Synthesis of lactosylated piperazinyl porphyrins and their hepatocyte-selective targeting

Article abstract of DOI:10.1007/s00044-007-9007-x

The aim of this work was the synthesis of a new family of lactosylated piperazinly porphyrins in which the galactoside piperazine moieties are linked to the tetra- and monophenyl ring by an amide bond. Two compounds were designed, synthesized, and characterized by ¹H-nuclear magnetic resonance (¹H-NMR), ultraviolet (UV)-visible, and matrix-assisted laser desorption/ionization (MALDI) mass spectra. The biological activity on cancer cells and the pharmacokinetics have also been evaluated, showing a very high liver-to-skin ratio and short retention time in tissues. It is suggested that such novel lactosylated piperazinyl porphyrins, as potential hepatocyte-selective targeting drugs, thus show promising activity in photodynamic therapy. Birkha?user Boston 2007.

Full text of DOI:10.1007/s00044-007-9007-x

Med Chem Res (2007) 16:28–38
DOI 10.1007/s00044-007-9007-x
ORIGINAL ARTICLE
Synthesis of lactosylated piperazinyl porphyrins and
their hepatocyte-selective targeting
He-Ping Li Æ Zhong Cao Æ Hua-Wu Xiao
Received: 2 November 2006 / Accepted: 16 March 2007 / Published online: 8 June 2007
Birkh a¨ user Boston 2007
ꢀ
Abstract The aim of this work was the synthesis of a new family of lactosylated
piperazinly porphyrins in which the galactoside piperazine moieties are linked to the
tetra- and monophenyl ring by an amide bond. Two compounds were designed,
1
1
synthesized, and characterized by H-nuclear magnetic resonance ( H-NMR),
ultraviolet (UV)-visible, and matrix-assisted laser desorption/ionization (MALDI)
mass spectra. The biological activity on cancer cells and the pharmacokinetics have
also been evaluated, showing a very high liver-to-skin ratio and short retention time
in tissues. It is suggested that such novel lactosylated piperazinyl porphyrins, as
potential hepatocyte-selective targeting drugs, thus show promising activity in
photodynamic therapy.
Keywords Hepatocyte-selective targeting drug ꢀ Lactosylated piperazinyl
porphyrin ꢀ Photodynamic therapy ꢀ Synthesis
Introduction
Recently, promising porphyrin photosensitizers have been developed for photody-
namic therapy (PDT) (Ali and van Lier, 1999; Allison et al., 2004; Venosa et al.,
2
006). Although a number of porphyrin-based photosensitizers have been approved
for clinical trials, very little is known about their mode of action. In addition, they
have demonstrated some obvious drawbacks. Because of their low selectivity, they
enter almost every tissue, and their slow clearance leads to a long-lasting
H.-P. Li (&) ꢀ Z. Cao
College of Chemical and Environmental Engineering, Changsha University of Science and
Technology, Changsha 410076, China
e-mail: lihepinghn@163.com
H.-W. Xiao
Xiangya Hospital, Central South University, Changsha 410083, China

Med Chem Res (2007) 16:28–38
29
photosensitivity, causing skin sensitivity for several weeks (Sharman et al., 1999).
Moreover, PDT treatment also was not suitable for therapy of cancer affecting deep
tissues (Yuan and Jie, 1984). As a result, new photosensitizers exhibit high
selectivity for neoplastic cells, fast elimination from healthy tissues, and strong light
absorption in the red region of the visible spectrum.
Although the exact mechanism of sensitizer uptake by tumor cells is still
undetermined, there is evidence that, besides the subtle balance between
hydrophobicity and hydrophilicity, charge distribution, symmetry, and configuration
of the molecules are important factors influencing the selective accumulation and
subcellular distribution. The asialoglycoprotein receptor (ASGR) is known to be
present only on hepatocytes at a high density of 500,000 receptors per cell, and
retained on several human hepatoma cell lines. This receptor system not only can
recognize terminal b-D-galactose or N-acetylgalactosamine residues, but can also
internalize them within membrane-bound vesicles or endosomes (Kim et al., 2004).
ASGR are considered as a particularly attractive target in many drug carrier studies.
The use of such nature molecules with galactosylated or lactosylated residues in
drug targeting carriers has resulted in significant targeting efficacy to the liver (Han
et al., 2001). Galactose is known as a specific adhesive ligand to ASGR of
hepatocyte. Lactobionic acid (LA), bearing a galactosyl group, is usually used as a
recognition moiety for the hepatocyte-targeting carrier (Gao et al., 2003; Wang
et al., 2006). Therefore, many amphiphilic porphyrins linked to sugar moieties have
been synthesized, and notably porphyrins bearing one or two carbohydrate
substituents gave promising results (Sylvian et al., 2002; Ahmed et al., 2004; Chen
et al., 2004; Tome et al., 2005; Sol et al., 2006). Such conjugates increase the water
solubility of the parent porphyrin, which contributes to their elimination from the
organism after treatment.
As part of our research program on the synthesis of such compounds (Guo et al.,
003; Li, 2006), we are interested in the preparation and biological evaluation of
2
lactosylated piperazinyl porphyrins obtained from synthetic precursors. In compar-
ison with lactosylated porphyrins, lactosylated piperazinyl porphyrins could
increase anticancer activity, because piperazine-containing compounds are widely
used in the pharmaceutical industry; some substituted piperazine compounds
especially have distinctive anticancer activity (Hagihara et al., 1999; Hepperle et al.,
1
999; Guo et al., 2003; Li, 2006). In the present article, the synthesis of two
lactosylated piperazinyl porphyrins and an evaluation of their biological activity are
reported.
Materials and Methods
Apparatus and reagents
Infrared spectra were recorded with a Nicolet Avator 360 spectrophotometer.
1
1
H-nuclear magnetic resonance ( H-NMR) spectra was recorded with a Varian FT-
0A spectrophotometer(400 MHz), and the chemical shifts was reported on the d
scale relative to trimethylsilane (TMS; solvent: CDCl ). Elemental analyses were
8
3

30
Med Chem Res (2007) 16:28–38
obtained via a PE 2400. Mass spectra were obtained via a QP-5000 mass
spectrophotometer. Ultraviolet (UV)-visible absorption spectra were measured with
an UV 2100 spectrophotometer. All fluorescence measurements were made with a
Hitachi M850 fluorescence spectrophotometer using a 1-cm quartz cell, and with
both excitation slit and emission slit at 10 nm. All pH measurements were made
with a digital pH and temperature meter 631 (Extech, Boston, MA).
Column chromatography was performed using alumina (200–300 mesh).
Lactobionic acid was purchased from Fluka Chemical (Buchs, Switzerland). 5-
0
Fluorouracil (5-Fu), N,N -Dicyclohexylcarbodiimide (DCC) and 3-(4,5-dim-
ethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma-Aldrich (St. Louis, MO). Three human tumor cell lines—Bel-7404, MCG
and HNE1—were obtained from Shanghai Institute of Cell Biology, Chinese
Academy of Science. The pyrrole and propionic acid were distilled before use. All
other chemicals were used as received.
Synthesis of N-phenylpiperazine
A mixture of aniline hydrochloride (0.04 mol) and diethanolamine hydrochloride
0.04 mol) was heated at about 220–2408C for 6–8 h. After cooling, the gummy
(
solid was treated with concentrated sodium hydroxide and the resulting oil was then
distilled under vacuum at 10 mm Hg ( b.p. 139–1418C). The light yellow oil was
obtained (3.9 g, 60%). MS (m/z): 162. IR (KBr): 3200 (–NH–), 1600, 1500 (Ar C–
ꢁ
1 1
H) cm . H NMR (CDCl ): d2.75–3.15 (m, 8H, NCH ), 6.85–7.38 (m, 5H, ArH).
3
2
0
Synthesis of N-lactobionylamino-N -phenylpiperazine
A mixture of N-phenylpiperazine (0.02 mol), lactobionic acid (0.05 mol),
tetramethylethylene diamine solution of DCC (0.04 mol), and benzene (100 ml)
was stirred at 28–408C for 72 h. The solvent was removed and the product was
purified by column chromatography (neutral alumina, 200–300 mesh; ethanol).
Yield, 7.2 g, 70%. MS (m/z): 516. IR (KBr): 3400 (O–H), 1657 (C=O), 1605, 1500
ꢁ1 1
(
Ar C–H), 755 (C–H) cm . H NMR (CDCl ): d2.35–3.27 (m, 10H, NCH ), 3.42–
3
2
4
.45 (m, 13H, lactobionyl), 6.65–7.57 (m, 5H, ArH).
0
Synthesis of N-lactobionylamino-N -(4-formylphenyl)piperazine
0
N-Lactobionylamino-N -phenylpiperazine (2.0 mmol) was dissolved in dimethyl-
formamide (10 ml). The solution was cooled in an ice bath. Phosphoryl trichloride
(
2.0 mmol) was added in small portions and the temperature was kept below 08C
with an ice bath. The mixtures were continuously stirred for 30 min at 08C, then
stirred at 908C for 1.5 h. The mixtures were poured into crushed ice (400 ml) with
vigorous stirring, neutralized to pH 5 with 5 M NaOH solution, and stored in the
refrigerator overnight. The mixtures were extracted with CHCl and the organic
3
layer was washed with water and dried with Na SO overnight. The product was
2
4
purified by column chromatography (neutral alumina, 200–300 mesh; ethanol).
ꢁ
1
1
Yield, 0.816 g, 75%. MS (m/z): 544. IR (KBr): 1670 (C=O) cm , H NMR

Med Chem Res (2007) 16:28–38
31
(
CDCl ), d2.40–3.27 (m, 10H, CH N), 3.45–4.55 (m, 13H, lactobionyl), 6.83–7.67
3
2
(
m, 4H, ArH), 9.76 (s, 1H, CHO).
0
Synthesis of 5,10,15,20-tetra[4-(4 -
lactobionylaminopiperazinyl)phenyl]porphyrin (TLPP)
0
A mixture solution of N-lactobionyl-N -(4-formylphenyl)piperazine (2.0 mmol),
pyrrole (2.0 mmol), and propionic acid (100 ml) was refluxed for 30 min and then
stored overnight at 08C. The purple precipitate was obtained. The precipitate was
purified via column chromatography (neutral alumina, 200–300 mesh; ethanol–
1
acetone, 20/80) to obtain TLPP as a purple solid (156 mg, 13.5%). H NMR
(
(
CDCl ):d8.62–8.66 (m, 8H, pyrrolic), 6.97–7.33 (m, 16H, ArH), 2.82–3.57
3
m, 32H, N-CH ), 4.22–4.54 (m, 52H, lactobionyl C–H), 2.08 (s, 32H, lactobionyl
ꢁ1
2
ꢁ3
ꢁ1
O–H), –2.46 (s, 2H, NH). UV-vis[kmax, nm (e · 10 cm mol L)] in CH Cl :
4
Anal. calcd. for C108H142N O : C, 56.10; H, 6.15; N, 7.27; O, 30.48%; found: C,
2
2
+
27 (204.9), 526 (17.6), 569 (22.1), 614 (19.2), 658 (10.8). MS (m/z): 2311(M +1).
12
44
ꢁ1
5
6.08; H, 6.18; N, 7.25; O, 30.49%. IR (KBr): 3324, 1640, 1606, 1512 cm .
0
Synthesis of 5-mono[4-(4 -lactobionylaminopiperazinyl)phenyl]-10,15,20-
triphenyl- porphyrin (MLPP)
0
N-Lactobionyl -N -(4-formylphenyl)piperazine (0.5 mmol ), benzaldehyde (1.5
1
mmol) and pyrrole (2.0 mmol) gave MLPP (90.3 mg, 17.4%). H NMR
(
(
CDCl ):d8.72–8.84 (m, 8H, pyrrolic), 7.03–7.34 (m, 19H, ArH), 2.85–3.59
3
m, 8H, N-CH ), 4.24–4.57 (m, 13H, lactobionyl C–H), 2.10 (s, 8H, lactobionyl
2
ꢁ
3
ꢁ1
ꢁ1
O-H), –2.69 (s, 2H, NH). UV-vis[kmax, nm (e · 10 cm mol L)] in CH Cl :
4
Anal. calcd. for C H N O : C, 69.36; H,5.59; N, 8.09; O, 16.96%; found: C,
2
2
+
24 (255.8), 524 (24.5), 562 (21.3), 617 (20.3), 662 (10.1). MS (m/z): 1039 (M +1).
6
0 58 6 11
ꢁ
1
6
9.47; H, 5.54; N, 8.01; O, 16.98%. IR (KBr ): 3332, 1639, 1605, 1514 cm
.
In vitro studies of TLPP
Irradiation. The light source used was that of a Kodak slide projector equipped with
a 150-W lamp. The light was filtered through a 3-cm water layer to absorb the heat
(
Alvarez et al., 2000).
Cell survival assay. The cytotoxic effects of some compounds on Bel-7404,
MCG, and HNE1 cells were determined via the MTT assay (Carystinos et al., 2001).
3
Cells were planted in 100 ml of medium at a concentration of 1 · 10 cells per well
in 96-well microtiter plates. Plates were then incubated for 18 h at 378C under an
atmosphere of air containing 5% CO . Medium (100 ml) containing the tested drug
2
dissolved in appropriate solvent was added to quadruplicate wells and incubated for
an additional 48 h. The medium was then removed from the wells and 200 ml of
MTT (1 mg/ml in complete medium) was added to each well followed by a 3-h
incubation. The formazan crystals were dissolved in 100 ml of dimethyl sulfoxide
buffered with 25 ml of glycine–NaCl solution (0.1 M glycine, 0.1 M NaCl, pH 10.5).
The absorbance was measured in an enzyme-linked immunoabsorbent assay plate

32
Med Chem Res (2007) 16:28–38
reader (Bio-Rad) at a wavelength of 570 nm. Based on concentration, 50% of cell
death (ID ) was determined for various compounds tested.
5
0
In vivo studies of PDT
Animal and tumor model. Female KM mice 6–8 weeks old (18–22 g) were
maintained in standard cages with free access to tap water and normal dietary chow.
When required, the animals were anesthetized with Ketalar (150 mg/kg, by i.p.
injection). The experimental tumors LM-2, originally obtained from Xiangya
Hospital (Changsha, China), were subcutaneously implanted by a sterile injection of
6
ca. 10 cells in phosphate-buffered saline into the right foreleg. The pharmacoki-
netic and/or phototherapeutic experiments were performed on the sixth day after
implantation, when the tumor was 0.6–0.7 cm in outer diameter. Spontaneous tumor
necrosis was minimal or absent for these tumor sizes.
Pharmacokinetic studies. TLPP (10 mg/kg) was administered by i.p. injection.
The animals were killed during different points of time (six mice at 24 h and three
mice at other points of time), varying between 1 h and 1 week after administration.
The skin, muscle, tumor, and the liver were recovered. About 200 mg of tissues
were homogenized in THF. The homogenates were centrifuged at 3000 rpm for 15
min, and the fluorescence of the supernatants was measured, setting the excitation
wavelength at 420 nm and recording the emission spectrum from 500 and 800 nm.
Serum samples, isolated from the blood by centrifugation, were diluted with suitable
volumes of 500 ml of tetrahydrofuran and the TLPP content was measured by
fluorescence. In all the cases, TLPP amounts were determined via interpolation of
emission intensity and TLPP concentration was plotted on a standard curve.
Results and Discussion
Synthesis and characterization of lactosylated piperazinyl porphyrins
An approach to porphyrin-based drugs makes use of a convenient reaction for
converting an aldehyde and pyrrole to the corresponding meso-substituted
porphyrin. The reaction provides a means for converting prefunctionalized
benzaldehyde to the corresponding porphyrin. In this study, the functional groups
selected were the drugs that are used in the treatment of cancers. We now report the
synthesis and the biological activity studies of lactosylated piperazinyl porphyrins.
The preparation of lactosylated piperazinyl porphyrins is summarized in Scheme 1.
While this work was in progress, different piperazinylporphyrins (Guo et al., 2003;
Li, 2006) have been reported.
The structures of lactosylated piperazinyl porphyrins were characterized by
1
elementary analysis, mass spectroscopy (MS), H-NMR, infrared (IR), and UV-vis.
1
The H NMR spectra at –2.46 to –2.69 ppm showed the characteristic single peak
for each porphyrin. Mass spectrometry of porphyrins was performed using the
matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) technique.
Positive ion mass spectra exhibited a base peak corresponding to the intact

Med Chem Res (2007) 16:28–38
33
CHO
NH
2
HN(CH
2
CH
2
OH)
2
DMF
POCl
DCC
LA
N
R
NH
N
R
3
N
1
3
2
R'
CHO
n
R'
CH CH COOH
3
2
N
HN
N
CHO
R
R'
NH
reflux 0.5 h
(4-n)
4
N
H
MLPP (n = 1, R= H)
TLPP (n = 4, R= R')
R
H
OH H
H
O
H
OH H H
N
HOOC
CH
2
OH
C
2
CH OH
R' =
N
OH H
HO
OH H
HO
O
OH
O OH
OH
OH
O
OH
OH
O
Lactobionic acid
LA)
(
OH
OH
Scheme 1 The synthesis of TLPP and MLPP
porphyrin and no fragment ions were detected. Analysis of the isotopic components
+
indicated the presence of a protonated species (M+H) with a minor contribution of
+
the radical cation M ꢀ, allowing the determination of the molecular mass with an
accuracy generally around 0.001%. Its structure was further confirmed by the
elementary analysis.
The key to the synthesis of lactosylated piperazinyl porphyrins is how to
introduce lactosylated piperazine to the porphyrin ring. TLPP is a symmetrical
lactosylated piperazinyl porphyrin, but MLPP is asymmetrical. We had tried to
couple iodoporphyrin with various lactosylated piperazine to synthesize target
molecules at room temperature according to the Buchwald method (Wolfe and
Buchwald, 1997). However, the reaction did not proceed, even after stirring over an
extended period of time. The coupling of aminophenylporphyrin with N,N-di(2-
bromoethyl)lactosamide at room temperature can produce the corresponding
lactosylated piperazinyl porphyrin, but the components of products are too
complicated to purify. In this study, synthesis of lactosylated piperazinyl porphyrins
was realized according to the method of Adler et al. (1967). The procedure consists
0
of the condensation of pyrrole (4 equiv) with N-lactobionylamino-N -(4-formyl-
0
phenyl)piperazine (4 equiv) or N-lactobionylamino-N -(4-formylphenyl)piperazine
(1 equiv) and benzaldehyde (3 equiv). In all cases, these reagents were added to dry
propionic acid as solvent at refluxed temperature, followed by flash chromatography

34
Med Chem Res (2007) 16:28–38
and purification on silica gel TLC, giving porphyrins TLPP and MLPP in 13.5% and
1
N-lactobionylamino-N -(4-formylphenyl)piperazine was prepared according to
7.4% yields, respectively.
0
0
the literature (Amaresh and Perumal, 1998). N-lactobionylamino-N -phenylpiper-
azine was synthesized via the formation of amide bonds between the secondary
amino group of the N-phenylpiperazine and the carboxylic acid group of lactobionic
acid according to the method reported by Bernkop-Schn u¨ rch and Thaler (2000). As
shown in reaction formula, the carboxylic moieties of lactobionic acid were
0
activated by N,N -dicyclohexylcarbodiimide (DCC), forming an intermediate
product, which reacted with the secondary amino group of N-phenylpiperazine.
0
The efficacy of the purification method for the resulting N-lactobionylamino-N -
phenylpiperazine could be verified by controls that were prepared in exactly the
same way as the conjugate but omitting DCC during the coupling reaction,
exhibiting a negligible amount of product. To optimize the synthesis of N-
0
lactobionylamino-N -phenylpiperazine, the influence of the coupling reaction time
was evaluated. Results demonstrated that the yield increased with increasing
reaction time, but when the reaction time lasted more than 72 h, the yield could not
increase obviously, indicating that the coupling reaction already came to equilib-
rium. Other factors that could influence the yield, such as pH of the reaction mixtures
and the weight ratio of N-phenylpiperazine to lactobionic acid, have already been
studied. As far as pH of the coupling reaction was concerned, alkaline pH benefits
the reaction to a greater extent than acidic pH. Therefore, a tetramethylethylene
diamine solution was selected as the catalyst of the coupling reaction.
In vitro anticancer activity
It is well known that substituted tetraphenylporphyrin can be used as a sensitizer in
PDT (Ali and van Lier, 1999; Allison et al., 2004; Venosa et al., 2006), and
nitrogenous heterocycle porphyrins have better anticancer activity than the
corresponding nitrogenous heterocycles in the absence of light (Guo et al., 2003;
Li, 2006). To study whether these synthetic lactosylated piperazinyl porphyrins
have anticancer activity, the cytotoxic effects of TLPP, MLPP, and a common
anticancer drug, 5-fluorouracil (5-Fu), on three human tumor cell lines—Bel-7404
(
a liver cancer cell), MCG (a stomach tumour cell), and HNE1 (a nasopharyngeal
carcinoma cancer cell)—were determined via the MTT assay (Carystinos et al.,
001) with and without irradiated conditions. The results of the prescreenings are
2
given in Fig. 1.
It can be seen from Fig. 1 that the death rates ranged from 24% to 19% for TLPP,
1
2% to 8% for MLPP, and 43% to 29% for 5-Fu when cells were incubated for 18 h
at 378C under an atmosphere of air containing 5% CO without light. Thus TLPP
2
and MLPP showed smaller cytotoxic effects than that of 5-Fu in this condition.
However, the death rates ranged from 96% to 98% for TLPP and 75% to 78% for
2
MLPP after 18 h incubation and 20 J/cm irradiation (Fig. 1), indicating the
presence of photosensitizing toxicity.
Structurally, TLPP and MLPP consist of two parts: tetraphenylporphyrin (TPP)
and a lactosylated piperazine compound. TLPP, MLPP, and two corresponding

Med Chem Res (2007) 16:28–38
35
Fig. 1 Anticancer activity of 5-
Fu, MLPP, and TLPP in cancer
cells (Bel-7404, MCG, HNE1)
determined via MTT assay.
120
HNE1
100
MCG
Bel-7404
Cancer cells were incubated for
1
(
8 h in solution (6.25 mg/ml).
0), without irradiation; (i),
represented with irradiation (at
80
60
40
20
0
2
650 nm and 20 J/cm )
5
-Fu(0) 5-Fu(i) TLPP(0) TLPP(i)MLPP(0)MLPP(i)
Table 1 ID50 values (mg/ml) of TLPP and other compounds
Compound
Bel-7404
MCG
HNE1
TLPP
29
43
35
48
33
46
MLPP
TPP
>50
>50
>50
>50
>50
>50
Lactobionylated piperazine
structural parts, porphyrin TPP and lactosylated piperazine, were also tested in vitro
against bel-7404 liver cancer cells via MTT (Carystinos et al., 2001) in the absence
of light. The ID50 values of the bel-7404 liver cancer cell in response to these
compounds are listed in Table 1.
The test results showed that TLPP and MLPP have smaller ID50 values than those
of TPP and the corresponding lactobionylated piperazine. This indicates that TLPP
and MLPP have better anticancer activity than that of either structure parts of
lactobionylated piperazine porphyrins. At the same time, it can be seen in Table 1
that for TLPP and MLPP, when the numbers of substitution group lactobionylated
piperazine increased, it increased the anticancer activity of the porphyrins. It is
interesting that TPP, which is not an anticancer drug, would be a good carrier for
toxic moieties to arrive at improved anticancer drugs. The possible reason might be
that TPP with anticancer drugs can intercalate strongly into the base pairs of DNA.
TLPP showed good anticancer activity toward cancer cells in the absence of light,
and might be a promising agent in chemotherapy and PDT. The molecular
mechanism for the anticancer activities of TLPP is being studied further.
Pharmacokinetics studies: biodistribution in tumor-bearing mice
The lipophilic and hydrophilic properties were characterized by the partition
coefficient of the compound between the two nonmiscible solvents octanol and
water. The octanol/water partition ratios were 18 for TLPP and 39 for MLPP,

36
Med Chem Res (2007) 16:28–38
Fig. 2 Biodistribution of TLPP
in tumor-bearing mice:
100
80
Recoveries of TLPP from
tumor-bearing female KM mice
injected with 10 mg/kg of drug.
Values represent the averages of
three experiments
3 h post-injection
24 h post-injection
144 h post-injection
60
4
0
0
0
2
liver
tumour
muscle
skin
indicating that TLPP was more hydrophilic than MLPP, but TPP is insoluble in
water. The distribution of TLPP was examined in different selected tissues of
tumor-bearing female KM mice at 3-, 24-, and 144-h postinjection periods (Li,
2
006). The results are shown in Fig. 2.
In vivo results indicate that TLPP is rapidly distributed in tissues and rapidly
eliminated. The highest concentrations were observed in the liver at 3 h
postinjection; the concentration of TLPP decreased rapidly in all tissues after 24
h, and very low sensitizer quantity was detected in these tissues after 144 h. The low
quantity detected in the skin was important, as it would cause photosensitization.
The shorter retention time of TLPP in tissues could decrease prolonged cutaneous
photosensitivity. The higher amounts of TLPP accumulated in the liver could be
explained by the presence of four lactosylated moieties that could recognize ASGR.
Although the efficiency of TLPP incorporation in tumor tissue was not high in
relation to other tissues, a high antitumor activity was observed when cells were
excited by visible light, since a high percentage of cells in apoptosis appeared in
tumors that were obtained from mice treated with TLPP and irradiated. In summary,
the present results show that TLPP is a potential tumor photosensitizer. The further
development of experimental protocols using a given sensitizer in vitro and in vivo
can contribute to the understanding of the photokilling effect in basic oncological
research and to assessment of the potential for clinical applications in the PDT of
cancer, and more extensive biological studies will be reported in the near future.
Conclusion
We have developed a practical and efficient approach to synthesize lactosylated
piperazinyl porphyrins based on the Adler method. Lactosylated piperazinyl
porphyrins showed good anticancer activity toward in the presence of light, and
would be promising agents in chemotherapy and PDT. The incorporation of
lactosylated residues into porphyrin enhanced the liver targetability. Moreover, the
results of intrahepatic distribution provided evidence that the recognition of

Med Chem Res (2007) 16:28–38
37
lactosylated residues of TLPP by ASGR on the surface of parenchymal cells
accounted for the liver accumulation of the drug. These results suggested that
porphyrin containing lactosylated residues could be a useful drug carrier system for
hepatocyteselective targeting.
Acknowledgments The project was supported by the Hunan Provincial Natural Science Foundation of
China (06JJ2038).
References
Adler AD, Longo FR, Finarelli JD, et al. (1967) A simplified synthesis for meso-tetraphenylporphyrin. J
Org Chem 32:476
Ahmed S, Davoust E, Savoie H, et al. (2004) Thioglycosylated cationic porphyrins-convenient synthesis
and photodynamic activity in vitro. Tetrahedron Lett 45:6045
Ali H, van Lier JE (1999) Metal complexes as photo- and radiosensitizers. Chem Rev 99:2379
Allison RR, Downie GH, Cuenca R, et al. (2004) Photosensitizers in clinical PDT. Photodiagn Photodyn
Ther 1:27
Alvarez MG, LaPenna M, Yslas EI, et al. (2000) Photrodamaging effects of porphyrin in a human
carcinome cell line. Chem Educat 5:24
Amaresh RR, Perumal PT (1998) A novel one-pot synthesis of 2-aminoquinolines from arylazidoketones
by cyclization under Vilsmeier conditions. Tetrahedron 54:14327
Bernkop-Schn u¨ rch A, Thaler SC (2000) Polycarbophil-cysteine conjugates as platforms for oral
polypeptide delivery system. J Pharm Sci 89:901
Carystinos GD, Alaoui-Jamali MA, Phipps J, et al. (2001) Upregulation of gap junctional intercellular
communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated
with a glutathione depletion and chemosensitivity in neuroblastom cells. Cancer Chemother
Pharmacol 47:126
Chen X, Hui L, Foster DA, et al. (2004) Efficient synthesis and photodynamic activity of porphyrin-
saccharide conjugates: targeting and incapacitating cancer cells. Biochemistry 43:10918
Gao S, Chen J, Xu X, et al. (2003) Galactosylated low molecular weight chitosan as DNA carrier for
hepatocyte-targeting. Int J Pharm 255:57
Guo CC, Li HP, Zhang XB (2003) Study on synthesis, characterization and biological activity of some
new nitrogen heterocycle porphyrins. Bioorg Med Chem 11:1745–1751
Hagihara M, Kashiwase H, Katsube T, et al. (1999) Synthesis and anti-HIV activity of arylpiperazinyl
fluoroquinolones: a new class of anti-HIV agents. Bioorg Med Chem Lett 9:3063
Han J, Lim SJ, Lee MK, et al. (2001) Altered pharmacokinetics and liver targetability of methotrexate by
conjugation with lactosylated albumins. Drug Deliv 8:125
Hepperle M, Eckert J, Gala D (1999) Sequential mono-N-arylation of piperazine nitrogens. Part 1: A
0
simplified method and its application to the preparation of a key N,N -biaryl piperazine antifungal
intermediate. Tetrahedron Lett 40:5655
Kim TH, Park IK, Nah JW, et al. (2004) Galactosylated chitosan/DNA nanoparticles prepared using
water-soluble chitosan as a gene carrier. Biomaterials 25:3783
Li HP (2006) Study on synthesis and biological activity of a galactosylated piperazinyl porphyrin. Bioorg
Med Chem Lett 16:6298
Sharman WS, Allen CM, van Lier JE (1999) Photodynamic therapeutics: basic principles and clinical
applications. Drug Discov Today 4:507
Sol V, Chaleix V, Champavier Y, et al. (2006) Glycosyl bis-porphyrin conjugates: synthesis and potential
application in PDT. Bioorg Med Chem 14:7745
Sylvain I, Zerrouki R, Granet R, et al. (2002) Synthesis and biological evaluation of thioglycosylated
porphyrins for an application in photodynamic therapy. Bioorg Med Chem 10:57
Tome JP, Neves MG, Tome AC, et al. (2005) Synthesis of glycoporphyrin derivatives and their antiviral
activity against herpes simplex virus types 1 and 2. Bioorg Med Chem 13:3878
Venosa G, Fukuda H, Batlle A, et al. (2006) Photodynamic therapy: regulation of porphyrin synthesis and
hydrolysis from ALA esters. J Photochem Photobiol B Biol 83:129

38
Med Chem Res (2007) 16:28–38
Wang S, Deng Y, Xu H, et al. (2006) Synthesis of a novel galactosylated lipid and its application to the
hepatocyte-selective targeting of liposomal oxorubicin. Eur J Pharm Biopharm 62:32
Wolfe JL, Buchwald SL (1997) Room temperature catalytic amination of aryl iodides. J Org Chem
62:6066
Yuan LB, Jie HN (1984) Porphyrin photosensitizers for cancer phototherapy. Huaxue Tongbao 8:25