Synthesis of Aza-BODIPY boron difluoride PDT agents to promote apoptosis in HeLa cells

Article abstract of DOI:10.2174/157017811796064377

BF₂ Chelated azadipyrromethene dyes fluoresce in the near infrared and have potential applications in photodynamic therapy. When irradiated above 600nm these aza-BODIPY compounds react with triplet O ₂ in the body to form a reactive

Full text of DOI:10.2174/157017811796064377

368
Letters in Organic Chemistry, 2011, 8, 368-373
Synthesis of Aza-BODIPY Boron Difluoride PDT Agents to Promote
Apoptosis in HeLa Cells
Ronny Priefer*,a, Justin R. Griffithsa, Janelle N. Ludwiga, Graham Skelhorne-Grossb and
Robert S. Greeneb
aDepartment of Chemistry, Biochemistry and Physics, Niagara University, NY 14109, USA
bDepartment of Biology, Niagara University, NY 14109, USA
Received December 10, 2010: Revised March 28, 2011: Accepted March 28, 2011
Abstract: BF2 Chelated azadipyrromethene dyes fluoresce in the near infrared and have potential applications in
photodynamic therapy. When irradiated above 600nm these aza-BODIPY compounds react with triplet O2 in the body to
form a reactive singlet oxygen species which leads to cell death. A small library has been synthesized of these potential
PDT agents via a four step process, with varying substituent’s on the aromatic ring of the starting benzaldehyde and
acetophenone. In vitro studies on HeLa cells have revealed an effective photosensitive compound with low dark
cytotoxicity and promotion of apoptotic cell death when exposed to light.
Keywords: Azadipyrromethene, chalcone, HeLa cells, Michael’s addition, photodynamic therapy.
INTRODUCTION
the energy can be transferred to the natural occurring triplet
state of oxygen which is thus excited to singlet oxygen [12].
The singlet oxygen can cause oxidative cellular damage,
ultimately leading to cell death [13]. The original PDT agent
can be re-excited as long as light is introduced. The short
half-life (0.6 X 10-6 sec) [14] of singlet oxidation allows this
technique to be very selective to the area irradiated.
Photodynamic therapy (PDT) is a very attractive
technique for the treatment of cancer as it is a non-invasive
procedure. Porfimer sodium, also known as Photofrin, was
the first PDT agent approved by the FDA in 1995. This drug
has been used in the treatment of cervical, stomach,
esophageal, bladder, endobroncheal, skin, and lung cancers
[1]. However, Photofrin exists as a mixture of dimeric and
oligomeric compounds. Other porphyrin based PDT that are
commercially available are Visudyne [2], Foscan [3],
Purlytin [4], Tookad [5], and Motexafin Lutetium [6]. In
addition, Metvix [7] and 5-aminolevulinic acid [8] have also
been used as PDT agents as they behave as pro-drugs, and
are metabolized to porphyrins within the body. To date, very
few non-porphyrin based PDT agents have been examined.
Studies have been performed on methylene blue [9] as well
as nile blue [10], however, both of these show dark toxicity
(i.e. demonstrated cytotoxic ability in the absence of a light)
which would impair their application as a PDT agent. Work
has been done using a boron core in a non-porphyrin based
PDT agent with promising results [11]. We wish to further
examine this approach and evaluate whether these agents are
active in PDT, show minimal dark toxicity, and can lead the
cell to death via apoptosis.
(iii)
1O2
Oxidative
cellular
damage
T1
E
(i)
(iv)
(ii)
3O2
Cell Death
s0
i) Absorption ii) Flourescence iii) Intersystem Crossing iv) Energy Transfer
Fig. (1). Proposed Mode of Action of PDT agents.
In order to have a truly effective PDT agent, three factors
must be optimized: 1) The ability of the agent to go through
an ISC so that it can cause singlet oxygen to be generated, 2)
have minimal-to-no dark toxicity, and 3) preferably induce
cell death apoptotically versus necrotically. We have
The general theory of PDT is based upon the principle of
in vivo synthesis of singlet oxygen (Fig. 1). When an
electron of the PDT agent is excited with visible or near-
visible light, the compound may liberate this energy in two
manners; either directly via fluorescence, which would bring
the agent back down to its ground state, or via an intersystem
crossing (ISC). If this latter approach occurs,
synthesized
a
small
library
of
BF2 chelated
azadipyrromethene PDT agents and evaluated their activity
on HeLa cancer cells.
RESULTS AND DISCUSSION
The synthesis (Scheme 1) of our azadipyrromethene
boron difluoride PDT agents began with the formation of a
diaryl ꢀ,ꢁ-unsaturated ketone (1A) [15]. The chalcone was
synthesized from benzaldehyde and acetophenone
derivatives which allows for the formation of a diverse
*Address correspondence to this author at the Department of Chemistry,
Biochemistry and Physics, Niagara University, NY 14109, USA;
Tel: +1 716 286 8261; Fax: +1 716 286 8254;
E-mail: rpriefer@niagara.edu
1570-1786/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

Synthesis of Aza-BODIPY Boron Difluoride PDT Agents
Letters in Organic Chemistry, 2011, Vol. 8, No. 6
369
O
O
O
EtOH
10% NaOH
0oC to Room Temp
H
+
Compound 1A
MeNO2, KOH
EtOH
Reflux, 12h
NO2
O
N
NH4OAc, BuOH
Reflux, 24h
NH
N
Compound 2A
Compound 3A
BF3-OEt2
iPr2EtN, CH2Cl2
Room Temp, 24h
N
N
N
B
F2
Compound 4A
Scheme 1. Synthetic scheme for the aza-BODIPY dye compounds.
library of final products with variations on the tetraphenyl
system. Michael adduct (2A) was then formed at nearly
quantitative yields, followed by ring closure/dimerization
with ammonium acetate (3A). Good yields were obtained
with the final chelation of the azadipyrromethenes with BF2
(4A) [11] affording the aza-borodipyrromethene (aza-
BODIPY).
electrophoresis of the detached cell DNA. The effect was
increased by approximately 30 percent in the presence of
light (Fig. 3). No effect was observed in the viable attached
Table 1. Synthesized aza-BODIPY dyes (4A-G)
R1
R1
A total of 7 different aza-BODIPY boron difluorides
were synthesized using this scheme (Table 1). The
compounds vary in electron withdrawing, electron donating,
non-polar and polar groups off the ortho, meta, and para
positions of the tetraphenyl system. This was to examine the
effects of different substituents on the molecules fluorescent
and cellular toxicity properties.
R2
R2
R3
R3
N
N
N
B
F2
HeLa cervical cancer cells were grown in the presence of
different aza-BODIPY compounds and exposed to either
light treatment for photoactivation or dark treatment to
determine toxicity affects. Apoptotic cells which detach from
the flask [13], and necrotic cells which remain attached, were
isolated and their DNA was extracted. Agarose gel
electrophoresis and gel imaging were used to characterize
and quantitate apoptotic and necrotic effects of the
compounds relative to their photodynamic activation or
toxicity.
R4
R4
R5
R5
4
R1
R2
R3
R4
R5
A
B
C
D
E
F
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N(CH3)2
H
H
OCH3
H
H
Results for all aza-BODIPY dyes tested showed similar
cell death by necrosis as the parent compound 4A with the
exception of compound 4B [16] (Fig. 2). Compound 4B (R1
= (CH3)2N) demonstrated apoptosis, as shown by the
characteristic laddering pattern rather than a smear after gel
H
Br
H
H
H
CH3
CH3
CH3
H
G
H

370 Letters in Organic Chemistry, 2011, Vol. 8, No. 6
Priefer et al.
Lane
Light
State
M
C
+
A
C
-
D
1
+
A
2
+
D
3
-
A
4
-
D
5
+
A
6
+
D
7
-
A
8
-
D
Fig. (2). Aza-BODIPY mediated necrosis and apoptosis in HeLa cells in culture. Lanes: M, Lambda marker DNA; C, control attached (A)
and detached (D) DNA; lanes 1-4, Compound 4B treated DNA isolation, attached (A) and detached (D) in the presence (+) and absence (-) of
light; lanes 5-8, Compound 4A treated DNA isolation, attached (A) and detached (D) in the presence (+) and absence (-) of light.
cell fraction for 4B; in contrast with compound 4A, where
cells were completely necrotic independent of light and with
no detectable detached cell fraction present. Compound 4B
has been previously synthesized, and although not examined
for its ability as a PDT agent, it was studied for its
absorbance and fluorescence profile [17]. It was revealed
there was a distictive red shift in absorbance at high solution
pH values. In addition, this scaffold has been covalently
linked to a solid support and examined as a fluorescent
sensor [18].
induce apoptosis on HeLa cells. All of these aza-BODIPY
compounds showed a classic necrotic cell death profile, with
the exception of 4B. Although the synthesis of 4B has been
recently reported [17], it has not been examined as a possible
PDT agent. Its ability to induce apoptosis opposed to
necrosis and a >30% selectivity in light versus dark
conditions has encouraged us to further examine this
particular amino moiety for future synthesis.
EXPERIMENTAL
All commercial reagents were obtained from Sigma-
Aldrich Chemical Company. Thin-layer chromatography
(SiliCycle Inc.) was performed on 0.25-mm silica-gel plates
with aluminum backing, visualized using short-wave UV
CONCLUSION
In summary, seven PDT agents have been synthesized
and were tested to look at their cytotoxicity and ability to
100
90
80
70
60
50
40
30
20
10
0
minus light
plus light
Fig. (3). Apoptosis and necrosis cell fraction percent distributions after treatment with Compound 4A and 4B and light.

Synthesis of Aza-BODIPY Boron Difluoride PDT Agents
Letters in Organic Chemistry, 2011, Vol. 8, No. 6
371
light. 1H and 13C-NMR were recorded on a Varian 400-MHz
instrument, and all spectra of synthesized compounds were
compared to previously reported spectra [11, 15, 17].
with water (3 x 50 mL). The organic layers were washed
with brine, dried over magnesium sulfate, filtered, and
evaporated to yield the desired oily residue.
4-nitro-1,3-phenylbutan-1-one (2A)
General Procedure for the Synthesis of Chalcone 1A-G
ꢀH (400MHz, CDCl3): 7.92 (d, 2H, J=7.2Hz), 7.58 (t, 2H,
J=7.6Hz), 7.46 (t, 2H, J=7.6Hz), 7.33 (d, 2H, J=7.6Hz), 7.29
(m, 2H), 4.84 (dd, 1H, J=6.0, 12.8Hz), 4.69 (dd, 1H, J=4.8,
12.8Hz), 4.23 (p, 1H, J=7.6Hz), 3.46 (dd, 1H, J=6.4Hz,
16.8Hz), 3.38 (dd, 1H, J=7.6Hz, 16.8Hz).
A solution of an acetophenone (1 eq) in absolute ethanol
(10 mL) was added to an aqueous solution of 10% NaOH
(30 mL) at 0°C. After stirring for 15 min, a benzaldehyde (1
eq) was added to the solution and stirred for an additional 15
min. The reaction mixture was then stirred at room
temperature for 24 hours. The product precipitated from the
solution over the course of the reaction. The solid was
collected by filtration and washed with water to yield the
desired chalcone. Some compounds did not form solids, but
an oil. This oil was concentrated, washed with ethyl acetate,
filtered, and evaporated to yield an oil. The product was used
without purification for the next step.
3-(4-dimethylaminophenyl)-4-nitro-1-phenylbutan-1-one
(2B)
ꢀH (400MHz, CDCl3): 7.91 (d, 2H, J=8.0Hz), 7.57 (t, 1H,
J=7.2Hz), 7.45 (t, 2H, J=7.6Hz), 7.12 (d, 2H, J=8.4Hz), 6.66
(d, 2H, J=7.2Hz), 4.77 (dd, 1H, J=5.2, 12.0Hz), 4.63 (dd,
1H, J=7.8, 12.0Hz), 4.12 (m, 1H), 3.41 (dd, 1H, J=6.8,
16.8Hz), 3.35 (dd, 1H, J=7.6, 16.8Hz), 2.95 (s, 6H).
1,3-diphenylprop-2-en-1-one (1A)
1-(4-methoxyphenyl)-4-nitro-3-phenylbutan-1-one (2C)
ꢀH (400MHz, CDCl3): 8.3 (d, 2H, J=7.6Hz), 7.8 (d, 1H,
J=15.6Hz), 7.65 (d, 2H, J=3.2Hz), 7.59 (t, 2H, J=8.0Hz),
7.56 (d, 1H, J=15.6Hz), 7.51 (t, 2H, J=8.0Hz), 7.42 (d, 2H,
J=4.0Hz).
ꢀH (400MHz, CDCl3): 7.90 (d, 2H, J=8.8Hz), 7.24-7.35
(m, 5H), 6.92 (d, 2H, J=8.8Hz), 4.84 (dd, 1H, J=6.4,
12.4Hz), 4.67 (dd, 1H, J=4.4, 12.4Hz), 4.21 (m, 1H), 3.40
(dd, 1H, J=6.0, 17.0Hz), 3.32 (dd, 1H, J=7.2, 17.0Hz), 3.87
(s, 3H).
3-(4-dimethylaminophenyl)-1-phenylprop-2-en-1-one (1B)
3-(3-bromophenyl)-4-nitro-1-phenylbutan-1-one (2D)
ꢀH (400MHz, CDCl3): 8.01 (d, 2H, J=6.0Hz), 7.79 (d, 1H,
J=15.6), 7.55 (m, 3H), 7.49 (t, 2H, J=7.2), 7.34 (d, 1H,
J=15.6Hz), 6.70 (m, 2H), 3.05 (s, 6H).
ꢀH (400MHz, CDCl3): 7.92 (d, 2H, J=7.2Hz), 7.59 (t, 1H,
J=7.2Hz), 7.38-7.49 (m, 4H), 7.2 (t, 2H, J=7.6Hz), 4.82 (dd,
1H, J=6.0, 12.8Hz), 4.66 (dd, 1H, J=4.8, 12.8Hz), 4.21 (m,
1H), 3.44 (dd, 1H, J=5.8, 17.2Hz), 3.37 (dd, 1H, J=6.8,
17.2Hz).
1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one (1C)
ꢀH (400MHz, CDCl3): 8.16 (d, 2H, J=8.4Hz), 7.92 (d, 1H,
J=15.6Hz), 7.76 (m, 2H), 7.67 (d, 1H, J=15.6), 7.52 (m, 3H),
7.09 (d, 2H, J=8.0Hz), 4.00 (s, 3H).
3-(4-bromophenyl)-4-nitro-1-phenylbutan-1-one (2E)
3-(3-bromophenyl)-1-phenylprop-2-en-1-one (1D)
ꢀH (400MHz, CDCl3): 7.91 (d, 2H, J=6.8Hz), 7.59 (t, 1H,
J=7.2), 7.44-7.49 (m, 4H), 7.18 (d, 2H, J=8.4Hz), 4.81 (dd,
1H, J=6.4, 12.8Hz), 4.66 (dd, 1H, J=4.0, 12.4Hz), 4.21 (m,
1H), 3.43 (dd, 1H, J=4.0, 17.6Hz), 3.34 (dd, 1H, J=5.2,
17.6Hz).
ꢀH (400MHz, CDCl3): 8.03 (d, 2H, J=7.9Hz), 7.81 (s,
1H), 7.73 (d, 1H, J=15.6), 7.61 (t, 1H, J=7.2Hz), 7.50-7.57
(m, 5H), 7.31 (t, 1H, J=7.6Hz).
3-(4-bromophenyl)-1-phenylprop-2-en-1-one (1E)
1-(3,4-dimethylphenyl)-4-nitro-3-phenylbutan-1-one (2F)
ꢀH (400MHz, CDCl3): 8.01 (d, 2H, J=7.2Hz), 7.75 (d, 1H,
J=16.0Hz), 7.50-7.60 (m, 8H).
ꢀH (400MHz, CDCl3): 7.89 (d, 2H, J=7.0Hz), 7.55 (t, 1H,
J=6.8Hz), 7.32-7.46 (m, 5H), 4.80 (dd, 1H, J=5.8, 12.8Hz),
4.63 (dd, 1H, J=4.2, 12.8Hz), 4.18 (m, 1H), 3.48 (dd, 1H,
J=5.2, 16.8Hz), 3.35 (dd, 1H, J=7.0, 16.8Hz), 2.45 (s, 3H),
2.41 (s, 3H).
1-(3,4-dimethylphenyl)-3-phenylprop-2-en-1-one (1F)
ꢀH (400MHz, CDCl3): 8.00 (d, 2H, J=7.2Hz), 7.75 (d, 1H,
J=15.8Hz), 7.51-7.64 (m, 6H), 7.46 (d, 1H, J=15.8Hz), 2.41
(s, 3H), 2.36 (s, 3H).
1-(4-methylphenyl)-4-nitro-3-phenylbutan-1-one (2G)
1-(4-methylphenyl)-3-phenylprop-2-en-1-one (1G)
ꢀH (400MHz, CDCl3): 7.82 (d, 2H, J=8.4Hz), 7.22-7.36
(m, 7H), 4.84 (dd, 1H, J=6.0, 12.4Hz), 4.68 (dd, 1H, J=4.4,
12.8Hz), 4.22 (m, 1H), 3.42 (m, 2H), 2.41 (s, 3H).
ꢀH (400MHz, CDCl3): 7.94 (d, 2H, J=8.0Hz), 7.80 (d, 1H,
J=15.6Hz), 7.64-7.75 (m, 7H), 7.54 (d, 1H, J=15.6Hz) 2.38
(s, 3H).
General Procedure for Synthesis of Azadipyrromethene
3A-G
General Procedure for the Synthesis of Michael’s Adduct
2A-G
The oily Michael adduct (2A-G) (1 eq), ammonium
acetate (35 eq), and butanol (10 mL) were added to a round
bottom flask and heated under reflux for 24 hours. After
cooling to room temperature the solution was evaporated to
approximately a quarter of its original volume, filtered, and
A solution of the chalcone (1A-G) (1 eq), nitromethane
(20 eq), and NaOH (0.2 eq) in absolute ethanol (1 mL) was
heated at 60 °C for 12 hours. After cooling to room
temperature, the solution was concentrated and the oily
residue obtained was dissolved in ethyl acetate and washed

372 Letters in Organic Chemistry, 2011, Vol. 8, No. 6
Priefer et al.
BF2 chelated [3-(4-dimethylaminophenyl)-5-phenyl-1H-
pyrrol-2-yl][3-(4-dimethylaminephenyl)-5-phenylpyrrol-2-
ylidene]amine (4B)
washed with absolute ethanol, to yield the desired blue-
purple azapyrromethane solid.
[3,5-diphenyl-1H-pyrrol-2-yl][3,5-diphenylpyrrol-2-
ylidene]amine (3A)
ꢀH (400MHz, CDCl3): 8.09 (d, 4H, J=8.8Hz), 8.01 (d, 4H,
J=7.2Hz), 7.34-7.51 (m, 6H), 6.82 (s, 2H), 6.78 (d, 4H,
J=8.8Hz), 3.10 (s, 12H).
ꢀH (400MHz, CDCl3): 8.07 (d, 4H, J=8.0Hz), 7.97 (d, 4H,
J=7.6Hz), 7.55 (t, 4H, J=7.6Hz), 7.48 (d, 2H, J=7.2Hz), 7.44
(t, 4H, J=8.4Hz), 7.37 (d, 2H, J=7.2Hz), 7.22 (s, 2H).
BF2 chelated [5-(4-methoxyphenyl)-3-phenyl-1H-pyrrol-2-
yl][5-(4-methoxyphenyl)-3-phenyl pyrrol-2-ylidene]amine
(4C)
[3-(4-dimethylaminophenyl)-5-phenyl-1H-pyrrol-2-yl][3-(4-
dimethylaminephenyl)-5-phenylpyrrol-2-ylidene]amine
(3B)
ꢀH (400MHz, CDCl3): 8.04-8.09 (m, 8H), 7.41-7.47 (m,
6H), 6.98-7.03 (m, 6H), 3.87 (s, 6H).
ꢀH (400MHz, CDCl3): 8.05 (d, 4H, J=8.8Hz), 7.95 (d, 4H,
J=7.2Hz), 7.52 (t, 4H, J=7.2Hz), 7.43 (t, 2H, J=7.6Hz), 7.06
(s, 2H), 6.79 (d, 4H, J=8.8Hz), 3.05 (s, 12H).
BF2 chelated [3-(3-bromophenyl)-5-phenyl-1H-pyrrol-2-
yl][3-(3-bromophenyl)-5-phenyl
(4D)
pyrrol-2-ylidene]amine
[5-(4-methoxyphenyl)-3-phenyl-1H-pyrrol-2-yl][5-(4-
methoxyphenyl)-3-phenyl pyrrol-2-ylidene]amine (3C)
ꢀH (400MHz, CDCl3): 8.18 (s, 2H), 8.01-8.05 (m, 6H),
7.56 (d, 2H, J=7.6Hz), 7.49-7.51 (m, 6H), 7.42 (t, 2H,
J=7.6Hz), 7.05 (s, 2H).
ꢀH (400MHz, CDCl3): 8.07 (d, 4H, J=8.0Hz), 7.90 (d, 4H,
J=8.0Hz), 7.43 (t, 4H, J=6.4Hz), 7.35 (t, 2H, J=6.0Hz), 7.15
(s, 2H), 7.06 (d, 4H, J=8.0Hz), 3.92 (s, 6H).
BF2 chelated [3-(4-bromophenyl)-5-phenyl-1H-pyrrol-2-
yl][3-(4-bromophenyl)-5-phenyl
(4E)
pyrrol-2-ylidene]amine
[3-(3-bromophenyl)-5-phenyl-1H-pyrrol-2-yl][3-(3-
bromophenyl)-5-phenyl pyrrol-2-ylidene]amine (3D)
ꢀH (400MHz, CDCl3): 8.03 (dd, 4H, J=4.0, 7.6Hz), 7.91
(d, 4H, J=8.4Hz), 7.61 (d, 4H, J=8.8Hz), 7.42-7.59 (m, 6H),
7.03 (s, 2H).
ꢀH (400MHz, CDCl3): 8.11 (s, 2H), 8.01 (d, 2H,
J=7.2Hz), 7.94 (d, 4H, J=7.2Hz), 7.49-7.55 (m, 6H), 7.37 (t,
4H, J=8.0Hz), 7.20 (s, 2H).
BF2 chelated [5-(3,4-dimethylphenyl)-3-phenyl-1H-pyrrol-
2-yl][5-(3,4-dimethylphenyl)-3-phenylpyrrol-2-
ylidene]amine (4F)
[3-(4-bromophenyl)-5-phenyl-1H-pyrrol-2-yl][3-(4-
bromophenyl)-5-phenyl pyrrol-2-ylidene]amine (3E)
ꢀH (400MHz, CDCl3): 8.06 (d, 4H, J=8.0Hz), 7.98 (d, 4H,
J=8.0Hz), 7.38-7.45 (m, 4H), 7.31 (d, 4H, J=8.4Hz), 7.04 (s,
2H), 2.57 (s, 6H), 2.43 (s, 6H).
ꢀH (400MHz, CDCl3): 7.91 (d, 4H, J=7.2Hz), 7.39-7.66
(m, 10H), 7.17 (t, 4H, J=8.0Hz), 6.78 (s, 2H).
[5-(3,4-dimethylphenyl)-3-phenyl-1H-pyrrol-2-yl][5-(3,4-
dimethylphenyl)-3-phenylpyrrol-2-ylidene]amine (3F)
BF2 chelated [5-(4-methylphenyl)-3-phenyl-1H-pyrrol-2-
yl][5-(4-methylphenyl)-3-phenylpyrrol-2-ylidene]amine
(4G)
ꢀH (400MHz, CDCl3): 8.02 (d, 4H, J=7.0H), 7.37-7.58
(m, 8H), 7.32 (t, 4H, J=7.6Hz), 7.18 (s, 2H), 2.46 (s, 3H),
2.39 (s, 3H).
ꢀH (400MHz, CDCl3): 8.06 (d, 4H, J=8.0Hz), 7.97 (d, 4H,
J=7.6Hz), 7.38-7.52 (m, 6H), 7.29 (d, 4H, J=8.0Hz), 7.04 (s,
2H), 2.42 (s, 6H).
[5-(4-methylphenyl)-3-phenyl-1H-pyrrol-2-yl][5-(4-
methylphenyl)-3-phenylpyrrol-2-ylidene]amine (3G)
ꢀH (400MHz, CDCl3): 8.06 (d, 4H, J=7.2H), 7.85 (d, 4H,
J=8.0Hz), 7.43 (t, 4H, J=7.2Hz), 7.35 (t, 6H, J=8.0Hz), 7.18
(s, 2H), 2.46 (s, 6H).
Procedure for Determining Apoptosis and Necrosis of
HeLa with Aza-BODIPY Compounds
HeLa cells were maintained at 37oC and 5% CO2. HeLa
cells were grown in 25cm2 TPP tissue culture flasks
containing 5 mL of RPMI + Glutamax and supplemented
with 10% fetal bovine serum (FBS). Flask media was grown
to 70% confluency prior to treatment at which time each
flask media was changed with 5 mL of fresh media, and
40uM concentrations of the aza-BODIPY compounds were
mixed directly into the media. Flasks were incubated for 24
hours at 37oC in the dark to allow the drug to accumulate
intercellularly. After 24 hours, media from all flasks was
replaced with fresh media. Dark toxicity flasks were
wrapped with aluminum foil to eliminate light exposure and
all flasks were placed under broad spectrum light for 60
minutes delivering 8 J/cm2. Flasks were returned to the
incubator for 24 hours before DNA isolation. All sample
assays were carried out in triplicate and averages of the
results were used for viability apoptosis and necrosis results.
General Procedure for Synthesis of Aza-BODIPY 4A-G
A
flame dried flask was charged with the
azapyrromethane (3A-G) (1eq), and flushed with argon. Dry
dichloromethane (30mL) and dry diisopropylethylamine
(11eq) were added, and the solution was stirred for 15 min,
whereby BF3•OEt2 (15.6eq) was slowly added. After stirring
for 24 h at room temperature, the mixture was washed with
water, the organic layer dried over magnesium sulfate, and
evaporated to give the target compound.
BF2 chelated [3,5-diphenyl-1H-pyrrol-2-yl][3,5-diphenyl-
pyrrol-2-ylidene]amine (4A)
ꢀH (400MHz, CDCl3): 8.03-8.08 (m, 8H), 7.43-7.5 (m,
12H), 7.04 (s, 2H).

Synthesis of Aza-BODIPY Boron Difluoride PDT Agents
Letters in Organic Chemistry, 2011, Vol. 8, No. 6
373
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Media plus detached cells were removed from all flasks and
the cells were washed with 5 mL 1% phosphate buffered
saline (PBS) on ice. The remaining attached cells were
trypsonized and washed with 5 mL of 1% PBS on ice and all
samples were centrifuged at 3000 rpms for 20 minutes at
5oC. After centrifugation, the supernatant was removed and
cell pellets were resuspended in 500 μL of ice cold DNA
Isolation Buffer (0.15 M NaCl, 0.015 M Na citrate, 10 mM
EDTA and 1% Na lauryl sarkosinate), and homogenized by
pipetting. Then 10 μL of Proteinase K was added to the
lysate, incubated for 1 hr at 50oC and 1 mL of ice cold 95%
ethanol was added to the tube. The samples were inverted
several times and then placed in a -20oC freezer for 20
minutes and centrifuged at 16G for 20 minutes at 5oC to
pellet the DNA. The supernatant was poured off and the
pellet dried for 1 hour. Pellets were mixed with 50μL of ice
cold 0.5% Tris/Borate/EDTA buffer by pipetting and then 5
μL of DNAse-free RNAse was added to the mixture.
Samples were vortexed for 30 seconds and incubated at 37oC
for 15 minutes then mixed with 5ꢀL of DNA loading dye.
Samples were loaded onto a 1 % agarose gel and run for 65
minutes at 65 Volts and DNA visualized and photographed
in a Bio Rad Gel imager. Cell distribution in the apoptosis
and necrosis samples was determined by counting using a
Hausser Scientific Counting Chamber. Following
trypsonization, a uniform suspension of cells was obtained
by pipetting and a 1:2 dilution of cell suspension in trypan
blue was prepared using 20 ꢀL of cell suspension.
Subsequently, 10 ꢀL of the mixtures were loaded onto the
hemocytometer and the total number of cells overlying each
of the four 1mm2 areas was counted for three replicates.
These numbers were averaged, divided by the dilution and
multiplied by the number of mL in the flask.
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Loudet, A.; Bandichhor, R.; Wu, L.; Burgess, K. Functionalized
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ACKNOWLEDGEMENTS
The authors thank the Niagara University Academic
Center for Integrated Science and the Rochester Academy of
Science for their financial support.
[16]
1H NMR (400 MHz, DMSO-d6) ꢀ 3.10 (s, 12H), 6.78 (d, 4H,
J=8.8Hz), 6.82 (s, 2H), 7.44 (m, 6H), 8.01 (d, 4H, J=7.2Hz), 8.09
(d, 4H, J=8.8Hz). 13C NMR (100 MHz, DMSO-d6) ꢀ 39.8, 109.4,
112.4, 123.5, 125.1, 129.0, 129.4, 131.4, 134.8, 141.3, 148.6,
151.8, 156.1. HR-MS (ESI: M+H+) Exact mass calculated for
C36H33N5BF2 584.2792, found 584.27899.
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