Synthesis and cancer cell cytotoxicity of water-soluble gold(III) substituted tetraarylporphyrin

Article abstract of DOI:10.1016/j.jinorgbio.2011.12.003

The synthesis of novel substituted gold(III) tetraarylporphyrins with aqueous solubility has been carried out. The analogs ClAuTPP(CH ₃Py⁺?I^-), ClAuTCPPNa, ClAuTPPCO ₂Na, ClAuTSPPNa and ClAuTPPNH₂?HCl were evaluated for their in vitro cytotoxic activity against sarcoma 180 mouse tumor and SGC-7901 human gastric cancer cell line panel. Compound ClAuTCPPNa exhibited significant growth inhibitory properties against sarcoma 180 mouse tumor and SGC-7901 human gastric cancer cell examined, and afforded IC₅₀ values < 25 μM for 66.63% of the cell lines in the panel. Compound ClAuTPPNH ₂?HCl was an effective inhibitor of sarcoma 180 mouse tumor and SGC-7901 human gastric cancer cell growth, but generally less effective as a cytotoxic agent. Thus, the substituted gold(III) porphyrin ClAuTCPP-Na ⁺ and ClAuTPPNH₂?HCl with aqueous solubility were regarded as useful lead compounds for further structural optimization.

Full text of DOI:10.1016/j.jinorgbio.2011.12.003

Journal of Inorganic Biochemistry 108 (2012) 47–52
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Short communication
Synthesis and cancer cell cytotoxicity of water-soluble gold(III) substituted
tetraarylporphyrin
Liang Sun a, Huasheng Chen b, Zonglei Zhang a, Qian Yang b, Haibo Tong a, Aihua Xu b, Cunde Wang, a,
⁎
a
School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China
b
School of Medicine, Yangzhou University, Yangzhou 225002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2011
Received in revised form 19 December 2011
Accepted 19 December 2011
Available online 27 December 2011
The synthesis of novel substituted gold(III) tetraarylporphyrins with aqueous solubility has been carried out.
The analogs ClAuTPP(CH3Py+·I−), ClAuTCPPNa, ClAuTPPCO2Na, ClAuTSPPNa and ClAuTPPNH2·HCl were
evaluated for their in vitro cytotoxic activity against sarcoma 180 mouse tumor and SGC-7901 human gastric
cancer cell line panel. Compound ClAuTCPPNa exhibited significant growth inhibitory properties against sar-
coma 180 mouse tumor and SGC-7901 human gastric cancer cell examined, and afforded IC50 values b25 μM
for 66.63% of the cell lines in the panel. Compound ClAuTPPNH2·HCl was an effective inhibitor of sarcoma 180
mouse tumor and SGC-7901 human gastric cancer cell growth, but generally less effective as a cytotoxic
agent. Thus, the substituted gold(III) porphyrin ClAuTCPP-Na+ and ClAuTPPNH2·HCl with aqueous solubility
were regarded as useful lead compounds for further structural optimization.
Keywords:
Gold(III) substituted porphyrins
Tetraaryl porphyrins
In vitro cytotoxicity
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
gold(III) complexes exhibit strong cytotoxic activity in vitro against
tumor cells and have tumor-inhibiting properties in vivo [15–18], a
New metal-containing anticancer drugs with preferential anti-
cancer activity have received special attention as promising candi-
dates for new anticancer drugs since the introduction of cis-
diamminedichloroplatinum(II) (cisplatin) into clinical use [1,2].
However, although cisplatin is the better anticancer agent so far, it
has several disadvantages, including obvious toxic side effects such
as neurotoxicity, nephrotoxicity, and emesis. In addition, some cancers
exhibit inherent resistance to cisplatin, whereas others develop resis-
tance after initial treatment, thereby limiting its clinical use [3,4]. These
particular disadvantages have driven the search for new compounds
exhibiting high cytotoxic activity along with reduced side effects and
no cross-resistance.
In recent years research has increasingly focused on the potential
of gold(III) complexes as anticancer drug candidates, because gold(III)
possesses the same isoelectronic configuration (d8) of the platinum(II)
ion; accordingly, the dominant coordination geometry for gold(III) com-
plexes is square planar tetra-coordination [5–10]. Some studies have
proven that polydentate ligands often enhance the stability of gold(III)
complexes in biological environments, and although the fact gold(III)
complexes are often structural analogs of cisplatin, they are widely
thought to impart tumor cell death via a different mechanism [11–14].
However, although there have been important developments in
the field of gold(III) complexes for anticancer chemotherapy, which
major problem hindering the development of gold(III) complexes
for medicinal application is their poor stability in aqueous solutions.
Consequently, some improved tetradentate ligands chelating gold(III)
strongly were designed and synthesized. However, these gold(III)
complexes are low cytotoxicity towards selected cancer cells with
IC50 values of >100 μM [19].
The structural analysis indicated that gold(III) complexes with
strongly chelating tetradentate ligands are difficult to carry the
metal to the cellular targets due to the over-stabilization of gold(III)
ions against reduction and demetalation.
Recently Che and co-workers have designed and synthesized
some modified tetraphenyllporphyrin ligands to chelate gold(III).
The gold(III) porphyrin compounds which behave as organic lipophilic
cations with a planar structure, are stable against demetalation under
physiological conditions. Further in vitro and in vivo studies revealed
that gold(III) porphyrin is a promising anticancer agent for the treatment
of colon and liver cancer [19–32], because the porphyrin ligand in the
complex can efficiently stabilize the gold(III) center, drastically reduces
its redox reactivity and oxidizing character. The use of gold(III) porphy-
rin complexes may provide an advantage for clinical application.
Although it has been demonstrated that various gold(III) porphy-
rin compounds have favorable anticancer properties, some gold(III)
porphyrins exhibit 30-fold to 100-fold stronger cytotoxicity than cis-
platin in several human cancer cell lines, including a cisplatin-
resistant human nasopharyngeal carcinoma cell line, in colon and
liver cancer. The major limitation of current gold(III) porphyrin com-
plexes is that few exhibit good solubility under physiological condi-
tions for clinical use [24–36]. Thus, there is an urgent need for the
⁎
Corresponding author. Tel.: +86 514 8797 5568; fax: +86 514 8797 5244.
E-mail address: wangcd@yzu.edu.cna (C. Wang,).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.12.003

48
L. Sun et al. / Journal of Inorganic Biochemistry 108 (2012) 47–52
Table 1
development of gold(III) porphyrin complexes with good solubility
and good stability under physiological conditions as new anticancer
drug candidates.
Preparation of substituted porphyrins (1–6).
R1
R2
H
X
Ligands (yield, %)
As the part of a drug discovery program to discover and develop
modified gold(III) porphyrins as potential anticancer agents, we
were interested in the substituted phenyl or pyridinyl with hydro-
philic group replacement of the phenyl moiety in the tetraphenylpor-
phyrin core skeleton. In continuation of our work on the design and
synthesis of substituted tetraphenylporphyrins and their gold(III)
complexes we focused on novel water-soluble gold(III) substituted
tetraphenylporphyrin complexes that incorporated electron donating
and electron withdrawing substituents in the aromatic ring of the tet-
raphenylporphyrin moiety.
H
H
C
N
C
C
C
C
23.0
6.5
22.0
9.0
64.2
74.9
(1)
(2)
(3)
(4)
(5)
(6)
CO2CH3
H
SO3H
H
CO2CH3
CO2CH3
SO3H
NO2
2.3. Procedure for the preparation 5,10,15,20-substitutedphenyl porphyrins
1–6
To investigate the effect of changing the water-soluble substituent
to gold(III) substituted tetraphenylporphyrin on bioactivity, different
aldehydes were used to synthesize the target ligands according to the
method described in Scheme 1 and Table 1. The synthesis of target
water-soluble gold(III) substituted tetraphenylporphyrin complexes
was described in Scheme 2 and Table 2.
A mixture of benzaldehyde (3.18 g, 30.0 mmol), substituted aro-
matic aldehyde (10.0 mmol) and freshly distilled pyrrole (2.68 g,
40.0 mmol) in propionic acid (140 mL) was refluxed for 3 h. After
completion of the reaction was checked by TLC, the mixture was
cooled in an ice-water bath and the crude product was filtered and
the filter cake was washed thoroughly with methanol (100 mL) and
dichloromethane (10 mL) respectively. The resulting purple crystals
are dried under air, the crude product was purified by column chro-
matography (silica gel, DCM/n-hexane: 1/5 V/V), re-crystallization
from ethanol gave pure products 1–4 (Table 1).
2. Materials and methods
2.1. Materials and reagents
The data of substituted porphyrin ligands were given:
All reagents and solvents were commercially available with ana-
lytical grade and used as received. Further purification and drying
by standard method were employed and distilled prior to use when
necessary. All evaporations of organic solvents were carried out
with a rotary evaporator in conjunction with a water aspirator. Melt-
ing points were taken on a hot-plate microscope apparatus and are
uncorrected. 1H NMR spectra were recorded with a Bruker AV-600
spectrometer. IR spectra were obtained on a Bruker Tensor 27 spec-
trometer (KBr disc). UV–Vis spectra were obtained on a UV 2501 PC
spectrometer. The MS spectra were obtained on a ZAB-HS mass spec-
trometer with 70 eV. Elemental analytical data were obtained by
using a model 240 elementary instrument. 5, 10, 15, 20-tetraaryl por-
phyrins 1–4 were synthesized by the reported method [20].
5,10,15,20-Tetraphenylporphyrin (compound 1) [33]: mp >
250 °C; 1H NMR (CDCl3, 600 MHz), δ (ppm): −2.74 (s, 2H, inner-
NH), 7.72–7.78 (m, 12H, Ph–CH ), 8.21 (d, J=6.6 Hz, 8H, Ph–CH),
8.83 (s, 8H, Por-CH); IR (KBr): υ 3438(s), 1560(w), 1472(w),
1437(w), 1348(w), 1070(w), 967(w), 797(m), 731(m), 697(w)
cm−1; MS (EI): 615 (M+1, 46%).
5,10,15-Triphenyl-20-(4-pyridinyl)porphyrin (compound 2): mp >
250 °C; 1H NMR (CDCl3, 600 MHz), δ(ppm): −2.82 (s, 2H, inner-NH),
7.75–7.80 (m, 9H, Ph-CH ), 8.17 (d, J=5.4 Hz, 2H, Py-CH), 8.21 (d,
J=6.6 Hz, 6H, Ph-CH), 8.79 (d, J=4.2 Hz, 2H, Py-CH), 8.86–8.90 (m,
6H, Por-CH), 9.03 (s, 2H, Por-CH); IR (KBr): υ 3446(s), 1634(w),
1590(w), 1473(w), 1396(w), 1351(w), 1070(w), 970(w), 798(m),
710(m), 657(w) cm−1; MS (EI): 616 ( M+1, 38%); UV–Vis (CH2Cl2)
λmax/nm (log ε) 416 (3.72), 513 (sh), 548 (2.37), 586 (4.38), 644
(4.20).
5,10,15,20-Tetra(4-methoxycarbonyl)phenylporphyrin (compound
3): mp >250 °C; 1H NMR (600 MHz, CDCl3), δ(ppm): −2.79 (s, 2H,
inner-NH), 4.11 (s, 12H, –COOCH3), 8.29 (d, J=7.8 Hz, 8H, Ph-CH),
8.44 (d, J=7.8 Hz, 8H, Ph-CH), 8.81 (s, 8H, Por-CH); IR (KBr): υ
3425(m), 2919(w), 1724(s), 1607(w), 1435(w), 1383(w), 1277(m),
1108(m), 965(w), 803(w), 762(w) cm−1; MS (EI): 735 (M+1, 87%);
UV–Vis (CH2Cl2) λmax/nm (log ε) 417 (3.08), 515 (sh), 549 (2.48),
588 (4.35), 645 (4.22).
Mycoplasma-free newborn calf serum was purchased from Hang-
zhou Sijiqing Biological Engineering Materials Co., Ltd. Cisplatin (Cis)
was the product of Jiangsu Hansen Pharmaceutical Co., Ltd. RPMI
1640, MTT, DMSO were purchased from Sigma-Aldrich Chemical Co.
2.2. Cell lines and cell culture
Sarcoma 180 mouse tumor cell line (S180) and SGC-7901 human
gastric carcinoma cell line (SGC-7901) were purchased from Shanghai
Institute of Cell Biology, Chinese Academy of Science. The cell line was
cultured in RPMI 1640 medium with 10% newborn calf serum. It was
maintained in a humidified incubator with an atmosphere of 95% air
and 5% CO2 at 37 °C. The cells were continuously passaged once every
3–4 days. Growing cells were collected on experiments.
5,10,15-Triphenyl-20-(4-methoxycarbonyl)phenyl porphyrin (com-
pound 4): mp >250 °C; 1H NMR (CDCl3, 600 MHz), δ(ppm): −2.79 (s,
2H, inner-NH), 4.11 (s, 3H, –COOCH3), 7.74–7.79 (m, 9H, Ph-CH), 8.21
(d, J=6.6 Hz, 6H, Ph-CH), 8.31 (d, J=7.8 Hz, 2H, Ph-CH), 8.44 (d,
Scheme 1. Reagents and conditions: (a) propionic acid, reflux, 2–4 h; (b) H2SO4, 120 °C, 4 h; (c) NaNO2, CF3CO2H, r.t. 1 min.

L. Sun et al. / Journal of Inorganic Biochemistry 108 (2012) 47–52
49
Scheme 2. Reagents and conditions: (i) KAuCl4, NaOAc, HOAc, reflux, 3 h from 2 to 4, 6 ; KAuCl4, H2O, reflux, 12 h then NaOH from 5 to 11 ; (ii) LiCl; (iii) CH3I, reflux, 3 h for 8; NaOH,
DMF, r.t. 2 h for 9, 10; SnCl2, HCl, r.t. 24 h for 12.
J=7.8 Hz, 2H, Ph-CH), 8.79 (d, J=4.2 Hz, 2H, Por-CH), 8.85 (s, 6H, Por-
CH); IR (KBr): υ 3442(s), 3319(w), 2924(w), 2853(w), 1812(m),
1722(s), 1604(w), 1472(w), 1437(w), 1394(w), 1353(w), 1279(s),
1182(w), 1106(w), 800(m), 739(w) cm−1; MS (EI): 645 (M+1, 88%);
UV–Vis (CH2Cl2) λmax/nm (log ε) 416 (3.12), 513 (sh), 548 (2.52), 589
(4.40), 646 (4.18).
gave pure product. 5,10,15-triphenyl-20-(4-nitro)phenyl porphyrin
(compound 6): mp >250 °C; 1H NMR (CDCl3, 600 MHz), δ (ppm):
−2.79 (s, 2H, inner-NH), 7.75–7.81 (m, 9H, Ph-CH), 8.21 (d, J=7.2 Hz,
6H, Ph-CH), 8.40 (d, J=8.4 Hz, 2H, Ph-CH), 8.64 (d, J=8.4 Hz, 2H, Ph-
CH), 8.74 (d, J=4.2 Hz, 2H, Por-CH), 8.86–8.90 (m, 6H, Por-CH);
IR(KBr): υ 3446(s), 2918(w), 2850(w), 1596(w), 1517(w), 1472(w),
1392(w), 1345(m), 1073(w), 840(w), 798(m), 706(m) cm−1; MS (EI):
660 (M+1, 58%); UV–Vis (CH2Cl2) λmax/nm (log ε) 418 (2.28), 514
(1.28), 549 (2.98), 588 (4.85), 645 (4.18).
5,10,15,20-Tetra(4-sulfo)phenylporphyrin (compound 5)
The mixture of 5,10,15,20-tetraphenylporphyrin (compound 1)
(1.0 g, 1.63 mmol) and 100% sulfuric acid (20 mL) was stirred at
120 °C for 4 h After the reaction mixture was cooled to room temper-
ature, the mixture was poured into ice-water (60 mL). The resulting
mixture was neutralized with 10 M NaOH to pH: 5, following concen-
trated to 20 mL. Then the mixture was cooled under 0 °C, the mix-
tures were filtrated and the solid was washed with methanol
(60 mL). The filtrate was diluted with methanol (100 mL) and the
solid Na2SO4 was removed by filter. The Na2SO4 was removed by re-
peating the foregoing operation three times and the resultant solu-
tion was concentrated to give a crude product. The product was
purified by re-crystallizing with a mixture solvent of methanol and
acetone. 5,10,15,20-Tetra(4-sulfo)phenylporphyrin (compound 5):
mp >250 °C; 1H NMR (600 MHz, DMSO-d6), δ(ppm): −2.98 (s, 2H,
inner-NH), 7.96 (d, J=6.0 Hz, 8H, Ph-CH), 8.08 (d, J=6.0 Hz, 8H,
2.4. Procedure for the preparation gold(III) substituted porphyrins 8–12
5,10,15-Triphenyl-20-(iodide N-methyl-4-pyridiniumyl) por-
phyrinato gold(III) chloride (8):A mixture of 5,10,15-triphenyl-20-
(4-pyridinyl)porphyrin (compound 2) (1.54 g, 2.5 mmol), K[AuIIICl4]
(2.82 g, 7.5 mmol) and sodium acetate (2.05 g, 25 mmol) in acetic
acid (5 mL) was refluxed for 3 h. After completion of the reaction
was checked by TLC, the crude product was obtained by removing
acetic acid. Next the solid product was washed thoroughly with
dichloromethane and water respectively. Then, the crude product
was purified by column chromatography (silica gel, DCM/methanol:
5/1 V/V), the purified product was dissolved in acetone, after filtering
the mixture solution was treated with LiCl in aqueous acetone,
5,10,15-triphenyl-20-(4-pyridinyl) porphyrinato gold(III) chloride
was obtained. The resultant solution of 5,10,15-triphenyl-20-(4-
pyridinyl)porphyrinato gold(III) chloride in chloroform (5 mL) was di-
luted with nitromethane (5 mL). To the mixture was added iodo-
methane (635 mg, 5.0 mmol), the reaction mixture was refluxed
under nitrogen for 6 h. After solvents and excess iodomethane were re-
moved by distillation, the residues were washed with chloroform
(10 mL). The crude product was purified by re-crystallizing to give an-
alytically pure gold(III) porphyrin compound 8 in 90.1% yield.
Ph-CH), 8.76 (s, 8H, Por-CH); IR (KBr):
υ 3449(m), 1635(w),
1448(s), 1385(w), 1192(m), 1126(w), 1042(w), 873(w), 800(w),
739(w), 638(w) cm−1; MS (EI): 935 (M+1, 19%); UV–Vis (CH2Cl2)
λmax/nm (log ε) 412 (2.54), 515 (1.68), 551 (2.68), 578 (4.39), 633
(4.08).
5,10,15-Triphenyl-20-(4-nitro)phenyl porphyrin (compound 6)
To the solution of 5,10,15,20-tetraphenylporphyrin (compound 1)
(245.7 mg, 0.40 mmol) in trifluoroacetic acid (10 mL) was added the
solution of NaNO2 (27.6 mg, 0.40 mmol) in trifluoroacetic acid (2 mL)
at room temperature in 1 min. The resultant mixture was stirred at
room temperature for 40s and was poured into water (100 mL). The
mixture was extracted with CH2Cl2 (3×50 mL), the organic phase was
washed with sat. Na2CO3 and water. The organic phase was dried over
anhydride Na2SO4. After removal of Na2SO4, the filtrate was concentrated
and the crude product was purified by column chromatography (silica
gel, CH2Cl2/n-hexane: 2/3 V/V), re-crystallization from CH2Cl2/ethanol
The data of compound 8 were given: mp 212–214 °C; 1H NMR
(CDCl3, 600 MHz), δ(ppm): 4.85(s, 3H),7.86–7.94 (m, 9H, Ph-CH),
8.27 (d, J=7.8 Hz, 6H, Ph-CH), 9.01 (s, 2H, Ph-CH), 9.42 (d,
J=4.2 Hz, 2H, Ph-CH), 9.32 (d, J=5.4 Hz, 2H, Por-CH), 9.34 (d,
J=5.4 Hz, 2H, Por-CH), 9.72 (s, 2H, Por-CH), 9.79 (s, 2H, Por-CH); IR
(KBr): υ 3393(s), 2924(m), 2853(w), 1637(w), 1460(w), 1359(w),
1317(w), 1261(w), 1078(w), 1033(w), 1021(w), 800(w), 755(w),
702(w) cm−1; MS: 952 (M, 100%); UV-Vis (CH2Cl2) λmax/nm (log ε)
407 (3.86), 519 (4.66); Anal. calcd. for C44H30ClIN5Au (%):C, 53.49; H,
3.06; N, 7.09; Found: C, 53.20; H, 3.00; N, 6.88.
5,10,15,20-Tetra(sodium benzoate-4-yl)porphyrinato gold(III)
chloride (9): A mixture of 5,10,15,20-tetra(4-methoxycarbonyl)phe-
nylporphyrin (compound 3) (2.12 g, 2.5 mmol), K[AuIIICl4] (2.82 g,
7.5 mmol) and sodium acetate (2.05 g, 25 mmol) in acetic acid
(5 mL) was refluxed for 3 h. After completion of the reaction was checked
by TLC, the crude product was obtained by removing acetic acid. Next the
solid product was washed thoroughly with dichloromethane and water
Table 2
Preparation of gold(III) substituted porphyrins (8–12).
Ligands
R3
R4
X
Gold(III) complex
(yield, %)
2
3
4
5
6
H
CH3
N+ −
I
90.1 (8)
61.3 (9)
88.1 (10)
70.1 (11)
82.0 (12)
CO2−Na+
CO2−Na+
CO2−Na+
SO3−Na+
NH3+Cl−
C
C
C
C
H
SO3−Na+
H

50
L. Sun et al. / Journal of Inorganic Biochemistry 108 (2012) 47–52
respectively. Then, the crude product was purified by column chromatog-
raphy (silica gel, DCM/methanol: 5/1 V/V), the purified product was dis-
solved in DMF (15 mL). To the foregoing solution was added NaOH
(0.60 g, 15 mmol), the resultant mixture was stirred at room tempera-
ture for 2 h. After the reaction mixture was diluted with water (35 mL),
the mixture was extracted with CH2Cl2 (3×10 mL), then the water
phase was neutralized with 1 M HCl to pH: 4. The acidic solution was
extracted with EtOAc (3×15 mL) and the EtOAc phase was washed
with water and brine, dried over anhydride Na2SO4. After removal of sol-
vent, the residue was dissolved in methanol (5 mL) and the solution was
neutralized carefully with 1 M NaOH to pH: 8. Solvents of above solution
were removed by reduced pressure to give a crude product. The crude
product was purified by re-crystallizing using methanol as solvent to
give analytically pure gold(III) porphyrin compound 9 in 61.3% yield.
Compound 9: mp >250 °C; 1H NMR (600 MHz, DMSO-d6), δ (ppm):
8.40 (d, J=6.0 Hz, 8H, Ph-CH), 8.47 (s, 8H, Por-CH), 9.34 (d, J=8.4 Hz,
8H, Ph-CH); IR (KBr): υ 3432(m), 2924 (w), 2364(w), 1709(s),
1607(m), 1384(w), 1264(m), 1175(w), 1110(w), 1024(w), 869(w),
792(w), 708(w) cm−1; MS: 1074.7 ( M+1-Cl−, 100%); UV–Vis (DMF)
λmax/nm (log ε) 411 (3.62), 524 (4.85); Anal. calcd. for C48H24ClN4O8-
Na4Au (%): C, 51.98; H, 2.18; N, 5.05; Found: C, 51.83; H, 2.06; N, 4.86
5,10,15-Triphenyl-20-(sodium benzoate-4-yl)porphyrinato gold(-
III) chloride (10):
Using the procedure for the preparation of 5,10,15,20-tetra(sodium
benzoate-4-yl)porphyrinato gold(III) chloride (10) with 5,10,15-tri-
phenyl-20-(4-methoxycarbonyl)phenyl porphyrin (compound 4) as a
starting material to give 5,10,15-triphenyl-20-(sodium benzoate-4-yl)
porphyrinato gold(III) chloride (10) in 88.1% yield. Compound 10:
mp >250 °C; 1H NMR (CDCl3, 600 MHz), δ (ppm): 7.84–7.90 (m,
9H, Ph-CH), 8.24 (d, J=6.0 Hz, 6H, Ph-CH), 8.51 (s, 2H, Ph-CH ),
8.72 (d, J=6.0 Hz, 2H, Ph-CH), 9.13 ( s, 2H, Por-CH), 9.25–9.28 (m,
6H, Por-CH); IR (KBr): υ 3448(s), 2922(m), 2853(w), 2371(w),
1713(w), 1632(m), 1384(m), 1260(w), 1089(w), 1028(w), 803(w),
706(w) cm−1; MS: 875.7 ( M+1-Cl−, 100%); UV–Vis (CH2Cl2) λmax/
nm (log ε) 412 (3.58), 523 (4.78); Anal. calcd. for C45H27ClN4O2NaAu
(%): C, 59.32; H, 2.99; N, 6.15; Found: C, 59.19; H, 2.78; N, 6.26.
respectively, dried over anhydride Na2SO4. Then, removal of solvent,
the crude product was purified by column chromatography (silica gel,
DCM/methanol: 5/1 V/V), the purified product was dissolved in ace-
tone, after filtering the mixture solution was treated with LiCl in aque-
ous acetone, analytically pure gold(III) porphyrin compound 12 are
obtained as chloride salts in 82.0% yield. Compound 12: mp 180–
181 °C; 1H NMR (CDCl3, 600 MHz), δ (ppm): 7.20 (d, J=8.4 Hz, 2H,
Ph-CH), 7.87–7.92 (m, 9H, Ph-CH), 7.96 (d, J=7.8 Hz, 2H, Ph-CH),
8.22 ( d, J=6.6 Hz, 6H, Ph-CH), 9.26–9.27 (m, 6H, Por-CH), 9.44 ( d,
J=5.6 Hz, 2H, Por-CH); IR (KBr): υ 3433(s), 2925 (w), 1603(m),
1492(w), 1442(w), 1383(w), 1360(w), 1247(w), 1180(w), 1082(w),
1031(m), 804(w), 758(w), 705(w) cm−1; MS: 825.7 (M+1-Cl−–HCl,
100%); UV–Vis (CH2Cl2) λmax/nm (log ε) 406 (3.50), 524 (4.78);
Anal. calcd. for C44H30Cl2N5Au (%): C, 58.94; H, 3.37; N, 7.81; Found: C,
58.82; H, 3.13; N, 7.98.
2.5. Cytotoxicity assay for Gold(III) substituted tetraarylporphyrin chloride
on the effect of S180 and SGC-7901 human cell proliferation [37]
Gold(III) substituted tetraarylporphyrin chloride was prepared at
the concentrations of 6.4×10−5 M, 3.2×10−5 M, 1.6×10−5 M,
8×10−6 M, 4×10−6 M and 2×10−6 M respectively. DMSO was
used as latent solvent with the highest concentration less than 0.1%
in solution of tetraarylporphyrin. The control groups of cisplatin,
blank (1640) and DMSO solvent were set up at the same time. The cy-
totoxicity of gold(III) substituted tetraarylporphyrin chloride was de-
termined by MTT cytotoxic assay [37]. S180 cells or SGC-7901 human
gastric carcinoma cells were plated in 96-well plates at 1×105/mL
and 100 μL/well in complete media. Then the prepared and various
amounts of gold(III) substituted tetraarylporphyrin chloride and cis-
platin were plated in 96-well plates contain S180 cells or SGC-7901
human gastric carcinoma cells and incubated commonly for 44 h.
100 μL supernatant liquid was sucked from each hole, 10 μL of MTT
(5 mg/mL) was added in and cultured for 4 h, the media was then re-
moved and add hydrochloric acid isopropanol at 100 μL/well. Surging
for 5 min using the micro oscillator to dissolve MTT crystal and OD
value was measured at 570 nm using a Model Elx 800 Autoplate reader
(Bio-Tek Instruments, U.S.A.).
5,10,15,20-Tetra(sodium
benzenesulfonate-4-yl)porphyrinato
gold(III) chloride (11): A mixture of 5,10,15,20-tetra(4-sulfo)phenyl-
porphyrin (compound 5) (2.34 g, 2.5 mmol), K[AuIIICl4] (2.82 g,
7.5 mmol) and NaOH (0.40 g, 10 mmol) in deionized water (10 mL)
was refluxed for 12 h. After completion of the reaction was checked
by TLC, the crude product was obtained by removing water. The prod-
uct was purified by re-crystallizing using acetone as solvent to give
analytically pure gold(III) porphyrin compound 11 in 70.1% yield.
Compound 11: mp >250 °C; 1H NMR (600 MHz, D2O), δ(ppm): 7.65
(d, J=6.0 Hz, 8H, Ph-CH), 8.22 (d, J=7.2 Hz, 8H, Ph-CH), 8.56 (s, 8H,
Por-CH); IR (KBr): υ 3436(s), 2923(w), 1633(w), 1507(w), 1390(w),
1190(m), 1123(m), 1039(m), 1007(w), 810(w), 740(w), 640(w)
cm− 1; MS: 1218.7 (M+1-Cl−, 100%); UV-Vis (water) λmax/nm
(log ε) 404 (2.79), 518 (4.80); Anal. calcd. for C44H24ClN4O12S4Na4Au
(%): C, 42.17; H, 1.93; N, 4.47; Found: C, 42.22; H, 1.75; N, 4.60
2.6. Statistical analysis of experimental data
All the data of the experiment were compiled and analysized
according to SPSS 15.0 software. Measurement data were expressed
as the mean S. D.
3. Results and discussion
The tetraarylporphyrins 1, and 3 (Scheme 1, Table 1) were synthe-
sized in 22–23% yield by treating the appropriately freshly distilled
pyrrole with benzaldehyde or various substituted benzaldehyde in
propionic acid utilizing the reported method [33,34].
5,10,15-Triphenyl-20-(chloride
anilinium-4-yl)porphyrinato
Using the similar procedure, the mono-substituted tetraarylpor-
phyrins 2, and 4 were obtained in 6.5–9.0% yield by treating the freshly
distilled pyrrole with benzaldehyde/various substituted benzaldehyde
(3/1, mol/mol) in propionic acid.
gold(III) chloride (12): A mixture of 5,10,15-triphenyl-20-(4-nitrophe-
nyl)porphyrin (compound 6) (1.65 g, 2.5 mmol), K[AuIIICl4] (2.82 g,
7.5 mmol) and sodium acetate (2.05 g, 25 mmol) in acetic acid (5 mL)
was refluxed for 5 h. After completion of the reaction was checked by
TLC, the crude product was obtained by removing acetic acid. Next the
solid product was washed thoroughly with dichloromethane and
water respectively. Then, the crude product was purified by column
chromatography (silica gel, DCM/methanol: 5/1 V/V) to give 5,10,15-
Triphenyl-20-(4-nitrophenyl)porphyrinato gold(III) chloride. The re-
sultant solution of 5,10,15-triphenyl-20-(4-nitrophenyl) porphyrinato
gold(III) chloride, SnCl2 (4.73 g, 25 mmol) and 36.5% HCl (5 mL) in
dichloromethane (25 mL) was stirred at room temperature for 24 h.
After the reaction mixture was diluted with dichloromethane (25 mL),
the organic phase was washed water, sat. NaHCO3 and brine
Sulfphonation of tetraphenylporphyrin (1) with 100% H2SO4 at
120 °C for 4 h afforded tetrakis(4-sulfonatophenyl)porphyrin (5)
(64.2%). Triphenyl(4-nitrophenyl) porphyrin (6) was readily obtained
by reaction of the tetraphenylporphyrin (1) and sodium nitrite pro-
moted by trifluoroacetic acid in 74.9% yield (Scheme 1, Table 1).
The general synthesis of water-soluble gold(III) porphyrin com-
pounds (8–12) was achieved through the route outlined in Scheme 2.
Firstly, gold(III) porphyrin compounds were synthesized by the treat-
ment of K[AuIIICl4] with the free-base porphyrin ligand in the presence
of NaOAc in acetic acid [20,30]. After purification with column chroma-
tography and metathesis reaction with LiCl in aqueous acetone, gold(III)

L. Sun et al. / Journal of Inorganic Biochemistry 108 (2012) 47–52
51
S180cell
SGC-7901cell
Table 3
80
60
40
20
0
The water-solubility of Gold(III) substituted tetra-aryl porphyrin chloride.
Symbol
Compound
Water-solubility
7
8
9
10
11
12
Cis
TPPAuCl[16]
−
AuClTPPPy+CH3·I−
ClAuTCPPNa
++
+++
++
+++
++
−
ClAuTPPCO2Na
ClAuTSPPNa
ClAuTPPNH2·HCl
Cisplatin
7
8
9
10
11
12
Cis
Fig. 1. Maximum inhibition rate of gold(III) substituted tetra-aryl porphyrin chloride against
sarcoma 180 mouse (left bar) and SGC-7901 human (right bar) tumor cell line in vitro.
porphyrin compounds are obtained as chloride salts in 60–70% yields.
Next, water-soluble gold(III) porphyrin compound 8 was obtained by
treatment of gold(III) triphenylpyridinylporphrin with methyl iodide.
Treatment of gold(III) tetrakis(4-sulfonatophenyl)porphyrin with
NaOH gave water-soluble gold(III) porphyrin compound 11 [31]. Hy-
drolysis of gold(III) tetrakis(4-methoxycarbonylphenyl)porphyrin and
gold(III) triphenyl (4-methoxycarbonylphenyl)porphyrin in the pres-
ence of NaOH gave water-soluble gold(III) porphyrin compound 9 and
10 respectively (Scheme 2, Table 2). Gold(III) triphenyl(4-nitrophe-
nyl)porphyrin was reduced by SnCl2 in the presence of 36% HCl to
give gold(III) triphenyl(4-aminophenyl)porphyrin, which was treated
with HCl to form water-soluble gold(III) porphyrin compound 12
(Scheme 2, Table 2) in 18% yield. Purification was achieved by flash
chromatography on silica gel. 1H NMR, electrospray ionization (ESI)
mass (+ mode) analysis spectrometry, and elemental analysis were
used to characterize all the synthesized compounds. The water-
solubility for these synthesized substituted gold(III) porphyrin com-
pounds 7–12 and cisplatin was tested (Table 3). The results illustrated
that the compounds 9 and 11 with four ion groups possess exceptionally
aqueous solubility. The compounds 8, 10 and 12 with an ion group re-
spectively have low aqueous solubility, but the TPPAuCl and Cisplatin
don't dissolve very nearly in water.
To analyze the potential of the compounds 8–12 as anti-tumor
agents, their cytotoxicity was evaluated (Table 4) towards the
sarcoma 180 mouse and SGC-7901 human tumor and for comparison
purposes the cytotoxicity of cisplatin and the TPPAuCl [20] was also
evaluated under the same experimental conditions. Because of low
aqueous solubility, the test compounds such as 7, 8, 10, 12 and cis
were dissolved in DMSO first and then serially diluted in complete cul-
ture medium such that the effective DMSO content did not exceed 1%.
The results showed that compounds 9 and 11 exerted significant
inhibitory effect on the growth of S180 and SGC-7901, and the
dose-dependent relationship was found between 1 and 32 μM in gen-
eral, while IC50 values of 9 was less than 25 μM in S180 cell line, and
IC50 values of 11 was less than 50 μM in S180 and SGC-7901 cell lines.
8, 10 and 12 had weaker inhibitory effect on S180 cells, IC50 value was
more than 50 μM in S180 cell line, while IC50 values of 8, 9 and 12
were less than 50 μM in SGC-7901 cell line, compound 12 exhibits
an IC50 value nearly two-fold lower than cisplatin on one cell line
(cell line SGC-7901) (Table 4, Figs. 1 and 2). The results showed that
aqueous ion groups could balance the aqueous solubility with liposolu-
bility, which was helpful to transport drugs into target cells to improve
the anti-tumor activity of complexes. From the results of in vivo assays,
the anti-tumor activity order was 7>9>cis>11>12>8>10 in S180
cell line and 7>12>9>8>cis>11>10 in SGC-7901.
As presented in Table 4, compounds 7, 9 and 11 exhibited signifi-
cant cytotoxic activity against sarcoma 180 mouse tumor cell line,
confirming the importance of water-soluble groups on the aromatic
ring for gold(III) substituted tetraphenylporphyrin complexs to cyto-
toxicity in the condition of clinical use. Comparison of the activity of
8, 10 and 12 suggests that just one water-soluble group including
acidic anion and ammonium or pyridinium salt of gold(III) substitut-
ed tetraphenylporphyrin complexs decreases this activity. In addi-
tion, a carboxylic acid anion group plays an important role in the
anti-tumor activity of these compounds. Sarcoma 180 mouse tumor
cells seemed more sensitive to compounds with symmetrical water-
soluble groups on the gold(III) substituted tetraphenylporphyrin
complexs in the para-position, as shown in compounds 7, 9 and 11.
A single para-substituted analog had lower anti-tumor potency com-
pared with these of the other same water-soluble group compounds.
But SGC-7901 human gastric carcinoma cell line (SGC-7901) seemed
more sensitive to compounds with cation water-soluble groups on
the gold(III) substituted tetraphenylporphyrin complexes in the
para-position, as shown in compounds 8 and 12. These results sug-
gested that symmetrical water-soluble groups and cation water-
soluble groups on the gold(III) substituted tetraphenylporphyrin
complexs may be crucial for anti-tumor activity.
To compare the potential of the compound ClAuTCPPNa (9) and its
ligand TCPPNa as anti-tumor agents, their cytotoxicity was evaluated
towards the sarcoma 180 mouse and SGC-7901 human tumor under
the same experimental conditions. Compound ligand TCPPNa exhib-
ited a little growth inhibitory properties against sarcoma 180 mouse
tumor and SGC-7901 human gastric cancer cell examined, and
afforded IC50 values=79 μM for 43.22% and 91 μM for 40.28% respec-
tively. The result showed that the anti-tumor activity of ligand
TCPPNa is lower than its gold(III) complex.
4. Conclusion
In summary, a number of new substituted gold(III) tetraarylporphyrin
with aqueous solubility were synthesized and evaluated for their in vitro
Table 4
Cytotoxicity of gold(III) substituted tetra-aryl porphyrin chloride against sarcoma 180 mouse and SGC-7901 human tumor cell line in vitro.
⁎
Symbol
Compound
Maximum inhibition rate (%)
IC50 (μM)
S180cell
SGC-7901cell
S180cell
SGC-7901cell
7
8
9
10
11
12
Cis
TPPAuCl[16]
AuClTPPPy+CH3I−
ClAuTCPPNa
ClAuTPPCOONa
TSPPAuCl
71.32
28.11
66.63
16.46
59.65
42.21
49.24
74.09
57.56
57.94
50.47
67.68
62.22
54.45
15.39 0.20
183.58 3.16
23.40 0.37
389.27 0.98
34.83 0.26
56.23 0.68
27.70 0.26
3.19 0.52
38.86 1.31
38.79 0.86
106.21 1.36
59.13 0.39
23.27 0.98
41.69 0.37
ClAuTPPNH2·HCl
Cisplatin
⁎
The concentration of the gold complexes is 32 μM.

52
L. Sun et al. / Journal of Inorganic Biochemistry 108 (2012) 47–52
S180cell
400
300
200
100
0
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Financial support of this research by the National Natural Science
Foundation of China (NNSFC 20872125) is gratefully acknowledged
by authors. A Project Funded by the Priority Academic Program De-
velopment of Jiangsu Higher Education Institutions.