and BPH-1 cell lines at a concentration of 100 μM was
assess by comparison with ALA (Fig. 1). It was found that after
incubation for 24h, compounds Z02–Z10 at this concentration
did not markedly enhance the synthesis of PpIX in these cells
as compared to ALA, some of them even led to the production
of less PpIX than ALA. However, compound Z01 was found to
significantly increase PpIX production at 100 μM in A549, KB
and LNCap cell lines, generating PpIX by 3.1, 2.9 and 4.0 times
higher than that induced by ALA, respectively. For the BPH-1
cell line, the fluorescence intensity of PpIX induced by Z01 was
only 1.5 time larger than that induced by ALA, indicating that
PpIX producing rate via heme biosynthesis pathway in tumor
cell is much higher than that in normal cell. Significant
differences of PpIX production was observed in these cell lines
(P < 0.05). After incubation with Z01, the highest level of PpIX
was detected in KB cell, the next in A549 cell. This difference
could be attributed to the different peptidase and/or esterase
bioavailability and activity in these cell lines, resulting in
different releasing rates of free ALA.
7
8
which were subjected to hydrogenation to remove benzyl
groups, generating ALA–HPO conjugates Z01-Z10 in excellent
yield (93-97%) (Scheme 2). All the compounds were fully
characterized by 1H NMR, 13C NMR, MS and HRMS.
2.2. Fluorescence Pharmacokinetics
In order to assess the efficacy of the structural designed ALA–
HPO conjugates Z01–Z10, at the onset of this study the ability
of these ALA–HPO conjugates to generate the active,
fluorescent photosensitizer PpIX was investigated by
measuring the fluorescence intensity of the peak emission at
635 nm after incubation with a range of tumor cell lines,
including A549 cell line (human lung carcinoma), MCF-7 cell
lines (human breast adenocarcinoma), KB cell line (human
mouth epidermal carcinoma cells), LNCap cell line (human
prostate cells derived from metastatic site lymph node) and
BPH-1 cell line (normal human prostate epithelial cells).
O
O
O
O
O
OBn
OBn
OBn
OH
a
b
c
N
R
CHO
N
R
O
2
1
3
4
O
OBn
d
5a: R=CH3
5b: R=CH3CH2
5c: R=n-C4H9
5d: R=n-C6H13
N
R
COOH
5e: R=CH2CH2OCH3
5
Scheme 1. Reagents and conditions
：(a) BnCl, NaOH, CH3OH,
Fig. 1.PpIX production in A549, KB, LNCap and BPH-1 cell lines
after incubation with 100 μM of ALA or ALA-HPO conjugates
for 24 h at 37 oC and humidified by 5% CO2. Bars represent
standard deviation (n = 3).
reflux, 7-8h; (b) RNH2, NaOH, CH3OH/H2O, reflux, 2.5h; (c)
SeO2, CH3COOH/(CH3CO)2O, reflux; (d) NH2SO3H, NaClO2,
acetone/H2O.
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**
PpIX production to that of ALA with the exception of Z01. The
lack of efficacy of generating PpIX from compounds Z02 Z10
(a)
3h
100
A549
-
**
90
80
70
60
50
40
30
20
10
0
6h
could partially be owing to their bioavailability within the cell
lines under investigation. It was clearly noticeable some
aggregation of those compounds on the surface of the
incubated cells. Probably, the use of serum free media (in
order to avoid releasing PpIX from the cells) containing the
compounds would not promote the solubility of the lipophilic
compounds, subsequently decreasing the intercellular
availability. It was reported that the aggregation problem with
the lipophilic compounds which led to reduction of uptake,
was much less appreciable in full media with serum.30 The
calculated partition coefficient (ClogP) value31 of Z01 was -
1.23, while the values of Z02-Z10 range from -1.40 to 1.22,
indicating that the lipophilicities of all these ALA-HPO
conjugates were increased as compared to ALA (ClogP -3.91).
However, it has been previously reported that increasing the
lipophilicity of ALA derivatives to certain level could negatively
influence the uptake of the compound.32,33 As this compound
series were all expected to penetrate the cell membrane by a
passive diffusion mechanism, it is reasonable to assume that
the relatively poor performance of derivatives (Z02-Z10) in
terms of PpIX production also reflects a slower rate of
24h
*
Compound Concentration (200 µM)
LNCap
3h
100
(b)
6h
90
80
70
60
50
40
30
20
10
0
**
**
24h
*
metabolism and
a corresponding lower affinity for the
peptidases and/or esterases involved in the release of ALA
from the prodrug. The superior activity of Z01 to the other
derivatives was probably due to its higher intercellular
bioavailability and its more rapid metabolism of the prodrug.
Compound Concentration (200 µM)
Fig. 2. PpIX fluorescence intensities produced after incubation
in tumor cell lines with 200 μM of ALA–HPO conjugates, co-
administration of ALA and CP94 (both 200 μM) for 3, 6 and 24
Owing to aggregation problem, we wished to evaluate the
potential of Z01 in more cell lines. KB was known to be
o
h at 37 C and humidified by 5% CO2. (a) incubation with A549
cell line; (b) incubation with LNCap cell line. Bars represent
standard deviation (n=3). *represents a significant difference
(P < 0.05), **represents a very significant difference (P <
0.001).
I
n this investigation, the efficacy of compounds Z01–Z10 to
produce PpIX in A549, LNCap, KB and PBH-1 cell lines was
compared with co-administration of ALA and CP94 by a time-
course study. In all the four cell lines, compounds Z02–Z10
were less efficient than the co-administration of ALA and CP94
to produce PpIX. However, Z01 was found to significantly
enhance the PpIX production in comparison with the
combination of ALA with CP94. It was found that PpIX
fluorescence intensity profile of A549 cell line was similar to
that of KB cell line at 200 μM at all incubation times. Thus, only
the results of A549 cell line were presented (Fig. 2a). After
incubation with the A549 cell line for 3, 6 and 24h, PpIX
fluorescence intensities generated by Z01 (200 μM) was
determined to be 27.7, 52.9 and 94.6 a.u., respectively, which
were 1.6, 1.3 and 1.2 times higher than those induced by ALA
in combination with CP94 (17.7, 42.1 and 77.8 a.u.
respectively). In the case of the LNCap cell line, after
incubation for 3, 6 and 24h, PpIX fluorescence intensities
generated by Z01 (200 μM) was determined to be 25.3, 47.1
and 58.4 a.u., respectively; whereas those induced by ALA in
combination with CP94 were 17.3, 31.3 and 46.0 a.u.,
respectively (Fig. 2b). The normal cell line PBH-1 produced a
lower PpIX level than that presented for LNCap in Fig. 2b (data
not shown). It is surprising that most of the newly synthesized
ALA-HPO derivatives showed a similar efficacy in intracellular
Fig. 3. Kinetics of PpIX fluorescence in (a) Hela cell line; (b)
MDA MB-435 cell line. Fluorescence measurements after
incubation with variable concentration (50–250 μM) of ALA,
ALA in combination with CP94, and Z01 for 5h at 37 oC
humidified by 5% CO2. Bars represent standard deviation
(n=3). (P < 0.001 to 0.05).
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100
(a)
90
Hela
80
70
60
50
40
30
20
10
ALA
ALA+CP94
Z01
0
0
50
100
150
200
250
Compound Concentration (µM)
100
(b)
90
MDA MB-435
80
70
60
50
40
30
20
10
ALA
ALA+CP94
Z01
0
0
50
100
150
200
250
Compound Concentration (µM)
Fig. 4. Photo-toxicity after incubation with ALA or ALA+CP94,
and ALA-HPO conjugates in a range of cell lines assessed by
MTT assay. Cells were incubated with the compounds for 4 h
and irradiated with blue light (2.5 J·cm-2). (a) compound
concentration 200μM; (b) compound concentration 100μM.
Figure 5.Photo-toxicity after incubation with ALA, ALA+CP94
and Z01 in tumor cell lines assessed by MTT assay. Cells were
incubated with the compounds for 4 h and irradiated with blue
light (2.5 J·cm-2).(a) Hela cell line; (b) MDAMB-435 cell line.
contaminated by Hela in all the stocks worldwide.34 Thus, Hela
cell line (human cervical cells) and MDA-MB 435 cell line
(breast cancer cells) were selected in the evaluation. As shown
in Fig. 3, compound Z01 exhibited a dose dependent response
of PpIX production at concentrations ranging between 50–250
μM in both Hela and MDA MB-435 cell lines. Again, Z01 was
more efficient in PpIX production in these two cell lines than
co-administration of ALA and CP94, which was found to
enhance PpIX levels when compared to ALA alone (P<0.001 to
0.05).
results were in agreement with those obtained from PpIX
fluorescence experiments. The comparison of cytotoxicity of
Z01-Z10 with co-administration of ALA and CP94 was also
investigated at both 100 μM (Fig. 4b) and 200 μM (data not
shown). At a concentration of 100 μM, Z01 was found to be
more efficient in cell killing than the combination of the ALA
with CP94 (P < 0.05). The survival percentages of A549, MCF-7,
LNCap and BPH-1 cell lines treated with Z01 were determined
to be 14.7, 21.1, 26.3 and 75.1%, respectively; those data in
the case of ALA combined with CP94 were 18.4, 26.7, 33.1 and
81.9%, respectively (Fig. 4b). Thus, at this concentration, both
Z01 and ALA in combination with CP94 were found to be much
less toxic on normal cells than on tumor cells. At 100 μM,
compounds Z02-Z10 were all found to be of low toxicity to the
tested tumor cell lines.
2.3. Cytotoxicity
The photo-toxicity of prodrugs Z01-Z10 (cells incubated with
the compounds and exposed to blue light) on the A549, KB,
LNCap, BPH-1 and MCF-7 cell lines was investigated (Fig. 4).
The percentage of cell survival with respect to the control cells
(without compounds) was calculated for the investigated
compounds. After the incubation of the tumor cells with 200
μM of Z01, followed by the irradiation with blue light, efficient
cell killing was observed. The survival percentages of A549, KB,
LNCap and BPH-1 cell lines treated Z01 were determined to be
16.3, 7.8, 13.5 and 12.4%, respectively; those data in the case
of ALA were 54.7, 52.6, 61.9 and 70.8%, respectively (Fig. 4a).
However, overall cytotoxicity of Z02-Z10 was not found to be
significantly enhanced as compared to ALA (Fig. 4a). These
The photo-toxicity of the most active conjugate Z01 on
additional two cell lines (Hela and MDA MB-435) in
comparison with ALA, and ALA+CP94 over
a range of
concentrations between 50-250 μM is presented in Fig. 5. The
cytotoxicity was dose dependent. It was found that Z01
exhibited the highest photo-toxicity, and ALA the lowest, on
these two cell lines. Significant differences between the
treatments were observed (P < 0.05). The survival percentages
of Hela cell line treated with Z01, ALA and ALA+CP94 at 250
μM were determined to be 15.3, 58.6 and 26.9% respectively.
For MDA MB-435 cell line incubated with Z01 and ALA (250
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μM), the survival percentages were 11.1, 61.4 and 28.9%, J=5.5Hz, 1H, NH), 9.61 (d, J=8.0Hz, 1H, NH). 13C NMR (DMSO-
respectively.
d6, 125MHz) δ 13.7, 21.8, 25.0, 27.1, 27.2, 30.4, 33.8, 37.4,
48.4, 51.3, 54.3, 56.2, 112.0 ,126.4, 128.1, 129.2, 136.2, 137.3,
138.1, 142.9, 158.9, 161.5, 170.7, 172.6, 205.1. ESI-MS: m/z
514 ([M+H]+). HRMS-ESI: calcd. for C27H36N3O7 514.2553
([M+H]+), found 514.2546.
The “dark” toxicity of the ALA-HPO conjugates Z01-Z10 (cells
incubated with the compounds without light exposure) on
A549, MCF-7, LNCap and BPH-1 cell lines as percentages of
control (cells without compounds and without light exposure)
was evaluated. The data indicated that there was no significant Z05
toxicity found for ALA or all the investigated compounds at 200 CH2COO), 2.71 (t, J= 5Hz, 2H, COCH2), 2.87 and 3.18 (m, 2H,
.
1H NMR (DMSO-d6, 500MHz) δ 2.50 (t, J=6.5Hz, 2H,
μM after 4h incubation (data not shown).
CH2), 3.15 (s, 3H, OCH3), 3.28-3.41 (m, 2H, OCH2), 3.58 (s, 3H,
COOCH3), 3.91 and 4.20 (m, 2H, CH2CO), 4.01(t, J= 6.0Hz, 2H,
NCH2), 4.88 (m, 1H, CH), 7.21-7.36 (m, 6H, Ph and buried C5-H
in pyridinone), 8.03 (d, J=7.0Hz, 1H, C6-H in pyridinone), 8.71
(t, J=5.5Hz, 1H, NH), 9.62 (d, J=8.5Hz, 1H, NH).13C NMR (DMSO-
d6, 125MHz) δ 27.1, 33.8, 37.3, 48.4, 51.4, 54.2, 55.4, 58.1,
69.7, 111.3, 126.5, 128.2, 129.1, 135.4, 137.3, 139.4, 143.2,
159.0, 162.4, 170.7, 172.6, 205.1. ESI-MS: m/z 488 ([M+H]+).
HRMS-ESI: calcd. for C24H30N3O8 488.2033 ([M+H]+), found
488.2023.
3. Experimental
The general synthetic procedures and spectral data for
allcompounds are given in the ESI.† The spectral data for the
final compounds Z01-10 are given below.
3.1. Spectral data for compounds Z01-10
Z01.
1H NMR (DMSO-d6, 500MHz) δ 2.50 (t, J=6.5Hz, 2H,
CH2COO), 2.70 (t, J=6.5Hz, 2H, COCH2), 2.87 and 3.17 (m, 2H,
CH2), 3.54 (s, 3H, COOCH3), 3.58 (s, 3H, NCH3), 3.97-4.08(m,
2H, CH2CO), 4.87-4.92 (m, 1H, CH), 7.18-7.36 (m, 6H, Ph and
buried C5-H in pyridinone), 8.14 (d, J=7.5Hz, 1H, C6-H in
pyridinone), 8.70 (t, J= 5.5Hz, 1H, NH), 9.51 (d, J=8.5Hz, 1H,
NH).13C NMR (DMSO-d6, 125MHz) δ 27.1, 33.9, 37.5, 42.8,
48.4, 51.4, 53.9, 111.7, 126.5, 128.2, 129.3, 136.3, 137.3,
138.9, 142.9, 158.9, 161.7, 170.6, 172.6, 205.1. ESI-MS: m/z
444 ([M+H]+). HRMS-ESI: calcd. for C22H26N3O7 444.1771
([M+H]+), found 444.1762.
1
Z06. H NMR (DMSO-d6, 500MHz) δ 0.91 (d, J=6.5Hz, 6H, CH3),
1.57 (m, 2H, CH2), 1.74 (m, 1H, CH), 2.49 (t, J=6.5Hz, 2H,
CH2COO), 2.71 (t, J=6.5Hz, 2H, COCH2), 3.57 (s, 3H, COOCH3),
3.91-4.05 (m, 5H, NCH3 and CH2 in NHCH2CO), 4.53 (q, J=7.5Hz,
1H, CH), 7.40 (d, J=7.0Hz, 1H, C5-H in pyridinone), 8.25 (d,
J=7.0Hz,1H, C6-H in pyridinone), 8.58 (t, J=5.5Hz, 1H, NH), 9.40
(d, J=8.0Hz, 1H, NH). 13C NMR (DMSO-d6, 125MHz) δ 21.3,
22.9, 24.2, 27.1, 33.9, 40.4, 43.4, 48.3, 51.3, 51.5, 111.8, 136.6,
138.9, 142.9, 159.0, 161.5, 171.6, 172.6, 205.1. ESI-MS: m/z
410 ([M+H]+). HRMS-ESI: calcd. for C19H28N3O7 410.1927
([M+H]+), found 410.1917.
1
Z02. HNMR (DMSO-d6, 500MHz) δ 1.13 (t, J=7.0Hz, 3H, CH3),
2.50 (t, J=6.5Hz, 2H, CH2COO), 2.71(t, J=6.5Hz, 2H, COCH2),
2.87 and 3.19 (m, 2H, CH2), 3.57 (s, 3H, COOCH3), 3.73 and 3.97
(m, 2H, CH2CO), 4.02 (q, J= 7.0Hz, 2H, NCH2), 4.93 (m, 1H, CH),
7.20-7.36 (m, 5H, Ph), 7.42 (d, J=7.0Hz, 1H, C5-H in
pyridinone), 8.26 (d, J=7.0Hz, 1H, C6-H in pyridinone), 8.72 (t,
J=5.5Hz, 1H, NH), 9.60 (d, J=8.5Hz, 1H, NH). 13C NMR (DMSO-
d6, 125MHz) δ 16.3, 27.1, 33.8, 37.5, 48.4, 51.4, 51.5, 54.1,
112.3, 126.5, 128.2, 129.2, 136.3, 137.3, 137.6, 142.8, 158.9,
161.4, 170.6, 172.6, 205.1. ESI-MS: m/z 458 ([M+H]+). HRMS-
ESI: calcd. for C23H28N3O7 458.1927 ([M+H]+), found 458.1924.
Z07. 1H NMR (DMSO-d6, 500MHz) δ 0.92 (d, J=6.5Hz, 6H, CH3),
1.38 (t, J= 7.5Hz, 3H, CH3), 1.57 (m, 2H, CH2), 1.73 (m, 1H, CH),
2.49 (m, 2H, CH2COO), 2.71 (m, 2H, COCH2), 3.57 (s, 3H,
COOCH3), 3.92-4.04(m, 2H, NCH2), 4.19-4.34 (m, 2H, CH2CO),
4.55 (q, J=7.5Hz, 1H, CH), 7.39 (d, J=6.5Hz, 1H, C5-H in
pyridinone), 8.29 (d, J=6.5Hz, 1H, C6-H in pyridinone), 8.57 (t,
J=5.5Hz, 1H, NH), 9.42 (d, J=8.0Hz, 1H, NH). 13C NMR (DMSO-
d6, 125MHz) δ 16.5, 21.3, 23.0, 24.2, 27.1, 33.8, 40.4, 48.3,
51.4, 51.5, 51.7, 112.2, 136.0, 137.8, 142.9, 159.0, 161.8,
171.5, 172.6, 205.1. ESI-MS: m/z 424 ([M+H]+). HRMS-ESI:
calcd. for C20H30N3O7 424.2084 ([M+H]+), found 424.2077.
Z03. 1H NMR (DMSO-d6, 500MHz) δ 0.75 (t, J=7.5Hz, 3H, CH3),
1.05 (m, 2H, CH2), 1.52 (m, 2H, CH2), 2.49 (t, J=6.5Hz, 2H,
CH2COO), 2.70 (t, J=6.5Hz, 2H, COCH2), 2.87 and 3.17 (m, 2H,
CH2), 3.57 (s, 3H, COOCH3), 3.69 and 4.06 (m, 2H, CH2CO), 4.01
(t, J=5.5Hz, 2H, NCH2), 4.86 (m, 1H, CH), 7.19-7.39 (m, 6H, Ph
and buried C5-H in pyridinone), 8.24 (d, J=7.0Hz, 1H, C6-H in
pyridinone), 8.73 (t, J=5.5Hz, 1H, NH), 9.60 (d, J=8.0Hz, 1H,
NH). 13C NMR (125MHz, DMSO-d6) δ 13.2, 18.7, 27.1, 32.4,
33.8, 37.3, 48.4, 51.4, 54.3, 55.8, 111.9, 126.5, 128.2, 129.2,
135.9, 137.3, 138.1, 143.1, 159.0, 161.7, 170.7, 172.6, 205.1.
ESI-MS: m/z 486 ([M+H]+). HRMS-ESI: calcd. for C25H32N3O7
486.2240 ([M+H]+), found 486.2234.
1
Z08. H NMR (DMSO-d6, 500MHz) δ 0.84 (t, J=7.5Hz, 3H, CH3),
0.91 (d, J=6.5Hz, 6H, CH3), 1.24 (m, 2H, CH2), 1.57 (m, 2H, CH2),
1.74 (m, 3H, CH2CH), 2.49 (t, J=6.5Hz, 2H, CH2COO), 2.72 (t,
J=6.5Hz, 2H, COCH2), 3.57 (s, 3H, COOCH3), 3.91-4.02 (m, 2H,
NCH2), 4.15-4.32 (m, 2H, CH2CO), 4.54 (q, J=7.0Hz, 1H, CH),
7.43 (d, J=7.0Hz, 1H, C5-H in pyridinone), 8.32 (d, J=7.0Hz, 1H,
C6-H in pyridinone), 8.60 (t, J=6.0Hz, 1H, NH), 9.45 (d, J=8.0Hz,
1H, NH).13C NMR (DMSO-d6, 125MHz) δ 13.2, 18.9, 21.2, 22.9,
24.1, 27.1, 32.7, 33.8, 40.4, 48.3, 51.3, 51.5, 56.1, 112.0, 136.3,
138.2, 143.0, 158.9, 161.6, 171.5, 172.6, 205.0. ESI-MS: m/z
452 ([M+H]+). HRMS-ESI: calcd. for C22H34N3O7 452.2397
([M+H]+), found 452.2392.
1
Z04. H NMR (DMSO-d6, 500MHz) δ 0.82 (t, J=7.0Hz, 3H, CH3),
0.98-1.14 (m, 4H, CH2), 1.19 (m, 2H, CH2), 1.54 (m, 2H, CH2),
2.49 (t, J=6.5Hz, 2H, CH2COO), 2.70 (t, J=6.5Hz, 2H, COCH2),
2.87 and 3.17 (m, 2H, CH2), 3.57 (s, 3H, COOCH3), 3.67 and 4.06
(m, 2H, CH2CO), 4.00 (t, J=6.0Hz, 2H, NCH2), 4.87 (m, 1H, CH),
7.20-7.37 (m, 5H, Ph), 7.43 (d, J=7.0Hz, 1H, C5-H in
pyridinone), 8.26 (d, J=7.0Hz, 1H, C6-H in pyridinone), 8.74 (t,
1
Z09. H NMR(DMSO-d6, 500MHz) δ 0.84 (t, J=6.5Hz, 3H, CH3),
0.91 (d, J=6.5Hz, 6H, CH3), 1.22 (m, 6H, CH2), 1.57 (m, 2H, CH2),
1.70-1.80 (m, 3H, CH2CH), 2.49 (t, J=6.5Hz, 2H, CH2COO), 2.71
(t, J=6.5Hz, 2H, COCH2), 3.57 (s, 3H, COOCH3), 3.91-4.03(m, 2H,
NCH2), 4.14-4.32 (m, 2H, CH2CO), 4.55 (q, J=7.0Hz, 1H, CH),
7.44 (d, J=7.0Hz, 1H, C5-H in pyridinone), 8.32 (d, J=7.0Hz, 1H,
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C6-H in pyridinone), 8.60 (t, J=6.0Hz, 1H, NH), 9.46 (d, J=7.5Hz, wavelengths, with slit widths set to 10 nm and an internal 515
1H, NH). 13C NMR (DMSO-d6, 125MHz) δ 13.7, 21.3, 21.9, 22.9, nm long-pass filter on the emission side. The spectral scans
24.2, 25.2, 27.1, 30.6, 30.7, 33.8, 40.4, 48.3, 51.3, 51.5, 56.3, were recorded between 600 and 750 nm, thereby selectively
112.0, 136.3, 138.2, 143.0, 158.9, 161.5, 171.5, 172.5, 205.0. identifying PpIX. The mean fluorescence intensity (expressed in
ESI-MS: m/z 480 ([M+H]+). HRMS-ESI: calcd. for C24H38N3O7 arbitrary units) was calculated after subtraction of the control
480.2710 ([M+H]+), found 480.2701.
values. PpIX fluorescence intensities were recorded over
periods of 3, 6, and 24 h with varied concentration of ALA
prodrugs (50–250 μM).
Z10. 1H NMR (DMSO-d6, 500MHz) δ 0.92 (d, J= 6.0Hz, 6H, CH3),
1.59 (m, 2H, CH2), 1.76 (m, 1H, CH), 2.51 (t, J=6.5Hz, 2H,
CH2COO), 2.73 (m, 2H, COCH2), 3.23 (s, 3H, OCH3), 3.58 (s, 3H, 3.2.3. Photodynamic Treatment
COOCH3), 3.60-3.73 (m, 2H, OCH2), 3.93-4.05 (m, 2H, NCH2),
Cells were seeded into 96-well plates at
a density of
4.39 (m, 1H, CH), 4.50 (m, 2H, CH2CO), 7.34 (d, J=6.5Hz, 1H, C5-
H in pyridinone), 8.12 (d, J=6.5Hz, 1H, C6-H in pyridinone), 7.91
(t, J=5.5Hz, 1H, NH), 8.81 (d, J=8.0Hz, 1H, NH). 13C NMR
(DMSO-d6, 125MHz) δ 21.2, 23.0, 24.2, 27.1, 33.8, 40.2, 48.3,
51.3, 51.7, 55.5, 58.1, 70.0, 111.4, 135.4, 139.4, 143.2, 159.1,
162.7, 171.6, 172.6, 205.1. ESI-MS: m/z 454 ([M+H]+). HRMS-
ESI: calcd. for C21H32N3O8 454.2189 ([M+H]+), found 454.2182.
approximately 5×104 per well. Following incubation for 48h,
the cells were washed with PBS and 100μL solutions containing
each compound at varying concentrations (between 50μM–
250 μM) were added to their designated wells for 4h
incubation periods. Each well plate contained three control
wells without the compound and the compound at five
different concentrations in triplicate. The plates were
irradiated with a fluence of (5 min) using a blue lamp, which
emits a uniform field. Peak output is 420 nm, which overlaps
with the PpIX soret band. Immediately following irradiation,
the medium was replaced and cells were incubated for further
18h. Cell cytotoxicity was determined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay: The cells were incubated with the compounds for 4h,
washed, and after 18h they were subjected to the MTT assay.
Cells were incubated with medium containing MTT (1mg/mL
dissolved in full media) for 2h. The insoluble end product
(formazan derivatives) was dissolved in 100 μL DMSO after
removing the medium. UV absorption was quantified at 490
nm using a 96-well plate reader (MR 700 Dynatech, Dynex).
The mean cell survival was calculated for each prodrug at
every concentration tested and expressed as a percentage of
control (cells only without the compounds but irradiated) cell
survival values. For determination of “dark” toxicity of the
compounds, well plates were prepared in the same manner as
above but without irradiation.
3.2. Biological assay
3.2.1. Cell Culture
The biological evaluation for ALA, ALA in combination with
CP94 as a mixture and ten novel ALA-HPO conjugates were
carried out in a range of human cell lines. A549 cell line
(human lung carcinoma), MDA MB-435 and MCF-7 cell lines
(human breast adenocarcinoma), and Hela cell line (human
cervical cells) were cultured in Dulbecco's Modified Eagle
Medium media (DMEM). Human mouth epidermal carcinoma
cell (KB cell line) was cultured in Eagle’s minimum essential
medium (EMEM). Human prostate cells derived from
metastatic site lymph node (LNCap cell line) and normal
human prostate epithelial cell (BPH-1 cell line) were both
cultured in Roswell Park Memorial Institute (RPMI 1460). All
types of media contained L-glutamine (20 μM) and phenol red,
supplemented with 10% fetal bovine serum (FBS) and
Gentamycine (500 units/mL; Life Technologies). They were
standardized to give an iron concentration between 450 and
600 μg/100 g. The cells were grown as monolayers in sterile,
vented-capped, angle-necked cell culture flasks (Corning) and
were maintained at 37 °C in a humidified 5% CO2 incubator (IR
Autoflow Water-Jacketed Incubator; Jencons Nuaire) until
confluent.
4. Conclusions
Although ALA is a useful therapeutical agent for PDT, it is
important to improve its bioavailability when administered
systemically and intravenously in order to overcome
distribution problems. Incorporation of iron chelating agents
to ALA prodrugs offers a promising way to enhance the cellular
PpIX accumulation by inhibiting ferrochelatase. In the present
study, a series of ALA–HPO conjugates have been synthesized.
Among these ALA prodrugs, Z01 exhibited the highest
efficiency in cellular PpIX accumulation in a range of cell lines.
Z01 was also found to be the most effective in enhancement of
tumor cell phototoxicity when compared to ALA and to a
combination of ALA with CP94. It is necessary to extend these
promising results in an in vivo setting in order to further
evaluate the potential of these ALA–HPO conjugates.
3.2.2. Fluorescence Pharmacokinetics
Cells were seeded into gama-sterilised 96 well plates (Orange
Scientific, Triple Red Laboratory Technologies) at a density of
approximately 5×104 cells per well for 48 hours. After
removing the culture medium, the wells were washed with
PBS. The cells were incubated with freshly prepared solutions
of ALA and ALA-HPO prodrugs: 100μL of serum-free medium
containing 5% DMSO and varying prodrug concentrations was
added to a designated series of wells. Each plate contained
control wells with cells without prodrug for determination of
the background reading, and reference wells containing cells
incubated with the same ALA concentrations. For drug
incubation, serum-free medium was used since serum is
known to cause release of PpIX from cells, thus resulting in loss
of the florescence signal. The fluorescence signal from each
well was measured with a fluorescence spectrometer plate
reader using 405 nm excitation and 635 nm emission
Acknowledgments
The authors thank Science Technology Departmentof Zhejiang
Province, China (No. 2013C24006) and Zhejiang Provincial
Natural Science Foundationof China (No. LY12B02014) for the
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Med. Sci., 2010, 25, 251–257.
financial support and ChemPharm Research LTD, UK. We also
acknowledge the assistance in the measurement of some of
the efficacy studies by Dr. Olivier Reefs at Department of
Pharmacy and Pharmacology, Bath University, UK.
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1
TABLE OF CONTENTS
2
3
4
Design and synthesis of 5-aminolaevulinic Acid / 3-hydroxypyridinone
conjugates for photodynamic therapy: enhancement of protoporphyrin IX
production and photo-toxicity in tumor cells
5
6
A series of novel 5-aminolaevulinic Acid (ALA)/3-hydroxypyridinone (HPO)
conjugates was prepared. One ALA-HPO conjugate was found to exhibit more
efficient in protoporphyrin IX (PpIX) production in a range of tumor cell lines and
higher photo-toxicity as compared to ALA alone and in combination with iron
7
8
9
10
chelator 1,2-diethyl-3-hydroxypyridin-4-one (CP94).
Cell
peptidase
penetration
HPO
ALA
HPO
AA
ALA
PpIX
AA
esterase
Noncharged prodrug
Inhibition
ferrochelatase
light
singlet oxygen
free radical
Haem
×
11
12