Synthesis of Novel Porphyrin Derivatives and Their Cytotoxic Activities against A431 Cells

Article abstract of DOI:10.1002/hlca.201500184

Three novel porphyrins, including two Schiff-bases porphyrins, 5,10,15-triphenyl-20-[4-(2-(4-formyl)phenoxy)ethoxy]phenyl porphyrin (H₂Pp(1)), 5,10,15-triphenyl-20-[4-(2-(4-hydroxyimino)phenoxy)ethoxy]phenyl porphyrin (H₂Pp(2)) and 5

Full text of DOI:10.1002/hlca.201500184

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4
Helv. Chim. Acta 2016, 99, 24 – 29
FULL PAPER
Synthesis of Novel Porphyrin Derivatives and Their Cytotoxic Activities against A431 Cells
a
b
a
a
a
by Ya-Hong Yao ) ), Yun Luo ), Jun Li* ), and Feng-Xing Zhang )
a) Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry &
Materials Science, Northwest University, Xiꢀan 710069, P. R. China (e-mail: junli@nwu.edu.cn)
b) College of Science, Xiꢀan University of Architecture and Technology, Xiꢀan 710055, P. R. China
Three novel porphyrins, including two Schiff-bases porphyrins, 5,10,15-triphenyl-20-[4-(2-(4-formyl)phenoxy)ethoxy]phenyl
porphyrin (H Pp(1)), 5,10,15-triphenyl-20-[4-(2-(4-hydroxyimino)phenoxy)ethoxy]phenyl porphyrin (H Pp(2)) and 5,10,15-
2
2
triphenyl-20-[4-(2-(4-m-hydroxyanilinodeneformyl)phenoxy)ethoxy]phenyl porphyrin (H Pp(3)), as well as three metallopor-
2
phyrins (CuPp (1a), ZnPp (1b), and CoPp (1c)) of porphyrin H Pp(1) were synthesized. Their molecular structures were
2
1
characterized by H-NMR, MS, UV/VIS, and FT-IR spectra. Furthermore, they were evaluated by their cytotoxicities against
human epidermal squamous cell carcinoma cell (A431) and normal human horn cells (HaCaT) in vitro with MTT assay.
Interestingly, these porphyrins and metalloporphyrins, which had a negligible cytotoxicity to HaCaT cells, showed highly
cytotoxicity against A431 cells with IC50 values in the range of 6.6 – 9.8 mm, and metalloporphyrins exhibited higher cytotoxicity
than that of metal-free porphyrins.
1. Introduction. – Human epidermal squamous cell tant role in a diversity of biological processes, including cell
carcinoma cell (A431) is a malignant tumor of the formed respiration, detoxification of xenobiotics, oxygen transport,
epidermis cutin cell [1][2] and is one of the most common fatty acid oxidation, and light harvesting [12]. They have
human non-melanoma skin malignancies. During the past been extensively exploited as platforms for the study of
decades, the incidence rate of this disease has tremendously synthetic and potential applications in a wide variety of
increased and become the main threat to human health of fields such as chemistry, physics, material science, engineer-
skin. Avariety of treatment modalities have been described ing, biology, and medicine [13]. Porphyrins have emerged
to treat human epidermoid carcinoma cells, including as hot topics of therapeutic drugs because of their selective
surgery, cryotherapy, topical chemotherapy, photodynamic accumulation in abnormal or hyperproliferative cells [14 –
therapy, and immune response modifiers. Surgical inter- 19], such as neoplastic tissue [20 – 23]. This selective
vention remains the only curative treatment and is often accumulation can result in effective ablation of the targeted
the first option in treatment [3][4]. Nevertheless, surgery tissue. For this reason, they produce less toxicity than
presents certain limitations: for example, it is poorly traditional antitumor drugs. Schiff bases are a class of
tolerated and unsuitable for the patient, and patients show organic compounds that contain a C¼N bond, such as the
distant metastases at the time of diagnosis, or it is cosmeti- imine and azomethine group, which have potential appli-
cally unacceptable. Additionally, curative surgery is per- cations as antibacterial, anticancer, and antiviral agents
formed more than just a few small circumscribed lesions [24][25]. It has been demonstrated that NH OH · HCl and
2
[
[
5][6]. Compared with the surgery removal, chemotherapy m-aminophenol are common pharmaceutical intermedi-
7 – 9] is also a main approach in the treatment of tumor for ates. Herein, they are chosen as peripheral substituents to
a long time. Unfortunately, the activity of most chemo- modify porphyrin through the C¼N bond which are
therapeutic agents attack not only cancer cells, but also expected to be bioactive compounds.
other healthy cells of the body, for example blood cells and
It has been known that metal ions involve in biological
lining, mouth, stomach, and intestines. Furthermore, they processes of life and have been a subject of interest. Among
can depress the immune system and cancer cells frequently metals, copper, zinc, and cobalt are all essential trace
develop resistance to many anticancer drugs which greatly elements presenting in living organisms. They exhibit
reduces their therapeutic usefulness [10][11]. Therefore, considerable and biochemical action as a constituent of
there is an urgent need to improve and develop more various exogenous administered compounds in humans.
effective therapeutic agents which combat cancer and are Current interest in metal complexes stem from their
able to evade drug resistance and other significant side potential use as antimicrobial, antitumor agents and
effects. Recent studies show that porphyrins have become enzyme inhibitors [26 – 28]. Especially the transition-metal
one of the new research targets as therapeutic drugs.
complexes provide enormously versatile platforms for drug
As we all know, porphyrins are a class of naturally design, for example a number of cisplatin and Cu(II)
occurring macrocyclic compounds, playing a very impor- complexes that exhibit cytotoxic activity through cell
DOI: 10.1002/hlca.201500184
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Helv. Chim. Acta 2016, 99, 24 – 29
25
apoptosis or enzyme inhibition have been reviewed [27 –
9]. These drugs have attracted researchers to synthesize
2
new transition metal compounds which can treat tumours
with a selective cytotoxicity to cancer cells. Today, a range
of metalloporphyrins due to a part of affinity with the
cancer cells, have been investigated as photosensitizers in
cancer treatment. For example, the Zn, Ca, Ru, and Co
complexes showed encouraging results because of their
interaction with biomolecules, mainly proteins and nucleic
acids [30 – 34]. In this work, we introduced transition metal
II
II
II
Cu , Zn , and Co into the porphyrin molecule to form a
metalloporphyrin. Therefore, this is emerging as a promis-
ing cytotoxic agent to conventional chemotherapy.
Because Schiff-bases are common pharmacophores in
the design and development of anticancer chemothera-
peutic agents, in this article, we report the synthesis of
two novel Schiff-bases porphyrins, 5,10,15-triphenyl-20-
Fig. 1. Structures of the porphyrins and metalloporphyrins
[
4-(2-(4-hydroxyimino)phenoxy)ethoxy]phenyl porphyrin
(
H Pp(2)) and 5,10,15-triphenyl-20-[4-(2-(4-m-hydroxy-
2
II
II
II
anilinodeneformyl)phenoxy)ethoxy]phenyl
porphyrin and transition metal ion Cu , Zn and Co . Their cytotoxic
(
H Pp(3); Fig. 1), by reacting aldehyde group-substituted activities against A431 cells and normal skin cells were
2
tailed porphyrin 5,10,15-triphenyl-20-[4-(2-(4-formyl)- studied in the dark.
phenoxy)ethoxy]phenyl porphyrin (H Pp(1); Fig. 1) with
2
NH OH · HCl and m-aminophenol respectively. More-
2. Results and Discussion. – 2.1. Synthesis of Porphyrins
2
over, three new metalloporphyrins (CuPp (1a), ZnPp (1b), and Metalloporphyrins. The synthetic routes to the por-
and CoPp (1c); Fig. 1) were also synthesized by chelation phyrins H Pp(1) – H Pp(3) and metalloporphyrins 1a – 1c
2
2
between the four N-atoms of the center of H Pp(1) ring were outlined in the Scheme. The porphyrins H Pp(1) was
2
2
Scheme. Synthetic Routes of the Porphyrins and Metalloporphyrins
a) Br(CH ) Br, K CO , acetone, 568. b) TPPOH, K CO , DMF, 608. c) M(Ac) , CH Cl , r.t. d) NH OH · HCl, Et N (for H Pp(2)); m-aminophenol,
2
2
2
3
2
3
2
2
2
2
3
2
EtOH, CH Cl , MgSO , r.t. (for H Pp(3))
2
2
4
2
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Helv. Chim. Acta 2016, 99, 24 – 29
synthesized by the reaction of TPPOH and 4-(2-bromo- the 512 – 646 nm region, characteristic of metal-free por-
ethoxy)benzaldehyde in the presence of K CO and DMF phyrin chromophore were presented in the absorption
2
3
at 608 with the yield around 85%.
spectra of H Pp(1) – H Pp(3). Compared to the metal-free
2 2
The Schiff-bases porphyrins H Pp(2) and H Pp(3) and porphyrins, a decrease in the number of Q bands on
2
2
metalloporphyrins were prepared from H Pp(1). The metalloporphyrins was found. The molecular electronic
2
H Pp(2) and H Pp(3) were obtained in 65% and 30% spectra of copper, zinc, and cobalt porphyrin complexes
2
2
yields by treating H Pp(1) with NH OH · HCl and m- exhibited one or two Q bands, respectively in the 538 –
2
2
aminophenol respectively in a mixture of CH Cl and 540 nm and 575 nm spectral ranges. Additionally, a distinct
2
2
EtOH at room temperature. The yields of H Pp(2) and blue shift (3 – 16 nm) of the absorption peak of metal-
2
H Pp(3) were improved by addition of MgSO to absorb loporphyrins was observed. From the UV/VIS spectra, it
2
4
the H O formed in the formation reaction of C¼N bond. can be demonstrated that the peripheral substituents of
2
Corresponding metalloporphyrins 1a – 1c were obtained by H Pp(1) – H Pp(3) had negligible influence on UV/VIS
2
2
reaction of H Pp(1) with a large excess of Cu(AcO) , spectra. Instead, insertion of a metal ion into the core of
2
2
Zn(AcO) , and Co(AcO) at room temperature, respec- porphyrin generated an obvious influence.
2
2
tively. After purification by column chromatography,
The FT-IR spectra of the porphyrins H Pp(1) –
2
metalloporphyrins 1a – 1c were obtained in 95, 85, and H Pp(3), and metalloporphyrins 1a – 1c are shown in
2
7
9% yield, respectively.
Fig. 3. A stretching vibration and a bending vibration for
À1
2
.2. Characterization of Porphyrins and Metallopor- the NÀH bond appear at 3314 – 3360 cm and around
À1
phyrins. The structures of the porphyrins H Pp(1) –
965 cm for metal-free porphyrins, respectively. A strong
2
À1
H Pp(3) and metalloporphyrins 1a – 1c were identified by absorption band at 1697 cm was assigned to the aldehyde
2
1
H-NMR spectroscopy, elemental analysis, UV/VIS spec- group of stretching vibration peaks for H Pp(1). It was
2
À1
troscopy, FT-IR spectra, and mass spectrometry.
shifted to a lower wave number below (1700 cm ) due to
The UV/VIS absorption spectra of the porphyrins conjugation of the C¼O group and the porphyrin ring. In
H Pp(1) – H Pp(3) and metalloporphyrins 1a – 1c in addition, the stretching vibrations for the C¼N bonds
2
2
À1
CH Cl as solvent are shown in Fig. 2. The maximum appeared at 1607 and 1602 cm in the FT-IR spectra of the
2
2
wavelengths and molar absorptivities of the porphyrin Schiff-bases H Pp(2) and H Pp(3), which indicated that
2
2
compounds were summarized in Table 1. An intense Soret the H Pp(2) and H Pp(3) were successfully synthesized by
2
2
band situated at 418 nm and four weak Q bands located in condensation of H Pp(1) with the selected NH OH · HCl
2
2
and m-aminophenol, respectively.
In the FT-IR spectra of the metalloporphyrins, a new
À1
band appeared around 1004 – 1018 cm , and it could be
assigned to a metal-dependent in-plane porphyrin defor-
mation mode. Instead, the disappearance of an NÀH
stretching vibration and a bending vibration in the metal-
free porphyrins indicated the coordination of N-atoms of
the porphyrinic core to the metal centers. This predicated
that the metalloporphyrins 1a – 1c were qualitatively pre-
pared.
1
The H-NMR spectra of the porphyrins H Pp(1) –
2
H Pp(3) have been recorded in CDCl . The spectra are
2
3
consistent with meso-substituted porphyrins, showing a
single peak around d(H) À 2.76 ppm due to the H-atoms
of the ÀNH group in porphyrinic core, and the chemical
shift of eight b-pyrrolic H-atom resonances at d(H) 8.84 –
8
9
.88 ppm. The aldehyde H-atom chemical shift is at
.94 ppm, and as expected, this peak is absent from the
spectra of H Pp(2) and H Pp(3) in which each a new single
Fig. 2. UV/VIS Absorption spectra of the porphyrins and metallopor-
2
2
phyrins
peak of H-atom for ÀOH was detected at 8.08 and
Table 1. UV/VIS Spectra Data of the Porphyrins and Metalloporphyrins (258, in CH Cl )
2
2
Compounds
Soret band [nm] (log e)
Q bands [nm] (log e)
H Pp(1)
417 (5.69)
418 (5.70)
418 (5.65)
415 (5.58)
415 (5.61)
415 (5.67)
515 (4.32), 550 (3.92), 590 (3.78), 646 (3.62)
515 (4.33), 550 (3.90), 590 (3.73), 646 (3.62)
512 (4.32), 554 (3.89), 591 (3.70), 645 (3.55)
539 (3.87)
540 (3.93), 575 (3.77)
538 (3.91)
2
H Pp(2)
2
H Pp(3)
2
CuPp (1a)
ZnPp (1b)
CoPp (1c)
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Helv. Chim. Acta 2016, 99, 24 – 29
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Table 2. Cytotoxicity of Porphyrins and Metalloporphyrins to A431
Cancer Cells after 24 h Incubation in the Dark
Compounds
IC50 [mm]
H Pp(1)
9.8 Æ 0.7
9.3 Æ 0.4
7.1 Æ 0.8
6.8 Æ 1.2
6.7 Æ 0.5
6.6 Æ 0.9
2
H Pp(2)
2
H Pp(3)
2
CuPp (1a)
ZnPp (1b)
CoPp (1c)
Comparably, the negligible cytotoxicities were detected in
HaCaT cells for all porphyrins with the cell viability of over
80%. The result revealed that these porphyrins could easily
and selectively accumulate in tumor cells and have more
cytotoxic effects on them.
2.4. Discussion. As described, all the compounds
exhibit high cytotoxicities against A431 cancer cells, but
only negligible cytotoxicities against HaCaT cells, which
indicate that these porphyrins have the ability to kill A431
cancer cells selectively. The introduction of Schiff base
C¼N bond in H Pp(2) and H Pp(3) increases the cytotox-
2
2
icity against A431 cancer cells compared with H Pp(1),
2
probably due to the intercalation to DNA of cancer cells
with more ease [35]. Among porphyrins H Pp(1) –
2
H Pp(3), H Pp(3) has a better inhibition effect, which
2
2
might be attributed to the attached phenolic group. The
phenolic group is easily oxidized to a benzoquinonyl group
in a cellular metabolic pathway [36], which finally causes
obvious cell damage. The high inhibition effect of the three
metalloporphyrins 1a – 1c, may be due to the effect of
coordinated metal ions that play a major role in mediating
potency of the complexes [26].
Fig. 3. IR Spectra of the porphyrins and metalloporphyrins
3
. Conclusion. – In summary, three novel porphyrins
II
II
II
8
.90 ppm, respectively. The formation of Schiff-base por- H Pp(1) – H Pp(3) and Cu (1a), Zn (1b), Co (1c)
2 2
1
phyrins was confirmed by comparing the H-NMR data of metalloporphyrins of H Pp(1) are synthesized and charac-
2
H Pp(1) with those of H Pp(2) and H Pp(3). In addition, terized. Their biological activities are evaluated by their
2
2
2
all compounds exhibited main peaks (protonated molecule cytotoxicities against human epidermal squamous cell
þ
[
M þ H] ) on high-resolution ESI mass spectroscopy.
carcinoma cell (A431). These compounds exhibit good
2
.3. Cytotoxicity Test. The cytotoxic activities of all the cytotoxicities towards A431 cancer cells, especially for the
porphyrins against A431 cells were evaluated in the metalloporphyrins. The results demonstrate that all the
absence of light after an incubation of 24 h. These porphyrins can inhibit effectively the A431 cancer cells,
porphyrins and metalloporphyrins demonstrated moderate with very low toxicities to HaCaT cells. It may provide
cytotoxicities with comparable values in the cell tests. All some valuable information for further deep research.
IC50 values were in the range of 6.6 – 9.8 mm (Table 2).
The authors acknowledge the research grant provided by the
National Nature Science Foundation of China (No. 21271148). We are
grateful to Prof. Cheng-Xin Li, Department of Dermatology, Xijing
Hospital, Fourth Military Medical University, Xiꢀan, 710032, P. R.
China, for the cytotoxicity experiments.
Three metalloporphyrins 1a – 1c were more effective in
stopping cell division and killing the tumor cells than the
porphyrins H Pp(1) – H Pp(3), with observed minimum
2
2
IC values 6.8 Æ 1.2, 6.7 Æ 0.5, and 6.6 Æ 0.9 mm, respective-
50
ly. The Schiff-base H Pp(3) was slightly less cytotoxic with
2
the IC of 7.1 Æ 0.8 mm than the metalloporphyrins, and the
5
0
H Pp(1) showed the smallest cytotoxicity. These results
2
Experimental Part
indicated that introduction of the Schiff base to porphyrin
molecules enhances cytotoxic activities against A431 cells.
General. 4-Hydroxybenzaldehyde, 4-(2-bromoethoxy)benzalde-
hyde, and pyrrole were obtained from Sinopharm Chemical Reagents
Company, MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide) were purchased from SigmaÀAldrich (Chiba, Japan).
The 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH) was
In particular, the cytotoxic activities of porphyrin H Pp(3),
2
which bears a phenolic group, against A431 cells were of
the same order of potency as that of metalloporphyrins.
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Helv. Chim. Acta 2016, 99, 24 – 29
synthesized according to the literature procedures described in [37],
and other reagents were obtained from Beijing Chemical Reagents
Company. Pyrrole was distilled before use. Dimethyl sulfoxide
obtained. Yield: 30%. M.p. > 2508. UV/VIS (CH Cl ): 418 (Soret
2 2
band), 512, 554, 591, 645 (Q bands). IR (KBr): 3361, 2925, 2609, 2362,
1
1602, 1176, 904, 686. H-NMR (CDCl , 400 MHz): 8.85 (s, 8 b-H of
3
(
DMSO) and N,N-dimethyl formamide (DMF) were dried (with
pyrrole); 8.22 (d, 6 o-H of phenyl); 8.90 (s, 1 H, OH); 7.74 – 7.77 (m, 9
m-H and p-H of phenyl); 7.55(s, 4 H of phenyl); 7.03 (t, 8 H of
substituted phenyl); 6.27 (s, 1 H, CH¼N); 4.32, 3.74 (2t, CH CH );
powdered MgSO ) and distilled at normal pressure before use.
4
UV/VIS Spectra: Shimadzu UV-1800 UV/VIS spectrophotometer;
2
2
À1
þ
lmax (log e) in nm. IR (4000 – 400 cm ) Spectra: samples in KBr matrix
for the title complexes on BEQUZNDX-550 series FT-IR spectropho-
tometer; n˜ in cm
À 2.77 (s, 2 H of imino). MS: 870.88 ([M þ H] ). Anal. calc. for
C H N O (870.0): C 81.41, H 4.96, N 8.08; found: C 81.45, H 4.98, N
59
43
5
3
À1
1
.
H-NMR Spectra: Varian Inova 400 MHZ
8.05.
spectrometer for metal-free porphyrins, in CDCl ; d in ppm rel. to
General Procedure for Synthesis of CuPp (1a), ZnPp (1b), and
CoPp (1c). H Pp(1) (0.078 g, 0.1 mmol) was dissolved in CH Cl
3
Me Si as internal standard, J in Hz. MS: carried out on a matrix assisted
4
2
2
2
laser desorption/ionization time of light mass spectrometer (MALDI-
TOF MS, Krato Analytical Company of Shimadzu Biotech, Man-
chester, Britain); in m/z. Elemental analysis (C, H and N): Vario EL-III
CHNOS instrument.
(20 ml), and Cu(AcO) (2 mmol) soln. in EtOH (5 ml) were added,
2
and the mixture was stirred for 24 h at r.t. The progress of the
complexation reaction was monitored by TLC. After completion of the
reaction, the mixture was filtered, and the solvent was removed under
reduced pressure. In order to separate the target compounds, the crude
product was dissolved in CH Cl and purified by SiO CC, using CH Cl
Synthesis of the Porphyrins H Pp(1) – H Pp(3). Synthesis of
2
2
5
,10,15-Triphenyl-20-[4-(2-(4-formyl)phenoxy)ethoxy]phenyl Porphyr-
2
2
2
2
2
in (¼4-{2-[4-(10,15,20-Triphenylporphyrin-5-yl)phenoxy]ethoxy}ben-
as eluent. The obtained violet soln. was concentrated and dried under
reduced pressure. 1a was obtained. The synthetic procedure for 1b and
1c was similar to that for 1a in almost quantitative yield by using
Zn(AcO) and Co(AcO) as the starting material, resp.
zaldehyde; H Pp(1)). TPPOH (0.126 g, 0.2 mmol) and 4-(2-bromoe-
2
thoxy)benzaldehyde (0.458 g, 2 mmol) were mixed and dissolved in
DMF (10 ml). The soln. was stirred in the presence of K CO (0.15 –
2
3
2
2
0
.2 g) for 12 h at 608 in the darkness. The progress of the reaction
Data of Copper(II) 5,10,15-Triphenyl-20-[4-(2-(4-formyl)phe-
was monitored by TLC. DMF was removed under reduced pressure
noxy)ethoxy]phenyl Porphyrin (¼ [4-{2-[4-(10,15,20-Triphenylpor-
4
21
22
23
24
after the reaction was finished, and the residue was dissolved in CH Cl2
phyrin-5-yl-k N ,N ,N ,N )phenoxy]ethoxy}benzaldehydato(2À)]-
2
and purified over a SiO column using CH Cl as eluent. The first violet
copper; 1a). Yield: 95%. M.p. > 2508. UV/VIS (CH Cl ): 415 (Soret
2
2
2
2
2
band was collected. After vaporizing and drying under vacuum,
band), 539 (Q bands). IR (KBr): 3437, 2925, 2803, 2730, 1685, 1601,
þ
H Pp(1) was obtained. Yield: 85%. M.p. > 2508. UV/VIS (CH Cl ):
1504, 1251, 1161, 1004, 827. MS: 841.71 ([M þ H] ). Anal. calc. for
2
2
2
4
2
4
6
18 (Soret band), 515, 550, 590, 646 (Q bands). IR (KBr): 3419, 2797,
C H CuN O (840.4): C 75.78; H 4.30, N 6.65, Cu 7.54; found: C 75.74,
53
36
4
3
734, 1687, 1601, 1577, 1507, 1253, 965, 830, 800. 1H-NMR (CDCl
,
H 4.32, N 6.67, Cu 7.56.
3
00 MHz): 9.94 (s, 1 H, aldehyde-H); 8.89 (s, 8 b-H of pyrrole); 8.25 (d,
o-H of phenyl); 8.13 – 8.15 (m, 2 H of phenyl); 7.77 – 7.84 (m, 9 m-H
Data of Zinc(II) 5,10,15-Triphenyl-20-[4-(2-(4-formyl)phenoxy)-
ethoxy]phenyl Porphyrin (¼ [4-{2-[4-(10,15,20-Triphenylporphyrin-5-
4
21
22
23
24
and p-H of phenyl); 7.13 (d, 2 H of substituted phenyl); 5.28 – 5.32 (m,
yl-k N ,N ,N ,N )phenoxy]ethoxy}benzaldehydato(2À)]zinc; 1b).
4
7
H of phenyl); 4.42, 3.61 (2t, CH CH ); À 2.76 (s, 2 H of imino). MS:
Yield: 85%. M.p. > 2508. UV/VIS (CH Cl ): 415 (Soret band), 540,
2
2
2
2
þ
79.71 ([M þ H] ) Anal. calc. for C H N O (778.89): C 81.71, H 4.96,
575 (Q bands). IR (KBr): 3440, 2853, 2736, 1668, 1598, 1161, 1004, 798.
53
38
4
3
þ
N 7.21; found: C 81.73, H 4.92, N 7.19.
MS: 843.31 ([M þ H] ) amu. Anal. calc. for C H N O Zn (842.2): C
53
36
4
3
Synthesis of 5,10,15-Triphenyl-20-[4-(2-(4-hydroxyimino)phenoxy)-
75.68; H 4.30, N 6.67, Zn 7.59; found: C 75.58, H 4.31, N 6.65, Zn 7.76.
Data of Cobalt(II) 5,10,15-Triphenyl-20-[4-(2-(4-formyl)phe-
noxy)ethoxy]phenyl Porphyrin ( ¼ [4-{2-[4-(10,15,20-Triphenylpor-
ethoxy]phenyl Porphyrin (¼ N-Hydroxy-1-(4-{2-[4-(10,15,20-triphenyl-
porphyrin-5-yl)phenoxy]ethoxy}phenyl)methanimine;
H Pp(2)).
2
4
21
22
23
24
H Pp(1) (0.078 g, 0.1 mmol) was dissolved in CH Cl (20 ml) with
phyrin-5-yl-k N ,N ,N ,N )phenoxy]ethoxy}benzaldehydato(2À)]-
2
2
2
anh. MgSO (0.5 g, 4 mmol), EtOH soln. (5 ml) with NH OH · HCl
cobalt; 1c). Yield: 79%. M.p. > 2508. UV/VIS(CH Cl ): 415 (Soret
4
2
2
2
(
0.07 g, 1 mmol), and Et N (0.5 ml, 0.004 mmol) were added into the
band), 538 (Q bands). IR (KBr): 3437, 2839, 2742, 1688, 1600, 1257,
3
þ
soln. The mixture was stirred for 24 h at r.t. Then, the mixture was
filtered off (MgSO ), and the solvent was removed. Further purifica-
tion of the residue was carried out over a SiO column with CH Cl as
1156, 1018, 802. MS: 836.7 ([M þ H] ). Anal. calc. for C H CoN O
53
36
4
3
(835.8): C 75.98; H 4.30, N 6.67, Co 7.12; found: C 76.16, H 4.34, N 6.70,
Co 7.05.
4
2
2
2
eluent. The second violet band was collected. After vaporizing and
4. Cell Culture Conditions. The A431 cells and HaCaT cells were
drying under vacuum, H Pp(2) was obtained. Yield: 65%. M.p. > 2508.
grown in plastic culture flasks at 378 (5% CO ) by using DMEM/F-12
2
2
UV/VIS(CH Cl ): 418 (Soret band), 515, 550, 590, 646 (Q bands). IR
medium with penicillin (100 UI/ml), streptomycin (100 mg/ml) and
10% heat-activated fetal bovine serum. The medium was replaced with
fresh every 48 h. When ca. 80% the confluence was reached, cells were
washed with 5 ml of phosphate buffer saline (PBS) for two times and
treated with trypsin (2 ml) to separate them from the flasks and
collected into a centrifuge tube containing 4 ml of the culture medium.
The flasks were further washed with 2 ml of PBS to remove the
remaining cells and this was transferred into a centrifuge tube. The tube
was centrifuged at 1000 rpm at r.t. for 5 min. The pellet was re-
suspended in an appropriate culture medium volume and seeded in
culture dishes before in vitro investigations.
2
2
1
(
KBr): 3427, 2924, 1607, 1512, 1240, 1174, 970, 800. H-NMR (CDCl ,
3
4
8
00 MHz): 8.84 – 8.86 (m, 8 b-H of pyrrole); 8.22 (d, 6 o-H of phenyl);
.08 (s, 1 H, OH); 7.73 – 7.79 (m, 9 m-H and p-H of phenyl); 7.43 (d, 4 H
of phenyl); 7.32 (d, 2 H of substituted phenyl); 7.07 (d, 2 H of
substituted phenyl); 6.92 (s, 1 H, CH ¼ N); 4.30, 3.64 (2t, CH CH ); À
2
2
þ
2
.77 (s, 2 H of imino). MS: 794.67 ([M þ H] ). Anal. calc. for
C H N O (793.91): C 80.21, H 4.98, N 8.77; found: C 80.18, H 4.95,
5
3
39
5
3
N 8.82.
Synthesis of 5,10,15-Triphenyl-20-[4-(2-(4-m-hydroxyanilinedene
formyl)phenoxy)ethoxy]phenyl Porphyrin (¼ 3-[(4-{2-[4-(10,15,20-Tri-
phenylporphyrin-5-yl)phenoxy]ethoxy}benzylidene)amino]phenol;
H Pp(3)). MgSO (0.5 g, 4 mmol) was added to the soln. of H Pp(1)
5. Evaluation of Cytotoxic Activity. Cytotoxic effects of metal-free
or metal porphyrins were evaluated by MTT method. Cells were
2
4
2
4
(0.078 g, 0.1 mmol) and m-aminophenol (0.218 g, 2 mmol) in a mixture
seeded at a density of 2 Â 10 cells/well in 96-well culture plates
of CH Cl2 and EtOH (25 ml). The resulting mixture was stirred
containing a 200 ml culture medium per well when cells were in the
exponential growth phase. Then cells were incubated for 24 h at 378 to
promote their adhesion to the plate. The medium was then removed,
replaced with the different metal-free porphyrins and metalloporphyr-
ins and incubated for 24 h in the dark. Every plate had three wells with
untreated cells as the control and three wells with cells treated with
each compound.
2
magnetically at r.t. for 24 h and monitored by TLC, then, the solvent
was removed in vacuo. The obtained violet product was dissolved in
CH Cl , and purified by CC (the SiO column was previously treated
2
2
2
with alkaline Et N to prevent the dissociation of the product) using a
3
binary eluent of CH Cl /EtOH (10 :1). The first violet band was
2
2
collected. After vaporizing and drying under vacuum, H Pp(3) was
2
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ꢁ 2016 Verlag Helvetica Chimica Acta AG, Zürich

Helv. Chim. Acta 2016, 99, 24 – 29
29
After 24 h incubation, 20 ml MTT (5 mg/ml) was added to each well
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and cells were incubated for 4 h at 378 and 5% CO . The culture
2
medium was removed and the formazan crystal was dissolved by adding
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50 ml of DMSO. The absorbance was measured using an enzyme-
linked immuneoabsorbent assay plate reader (Bio-Rad) at 680 nm. The
cell viability was expressed by the absorbance changes, and the survival
rate was given as the percentage compared to the untreated cells. The
concentration required for 50% inhibition of cell viability (IC ) was
determined for the various porphyrins tested. HaCaT cells were treated
in the same conditions just for comparision.
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2
010, 104, 62.
Accepted October 29, 2015
ꢁ
2016 Verlag Helvetica Chimica Acta AG, Zürich
www.helv.wiley.com