New 5-Aminolevulinic Acid Esters-Efficient Protoporphyrin Precursors for Photodetection and Photodynamic Therapy

Article abstract of DOI:10.1562/0031-8655(2003)078<0481:NAAEPP>2.0.CO;2

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.

Full text of DOI:10.1562/0031-8655(2003)078<0481:NAAEPP>2.0.CO;2

New 5-Aminolevulinic Acid Esters—Efficient Protoporphyrin Precursors for
Photodetection and Photodynamic Therapy
Author(s): H. Brunner, F. Hausmann, R. Knuechel
Source: Photochemistry and Photobiology, 78(5):481-486.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2003)078<0481:NAAEPP>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282003%29078%3C0481%3ANAAEPP
%3E2.0.CO%3B2
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and
environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published
by nonprofit societies, associations, museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of
BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries
or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research
libraries, and research funders in the common goal of maximizing access to critical research.

Photochemistry and Photobiology, 2003, 78(5): 481–486
New 5-Aminolevulinic Acid Esters—Efficient Protoporphyrin Precursors
for Photodetection and Photodynamic Therapy
{
1
H. Brunner* , F. Hausmann and R. Knuechel
1
2
1
Institute of Inorganic Chemistry, University of Regensburg, Regensburg, Germany and
Institute of Pathology, University of Regensburg, Regensburg, Germany
2
Received 14 May 2003; accepted 10 August 2003
derivative, PPIX, applied systemically, topically or synthesized
from ALA, shows a very short half-life in the body, and photo-
sensitizing effects do not last much longer than 24 h (2). How-
ever, ALA-induced PPIX accumulation is limited by the high
hydrophilicity of ALA, and therefore fairly high doses are needed
to reach clinically relevant concentrations of PPIX. Hydrophilic
compounds such as ALA poorly cross biological barriers like
cellular membranes (3,4). To overcome this problem, ALA esters
have been used as prodrugs instead of ALA itself. Within the cells
the ALA esters are hydrolyzed by unspecific esterases to give the
parent compound ALA. The improvement of bioavailability of
ALA by application of ALA esters is determined by two processes:
the rate of diffusion of the prodrug through the cell membrane
and its rate of enzymatic conversion into ALA (5). Esterified
derivatives of ALA are currently being investigated as a favorable
alternative to ALA to increase cellular PPIX contents. In addition
to dermatology, possible fields of application of ALA ester–
induced PPIX production for PD and PDT are neurosurgery,
pneumology, gastroenterology, gynecology, urology and oral
surgery.
ABSTRACT
Photodetection (PD) and photodynamic therapy (PDT) with 5-
aminolevulinic acid (ALA)–induced protoporphyrin IX
(
PPIX) accumulation are approaches to detect and treat
dysplasia and early cancer in the gastrointestinal tract and in
the urinary bladder. Because ALA-induced PPIX production
is limited, we synthesized ALA ester hydrochlorides 3–22 and
tested them in two different in vitro models (gastrointestinal
tract: HT29–CCD18; urinary bladder: J82–UROTSA). PPIX
accumulation after incubation with 0.12 mmol/L for 3 h and
PPIX accumulation as a function of different incubation times
were measured using flow cytometry. 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide assays were performed
to check cellular dark toxicity. Phototoxicity after irradiation
was tested. ALA nonafluorohexylester hydrochloride 11, ALA
thiohexylester hydrochloride 13 and ALA dibenzyldiester di-
hydrochloride 19 induced appreciably increased PPIX levels
and showed improved phototoxicity compared with the
references ALA hydrochloride 1, ALA hexylester hydrochlo-
ride 3 and ALA benzylester hydrochloride 4. Thus, the new
compounds 11, 13 and 19 are promising compounds for PD
and PDT.
ALA hydrochloride 1 is increasingly replaced by its esterified
derivatives because of their higher PPIX accumulation. In derma-
tology, ALA methylester hydrochloride 2 is used for PDT of basal
cell carcinoma and actinic ceratosis (6). ALA hexylester hydro-
chloride 3 and ALA benzylester hydrochloride 4 are in clinical
trials for PD and PDT of the gastrointestinal tract and the urinary
bladder (Fig. 1).
INTRODUCTION
Photodetection (PD) and photodynamic therapy (PDT) are
techniques currently under clinical assessment for observation
and local destruction of malignant tumors and premalignant
lesions. After application of 5-aminolevulinic acid (ALA), an
increased metabolic formation of protoporphyrin IX (PPIX) is
induced rather specifically in neoplastic tissue. During blue-light
excitation, PPIX with its distinct red fluorescence can be used
for photodetection or applying higher light doses for PDT (1).
In contrast to other photosensitizers such as hematoporphyrin
To enhance PPIX accumulation beyond the level achievable
with ALA hexylester hydrochloride 3 and ALA benzylester
hydrochloride 4, we synthesized the ALA oxaalkylester hydro-
chlorides 5–8, the fluorinated ALA alkylester hydrochlorides 9–12,
the ALA thiohexylester hydrochloride 13, the ALA benzylester
hydrochlorides 14–19 and miscellaneous other ALA ester hydro-
chlorides 20–22. PPIX accumulation and phototoxicity were
evaluated by in vitro studies (7) (Fig. 2).
{
Posted on the website on 2 September 2003
MATERIALS AND METHODS
*
To whom correspondence should be addressed at: Institute of Inorganic
Chemistry, University of Regensburg, D-93040 Regensburg, Germany.
Fax: 49-941-943-4439; e-mail: henri.brunner@chemie.uni-regensburg.de
The synthesis of compounds 2–6 has been described by Takeya (8) and
Berg et al. (9).
Abbreviations: ALA, 5-aminolevulinic acid; BOC, t-butyloxycarbonyl;
DMSO, dimethyl sulfoxide; FCS, fetal calf serum; LD50, lethal dose
Thionyl chloride method (8). An excess of the dry commercially
available alcohol (20 mL) was cooled to 08C, and freshly distilled thionyl
chloride (3.0 mL, 41.4 mmol) was added. ALA hydrochloride (0.5 g, 3
mmol) was added to the solution. The solution was stirred at 608C for 5 h.
Then, the excess of alcohol was evaporated in vacuo. After recrystallization
from methanol–diethylether atꢀ208C, the pure ALA esters 3, 4, 5–8 and 15
were obtained as the hydrochloride salts (Fig. 3).
(
50%); MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; PBS,
phosphate-buffered saline; PD, photodetection; PDT, photodynamic
therapy; PPIX, protoporphyrin IX.
Ó 2003 American Society for Photobiology 0031-8655/03
$5.00þ0.00
4
81

4
82 H. Brunner et al.
Figure 1. ALA hydrochloride 1, ALA methylester hydrochloride 2, ALA
hexylester hydrochloride 3 and ALA benzylester hydrochloride 4.
Figure 3. Synthesis of ALA ester hydrochlorides with thionyl chloride.
Carbodiimide coupling. ALA ester hydrochlorides 9–14 and 16–22 were
prepared in a three-step synthesis consisting of t-butyloxycarbonyl (BOC)
protection of ALA hydrochloride (9), esterification (10) and deprotection.
An aqueous solution of ALA hydrochloride (420 mg, 2.5 mmol) in 5 mL of
water was adjusted to pH 8.5 with 1 N NaOH. After addition of di-tert-
butyldicarbonate (1.16 g, 3 mmol) in 5 mL of dioxan, the solution was
stirred for 18 h at room temperature. Excess of di-tert-butyldicarbonate was
removed by extraction with diethylether. After acidifying the aqueous
solution with 1 N HCl and extraction with ethyl acetate, 5-tert-butyloxy-
carbonyl aminolevulinic acid was obtained as a colorless oil.
acetate–petroleum ether) afforded the pure water-soluble ALA ester
hydrochlorides (Fig. 4).
1
H-nuclear magnetic resonance and mass spectrometry data. Com-
1
pound 7: H-nuclear magnetic resonance (NMR) (dimethyl sulfoxide
[DMSO-D ]): d 1.56 (m, 4H, 2CH ), 2.55/2.80 (AA9BB9 system, 4H,
6
2
2CH
2
), 3.22 (s, 3H, CH
3
2 2
), 3.32 (m, 2H, CH ), 3.95 (s, 2H, CH ), 4.02 (m,
2H, CH
20ClNO
NMR (DMSO-D
(m, 7H, 2CH , CH
CH ), 8.05 (3H, NH ). MS PI-DCI (NH ) C10H20ClNO (269.7) m/z (%):
2
), 8.31 (s, 3H, NH
(253.7) m/z (%): 218.1 (MH-HCl, 100). Compound 8: H-
): d 2.57/2.79 (AA9BB9 system, 4H, 2CH ), 3.30–3.60
), 3.77 (m, 2H, CH ), 3.97 (s, 2H, CH ), 4.11 (m, 2H,
3
). Mass spectrometry (MS) PI-DCI (NH )
3
1
C
10
H
4
6
2
2
3
2
2
To an ice-cooled solution of 300 mg (1.3 mmol) of 5-tert-butyloxy-
carbonyl aminolevulinic acid, 120 mg (1 mmol) of dimethylaminopyridine,
2
3
3
5
1
234.0 (MH-HCl, 100). Compound 9: H-NMR (D O): d 2.73/2.85
2
1
(
.5 mmol of the corresponding commercially available alcohol and 257 mg
1.6 mmol) of 1-ethyl-3-[3-dimethyl(aminopropyl)]carbodiimide hydro-
chloride were added. The solution was stirred for 2 h at 08C and then for
8 h at room temperature. After evaporation of the solvent, the residue was
dissolved in water. Extraction with ethyl acetate, washing the organic layer
with 10% aqueous NaHCO solution and removal of the solvent gave the
BOC-protected ALA esters, which were purified by column chromatogra-
phy (SiO , MeOH–CH Cl 40:1).
(AA9BB9 system, 4H, 2CH
CH ). MS PI-DCI (NH ) C10
HCl, 100). Compound 10: H-NMR (D
2
4H, 2CH ), 4.10 (s, 2H, CH ), 4.57–4.68 (t, J(H-F) 5 13.7 Hz, 2H, CH ),
2
), 4.10 (s, 2H, CH
12ClF NO (349.6) m/z (%): 314.2 (MH-
O): d 2.85/2.90 (AA9BB9 system,
2
), 4.57–4.68 (m, 2H,
2
3
H
8
3
1
2
3
4
2
2
2
3
6.13–6.67 (tt, J(H-F) 5 51.1 Hz, J(H-F) 5 5.5 Hz, 1H, CH). MS PI-DCI
3
(NH ) C10H12ClF NO (381.7) m/z (%): 346.3 (MH-HCl, 100). Compound
3
8
3
1
3
3
11: H-NMR (D
2H, CH ), 2.62/2.79 (AA9BB9 system, 4H, 2CH
(t, J(H-H)56.1 Hz, 2H, CH ). MS PI-DCI (NH
2
O): d 2.40–2.57 (tt, J(H-H) 5 6.1 Hz, J(H-F) 5 19.4 Hz,
2
2
2
2
2
), 4.00 (s, 2H, CH
) C11 13ClF NO
O): d 2.71/
), 4.76 (m, 2H, CH ).
MS PI-DCI (NH ) C12H11ClF19NO3 (499.3) m/z (%): 464.2 (MH-HCl,
2
), 4.34
(413.7)
3
To an ice-cooled solution of 0.25 mmol BOC-protected ALA ester in
mL of ethyl acetate, 5 mL of HCl-saturated ethyl acetate was added.
2
3
H
9
3
1
5
m/z (%): 378.3 (MH-HCl, 100). Compound 12: H-NMR (D
2.84 (AA9BB9 system, 4H, 2CH ), 4.00 (s, 2H, CH
2
After 30 min, the ice bath was removed, and the solution was stirred for
h at room temperature. Removal of the solvent and recrystallization (ethyl
2
2
2
3
3
Figure 2. ALA ester hydrochlorides 5–22.

Photochemistry and Photobiology, 2003, 78(5) 483
Figure 5. PPIX accumulation measured by flow cytometry in colon
carcinoma cell line HT29 induced by the references ALA hydrochloride 1,
ALA hexylester hydrochloride 3, ALA benzylester hydrochloride 4 and
ALA ester hydrochlorides 5–22 (0.12 mmol/L, 3 h).
Figure 4. Synthesis of ALA ester hydrochlorides using carbodiimide
coupling.
1
3
1
00). Compound 13: H-NMR (D
CH ), 1.20–1.40 (m, 6H, 3CH ), 1.55 (m, 2H, CH
CH ), 4.27 (s, 2H, CH ), 8.17 (br. s, 3H, NH
22ClNO S (267.8) m/z (%): 232.2 (MH-HCl, 100). Compound 14: H-
NMR (D O): d 2.70/2.82 (AA9BB9 system, 4H, 2CH ), 3.99 (s, 2H, CH ),
.16 (s, 2H, CH ), 7.48/8.13 (AA9BB9 system, 4H, 4CH). MS PI-DCI
NH ) C12 15ClN (302.7) m/z (%): 267.3 (MH-HCl, 100). Compound
5: H-NMR (D O): d 2.62/2.83 (AA9BB9 system, 4H, 2CH ), 3.98 (s, 2H,
), 5.08 (s, 2H, CH ), 7.19/7.41 (AA9BB9 system, 4H, 4CH). MS PI-
DCI (NH ) C12 15ClFNO (275.7) m/z (%): 240.3 (MH-HCl, 66), 133.1
100). Compound 16: H-NMR (D O): d 2.65/2.80 (AA9BB9 system, 4H,
), 5.10 (s, 2H, CH ), 7.45/7.62 (AA9BB9 system,
H, 4CH). MS PI-DCI (NH ) C13 15ClF NO (325.7) m/z (%): 290.4
O): d 2.65/2.80 (AA9BB9
), 5.10 (s, 2H, CH ), 7.12 (m, 1H, CH).
12ClF NO (329.7) m/z (%): 294.0 (MH-
HCl, 100). Compound 18: H-NMR (D O): d 2.59/2.79 (AA9BB9 system,
H, 2CH ), 3.98 (s, 2H, CH ), 5.17 (s, 2H, CH ). MS PI-DCI (NH
11ClF NO (347.7) m/z (%): 312.1 (MH-HCl, 62.2); 133.1 (100).
Compound 19: H-NMR (D
.98 (s, 4H, 2CH ), 5.05 (s, 4H, 2CH
CH OH, 1% HAc) C18 26Cl (437.3) m/z (%): 365.0 (MH-2HCl,
00). Compound 20: H-NMR (D O): d 2.72/2.87 (AA9BB9 system, 4H,
CH ), 3.22–3.28 (m, 4H, 2CH ), 3.98 (m, 1H, CH), 4.02 (s, 2H, CH ). MS
OH, 1% HAc) C 20Cl (312.6) m/z (%): 203.8 (MH-3HCl,
00). Compound 21: H-NMR (D O): d 2.62/2.81 (AA9BB9 system, 8H,
CH ), 4.15 (s, 4H, 2CH ), 4.23 (s, 4H, 2CH ). MS ESI (CH OH, 1% HAc)
22Cl (361.2) m/z (%): 288.9 (MH-2HCl, 100). Compound 22:
H-NMR (D O): d 2.65/2.83 (AA9BB9 system, 16H, 8CH ), 3.97–4.15 (m,
6H, 8CH ). MS ESI (CH OH/H O, 1% HAc) C25 44Cl 12 (734.5) m/
z (%): 589.2 (MH-4HCl, 15); 295.2 (1/2(M-4HCl) þ H, 100).
2
O): d 0.88 (t, J(H-H) 5 6.8 Hz, 3H,
), 2.79–3.14 (m, 6H,
). MS PI-DCI (NH
FCS. Cells were cultivated in six well plates and incubated with ALA and
ALA esters for 5–180 min at 378C in the dark using an incubation volume
of 2 mL. In subsequent handling, care was taken to avoid exposure to light.
Determination of ALA-induced fluorescence by flow cytometry. After
incubation, cells were trypsinized, removed from the six well plates and
resuspended in medium without FCS to yield a cell concentration of about
3
2
2
3
C
2
2
3
3
)
1
11
H
2
2
2
2
5
(
1
CH
2
5
3
1
H
2
O
5
5310 cells/mL. The cellular fluorescence was quantified by a FACScalibur
2
2
cytometer (Becton-Dickinson, Heidelberg, Germany). ALA-induced fluo-
rescence was excited by an argon ion laser emitting at 488 nm and collected
by a photomultiplier after passing through a 670 nm long-pass filter. The
2
2
3
H
3
1
4
(
2
flow rate was adjusted to about 2000 events/s, and 2 3 10 events were
2
4
CH
2
), 4.08 (s, 2H, CH
2
2
recorded for each sample. The same instrument settings were used for all
experiments. The stability of the cytometer was maintained by weekly
calibration using the AUTOCOMP software (Becton-Dickinson). Data were
recorded and analyzed with the CellQuest program (Becton-Dickinson).
Debris and cell aggregates were excluded from analysis using forward
and side scatter signals. To compensate for autofluorescence, the mean
fluorescence of ALA–ALA ester-incubated cells was determined by cor-
recting with the mean fluorescence of sham-treated control cells in each
individual experiment. To relate fluorescence intensities with PPIX con-
centrations, PPIX levels (ng/mg protein) were calculated by correlating
from a calibration curve obtained by an extraction method as described by
Krieg et al. (11)
Screening of compounds. To identify promising ALA derivatives for PD
and PDT, a screening of ALA hydrochloride 1 and ALA ester hydro-
chlorides 3–22 was performed. The four human cell lines (HT29–CCD18,
J82–UROTSA) were incubated for 3 h with ALA hydrochloride 1 and ALA
ester hydrochlorides 3–22 using a concentration of 0.12 mmol/L. PPIX
accumulation was quantified using flow cytometry. 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out as
described by Hausmann (7) to ensure that ALA and ALA esters did not
show cellular toxicity.
3
H
3
3
1
(
MH-HCl, 100). Compound 17: H-NMR (D
system, 4H, 2CH ), 4.08 (s, 2H, CH
MS ESI (CH OH, 1% HAc) C12
2
2
2
2
3
H
4
3
1
2
4
C
2
2
2
3
)
12
H
5
3
1
2
O): d 2.64/2.81 (AA9BB9 system, 8H, 4CH
2
),
3
(
1
2
2
2
), 7.32 (s, 4H, 4CH). MS ESI
3
H
2 2 6
N O
1
2
2
2
2
ESI (CH
1
4
3
8
H
3 3 3
N O
1
2
2
2
2
3
C
12
H
2 2 6
N O
1
2
2
1
2
3
2
H
4 4
N O
Cell lines. The human adenocarcinoma cell line HT29 and the human
colonic fibroblast cell line CCD18 were used as an in vitro model for the
gastrointestinal tract. For the urothelium the human urothelial carcinoma
cell line J82 and the human urothelial cell line UROTSA were chosen as an
in vitro model. The cell line HT29 [G2] was maintained in Dulbecco
modified Eagle medium (Sigma, Deisenhofen, Germany) supplemented
with 5% fetal calf serum (FCS; Sigma). The cell line CCD18 was
maintained in modified Eagle medium þ 10% FCS. The urothelial cell lines
J82 [G3] and UROTSA were maintained in Roswell Park Memorial
Institute 1640 medium (Biochrom, Berlin, Germany) supplemented with
Time-dependent measurements. To optimize the use of ALA esters in
PD and PDT, the carcinoma cell lines HT29 and J82 were incubated with
ALA hydrochloride 1 and ALA ester hydrochlorides 3, 4, 11, 13 and 19
using different incubation times (5, 30, 180 min, 0.12 mmol/L). After the
respective incubation periods, the cells were washed with PBS, and new
culture medium without ALA or ALA esters was added. After altogether
3 h of metabolization, PPIX accumulation was measured using flow
cytometry.
Phototoxicity measurements. For quantification of PPIX-induced photo-
toxicity, the cell lines HT29 and CCD18 were incubated (0.12 mmol/L, 3 h)
with ALA hydrochloride 1 and ALA ester hydrochlorides 3, 11, 13 and 19
in medium without FCS. After incubation, the cells were irradiated using an
incoherent xenon light source (k 5 590–700 nm). Energy densities of 0–30
5
% FCS, 1% L-glutamine and 1% sodium pyruvate (GIBCO, Eggenstein,
Germany) and were kept at 378C in a humidified atmosphere containing 5%
carbon dioxide. Cells were subcultured before reaching plateau growth
using 0.1% trypsin–0.04% ethylenediaminetetraacetic acid (GIBCO) in
phosphate-buffered saline (PBS; Biochrom). Experiments were performed
with postconfluent cells (cell density/growth period: HT29, 5 3 10 /cm /7
days; CCD18, 1310 /cm /8 days; J82, 6310 /cm /8 days; UROTSA, 63
2
J/cm were applied. Irradiated cells were cultivated for 24 h. Then, they
were trypsinized and seeded in 96 well plates. After 24 h of growth, MTT
tests were performed to examine cellular vitality.
4
2
4
2
3
2
3
2
0 /cm /10 days).
1
Data analysis and statistics. All experiments were performed at least
three times. Data are presented as mean and standard deviation of the
mean. Fluorescence kinetics were fitted according to the Michaelis Menten
model using Sigma Plot 2.0 (Jandel Corporation, San Raphael, CA). For
quantification of phototoxicity, the cellular vitalities in dependence on
applied energy densities were fitted according to a four-parametric function
Incubation. Stock solutions of ALA hydrochloride 1 and ALA ester
hydrochlorides 3–22 were prepared in deionized water at a concentration of
0
.6 mol/L and stored at ꢀ208C. For each experiment, the stock solution was
diluted with culture medium without FCS. Before addition of ALA–ALA
ester solutions, the cell layer was rinsed with PBS to remove remaining

4
84 H. Brunner et al.
Figure 6. PPIX accumulation measured by flow cytometry in urothelial
carcinoma cell line J82 induced by the references ALA hydrochloride 1,
ALA hexylester hydrochloride 3, ALA benzylester hydrochloride 4 and
ALA ester hydrochlorides 5–22 (0.12 mmol/L, 3 h).
Figure 8. PPIX ratios (J82–UROTSA) induced by the references ALA
hydrochloride 1, ALA hexylester hydrochloride 3 and ALA ester hydro-
chlorides 11, 13 and 19 (0.12 mmol/L, 3 h).
using Sigma Plot 2.0. Using these data, values of lethal dose (50%) (LD50
were determined.
)
Compared with HT29 the colon fibroblast cell line CCD18 led to
very low PPIX accumulation levels, ranging from 3.9 to 6.3 ng
PPIX/mg protein (7) (data not shown). For the most successful com-
pounds 11, 13 and 19 the PPIX accumulation in CCD18 cells can be
calculated from the HT29–CCD18 ratios given together with those
of the reference compounds 1 and 3 in Fig. 7 (see below).
PPIX levels induced in J82 cells by ALA hydrochloride 1 and
ALA ester hydrochlorides 3–22 ranged from 14.2 to 69.8 ng PPIX/
mg protein as shown in Fig. 6. It is obvious that the overall trend
is very similar to the results for the HT29 cells shown in Fig. 5.
Importantly, the fluorinated ALA ester hydrochlorides 10–12, ALA
thiohexylester hydrochloride 13 and ALA benzyldiester dihydro-
chloride 19 exceeded the reference compounds 3 and 4. Thus,
compared with the HT29 cells in the experiments with J82 cells the
fluorinated compound 12 improved and the benzylester 14 fell
back, leaving 11, 13 and 19 as the most promising compounds
successful in both systems.
RESULTS
In a flow cytometric screening, the PPIX accumulation efficiency
of ALA hydrochloride 1 and ALA ester hydrochlorides 3–22 was
examined. The resulting PPIX levels in HT29 after incubation with
0
.12 mmol/L for 3 h are presented in Fig. 5. PPIX accumulation
ranged from 9.3 to 292.1 ng PPIX/mg protein. ALA hydrochloride
induced a very low PPIX level, whereas the commercial ALA
1
hexylester hydrochloride 3 and ALA benzylester hydrochloride 4
established PPIX levels of the order of 180–195 ng PPIX/mg
protein, which successful new ALA esters have to exceed. ALA
oxaalkylester hydrochlorides 5–8 and ALA ester hydrochlorides
2
0–22 were not able to generate significant PPIX levels except 21,
which matched the levels generated by 3 and 4.
In the series of the fluorinated ALA alkylester hydrochlorides
PPIX ratios between tumor cells and normal cells were formed
for the pairs HT29–CCD18 (gastrointestinal tract model) and J82–
UROTSA (urinary bladder model). Enrichment factors for 11, 13
and 19 are 45–55 for HT29–CCD18 (Fig. 7) and 1.7–2.2 for J82–
UROTSA (Fig. 8) in both systems, being higher than for the
reference compounds 1 and 3.
MTT assays were performed to ensure that the new ALA ester
hydrochlorides did not show dark toxicity. No significant cellular
dark toxicity was obtained for any MTT-tested compound (data not
shown), when compared with sham-treated controls (7).
9
–13, the short-chain C derivative did not achieve the level of 3
4
and 4, whereas the long-chain C
middle-chain C5- and C derivatives 10 and 11 definitely
outperformed references 3 and 4 in the PPIX accumulation (Fig.
7
derivative did. Interestingly, the
6
5
). The same was true for ALA thiohexylester hydrochloride 13.
In the group of the ALA benzylester hydrochlorides 14–19
the fluorinated ALA benzylester hydrochlorides 15–18 generated
similar PPIX contents as 3 and 4. Interestingly, ALA nitro-
benzylester hydrochloride 14 and ALA dibenzyldiester dihydro-
chloride 19 surpassed 3 and 4 appreciably (Fig. 5).
Figure 9. Time-dependent PPIX accumulation in HT29 induced by the
references ALA hydrochloride 1, ALA hexylester hydrochloride 3, ALA
benzylester hydrochloride 4 and ALA ester hydrochlorides 11, 13 and 19.
Abscissa: incubation time with ALA derivatives followed by 3 h of
metabolization as described in the Materials and Methods.
Figure 7. PPIX ratios (HT29–CCD18) induced by the references ALA
hydrochloride 1, ALA hexylester hydrochloride 3 and ALA ester hydro-
chlorides 11, 13 and 19 (0.12 mmol/L, 3 h).

Photochemistry and Photobiology, 2003, 78(5) 485
Figure 12. LD50 ratios (HT29–CCD18) induced by the references ALA
hydrochloride 1, ALA hexylester hydrochloride 3 and ALA ester hydro-
chlorides 11, 13 and 19.
Figure 10. Time-dependent PPIX accumulation in J82 induced by the
references ALA hydrochloride 1, ALA hexylester hydrochloride 3, ALA
benzylester hydrochloride 4 and ALA ester hydrochlorides 11, 13 and 19.
Abscissa: incubation time with ALA derivatives followed by 3 h of
metabolization as described in the Materials and Methods.
hexylester hydrochloride 3 induced a remarkable decrease in
cellular vitality already at about 3.5 J/cm . For the new compounds
2
1
using energy densities higher than 1 J/cm .
1, 13 and 19, a steep decrease in cellular vitality was observed
The time dependence of PPIX accumulation is shown in Fig.
2
9
(HT29) and Fig. 10 (J82). In the measurement of the time-
The higher phototoxicity of ALA ester hydrochlorides 11, 13
and 19 with respect to ALA 1 and ALA hexylester hydrochloride
3 is a consequence of higher PPIX accumulation. Thus, energy
densities necessary for reducing cellular vitality (LD50 values)
dependent PPIX accumulation in HT29 cells (Fig. 9), ALA 1 gave
low PPIX levels, whereas ALA hexylester hydrochloride 3 and
ALA benzylester hydrochloride 4 induced a steep increase in PPIX
formation. Interestingly, the new compounds 11 and 13 showed the
same steep increase, achieving PPIX levels clearly above those
achieved by 3 and 4. The curve of 19 grew more slowly. However,
after 10 min it exceeded the curves of 3 and 4 and continued to
grow after 50 min, producing the highest PPIX levels of all the
compounds tested.
could be minimized by the new compounds 11, 13 and 19 (LD :
5
0
2
1
.8–2.1 J/cm ) compared with the references ALA 1 (LD50: 9.7 J/
2
2
cm ) and ALA hexylester hydrochloride 3 (LD50: 4.3 J/cm ).
The ratios of LD50 values between tumor cells and normal cells
(HT29–CCD18) were calculated (Fig. 12). Small LD50 ratios mean
a predominant destruction of cancerous cells compared with
normal tissue under identical conditions. In this regard, LD50 ratios
were greatly reduced by ALA ester hydrochlorides 11, 13 and 19
In the time-dependence studies of PPIX accumulation in the cell
line J82 (Fig. 10), ALA 1 formed more PPIX than in the HT29 cell
line, although it still strongly fell behind all the esters that show
a behavior similar to that shown in Fig. 9. After about 30 min, the
curves of the new esters 13 and 19 exceeded the curves of 3 and 4.
The curve of the fluorinated ALA ester hydrochloride 11 showed the
steepest increase in PPIX accumulation and stayed above the curves
of all the other tested compounds within the time period of 3 h.
To examine the efficiency of the new compounds for PDT,
phototoxicity experiments were performed. In Fig. 11, phototox-
icity curves of ALA hydrochloride 1, ALA hexylester hydrochlo-
ride 3 and the new ALA ester hydrochlorides 11, 13 and 19 are
shown for HT29. Cellular vitalities were obtained using MTT
assays (7) correlated to nonirradiated controls. The curve of ALA 1
demonstrated that no decrease in the cellular vitality occurred for
(0.11–0.18). The references ALA hydrochloride 1 (0.52) and ALA
hexylester hydrochloride 3 (0.32) showed much higher ratios.
DISCUSSION
In vitro studies of ALA esters had shown that ALA hexylester
hydrochloride 3 and ALA benzylester hydrochloride 4 induced
high intracellular PPIX levels (6). Lengthening or shortening as
well as branching the alkyl chain of the esters led to a reduction in
PPIX accumulation (12). The present study examines the effects of
new ALA esters on ALA-induced PPIX accumulation in two in
vitro models for the gastrointestinal tract and the urinary bladder.
Several factors contribute to the success of ALA esters. Fast
cellular uptake and rapid ester hydrolysis are prerequisites for high
intracellular PPIX levels induced by ALA esters. Both diffusion
across cell membranes and enzymatic conversion into ALA are
influenced by steric and electronic effects.
2
applied energy densities smaller than 5 J/cm , whereas ALA
Depending on the solubility of ALA hydrochloride 1 in the
corresponding alcohol, two different procedures, the thionyl
chloride method and the carbodiimide coupling involving BOC
protection, esterification and deprotection, were used to synthesize
ALA ester hydrochlorides 3–22 in good yields.
Fluorinated ALA alkylesters, especially ALA nonafluorohexy-
lester hydrochloride 11, induced high PPIX levels. Obviously, the
electron-withdrawing effect of the fluoro substituents activated the
esters for nucleophilic attack, and ester hydrolysis occurred more
rapidly than in nonactivated esters, leading to PPIX contents,
which exceeded those achievable with the references ALA
hexylester hydrochloride 3 and ALA benzylester hydrochloride
4. Because ALA amides afforded only low PPIX levels (7), we
Figure 11. Photoxicity induced by the references ALA hydrochloride 1,
ALA hexylester hydrochlorides 3 and ALA ester hydrochlorides 11, 13 and
19 in HT29.

4
86 H. Brunner et al.
investigated ALA thiohexylester hydrochloride 13, the thio
analogue of ALA hexylester hydrochloride 3, which also should
be hydrolysed easily. In fact, ALA thiohexylester hydrochloride 13
gave a distinctly higher PPIX accumulation than ALA hexylester
hydrochloride 3. In this case, a beneficial effect for PPIX
accumulation could be the stimulation of ALA dehydrogenase by
the hexanethiol formed during hydrolysis (13). In our ester
screening, ALA dibenzyldiester dihydrochloride 19, containing
two times the effective benzylester substructure, turned out to be
another promising prodrug exceeding the PPIX levels established
by the references 3 and 4.
2. Kennedy, J. C., R. H. Pottier and D. C. Pross (1990) Photodynamic
therapy with endogenous protoporphyrin IX: basic principles and
present clinical experience. Photochem. Photobiol. 6, 143–148.
3
. Eleouet, S., N. Rousset, J. Carre, L. Bourre, V. Vonarx, Y. Lajat,
G. M. J. Beijersbergen van Henegouwen and T. Patrice (2000) In vitro
fluorescence, toxicity and phototoxicity induced by delta-aminolevu-
linic acid (ALA) or ALA-esters. Photochem. Photobiol. 71, 447–454.
4. Kloek, J. and G. M. J. Beijersbergen van Henegouwen (1996) Prodrugs
of 5-aminolevulinic acid for photodynamic therapy. Photochem.
Photobiol. 64, 994–1000.
5
6
. Buundgard, H. (1985) Design of Prodrugs. Elsevier, New York.
. Gierskcky, K. E., J. Moan, Q. Peng, H. Steen, T. Warloe and A.
Bjorseth (1996) Preparation of esters of 5-aminolevulinic acid as
photosensitizing agents in photochemotherapy. PCT Int. Appl. WO
Summarizing, in a series of experiments, new ALA ester
hydrochlorides, in particular 11, 13 and 19, proved to be superior
to ALA hydrochloride 1 as well as its hexyl and benzyl esters 3 and
9
628412.
7
8
9
. Hausmann, F. (2003) 5-Aminol a¨ vulins a¨ ureester in Tumortherapie und
Tumordiagnostik. Ph.D. thesis, University of Regensburg, Regensburg,
Germany.
. Takeya, H. (1992) Preparation of 5-aminolevulinic acid alkyl esters as
herbicides. Jpn. Kokai Tokkyo Koho, JP4009360. [Chem. Abstr. 116,
P189633]
. Berg, Y., A. Greppi, A. O. Siri, R. Neier and L. Juillerat-Jeanneret
(2000) Ethylene glycol and amino acid derivatives of 5-aminolevulinic
acid as new photosensitizing precursors of protoporphyrin IX in cells.
4, respectively. Measurement of PPIX accumulation (0.12 mmol/L
for 3 h) and determination of time-dependent PPIX formation
showed that ALA ester hydrochlorides 11, 13 and 19 induced higher
PPIX levels compared with compounds 1, 3 and 4. Furthermore,
PPIX ratios between tumor cells and normal cells were increased for
11, 13 and 19. As a consequence of the higher PPIX accumulation,
the new ALA ester hydrochlorides 11, 13 and 19 afforded improved
phototoxicity with respect to the reference substances, resulting in
lower LD50 values. Thus, the new ALA ester hydrochlorides 11, 13
and 19 exhibiting no dark toxicity are promising candidates for
photodynamic diagnosis and PDT. Further investigations on animal
models such as ulcerative colitis will have to demonstrate the
suitability of the new esters 11, 13 and 19 (14). It is well known that
in vitro properties of ALA esters are quite different from their in vivo
properties. Therefore, esters poorly performing in this study might
show better performance in in vivo studies.
J. Med. Chem. 43, 4738–4746.
10. Dhaon, M. K., R. K. Olson and K. Ramasamy (1982) Esterification
of N-protected a-amino acids with alcohol/carbodiimide/4-(dimethyl-
amino)-pyridine. Racemization of aspartic and glutamic acid deriva-
tives. J. Org. Chem. 47, 1962–1965.
1. Krieg, R. C., S. Fickweiler, O. S. Wolfbeis and R. Knuechel (2000)
Cell-type specific protoporphyrin IX metabolism in human bladder
cancer in vitro. Photochem. Photobiol. 72, 226–233.
12. Brunner, H., F. Hausmann, R. C. Krieg, E. Endlicher, J. Sch o¨ lmerich,
R. Kn u¨ chel and H. Messmann (2001) The Effects of 5-aminolevulinic
acid esters on protoporphyrin IX production in human adenocarcinoma
cell lines. Photochem. Photobiol. 74, 721–725.
1
1
3. H u¨ ckel, D. and D. Beyersmann (1979) Rapid purification and direct
spectrophotometric assay for 5-aminolevulinic acid dehydratase. Anal.
Biochem. 97, 277–281.
Acknowledgement—This study was supported by grant 96.081.2 from the
Wilhelm-Sander-Stiftung.
1
4. Endlicher, E., P. R u¨ mmele, F. Hausmann, R. Krieg, R. Kn u¨ chel,
H. C. Rath, J. Sch o¨ lmerich and H. Messmann (2001) Protoporphyrin
IX distribution following local application of 5-aminolevulinic acid
and its esterified derivatives in the tissue layers of the normal rat
colon. Br. J. Cancer 85, 1572–1576.
REFERENCES
1
. Doughtery, T. J., J. E. Kaufman, A. Goldfarb, K. R. Weishaupt, D. G.
Boyle and A. Mittleman (1978) Photoradiation therapy for the treatment
of malignant tumors. Cancer Res. 38, 2628–2635.