Y. Wang, Z. Pan, X.-L. Cheng et al.
European Journal of Medicinal Chemistry 209 (2021) 112867
TBPoSeOHetreated B16/A375 cells was significantly increased af-
ter photo-irradiation. Then, many hydrolytic enzymes (e.g.
cathepsin B and cathepsin D, etc.) are released into the cytoplasm
when the lysosomal membrane permeabilization is disturbed by
ROS [31]. This subsequently induces the dimerization of Bak and
Bax and forms pore channels on the mitochondrial membrane,
which promotes release of cytochrome C, leading to cell apoptosis
[32,33]. Consistent with these previous reports, our data showed
that TBPoS-OH could increase the ROS level, and subsequently
induce cell apoptosis via the lysosomal-mitochondrial pathway.
We then further validated the photodynamic therapy of TBPoS-
OH with two xenografted mouse models of melanoma A375 and
B16 cells. This further confirmed the excellent effect of TBPoS-OH
against tumor. Furthermore, an ideal PDT agent should possess a
ethan-1-ol, benzenesulfonyl chloride, and aluminum chloride (AlCl3)
were purchased from TCI (Shanghai). Tetrabutylammonium hexa-
fluorophosphate (Bu4NPF6) was obtained from Fluka and was
recrystallized in ethanol before use. Tetrahydrofuran (THF),
dichloromethane (DCM), dimethyl formamide (DMF) and pyridine
were dried over calcium hydride (CaH2). Solvents was purchased
from DUKSAN and used as received unless otherwise specified. All
reactions were monitored by analytical thin-layer chromatography
(TLC) on 0.25 mm silica gel plates with fluorescent indicator (GF254)
and visualized under UV light. Column chromatography was per-
formed with Fluka silica gel 60 (200e400 mesh ASTM) with the
solvent mixtures specified in the corresponding experiment.
The 1H and 13C NMR spectra were recorded on a Bruker NMR
400 MHz (1H: 400 MHz, 13C:100 MHz) spectrometer using tetra-
methylsilane (TMS) as internal standard at 25 ꢁC. Samples were
prepared as solutions in deuterated solvent. The following abbre-
viations were used to indicate the observed spin multiplicities on
NMR spectra: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
dd ¼ doublet of doublets, m ¼ multiplet, and br ¼ broad. High
resolution mass spectra (HRMS) were recorded on Bruker Autoflex
MALDI-TOF mass spectrometer. Purity of all final compounds was
95% or higher as determined by high performance liquid chroma-
tography (HPLC) (SHIMADZU Labsolutions) analysis on the Aglilent
high photo-therapeutic index (PTI
¼
dark cytotoxicity/photo-
cytotoxicity), i.e., a strong photo-cytotoxicity together with a low
dark cytotoxicity [34]. Thus, the cytotoxicity of TBPoS-OH and TBPoS-
2OH was further measured without photo-irradiation on the afore-
mentioned four cancer cell lines. The cells were exposed to
increasing concentrations of these compounds up to their maximum
solubility, and the dark LD50 values of the two porphycene-based
photosensitizers indicated no significant cytotoxicity for TBPoS-OH
towards the four cancer cells without photo-irradiation. Moreover,
we also found that TBPoS-OH had no obvious immunogenicity and
toxicity to blood cells and major organs in C57BL/6 mice.
C18 column (4.6 ꢂ 250 mm, 5
mm) using gradient elution (Mobile
Phase: A Phase ¼ ACN, B Phase ¼ 0.3% H3PO4 in H2O) at a flow rate
of 1.0 mL/min. All electrochemical experiments were performed on
a CHI 630C electrochemical Analyzer (CH Instruments, Inc., USA).
However, some limitations in this study should be noted. Firstly,
the mechanism of photodynamic treatment of TBPoS-OH and
TBPoS-2OH under hypoxic condition still needs to be explored.
Recent studies demonstrated phosphorylated proteins (e.g.
g-
4.2. Preparation of 1-(phenylsulfonyl)-1H-pyrrole (1)
H2AX) participate the generation of hydroxyl radicals, which
inducing severe oxidative damage to DNA and leading to cell
apoptosis [35]. Quantitative phosphoproteomic and metabonomic
analyses should be used in future studies to explore the mechanism
of photodynamic treatment of the photosensitizers under hypoxic
condition. Secondly, triplet exciton lifetimes of TBPoS-OH/TBPoS-
2OH, and the lifetime of single oxygen produced by TBPoS-OH/
TBPoS-2OH should be measured. Finally, the photocytotoxicity of
TBPoS-2OH is more sensitive than TBPoS-OH in KYSE-70, A375, and
B16 cells under normoxic/hypoxic conditions, but it is reversed in
HeLa cells. This difference still needs to be explored in the further
study.
A solution of benzenesulfonyl chloride (2.82 mL,18.5 mmol) was
added to a solution of pyrrole (1.00 g, 19.9 mmol) and NaOH (2.40 g,
0.06 mol) in dichloromethane (DCM, 50 mL) at 0 ꢁC. After stirring
for 30min, the reaction mixture was allowed to warm to room
temperature and stirred overnight. The mixture was quenched with
water and the products were extracted with DCM. The extract was
washed several times with water, dried over Na2SO4, and evapo-
rated under reduced pressure to give product 1 as a gray solid
(2.78 g, 90%). Recrystallization from methanol gave colorless cube-
like crystal. 1H NMR (400 MHz, CDCl3):
d
¼ 7.83e7.88 (m, 2H),
7.56e7.62 (m, 1H), 7.47e7.52 (m, 2H), 7.17 (t, J ¼ 2.3 Hz, 2H), 6.30 (t,
J ¼ 2.3 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d
¼ 113.7, 120.8, 126.7,
Collectively, this study collectively represents two novel
porphyrin-based PDT candidates with effective anti-tumor effects
under hypoxia, low dose, red LED light excitation
129.3, 133.8, 139.1. MS (ESI): [MþH]þ: calculated 208.3, found 208.1.
(
lem ¼ 640
15 nm), and a high photo-therapeutic index. The
4.3. Preparation of 3-(tert-butyl)-1-(phenylsulfonyl)-1H-pyrrole (2)
TBPoS-OH is particularly promising and will be evaluated in depth
in subsequent studies.
1 (2.07 g, 10 mmol) was added to a stirred mixture of aluminum
chloride (4.81 g, 36 mmol) in cooled DCM (30 mL) in an ice bath. A
solution of tert-butyl chloride (1.38 g,15 mmol) in DCM (20 mL) was
added dropwise to the mixture, which was then allowed to warm to
room temperature, stirred for 5 h, and poured into crushed ice.
Following the usual work-up, the solvent was removed to afford the
product as yellow oil that slowly crystallized. Recrystallization from
methanol gave 2 (2.42 g, 92%). 1H NMR (400 MHz, CDCl3):
4. Experimental section
4.1. Materials and instrumentation
The well-known hypoxic photo-sensitizer EtNBS (used as refer-
ence in this study) was prepared according to the literature [36].
Titanium tetrachloride (TiCl4), phosphorus (V) oxychloride (POCl3),
zinc power, n-butyl lithium (n-BuLi) and t-butyl chloride were pur-
chased from Sigma-Aldrich. Cuprous chloride (CuCl), magnesium
(Mg), sulfurochloridic acid, and 2,2,6,6-tetramethylpiperidine
(TMPP) were purchased from ACROS. The 2-(2-aminoethoxy)
d
¼ 7.87e7.80 (m, 2H), 7.64e7.52 (m, 1H), 7.50 (dd, J ¼ 10.5, 4.8 Hz,
2H), 7.08 (dd, J ¼ 3.2, 2.3 Hz, 1H), 6.89 (t, J ¼ 2.0 Hz, 1H), 6.25 (dd,
J ¼ 3.2, 1.7 Hz, 1H), 1.18 (s, 9H); 13C NMR (100 MHz, CDCl3):
d
¼ 140.6, 139.3, 133.5, 129.2, 126.6, 120.8, 115.1, 112.7, 30.8, 30.7. MS
(ESI): [MþH]þ: calculated 264.4, found 264.1.
apoptotic rates of A375 and B16 cells after treatment with TBPoS-OH. (d) Quantitative analysis of the apoptotic rates of A375 and B16 cells after treatment with TBPoS-OH. (e) The
changes in mitochondrial membrane potential in TBPoSeOHetreated A375 and B16 cells were examined by JC-1 staining. (f) Western blot analysis for levels of Bid, Bad, Bax, and
Bcl-2 in A375 and B16 cells after treatment with TBPoS-OH. (g-h) Western blot analysis for the levels of cytoplasm cytochrome C, caspase-3 cleaved caspase-3, caspase-9, and
cleaved caspase-9 in A375 and B16 cells after treatment with TBPoS-OH. The data are shown as the mean standard deviation; n ¼ 3 per group. **P < 0.01. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
10