Design, synthesis and biological evaluation of 5-aminolaevulinic acid/3-hydroxypyridinone conjugates as potential photodynamic therapeutical agents

Article abstract of DOI:10.1016/j.bmcl.2014.12.018

5-Aminolaevulinic acid (ALA) prodrugs have been widely used in photodynamic therapy (PDT) as precursors to the natural photosensitizer, protoporphyrin IX (PpIX). The main disadvantage of this therapy is that ALA is poorly absorbed by cells due to its high hydrophilicity. In order to improve the therapeutical effect and induce higher yields of PpIX, a range of prodrugs of ALA conjugated to 3-hydroxypyridin-4-ones (HPO) were synthesized. Pharmacokinetic studies indicated that some of the ALA-HPO conjugates are more efficient than ALA for PpIX production in the human breast adenocarcinoma cell line (MDA-MB-468). The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure, suggesting the potential application of the ALA-HPO conjugates in photodynamic therapy.

Full text of DOI:10.1016/j.bmcl.2014.12.018

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of 5-aminolaevulinic
acid/3-hydroxypyridinone conjugates as potential photodynamic
therapeutical agents
Chun-Feng Zhu a, Sinan Battah b, Xiaole Kong c, Brandon J. Reeder b, Robert C. Hider c, Tao Zhou a,
⇑
a School of Food Science and Biotechnology, Zhejiang Gongshang University, 18 Xuezheng Street, Xiasha, Hangzhou, Zhejiang 310018, PR China
b Biological Sciences Department, University of Essex, Wivenhoe Park CO4 3SQ, United kingdom
c Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Aminolaevulinic acid (ALA) prodrugs have been widely used in photodynamic therapy (PDT) as precur-
sors to the natural photosensitizer, protoporphyrin IX (PpIX). The main disadvantage of this therapy is
that ALA is poorly absorbed by cells due to its high hydrophilicity. In order to improve the therapeutical
effect and induce higher yields of PpIX, a range of prodrugs of ALA conjugated to 3-hydroxypyridin-
4-ones (HPO) were synthesized. Pharmacokinetic studies indicated that some of the ALA–HPO conjugates
are more efficient than ALA for PpIX production in the human breast adenocarcinoma cell line
(MDA-MB-468). The intracellular porphyrin fluorescence levels showed good correlation with cellular
phototoxicity following light exposure, suggesting the potential application of the ALA–HPO conjugates
in photodynamic therapy.
Received 19 September 2014
Revised 4 December 2014
Accepted 9 December 2014
Available online xxxx
Keywords:
Photodynamic therapy
5-Aminolaevulinic acid
Iron chelators
Hydroxypyridinone
Protoporphyrin IX
Ó 2014 Published by Elsevier Ltd.
5-Aminolevulinic acid (ALA) is a precursor in the heme biosyn-
thesis pathway and is metabolized to fluorescent protoporphyrin
IX (PpIX) before being converted to photo inactive heme.1 The pho-
tosensitiser, PpIX accumulates, temporarily in malignant tissue
after exogenous application of ALA which upon exposure to spe-
cific light generates singlet oxygen and free radicals that are toxic
to tumor cells. ALA prodrugs have been widely used in photody-
namic therapy (PDT) for the treatment of actinic keratosis, squa-
mous cell carcinoma, and Bowen’s disease, as well as cutaneous
microbial infections such as acne, onychomycosis, and verru-
cae.2–5 The main advantages of ALA–PDT are the short half-life of
the photosensitizing effects which limits the duration of skin
photosensitivity and its efficacy using topical administration.6
ALA-induced PDT can also be used as a diagnostic tool for the visu-
alization of precancerous changes in the mucosae by fluorescence
spectroscopy.7,8 However, due to the high hydrophilicity, ALA is
poorly absorbed by cells, which greatly reduces the bioavailability
of ALA.9,10 In order to improve the therapeutic effect of ALA, a num-
ber of ALA prodrugs with higher lipophilicity have been investi-
gated, for instance, esters,11,12 peptide derivatives.13 Dendrimers
containing ALA moieties have also attracted interest by virtue of
their ability to improve the delivery of ALA.14,15
The therapeutic effect of ALA–PDT depends on the level of PpIX
in the tumor cells to a great extent. The accumulation of PpIX in
cells depends not only on the concentration of ALA entering the
cell, but also on the activity of ferrochelatase, which insert iron(II)
into PpIX, forming light inactive heme.16 Ferrochelatase can be
inhibited by chelators through scavenging the intracellular labile
iron pool. Thus, ALA–PDT can be modulated in the presence of iron
chelators such as EDTA,17,18 and desferioxamine.19,20 Several stud-
ies have also demonstrated enhancement of PpIX concentration in
cells or tissues exposed to a combination of ALA and the membrane
permeable iron chelator 1,2-diethyl-3-hydroxypyridin-4-one
(CP94).21–23 In this study, a range of ALA–HPO conjugates were
designed and synthesized. In these molecules, ALA moiety was
linked to the HPO moiety via an ester bond, ALA is also linked with
either phenylalanine or leucine through a peptide bond which are
anticipated to be hydrolyzed by the esterase and peptidase, liber-
ating ALA and HPO. The photo-toxicity of these ALA–HPO conju-
gates has been investigated.
The synthetic route of benzyl protected 3-hydroxypyridinone
containing a free hydroxyl group (5) is presented in Scheme 1.
The benzylation of the starting material, 3-hydroxy-2-methyl-
4H-pyran-4-one (1) was achieved by the treatment with benzyl
chloride to obtain 2 in 86% yield. Treatment of 2 with primary
amines under basic conditions provided the HPO derivatives 3 in
good yield (85–90%), which underwent selective oxidation of the
⇑
Corresponding author. Tel.: +86 571 88071024 7587; fax: +86 571 88905733.
E-mail address: taozhou@zjgsu.edu.cn (T. Zhou).
http://dx.doi.org/10.1016/j.bmcl.2014.12.018
0960-894X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Zhu, C.-F.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.12.018

2
C. -F. Zhu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
O
O
OBn
c
a
OBn
OH
b
N
R
O
2
1
3
O
O
5a R= C2H5
OBn
OBn
OH
d
5b R= n-C4H9
5c R= n-C6H13
N
R
4
CHO
N
R
5d R= CH2CH2OCH3
5e R= CH2CH2OBn
5
Scheme 1. Synthesis of benzyl-protected HPOs containing free hydroxyl group (5).
aReagents and conditions:(a) BnCl, NaOH, CH3OH, reflux, 7–8 h, 86%; (b) RNH2,
NaOH, CH3OH/H2O, reflux, 2.5 h, 85–90%; (c) SeO2, CH3COOH/(CH3CO)2O, reflux,
71–80%; (d)NaBH4, rt, 63–70%.
methyl group at 2-position with selenium dioxide in acetic anhy-
dride to give aldehyde 4 in moderate yield.24 Reduction of 4 was
carried out using sodium borohydride as a reducing agent, produc-
ing alcohol 5 in 63–70% yield. Coupling reaction using methyl
5-aminolaevulinate (6) with N-Cbz amino acid (N-Cbz-AA, 7)
proceeded in the presence of 2-(6-chloro-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU) gave
product 8, which was hydrolyzed with lithium hydroxide to car-
boxylic acid 9 (Scheme 2). Conjugation of 9 and 5 was carried
out in the presence of N,N0-dicyclohexylcarbodiimide (DCC) and
catalytic amount of 4-dimethyaminopyridine (DMAP) to give 10,
which were subjected to hydrogenation to remove protecting
groups at both the amino and hydroxyl groups, generating ALA–
HPO conjugates 11.25 Acetylation of 11 was achieved by the treat-
ment with acetic anhydride in pyridine at 0 °C, providing another
group of ALA–HPO conjugates 12 in quantitative yield.26 All the
compounds were fully characterized by 1H NMR, 13C NMR, MS
and HRMS.
In order to investigate the ability of ALA–HPO conjugates to
generate the fluorescent photosensitizer PpIX, the fluorescence
intensity of the peak emission at 635 nm was measured after incu-
bation of the conjugates 11a–j and 12a–j with the human breast
adenocarcinoma cell line (MDA-MB-468) for 4 and 24 h.27 As
shown in Figure 1A, compounds 11 did not markedly enhance
the synthesis of PpIX in MDA-MB-468 cells as compared with
ALA, some of them even led to the production of less PpIX than
ALA. However, compounds 12i, 12f and 12j were found to signifi-
Figure 1. PpIX fluorescence intensities produced after incubation in MDA-MB-468
cell line with 100 mol/L of ALA, ALA–HPO conjugates 11 or 12 for 4 and 24 h at
37 °C and humidified by 5% CO2. (A) Incubation with ALA and compounds 11; (B)
incubation with ALA and compounds 12. Bars represent standard deviation (n = 3).
l
higher than PpIX induced by ALA after 4 h incubation, respectively.
While 24 h incubation periods led to PpIX levels of approximately
3.5, 2.8 and 1.9 times higher by 12i, 12f and 12j than that gener-
ated by ALA. As indicated by the ClogP values28 (Table 1), lipophi-
licities of all the compounds were improved in comparison with
ALA which was expected to facilitate the cellular uptake. It was
reported that increasing the lipophilicity of ALA derivatives to cer-
tain level could negatively influence the uptake of the compound,
leading to accumulation within membranes.29,30 Acetylated com-
pounds (Group 12) are generally found to be more active than
non-acetylated compounds (Group 11), indicating that compounds
12, with higher lipophilicity and non-charged nature, can pene-
trate cell membrane by non-facilitated transport more easily than
cantly increase PpIX production at 100lM (Fig. 1B). According to
PpIX fluorescence intensities, PpIX production in MDA-MB-468
cells treated with 12i, 12f and 12j were 6.1, 4.1 and 4.5 times
O
O
c
O
O
O
O
R1
b
R1
N
H
OH
R1
a
N
H
O
OH
O
NH3
Cl
+
O
NHCbz
O
O
NHCbz
8
NHCbz
7
6
9
R1 = PhCH2
11a: R=C2H5
11b: R=n-C4H9
R1 = Me2CHCH2
11f: R=C2H5
O
O
HO
BnO
O
NHCbz
NH2
O
O
d
H
N
O
11g: R=n-C4H9
H
N
O
R1
N
R
11c: R=n-C6H13
11h: R=n-C6H13
11i: R= (CH2)2OCH3
11j: R= (CH2)2OH
R1
N
R
11d: R= (CH2)2OCH3
11e: R= (CH2)2OH
O
O
O
11
10
R1 = Me2CHCH2
R1 = PhCH2
O
O
HO
12a: R=C2H5
12f: R=C2H5
NH
O
e
H
N
12b: R=n-C4H9
12c: R=n-C6H13
12g: R=n-C4H9
O
O
R1
N
R
12h: R=n-C6H13
12d: R= (CH2)2OCH3
12i: R= (CH2)2OCH3
12j: R= (CH2)2OCOCH3
O
12e: R= (CH2)2OCOCH3
12
Scheme 2. Synthesis of ALA–HPO conjugates 11 and 12. aReagents and conditions: (a) HCTU, Et3N, rt, overnight, 78–81%; (b) (i) LiOH, CH3OH/H2O; (ii) Amberlite IR-120 resin,
84–89%; (c) 5, DMAP, DCC, DMF/CH2Cl2, 66–79%; (d) H2, Pd/C, 3 h, 94–98%; (e) (CH3CO)2O, pyridine/0 °C, 5 min, quantitative.
Please cite this article in press as: Zhu, C.-F.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.12.018

C. -F. Zhu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
with the various prodrugs, efficient cell killing was observed at con-
centrations ranging from 10 to 200 M (Fig. 3). Such phototoxic data
LD50 values and ClogP of compounds 12 and 11
l
is important as there is a strong correlation between PpIX genera-
tion and cell survival. The ALA–HPO conjugates that exhibited a
marked enhancement of PpIX fluorescence also exhibited high
photo-toxicity. The percentage of cell survival with respect to the
control was calculated for various concentrations of the investigated
compounds. Associated LD50 values were calculated for all the
investigated compounds 12 (Table 1). Compounds 12i, 12j and 12f
were found to be the most efficient phototoxic conjugates on the
Compd
LD50
(lM)
ClogP
Compd
ClogP
ALA
12a
12b
12c
12d
12e
12f
12g
12h
12i
195
195
207
205
175
192
90
175
185
37
ꢀ3.911
ꢀ0.93
11a
11b
11c
11d
11e
11f
11g
11h
11i
ꢀ1.2
0.132
ꢀ0.138
1.143
0.872
ꢀ1.321
ꢀ1.234
ꢀ1.081
ꢀ0.019
0.991
ꢀ1.592
ꢀ2.208
ꢀ1.351
ꢀ0.289
0.721
MDA-MB-468 cell line, with LD50 values of 37, 62 and 90
lM, while
ꢀ1.472
ꢀ1.743
LD50 of ALA was calculated to be 195 M. The dark toxicity of Groups
l
12j
62
ꢀ1.385
11j
ꢀ2.359
11 and 12 showed no significant cytotoxicity in the absence of light
exposure, regardless of employing higher concentrations than
adopted for the photo-toxicity study. Cells were incubated with
compounds 11, which will adopt a positive charge under physio-
logical conditions. Within Group 12, compounds 12a–12e are less
active than compounds 12f–12j, indicating that the peptide bond
between ALA and phenylalanine is more difficult to be cleaved
by intracellular peptidases than that between ALA and leucine. This
is probably due to the peptide bond between ALA and leucine
being less sterically hindered for cleavage. Surprisingly, among
compounds 12f–12j, compounds 12h and 12g with a relatively
higher lipophilicity, were found to be the least effective. Thus,
hydrophobic side chains at position-1 on pyridinone lead to a
lower rate of cleavage of the ester bond between ALA and HPO.
This may reflect the selectivity of cytosolic esterases. Although
12j has a similar ClogP value to 12g, the acetyl group at the side
chain on HPO is expected to be removed easily by esterases, gener-
ating a hydrophilic hydroxyl group.
Co-administration of ALA and HPO separately has been
demonstrated to enhance PpIX levels in tumor cell lines compared
to ALA used alone.23 In this paper, further investigation on
compounds 12i, 12f and 12j for their efficacy of producing PpIX
in comparison with ALA and deferiprone (3-hydroxy-1,2-dimethyl-
pyridin-4(1H)-one), a clinically used iron chelator, was undertaken.
The compounds exhibited a dose dependent response of porphyrin
the various compounds (500 lM) and maintained in the dark for
24 h before the MTT test. The viabilities of the cells for all the com-
pounds, including ALA were calculated to be greater than 98% of the
viability of the cells with no treatment. Similar results were
obtained for human cervical cancer cells (Hela) (data not shown).
In summary, although ALA is a useful therapeutical agent for
PDT, it is important to improve its efficiency when administered
systemically and intravenously in order to overcome distribution
problems. The enhancing influence of iron chelating agents offers
a promising way to improve ALA–PDT by increasing the cellular
PpIX accumulation. In the present study, a series of ALA–HPO con-
jugates have been synthesized. Lipophilicities of these molecules
were increased as compared to ALA. However, the results revealed
a lack of correlation between lipophilicity of prodrug and efficiency
of PpIX production. Some of ALA–HPO conjugates showed signifi-
cantly elevated cellular PpIX accumulation in the MDA-MB-468
cell line when compared to ALA and a combination of ALA with
production at concentrations ranging between 10–200 lM. Com-
pounds 12i, 12f and 12j were found to be more efficient in PpIX
production compared to the combination of ALA and deferiprone
(Fig. 2). This is understandable because although deferiprone can
penetrate cell membrane and assist in the accumulation of PpIX
induced by ALA, it is unable to enhance the ability of ALA to pene-
trate cell membranes.
The photo-toxicity of prodrugs 12 on the MDA-MB-468 cell line
was also investigated.31 Following illumination of cells incubated
60
50
40
30
20
10
0
ALA+deferiprone
12f
12i
12j
0
50
100
150
200
Prodrug concentration (µM)
Figure 3. Phototoxicity after incubation with ALA and ALA–HPO conjugates 12 at a
range of concentrations in MDA-MB-468 cell line. Cells were incubated with the
compounds for 4 h and irradiated with blue light (5 J cmꢀ2). Cells were assessed by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
24 h after light exposure. (A) Incubation with ALA and compounds 12a–e; (B)
incubation with ALA and compounds 12f–j (n = 5).
Figure 2. Kinetics of PpIX fluorescence in MDA-MB-468 cell line. Fluorescence
measurements after incubation with variable concentration (20–100 mol/L) of
ALA, 12f, 12i and 12j for 6 h, at 37 °C humidified by 5% CO2. Bars represent standard
deviation (n = 5).
l
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C. -F. Zhu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
160.7, 144.8, 139.8, 135.3, 111.4, 60.1, 57.6, 55.5, 50.8, 48.1, 33.9, 27.1, 23.4,
22.4, 22.1. ESI-MS: m/z 412 ([M+H]+); HRMS: calcd. for C19H30N3O7 ([M+H]+),
412.2078; found, 412.2065.
deferiprone. Compound 12i was found to be the most effective in
both enhancement of cellular PpIX level and tumor phototoxicity.
We plan to extend these promising results in an in vivo setting.
26. General procedure for preparation of 12. A mixture of compounds 11 (50 mg),
pyridine (4 mL) and acetic anhydride (0.8 mL) was stirred over an ice-bath for
5 min. 4 mL of ice-water was then added. The stirring was continued for
additional 5 min. The reaction mixture was freeze dried, providing compounds
12 in quantitative yield. Data for 12f: 1H NMR (CD3OD, 500 MHz) d 0.95 (d,
J = 7.0 Hz, 3H, CH3), 0.99 (d, J = 7.0 Hz, 3H, CH3), 1.53 (t, J = 7.5 Hz, 3H, CH3),
1.62 (m, 2H, CH2), 1.72 (m, 1H, CH), 2.02 (s, 3H, CH3), 2.66 (t, J = 6.5 Hz, 2H,
CH2), 2.83 (t, J = 6.5 Hz, 2H, CH2), 4.06 (t, J = 6.5 Hz, 2H, CH2), 4.38 (q, J = 7.5 Hz,
2H, CH2), 4.44 (m,1H, CH), 5.47 (s, 2H, CH2), 6.96 (d, J = 6.5 Hz, 1H, C5-H in
pyridinone), 8.10 (d, J = 6.5 Hz, 1H, C6-H in pyridinone); 13C NMR (125 Hz,
CD3OD) d 205.0, 172.3, 171.2, 144.6, 143.7, 139.6, 136.8, 111.7, 56.1, 52.6, 51.9,
44.2, 39.1, 33.4, 33.2, 31.9, 30.0, 24.8, 17.1, 14.1. ESI-MS: m/z 438 ([M+H]+);
HRMS: calcd. for C21H32N3O7 ([M+H]+) 438.2240; found 438.2226. 12i: 1H NMR
(CD3OD, 500 MHz) d 0.95 (d, J = 6.5 Hz, 3H, CH3), 0.99 (d, J = 6.5 Hz, 3H, CH3),
1.62 (m, 2H, CH2), 1.72 (m, 1H, CH), 2.02 (s, 3H, CH3), 2.65 (t, J = 6.0 Hz, 2H,
CH2), 2.83 (t, J = 6.0 Hz, 2H, CH2), 3.35 (s, 3H, CH3), 3.81 (t, J = 5.0 Hz, 2H, CH2),
4.04 (m, 2H, CH2), 4.42 (m,1H, CH), 4.68 (t, J = 5.0 Hz, 2H, CH2), 5.54 (s, 2H,
CH2), 7.22 (d, J = 7.0 Hz, 1H, C5-H in pyridinone), 8.26 (d, J = 7.0 Hz, 1H, C6-H in
pyridinone); 13C NMR (125 Hz, CD3OD) d 204.9, 174.0, 172.0, 145.8, 142.8,
140.1, 136.5, 111.2, 70.7, 58.0, 56.1, 55.4, 51.9, 40.5, 39.1, 33.8, 27.0, 24.6, 22.0,
21.1, 20.5. HRMS: calcd. for C22H34N3O8 ([M+H]+) 468.2346, found 468.2331;
calcd. for C22H33N3O8Na ([M+Na]+) 490.2165, found 490.2152. 12j: 1H NMR
(CD3OD, 500 MHz) d 0.95 (d, J = 6.5 Hz, 3H, CH3), 0.99 (d, J = 6.5 Hz, 3H, CH3),
1.63 (m, 2H, CH2), 1.72 (m, 1H, CH), 2.02 (s, 3H, CH3), 2.05 (s, 3H, CH3), 2.66 (t,
J = 6.0 Hz, 2H, CH2), 2.83 (t, J = 6.0 Hz, 2H, CH2), 3.92 (t, J = 5.0 Hz, 2H, CH2), 4.05
(m, 2H, CH2), 4.44 (m,1H, CH), 4.54 (t, J = 5.0 Hz, 2H, CH2), 5.48 (s, 2H, CH2),
6.83 (d, J = 7.0 Hz, 1H, C5-H in pyridinone), 8.20 (d, J = 7.0 Hz, 1H, C6-H in
pyridinone); 13C NMR (125 Hz, CD3OD) d 206.0, 204.9, 174.1, 172.2, 146.5,
144.1, 143.4, 139.8, 111.7, 75.1, 66.2, 64.8, 51.9, 40.5, 39.1, 33.9, 27.2, 24.6,
22.0, 21.5, 21.1, 20.5. ESI-MS: m/z 496 ([M+H]+); HRMS: calcd. for C23H34N3O9
([M+H]+) 496.2295, found 496.2286.
Acknowledgments
The authors thank Zhejiang Provincial Natural Science Founda-
tion of China (No. LY12B02014) and Science Technology Depart-
ment of Zhejiang Province, China (No. 2013C24006) for the
financial support and ChemPharm Research LTD, UK.
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c-sterilized 96-well plates (Orange Scientific, Triple Red
Laboratory Technologies) at a density of approximately 5 ꢁ 104 per well for
48 h. The culture medium was removed and the cells were washed with PBS.
The cells were incubated with freshly prepared solutions of ALA and ALA–HPO
18. Liu, F.; Xu, S.; Zhang, C. R. Chin. Med. J. 2004, 117, 922.
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24. Zhang, M. X.; Zhu, C. F.; Zhou, Y. J.; Kong, X. L.; Hider, R. C.; Zhou, T. Chem. Biol.
Drug Des. 2014, 84, 659.
conjugates: 100
concentrations was added to
l
L
serum-free medium containing varying prodrugs
designated series of wells. Each plate
a
contained wells with cells without prodrug as control for subtracting the
natural PpIX fluorescence reading. PpIX fluorescence induced by ALA and ALA–
HPO derivatives was measured from each well with a Perkin-Elmer VICTOR™ X
fluorescence spectrometer plate reader using 405 nm excitation and 635 nm
emission wavelengths with slit widths set to 10 nm and the internal 515 nm
long-pass filter used on the emission side. The spectral scans were recorded
between 600 and 750 nm, identifying PpIX from other porphyrins, if any were
present. The mean fluorescence intensity (expressed in arbitrary units) was
calculated after subtraction the control values. PpIX fluorescence intensities
were recorded over periods of 4, 6, and 24 h with varied concentration of ALA
25. General procedure for preparation of 11. To a suspension of 10 (1 mmol) and
benzyl chloride (2.4 mmol) in MeOH (30 mL) was added 5% palladium/charcoal
(0.15 g). Hydrogenation was carried out at 30 psi H2 for 5–6 h. After filtration
to remove the catalyst, the filtrate was concentrated to dryness. The residue
was purified by crystallization from methanol–diethyl ether. ALA–HPO
conjugates 11 were obtained as hydrochlorides. Data for 11f: Yield: 98%, 1H
NMR (DMSO-d6, 400 MHz) d 0.91 (t, J = 7.2 Hz, 6H, CH3), 1.42 (t, J = 6.8 Hz, 3H,
CH3), 1.58 (t, J = 6.8 Hz, 2H, CH2), 1.73 (m, 1H, CH), 2.56 (t, J = 6.0 Hz, 2H, CH2),
2.78 (t, J = 6.0 Hz, 2H, COCH2), 3.82 (m, 1H, CH), 4.00 and 4.11 (m, 2H, CH2),
4.37 (q, J = 7.2 Hz, 2H, CH2), 5.34 (s, 2H, CH2), 7.44 (d, J = 6.4 Hz, 1H, C5-H in
pyridinone), 8.37 (m, 4H, NH+3 and C6-H in pyridinone), 8.97 (br, 1H, NH); 13C
NMR (100 Hz, DMSO) d 203.9, 171.1, 168.8, 160.2, 145.0, 138.7, 134.8, 111.9,
59.1, 55.4, 50.8, 48.1, 33.9, 27.0, 23.4, 22.4, 22.1, 16.2. ESI-MS: m/z 396
([M+H]+); HRMS: calcd. for C19H30N3O6 ([M+H]+), 396.2129; found, 396.2131.
11i: Yield: 98%, 1H NMR (DMSO-d6, 400 MHz) d 0.90 (t, J = 7.2 Hz, 6H, CH3),
1.58 (t, J = 7.2 Hz, 2H, CH2), 1.73 (m, 1H, CH), 2.56 (t, J = 6.4 Hz, 2H, CH2), 2.77 (t,
J = 6.4 Hz, 2H, CH2), 3.23 (s, 3H, CH3), 3.69 (t, J = 4.8 Hz, 2H, CH2), 3.83 (m, 1H,
CH), 3.99 and 4.10 (m, 2H, CH2), 4.56 (t, J = 4.8 Hz, 2H, CH2), 5.34 (s, 2H, CH2),
7.43 (d, J = 7.2 Hz, 1H, C5-H in pyridinone), 8.27 (d, J = 7.2 Hz, 1H, C6-H in
pyridinone), 8.37 (br, 3H, NH3+), 8.99 (t, J = 5.6 Hz, 1H, NH); 13C NMR (100 Hz,
DMSO) d 203.9, 171.1, 168.8, 161.5, 145.0, 139.8, 134.4, 111.4, 70.4, 58.2, 55.5,
54.9, 50.7, 48.1, 33.9, 27.1, 23.4, 22.4, 22.1. ESI-MS: m/z 426 ([M+H]+); HRMS:
prodrugs (10–200 lM).
28. http://www.molinspiration.com/cgi-bin/properties.
29. De Rosa, F. S.; Lopez, R. F.; Thomazine, J. A.; Tedesco, A. C.; Lange, N.; Bentley,
M. V. Pharm. Res. 2004, 21, 2247.
30. De Rosa, F. S.; Tedesco, A. C.; Lopez, R. F.; Pierre, M. B.; Lange, N.; Marchetti, J.
M.; Rotta, J. C.; Bentley, M. V. J. Controlled Release 2003, 89, 261.
31. Cells were seeded into 96-well plates at a density of ꢂ10 ꢁ 104 per well.
Following incubation for 48 h, the cells were washed with PBS and 100 ll
solutions containing each compound at varying concentrations (between
10 M and 200 M) were added to their designated wells for 4 h incubation
l
l
periods. Each well plate contained three control wells without the compound
and the compound at five different concentrations. The plates were irradiated
with a fluence of 5 J/cm2 using blue light source (Phantom 300 w, Shenzhen
CIDLY Optoelectronic Technology, China). Peak output is ꢂ420 nm, which
overlaps with the PpIX Soret band. Immediately following irradiation, the
medium was replaced and cells were incubated for further 18 h. Cell
cytotoxicity was determined using MTT assay: cells were incubated with
medium containing MTT (1 mg/mL dissolved in full RPMI 1460 for 2 h. The
insoluble end product (formazan derivatives) was dissolved in 100 lL 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 (incubated with the compounds but not 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. The
cells were incubated with the compounds for 4 h, washed, and after 18 h they
were subjected to the MTT assay.
calcd. for C20H32N3O7 ([M+H]+), 426.2235; found, 426.2255. 11j: Yield: 96%, 1
H
NMR (DMSO-d6, 400 MHz) d 0.89 (t, J = 7.2 Hz, 6H, CH3), 1.57 (t, J = 7.2 Hz, 2H,
CH2), 1.72 (m, 1H, CH), 2.54 (t, J = 6.4 Hz, 2H, CH2), 2.76 (t, J = 6.4 Hz, 2H, CH2),
3.74 (t, J = 4.8 Hz, 2H, CH2), 3.82 (m, 1H, CH), 3.98 and 4.08 (m, 2H, CH2), 4.46 (t,
J = 4.8 Hz, 2H, CH2), 5.36 (s, 2H, CH2), 7.48 (d, J = 6.8 Hz, 1H, C5-H in
pyridinone), 8.32 (d, J = 6.8 Hz, 1H, C6-H in pyridinone), 8.36 (br, 3H, NH+3),
8.99 (t, J = 5.6 Hz, 1H, NH); 13C NMR (100 Hz, DMSO) d 203.8, 171.0, 168.8,
Please cite this article in press as: Zhu, C.-F.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.12.018