Discovery of a Monoiodo Aza-BODIPY Near-Infrared Photosensitizer: In vitro and in vivo Evaluation for Photodynamic Therapy

Article abstract of DOI:10.1021/acs.jmedchem.0c00882

Photodynamic therapy (PDT) as a rising platform of the cancer treatment method is receiving increased attention. Through systematic evaluation of halogen substitution on aza-4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY), we have found that monoiodo-derived aza-BODIPYs provided greater efficacy than other halogenated aza-BODIPY PSs. 4 and 15 as monoiodinated aza-BODIPY dyes containing p-methoxyphenyl moiety were identified to be potent NIR aza-BODIPY-type PSs with IC50 values against HeLa cells at a light dose of 54 J/cm2 as low as 76 and 81 nM, respectively. 4 possessed superior phototoxicity, low dark toxicity, and good thermal/photostability and distributed majorly in mitochondria in cells. Apoptosis was verified to be the main cell death pathway, and in vitro reactive oxygen species generation was demonstrated. In vivo whole-body fluorescence imaging and ex vivo organ distribution studies suggested that 4 afforded an excellent PDT effect with a low drug dose under single-time light irradiation and revealed advantages over known PSs of ADPM06 and Ce6.

Full text of DOI:10.1021/acs.jmedchem.0c00882

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Article
Discovery of Monoiodo Aza-BODIPY Near-Infrared Photosensitizer:
in vitro and in vivo Evaluation for Photodynamic Therapy
zhiliang yu, Junliang Zhou, Xin Ji, Guangyu Lin, Shuang Xu, Xiaochun Dong, and Weili Zhao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00882 • Publication Date (Web): 11 Aug 2020
Downloaded from pubs.acs.org on August 13, 2020
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Discovery of Monoiodo Aza-BODIPY Near-Infrared
Photosensitizer: in vitro and in vivo Evaluation for
Photodynamic Therapy
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Zhiliang Yu#, ac, Junliang Zhou , Xin Ji , Guangyu Lin , Shuang Xu , Xiaochun
#, a
a
a
a
a
ab
Dong* , and Weili Zhao*
aSchool of Pharmacy, Fudan University, Shanghai, 201203, P. R. China.
bKey Laboratory for Special Functional Materials of the Ministry of Education, Henan
University, Kaifeng, 475004, P. R. China.
cShanghai Skin Disease Hospital, Shanghai, 200443, P. R. China.
ABSTRACT: Photodynamic therapy as a rising platform of cancer treatment method is
receiving increased attention. Through systematic evaluation of halogen substitution on
aza-BODIPY, we have found that monoiodo-derived aza-BODIPYs provided greater
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efficacy than other halogenated aza-BODIPY PSs. 4 and 15 as monoiodinated aza-
BODIPY dyes containing p-methoxyphenyl moiety were identified to be potent NIR aza-
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BODIPY type PSs with IC values against HeLa cells at a light dose of 54 J/cm as low
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as 76 nM, 81 nM respectively. 4 possessed superior photo-toxicity, low dark-toxicity and
good thermal/photo stability and distributed majorly in mitochondria in cells. Apoptosis
was verified to be the main cell death pathway and in vitro ROS generation was
demonstrated. In vivo whole-body fluorescence imaging and ex vivo organ distribution
studies suggested that 4 afforded excellent PDT effect with low drug dose under single
time light irradiation and revealed advantages over known PSs of ADPM06 and Ce6.
1. INTRODUCTION
Photodynamic therapy (PDT) is a rising platform of noninvasive and clinically approved
protocol for the treatment of multiple types of cancers.1-3 Three pivotal components are
usually involved in PDT: photosensitizer (PS), light, and oxygen. The light-activated PS
transfers its excited-state energy to either molecular oxygen (in triplet ground state) to
1
form O (type II reaction), or to react with a biological substrate to form superoxide anion
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radical, hydroxyl radical, or hydrogen peroxide (type I reaction). The combined cytotoxic
reactive oxygen species (ROS) result in damage of tumor cells, tumor-associated
vasculature infarction, and activation of immune response system.4-6 Due to different
microenvironment of tumor tissue from normal tissue, the PS administered intravenously,
intraperitoneally or locally, may be selectively retained in the tumor tissue after being
distributed for a period of time in the body.7-9
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Since the clinical approval of Porfimer sodium (Photofrin), a complex mixture of
hematoporphyrin derivatives, as PDT agent for the treatment of early and advanced
cancers,10 many efforts have been focused on discovery of new photosensitizers to
overcome some deficiencies in Photofrin such as low molar absorption coefficient, poor
bioavailability, and long-lasting cutaneous photosensitivity.11 Various cyclic tetrapyrrole
type PSs such as porphyrin, chlorin, bacterial chlorophyll, as well as phthalocyanine were
developed. Some of them have been widely used as the second-generation of PSs in
clinical treatments of cancers with improved efficacy and reduced side-effect (Fig. 1).12-14
However, talaporfin and chlorin e6 (Ce6) suffered from poor photostability. Temoporfin,
photochlor and verteporfin are fairly hydrophobic with the defect of serious self-
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aggregation upon administration.15,16 In addition, these tetrapyrrole-based
photosensitizers, as well as phthalocyanines (Pc 4 and AlPcS ), are difficult to synthesize
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and purify, therefore restricting the fine-tuning of their photophysical and chemical
properties. Consequently, there is interest to develop non-porphyrin photosensitizers with
ease of preparation. The focus on non-porphyrin PSs had been primarily examined on
cationic structures such as methylene blue, Nile blue, Nile red analogues, and the
chalcogenopyrylium class of photosensitizers. These classes of compounds, however,
suffer from major drawbacks of inherent dark cytotoxicity and short absorption maxima.
In recent years, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) have emerged
as a new class of PDT agent with many favorable characteristics.17-25
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OH
O
NH
N
N
NH
N
N
HO
NH
N
N
HN
NH
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N
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COOH
COOH
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COOH
COOH
O
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OH
HOOC
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O
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COOH
HO
Temoporfin
Talaporfin
Chlorin e6
Photochlor
O
Si
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MeO
MeO
HO3S
SO3H
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HN
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O
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Si
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NH
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Cl Al
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(
H3CO)HO
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OCH3(OH)
O
O
HO3S
3
SO H
OH
Verteporfin
(mixture of two isomers)
Pc 4
AlPcS4
ADPM06
Figure 1. Representative photosensitizers.
BODIPYs possess many advantages of ideal photosensitizer characteristics such as
excellent photostability, high molar extinction coefficient, high photo-dark toxicity ratio,
and easy structural variation.20,22,26,27 Since the early discovery of Nagano and coworkers
through introduction of 2,6-diiodo onto 1,3,5,7-tertramethyl BODIPY to facilitate
intersystem crossing to afford a potent photosensitizer with higher efficiency of singlet
oxygen production, as well as favorable photostability,28 various BODIPY analogues
carrying two or more heavy atoms have been reported and demonstrated useful
performances on in vitro and in vivo anti-tumor studies.29-34 Some of the non-halogenated
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BODIPYs behaved as PDT agents, unfortunately, their efficacies were fairly weak in polar
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environment. Despite extraordinary efforts on modifications on BODIPY type PSs and
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nano-photosensitizers, they are not featured in clinical photodynamic therapy.17,18,36 More
recently, aza-BODIPYs, having the BODIPY meso-carbon replaced by nitrogen, attracted
much attention due to large absorption coefficient and red-shifted absorption to fit
therapeutic window (650900 nm) which is clinically favored with improved tissue
22
penetration of light. One landmark for aza-BODIPY PS was disclosed by O’Shea and
coworkers by attaching 2,6-dibromo onto 3,5-di(p-methoxyphenyl)-1,7-diphenyl aza-
BODIPY as efficient NIR PS (ADPM06) as shown in Fig.1.37-38 It exhibited potent
efficacies towards a panel of cancer cell lines, as well as effective eradication of tumor
with comparable to ‘‘cure-rates’’ in mice xenograft models.37-40 Various examples of aza-
BODIPY dyes carrying two or more halogen atoms were evaluated as PSs and anti-tumor
or anti-microbial studies were examined.41-50 However, incorporation of halogen to
facilitate intersystem crossing to produce singlet oxygen may lead to increased dark
toxicity. So far, the bottleneck to limit these aza-BODIPYs moving into clinical stage as
non-porphyrin PSs for cancer treatment is still unclear. Up to now, there is no systemic
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investigation of the halogen effect and substitution variation on aza-BODIPY as PS for
anti-tumor study from medicinal chemistry point of view.
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In this paper, a series of bromo and iodo aza-BODIPYs were synthesized based on
3,5-di(p-methoxyphenyl)-1,7-diphenyl aza-BODIPY scaffold and their physical-chemical
properties were carefully evaluated. We extended our discovery in more broad range and
came to the conclusion that monoiodo derivatives of aza-BODIPY possessed various
advantages of ideal PS with excellent photostability, chemical stability and photo
cytotoxicity. The superior antitumor efficacy was demonstrated both in vitro and in vivo.
2. RESULTS AND DISCUSSION
2.1. Design and Synthesis.
Various symmetric and non-symmetric aza-BODIPY fluorescent dyes were easily
achieved using our previous optimized methods.51-52 The general known strategy to
transform aza-BODIPY dyes into PSs was to introduce heavy atoms (e.g. Br, I) onto both
2- and 6- positions of aza-BODIPYs.37-50 The focus of our design was the comparison of
halogen effect by installation of mono-, di-, or mixed heavy atom(s) onto aza-BODIPY
core. Therefore, we initially selected the most advanced aza-BODIPY PS of ADPM06 as
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leading compound. Various selective halogenation methods including monohalogenation,
dihalogenation, mixed halogenation were successfully explored using 3,5-di(p-
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methoxyphenyl)-1,7-diphenyl aza-BODIPY (compound A) as the key scaffold (Scheme
37
1
). Both dibromo PS (1, namely ADPM06 by O’Shea et al. ) and diiodo PS (2) were
prepared in excellent yields (91.3%, 90.4% respectively). Monobromo PS (3) and
monoiodo PS (4) were also obtained nicely (93.5%, 89.2% respectively). Moreover, 2-
bromo-6-iodo aza-BODIPY (5) as mixed halogenated PS was also synthesized in high
yield (88.7%).
Scheme 1. Selective halogenation of aza-BODIPY dyes.
Ph
Ph
N
B
N
N
F
F
R
R
A (R = OMe); B (R = Me)
iv)
N
i)
ii)
iii)
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Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
N
B
N
B
N
N
B
N
B
Br
Br
I
I
Br
I
iii)
Br
I
N
N
N
N
N
N
N
N
N
B
F
F
F
F
F
F
F
F
F F
R
1: R = OMe
6: R = Me
R
R
2: R = OMe
7: R = Me
R
R
3: R = OMe
8: R = Me
R
R
4: R = OMe
9: R = Me
R
R
5: R = OMe
R
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Reagents and conditions: i) NBS (2.2 equiv), AcOH, DCM, r.t., 30 min; ii) NIS (2.2 equiv),
AcOH, DCM, r.t., 30 min; iii) NBS (1.0 equiv), AcOH, DCM, r.t., 30 min; iv) NIS (1.0 equiv),
AcOH, DCM, r.t., 30 min.
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We further examined the halogen effect on other NIR aza-BODIPY PSs characterized
with electron-donation groups in broader range using 3,5-di(p-tolyl)-1,7-diphenyl aza-
BODIPY (compound B) as key scaffold and the halogenated PSs were similarly prepared
as shown in Scheme 1. The generality of halogen effect was also investigated based on
3,5-di(5’-methylthiophenyl-2-yl)-1,7-diphenyl aza-BODIPY (compound C) and the
halogenated derivatives were prepared as shown in Scheme 2. All PSs were obtained in
excellent yields.
Scheme 2. Synthesis of mono or dihalogenated aza-BODIPY photosensitizers.
Ph
Ph
Ph
Ph
Ph
Ph
N
B
N
B
N
B
I
I
I
ii)
N
N
N
N
i)
N
N
F
F
F
F
F F
S
S
S
S
S
S
Me
Me
Me
Me
Me
Me
10
C
11
Reagents and conditions: i) NIS (2.2 equiv), AcOH, DCM, r.t., 30 min; ii) NIS (1.0 equiv),
AcOH, DCM, r.t., 30 min.
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We finally paid our attention on conformationally restricted aza-BODIPY dyes with
favorable long absorption maxima and greater absorption coefficient.51-52 Various
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electron-donating aromatic and heteroaromatic groups were included to favor
requirement of the deep tissue penetration. The non-symmetric dyes (D~G) were easily
obtained.51-52 Mono-brominated derivatives (12~14) and mono-iodinated species (15~18)
were all prepared in excellent yields using the conditions shown in Scheme 3.
Scheme 3. Preparation of conformationally restricted aza-BODIPY photosensitizers.
Ph
Ph
Ph
Ph
Ph
Ph
N
B
N
B
N
B
Br
Br
Br
i)
N
N
N
N
N
N
or
or
Ph
Ph
Ar
N
B
F
F
F
F
F F
S
N
N
MeO
12
OMe
MeO
13
MeO
14 Me
F
F
Me
MeO
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
D (Ar = p-Methoxyphenyl);
E (Ar = p-tolyl);
N
B
N
B
N
N
B
I
I
I
I
N
N
N
N
ii)
N
N
N
N
F (Ar = 5-Methylthiophen-2-yl);
or
or
or
B
F F
F
F
F
F
G (Ar = thiophen-2-yl)
F
F
S
S
MeO
OMe MeO
Me
MeO
17 Me
MeO
18
15
16
Reagents and conditions: i) NBS (1.0 equiv), AcOH, DCM, r.t., 30 min; ii) NIS (1.0 equiv),
AcOH, DCM, r.t., 30 min.
1
2
.2. Spectroscopic, Photophysical Properties, and O Generation Efficiencies.
2
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The spectroscopic and photophysical properties of aza-BODIPY PSs were frequently
evaluated in non-polar solvents in literatures.37-50 The electronic absorption and
fluorescence spectra of our aza-BODIPY dyes (1~5) were initially recorded in chloroform
using compound A as reference dye and the spectroscopic data were collected in Table
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
. The absorption maxima ( ) of halogenated aza-BODIPY displayed hypsochromic
abs
shift around 9 nm compared with the parent dye (compound A with abs of 688 nm). In
contrast, the emission maxima ( ) of halogenated aza-BODIPY displayed only minor
em
hypsochromic shift (2~5 nm) compared with the parent dye (compound A with em of 725
nm). The monohalogenated aza-BODIPYs presented minor decrease of absorption

1
1
coefficient ( of 81,000, 97,000 M cm for 3 and 4, respectively) comparing with parent

1
1
1
1
dye A ( of 99,000 M cm ), whereas dihalogenated species ( of 77,000, 74,000 M cm
for 1 and 2, respectively) showed much decreased absorption coefficient. The mixed

1
1
halogenated species (5 with  of 76,000 M cm ) behaved as dihalogenated species.
Interestingly, while monobromination only led to trivial decline of fluorescence quantum
yield ( ) (comparing 3 with  of 0.34 to A with  of 0.36), dibrominated aza-BODIPY
f
f
f
(1) revealed much decreased fluorescence quantum yield ( = 0.17). In contrast,
f
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
monoiodo derivative (4) exhibited even smaller value of  (0.08) than 1. Diiodination
f
resulted in much reduced fluorescence quantum yield ( = 0.03) as shown from the data
f
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of 2. As expected, mixed halogenation (5) afforded significantly diminished fluorescence
emission ( = 0.07). In PDT application, PSs were usually applied in aqueous
f
environments. Due to the hydrophobic nature of aza-BODIPY dye, the photosensitization
of aza-BODIPY type PS may behave significantly different in polar environment from non-
polar solvent because of possible aggregation. We therefore examined the spectroscopic
i
and photophysical properties of PSs in isopropanol (PrOH), acetonitrile (CH CN), N,N-
3
dimethylformamide (DMF), as well as PBS buffer containing 1% Cremophor EL
(CrEL)/1,2-propanediol (10:3, v:v). The absorption spectra of aza-BODIPY PSs in
emulsified PBS buffer and in other polar solvents were shown in Fig. S1 (supporting
information). The spectroscopic data in various polar environments were presented in
Table S1 (supporting information). It can be found that in various solvents, all these
halogenated compounds revealed intense Q-band with absorption maxima around
665~685 nm, which falls into the body’s therapeutic window (650~900 nm). In consistence
with the trend observed in chloroform, all PSs showed declined absorption coefficients
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compared with the parent dye compound A. Notably, the absorption coefficients () of all
1
1
dyes investigated revealed much declined values in PBS (34,000~59,000 M cm )
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

1
1
compared with those in organic media (59,300~105,000 M cm ). Concurrently, the Full-
Width Half-Maximum (FWHM) values in aqueous environment (87~90 nm) were
significantly greater than those (56~67 nm) in organic solvents (Table S1).
Table 1. Photophysical data for the first round BODIPY photosensitizers.
Ph
Ph
N
B
X
Y
N
N
F
F
MeO
OMe
abs (nm)a
ε (M−1 cm−1)a
λ
Φ
a
rel. rateb
rel. ratec
rel. rated
Φ
Δ
d
PS
λ
em
f
A (X=Y=H)
688
679
679
679
678
679
99000
77000
74000
81000
97000
76000
725
720
723
720
721
720
0.36
0.17
0.03
0.34
0.08
0.07
0.28
4.5
7.0
1.1
7.3
6.8
0.30
1.9
0.04
0.36
0.66
0.19
1.3
0.02
0.14
0.24
0.10
0.52
0.29
1
2
3
4
5
(X=Y=Br)
(X=Y=I)
2.2
(X=H; Y=Br)
(X=H; Y=I)
(X=Br; Y=I)
0.83
2.4
2.3
0.65
a
b
i
In Chloroform; Relative rate of degradation of DPBF to methylene blue in PrOH;
cRelative rate of degradation of DPBF to methylene blue in PBS containing 1%
d
Cremophor EL (CrEL)/1,2-propanediol (10:3, v:v); Measured in DMF using ZnPc (Φ =
Δ
0.56 in DMF) as standard.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To evaluate the photosensitizing abilities of these aza-BODIPY dyes, their singlet
oxygen generation efficiencies were studied using 1,3-diphenylisobenzofuran (DPBF) as
singlet oxygen scavenger. The rate of photodegradation of this quencher was measured
by monitoring the decrease in absorbance at 411 nm with time upon irradiation with light.
Different from previous publications which evaluated photosensitizing efficiency
frequently in non-polar solvent (e.g. toluene), we focused our investigations in polar
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
environments (PrOH, DMF, PBS buffer containing Cremophor EL) in order to find useful
information for optimal PS. The photosensitizing capabilities of individual PS were carried
out under irradiation with filtered light (λ > 590 nm from halogen lamp with energy power
2
of 90 mW/cm ). Time-dependent changes of absorption spectra of various aza-BODIPY
dyes containing DPBF under light irradiation were shown in Fig. S2. The relative rates of
i
degradation of DPBF monitored at 411 nm in PrOH, DMF, PBS buffer containing 1%
Cremophor EL (CrEL)/1,2-propanediol (10:3, v:v) were illustrated in Fig. S3 and the data
calculated were collected in Table 1. From Fig. S2 and Fig. S3, it can be found that the
photosensitizing efficiencies of these PSs were solvent-dependent.
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9
As expected, the parent fluorescent dye A without any heavy atom in the molecule
1
exhibited poor O generation ability in all solvents applied. In contrast, all halogenated
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
aza-BODIPY PSs (1~5) revealed potent photosensitizing capability in PrOH. In
consistent with previous reports,37-50 dibrominated aza-BODIPY (1) or diiodinated aza-
i
BODIPY (2) afforded potent photosensitization in PrOH and 2 was more effective than 1.
We also observed that 5 as mixed halogenated PS possessed nice photosensitizing
ability as well (1 < 5 < 2). Though monobromination enabled photosensitizing capability
(see 3) similar to the reference PS of methylene blue (MB), significant improvement of
singlet oxygen generation was astonishingly found for monoiodo derivative (4) with up to
i
7-fold rate relative to MB in PrOH for degradation of DPBF. It worth to note that monoiodo
PS 4 exhibited much more potent photosensitizing ability than dibromo PS 1 and even
more effective than mixed halogenated PS 5 and diiodo PS 2. Such trend of halogenation
effects were also observed in PBS buffer containing 1% Cremophor EL (CrEL)/1,2-
propanediol (10:3, v:v) though degradation of DPBF was less efficient due to the
hydrophobic nature of the aza-BODIPY PSs. Unexpectedly, when evaluation of
photosensitizing capability was performed in dipolar aprotic solvent such as DMF or
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DMSO, it was found that obvious degradation of PS for halogenated aza-BODIPY
illustrated in the decrease of the absorbance of the NIR band was generally observed as
shown in Fig. S2. To further evaluate the halogenation-related degradation, we selected
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ZnPc (Φ = 0.56 in DMF) as standard to compare the O generation efficiencies of PSs
Δ
2
in DMF. In fact, dihalogenated aza-BODIPY (1, 2) or mixed halogenated species (5)
encountered significant degradation and afforded concurrently declined efficiency of
1
production of O (Φ = 0.14 for 1; 0.24 for 2; 0.29 for 5). While monobromo PS 3 (Φ =
2
Δ
Δ
0.10) was weaker than those PSs with two halogen atoms, the monoiodinated aza-
1
BODIPY (4) revealed excellent O production with Φ = 0.52 in DMF. We believed that
2
Δ
the difference of photosensitizing ability among the aza-BODIPY PSs was amplified in
DMF and such a solvent was used to evaluate other PSs in our subsequent modifications.
The next round modification intended to inspect the generality of the trend observed
above. To enable long absorption maxima, we installed p-tolyl moiety or 5-thiophenyl-2-
yl group onto 3- and 5- positions of aza-BODIPY and the corresponding dyes were termed
B/C respectively. The halogenated derivatives were listed in Table 2 and their properties
were collected as well. It can be found that B derived PS series displayed slightly shorter
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absorption maxima than A derived series whereas C derived PSs possessed longer
absorption maxima than compound A derived PSs as expected based on the electron-
donating ability of the substituent. Unfortunately, though monoiodination resulted in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
advantageous photosensitizing ability over monobromination, dibromination, or
1
diiodination (comparing 9 with 8, 6, 7; comparing 11 with 10, Table 2), the O production
2
quantum yields in DMF were relatively low for either 9 (Φ = 0.14) or 11 (Φ = 0.17). These
Δ
Δ
1
data implied that substituent on 3-/5- position of aza-BODIPY affected the O production
2
and p-methoxyphenyl group was advantageous over p-tolyl or 5-thiophenyl-2-yl.
Table 2. Photophysical data for aza-BODIPY photosensitizers prepared according to
Scheme 2.
Ph
Ph
Ph
Ph
N
B
N
B
X
Y
X
Y
N
N
N
N
F
F
F F
S
S
Me
Me
Me
Me
6~9
10, 11
a
rel. rateb
b
Entry
λ
abs (nm)a
 (M−1 cm−1)a
λ
em (nm)a
Φ
f
Φ
Δ
6
7
8
(X=Y=Br)
658
662
658
659
79900
92000
83600
89600
685
688
685
690
0.04
0.02
0.19
0.04
0.22
0.28
0.12
0.26
0.10
0.12
0.05
0.14
(X=Y=I)
(X=H; Y=Br)
9 (X=H; Y=I)
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0 (X=Y=I)
732
732
91300
71700
768
766
0.07
0.11
0.36
0.32
0.14
0.17
11 (X=H; Y=I)
a
b
In chloroform; Relative rate of degradation of DPBF and singlet oxygen quantum yield
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Φ ) with reference to ZnPc (Φ = 0.56) in DMF.
Δ
Δ
Table 3. Photophysical data for aza-BODIPY photosensitizers prepared according to
Scheme 3.
Ph
Ph
Ar
Ph
Ph
Ar
N
B
N
B
Br
I
N
N
N
N
F
F
F F
MeO
12~14
MeO
15~18
abs(nm)a
 (M−1 cm−1)a
em(nm)a
a
rel. rateb
b
Entry
λ
λ
Φ
f
Φ
Δ
1
1
1
1
2 (Ar=p-MeOPh)
692
90300
104400
73700
101300
110000
76900
81500
724
709
753
725
709
756
740
0.44
0.60
0.24
0.14
0.17
0.16
0.16
0.17
0.14
0.14
1.2
0.08
0.08
0.11
0.51
0.16
0.25
0.19
3 (Ar=p-MePh)
682
714
689
682
716
695
4 (Ar=5-Me-thiophen-2-yl)
5 (Ar=p-MeOPh)
16 (Ar=p-MePh)
0.25
0.42
0.37
17 (Ar=5-Me-thiophen-2-yl)
1
8 (Ar=thiophen-2-yl)
a
b
In chloroform; Relative rate of degradation of DPBF and singlet oxygen quantum yield
(
Φ ) with reference to ZnPc (Φ = 0.56) in DMF.
Δ
Δ
1
The established advantageous O production with monoiodo aza-BODIPY derivatives
2
shown in Tables 1 and 2 promoted us to apply various monoiodination on
1
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conformationally-restricted
aza-BODIPYs
using
4,5-dihydro-7-methoxy-3-
phenylbenzo[g]indole and 2-aryl-4-phenyl pyrrole to constitute the parent aza-BODIPY
core as shown in Scheme 3 in an attempt to further improve light penetration capability.51
Among the designed PSs, p-tolyl, p-methoxyphenyl, 5-methyl-thiophene-2-yl, and
thiophene-2-yl were selected as the candidates of Ar in the pyrrole scaffold (The aza-
BODIPYs constructed were termed as compounds D, E, F, and G respectively). Mono-
brominated derivatives (12~14) and mono-iodinated species (15~18) were all examined
the photophysical properties and photosensization capabilities and the results were
collected in Table 3. As a general rule, the singlet oxygen quantum yields of
monobrominated PSs (0.08~0.11) were inferior to monoiodinated PSs (0.16~0.51).
Though thienyl (in 18 with Φ of 0.19) and 5-methyl-thiophene-2-yl (17 with Φ of 0.25)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Δ
Δ
led to long absorption maxima of the PSs, the photosensitizing capabilities were
1
unfortunately suboptimal. p-Tolyl modified PSs (13 and 16) did not provide enough O
2
generation quantum yields (Φ of 0.08, 0.16 respectively) and beneficial absorption
Δ
maxima (abs of 682 nm) over other conformationally-restricted PSs. Pleasantly, we
discovered that 15 carrying p-methoxyphenyl moiety possessed nice photosensitization
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
efficiency (Φ of 0.51), favorable absorption maximum ( of 689 nm), and absorption
Δ
abs
1
1
coefficient ( of 101,300 M cm ).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.3 Cytotoxicity assays.
The in vitro photodynamic activities of aza-BODIPYs in Cremophor emulsion were
investigated against cervical carcinoma cell line (HeLa), breast cancer cell line (MCF-7),
and colon carcinoma cell line (SW480) under irradiation with light > 590 nm using MTT
assay with Ce6 as reference PS (Table 4). Parallel assays without light irradiation were
also performed to determine the dark toxicity. As shown in Table 4, all the PSs have
2
negligible dark toxicity up to 50 μM. When irradiated with light dose of 54 J/cm , IC
50
values of various PSs exhibited broad range of activity from micromolar to nanomolar
level. In the series of aza-BODIPY A derived PSs, dibromo species 1 (ADPM06)
possessed good light-induced toxicity (0.13~0.16 μM) for the investigated cell lines in
consistence with the literature reports.39 Diiodo PS 2 which efficiently generated high
singlet oxygen in organic media was found to have unsatisfied potency with light toxicity
around 1 μM. Similarly, the mixed halogenated PS 5 presented suboptimal light toxicity
around 0.5 μM. To our surprise, monoiodo PS 4 displayed potent inhibition for all cell lines
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(
up to 76 nM for HeLa cells) in consistent with efficient production of O . For B and C
2
derived series wherein p-tolyl and 5-methylthiophene-2-yl were installed onto the 3,5-
position of aza-BODIPY dye respectively, the monoiodo substituted photosensitizers (9,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1) exhibited better efficacy than monobrominated (8) and dihalogenated (6, 7, 10) PSs
in cell-based assays, even though the potency was relatively low. Among the
conformationally restricted system, we were pleased to find that monoiodo derivatives
(
15~18) were generally more active than monobromo derivatives (12~14) and p-
methoxyphenyl derived monoiodo PS (15) showed more potent inhibition than p-tolyl, 5-
methyl-thiophene-2-yl, thiophene-2-yl derivatized PSs (16, 17, 18 respectively).
Table 4. Photo and dark cytotoxicity of PSs in HeLa, MCF-7 and SW480 tumor cell lines.
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ar
Ph
Ph
Ar
N
B
N
B
N
B
N
B
N
B
N
B
X
Y
X
Y
X
Y
Br
I
N
N
N
N
N
N
N
N
N
N
N
N
F
F
F
F
F
F
F F
F
F
F F
S
S
Me
Me
MeO
OMe MeO
OMe Me
Me
A
1~5
6~9
10, 11
MeO
12~14
MeO
15~18
PS
IC50 (μM)
HeLa
MCF-7
SW480
J/cm2
54 J/cm2
0 J/cm2
54 J/cm2
0 J/cm2
54 J/cm2
0
A (X=Y=H)
>50
11.6±2.0
>50
9.4±1.3
>50
12.1±1.8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
(X=Y=Br)
(X=Y=I)
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.13±0.006
0.59±0.12
0.27±0.05
>50
>50
>50
0.15±0.01
0.66±0.07
0.35±0.06
0.10±0.01
0.42±0.08
1.5±0.20
5.3±0.43
2.6±0.18
1.2±0.14
16.4±2.9
7.7±1.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.16±0.01
1.3±0.11
0.28±0.04
0.086±0.006
0.53±0.06
16.1±2.2
>25
(X=H; Y=Br)
(X=H; Y=I)
(X=Br; Y=I)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.076±0.004 >50
0.31±0.05
4.3±0.89
13.4±2.3
7.2±1.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
6 (X=Y=Br)
7 (X=Y=I)
8 (X=H; Y=Br)
>25
9
1
1
1
1
1
1
(X=H; Y=I)
2.6±0.46
>25
12.4±2.0
>25
0 (X=Y=I)
1 (X=H; Y=I)
15.6±3.2
0.83±0.17
1.2±0.31
22.6±3.3
0.081±0.01
0.11±0.02
12.3±1.9
0.46±0.09
20.6±3.2
1.9±0.26
15.6±3.4
>25
2 (Ar=p-MeOPh)
3 (Ar=p-MePh)
4 (Ar=5-Me-thiophen-2-yl)
5 (Ar=p-MeOPh)
4.5±0.41
8.1±1.3
>25
0.14±0.02
0.41±0.06
16.6±3.8
0.75±0.11
0.12±0.01
0.66±0.08
21.4±4.6
0.83±0.14
16 (Ar= p-MePh)
17 (Ar=5-Me-thiophen-2-yl)
18 (Ar=thiophen-2-yl)
2
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Ce6
>50
1.92±0.22
>50
3.4±0.29
>50
3.1±0.46
Through comparison of various aza-BODIPY PSs in solution-based and cell-based
activities, we came to the conclusion that dihalogenation of aza-BODIPYs did not behave
as expected to lead to potent PDT effect according to general trend of heavy atom
effect.17,18,20 Actually, two monoiodinated aza-BODIPY dyes (4, 15) containing p-
methoxyphenyl moiety among the investigated PSs offered excellent photosensitization
and behaved as favorable PSs with intense long absorption, high phototoxicity and low
dark toxicity. Therefore, our subsequent efforts focused on detailed studies of 4 as a nice
example to confirm the advantage of monoiodination in comparison with known leading
standard of ADPM06 (compound 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The cytotoxic effects under or without light irradiation for 4 and 1 against HeLa cells
were shown in Fig. 2A and Fig. 2B, respectively. From Fig. 2A it can be concluded that 4
displayed better PDT activity than 1 over the investigated concentration range. As shown
in Fig. 2B, though both PSs exhibited minimal dark toxicity against HeLa cells without
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6
light irradiation, the cell viability evaluations clearly suggested the dibromination led to
greater dark toxicity than monoiodination.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Photo toxicity (A) and dark toxicity (B) of photosensitizers 4 and ADPM06
2
against HeLa cells. Light dose: 54 J/cm , λ > 590 nm. *p<0.05, **p<0.01.
To further investigate the PDT behavior of 4, light dose-dependent phototoxicity assays
were performed and the results were displayed in Fig. S4. From Fig. S4, we were pleasant
to find that 4 exhibited light dose-dependent antitumor activity against HeLa cells and
much greater cell killing ability was achieved by 4 under stronger exposure of light
irradiation.
2.4. Photostability and thermal stability studies.
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Upon evaluation of photosensitivity, we have noticed that some PSs encountered
degradation during light irradiation (Fig. S2). We suspected that the observed in vitro
efficacy may significantly rely on the stability of PS. To investigate the photostability
behavior comprehensively and establish the relationship between the stability and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
potency, we selected A-derived series and monitored the absorbance of PSs in N,N-
i
dimethylformamide (DMF), acetonitrile (CH CN), isopropanol (PrOH) and PBS buffer
3
containing 1% Cremophor EL (CrEL)/1,2-propanediol (10:3, v:v) respectively under
2
irradiation with light (λ > 590 nm, light power: 90 mW/cm ). As shown in Fig. 3, all PSs
encountered obvious degradation in DMF under irradiation and followed the trend of
stability as A > 3 > 4 > 1 > 5 > 2. Same trend was also found in other solvent e.g. CH CN,
3
iPrOH, or emulsified PBS buffer, however, the degradation of PS was insignificant.
Meanwhile, the thermal stabilities in the dark without irradiation in CH CN and emulsified
3
PBS buffer (containing minimum amount of detergent) were investigated as well and the
results were displayed in Fig. S5. Slow degradation of PS in the dark was discovered for
all dyes investigated both in CH CN and emulsified PBS buffer and followed the same
3
trend as light-induced degradation (Fig. 3). As demonstrated in Fig. 3 and Fig. S5,
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6
monohalogenated PSs possessed much better stability than dihalogenated and mixed
halogenated PSs. Therefore, the stability studies may provide a plausible explanation for
the observed potent PDT effect of 4.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Normalized absorbance-irradiation time histogram of dyes and photosensitizers
i
in different solvents: (A) DMF; (B) CH CN; (C) PrOH; (D) PBS buffer containing 0.01%
3
2
Cremophor EL (CrEL)/1,2-propanediol (10:3, v:v). Light power: 90 mW/cm (λ > 590 nm).
2.5. Subcellular localization.
The subcellular localization of photosensitizer is critical for photodynamic effect and
5
plays critical role for the mode of cell death. Since singlet oxygen has short half-life (~3.5
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μs) and limited diffusion distance (< 0.02 μm),53-55 the sites where the photosensitizers
accumulated in tumor cells coincide with the photo-damaged cellular structures. Actually,
some examples of subcellular-targeted photosensitizers were reported and it was well-
known that subcellular localization of photosensitizers could strongly affect the cell death
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
56
pathways. Mitochondria perform critical cellular functions in regulating multiple signaling
cascade reactions, such as energy production, intrinsic apoptotic mediated by caspase
9
/caspase 3 pathway and cell cycle regulation, thus acting as a crucial target for PDT.57
Localization of photosensitizers in mitochondria can cause in situ damage, maximize the
killing effect of the photosensitizer, lead to the destruction of the cells' energy supply
system and ultimately cell death.58-60
To investigate the subcellular localization behavior of 4, laser confocal microscopy
studies were performed on HeLa cells incubated with 4 together with commercially
available organelle-targeted dyes (Mito Tracker Green FM, Lyso Tracker Green, or ER
Tracker Green) and their co-localization patterns were displayed in Fig. 4. The overlapped
images indicated that 4 revealed good overlap with Mito Tracker Green FM (Fig. 4D1/E1),
as well as partial colocalization with Lyso Tracker Green (Fig. 4D2/E2) and ER Tracker
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Green (Fig. 4D3/E3), indicating that 4 was mainly localized in mitochondria, and
distributed a part in endoplasmic reticulum and lysosomes. The subcellular localization
studies of 4 may afford reasonable explanation for its excellent phototoxicity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Subcellular location of 4 in HeLa cells at 10 μM for 2 h: (A1, A2, A3) bright field;
(B1) fluorescence of Mito Tracker Green (250 nM); (B2) fluorescence of Lyso Tracker
Green (160 nM); (B3) fluorescence of ER Tracker Green (2 μM); (C1, C2, C3) the
corresponding red fluorescence of 4; (D1, D2, D3) the corresponding superimposed
images of organelle trackers and 4; (E1, E2, E3) Luminescence intensity profiles of
regions of interest (ROI) across HeLa cells in (D1, D2, D3) respectively. Scale bar: 5 μm.
2.6. Investigation on cell death pathway.
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Apoptosis is an important manner of cell death after illumination during photodynamic
therapy.61,62 In early apoptotic cells, phosphotidylserine exposes to the outside of the cell
membrane, which can bind to Annexin-V with high affinity. Propidium iodide (PI) is able
to stain nucleus of late apoptotic and necrotic cells. Since 4 located majorly in
mitochondria, we were looking forward to know whether apoptosis is the major way of cell
death. Therefore, Annexin V-FITC/PI double staining flow cytometry assay was
conducted to evaluate the effect caused by 4 in HeLa cells. As is shown in Fig. 5, no
obvious apoptotic cells was detected in control experiment wherein photosensitizers and
light irradiation were not treated (Fig. 5A). When the cells were incubated with 40 nM of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4 and irradiated with 54 J/cm of light (λ > 590 nm), the proportion of apoptotic cells was
2
about 20.99% (Fig. 5B). Using 200 nM of PS and exposed to 27 J/cm of light increased
2
the apoptosis to 35.44% (Fig. 5C). Upon increment of light dose to 54 J/cm , the apoptosis
reached to 89.64% (Fig. 5D). These results demonstrated that 4 induced major death of
cells through early/late apoptosis and also resulted in some necrosis under light
irradiation.
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