Study on synthesis and biological activity of a galactosylated piperazinyl porphyrin

Article abstract of DOI:10.1016/j.bmcl.2006.09.020

In order to obtain an carcinoma-selective drug, the synthesis and characterization of 5,10,15,20-tetra[4-(4′-galactosylpiperazinyl)phenyl]porphyrin (TGPP) is reported. The biological activity on cancer cells and the pharmacokinetics are also reported as preliminary results showing a very high liver to skin ratio and short retention time in tissues, and thus promising activity in photodynamic therapy.

Full text of DOI:10.1016/j.bmcl.2006.09.020

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6298–6301
Study on synthesis and biological activity of a galactosylated
piperazinyl porphyrin
He-Ping Li*
College of Chemical and Environmental Engineering, Changsha University of Science and Technology, Changsha 410076, China
Received 14 June 2006; revised 3 September 2006; accepted 7 September 2006
Available online 25 September 2006
Abstract—In order to obtain an carcinoma-selective drug, the synthesis and characterization of 5,10,15,20-tetra[4-(40-galactosylpip-
erazinyl)phenyl]porphyrin (TGPP) is reported. The biological activity on cancer cells and the pharmacokinetics are also reported as
preliminary results showing a very high liver to skin ratio and short retention time in tissues, and thus promising activity in
photodynamic therapy.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Recently, promising porphyrin photosensitizers have
been developed for photodynamic therapy (PDT).1–3
Numerous porphyrins have been synthesized and tested
for in vitro and in vivo phototoxicity, but reasons for
selectivity of the compound remain recondite. Besides
the subtle balance between hydrophobicity and hydro-
philicity, charge distribution, symmetry and configura-
tion of the molecules were suggested as important
factors influencing the selective accumulation and sub-
cellular distribution. The glycosylated porphyrins owing
to good solubility in aqueous solution and specific mem-
brane interaction have been applied in cancer PDT as
promising photosensitizers.4–6 In such compounds, the
nature and number of carbohydrate residues and hydro-
phobic substituents linked to the macrocycle allow a
large variability in the hydrophilic or hydrophobic char-
acters. Although a number of porphyrin-based photo-
sensitizers have been approved for clinical trials,
however, they are not very selective, causing skin sensi-
tivity for some weeks.7 Moreover, PDT treatment also
was not suitable for therapy of the cancer locating at
the deeper tissue of the body.8
drugs, including piperazine-containing drugs, the cyto-
toxicity of drugs on normal cells is caused for the drugs
could not efficiently concentrate in tumour tissue. The
side effects exist inevitably for patients, the dosages of
the drugs have to be reduced in clinical to mitigate the
side effects of drugs. But this will prevent the drugs from
bringing their efficacy into play and results in the cura-
tive effect decreasing. Therefore, it is very promising to
synthesize some new compounds as anticancer drugs
that can accumulate to higher concentrations in malig-
nant tumours than in normal tissue. It has been reported
that nitrogenous heterocycle porphyrins have better
anticancer activity than the corresponding nitrogenous
heterocycles in the absence of light.15 We sought to
develop an anticancer drug approach that can accumu-
late in neoplastic tissue to higher concentrations than in
surrounding normal tissue.
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 hepato-
ma cell lines. This receptor system can not only recog-
nize terminal b-D-galactose or N-acetylgalactosamine
residues, but can also internalize them within mem-
brane-bound vesicles or endosomes.16 ASGR are con-
sidered a particularly attractive target in many drug
carrier studies. The use of such nature molecules with
galactosylated or lactosylated residues in targeting drug
has resulted in significant targeting efficacy to the liver.17
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
Piperazine-containing compounds are widely used in
pharmaceutical industry. Especially, some substituted
piperazine compounds have distinctive anticancer
activity.9–14 But so far, all clinical chemical anticancer
Keywords: Porphyrin; Galactosyl; Piperazinyl; Synthesis; Biological
activity.
*
Tel./fax: +86 731 2618234; e-mail: lihepinghn@163.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.020

H.-P. Li / Bioorg. Med. Chem. Lett. 16 (2006) 6298–6301
6299
moiety for the hepatocyte-targeting drug.18,19 It seems a
good idea to link a galactose-guided drug moiety to
porphyrin ring to avoid or mitigate the side effect of
drugs. We now report the synthesis and the biological
activity studies of 5,10,15,20-tetra[4-(40-galactosylpiper-
azinyl)phenyl]porphyrin (TGPP), a structural change
which is accompanied by a short retention time in tissues
together with a promising activity. Cell uptake and pho-
todynamic properties are compared to those of the
structurally related 5,10,15,20-tetra(4-aminophenyl)por-
phyrin (TAPP), a drug which is one of the most potent
photosensitizers discovered to date.
N-galactosylamino-N0-phenylpiperazine could be veri-
fied by controls which were prepared in exactly the
same way as the conjugate but omitting DCC during
the coupling reaction, exhibiting a negligible amount
of product. In order to optimize the synthesis of N-galac-
tosylamino-N0-phenylpiperazine, the influence of the
coupling reaction time was evaluated. Results demon-
strated 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 equilibrium. Other
factors that can influence the yield, such as pH of the
reaction mixtures and the weight ratio of N-phenylpiper-
azine to lactobionic acid, were already studied, as far as
pH of coupling reaction was concerned, alkaline pH
would benefit the reaction greater than acidic pH. There-
fore, tetramethylethylene diamine solution was selected
to the catalyst of the coupling reaction. Our 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 benz-
aldehyde to the corresponding porphyrin. In this paper,
the functional groups selected are those drugs that be
used in the treatment of cancers. The synthetic route of
TGPP is summarized in Figure 1. Whilst this work was
in progress, a different piperazinylporphyrin15 has been
reported.
TGPP is a symmetrical-galactosylated piperazinylporph-
yrin. The key of the synthesis of TGPP is how to intro-
duce galactosylated piperazine to the porphyrin ring.
We tried to couple iodoporphyrin with various galac-
tosylated piperazines to synthesize target molecules at
room temperature according to Buchwald method.20
However, the reaction did not proceed, even after stir-
ring over an extended period of time. The coupling of
TAPP with N,N0-di(2-bromoethyl)galactosamide at
room temperature can obtain the corresponding galac-
tosylated piperazinyl porphyrin, but the components
of products are too complicated to purify. TGPP is syn-
thesized by the direct condensation of pyrrole with the
appropriate benzaldehyde (3) under Adler’s condi-
tions.21 Benzaldehydes bearing substituted piperazines
were prepared according to the literature.22–24 N-galac-
tosylamino-N0-phenylpiperazine was synthesized by the
formation of amide bonds between the secondary amino
group of the N-phenylpiperazine25 and the carboxylic
acid group of lactobionic acid according to the method
The structure of TGPP is characterized by elementary
1
analysis, MS, H NMR, IR and UV–vis.26 The 1H
NMR spectra at ꢀ2.46 ppm show the characteristic
single peak for porphyrin. The molecular ion peak and
elementary analysis further confirm its structure.
reported by Bernkop-Schnurch et al.25 As shown in reac-
¨
tion formula, the carboxylic moieties of lactobionic acid
were activated by N,N0-dicyclohexylcarbodiimide
(DCC), forming an intermediate product, which reacted
with the secondary amino group of N-phenylpiperazine.
The efficacy of the purification method for the resulting
As expected from the presence of four galactosylated
groups, the compound is soluble in polar solvent such
as methanol and water. The lipophilic and hydrophilic
properties were characterized by the partition coefficient
CHO
NH2
DMF
N
HN(CH2CH2OH)2
DCC
LA
NH
POCl3
N
R
N
3
1
2
R'
R'
CHO
R'
CH3CH2COOH
reflux 0.5 h
N
HN
N
R'
R'
NH
H
N
TGPP
R'
OH H H
O
C
H
H
OH H
H
CH2OH
HOOC
CH2OH
OH H
HO
OH H
O
OH
O
OH
N
HO
R
R' =
N
R- =
OH
OH
O
OH
O
OH
Lactobionic acid
( LA)
OH
OH
Figure 1. The synthesis of TGPP.

6300
H.-P. Li / Bioorg. Med. Chem. Lett. 16 (2006) 6298–6301
TGPP have better anticancer activity than galactosylat-
100
80
60
40
20
0
ed piperazine. It is interesting that TAPP, which is an
anticancer drug too, would be a good carrier for toxic
moieties to arrive at improved anticancer drugs. The
possible reason might be that TAPP with anticancer
drugs can intercalate into the base pairs of DNA strong-
ly. The molecular mechanism for the anticancer activi-
ties of TGPP is being further studied.
HNE1
MCG
Bel-7404
The distribution of TGPP was examined in different
selected tissues of tumour-bearing female KM mice.
TGPP (10 mg/kg) was administered by ip injection.
The animals were sacrificed 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, tumour, and the liv-
er were recovered. About 200 mg of tissues was
homogenized in THF. The homogenates were centri-
fuged at 3000 rev/min for 15 min, and the fluorescence
of the supernatants was measured, setting the excita-
tion wavelength at 420 nm and recording the emission
spectrum from 500 and 800 nm. Serum samples, iso-
lated from the blood by centrifugation, were diluted
with suitable volumes of 500 ll THF and the TGPP
content was measured by fluorescence. In all the
cases, TGPP amounts were determined by the inter-
polation of emission intensity and TGPP concentra-
tion plotted on a standard curve. The results are
shown in Figure 3.
cis-Pt(0) cis-Pt(i) 5-Fu(0) 5-Fu(i) TGPP(0)TGPP(i)
Figure 2. Anticancer activity of cis-Pt, 5-Fu and TGPP in cancer cells
(Bel-7404, MCG, and HNE1) determined by MTT assay. Cancer cells
were incubated for 18 h in solution (6.25 lg/ml). ‘0’ represented
without irradiation, and ‘i’ represented with irradiation (at 650 nm and
20 J/cm2).
of the compound between the two non-miscible solvents
octanol and water. The octanol/water partition ratios
were 18 for compound TGPP and 42 for TAPP indicat-
ing that TGPP was more hydrophilic than TAPP. In
order to study the anticancer activity of TGPP, with
and without irradiation, the cytotoxic effects of TGPP
and two common anticancer drugs, cis-Platinum (cis-
Pt) and 5-fluorouracil (5-Fu), on three human tumour
cell lines, Bel-7404 (a liver cancer cells), MCG (a stom-
ach tumour cells) and HNE1 (a nasopharyngeal carci-
noma cancer cells), were determined using the MTT
method.15,27 The results of the pre-screenings are given
in Figure 2.
This suggests that TGPP is rapidly distributed in tissues
and rapidly eliminated. The highest concentrations were
observed in the liver at 3 h post-injection treatment; the
concentration of TGPP 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 could be favourable, as it would cause photo-
sensitization. The higher quantity of TGPP in liver
can be explained by the presence of four galactosylated
moieties which can recognize ASGR. The shorter reten-
tion time of TGPP in tissues can decrease prolonged
cutaneous photosensitivity. Further developments and
One can see from Figure 2 that the death rates ranged
from 24% to 19% for TGPP, 40–33% for cis-Pt and
43–29% for 5-Fu when cells were incubated for 18 h at
37 ꢁC under an atmosphere of air containing 5%
CO2without light. The results showed that the dark tox-
icity of TGPP was weaker to those of cis-Pt and 5-Fu in
this condition. However, the death rates ranged from
96% to 98% for TGPP after 18 h incubation and
20 J/cm2 irradiation (Fig. 2), showing presence of photo-
sensitizing toxicity.
100
Structurally, TGPP consists of two parts: TAPP and
galactosylated piperazine compound. In order to study
whether TGPP and two corresponding structure parts
(galactosylated piperazine and TAPP) have anticancer
activity to cancer cell, they were tested in vitro against
cancer cell by MTT method in the absence of light.15
The ID50 (50% of cell death) values of the cancer cell
to these compounds are listed in Table 1.
80
3 h
24 h
144 h
60
40
20
0
Table 1 shows that TGPP have smaller ID50 values than
that of galactosylated piperazine. This indicates that
Table 1. ID50 values in lg/ml of TGPP and some compounds
Compound
Bel-7404
MCG
HNE1
liver
tumour
muscle
skin
TGPP
TAPP
Galactosylated piperazine
29
33
35
24
33
28
Figure 3. Biodistribution of TGPP in tumour-bearing mice: Recover-
ies of TGPP from tumour-bearing KM mice injected with 10 mg/kg of
drug. Values represent the average of the three experiments.
>50
>50
>50

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