Novel porphyrin-Schiff base conjugates: Synthesis, characterization and: In vitro photodynamic activities

Article abstract of DOI:10.1039/c6ra05682b

Three novel porphyrin-Schiff base conjugates derived from tetra(4-aminophenyl)porphyrin (TAPP), namely, tetra[4-(4-hydroxy benzylideneamino)]phenyl porphyrin (3a), tetra[4-(2-thienyldeneamino)]phenyl porphyrin (3b) and tetra[4-(2-pyridyldeneamino)]phenyl porphyrin (3c), were synthesized and characterized by IR, UV-vis, ¹H NMR, HRMS and elementary analysis. Their biological activities against human epidermoid carcinoma (A431) cells were evaluated with an MTT assay. As we expected, the porphyrin conjugates showed negligible cytotoxicity to A431 cells in the absence of light, while their phototoxic activities were improved after irradiated with LED lamp (425 nm) and increased significantly with increased doses. The fluorescence microscope pictures revealed that the three porphyrin-Schiff conjugates could diffuse into skin cancer cells, demonstrating that these compounds are potential candidates for photodynamic therapy agents.

Full text of DOI:10.1039/c6ra05682b

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Page 1 of 22
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Novel porphyrin-Schiff base conjugates: synthesis, characterization
and in vitro photodynamic activities
YaꢀHong Yaoa,b, Jun Lia,*, LongꢀFei Yuana, ZengꢀQi Zhanga and FengꢀXing Zhanga
a) Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
Ministry of Education, College of Chemistry & Materials Science, Northwest
University, Xi’an, Shaanxi 710069, China)
b) College of Science, Xi’an University of Architecture and Technology, Xi’an,
Shaanxi 710055, China
*corresponding author, eꢀmail: junli@nwu.edu.cn
Abstract
Three novel porphyrinꢀSchiff base conjugates derived, namely, tetra[4ꢀ(4ꢀhydroxy
benzylideneamino)] phenyl porphyrin (3a), tetra[4ꢀ(2ꢀthienyldeneamino)]phenyl
porphyrin (3b) and tetra [4ꢀ(2ꢀpyridyldeneamino)]phenyl porphyrin (3c) from the
tetra (4ꢀaminophenyl) porphyrin (TAPP) were synthesized and characterized by IR,
1
UVꢀvis, H NMR, HRMS and elementary analysis. Their biological activities against
human epidermoid carcinoma (A431) cells were evaluated with a MTT assay. As we
expected, the porphyrin conjugates showed negligible cytotoxicity to A431 cells in the
absence of light. While their phototoxic activities were improved after irradiated with
LED lamp (425nm) and increased significantly with the increase of doses. The
fluorescence microscope pictures revealed that three porphyrinꢀSchiff conjugates
could diffuse into skin cancer cells, demonstrating that these compounds are potential
candidates for photodynamic therapy agents.
Keywords: Porphyrin; Schiff base; Antiꢀtumor activity; Photodynamic therapy;
Cytotoxicity

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1. Introduction
Cancer is one of the deadliest diseases of in nowadays. The traditional cancer
treatments, including surgery, chemotherapy and radiation therapy, may cause serious
1,
side effects resulted from the loss of normal organ function
2. In contrast,
photodynamic therapy (PDT) can selectively destroy malignant cells by the
administration of a nonꢀtoxic photosensitizer and hold the potential for minimal side
3,
effects
4. Upon irradiation with light of specific wavelength, photosensitizer
transfers energy to triplet oxygen (3O2), and then generates singlet oxygen (1O2) and
other reactive oxygen species (ROS) near the tumor. These generated molecules can
cause intracellular oxidative damages and cell death through apoptosis or necrosis 5, 6
.
Porphyrins and their derivatives, as a promising photosensitizer, have attracted
extensive attention in the field of PDT for cancer treatment 7ꢀ10. This is due to the
obvious advantages of porphyrins, such as favourable wavelength, intense absorption
11
in visible region of the electromagnetic spectrum and low intrinsic toxicity
.
Moreover, it has been demonstrated that porphyrins prefer to accumulate in tumor
cells in vitro and in vivo rather than in normal tissue. Notably the firstꢀgeneration
photosensitizer Photofrin is a complex mixture of hematoporphyrins derivatives and
has been approved by the United States Food and Drug Administration (FDA) for
PDT treatment of various cancers such as oesophageal cancer, earlyꢀand lateꢀstage
lung cancer, bladder cancer and malignant, nonmalignant and earlyꢀstage cervical
cancer 12ꢀ14. There has been a growing interest in the synthesis and functionalization
of porphyrins for the chemists 15, 16
.
Schiff bases were an important class of organic compounds, and were reported to
have a wide range of biological activities including antiviral, anticancer, cytotoxic,
antimicrobial, etc. 17, 18. Thus, development and synthesis of novel Schiff base
derivatives as potential chemotherapeutics attracted the attention of organic and
medicinal chemists 19. Furthermore, hydroxyl group endows compounds good
solubility in aqueous environment, and is a commonly used hydrophilic moiety in
dissolution of some photosensitizers. Thereby porphyrin conjugate with hydroxyl
groups was expected to enhance its therapeutic efficacy in vitro 20ꢀ22. Besides,

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heterocyclic rings such as thiophene, furan and pyrrole in the compounds, have played
a major role in expanding their pharmacological properties. For example,
pregnenolone derivatives containing heterocyclic moieties were found to exhibit
anticancer activity 23, 24. In addition, suitable candidates for photosensitizer were
obtained by attaching various substituents to the peripheral positions of the porphyrin
core.
Herein, we reported the design and synthesis of the porphyrinꢀSchiff base
conjugates, which bearing hydroxyl groups, thienyl and pyridyl groups on porphyrin
peripheral substituents positions 3a-3c (Sheme 1). In addition, we evaluated their
cytotoxic activity against human skin cancer cell lines and normal skin cells.
Furthermore, the photodynamic therapy and cellular uptake of the porphyrins in the
tumor cells are also investigated.
2. Experimental
2.1 Materials and Instruments
1HꢀNMR spectra were collected in a Varian Inova 400 MHz NMR spectrometer.
Deuterated dimethylsulfoxide (d6ꢀDMSO) was used as solvent and TMS as internal
reference. Elemental analyses (C, H and N) were performed on a Vario ELꢀIII
CHNOS instrument. Mass spectra (MS) were recorded on a matrix assisted laser
desorption/ionization time of flight mass spectrometer (MALDIꢀTOF MS, Krato
Analytical Company of Shimadzu Biotech, Manchester, Britain). FTꢀIR (4000ꢀ400
cmꢀ1) spectra were obtained with samples in KBr matrix for the title complexes on A
BEQUZNDXꢀ550 series FTꢀIR spectrophotometer. The UVꢀvis spectra were recorded
on a Shimadzu UV1800 UVꢀvisꢀNIR spectrophotometer using CHCl3 as solvent.
Fluorescence spectra were recorded in CHCl3 with a HITACHI Fꢀ4500
spectrophotometer. Chromatographic purification was performed with silica gel (100–
120 mesh). 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium bromide (MTT)
were purchased from SigmaꢀAldrich Japan (Chiba, Japan). Pyrrole and DMF were
distilled before use. All the other solvents and reagents were used without further
purification.

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2.2 Synthesis of porphyrins
2.2.1 Tetra (4ꢀnitrophenyl) porphyrin (TNPP)
The synthetic method of TNPP was presented in the previous report 25, 26, as shown
in Scheme 1. In the 250 mL threeꢀnecked flask, pꢀnitrobenzaldehyde (5.5 g, 36 mmol)
and acetic anhydride (6 mL, 64 mmol) were added to a refluxing mixture of propionic
acid (150 mL), then freshly distilled pyrrole (2.5 mL, 36 mmol) in 10 mL of propionic
acid was dropwise added (ca. 30 min) to the mixture with stirring. The reaction
mixture was brought to reflux for 2 h. The mixture was allowed to cool overnight for
efficient precipitation, and a dark residue was collected by filtration. The dark solid
was washed with water and dried in vacuum. The solid was purified in 40 mL of
refluxing pyridine for 1 h. After that, the solution was cooled to room temperature and
stored at ꢀ4 ºC overnight. Then the mixture was filtered under vacuum and washed
with acetone till the eluent became colorless. The yield of the purified red brown
product was 12%.
2.2.2 Tetra (4ꢀaminophenyl) porphyrin (TAPP)
The above obtained TNPP (1.5 g, 1.9 mmol) was dissolved in 75 mL of
concentrated HCl solution, and the aqueous solution was heated to 75~80 ºC in a
water bath. Then another concentrated HCl solution (25 mL) with SnCl2·2H2O (10.0 g,
44 mmol) was added slowly into the aforementioned solution under stirring, and
followed by heating to 75~80 ºC for 2 h. After finishing the heating treatment, the
reaction mixture was cooled with a coldꢀwater bath and then an ice bath, and
neutralized to weak base by adding concentrated NH3·H2O slowly. Then solid was
collected by filtration, and mixed vigorously with 5% NaOH (150 mL). The mixture
was filtered and washed with distilled water repeatedly. The residue was extracted by
Soxhlet extraction with 200 mL of chloroform and then removed the solvent under
vacuum. The crude product was purified by SiO2 column chromatography
(CH2Cl2/EtOH=2:1) to give purple porphyrin TAPP. Yield: 30.0%. M.p. >250 ºC;
Anal. Calcd. for C44H34N8(%): C, 78.32; H, 5.08; N, 16.60. Found: C, 77.89; H, 5.14;
N, 16.27. MS, m/z : 676.00 [M+H]+amu; UVꢀvis (CHCl3) λmax: nm 428 (Soret band),

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523 (Q ), 563 (Q ), 604 (Q ), 655 (Q ); IR (KBr)/cmꢀ1: 3443 (νvs NꢀH, amino), 3375
Ⅰ
Ⅱ
Ⅲ
Ⅳ
1
(νNꢀH, pyrrole), 1512 (δNꢀH, amino), 1465 (νC=N, pyrrole), 1288 (νCꢀN, amino). H
NMR (d6ꢀDMSO, 300 MHz): H, ppm 8.89 (s, 8H, position of the pyrrole moiety),
β
7.86 (d, 8H, ArH), 7.01 (d, 8H, ArH), 5.58 (s, 8H, amine), ꢀ2.74 (s, 2H, innerNH).
2.2.3 General procedure for the synthesis of the porphyrinꢀSchiff base conjugates 3a–
3c
TAPP (0.2 g, 0.2959 mmol), dissolved in 20 mL of anhydrous ethanol, was heated
to reflux, and another ethanol solution (30 mL) with aldehyde (9 mmol) was added
dropwise into the solution, followed by refluxing the mixture for 3 h. The process of
the reaction was monitored by TLC. After finishing the reaction, the mixture was fully
cooled down and the precipitate was filtered off and washed with ethanol repeatedly.
The solid was dried under vacuum. The obtained violet product was dissolved in
dichloromethane, and purified by column chromatography (the silica gel column was
pretreated with alkaline triethylamine to prevent the dissociation of the product) using
a binary eluent of dichloromethane and ethanol.
Tetra [pꢀ(pꢀhydroxy benzylideneamino)] phenyl porphyrin
（3a）
The compound 3a was prepared from compound TAPP (0.2 g, 0.2959 mmol), and
pꢀhydroxybenzaldehyde (1.1 g, 9 mmol) according to the above given procedure.
Yield 72%. M. p. >250 ºC; Anal. Calcd. for C72H50N8O4(%): C, 79.25; H, 4.62; N,
10.27. Found: C, 78.91; H, 4.27; N, 9.91. MALDIꢀTOFꢀMS: m/z 1091.4018 for
[M+1]+ (calcd 1091.3955); UVꢀvis (CHCl3) λmax: nm 426 (Soret band), 521 (Q ), 559
Ⅰ
(Q ), 603 (Q ), 654 (Q ); IR (KBr)/cmꢀ1: 3314 (νNꢀH, pyrrole), 3423 (νOꢀH, phenyl),
Ⅱ
Ⅲ
Ⅳ
1
1580 (νC=N, Schiff base), 1512 (νC=C, phenyl), 1159 (δCꢀN, pyrrole). H NMR
(d6ꢀDMSO, 300 MHz): H, ppm 10.22 (s, 4H, ꢀOH), 8.92 (s, 8H, β position of the
pyrrole moiety), 8.83 (s, 4H, Schiff base), 8.20ꢀ8.22 (d, J=7.1, 8H, phenyl), 7.94ꢀ7.96
(d, J=7.1, 8H, phenyl), 7.62ꢀ7.64 (d, J=7.1, 8H, phenyl), 6.97ꢀ6.99 (d, J=7.8, 8H,
phenyl), ꢀ2.83 (s, 2H, inner NH).
Tetra [pꢀ(2 ꢀ thienyldeneamino)] phenyl porphyrin
（3b）
The compound 3b was prepared from compound TAPP (0.2 g, 0.2959 mmol), and
2ꢀthenaldehyde (1.0 g, 9 mmol) according to the procesures similar to that of

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compound 3a. Yield 48%. M.p.>250 ºC; Anal. Calcd. for C64H42N8S4 (%): C, 73.11; H,
4.03; N, 10.66. Found: C, 73.58; H, 3.95; N, 10.28. MALDIꢀTOFꢀMS: m/z 1051.4948
for [M+1]+ (calcd 1050.2415); UVꢀvis (CHCl3) λmax: nm 425 (Soret band), 519 (Q ),
Ⅰ
558 (Q ), 596 (Q ), 651 (Q ); IR (KBr)/cmꢀ1: 3445 (ν NꢀH, pyrrole), 1618 (νC=N
,
Ⅱ
Ⅲ
Ⅳ
1
Schiff base), 1585 (νC=C, phenyl), 1169 (δCꢀN, pyrrole), 716 (νCꢀS, thienyl). H NMR
(d6ꢀDMSO, 300 MHz): H, ppm 9.18 (s, 4H, Schiff base), 8.94 (s, 8H, β position of the
pyrrole moiety), 8.22ꢀ8.26 (d, J=7.3, 8H, phenyl), 7.92ꢀ7.94 (d, J=5.0, 4H, thienyl),
7.85 (s, 4H, thienyl), 7.72ꢀ7.74 (d, J=7.3, 8H, phenyl), 7.33 (s, 4H, thienyl), ꢀ2.84 (s,
2H, inner NH).
Tetra [pꢀ(2 –Pyridyldeneamino)] phenyl porphyrin
（3c）
The compound 3c was also prepared from compound TAPP (0.2 g, 0.2959 mmol),
and 2ꢀpyridinecarboxaldehyde (1.0 g, 9 mmol) according to aforementioned
procedures. Yield 72%. M.p. >250 ºC; Anal. Calcd. for C68H46N12(%): C, 79.20; H,
4.50; N, 16.30. Found: C, 78.97; H, 4.27; N, 16.46. MALDIꢀTOFꢀMS: m/z 1032.3773
for [M+1]+ (calcd 1031.1732); UVꢀvis (CHCl3) λmax: nm 420 (Soret band), 520 (Q ),
Ⅰ
558 (Q ), 596 (Q ), 649 (Q ); IR (KBr)/cmꢀ1: 3437 (νNꢀH, pyrrole), 3053 (νCꢀH
Ⅱ
Ⅲ
Ⅳ
pyridyl), 1628 (νC=N, Schiff base), 1569 (νC=C, phenyl), 1173 (δCꢀN, pyrrole), 800 (δCꢀH
,
1
pyridyl). H NMR (d6ꢀDMSO, 300 MHz): H, ppm 8.93ꢀ8.98 (d d, J=6.5 J=6.2, 8H, β
position of the pyrrole moiety), 8.83 (s, 4H, Schiff base), 8.31ꢀ8.37 (m, 8H, phenyl),
8.03ꢀ8.09 (d, J=7.5, 4H, pyridyl), 7.88ꢀ7.94 (m, 4H, pyridyl), 7.83 (s, 8H, phenyl),
7.72 (t, 4H, pyridyl), 7.63 (t, 4H, pyridyl), ꢀ2.83 (s, 2H, inner NH).
2.3 Photodynamic activity experiments in vitro
2.3.1 Cell culture and incubation conditions
The A431cells and human keratinocyte (HaCaT) cells were cultured in a
humidified incubator at 37oC with 5% CO2 in DMEM/Fꢀ12 medium supplemented
with 10% fetal bovine serum, 100 U/mL penicillin and 100 ꢁg/mL streptomycin. The
medium was replaced with fresh cell culture medium every 48 h. When ~80% the
confluence was reached, the medium was removed and washed with 5mL of
phosphate buffer saline (PBS) for three times, and then cells were replaced with fresh
medium for subculture. The porphyrinꢀSchiff base conjugates were dissolved in

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DMSO at a concentration of 5 mM as the stock solution and then diluted with
complete medium to the required concentration immediately prior to use. The final
DMSO concentration was always below 0.5 % v/v, which showed no effects on cell
viability.
2.3.2 Cellular treatment with porphyrins 3a-3c
To evaluate the cytotoxicity of compounds 3aꢀ3c in dark (cytotoxicity without light
exposure), cells in the exponential growth phase were seeded at a density of 2×104
cells/mL in 96ꢀwell culture plates containing a 200 ꢁL culture medium per well. Then
cells were incubated for 24 h at 37 ºC to allow for cell adherence. The medium was
then replaced with the 3a–3c solutions in cell culture medium (10 ꢁM) and incubated
for 24 h in the dark. Every plate had three wells without complexes as the control and
cells treated with each compound was assessed in three copies. HaCaT cells were
treated in the same conditions just for comparison. Phototoxicity experiments were
performed with A431 cells in 96ꢀwell plates similar to the procedures as above
described. After then, the compounds solutions were removed, and the cells were
washed with 5 mL of PBS for three times and exposed to blue light.
2.3.3 Irradiation
Irradiation was carried out using a blue light source, which was a LED equipped
with a 150W lamp. A wavelength range between 400 and 450 nm was selected using
optical filters. The light intensity at the treatment site was 100mW/cm2. Exposure
time was 0s and 480s, in which cells were delivered 0 J/cm2 and 48 J/cm2.
2.3.4 Determination of cytotoxicity and phototoxicity of the porphyrinꢀSchiff base
conjugates
The determination of cell survival was documented by the MTT assay27, 28. Briefly,
this method is based on the reduction of MTT to a coloured formazan compound
(λmax=570 nm) by the active mitochondrial hydrogenase in living cells. After 24 h
incubation with each compound, 20 ꢁL MTT (5 mg/mL) in PBS was added to each
well and incubated for another 4 h in a humidified incubator with 5% CO2 at 37 °C.
The MTT solution was then removed and 150 ꢁL DMSO was added to each well to
lyse the released formazan dye. The data were recorded by optical density (OD) at

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570 nm. The relative cell viability was expressed as the OD absorbance of tested
compound versus that of the control. The same procedures were carried out without
irradiation to determine the toxicity of normal skin cells in dark.
2.4 Cellular uptake of porphyrin conjugates
The HaCaT and A431 cells were seeded on 24ꢀwell culture plates in complete
culture medium and allowed to adhere for overnight. The porphyrinꢀSchiff base
conjugates (10 µM) were added to each well and incubated at 37 °C in the dark for 24
h, followed by washing twice with PBS. The cellular uptake of porphyrinꢀSchiff base
conjugates was examined under fluorescence microscopy with a filter set at 535 nm
excitation light (BP 515–560, FT 580, LP 590).
3. Results and discussion
3.1. Synthesis of porphyrin-Schiff base conjugates
Three novel porphyrinꢀSchiff conjugates 3a-3c were successfully synthesized
according to the synthetic route in Scheme 1 and characterized by elemental analysis,
1
mass spectroscopy, UVꢀvis spectroscopy and H NMR spectroscopy. In order to get
the target porphyrinꢀSchiff base conjugates, TNPP was firstly obtained through the
reaction of pyrrole and pꢀnitrobenzaldehyde with the molar ratio of 1:1 under refluxing
propionic acid by Adler’s strategy. Then TNPP was reduced by SnCl2·2H2O in
concentrated HCl solution to get the key intermediate TAPP. Finally, the three
conjugates were obtained from condensation of amine groups of TAPP with different
aldehydes in a mixture of dichloromethane and ethanol under reflux. The formation of
C=N bond can be greatly accelerated by addition of anhydrous magnesium sulfate to
the solution, which improved the yields of conjugates in the meanwhile.
The synthesis of three porphyrinꢀSchiff base conjugates was easyꢀoperation and
fast reaction, while their separation and purification was difficult by column
chromatography because of the breakdown of C=N bond in acid medium. Therefore,
the silica gel column was treated with alkaline triethylamine in advance to prevent the
dissociation of the product and exhibit pretty good results.
3.2. Spectral properties of porphyrin-Schiff base conjugates

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The UVꢀvis absorption spectra of 3a-3c in dichloromethane solution showed one
intense Soret band around 426 nm, followed by four weak Qꢀbands in the visible
spectral region of 519ꢀ652 nm (Fig 1). Their spectra data were collected and
summarized in Table 1. It can be seen that, the absorption peaks of porphyrins 3aꢀ3c
exhibited no obvious wavelength difference and appeared slightly blue shifts
compared with TAPP. This might be attributed to the chemical groups change from
primary amine to Schiff base. The electronic effect from –N=C– and phenyl groups in
porphyrinꢀSchiff base conjugates was similar to that of amino groups and phenyl
groups in TAPP 29, and in the meanwhile the pꢀπ conjugation between N atoms and
porphyrin ring was weakened, which caused the blue shifts of Soret and Q band.
Furthermore, the changes were almost the same, which indicated that the position of
peripheral substituents was far from the porphyrin ring and had little effects on the
distribution of electrons. Therefore, it did not produce noticeable effects upon the
UVꢀvis absorption properties of TAPP 26.
The fluorescence emission spectra of TAPP and 3a-3c in chloroform were obtained
upon excitation at λex=415nm, as shown in Fig 2. All compounds gave their emission
spectra with different λem (Table 1) and the λem of the porphyrins conjugates were
shorter than that of TAPP, which was owned to the effect of the peripheral
substituents around the porphyrin chromophores. After changing from primary amine
to Schiff base，the pꢀπ conjugation between N atoms and porphyrin ring was changed
to π–π conjugation and the decrease of πꢀelectron cloud density of porphyrin ring
resulted in a blue shift of λem. Comparing with 3b and 3c, the outermost substituted
hydroxyl groups (–OH) in the compound 3a had contribution to the electron cloud
density of the conjugated porphyrin ring by electron donating effects, making the
longer emission wavelength. The new weak emission peaks of the porphyrins
conjugates around 712nm were appeared, while those peaks are absent on the spectra
of TAPP, which might be attributed to electron transition from central H atom to the
conjugated system of porphyrins 29, 30
.
The vibrational spectroscopy can provide ample information about the structure of
porphyrin. The tentative IR assignments of the TAPP and porphyrinꢀSchiff base

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conjugates were reported in Table 2. The characteristic amino bands were found in
TAPP at 1288 and 3443 cmꢀ1, while the characteristic amino bands were absent in the
compounds 3a-3c. However, three new bands ascribed to the stretching vibration of –
C=N– at 1580, 1618 and 1628 cmꢀ1 were observed in the IR spectra of compounds
3aꢀ3c respectively, which indicated that the expected porphyrinꢀSchiff base
conjugates were successfully synthesized by the condensation of TAPP with the
selected pꢀhydroxybenzaldehyde, 2ꢀthenaldehyde and 2ꢀpyridinecarboxaldehyde,
respectively. A broad band appeared at 3423 cmꢀ1 in the spectra of 3a was assigned to
ν
OꢀH of the phenolic. The band at 716 cm ꢀ1 was ascribed to ν CꢀS of thiophene group of
3b. The typical CꢀH peak of pyridine ring of 3c was observed at 3053 and 800 cmꢀ1.
In addition, all of the obtained compounds, including TAPP and porphyrinꢀSchiff
base conjugates, showed a broad absorption band in the range 3445–3314 cmꢀ1, which
was belonged to ν (NH) of porphyrin ring.
1H NMR spectra of the compounds (TAPP and 3a-3c) were recorded in d6ꢀDMSO
and the peak assignment was summarized in (Table 2). As for TAPP, a sharp singlet
was observed at 5.58 ppm and was assigned to Hꢀatoms of NH2 groups. Whereas, this
1
amino proton signal was absent in the H NMR spectra of compounds 3aꢀ3c. On the
contrary, the azomethine protons (–CH=N–) appeared as a singlet at 8.83–9.18 ppm,
which revealed that porphyrinꢀSchiff base derivatives were successfully synthesized
1
by the condensation reaction. In addition, H NMR spectra of the compound 3a
31
showed a peak at 10.22 ppm, which belonged to hydroxyl protons of the phenol
.
Similarly, the multiplets for thiophene protons of 3b and pyridyl protons of 3c were
detected in the range of 7.33–7.94 ppm and 7.63–8.09 ppm respectively32.
Furthermore, the broad multiplet observed in the range 6.97–8.37 ppm for all of the
1
obtained compounds was assigned to the aromatic protons. The H NMR spectra of
TAPP and the porphyrinꢀSchiff base derivatives all exhibited a weak singlet at
approximately ꢀ2.83 ppm, which was the characteristic signal of the inner protons of
porphyrin ring. The formations of all compounds were also confirmed by their mass
spectral data given in the experimental section.
3.3 Photostability of compounds

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The rate of photodegradation exhibit by a compound under light conditions is a
very important parameter to assess when we consider their potential application as
photosensitizer 33, 34. In order to establish if porphyrinꢀSchiff base conjugates 3aꢀ3c
could undergo photobleaching, we performed photostability studies in the same
conditions of irradiation used for photocytotoxicity in the A431 cells (fluence rate 100
mW/cm ꢀ2). The UV–vis and fluorescence emission spectra of each porphyrin (1 ꢁM)
were measured after different times of irradiation (0, 5, 10, 15, 20, 25, and 30 min).
However, we observed minimal decay of absorption and emission intensity. As an
example, the UV–vis and fluorescence emission spectra of porphyrinꢀSchiff base
conjugates 3a at different irradiation times are shown in Fig 3. The results point out
that all compounds (3aꢀ3c) showed high photostability under the conditions of the
experiment, which is the desired property of a PDT photosensitizer.
3.4. Cytotoxicity assay
The cytotoxicity of the porphyrins 3aꢀ3c was evaluated by two kind of human skin
cell lines, human keratinocyte HaCaT and epidermoid carcinoma A431. The cells
seeded in well plates were treated with 10 ꢁM of 3a–3c solutions in the dark. As
shown in Fig 4, the relative cell viability of A431 cells for 3a–3c was in the range of
74.8%ꢀ75.5%, while that of HaCaT cells for 3a–3c maintained from 86.5% to 109.8%.
From the histogram of the cytotoxicity comparison in Fig 4, it was not hard to see that
the cell viability of HaCaT and A431 cells was almost the same and maintained
zzaround 92% and 82%, when cultured with TAPP, revealing that TAPP was almost
harmless to both normal cells and cancer cells. However, porphyrins 3a–3c showed
higher cytotoxicity in dark to A431 cancer cells when compared with TAPP,
especially for the porphyrin 3c, which showed the highest cytotoxicity with the A431
cell viability of 74.8%. Conversely, negligible cytotoxicity was detected on HaCaT
cells for compounds 3a–3c with the cell viability of over 80% and even exceeded
100%. It was worth noting that 3b and 3c was observed without cytotoxicity in dark
for normal HaCaT cells and 3a showed very slight cell damage. The cell survival
histogram demonstrated that the synthesized porphyrin conjugates were more

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effective to kill the cancerous A431 cells than the normal HaCaT cells at the
concentration of 10 ꢁM after 24 h of incubation and the antitumor activities of all of
the porphyrinꢀSchiff base conjugates were evidently higher than that of TAPP with
the same culture conditions. The results were attributed to the presence of the Schiff
base, which showed conspicuous inhibitory to the growth of cancer cells.
3.5. Photodynamic effect
The in vitro phototoxicity studies were performed using A431 cells. The A431 cells
adhered on the culture plate were incubated with porphyrins conjugates 3a-3c
solutions at the concentration of 10 ꢁM for 24 h, respectively. After finishing the
incubation, the samples were irradiated with blue light with the wavelength of 400
ꢀ450 nm, a fluence rate of 100 mW/cm2 and light doses from 0 to 48 J/cm2. Cell
viability was determined by MTT assay. As shown in Fig 5, all of the tested
compounds exhibited strong photocytotoxicity after exposure to the blue light for
0ꢀ480s, and the cell viability decreased dramatically with the increase of the light dose.
The cell survival reduced sharply at the beginning of irradiation, and the decreasing
tendency of cell viability became leveling off when the irradiation dose exceeded 12
J/cm2.
The cells lines treated with 3a-3c at different concentrations were irradiated with
blue light for 360s. The compounds showed remarkable photodynamic effects at all
concentrations. Fig 6 showed the histogram of the cell viability at different compound
concentrations, which indicated the viability of tumoral A431 cells reduced
significantly with the increase of porphyrins concentration. The IC50 values, defined
as the porphyrins concentration at which 50% of tumor cells were killed after the
photoirradiation as compared to the control, were calculated based on the results of
Fig 6 and summarized in Table 3. From the IC50 values listed in Table 3, it was
obvious that the conjugate 3a was the most phototoxic under the above irradiation
conditions with an IC50 of 0.57 µM, which was followed by 3b with an IC50 of 1.19
µM and then 3c with an IC50 of 1.31 µM. Notably, 3a showed a 2.4 times enhanced
photocytotoxicity compare to TAPP, which might be attributed to the attached
phenolic group. The phenolic group is easily oxidized to benzoquinonyl group in

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cellular metabolic pathway 35ꢀ37, which finally causes obvious cell damage.
3.6 Cellular uptake of porphyrin conjugates
It was a quite crucial question whether photosensitive compound could penetrate
into the tumor cells for the therapeutic effect of PDT. Cellular uptake of porphyrins
could be directly observed by fluorescence microscope without fluorescence labeling
38ꢀ40, for porphyrins uptaken by tumor cells would emit red fluorescence when being
excitation. Based on the point, we could judge whether the porphyrins entered into the
cells. In the fluorescence microscopic images (summarized in Fig 7), it was obvious
that all the compounds could enter into the A431 cells causing intracellular
fluorescence. The 3b and 3c samples displayed slightly brighter red fluorescence
spots, while cells incubation with 3a did not reveal enough fluorescence plots. These
findings indicated that the conjugates 3aꢀ3c were different in cellular uptake and not
in the nucleus. Furthermore, these intracellular levels of conjugates resulted in
different photodynamic activities. Interestingly, the conjugate 3a with the greatest
photodynamic activity corresponded with the lowest intracellular uptake of all the
conjugates tested.
4. Conclusion
In conclusion, three novel porphyrin conjugates 3aꢀ3c have been synthesized with
excellent yields and their biological activities were evaluated by A431cells with a
MTT assay. The conjugates 3b and 3c exhibited high selective inhibition against
A431 cells in the absence of light. The result revealed that these conjugates could
easily and selectively accumulate in tumor cells and had more cytotoxic effects on
them. Incubation of A431 cells gave rise to an efficient cellular uptake of porphyrin
conjugates, which was determined by fluorescence microscopy. The fluorescent
image revealed that cells displayed an uptake of conjugates and appeared to be
localized in the cytoplasm. The result was consistent with the strong photocytotoxic
activity of porphyrin compounds after irradiation. The facts indicated that these
porphyrin conjugates was a potential candidate of anticancer drugs in PDT, but their
promising behavior needed further investigated investigation in vitro and in vivo
experiments.

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Acknowledgements
The authors acknowledge the research grant provided by the National Nature
Science Foundation (21271148).
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FIGURES AND SCHEME CAPTIONS
Table 1. Optical data for TAPP and 3a-3c (CHCl3, 25ºC)
Table 2 1HꢀNMR and IR spectral bands of TAPP and 3a-3c
Table 3. IC50 value of TAPP and 3a–3c for A431 cells
Scheme 1. Synthetic routes of TAPP and 3a–3c
Fig 1 UVꢀvis absorption spectra of TAPP and 3a–3c
Fig 2 The fluorescence spectra of TAPP and 3a–3c. Concentrations of the porphyrins
were 1×10ꢀ6 mol/L in CHCl3
Fig 3 UVꢀVis absorbance spectra (A) and Emission spectra (B) of porphyrin 3a after
different times of irradiation
Fig 4 Cytotoxicity of TAPP and 3a–3c on A431 cells and normal skin cells
Fig 5 Relative cell viability of A431 cells irradiated with light doses from 0 to 48
J.cmꢀ2
Fig 6 Relative cell viability of A431 cells at different concentrations of porphyrins
TAPP and 3a–3c
Fig 7 Fluorescence microscopy of A431 cells incubated with TAPP and 3aꢀ3c (10µM,
24h) (Left) white light and (Right) fluorescence

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Table 1 Photophysical data of TAPP and 3a-3c (CHCl3, 25ºC)
Compounds Soret band (nm)
Q bands (nm)
λem(nm)
665
428.0
426.5
426.5
425.5
523.0, 563.5, 598.0, 656.0
TAPP
3a
520.5, 559.0, 594.5, 652.0 657, 711
520.0, 557.0, 594.0, 650.5 655, 712
519.5, 557.0, 593.5, 651.0 655, 712
3b
3c
Table 2 1HꢀNMR and IR spectral bands of TAPP and 3a-3c
1HꢀNMR spectra (ppm) IR spectra (cmꢀ1)
C=N(imine) νC=N(ring)
Comp.
δinnerNH
δArꢀH
8.89 7.01ꢀ7.86
ꢀ2.83 8.92 6.97ꢀ8.22
δNH or δCH=N
5.58
δβ
νNH
ν
ꢀH
3375
3314
3445
3437
ꢀꢀ
1465
TAPP ꢀ2.74
3a
3b
3c
8.83
1580
1618
1628
1468
1425
1470
ꢀ2.84
ꢀ2.83
8.94 8.22ꢀ8.26
8.94 7.82ꢀ8.37
9.18
8.83
Table 3 IC50 value of TAPP and 3a–3c for A431 cells
Compounds
IC50(µM)
1.36
TAPP
3a
0.57
1.19
3b
1.31
3c

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Scheme 1 Synthetic routes of TAPP and 3a–3c
Fig 1 UVꢀvis absorption spectra of TAPP and 3a–3c

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Fig 2 The fluorescence spectra of TAPP and 3a-3c. Concentrations of the porphyrins
were 1×10ꢀ6 mol/L in CHCl3
Fig 3 UVꢀVis absorbance spectra (A) and Emission spectra (B) of porphyrin 3a after
different times of irradiation
Fig 4 Cytotoxicity of TAPP and 3a–3c on A431 cells and normal skin cells

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Fig 5 Relative cell viability of A431 cells irradiated with light doses from 0 to 48
J.cmꢀ2
Fig 6 Relative cell viability of A431 cells at different concentrations of porphyrins
TAPP and 3a–3c

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Fig 7 Fluorescence microscopy of A431 cells incubated with TAPP and 3aꢀ3c (10µM,
24h) (Left) white light and (Right) fluorescence

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Three novel porphyrin-schiff base conjugates were synthesized and characterized.
Their phototoxic activities were improved after irradiation and increased significantly
with the increase of doses against A431 cells.