Novel phosphoramidates with porphine and nitrogenous drug: One-pot synthesis and orientation to cancer cells

Article abstract of DOI:10.1016/j.ejmech.2009.11.027

One-pot synthesis of novel phosphoramidates with porphine and nitrogenous drug was accomplished. In the absence of light, MTT test showed that they killed the BEL-7402 liver cancer cells effectively in vitro. The cell viability studied on normal liver and

Full text of DOI:10.1016/j.ejmech.2009.11.027

European Journal of Medicinal Chemistry 45 (2010) 890–895
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel phosphoramidates with porphine and nitrogenous drug: One-pot synthesis
and orientation to cancer cells
*
Zhi-Wei Wang, Can-Cheng Guo , Wen-Zhong Xie, Chao-Zhou Liu, Chun-Guang Xiao, Ze Tan
College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2009
Received in revised form
9 November 2009
Accepted 12 November 2009
Available online 18 November 2009
One-pot synthesis of novel phosphoramidates with porphine and nitrogenous drug was accomplished. In
the absence of light, MTT test showed that they killed the BEL-7402 liver cancer cells effectively in vitro.
The cell viability studied on normal liver and cancer cells showed that porphine phosphoramidates
selectively kill the cancer cells, which was in sharp contrast with the non-porphine containing
compound 4-formylphenyl N,N-bis(2-chloroethyl)-phosphoramidate. These results, coupled with the cell
uptake test showing that they could differentiate the tumor cells from the normal cells by their selective
accumulation in cancer cells, gave strong support to the notion that the introduction of porphine moiety
in these molecules was responsible for the effectiveness and cell differentiability of these porphine
phosphoramidates.
Keywords:
Phosphoramidate
Porphine
Ó 2009 Published by Elsevier Masson SAS.
Ifosfamide mustard
Synthesis
Bioactivity
1. Introduction
yields, the poor solubility of porphines in both water and organic
solvents make the synthesis of drugs bearing porphines moiety
Phosphorates are widely present in life forms as important
physiological function molecules [1–3]. Researchers have found
that phosphoramidates could also be used as drugs because of their
good bioactivities [4–6]. For example, cyclophosphamide and
ifosfamide have been used as anticancer drugs in the treatment of
breast cancer, lung cancer, ovarian cancer, etc. [7,8]. These drugs
could kill tumor cells effectively. However, one major problem
associated with them is that they can not distinguish tumor cells
from normal cells. Thus, improving the recognition ability of these
phosphoramidate drugs to tumor cells has become one of the major
challenges for medicinal chemists [9–11].
Porphines could selectively accumulate in tumor tissues than in
normal tissues. Based on this fact, scientists have used porphines in
photodynamic therapy (PDT) as sensitizers [12–18]. Recognizing
this unique property of porphines, scientists have introduced por-
phines moiety into the drugs to improve the selectivity of the drugs
for the tumor cells. As a result the side effects of the drug could
potentially be alleviated when the drug is administrated at the same
dosage. Though there have been many synthetic methods available
for the synthesis of substituted porphines [19], the typical low
quite challenging. In addition the sensitive nature of some drugs
toward acids and/or bases can make their synthesis even more
complicated. In recent years, the synthesis of anticancer drugs
bearing porphines has attracted a lot of interest from chemists and
medicinal scientists alike because of their high affinity to tumor cell
and also for their synthetic challenges [20–24]. For example, Napoli
synthesized meso-tetrakis(4-phosphonatophenyl)porphine, a type
of porphine containing phosphates, by the reaction of dieth-
yl(4-formylphenyl)phosphorate and pyrrole in refluxing propanoic
acid [25]. Ganesan prepared 5,10,15,20-tetrakis (4-phosphonoxy
phenyl)porphine by the oxidation of 5,10,15,20-tetrakis(4-phosphi-
tephenyl)porphine, which was synthesized by the reaction of
5,10,15,20-tetrakis(4-hydroxyphenyl)porphine and di-tert-butyl-
N,N-diethylphosphoramidite [26]. However, as far as we know, there
is still nothing reported on the preparation of porphine containing
phosphoramidates.
In this paper, we synthesized five phosphoramidates with meso-
substituted AB3 porphine and ifosfamide mustards (1–5) shown in
Scheme
1 by a simple one-pot phosphonation of hydroxyl
substituted porphine with POCl3, and subsequent amidation with
2-chloroethylamine. The biological activities of porphine phos-
phoramidates against normal liver cells (L-02) and cancer cells
(BEL-7402) were tested by the MTT method, which were compared
with those of the corresponding phosphoramidates without
* Corresponding author. Tel.: þ86 731 88821314; fax: þ86 731 88821488.
E-mail address: ccguo@hnu.cn (C.-C. Guo).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.11.027

Z.-W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 890–895
891
Indeed, when the hydroxyl porphine 7 was reacted with 20 equiv
POCl3, the starting material 7 disappeared completely after 2 h, and
porphine phosphorodichloridate 8 was formed. However, the
attempt to remove the excess of POCl3 from the reaction mixture by
vacuum distillation failed because the amines within the porphine
structure reacted with the POCl3 at the reflux temperature [33].
This result was confirmed with LC-MS analysis of the reaction
mixture. The molecular ion peak of (5,10,15,20-tetraphenylporphi-
nato) dichlorophosphorous chloride at m/z 713 showed that only
the hydroxyl group of 5,10,15,20-tetraphenylporphine could react
with POCl3 at room temperature, and more complicated products
were observed at 50 ꢀC. Above results suggested that it was difficult
to remove the excess of POCl3 from the mixtures of porphine
R
Cl
HN
P
1, R=Br
NH
N
O
O
R
2, R=CH3
N HN
HN
3, R=OCH3
4, R=Cl
Cl
5, R=H
R
Scheme 1. The structures of porphine phosphoramidates (1–5).
phosphorodichloridate
8 and POCl3 by vacuum distillation.
Attempts to purify the reaction mixture via column chromatog-
raphy failed too because of the instability of porphine phosphor-
odichloridate 8 on column.
porphine. These results, coupled with cell viability test and the cell
uptake test show that phosphoramidates bearing porphine not only
had considerable cytotoxicity against liver cancer cell but they can
also differentiate the tumor cells from the normal cells by their
selective accumulation in cancer cells.
Fortunately, much to our delight, the phosphonation of the
hydroxyl porphine 7 with 1.2 equiv POCl3 and 1.8 equiv triethyl-
amine gave only one product, i.e., porphine phosphorodichloridate
8 after 4 h at ꢁ20 ꢀC. It was also found that the compound 8 could
be used without further purification for the amidation step. Addi-
tion of 3 equiv of 2-chloroethylamine hydrochloride and 6 equiv of
triethylamine to the in situ generated porphine phosphorodi-
chloridate 8 gave porphine phosphoramidate 1 in 5% yield after 4 h
at ꢁ20 ꢀC. The yield of porphine phosphoramidate 1 was increased
to 10% when the temperature was raised from ꢁ20 ꢀC to 20 ꢀC.
subsequent investigation revealed that the yield of porphine
phosphoramidate 1 was profoundly influenced by the amount of
2-chloroethylamine hydrochloride and triethylamine and the
results are summarized in Table 1.
As shown in Table 1, the yield of porphine phosphoramidate
mustard 1 was increased from 10% to 50% when the amounts of
2-chloroethylamine and triethylamine increased from 3 equiv to
6 equiv–5 equiv and 10 equiv, respectively (Run 3). Prolonging the
reaction time from 8 h to 24 h had little effect on the yield (Run 4).
Further increasing the amounts of 2-chloroethylamine hydrochlo-
ride and triethylamine did not raise the yield of porphine phos-
phoramidate 1 either (Run 5).
2. Results and discussion
2.1. Chemistry
Since Adler–Longo method and Lindsey method are the reac-
tions of choice for the synthesis of AB3 porphines [27,28], initially,
we planned to synthesize the desired porphine phosphoramidates
through the direct condensation of pyrrole with two different types
of substituted benzaldehyde, one bearing the desired phosphor-
amidates, under acid conditions. (Scheme 2).
As such, 4-formylphenyl N,N-bis (2-chloroethyl)phosphor-
amidate (6) has to be prepared first (Scheme 3). Compound 6 was
readily synthesized by treating p-hydroxybenzaldehyde with POCl3
and followed by amidation with chloroethylamine according to the
literature procedures [29–31].
Unfortunately, the synthesis of the porphine phosphoramidate
(1) by the reaction of compound 6, 4-bromobenzaldehyde and
pyrrole according to the Adler–Longo procedure in refluxing
propanoic acid failed. The failure might be due to the fact that the
P–N bonds of 6 were not stable under the acid conditions at high
temperature [32].
With a satisfactory protocol for the synthesis of the desired
porphine containing ifosfamide mustard in hand, compounds 1–5
were synthesized accordingly from their corresponding mono-
hydroxyl substituted tetraarylporphines in yields of 42–50%.
Next, compound 1 was attempted by using Lindsey procedure at
room temperature. We were delighted to discover that compound 6
was stable under standard Lindsey conditions at room temperature.
However, the reaction of compound 6, 4-bromobenzaldehyde and
pyrrole under the Lindsey conditions, gave 5, 10, 15, 20-tetra(4-
bromophenyl)porphine as the main product.
Finally, we decided to synthesize compound 1 according to
a different route by utilizing the monohydroxyl substituted tetra-
phenylporphine 7 as shown in Scheme 4, since monohydroxyl
substituted tetraphenylporphines are well known and can be easily
synthesized. Phosphonation of the hydroxyl groups on the mono-
hydroxyl substituted tetraphenylporphines with POCl3 and ami-
dation with 2-chloroethylamine would produce the desired
porphine containing ifosfamide mustards. By simply changing the
substituents on the monohydroxyl tetraphenylporphine from Br to
Me, MeO, Cl, and H, we can synthesize all five different porphine
phosphoramidates via a single strategy.
2.2. Cytotoxicity assays
In the absence of light, the porphine phosphoramidates 1–5, 4-
formylphenyl N,N-bis(2-chloroethyl)phosphoramidate 6, and hae-
matoporphine dihydrochloride (HPD) were tested in vitro against
liver cancer cell BEL-7402 by the MTT method [23,24] and the IC50
values of the liver cancer cell BEL-7402 were listed in Table 2.
Compound 6 and HPD were used as the references to truly gauge
the effectiveness of the five porphine phosphoramidates since
compound 6 had the same phosphoramide structure as 1–5, and
HPD was a known drug used in the treatment of cancer. Test results
showed that porphine phosphoramidates 1–5 had much lower IC50
values than 4-formylphenyl N,N-bis (2-chloroethyl)phosphor-
amidate 6, and were nearly as effective as HPD while the Br-
substituted compound was found to be the most effective one,
affording a IC50 value at 8.41 mM. Other four compounds gave values
The reaction of 5, 10, 15-tri(4-bromophenyl)-20-(4-hydrox-
yphenyl)porphine (7) with excess amount of POCl3 should give the
tetraphenylporphine phosphorodichloridate (8). It was reported
that 20 equiv of POCl3 was needed and the excess POCl3 had to be
removed by vacuum distillation for such transformation [29–31].
ranging from 11.42 to 20.32. The low IC50 values observed for these
five compounds suggested they all can be potential anticancer drugs.
To compare the toxicity of the porphine phosphoramidates to
the tumor and normal cells, porphine phosphoramidates 1–5 and
compound 6 were tested via MTT test in liver cancer cells BEL-7402

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Z.-W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 890–895
Br
OHC
OHC
R
6
Cl
HN
P
H
NH
N
N
R
Br
Br
R=
O
O
HN
HN
Cl
H
N
Br
1
Scheme 2. Synthetic route of porphine phosphoramidate via Adler or Lindsey procedures.
and normal liver cells L-02 (in the absent of light), respectively, and
the cell viability results were shown in Scheme 5.
not only can selective accumulate in cancer cells but also can
selective kill the tumor cells. Investigations are currently under way
to elucidate the molecular mechanism for the intracellular accu-
mulation of these porphine phosphoramidates in liver cancer cells
BEL-7402.
In the case of compound 6, we noted that the cell viability of the
normal liver cells L-02 was lower than the liver cancer cells BEL-
7402, which was expected because tumor cells tends to be more
difficult to kill than normal cells. However, with compounds 1–5,
cell viabilities of normal liver cells L-02 were higher than those of
the liver cancer cells BEL-7402 when compounds 1–5 were given at
the same dosage as in the case of 6. This reversal of the trend
showed that porphine phosphoramidates could selectively kill the
cancer cells. Clearly, the inclusion of a porphine structure motif is
responsible for this.
4. Experimental section
4.1. Chemistry
1H NMR and 31P NMR spectra were recorded on a Varian NOVA-
400 spectrometer (400 MHz 1H), and the chemical shifts were
reported with tetramethylsilane (TMS) as the internal standard in
CDCl3 solvent. Mass spectra were obtained on a Shimadzu QP-5000
mass spectrometer and Agilent 1100 LC-MS. Elemental analyses
were obtained on a Vario El III elementary analyzer. Fluorescence
emission was recorded on a Hitachi F-4500 Fluorescence Spectro-
photometer. Cell numbers were measured on a CASY Cell Counter
and Analyzer. CH2Cl2, pyrrole and POCl3 were distilled before use.
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was from Sigma Chemical Co. (USA). The cell lines BEL-7402
and L-02 were obtained from Shanghai Institute of Cell Biology,
Chinese Academy of Science. Other chemicals were used as
received.
2.3. Cancer cell selectivity
In order to further understand the cancer cell-oriented accu-
mulation of the synthetic porphine phosphoramidates 1–5, their
accumulations in the BEL-7402 tumor and the normal cells L-02
were investigated by the cell uptake tests [34]. The amounts of the
intracellular porphine phosphoramidates 1–5 absorbed by the liver
cancer cells BEL-7402 and the normal cells L-02 were shown in
Scheme 6.
As shown in Scheme 6, significant concentration differences of
the porphine phosphoramidates between the liver cancer cells
BEL-7402 and normal cells L-02 were observed. The fact that the
uptakes of liver cancer cells BEL-7402 were higher than liver
normal cells L-02 gave proof to that the selective accumulation of
porphine phosphoramidates in liver cancer cells BEL-7402 did
occur. This could be the reason for the selective killing of the tumor
cells.
4.1.1. General procedure for the synthesis of porphine
phosphoramidate (1–5, Scheme 4)
A solution of hydroxyl porphine (0.1 mmol, 1.0 equiv) and POCl3
(0.12 mmol, 1.2 equiv) in CH2Cl2 under nitrogen was cooled to
ꢁ20 ꢀC. NEt3 (0.18 mmol, 1.8 equiv) in CH2Cl2 was added dropwise
while maintaining the temperature at ꢁ20 ꢀC. The reaction mixture
was stirred for 4 h at ꢁ20 ꢀC before dry 2-chloroethylamine
hydrochloride (0.5 mmol, 5 equiv) was added. The final mixture
was stirred at ꢁ20 ꢀC for 15 min and then warmed slowly to room
temperature for another 8 h. The solution was washed once with
aqueous saturated NaHCO3, once with water and dried over anhy-
drous Na2SO4. After removing the solvent under vacuum, the crude
product was purified by column chromatography (CH2Cl2:ethyl
acetate, 5:1).
3. Conclusion
In conclusion, we have developed a practical and efficient one-
pot approach to synthesize porphine containing ifosfamide
mustard and 5 different porphine phosphoramidates are synthe-
sized. In the absence of light, the bioactivity tests show that the
porphine phosphoramidates are potent anticancer reagents, with
one being as effective as the commercial anticancer drug HPD. They
Cl
HN
P
O
NH2CH2CH2Cl HCl
NEt3
Cl
POCl3
O
P
OHC
OH
O
OHC
O
OHC
NEt3
HN
Cl
Cl
6
Scheme 3. Synthetic routes of 4-formylphenyl N,N-bis(2-chloroethyl)phosphoramidate.

Z.-W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 890–895
893
Br
Br
1.2eq
POCl3
NEt3
Cl
NH
N
N
NH N
N HN
OH
Br
O
Br
P
O
HN
1.8eq
Cl
-20°C
Br
Br
8
7
Br
Cl
HN
NH N
N HN
10eq
5eq
NEt3
P
O
O
Br
HN
NH2CH2CH2Cl HCl
20°C
Cl
Br
1
Scheme 4. Synthetic routes of porphine phosphoramidates.
4.1.1.1. 5,10,15-tri(4-Bromophenyl)-20-(4-phenyl)porphine N,N-bis(2-
chloroethyl) phosphoramidate (1). The compound was prepared
according to the general procedure mentioned above, purified and
isolated as a purple solid; yield 50%. MS: m/z 1070. 1H NMR (CDCl3):
4.1.1.4. 5,10,15-tri(4-Chlorophenyl)-20-(4-phenyl)porphine N,N-bis(2-
chloroethyl) phosphoramidate (4). The compound was prepared
according to the general procedure mentioned above, purified and
isolated as a purple solid; yield 49%. MS: m/z 937. 1H NMR (CDCl3):
d
, ppm, 8.84 (s, 8H), 8.17 (d, 2H, J ¼ 8 Hz), 8.06 (d, 6H, J ¼ 8 Hz), 7.90
d
, ppm, 8.84–8.87 (m, 8H), 8.18 (d, 2H, J ¼ 8 Hz), 8.13 (d, 6H,
(d, 6H, J ¼ 8 Hz), 7.63 (d, 2H, J ¼ 8 Hz), 3.77 (t, 4H, J ¼ 5.2 Hz), 3.52–
J ¼ 8 Hz), 7.75 (d, 6H, J ¼ 8 Hz), 7.64 (d, 2H, J ¼ 8 Hz), 3.77 (t, 4H,
3.59 (m, 4H), 3.43–3.48 (m, 2H), ꢁ2.87 (s, 2H). 31P NMR (CDCl3):
d,
J ¼ 5.2 Hz), 3.53–3.60 (m, 4H), 3.44–3.49 (m, 2H), ꢁ2.86 (s, 2H). 31P
11.77. Anal.Calcd. for C48H36Br3Cl2N6O2P, %: C, 53.86; H, 3.39; N,
7.85; Found: C, 54.03; H, 3.42; N, 7.64.
NMR (CDCl3): d, 11.32. Anal.Calcd. for C48H36Cl5N6O2P %: C, 61.52; H,
3.87; N, 8.97. Found: C, 61.38; H, 3.79; N, 9.08.
4.1.1.2. 5,10,15-tri(4-Methylphenyl)-20-(4-phenyl)porphine N,N-bis(2-
chloroethyl) phosphoramidate (2). The compound was prepared
according to the general procedure mentioned above, purified and
isolated as a purple solid; yield 45%. MS: m/z 876. 1H NMR (CDCl3):
4.1.1.5. 5,10,15-Triphenyl-20-(4-phenyl)porphine N,N-bis(2-chlor-
oethyl)phosphoramidate (5). The compound was prepared accord-
ing to the general procedure mentioned above, purified and
isolated as a purple solid; yield 46%. MS: m/z 834. 1H NMR (CDCl3):
d
, ppm, 8.86 (s, 6H), 8.83 (s, 2H), 8.18 (d, 2H, J ¼ 8 Hz), 8.09 (d, 6H,
d
, ppm, 8.83–8.86 (m, 8H), 8.21 (dd, 6H, J ¼ 1.2, 8 Hz), 8.18 (d, 2H,
J ¼ 8 Hz), 7.61 (d, 2H, J ¼ 8 Hz), 7.56 (d, 6H, J ¼ 8 Hz), 3.75 (t, 4H,
J ¼ 8 Hz), 7.72–7.78 (m, 9H), 7.60 (d, 2H, J ¼ 8 Hz), 3.73 (t, 4H,
J ¼ 5.2 Hz), 3.52–3.61 (m, 4H), 3.43–3.48 (m, 2H), ꢁ2.81 (s, 2H). 31P
J ¼ 5.2 Hz), 3.51–3.60 (m, 4H), 3.42–3.46 (m, 2H), ꢁ2.80 (s, 2H). 31P
NMR (CDCl3):
d, 10.80. Anal.Calcd. for C51H45Cl2N6O2P, %: C, 69.94;
NMR (CDCl3): d, 11.01. Anal.Calcd. for C48H39Cl2N6O2P, %: C, 69.15; H,
H, 5.18; N, 9.60; Found: C, 70.02; H, 5.09; N, 9.75.
4.71; N, 10.08. Found: C, 69.44; H, 4.91; N, 9.89.
14.1.2. Synthesis of 4-formylphenyl N,N-bis
(2-chloroethyl)]phosphoramidate (6, Scheme 3)
4.1.1.3. 5,10,15-tri(4-Methoxyphenyl)-20-(4-phenyl)porphine N,N-bis(2-
chloroethyl) phosphoramidate (3). The compound was prepared
according to the general procedure mentioned above, purified and
isolated as a purple solid; yield 42%. MS: m/z 924. 1H NMR (CDCl3):
A solution of NEt3 (3 mmol, 1.0 equiv) was slowly added to
a mixture of 4-hydroxybenzaldehyde (3 mmol, 1.0 equiv) and POCl3
(60 mmol, 20 equiv) in CH2Cl2. The mixture was stirred for 1 h at
20 ꢀC. Subsequently 10 mL of ether was added. The solids were
removed by filtration. The solvents were removed, and 10 mL of
ether was added again. The remaining solids were removed, and
the solvent was evaporated. The excess of POCl3 was removed in
vacuum. A greenish oil (4-formylphenyl phosphorodichloridate)
was obtained in the yield of 51%.
d
, ppm, 8.88 (s, 6H), 8.83 (s, 2H), 8.19 (d, 2H, J ¼ 8 Hz), 8.12 (d, 6H,
J ¼ 8 Hz), 7.62 (d, 2H, J ¼ 8 Hz), 7.30 (d, 6H, J ¼ 8 Hz), 3.77 (t, 4H,
J ¼ 5.2 Hz), 3.53–3.59 (m, 4H), 3.43–3.46 (m, 2H), ꢁ2.80 (s, 2H). 31P
NMR (CDCl3): d, 10.80. Anal.Calcd. for C51H45Cl2N6O5P, %: C, 66.31;
H, 4.91; .N, 9.10. Found: C, 66.41; H, 4.75; N, 9.24.
Table 1
Reaction condition screening.
Subsequently NEt3 (1 mmol, 10 equiv) and 2-chloroethylamine
hydrochloride (0.5 mmol, 5 equiv) were added at 20 ꢀC. 4 h later
the volatiles were removed, and ether was added again. The
remaining solids were removed, and the solvent was evaporated.
The crude product was purified by column chromatography and
isolated as a yellow liquid. Yield 52%. MS: m/z 325, 1H NMR (CDCl3):
Reaction 2-chloroethylamine/equiv Triethylamine/equiv Time/h Yield %
run
1
2
3
4
5
3
3
5
5
10
6
6
10
10
20
8
24
8
24
8
10
13
50
50
50
d, ppm,9.95 (s, 1H), 7.87 (d, 2H, J ¼ 8.4 Hz), 7.40 (d, 2H, J ¼ 8.4 Hz),
3.59–3.62 (m, 6H), 3.35–3.40 (m, 4H). 31P NMR (CDCl3):
d, 11.65.

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Table 2
IC50 values of five porphine phosphoramidates.a
IC50
1
2
3
4
5
6
HPD
m
M
8.41(7.48 ꢁ 9.35)
11.42(9.13 ꢁ 13.70)
17.33(16.25 ꢁ 19.50)
20.32(18.18 ꢁ 22.46)
14.41(10.80 ꢁ 15.61)
>150
8.93(7.44 ꢁ 10.42)
a
IC50 values represent the mean from at least three independent experiments.
4.1.3. General procedure for the synthesis of monohydroxyl
substituted porphines (7,9–12)
procedure mentioned above, purified and isolated as a purple solid;
yield 7.0%. MS: m/z 734. 1H NMR (CDCl3):
, ppm, 8.90 (d, 2H,
d
The reactions was performed in a 500 mL three neck round
bottom flask, which was charged with propionic acid (300 mL),
benzaldehyde (26 mmol), substituted benzaldehyde (60 mmol),
and pyrrole (80 mmol). The resulting solution was magnetically
stirred in refluxing propionic acid for 35 min. The crude product
was recrystallized from cold propionic acid and further purified by
column chromatography (CH2Cl2).
J ¼ 4.4 Hz), 8.83 (s, 6H), 8.14 (d, 6H, J ¼ 8 Hz), 8.06 (d, 2H, J ¼ 8 Hz),
7.75 (d, 6H, J ¼ 8 Hz), 7.21 (d, 2H, J ¼ 8 Hz), ꢁ2.82 (s, 2H). Anal.Calcd.
for C44H27Cl3N4O %: C, 71.99; H, 3.71; N, 7.63. Found: 72.12; H, 3.76;
N, 7.43.
4.1.3.5. 5,10,15-Triphenyl-20-(4-hydroxyphenyl)porphine (12). The
compound was prepared according to the general procedure
mentioned above, purified and isolated as a purple solid; yield 4.9%.
4.1.3.1. 5,10,15-tri(4-Bromophenyl)-20-(4-hydroxyphenyl)porphine
(7). The compound was prepared according to the general proce-
dure mentioned above, purified and isolated as a purple solid; yield
MS: m/z 631. 1H NMR (CDCl3):
d, ppm, 8.84–8.89 (m, 8H), 8.22 (dd,
6H, J ¼ 1.2, 8 Hz), 8.08 (d, 2H, J ¼ 8 Hz), 7.73–7.78 (m, 9H), 7.22 (d,
2H, J ¼ 8 Hz), ꢁ2.78 (s, 2H). Anal.Calcd. for C44H30N4O,%: C, 83.79;
H, 4.79; N, 8.88. Found: C, 83.94; H, 4.62; N, 9.03.
7.1%. MS: m/z 867. 1H NMR (CDCl3):
8.81–8.83 (m, 6H), 8.05–8.07 (m, 8H), 7.88 (dd, 6H, J ¼ 2, 8 Hz), 7.16
(d, 2H, J ¼ 8 Hz), ꢁ2.86 (s, 2H). Anal.Calcd. for C44H27Br3N4O %: C,
60.92; H, 3.14; N, 6.46; Found: C, 61.11; H, 3.40; N, 6.64.
d
, ppm, 8.89 (d, 2H, J ¼ 4.8 Hz),
4.2. Bioactivity
4.2.1. Cytotoxicity assays
4.1.3.2. 5,10,15-tri(4-Methylphenyl)-20-(4-hydroxyphenyl)porphine
(9). The compound was prepared according to the general proce-
dure mentioned above, purified and isolated as a purple solid; yield
The cytotoxicity effects of the compounds on BEL-7402 cell were
determined by using the MTT assay. BEL-7402 cells were plated at
a concentration of 5000 cells per well on a Costar 96-well plate and
allowed to attach overnight. Conjugate stock solutions were
prepared in DMSO at a concentration of 50 mM. These stocks were
6.7%. MS: m/z 673. 1H NMR (CDCl3):
d, ppm, 8.86 (s, 8H), 8.09 (d, 6H,
J ¼ 8 Hz), 8.03 (d, 2H, J ¼ 8 Hz), 7.54 (d, 6H, J ¼ 8 Hz), 7.10 (d, 2H,
J ¼ 8 Hz), 2.70 (s, 9H), ꢁ2.19 (s, 2H). Anal.Calcd. for C47H36N4O,%: C,
83.90; H, 5.39; N, 8.33. Found: C, 83.62; H, 5.59; N, 8.30.
diluted to give a final concentration range from 2.5
The cells were then incubated 48 h in the dark. 20 L MTT (5 mg/mL
mM to 100 mM.
m
in PBS) was added to each well followed by 3 h incubation. The
formazan crystals were dissolved in DMSO. The absorbance was
measured in an enzyme linked immunoabsorbent assay plate
reader (Bio-Tek) at a wavelength of 570 nm. Concentration 50%
cell death (IC50) was determined for the various compounds tested.
L-02 cells were tested as above.
4.1.3.3. 5,10,15-tri(4-Methoxyphenyl)-20-(4-hydroxyphenyl)porphine
(10). The compound was prepared according to the general
procedure mentioned above, purified and isolated as a purple solid;
yield 7.4%. MS: m/z 721. 1H NMR (CDCl3):
d, ppm, 8.87 (s, 6H), 8.52
(d, 2H, J ¼ 8 Hz), 8.47 (s, 2H), 8.13 (d, 6H, J ¼ 8 Hz), 7.53 (d, 2H,
J ¼ 8 Hz), 7.29 (d, 6H, J ¼ 8 Hz), 4.16 (s, 3H), 4.11 (s, 6H), ꢁ2.76 (s,
2H). Anal.Calcd. for C47H36N4O4, %: C, 78.31; H, 5.03; N, 7.77; Found:
C, 78.20; H, 4.87; N, 8.01.
4.2.2. Cell uptake assays
The uptake test for liver cancer cells BEL-7402 and normal liver
cells L-02 was carried out according to the following processes.
Liver cancer cells BEL-7402 (or normal liver cells L-02) were seeded
on a Costar 24-well plate and allowed to attach overnight, and then
4.1.3.4. 5,10,15-tri(4-Chlorophenyl)-20-(4-hydroxyphenyl)porphine
(11). The compound was prepared according to the general
100
BEL-7402
0.045
L-02
BEL7402
L-02
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
80
60
40
20
0
1
2
3
4
5
6
1
2
3
4
5
Scheme 5. Cell viability of BEL-7402 and normal L-02 cells in the presence of 10
porphine phosphoramidates 1–5 and 4-formylphenyl N,N-bis(2-chlor-
oethyl)phosphoramidate 6.
mM
Scheme 6. Uptake of 5 porphine phosphoramidates at 5 mM by BEL-7402 cells and L-
02 cells for 24 h.

Z.-W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 890–895
895
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exposed to 5 mM porphine in medium for 24 h. At the end of the
loading period, the medium was removed, and the cells were
washed three times with ice-cold PBS (phosphate buffered saline)
and dissociated from the plate using enzyme free cell dissociation
solution and measured in 1.5 mL DMSO solution. Standard
concentration–absorbency curve are established on a Hitachi
F-4500 Fluorescence Spectrophotometer using excitation/emission
wavelengths of 420 and 650 nm, respectively. So the intracellular
accumulation of conjugate was determined by measuring the
fluorescence emission of porphines and the data were the average
of at least three independent experiments. Cell numbers were
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