Synthesis and in vitro PDT activity of miscellaneous porphyrins with amino acid and uracil

Article abstract of DOI:10.1016/j.bmc.2008.03.063

The synthesis of a series of modularized porphyrins bearing bioactive molecule is described. Starting with meso-tetraphenylporphyrin, the compounds with two nitro functional groups were synthesized via regiospecific nitration reaction. After reduction to the amino group and subsequent coupling with l-phenylalanine or 1-carboxylmethyl-5-fluorouracil (5-Fu acid), the functionalized porphyrins were metallized with Co(II) or Mn(II) to form miscellaneous porphyrins in good yields. The spectra of all the porphyrins were furnished. In vitro photodynamic therapy of the porphyrins against Ec9706 cell line was evaluated by standard cytotoxicity assays.

Full text of DOI:10.1016/j.bmc.2008.03.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5665–5671
Synthesis and in vitro PDT activity of miscellaneous
porphyrins with amino acid and uracil
Shi Weimin, Zhang Gen, Dai Guifu, Zhang Yunxiao, Zhao Jin and Tao Jingchao*
Department of Chemistry, Zhengzhou University, Zhengzhou 450052, China
New Drug Research and Development Center, Zhengzhou University, Daxue Road, Zhengzhou 450052, China
Received 26 January 2008; revised 22 March 2008; accepted 25 March 2008
Available online 30 March 2008
Abstract—The synthesis of a series of modularized porphyrins bearing bioactive molecule is described. Starting with meso-
tetraphenylporphyrin, the compounds with two nitro functional groups were synthesized via regiospecific nitration reaction. After
reduction to the amino group and subsequent coupling with L-phenylalanine or 1-carboxylmethyl-5-fluorouracil (5-Fu acid), the
functionalized porphyrins were metallized with Co(II) or Mn(II) to form miscellaneous porphyrins in good yields. The spectra
of all the porphyrins were furnished. In vitro photodynamic therapy of the porphyrins against Ec9706 cell line was evaluated by
standard cytotoxicity assays.
ꢀ
2008 Published by Elsevier Ltd.
2
3
1. Introduction
for better anticancer activity. Texaphytinato-Lu, Chlo-
2
4
23
rin
and Bacteriochlorophyll
exhibited a strong
Photodynamic therapy (PDT), a binary therapy of can-
cer, is a new modality for treatment of solid tumor based
on the selection aggregation of photosensitizers such as
absorption band in the red wavelength area. Interest has
also been focused on porphyrins bearing positively
charged groups permitting water solubility.
2
5,26
1
–3
porphyrin in tumor tissues.
Irradiation of the neo-
plastic region with light leads to activation of photosen-
sitizer. Thereby, the generated singlet oxygen ruins
abnormal tissues while sparing healthy adjacent areas.
The first generation of pophyrin photosensitizer, Photo-
However, a strategy implicated in ‘miscellaneous’ por-
phyrins with verified biological potent molecule essence
to ‘add-on’ the sole attributes like intracellular recogni-
tion, tissue selectivity, shift chromophores, and aqueous
ꢁ
27
frin is widely used in many countries for treatment of
characteristic has accepted little attention. Our re-
2
8
diverse cancers. Further works in developing new photo-
sensitizer with higher photoactivity and higher therapeu-
tic index has resulted in a vast amount of second
port in pophyrins field has suggested that amino acidic
porphyrin displayed more PDT efficiency than their pre-
cursors, and amino acid has been approved to amplify
4
–7
29
generation porphyrins.
the affinity of porphyrin with cell membrane. On the
other hand, widely used chemical anticancer reagents,
such as uracil-containing compounds, suffer from side
Recently much effort has been aimed at several properties
of modified porphyrins. It has been believed that the pres-
3
0
effects, thus we got better cytotoxicity of 5-fluorouracil
derivatives by the high compatibility of the high fatty
8
–11
ence of the carbohydrates moiety,
14–16
amino acidic resi-
etc., could enhance the membrane
1
2,13
31
dues,
steroid,
acid with cell membrane. Therefore it is very promis-
interaction to increase tumor selectivity. Porphyrins
equipped with chemical therapeutic agents, nitrogen mus-
ing to synthesize novel compounds that can accumulate
higher concentrations in malignant tumors than in nor-
mal tissues.
1
7,18
tard analogues,
1
boron neutron capture therapy
22
and nitroxyl derivatives were synthesized
9–21
(
BNCT),
At present, development of new methodologies to func-
tionalize porphyrins at porphyrin periphery, and b of
pyrrole and aryl at meso sites is still an active and excit-
Keywords: Miscellaneous porphyrins; Functionalization; Synthesis;
Photodynamic therapy.
3
2–34
ing field
due to providing a variety of new photosen-
*
Corresponding author. Tel./fax: +86 371 67767200; e-mail: jctao@
zzu.edu.cn
sitizers. However, practical and efficient methods
for unsymmetrically substituted porphyrins are still
0
968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.03.063

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S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
3
7
remarkably absent because of feazing, puzzling hetero-
geneous condensation of pyrroles and mixed aldehydes.
Previously, a vast majority of unsymmentric porphyrins
linked with bioactive molecule residues have been syn-
thesized by condensation of several aldehydes with pyr-
Alder–Long method. We have reported regiospecific
2
2
nitration of para position of phenyl at porphyrin.
Herein we present a more convenient operation. A B
porphyrin 2 was obtained in good yield by nitration of
TPP with Cl CCOOH/ HNO in CHCl at room tem-
3
perature for a few minutes under air. Corresponding
amino porphyrin 3 was readily synthesized by the treat-
2
2
3
3
8
,12,15,17
role
purification in poor yields. Recently, import of disparate
followed by tedious chromatographic
3
5,36
groups to prepared porphyrins
need.
released the pressing
ment of 2 with SnCl /HCl at 60 ꢂC over 1 h in yield of
2
90%.
Herein we describe a modified method of nitration of
aryl at porphyrin periphery which is convenient for the
Although the formation of amide bond is very common
by condensation of carboxyl acid or carbonyl chloride
3
8,39
synthesis of unsymmentric porphyrins.
of substituted porphyrins with L-phenylalanine or
-carboxylmethyl-5-fluorouracil (5-Fu acid) as func-
A
series
with amine,
carboxylations of the amino groups at
porphyrin are special due to the step by step procedure.
The condensations of the amino porphyrins with car-
boxyl acid were realized in moderate yield with excess
1
tional substituent were synthesized in good yield, and
their metal derivatives were also prepared. The primary
in vitro photocytotoxicity of these compounds toward
human esophageal cancer cell line was investigated.
0
of N,N -dicyclohexylcarbodiimide (DCC) as dehydrant,
while the attempt was successful to condense mono-
amino porphyrin with carboxyl of Boc-valine in 90%
2
8
yield. The exact mechanism of the impediment of the
amide bond to subsequent coupling is unknown, but this
will benefit the synthesis of ‘miscellaneous’ porphyrins
containing a variety of side chains via a series of
functional group conversions step by step.
2
. Results and discussion
2
.1. Synthesis of functionalized porphyrins
Porphyrin derivatives 3–11 were synthesized via the pro-
cedure shown in Figure 1.
In this case, we preferred introducing L-phenylalanine
moiety first instead of 5-Fu acid moiety, since the poor
solubility of the earlier synthesized uracil porphyrin
would prevent the subsequent carboxylation. The car-
boxyl moiety of Boc-L-phenylalanine was activated by
DCC, producing an intermediate species which reacted
with the amino group of porphyrin 3. By condensation
of Boc-L-phenylalanine with 3, under the given condi-
tions over a period of 48 h in CH Cl at room tempera-
So far, functionalization of synthetic porphyrins has
been an alternative method for unsymmetrical porphy-
rins. We chose to improve the nitration reaction for
substituting the para-H of phenyl at meso-tetraphenyl-
porphyrin (TPP), which was readily prepared using
2
2
ture, 40% of 4 and 46% of 5 were obtained.
R1
R2
1
2
3
R1 = R2 = H
In addition, porphyrin 3 was added to the reaction mix-
ture following reaction of Boc-L-phenylalanine with
DCC for 30 min at low temperature, and only one
Boc-L-phenylalanine was mounted to compound 3, giv-
ing compound 4 in 82% yield.
R1 = R2 = NO2
N
N
R1 = R2 = NH2
R1 = X
NH
HN
4
R2 = NH2
5
6
R1 = R2 = X
R1 = Y
R2 = NH2
By application of the similar condensation procedure in
DMF, porphyrin bearing one Boc-L-phenylalanine resi-
due was converted into ‘miscellaneous’ porphyrin 10
with uracil residue on another amino group in 95%
yield.
R1 = R2 = Y
7
8
R1 = Z
R2 = NH2
8
i
ii
iii
9
R1 = R2 = Z
1
2
5
3
9
10
R1 = X
R2 = Z
R2 = Z
iv
1
1 R1 = Y
v
7
v
One and double 5-Fu acids were appended on porphyrin
to result in 8 and 9 with total yield of 95% by condensa-
tion of carboxyl of 5-Fu acid with the amino group of
porphyrin 3 in DMF under the given conditions. Sepa-
ration of the crude porphyrins on silica gel gave 8 in
47% yield, and 9 in 48% yield.
6
4
v
iii
1
0
11
O
F
X =
Y =
Z =
HN
H
H N
2
O
HN
N
BOC-NH
HN
H
All the porphyrins equipped with Boc-L-phenylalanine
were well deprotected by CF COOH in DMF for 2 h
in good yields.
O
HN
O
3
O
Figure 1. Preparation of porphyrin derivatives. Reaction conditions:
2
.2. Preparation of the metal porphyrins
(
(
(
i) CCl
iii) 1-carboxylmethyl-5-fluoro-uracil, DCC, DMAP, DMF, rt, 48 h;
iv) L-phenylalanine, DCC, DMAP, CH Cl , 48 h; (v) CF COOH,
Cl , 2 h.
3 3 3 2
COOH/HNO , CHCl , 5 min; (ii) SnCl /HCl, 60 ꢂC, 60 min;
Metalations were performed in good yield according to
3
the literature method. As shown in Figure 2, mixture
2
2
3
8
CH
2
2

S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
5667
R1
R2
R1
R2
of amino acid or 5-Fu acid with porphyrin makes the
change of kmax by a few amounts, for example, a blue
shift of 3.0 nm is present between the peak of Soret band
of porphyrin 4 and that of the precursor 3. Metals
induce greater shift than the introduction of side chain.
The difference between Q band of 3 and its metal
derivatives 3a or 3b is approximately 30 nm.
N
N
N
M
N
MCl ,DMF
2
NH
HN
N
N
reflux
Compared to the metal porphyrins, it was found that
substituents took opposite effects on free porphyrins.
As can be seen in Table 1, introduction of 5-Fu acid
or L-phenylalanine fetches blue shift of Soret band of
free porphyrins. In contrast, red shift occurs when side
modification on metal porphyrins is carried out.
a M = Co
b M = Mn
Figure 2. Metallization of porphyrins.
of the relative transition metal chloride and solution of
porphyrins in DMF were refluxed for 2 h to give corre-
sponding metallized porphyrins.
2
.4. In vitro photocytotoxicity of porphyrins
A standard MTT assay was performed at a fixed por-
phyrin concentration. Inhibition of porphyrin 4–12
against Ec9706 (human esophageal cancer cell line)
was measured after irradiation for 2.5 h and a further
24 h of incubation in the dark. In vitro Photocytotoxic-
ity data are summarized in Table 2.
2
.3. Characterization of the porphyrin derivatives
1
HNMR, HRMS, and UV were obtained for all free
porphyrins, and the structures of metallized porphyrins
were confirmed by these spectra except for HNMR.
1
As can be seen from Table 2, the photocytotoxicity of
the vast majority of free porphyrins was improved by
introduction of the bioactive molecules. Unlike Co(II),
Mn(II) likely had advantageous effect on the anticancer
therapy of porphyrins.
The synthesized free base porphyrins and their metal-
lized derivatives were characterized with UV–vis spec-
tra, comprising a Soret band and a Q band.
As shown in Table 1, side modifications on porphyrin
structure have little effect on the photophysical proper-
ties of the chromophore, while insertion of a metal into
the core of a porphyrin alters the optical spectrum dras-
L-phenylalanine took notable effect on photocytotoxic-
ity of porphyrins. As shown in Table 2, inhibition per-
centage was decreased from 66% to about 40% when
single Boc-L-phenylalanine or L-phenylalanine was
introduced onto 3. However, inhibitions were enhanced
to over 96% when two Boc-L-phenylalanines or L-phen-
4
tically. The result agrees with the literature. Coupling
Table 1. UV–vis spectra of porphyrin derivatives
ylalanines were introduced. This result is different from
8,12,22
Porphyrins
Soret band
422.0
Q band
the reported result,
in which inhibitions of porphy-
3
516.0, 557.5, 597.0, 650.0
543.0, 581.0
472.5, 573.5, 612.0
rins were enhanced by introduction of Boc-amino acid.
Previous reports showed that amino acids, L-valine
and L-alanine both evidently enhanced the anticancer
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
9
9
9
1
1
1
1
1
1
a
308.0
381.5, 399.0
430.0
425.5
419.0
430.0
423.0
415.5
428.5
421.5
421.5
435.5
422.5
417.5
434.0
419.0
416.5
433.0
423.0
416.0
428.0
418.5
416.0
428.5
421.5
416.5
433.5
422.0
b
516.0, 554.0, 597.0, 648.0
544.0, 582.0
12,13
activity of porphyrin by membrane recognition.
a
b
316.0
381.0, 401.0
470.5, 520.0, 564.0, 606.5
513.0, 548.5, 590.0, 645.0
467.0, 543.5, 582.0
5
-Fu acid was confirmed to be profitable to photoactiv-
ity of porphyrins. Porphyrins with either one or two
-Fu acid exhibited higher bioactivity than the
a
b
5
382.5, 400.5
468.5, 520.0, 565.0, 601.0
516.5, 556.0, 597.0, 648.0
549.0, 585.5
precursor, suggesting that 5-Fu acid could increase the
bioactivity of porphyrins. Parallel to other chemical
a
b
314.0
381.5, 400.0
470.5, 522.0, 571.0, 606.0
513.0, 551.5, 593.5, 645.5
546.0, 585.0
a
b
323.0
380.0, 400.5
Table 2. Inhibition of porphyrins against Ec9706 (human esophageal
cancer cell line)
468.0, 522.0, 567.5, 601.5
516.0, 553.0, 592.5, 648.0
544.0, 585.5
Porphyrins Free
Co (II)
Mn (II)
porphyrins (%) porphyrins (%) porphyrins (%)
a
b
316.0
381.0, 401.0
470.5, 571.0, 607.0
513.0, 553.0, 593.0, 645.0
544.0, 585.0
3
4
5
6
7
8
9
1
1
66.60
47.21
100.00
37.94
96.13
97.09
100.00
88.65
69.45
51.98
55.80
42.48
56.82
74.93
44.54
49.32
20.82
5
100.00
99.81
42.22
77.73
83.31
57.94
56.77
55.29
58.00
a
313.0
381.5, 401.0
b
468.5, 522.0, 565.5, 602.0
513.0, 551.0, 590.0, 645.0
543.0, 575.0
0
0a
0b
1
313.5
381.0, 401.0
468.5, 518.0, 565.5, 601.5
513.0, 553.0, 594.0, 648.0
544.0, 585.0
0
1
1a
1b
315.5
381.0, 400.5
468.5, 522.0, 567.0, 601.0

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S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
1
7–21
therapeutic agents,
5-fluoro uracil assisted the por-
4.1.1. 5,10-Di(4-aminophenyl)-15,20-diphenylporphyrin
(3). To a solution of porphyrin 1 (1 g, 1.6 mmol) and tri-
chloroacetic acid (50 g) chloroform (100 mL) was drop-
wise added HNO3 (7.50 mL, 75%, 15.5 mmol) over
2 min at room temperature under atmosphere. The mix-
ture was stirred for 5 min and then quenched with water,
and neutralized with ammonium hydroxide aqueous solu-
tion to pH ꢀ7. Chloroform (100 mL) was added, and the
organic layer was washed with water (4· 150 mL), and
dried over magnesium sulfate. Solvent was removed with
evaporation under reduced pressure. The residue was dis-
solved in concentrated hydrochloric acid (30 mL), to
which SnCl Æ 2H O (6 g, 0.028 mol) was added. The mix-
phyrin for photocytotoxicity via its strong cytotoxicity.
It is possible that the selective congregation of porphyrins
in tumor tissue was incorporated with high cytotoxicity of
5
-Fu, resulting in magnified inhibitions of porphyrins.
Porphyrins decentralized the cultures with cells at 50 lg/
mL. After irradiation for 2 h by red wave, kept at 37 ꢂC
in dark for 24 h. Inhibitions were measured by standard
MTT methods. a: porphyrinate Co(II); b: porphyrinate
Mn(II).
As shown in Table 2, all of the porphyrin complex with
Mn(II) showed higher photocytotoxicity than those with
Co (II). Inhibition against Ec9706 cell lines for porphyrins
with Co (II) spread from 44% to 74%, while those for por-
phyrins with Mn(II) from 55% to 100%. It is interesting
that insertion of metal ion enhances the activity of
mono-substituted porphyrins 4, 7, and 9 and in contrast
decreases that of di-substituted porphyrins 5, 6, 8.
2
2
ture was heated to 60 ꢂC and maintained for 1 h. After
neutralization with ammonium hydroxide aqueous solu-
tion to pH ꢀ7 under cooling, chloroform (200 mL) was
added. The organic layer was washed with water (4·
200 mL) and dried over magnesium sulfate. Solvent was
evaporated in vacuum. The crude product was purified
by column chromatography (silica, eluent/chloroform)
1
to provide 3 (370 mg, 36%). H NMR (CDCl3,
4
00 MHz) d (ppm) = 8.92 (s, 4H, b-pyrrole), 8.82 (s, 4H,
b-pyrrole), 8.21 (d, 4H, J = 6.2 Hz, 2, 6-H phenyl), 7.98
d, 4H, J = 8.1 Hz, 2, 6-H aminophenyl), 7.77–7.71 (m,
3. Conclusion
(
Functionalized porphyrins were synthesized using a mod-
ified nitration at para position of phenyl at porphyrin,
reduction with SnCl /HCl, and then coupling with some
2
6H, 3, 5-H phenyl), 7.06 (d, 4H, J = 8.2 Hz, 3, 5-H amino-
phenyl), 3.67 (s, 4H, NH ), ꢁ2.73 (s, 2H, NH-pyrrole).
2
UV–vis (k , nm) in CHCl : 422.0, 516.0, 557.5, 597.0,
max
3
bioactive molecules. A series of porphyrins bearing 5-Fu
or L-phenylalanine were synthesized by coupling 5-Fu
acid or Boc-L-phenylalanine with amino porphyrins in
good yields. The in vitro photocytotoxicities of the func-
tionalized porphyrins against Ec9706 cell lines were
tested, and the results suggested that photocytotoxicities
of porphyrin were improved remarkably by introduction
of 5-fluorouracil and L-phenylalanin, whereas transition
metals Co(II) and Mn(II) brought distinct effects on
photo activity of porphyrins. Further developments of
this strategy are in progress, including other characteris-
tics of photocytotoxicities of porphyrins.
650.0. HRMS: calcd for C H N : 644.2689; found:
44 32 6
+
667.2562 [M+Na] .
4.1.1.1. Compound 3a. Compound 3 (100 mg,
0.16 mmol) and CoCl (100 mg, 0.78 mmol) in CHCl
2
3
(10 mL) gave 90 mg of 3a (83%). UV–vis (kmax, nm) in
CHCl : 308.0, 430.0, 543.0; HRMS: calcd for C H
3
44 30
+
CoN : 701.1864; found: 724.1765 [M+Na] .
6
4.1.1.2. Compound 3b. Compound
0.16 mmol) and MnCl (97 mg, 0.78 mmol) in CHCl
3
(100 mg,
2
3
(10 mL) gave 99 mg of 3a (92%). UV–vis (kmax, nm) in
CHCl : 381.5, 399.0, 425.5, 472.5, 573.5, 612.0. HRMS:
calcd for C H MnN : 697.1912. found: 720.1817
3
4
4
30
6
+
[M+Na] .
4
. Experimental methods
5
method.
-Fu acid was synthesized according to the literature
4
4.1.2. 5-{4-[N-(t-Butyloxycarbonyl)-3-phenyl-L-alanylam-
ido]phenyl}-10-(4-aminophenyl)-15,20-diphenylporphyrin
(4). To a solution of Boc-L-phenylalanine (41 mg,
0.155 mmol) in dried chloroform (30 mL) were added
DCC (17 mg, 0.35 mmol) and DMAP (20 mg,
0.35 mmol). The mixture was stirred at 0–5 ꢂC for
60 min, and 3 (100 mg, 0.155 mmol) was subsequently
added. The mixture was maintained at room temperature
for 24 h, and then in ice bath overnight. Most of DCU was
removed by filtration, oxalic acid was added to consume
the remaining DCC. Solvent was evaporated in vacuum.
The crude product was purified by column chromatogra-
0,41
Boc-phenylalanine and pyrrole were pur-
chased from Aldrich, and distilled under reduced pressure
before use. All other reagents and solvents were used as
received. Solvents were of reagent grade unless otherwise
specified and were dried and distilled by the standard
method. The UV–vis spectra were obtained on a Perkin-
1
Elmer LS-5B spectrophotometer. H NMR spectra were
recorded on Bruker AVANCE DPX-400 spectrometer.
HRMS (high-resolution mass spectra) were taken with a
Q-Tof Micromass spectrometer.
4
.1. General metallization of porphyrins
phy (silica, eluent: chloroform/methanol = 10:1) to give 4
1
in 82% yield. H NMR (CDCl ) d 8.83–8.80 (m, 8H, b-
3
Porphyrins (1 equiv) were dissolved in CHCl , then
3
MCl (5 equiv) was added. The mixture was stirred,
2
and refluxed for 2 h. Washed with water, the organic
layer was dried on magnesium sulfate. Solvent was evap-
orated in vacuum. The crude product was purified by
column chromatography.
pyrrole), 8.22–8.14 (m, 6H, 3,5-H amidophenyl and 2,6-
H phenyl), 7.88 (d, 2H, J = 8.12 Hz, 3, 5-H amidophenyl),
7.80 (d, 2H, J = 8.04 Hz, 2, 6-H aminophenyl), 7.75–7.71
(m, 6H, 3, 4, 5-H phenyl), 7.40 (d, 4H, J = 8.25 Hz, 2, 3, 5,
6-H alkylphenyl), 7.24 (s, 1H, 4-H alkylphenyl), 6.98 (s,
2H, 3, 5-H aminophenyl), 5.32 (s, 1H, N-CH), 3.31 (m,

S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
5669
2
H, Ph-CH ), 1.51(s, 9H, C-CH ), ꢁ2.73 (s, 2H, NH-pyr-
(113 mg, 0.6 mmol) and DCC (85 mg, 0.7 mmol), and
DMAP (135 mg, 0.7 mmol) in CH Cl (6 mL) gave
100 mg of 8 (40%) and 140 mg of 9 (46%). The product
2
3
role). UV–vis (k , nm) in CHCl : 419.0, 516.0, 554.0,
597.0, 648.0. HRMS: calcd for C H N O : 891.3897.
found: 914.3791 [M+Na] .
max
3
2
2
5
8
49
7
3
+
was obtained by column chromatography (silica, eluent:
1
chloroform/methanol = 9:4). 8 H NMR (CDCl ) d
3
4
.1.2.1. Compound 4a. Compound
4
(100 mg,
10.87 (s, 1H, NH-uracil), 8.97 (s, 4H,b-pyrrole), 8.80 (d,
4H, J = 4.9 Hz, b-pyrrole), (m, 4H,b-pyrrole), 8.23–8.17
(m, 6H, 3, 5-H amidophenyl and 2, 6-H phenyl), 8.06 (d,
2H, J = 9.6 Hz, 2, 6-H amidophenyl), 7.88–7.82 (m,
10H, 2, 3, 5, 6-H aminophenyl and 3, 4, 5-H phenyl),
0
.11 mmol) and CoCl (72 mg, 0.56 mmol) in CHCl
2 3
(
CHCl : 316.0, 430.0, 544.0, 582.0; HRMS: calcd for
10 mL) gave 90 mg of 4a (85%). UV–vis (kmax, nm) in
3
+
C H CoN O : 948.3072. found: 971.2972 [M + Na ].
5
8
47
7
3
4
.71 (s, 2H, N-CH ), ꢁ2.84 (s, 2H, NH-pyrrole). UV–
2
4
.11 mmol) and MnCl (70 mg, 0.56 mmol) in CHCl3
.1.2.2. Compound 4b. Compound
4
(100 mg,
vis (k , nm) in CHCl :416.5, 516.0, 553.0, 592.5,
max 3
0
648.0. HRMS: calcd for C H FN O : 814.2816. found:
50 35 8 3
2
+
837.2718 [M+Na] .
(
CHCl : 381.0, 401.0, 423.0, 470.5, 520.0, 564.5, 606.5.
10 mL) gave 94 mg of 4b (92%). UV–vis (kmax, nm) in
3
HRMS: calcd for C H CoN O : 944.3121. found:
5
9
4.2.2.1. Compound 8a. Compound
0.098 mmol) and CoCl (63 mg, 0.49 mmol) in CHCl
8
(80 mg,
8
47
7
3
+
67.3025 [M+Na ].
2
3
(5 mL) gave 68 mg of 8a (80%). UV–vis (kmax, nm) in
4
(
.2. General procedure for synthesis of porphyrins (5), or
8),(9), (10) containing with amino acid and/or uracil
CHCl : 316.0, 433.0, 544.0, 585.5; HRMS: calcd for
3
+
C H CoFN O : 871.1992, found: 894.1896 [M+Na] .
5
0
33
8
3
Porphyrin 3 was dissolved in CH Cl , and DCC (1–2.3
2
4.2.2.2. Compound 8b. Compound
0.098 mmol) and MnCl (61 mg, 0.49 mmol) in CHCl
(5 mL) gave 78 mg of 8b (91%). UV–vis (kmax, nm) in
8
(80 mg,
2
equiv), DMAP (1–2.3 equiv), Boc-L-phenylalanine (2.5
equiv) or 5-Fu acid (1–2 equiv) were added. The mixture
was stirred at room temperature for 48 h, then DCU
was removed by filtration followed by addition of oxalic
acid. Solvent was evaporated in vacuum. The product
was obtained by purification on silica gel.
2
3
CHCl : 381.5, 401.0, 423.0, 470.5, 571.0, 607.0; HRMS:
calcd for C H MnFN O : 867.2040. found: 890.1945
3
5
0
33
8
3
+
[M+Na] .
1
.2.2.3. Compound 9. Compound 9 H NMR (CDCl )
4
3
4
.2.1. 5,10-Di{4-[N-(t-butyloxycarbonyl)-3-phenyl-L-ala-
d 10.86 (2H, s, NH-uracil), 10.86 (s, 2H, NH-uracil),
8.88–8.82 (m, 8H,b-pyrrole), 8.25–8.18 (m, 10H, 3, 5-H
amidophenyl-H and 2, 6-H phenyl), 8.06 (d, 4H,
J = 8.44 Hz, 2, 6-H amidophenyl), 7.87–7.80 (m, 6H,
nylamido]phenyl}-15,20-di-phenylporphyrin (5). Com-
pound 3 (100 mg, 0.155 mmol), Boc-L-phenylalanine
(
(
(
100 mg, 0.377 mmol), DCC (43 mg, 0.35 mmol), DMAP
68 mg, 0.35 mmol) in CH Cl (3 mL) gave 157 mg of 5
89%). The product was obtained by column chromatog-
H
3, 4, 5-H phenyl), 4.71 (s, 4H, N-CH ), ꢁ2.90 (s, 2H,
2
2
2
NH-pyrrole). UV–vis (k , nm) in CHCl : 416.0,
max
3
1
raphy (silica, eluent: chloroform/methanol = 6:1).
513.0, 553.0, 593.5, 645.0. HRMS: calcd for C H
56 38
+
F N O : 984.2944. found: 1007.2835 [M+Na] .
NMR (CDCl ) d 8.83–8.80 (m, 8H, b-pyrrole), 8.19–
3
2
10
6
8
7
7
2
2
.11 (m, 6H, 3, 5-H amidophenyl and 2, 6-H phenyl),
.79–7.68 (m,10H, 2, 5-H amidophenyl, 3, 4, 5-H phenyl),
.41 (d, 8H, J = 5.6 Hz, 2, 3, 5, 6-H alkylphenyl), 7.25 (d,
H, J = 5.6 Hz, alkylphenyl), 5.33 (s, 1H, N-CH), 3.30 (m,
H, Ph-CH ), 1.51 (s, 18H, CCH ), ꢁ2.73 (s, 2H, NH-pyr-
4.2.2.4. Compound 9a. Compound
9
0.081 mmol) and CoCl (52 mg, 0.41 mmol) in CHCl
(80 mg,
2
3
(5 mL) gave 71 mg of 9a (84%). UV–vis (kmax, nm) in
CHCl : 313.0, 428.0, 544.0, 585.0; HRMS: calcd for
2
3
3
+
C H CoF N O : 1041.2119. found: 1064.2025 [M+Na] .
role). UV–vis (k , nm) in CHCl : 415.5, 513.0, 548.5,
max
3
56 36
2
10 6
590.0, 645.0. HRMS: calcd for C H N O : 1138.5105.
found: 1161.5006 [M+Na] .
72 66 8 6
+
4.2.2.5. Compound 9b. Compound
9
(80 mg,
.081 mmol) and MnCl (51 mg, 0.41 mmol) in CHCl
0
2
3
4
.2.1.1. Compound 5a. Compound
5
(100 mg,
.088 mmol) and CoCl (57 mg, 0.44 mmol) in CHCl3
(5 mL) gave 76 mg of 9b (90%). UV–vis (kmax, nm) in
0
CHCl : 381.5, 401.0, 418.5, 468.5, 522.0, 565.5, 602.0;
HRMS: calcd for C H MnF N O : 1037.2168.
2
3
(
CHCl : 428.5, 467.0, 543.5, 582.0; HRMS: calcd for
5 mL) gave 86 mg of 5a (82%). UV–vis (kmax, nm) in
56
+
found: 1060.2068 [M+Na] .
36
2
10
6
3
+
C H CoN O : 1195.4281, found: 1218.4171 [M+Na] .
7
2
64
8
6
4.2.3. 5-{4-[2-(5-Fluoro-uracil)-acetylamido]phenyl}-10-{4-
4
.2.1.2. Compound 5b. Compound
5
(100 mg,
.088 mmol) and MnCl (55 mg, 0.44 mmol) in CHCl3
[N-(t-butyloxycarbonyl)-3-phenyl-L-alanylamido]phenyl}-
15,20-diphenylporphyrin (10). Compound 4 (100 mg,
0.11 mmol), 2-(5-Fu acid)-acetic acid (13 mg, 0.11 mmol)
and DCC (23 mg, 0.12 mmol), and DMAP (15 mg,
0.12 mmol) in CH Cl (3 mL) gave 110 mg of 10 (95%).
0
2
(5 mL) gave 99 mg of 5b (95%). UV–vis (kmax, nm) in
CHCl : 382.5, 400.5, 421.5, 468.5, 520.0, 565.0, 601.0.
3
HRMS: calcd for C H MnN O : 1191.4329. found:
8
7
2
64
6
2
2
+
214.4231 [M+Na] .
1
The product was obtained by column chromatography
(
1
silica, eluent: chloroform/methanol = 5:1). H NMR
4
.2.2. 5-{4-[2-(5-Fluoro-uracil)-acetylamido]phenyl}-10-(4-
(CDCl ) d 10.80 (s, 1H, NH-uracil), 8.90 (d, 4H,
3
aminophenyl)-15,20-diphenylporphyrin (8) 5,10-di{4-[2-(5-
fluoro-uracil)-acetylamido]phenyl}-15,20-diphenylporph-
yrin (9). Compound 3 (200 mg, 0.31 mmol), 5-Fu acid
J = 11.7 Hz, b-pyrrole), 8.83 (s, 4H,b-pyrrole), 8.24–8.18
(m, 8H, 3, 5-H amidophenyl and 2, 6-H phenyl), 8.10–
8.05 (m, 4H, amidophenyl), 7.84 (d, 8H, J = 6.04 Hz, 3,

5670
S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
4, 5-H phenyl), 7.37–7.47 (m, 4H, 2, 3, 5, 6-H alkylphe-
nyl), 7.27 (s, 1H, 4-H alkylphenyl), 4.71 (s, 2H, N-CH2),
4.3.2. 5,10-Di[4-(3-phenyl-L-alanylamido)phenyl]-15,20,-
diphenyl-porphyrin (7). Compound 4 (50 mg, 0.056 mmol)
with TFA (0.8 mL, 8.5 mmol) gave 48 mg of 7 (91%). The
4
9
.53 (1H, s, N-CH), 3.21–3.17 (m, 2H, Ph-CH ), 1.40 (s,
2
H, C-CH ), ꢁ2.90 (s, 2H, NH-pyrrole). UV–vis (kmax,
product was obtained by column chromatography (silica,
3
1
nm) in CHCl : 416.0, 513.0, 551.0, 590.0, 645.0. HRMS:
3
eluent: chloroform/methanol = 4:1). H NMR (CDCl ) d
3
calcd for C H FN O : 1061.4025. found: 1084.3926
6
[
8.89(s, 4H, b-pyrrole), 8.83 (s, 4H, b-pyrrole), 8.24–8.22
(4H, m, 2, 6-H phenyl), 8.16 (d, 4H, J = 8.52 Hz, 3, 5-H
amidophenyl), 8.11(d, 4H, J = 8.52 Hz, 2, 6-H amidophe-
nyl-H at meso), 7.85–7.83 (m, 6H, 3, 4, 5-H phenyl), 7.40–
7.35 (m, 8H, 2, 3, 5, 6-H alkylphenyl), 7.27 (s, 2H, 4-H
alkylphenyl), 3.77–3.81 (m, 1H, N-CH), 3.24–3.16 (m,
4
52
9
6
+
M+Na] .
4
.075 mmol) and CoCl (48 mg, 0.38 mmol) in CHCl
.2.3.1. Compound 10a. Compound
9
(80 mg,
0
2
3
(
CHCl : 313.5, 428.5, 543.0, 575.0; HRMS: calcd for
5 mL) gave 71 mg of 9a (84%). UV–vis (kmax, nm) in
4H, Ph-CH ), ꢁ2.90 (s, 2H, NH-pyrrole). UV–vis (kmax,
3
2
+
C H CoFN O : 1118.3200. found: 1141.3093 [M+Na] .
nm) in CHCl : 417.5, 513.0, 551.5, 593.5, 645.5. HRMS:
3
6
4
50
9
6
calcd for C H N O : 938.4057. found: 961.3958
2
6
2
50
8
+
[M+Na] .
4
.075 mmol) and MnCl (47 mg, 0.38 mmol) in CHCl
.2.3.2. Compound 10b. Compound
9
(80 mg,
0
2
3
(
CHCl : 381.0, 401.0, 421.5, 468.5, 518.0, 565.5, 601.5.
5 mL) gave 74 mg of 9a (88%). UV–vis (kmax, nm) in
4.3.2.1. Compound 7a. Compound
0.085 mmol) and CoCl (55 mg, 0.43 mmol) in CHCl
3
7
(80 mg,
3
2
HRMS: calcd for C H MnFN O : 1114.3249. found:
9
1
(5 mL) gave 68 mg of 7a (80%). UV–vis (kmax, nm) in
6
4
50
6
+
137.3142 [M+Na] .
CHCl : 323.0, 434.0, 546.0, 585.0; HRMS: calcd for
3
+
C H CoN O : 995.3232, found: 1018.3134 [M+Na] .
6
2
48
8
2
4
.3. General deprotection of Boc
4
.3.2.2. Compound 7b. Compound
7
(80 mg,
0.085 mmol) and MnCl (65 mg, 0.43 mmol) in CHCl
To a solution of Porphyrins (1 equiv) with Boc group in
dried DMF was added trifluoro-acetic acid (TFA, 20–63
equiv) at 0–5 ꢂC. The mixture was kept at room temper-
ature for 2 h. After being neutralized with ammonium
hydroxide aqueous solution, the mixture was extracted
by CH Cl . Combined organic layer was washed with
2
3
(5 mL) gave 74 mg of 7 b (88%). UV–vis (kmax, nm) in
CHCl : 380.5, 400.5, 419.0, 468.0, 522.0, 567.5, 601.5.
HRMS: calcd for C H MnN O : 991.3281. found:
3
6
2
48
8
2
+
1014.3186 [M+Na] .
2
2
saturated brine, and then dried over magnesium sulfate.
Solvent was removed by evaporation under reduced
pressure. The product was obtained by purification on
silica gel.
4.3.3. 5-{4-[2-(5-Fluoro-uracil)-acetylamido]phenyl}-10-
[4-(3-phenyl-L-alanylamido)phenyl]-15,20-diphenylporph-
yrin (11). Compound 4 (90 mg, 0.085 mmol) with TFA
(0.4 mL, 8.5 mmol) gave 78 mg of 11 (96%). The product
was obtained by column chromatography (silica, eluent:
1
4
.3.1. 5-[4-(-3-Phenyl-L-alanylamido)phenyl]-10-(4-ami-
nophenyl)-15,20-diphenyl-porphyrin (6). Compound 4
50 mg, 0.056 mmol) with TFA (0.4 mL, 4.2 mmol) gave
0 mg of 6 (91%). The product was obtained by column
chloroform/methanol = 3:1). H NMR (CDCl ) d 10.94
3
(s, 1H, NH-uracil), 8.89(s, 4H, b-pyrrole), 8.83 (s, 4H,
b-pyrrole), 8.24–8.06 (m, 12H, amidophenyl and 2, 6-H
phenyl), 7.84–7.80 (m, 6H, 3, 4, 5-H phenyl), 7.40–7.34
(m, 4H, 2, 3, 5, 6-H alkylphenyl), 7.27 (s, 1H, 4-H alkyl-
(
4
chromatography (silica, eluent: chloroform/metha-
nol = 7:1). H NMR (CDCl ) d 8.92 (s, 2H, b-pyrrole),
1
phenyl), 4.69 (s, 2H, C-CH ), 3.80–3.77 (m, 1H, N-CH),
3
3
8
8
.87 (s, 2H, b-pyrrole), 8.80 (d, 4H, J = 5.3 Hz, b-pyrrole),
.33–8.20 (m, 4H, 3, 5-H phenyl), 8.16 (2H, d,
3.23–3.16 (m, 2H, Ph-CH ), ꢁ2.90 (s, 2H, NH-pyrrole).
2
UV–vis (k , nm) in CHCl : 416.5, 513.0, 553.5, 594.0,
max
3
J = 8.64 Hz, 3, 5-H amidophenyl), 8.10 (d, 2H,
J = 8.48 Hz, 2, 6-H amidophenyl), 7.88–7.83 (m, 6H, 2,
648.0. HRMS: calcd for C H FN O : 961.3500. found:
59 44 9 4
+
984.3404 [M+Na] .
6
-H aminophenyl and 3, 4, 5-H phenyl), 7.37 (d, 4H,
J = 6.64 Hz, 2, 3, 5, 6-H alkylphenyl), 7.27 (s, 1H, 4-H
phenyl), 7.01 (d, 2H, J = 8.24 Hz, 3, 5-H aminophenyl),
4.3.3.1. Compound 11a. Compound 11 (80 mg,
0.083 mmol) and CoCl (53 mg, 0.42 mmol) in CHCl
2
3
3
.80–3.77 (m, 1H, N-CH), 3.20–3.16 (m, 2H, Ph-CH2),
2.90 (s, 2H, NH-pyrrole). UV–vis (kmax, nm) in CHCl3:
21.5, 516.5, 556.0, 597.0, 648.0. HRMS: calcd for
(5 mL) gave 72 mg of 11a (85%). UV–vis (kmax, nm) in
ꢁ
CHCl : 315.5, 433.5, 544.0, 585.0; HRMS: calcd for
3
+
C H CoFN O : 1018.2676. found: 1041.2576 [M+Na] .
4
5
9
42
9
4
+
C H N O: 791.3373. found: 814.3274 [M+Na] .
5
3
41
7
4.3.3.2. Compound 11b. Compound 11 (80 mg,
0.083 mmol) and MnCl (52 mg, 0.42 mmol) in CHCl
(5 mL) gave 74 mg of 11b (88%). UV–vis (kmax, nm) in
4.3.1.1. Compound 6a. Compound 6 (80 mg, 0.1 mmol)
and CoCl (65 mg, 0.5 mmol) in CHCl (5 mL) gave
2
3
2
3
6
4
8
9 mg of 6a (81%). UV–vis (kmax, nm) in CHCl : 314.0,
CHCl : 381.0, 400.5, 422.0, 468.5, 522.0, 567.0, 601.0;
HRMS: calcd for C H MnFN O : 1014.2724. found:
3
3
35.5, 549.0, 585.5; HRMS: calcd for C H CoN O:
5
3
39
7
59 42
9
4
+
48.2548. found: 871.2453 [M+Na] .
+
1037.2628 [M+Na] .
4.3.1.2. Compound 6b. Compound 6 (80 mg, 0.1 mmol)
and MnCl (63 mg, 0.5 mmol) in CHCl (5 mL) gave
Acknowledgements
2
3
7
9 mg of 6a (93%). UV–vis (kmax, nm) in CHCl : 381.5,
3
400.0, 422.5, 470.5, 522.0, 571.0, 606.0; HRMS: calcd
for C H MnN O: 844.2597. found: 867.2498 [M+Na] .
This project was supported by National Natural Science
Foundation of China (20772113, 20505015) and Natural
+
5
3
39
7

S. Weimin et al. / Bioorg. Med. Chem. 16 (2008) 5665–5671
5671
Science Foundation Henan Education Department
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