Synthesis and photo-physical properties of a series of BODIPY dyes

Article abstract of DOI:10.1016/j.tet.2013.04.064

A series of 39 boron-dipyrrolylmethenes (BODIPYs) have been synthesized and characterized. Their spectroscopic properties, degree of lipophilicity, chemical stability under irradiation, and singlet-oxygen generation rate have also been studied. These compounds differ in the presence of ethyl groups (group A), hydrogens (group B) or iodines (group C) on the 2,6 positions; these appendices confer particular characteristics to each group. The presence of an aromatic substituent or hydrogen on the indacene 8 position produces 13 different molecules of each group. Besides the effects exerted by the group or atom on the 2,6 positions, the substituent on the 8 position exerts a further effect on the physico-chemical parameters, thus the desired properties of BODIPYs, concerning fluorescence, lipophilicity, and singlet oxygen production can be modulated accordingly.

Full text of DOI:10.1016/j.tet.2013.04.064

Tetrahedron 69 (2013) 4845e4856
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and photo-physical properties of a series of BODIPY dyes
,
Stefano Banfi a, Gianluca Nasini b, Stefano Zaza c, Enrico Caruso a
*
a Department of Theoretical and Applied Sciences (DiSTA), University of Insubria, Via J. H. Dunant 3, 21100 Varese, Italy
b Department of Chemistry, Materials and Chemical Engineering, Polytechnic of Milano, CNR-ICRM, Via Mancinelli 7, Milano, Italy
c Shimadzu Italia srl Via G. B. Cassinis 7, 20139 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 39 boron-dipyrrolylmethenes (BODIPYs) have been synthesized and characterized. Their
spectroscopic properties, degree of lipophilicity, chemical stability under irradiation, and singlet-oxygen
generation rate have also been studied. These compounds differ in the presence of ethyl groups (group
A), hydrogens (group B) or iodines (group C) on the 2,6 positions; these appendices confer particular
characteristics to each group. The presence of an aromatic substituent or hydrogen on the indacene 8
position produces 13 different molecules of each group. Besides the effects exerted by the group or atom
on the 2,6 positions, the substituent on the 8 position exerts a further effect on the physico-chemical
parameters, thus the desired properties of BODIPYs, concerning fluorescence, lipophilicity, and singlet
oxygen production can be modulated accordingly.
Received 23 October 2012
Received in revised form 3 April 2013
Accepted 15 April 2013
Available online 18 April 2013
Keywords:
BODIPY
Fluorescence
Singlet oxygen
Lipophilicity
IC50
Ó 2013 Elsevier Ltd. All rights reserved.
Photodegradation
1. Introduction
The absorption and emission characteristics of BODIPYs can be
tuned by introducing appropriate substituents onto the BODIPY
After their introduction in 1968,1 the 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene chromophores (Fig. 1), also known as boron dipyr-
rolylmethenes but commonly named with the acronymBODIPY, have
shown phenomenal growth in their applications and synthetic vari-
ants since the early nineties.2 Currently, BODIPY derivatives are
employed in a wide range of applications, e.g., as tunable laser dyes,3
chemosensors,4 fluorescent switches,5 and biological labels.6 BODIPY
dyes possess many distinctive and biologically favorable properties,
such as high molar absorption coefficients, high fluorescent quantum
yields, and long wavelengths of emission.7 Moreover, photochemical
properties and chemical stabilities of the boron dipyrrolylmethene
chromophore are higher than those of other dyes.
framework. The effect is particularly evident when the substituent
is linked to a carbon atom of a pyrrole ring.8 On the contrary, the
presence of an aromatic chemical group tethered on position 8
(meso) exerts a very weak effect, however it has been reported that
the presence of a strong electron-withdrawing group, such as CF3,
causes a deep bathochromic shift as compared to congeners with
an aryl substituent on the same position.9
The weak effect of the aromatic ring is explained by the feeble
electronic interaction between the aromatic ring and the BODIPY
framework. In fact the two moieties are almost orthogonal to each
other.10 Therefore, any structural difference on the meso-aryl group
does not noticeably influence the intensity and wavelength of the
absorption and fluorescence bands, however the degree of polarity
of this moiety could modulate the lipophilic/hydrophilic balance of
the molecule.
8 = meso
1
7
positions
β
The presence of substituents on the 2,6 positions of the pyrrole
units (both are b-pyrrole positions) exerts some evident effects; for
instance the presence of alkyl groups or heavy atoms has a direct
influence on the energy of the HOMOeLUMO orbitals, and on the
population of the triplet state thus influencing the phenomena of
absorption and emission of UVevis radiation.11
2
6
N
N
B
5
4
position
α
3
F
F
4,4-difluoro-4-bora-3a,4a-s-indacene
Fig. 1. Representation and numbering of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY).
Beside the well known fluorescence property that makes these
molecules very useful as biomarkers, an important new field of
application of BODIPYs concerns photodynamic therapy (PDT) as
they can be used as photosensitizers once opportunely modified.12
PDT is an emerging modality for the eradication of some kind of
* Corresponding author. Tel.: þ39 0332 421550; fax: þ39 0332 421554; e-mail
address: enrico.caruso@uninsubria.it (E. Caruso).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.064

4846
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
small solid cancers.13 It is based on the intravenous or topical ad-
ministration of a drug (a photosensitizer, PS) followed by irradia-
tion, after a predetermined incubation period, with a visible light of
an appropriate wavelength, which leads the formation of a PS in the
excited state. The absorbed energy is then transferred to the
neighboring molecules, especially to intracellular molecular oxy-
gen, thus producing toxic oxygen radical species able to induce cell
death. The basic factor influencing PDT outcome is the capability of
the photosensitizer to produce singlet oxygen, the main cytotoxic
agent involved in the killing of tumor cells in the PDT therapeutic
process.14 The rate of singlet oxygen production of a PS is regulated
by the efficiency of a spin-forbidden electronic transition, indicated
as intersystem crossing (ISC), in which the electrons switch from
the singlet to the triplet excited state. The introduction on the
BODIPY frame of bulky and electron rich atoms, such as the large
halogens Br and I, is known to have an influence over the rates of
ISC; this is named the heavy-atom effect.15 In fact, two different
type of heavy-atom effect are recognized: the internal heavy-atom
effect, in which heavy atoms are directly attached to the molecule,16
and the external heavy-atom effect, in which the photosensitizer is
positioned in a heavy atom-containing environment, the first being
much more effective than the second one.17
been characterized and studied for their different physico-chemical
properties, such as lipophilicity, absorption, and emission spec-
troscopies, chemical stability and, finally, the relative rate of singlet
oxygen (1O2) generation. In particular the fluorescence quantum
yields are measured in three solvents of different polarity, and the
results discussed on the basis of the level of the HOMO energy of the
aromatic moiety present on the 8 position of the BODIPY skeleton.
Preliminary in vitro photodynamic studies, concerning the mole-
cules characterized by the presence of iodine atoms on the dipyr-
rolylmethene frame, are also discussed in our text.
2. Results and discussion
2.1. Synthesis
The BODIPY derivatives were synthesized via acid catalyzed
condensation of aromatic aldehydes and pyrroles following the
general methods described by Akkaya and Liu.18 Two differently
substituted pyrroles, the 2,4-dimethylpyrrole and the 3-ethyl-2,4-
dimethylpyrrole, were used in the condensation reactions with
aromatic aldehydes, carried out in dichloromethane (DCM) or tet-
rahydrofuran (THF) in the presence of trifluoroacetic acid (TFA). The
obtained dipyrrolylmethane intermediate was subsequently oxi-
dized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and
then treated with BF3$Et2O in the presence of Et3N, in the same
reaction flask, yielding the desired pure BODIPY in fairly good
yields after a single chromatographic separation.
In this manuscript we report the synthesis of a wide series of
BODIPY derivatives featuring alkyl, hydrogen or iodine substituents
on the 2,6-pyrrolylmethene positions, thus forming three different
groups of molecules. Thirty-six of these compounds bear an aro-
matic ring with variable structural characteristics on the meso po-
sition, whereas three compounds have an unsubstituted meso
position featuring only a hydrogen atom. All of these molecules have
Following this synthetic pathway (Scheme 1), we obtained
BODIPYs characterized by the presence of ethyl appendices on the
Ar
H3C
CH3
1) TFA
2) DDQ
H3C
N
N
B
R
.
3) BF3 Et2O; Et3N
H3C
CH3
NH
F
F
+
R1
O
H
group A
CH2Cl2 o THF
CH3
Ar
B
Ar
B
H3C
H3C
CH3
H3C
H3C
CH3
CH3
R1
= H, Et
I2, HIO3
H
H
I
I
N
N
N
N
EtOH, RT
15-18 h
CH3
F
F
F
F
group B
group C
OMe
OMe
OMe
OH
I
OMe
OMe
OH
[4,5-(MeO)2,3-I]-Ph
4-MeO-Ph
3-MeO-Ph
4-OH-Ph
4-MeO-Naphth
3-OH-Ph
4a-b
3a-c
5a-b
6a-c
1a-c
2a-c
*
*
Ar =
OMe
F
N
F
F
F
N
Cl
Cl
OMe
F
4,7-(MeO)2-Naphth
7a-c
2,6-Cl2-Ph
8a-c
F5-Ph
3-Py
4-Py
9a-c
11a-c
12a-c
Scheme 1. Synthesis, structures, and acronyms of the BODIPY dyes (* for the structure of the per-iodinated compounds 4c and 5c see Fig. 3).

S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
4847
2,6 positions of the dipyrrolylmethene (group A), and those bearing
2.2. Analytical data
hydrogen atoms on the same positions (group B).
In this synthetic procedure, it is worth noticing that, in almost
every acid catalyzed condensation between pyrrole and aromatic
aldehyde, a low percentage (3e7%) of the unsubstituted BODIPYs
13a,b may be isolated as a byproduct (Fig. 2). It seems that no
correlation can be found between the nature of the aromatic
moiety and the isolated yield of compounds 13a,b.
2.2.1. HPLC retention time. The lipophilicity of organic compounds
is commonly evaluated measuring the molecule partition co-
efficient (P) between 1-octanol and water. The values obtained
following UVevis analysis of both the organic and the aqueous
phase are generally expressed as Log P; in this case values >3 in-
dicate highly lipophilic molecules. This method cannot be applied
when highly lipophilic (or, vice versa, hydrophilic) compounds are
considered, as the absorbance of one or the other phase is too low
to be spectrophotometrically detected. When spectroscopic
methods cannot be applied, a relative scale of the lipophilicity of
chemical compounds belonging to a homogeneous series can be
determined measuring the HPLC retention time (tR). Performing the
analyses on a reversed phase column (C18 is the most commonly
used), under standardized conditions (eluant and flux), the most
lipophilic compound will show the highest tR. The average value of
H
B
H
H3C
H3C
CH3
CH3
H3C
H3C
CH3
H
H
N
N
N
N
B
F
F
CH3
F
F
the tR ðM Þ of each BODIPY group clearly indicates that the bulky
13a
tR
13b
and low polarity iodine atoms confer high lipophilicity to all the
BODIPYs of the group C ðMt Þ ¼ 1305400; the group A molecules,
Fig. 2. Chemical structure of the byproducts 13a and 13b.
R
featuring the ethyl, show a ðMt Þ ¼ 901000 while the H substituted
R
BODIPYs (group B) are characterized by the shortest retention time
The BODIPYs characterized by hydrogen-bearing 2,6-positions
(R1¼H, Group B) are modified by the insertion of two iodine
atoms (Scheme 1) leading to the compounds of Group C. In the
iodination process, we found that the conditions reported in the
literature (I2, HIO3, 60 ꢀC, 20 min)19 cause partial degradation of the
boronedipyrrolylmethenes skeleton, leading to extremely low
yields of the desired compound. Lowering the temperature to
20e25 ꢀC and increasing the reaction time to 15e18 h allows the
recovery of the iodinated derivatives in fairly good yields (>80%).
It must be considered that, in the presence of a phenolic group
on the meso position of the BODIPY skeleton, iodination also oc-
curs on the aromatic ring. This undesired reaction cannot be avoid
as the hydroxyl substituent promotes electrophilic aromatic sub-
stitution, which occurs on the phenyl contemporaneously with the
iodination of the 2,6 free positions. More specifically, two iodine
atoms have been introduced on the 30,50 positions, when the ar-
omatic ring was characterized by the presence of the hydroxyl
group at the 40 position (compound 4c), whilst in the case of
a phenyl bearing the hydroxyl on position 30 the introduction of
only one iodine atom at the 40 position has been observed (com-
pound 5c) (Fig. 3).
with a ðMt Þ ¼ 700800: These data indicate the possibility to modify
R
the degree of lipophilicity of this class of molecules according to the
nature of the group or atom placed on the 2,6 positions; as we will
see later on (paragraph 2.2.4), the effect of having aromatic group in
the meso position can further partially modify the hydrophilic/li-
pophilic nature of BODIPYs.
2.2.2. Absorption and fluorescence spectroscopy. Both the max-
imumeminimum values range and the average values of the ex-
tinction coefficients at l maximum indicate the highest absorbance
of the hydrogen substituted compounds (group B) with a mean
value of 84,300, followed by those featuring the ethyl groups
(group A, mean 76,300), while the presence of the iodine atoms
further decreases the mean value of absorbance (group C, mean
73,000) although to a low extent. In every group, the absorbances in
water (W) are lower than those registered in DCM, with the ratios
AbsW/AbsDCM ranging from 0.50 to 0.80 for most of the molecules.
This occurrence is easily ascribable to the formation of aggregates
of the lipophilic compounds in the aqueous medium. Indeed the
largest decrease of absorbance is always associated with a broad-
ening of the peak, as evidenced by the two or three times greater
values of the full width at half maximum (FWHM) in water with
respect to those in DCM. In particular, the BODIPYs of group B show
the highest variability in the aqueous phase, indeed the highest
value of FWHM is observed for the compound bearing a highly li-
pophilic naphthyl group (7b, FWHM¼80) and the lowest for the
more polar, hydroxy substituted, compounds 4b and 5b
(FWHM¼27 and 28, respectively).
OH
I
4'
4'
I
OH
I
3'
3
5'
6'
5
6
2
2
1'
8
1'
8
1
3
1
3
7
7
As far as groups A and C are concerned the presence of the non-
polar and larger substituents on the 2,6 positions makes these
compounds generally more lipophilic with respect to those of
group B. It is conceivable that the meso-aryl group exerts a lower
effect on the absorption band broadening in the water phase.
6
6
2
2
I
I
I
I
4
B
4
B
N
N
N
N
5
5
F
F
F
F
4c
5c
The
l max values are instead well correlated to the presence of
the particular substituents on the 2,6 positions of the dipyrro-
lylmethene skeleton, as the presence of ethyl groups causes
a bathochromic shift of about 20 nm, and the iodines produce
a 30 nm shift with respect to the values recorded for the hydrogen
Fig. 3. Chemical structure of the two per-iodinated meso phenyl BODIPYs 4c and 5c.
Compounds 10aec were synthesized from the corresponding
compounds 9aec via nucleophilic aromatic substitution of the
fluorine in position 40 of the pentafluorophenyl group with 4-
methoxybenzylmercaptan. The reaction was carried out in THF, at
reflux for 30 min, in the presence of K2CO3 as base (Scheme 2).
In Tables 1e3 are reported the physico-chemical properties of
the synthesized BODIPYs.
bearing BODIPYs (group B). This latter shows
l max range between
500 and 518 nm, whereas the max of compounds listed in groups
l
A and C can be found in the range 524e536 nm and 532e548 nm,
respectively. The low difference about the observed position of the
l
max between the compound bearing two ethyls and those bear-
ing two iodines (about 10 nm of difference) does not account for the

4848
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
CH
O
F
S
F
F
F
F
F
F
F
F
CH
O
H C
H C
CH
CH
H C
H C
CH
CH
K2CO
+
THF
R
R
N
N
R
R
N
N
B
B
F
HS
F
F
F
R = Et, H, I
(4-BzS)-F
F -Ph
9a-c
10a-c
Scheme 2. Nucleophilic aromatic substitution on pentafluorophenyl substituted BODIPYs 9aec with 4-methoxybenzylmercaptan, affording compounds 10aec.
Table 1
Physico-chemical properties of the compounds of group A (2,6-ethyl substituted BODIPYs)
1O2
a
‘meso’ substituent
3
(DCM) 524<
l<536
FWHM
3
(W) 520<
l<532
FWHM (W)
HPLC tRe(RP C18)
(DCM)
(
3
Ratio W/DCM)
(Ratio W/DCM)
(min)
1a
2a
3a
4a
5a
6a
7a
8a
[4,5-(MeO)2,3-I]ePh
4-MeOePh
3-MeOePh
4-OHePh
92,600
66,200
84,300
63,400
72,000
80,500
83,100
72,100
56,800
60,200
71,400
57,000
133,000
27
27
27
27
27
23
23
28
28
29
27
29
23
75,100 (0.81)
55,400 (0.83)
60,100 (0.73)
48,700 (0.77)
58,900 (0.82)
61,500 (0.76)
64,300 (0.77)
41,000 (0.57)
44,100 (0.77)
42,400 (0.70)
57,400 (0.80)
53,800 (0.94)
57,600 (0.44)
57 (2.1)
62 (2.3)
62 (2.3)
61 (2.3)
62 (2.3)
62 (2.7)
57 (2.5)
65 (2.3)
65 (2.3)
60 (2.0)
62 (2.3)
65 (2.2)
75 (2.2)
1002700
905700
905000
704400
800000
1201200
1102400
401700
804100
1001400
805200
802700
801900
4.92ꢂ10ꢁ2
5.75ꢂ10ꢁ2
6.81ꢂ10ꢁ2
5.83ꢂ10ꢁ2
6.81ꢂ10ꢁ2
6.69ꢂ10ꢁ2
5.18ꢂ10ꢁ2
1.17ꢂ10ꢁ1
5.98ꢂ10ꢁ2
1.80ꢂ10ꢁ1
9.17ꢂ10ꢁ2
1.06ꢂ10ꢁ1
5.50ꢂ10ꢁ2
3-OHePh
4-MeOeNaphth
4,7-(MeO)2eNaphth
2,6-Cl2ePh
F5ePh
(4-BzS)eF4ePh
4-Py
9a
10a
11a
12a
13a
3-Py
H
Range
Mean
56.8O133ꢂ103
41O75ꢂ103
401700O1201200
901000
4.92O18ꢂ10ꢁ2
76,300
55,400
7.92ꢂ10ꢁ2
1O2 Comparative singlet-oxygen generation relative to Rose Bengal.
a
Table 2
Physico-chemical properties of the compounds of group B (2,6-hydrogen substituted BODIPYs)
1O2
a
‘meso’ substituent
3
(DCM) 500<l<518
FWHM
3
(W) 496<l<510
FWHM (W)
HPLC tRe(RP C18)
(DCM)
(ratio W/DCM)
(Ratio W/DCM)
(min)
1b
2b
3b
4b
5b
6b
7b
8b
[4,5-(MeO)2,3-I]ePh
4-MeOePh
3-MeOePh
4-OHePh
109,800
72,900
62,200
65,200
79,800
103,300
94,300
91,100
61,500
85,700
85,700
69,800
115,000
19
21
20
21
21
20
19
22
22
34
28
29
17
74,900 (0.68)
68,000 (0.93)
48,600 (0.79)
63,400 (0.96)
79,000 (0.99)
76,800 (0.74)
61,000 (0.64)
43,000 (0.47)
49,700 (0.81)
54,600 (0.63)
60,100 (0.69)
46,800 (0.67)
71,100 (0.62)
52 (2.7)
63 (3.0)
57 (2.8)
28 (1.3)
27 (1.3)
58 (2.9)
80 (4.2)
67 (3.0)
66 (3.0)
60 (1.8)
71 (2.5)
61 (2.1)
68 (4.0)
704800
703100
702800
602200
602900
805900
801600
400800
604800
705000
605300
604000
603100
1.17ꢂ10ꢁ2
8.89ꢂ10ꢁ3
8.91ꢂ10ꢁ3
8.13ꢂ10ꢁ3
1.90ꢂ10ꢁ2
2.25ꢂ10ꢁ2
4.95ꢂ10ꢁ2
1.86ꢂ10ꢁ2
4.28ꢂ10ꢁ2
3.98ꢂ10ꢁ2
9.96ꢂ10ꢁ2
1.68ꢂ10ꢁ2
1.64ꢂ10ꢁ2
3-OHePh
4-MeOeNaphth
4,7-(MeO)2eNaphth
2,6-Cl2ePh
F5ePh
(4-BzS)eF4ePh
4-Py
9b
10b
11b
12b
13b
3-Py
H
Range
Mean
61.5O115ꢂ103
43O79ꢂ103
400800O805900
700800
0.81O9.96ꢂ10ꢁ2
84,300
61,300
2.79ꢂ10ꢁ2
1O2 Comparative singlet-oxygen generation relative to Rose Bengal.
a
different electronic effect of the two moieties, as the former exert
only a weak positive inductive (þI) effect while the latter has
a weak electron-withdrawing effect (ꢁI) and a positive mesomeric
(þM) effect due to the electron pairs present on the iodine atoms,
thus it seems that the bulkiness of the substituent is the main pa-
rameter affecting the max.
The fluorescence data, reported in Table 3 for the 2,6-iodine
substituted BODIPYs and in Table 4 for the other BODIPYs, have
l

S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
4849
Table 3
Physico-chemical properties of the compounds of group C (2,6-iodine substituted BODIPYs)
1O2
a
b
‘meso’ substituent
3
(DCM) 532<l<548
FWHM
3
(W) 526<l<540
FWHM (W)
HPLC tRe(RP C18)
Ffluo
(
l, nm)
(DCM)
(
3
ratio W/DCM)
(Ratio W/DCM)
(min)
(DCM)
1c
2c
3c
4c
5c
6c
7c
8c
[4,5-(MeO)2,3-I]ePh
4-MeOePh
3-MeOePh
4-OHePh
89,300
61,500
69,900
73,700
67,700
72,000
97,800
76,400
50,500
48,300
78,800
67,100
96,500
29
30
23
25
22
28
28
29
43
32
35
36
37
68,200 (0.76)
35,000 (0.57)
49,800 (0.71)
38,600 (0.50)
53,000 (0.77)
51,000 (0.70)
73,200 (0.74)
54,300 (0.71)
38,600 (0.78)
33,400 (0.68)
49,500 (0.62)
30,400 (0.44)
60,300 (0.62)
56 (1.9)
77 (2.6)
61 (2.6)
70 (2.8)
77 (3.5)
58 (2.1)
58 (2.1)
77 (2.6)
90 (2.1)
70 (2.2)
75 (2.1)
87 (2.4)
71 (1.9)
1500400
1404200
1402700
1305000
1202100
2004400
1703200
503300
120080
1502700
1200600
1100700
1100500
0.01 (556)
0.02 (551)
0.02 (552)
0.04 (555)
0.05 (551)
0.02 (555)
0.01 (556)
0.02 (555)
0.01 (578)
0.04 (566)
0.02 (562)
0.03 (560)
0.02 (555)
1.27
1.04
1.27
1.45
1.48
1.27
1.33
1.53
1.20
1.17
1.99
1.81
1.53
3-OHePh
4-MeOeNaphth
4,7-(MeO)2eNaphth
2,6-Cl2ePh
F5ePh
(4-BzS)eF4ePh
4-Py
9c
10c
11c
12c
13c
3-Py
H
Range
Mean
97.8O48.3ꢂ103
30.4O73.2ꢂ103
503300O2004000
1305400
0.01O0.05
2.38ꢂ10ꢁ2
1.04O1.99
73,000
48,900
a
F
represents quantum efficiency of fluorescence with fluorescein (0.1 M NaOH, 0.85) as reference.
b
1O2 Comparative singlet-oxygen generation relative to Rose Bengal.
Table 4
HOMO energy levels and quantum yield of fluorescence in various solvents of the 2,6-ethyl substituted BODIPYs (part A) and of the 2,6-hydrogen substituted BODIPYs (part B)
HOMO energy (eV)
Solvent
lem (nm)
Ffluo
HOMO energy (eV)
Solvent
lem (nm)
Ffluo
1
2
3
4
5
6
7
ꢁ8.8353
ꢁ9.1150
ꢁ9.1150
ꢁ9.1745
ꢁ9.1745
ꢁ8.5161
ꢁ8.4026
a
b
a
b
a
b
a
b
a
b
a
b
a
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
542
544
542
519
518
517
541
539
538
513
513
511
540
541
538
512
513
512
537
539
539
517
513
513
540
540
538
515
514
512
543
542
541
517
517
515
542
543
541
0.38
0.45
0.27
0.37
0.50
0.41
0.40
0.50
0.43
0.59
0.74
0.31
0.37
0.48
0.34
0.51
0.67
0.30
0.41
0.58
0.39
0.65
0.70
0.57
0.47
0.50
0.32
0.49
0.62
0.59
0.54
0.53
0.33
0.67
0.70
0.12
0.39
0.44
0.32
8
ꢁ9.4204
a
b
a
b
a
b
a
b
a
b
a
b
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
MeCN
Hexane
DCM
551
548
548
527
526
523
559
561
558
528
531
530
558
557
557
527
533
525
546
541
545
520
516
518
542
541
542
521
514
510
543
544
540
514
514
510
0.52
0.65
0.49
0.79
0.88
0.62
0.29
0.34
0.20
0.68
0.74
0.60
0.42
0.56
0.38
0.71
0.81
0.59
0.35
0.50
0.33
0.66
0.83
0.53
0.40
0.50
0.42
0.61
0.62
0.56
0.24
0.34
0.43
0.87
0.88
0.76
9
ꢁ10.5472
ꢁ9.2170
10
11
12
13
ꢁ10.1047
ꢁ10.1047
MeCN
Hexane
DCM
MeCN
MeCN
b
Hexane
DCM
MeCN
518
518
517
0.45
0.51
0.009
All data were measured in the presence of up 1% acetone as a cosolvent. The quantum yield of fluorescence (Ffluo) was determined using fluorescein as a standard (0.85 in 0.1 M
NaOH). HOMO energy levels of the corresponding benzene moieties were calculated with HYPERCHEM software after minimization to their lowest energy conformation using
the semi-empirical PM3 method in HYPERCHEM software too.

4850
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
been normalized with respect to the value obtained using fluo-
rescein as standard.20 It is known that the fluorescent properties of
such dyes are controlled by electron transfer from the donor moiety
(the aryl substituent in the meso position in our case) to the ac-
ceptor fluorophore (BODIPY core), therefore a peculiar dependence
on solvent polarity could be observed.4a,21 Consequently we have
measured the quantum yield of fluorescence of groups A and B in
three different solvents, hexane, dichloromethane (DCM), and
acetonitrile (MeCN), characterized by particularly different di-
electric constants, 1.89, 9.14, and 37.5, respectively. The BODIPYs
included in group C were not considered in this study, as the iodine
atoms exert a well known ISC effect, thus shifting the singlet ex-
cited state into the lower energetic triplet state causing a nearly
complete inhibition of fluorescence to the advantage of a very high
rate of singlet-oxygen generation.
chosen to ensure a good overlap between the lmax of absorbance of
these BODIPYs and the emission wavelength of the light, thus
obtaining a maximum excitation power.
The values reported in Tables 1e3 show the great difference
between the iodinated BODIPYof group C and the other two groups.
All the BODIPYs of group C are largely more efficient than those
belonging to groups A and B that indeed show a very poor 1O2
outcome. It must be pointed out that the rate of singlet oxygen
production, higher than the value obtained with Rose Bengal,
measured for the BODIPYs belonging to the group C, make these
molecules particularly interesting for their application in photo-
dynamic therapy. In particular an impressive rate of 1O2 production
is observed for the pyridyl bearing compounds 11c and 12c that
show a 2-fold increased rate with respect to Rose Bengal.
The last comment concerns the influence of external iodine
atoms on the efficacy of 1O2 production rate; among the reported
BODIPYs four of them feature one extra iodine atom on the meso
phenyl (1aec, 5c) and one molecule bears two iodine atoms (4c). In
principle, an increased 1O2 production rate might be expected, due
to an external heavy-atom effect, however the values calculated for
these compounds are slightly higher than the mean value of the
corresponding group, thus indicating the absence of an external
heavy-atom effect.
The comparison of
that all compounds belonging to group B show a
of group A and that in the whole set of compounds DCM allows
Ffluo higher than hexane and MeCN. Among these last two sol-
F
fluo obtained in the three solvents indicates
F
fluo> than those
a
vents the more polar acetonitrile inhibits the fluorescence to
a greater extent with respect to hexane, however only when the
aryl meso substituent had a higher HOMO energy level was an
important inhibiting effect on fluorescence observed, as evidenced
by the values obtained for compound 7b and, partially, for 6b.
The inhibiting effect of polar solvents is absent in BODIPYs
characterized by the presence of an aromatic ring in the meso po-
sition when its HOMO energy is very low, i.e., when electron-
withdrawing groups are present on the aryl.
2.2.4. Effect of the substituent on position 8 (meso position) on the
lipophilic/hydrophilic balance. The first observation is the compa-
rable effect exerted by the meso substituent in each group, irre-
spective of the different substituent present in the 2,6 BODIPY
positions. Besides the effect on absorbance, the aromatic group
partially modifies the hydrophilicity of these molecules. Concern-
ing the HPLC retention times, the aromatic moieties confer a higher
lipophilicity to the BODIPYs as compared with the meso H
substituted compounds. More precisely the molecules featuring the
lipophilic naphthyl groups (6aec, 7aec) show a greater affinity for
the reversed phase HPLC column (C18), as witnessed by the highest
tR in each group. A similar effect was also found for compounds
10aec, all characterized by the presence of a large and non-polar
aromatic appendix. In the iodinated compounds of group C, the
presence of one or two more iodine atoms on the meso phenyl ring
(compounds 4c and 5c) does not seem to increase the lipophilicity,
probably because the iodine in the 2,6 positions already confers
a high hydrophobic character to these molecules. The presence of
electron-withdrawing atoms or the presence of a hydroxyl group
on the meso aromatic rings makes the BODIPY more polar, as
shorter tR were observed (compounds 4, 5, 9, 11, and 12 in every
group). Particularly striking is the effect of the two chlorine atoms
present in compounds 8aec, conferring the highest hydrophilicity
to these molecules, as witnessed by the low affinity for the sta-
tionary phase used. The tR of compounds 8aec are by far the
shortest in each group and do not differ greatly with the nature of
the group present on the 2,6 pyrrole positions.
Compounds 8a and 8b show the highest Ffluo values in groups A
and B, respectively. This can be explained by considering the
presence of two chlorine atoms, bulky groups, on the ortho and
ortho0 positions of the 8-aryl substituted BODIPYs. In fact, it has
been reported that the molecular rigidity arising from the in-
troduction of bulky groups at the ortho positions minimizes the
decrease in fluorescence intensity due to a nonradiative pathway.22
A particular case concerns compound 13b that shows the same
high Ffluo value of 8b, although it features the greatest number of
hydrogen atoms in the indacene structure (positions 2, 6, and 8).
2.2.3. Singlet oxygen (1O2) production rate. For BODIPYs to be used
as biological markers,6 their fluorescence should be at the possible
utmost or, alternatively, if they have to act as photosensitizers then
a high singlet oxygen production is required. Both processes take
place after light absorption, and the structural differences in the
BODIPY skeleton allow the different outcome of the energy loss
from the excited state. In the first case the energy loss occurs from
the singlet excited state through a strong light emission, possibly in
the red visible region of light. On the contrary, when a massive
production of singlet oxygen is required to impair the close bi-
ological structures, the triplet excited state must be involved from
which the energy loss via light emission becomes a forbidden
process; as a consequence this excited state can be lost with dif-
ferent pathways dissipating the energy to neighboring molecules.
Among them, molecular oxygen is one of the favored targets, thus
producing 1O2, a short living but highly toxic compound when
generated inside cells. The relative rates of singlet oxygen pro-
duction are generally measured by comparing the results with
a reference molecule; in this work Rose Bengal, a widely recognized
singlet oxygen producer, was used as standard.23 The amount of 1O2
produced by each photosensitizer was indirectly determined
measuring the disappearance of the absorption band, at 410 nm, of
the 1,3-diphenylisobenzofuran (DPBF), a well known 1O2 scaven-
ger.24 The experiments were carried out following a literature
The last aspect to be considered is the 1O2 production that is
remarkable for the BODIPYs of group C only. In this group, the
fluorescence is inhibited and the formation of the triplet excited
state is favored by the presence of the iodine atoms on the 2,6 po-
sitions. An impressive rate of 1O2 is observed for the pyridyl bearing
compounds 11c and 12c, which show a 2-fold increased rate with
respect to Rose Bengal, the reference compound known for its effi-
cacy in singlet oxygen production. These last molecules should be
considered in the application of BODIPYs in the field of PDT.
2.3. Photodegradation of BODIPY dyes
method, using isopropanol as solvent with a 50
centration of DPBF and in the presence of 2.5 M of BODIPY. Irra-
diation was obtained with a green LED device. The green light was
m
M initial con-
In in vivo PDT applications, the photodegradation of the pho-
tosensitizer plays an important role. Indeed, any PS with a high
stability can turn out to be highly phototoxic for a prolonged time
m

S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
4851
after the treatment, particularly when it is localized in the skin
3. Conclusion
where sunlight can produce serious damage. On the other hand,
a PS quickly decomposed by light can hardly be applied in therapy,
as its efficacy could end before complete treatment of the diseased
tissue. For this reason we have evaluated the stability of group C
BODIPYs, those producing a good amount of 1O2, and, for the sake of
comparison, some analogues of the two other groups. Thus the
stability of the series of compounds 5aec, 7aec, and 11aec were
The data presented in this work allows us to choose the reagents
to synthesize BODIPYs suitable either for diagnostic purposes or for
photodynamic therapy in which the fundamental features are de-
termined by the presence of peculiar groups or atoms on the 2,6
positions. These characteristics can be further finely tuned by in-
troducing aromatic rings with electron-withdrawing or electron-
donating effects. In particular, the polarity of the BODIPY frame
can be increased or diminished according to the groups hanging
from the meso-aryl substituent, thus making these molecules more
or less prone to cell penetration and, possibly, also with a particular
internal distribution in the various cell organelles. We have recently
demonstrated that a few of the molecules here reported have
shown interesting photosensitizing capabilities against human
colorectal carcinoma (HCT116) cells in in vitro assays.12 Further-
more compound 11c can be alkylated on the pyridyl nitrogen thus
affording cationic BODIPYs, which have been found to be suitable
for a photo induced killing effect against bacteria.25 The systematic
study of these kind of molecules can bring further insight to the
development of new photosensitizers suitable for both cancer cell
killing or the eradication of localized infections.
studied irradiating an aqueous 10 mM solution of each BODIPY with
the green LED and recording the absorbance every 15 min. The
results were plotted (Abs vs time) and the values of half-
degradation time evaluated with an exponential equation. As re-
ported in Fig. 4 for a selected example with BODIPYs 7aec, it is
evident that the degradation process rapidly occurs at high con-
centrations, whereas it slows down when the majority of the PS is
already decomposed, thus indicating that a bimolecular process
occurs under irradiation. It must be considered that the PS con-
centrations used in these analyses are much higher than those used
in in vitro PDT experiments because of the necessity to start with
the absorbance values at about 0.5e0.6 for each compound. The
average half-degradation time is 30 min for group A BODIPYs, and
50e60 min for the compounds of both group B and C, thus we
decide to lengthen the time of irradiation to 120 min, as the cell
killing experiments rely on the presence, although it could be in
low percentage, of intact PS up to the end of the irradiation period.
4. Experimental section
4.1. General
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7a
7b
7c
IR spectra were measured on a Nicolet Avatar 360 FT-IR. 1H NMR
and 13C NMR spectra were recorded on a Bruker 400 MHz spec-
trometer in CDCl3 or DMSO-d6; chemical shifts are expressed in
parts per million relative to chloroform (7.28) and are reported as s
(singlet), d (doublet), t (triplet), m (multiplet), br s (broad singlet).
Mass spectrometric measurements were performed on a Bruker
Esquire 3000 Plus. Elemental analyses were performed on a Ther-
moQuest NA 2100, C, H, N analyzer, equipped with an electronic
mass flow control and thermal conductivity detector. Merck 60 F254
silica gel precoated sheets (0.2 mm thick) were used for analytical
thin-layer chromatography (TLC). Silica gel 60 (70e230 mesh,
Merck) was used for column chromatography. Aromatic aldehydes,
2,4-dimethyl-3-ethylpyrrole, and 2,4-dimethylpyrrole were com-
mercial products (SigmaeAldrich) and used as received. Dichloro-
methane used for BODIPY synthesis was distilled from CaCl2
directly into the reaction flask. HPLC analyses were conducted with
an Agilent 1100 Series instrument coupled with a diode array de-
tector. The instrument was fitted with a 250ꢂ4.6 mm column
(Supelco, Ascentis) packed with C-18 reversed-phase particles
0
20
40
60
80
100
120
time (min)
Fig. 4. Plot of photodegradation rate of the 7aec BODIPYs.
2.4. In vitro preliminary evaluation of the photodynamic
activity
The BODIPYs of group C were tested to study their phototoxic
activity against ovarian carcinoma SKOV3 cells. For this purpose, cell
cultures were treated with increasing concentrationsof the BODIPYs.
After 24 h of dark incubation, they were irradiated with a green LED
device for 2 h (fluence 33J/cm2) and theefficacyof the treatment was
determined with the MTT assay. The intrinsic effects of the PSs were
assessed by omitting the irradiation step from the treatment pro-
(5 mm) and operated with an isocratic elution with MeCN at 0.5 mL/
min. The green LED array is composed of 12ꢂ3 W diodes (lmax of
emission at 525 nm, width at half maximum of 70 nm), placed on
an 11 cm diameter disk, and equipped with a heatsinker. The
electric supply was ensured by a 50 W current transformer. This
array was placed above the plate at such a distance as to produce
a homogeneous irradiated area with a fluence rate (irradiance) of
tocol; it was found negligible in all cases up to 5 mM, thus indicating
photoactivation to be an absolute requirement for the activity of
these molecules. The 13 compounds of group C were found to be
particularly active (IC50 values in the nM range), however the one
featuring the hydrogen on the meso position was less active (13c,
IC50¼28.75 nM) with respect to the meso-aryl derivatives
(0.65<IC50<8.03 nM). From these results it is understood that the
aromatic ring positioned in the 8 position of the dipyrrolylmethene
skeleton exerts an important effect on the BODIPYs activity. In par-
ticular, compounds 8c and 2c showed interestingly high photody-
namic efficiencies as the IC50 values were found 0.772 and 0.650 nM,
respectively. To emphasize this, we are currently evaluating the re-
lationship between chemical structure and photodynamic activity of
these molecules with QSAR studies in order to plan the synthesis of
new structure with, possibly, higher PDT efficacy.
1.31ꢂ10ꢁ4 W/cm2 at 525 nm (
l of maximum emission), as de-
termined with an LI-COR 1800 spectroradiometer.
4.2. Synthesis
General comment on BODIPY synthesis. The acid catalyzed
condensation step between pyrroles and aromatic aldehydes al-
ways produces the compound lacking the aromatic ring in variable
yields ranging from 3 to 7%. This arises from the loss of the aromatic
moiety from the aldehyde carbon instead of the hydrogen in the
oxidation process from dipyrrolylmethane to the corresponding
methene.

4852
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
As an example, the reaction of 3-ethyl-2,4-dimethylpyrrole with
533.01 calcd for C21H22BF2IN2O2 510.12; HPLC: retention time-
3-iodo-4,5-dimethoxybenzaldehyde gives 2,6-diethyl-1,3,5,7-
tetramethyl-8-H-4,40-difluoroboradiazaindacene (compound 13a)
as byproduct of 1a.
¼704800 (99.8%).
Compound 13b, mp 214 ꢀC; EA: calcd for C13H15BF2N2: C, 62.94;
H, 6.09; N, 11.29; found: C, 63.02; H, 6.10; N, 11.33; UV ꢁ vis
:
ðCH2Cl2Þ
2,6-Diiodo-1,3,5,7-tetramethyl-8-H-4,40-difluoroboradiazainda-
cene (13c),12a 2,6-diethyl-1,3,5,7-tetramethyl-8-(4-methoxyphe-
nyl)-4,40-difluoroboradiazaindacene (2a),12b 1,3,5,7-tetramethyl-8-
(4-methoxyphenyl)-4,40-difluoroboradiazaindacene (2b),12b 2,6-
diiodo-1,3,5,7-tetramethyl-8-(4-methoxyphenyl)-4,40-difluorobor-
adiazaindacene (2c),12b 2,6-diethyl-1,3,5,7-tetramethyl-8-(4-hyd-
roxyphenyl)-4,40-difluoroboradiazaindacene (4a),12b 1,3,5,7-tetra-
methyl-8-(4-hydroxyphenyl)-4,40-difluoroboradiazaindacene (4b),12b
2,6-diethyl-1,3,5,7-tetramethyl-8-(3-hydroxyphenyl)-4,40-difluorob-
oradiazaindacene (5a),12b 1,3,5,7-tetramethyl-8-(3-hydroxyphenyl)-
4,40-difluoroboradiazaindacene (5b),12b 1,3,5,7-tetramethyl-8-(4-pyr-
idyl)-4,40-difluoroboradiazaindacene (11b),25 2,6-diiodo-1,3,5,7-
506 nm (
3
¼115,000); IR (KBr, cmꢁ1) 2968 (CH), 2920 (CH), 2865
(CH), 1529, 1184, 974; 1H NMR (CDCl3)
d
2.17 (s, 6H, 2ꢂ CH3), 2.47 (s,
6H, 2ꢂ CH3), 5.96 (s, 1H), 6.95 (s, 2H); 13C NMR (CDCl3)
d 14.65,
127.29,134.01,137.63,139.78,154.01; MS (ESI): MNaþ found: 270.96
calcd for C13H15BF2N2 248.08; HPLC: retention time¼603100 (100%).
4.2.3. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(3-iodo-4,5-dimethoxyphe-
nyl)-4,40-difluoroboradiaza
indacene
(1c). Compound
1b
(0.069 mmol), I2 (0.138 mmol), and HIO3 (0.138 mmol) were dis-
solved in EtOH (5 mL). This mixture was stirred for 15e18 h at rt.
The solution was then washed with water and extracted three
times with CH2Cl2, the organic phase was dried over Na2SO4, and
evaporated to dryness. The crude product was purified by column
chromatography (SiO2, petroleum ether/CH2Cl2, 1:1) affording
28 mg (yield: 53%) of 1c as red needles, mp>300 ꢀC; EA: calcd for
C21H20BF2I3N2O2 C, 33.10; H, 2.65; N, 3.68; found: C, 33.21; H, 2.67;
tetramethyl-8-(4-pyridyl)-4,40-difluoroboradiazaindacene
(11c),25
were synthesized as previously described.
4.2.1. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(3-iodo-4,5-dimethoxyphe-
nyl)-4,40-difluoroboradiaza indacene (1a). 3-Iodo-4,5-dimethoxy-
benzaldehyde (1.5 mmol) and 2,4-dimethyl-3-ethylpyrrole
(3.3 mmol) were dissolved in absolute CH2Cl2 (30 mL) under N2
atmosphere, 10 drops of TFA were added and the solution was
stirred at rt overnight or until TLC analysis showed complete con-
sumption of the aldehyde. At this time of DDQ (2.5 mmol) were
added and stirring continued for 20 min. Then, Et3N (4 mL) and
BF3$OEt2 (4 mL) were added. The mixture was stirred for 12 h then
the organic layer containing the crude product was subsequently
washed three times with water; the organic solution was dried
over Na2SO4, and evaporated to dryness. The raw material was
chromatographed (SiO2, petroleum ether/CH2Cl2, 6:4) affording
280 mg (yield: 33%) of 1a in the form of orange needles, mp
220 ꢀC. EA: calcd for C25H30BF2IN2O2: C, 53.03; H, 5.34; N, 4.95;
N, 3.66; UV ꢁ vis
: 538 nm (
3
¼89,300); IR (KBr, cmꢁ1) 2922
ðCH2Cl2Þ
(CH), 2851 (CH), 1530, 1174, 994, 524 (CI); 1H NMR (CDCl3)
d 1.48 (s,
6H, 2ꢂ CH3), 2.58 (s, 6H, 2ꢂ CH3), 3.76 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 6.70 (d, J¼2 Hz, 1H), 7.21 (d, J¼2 Hz, 1H); 13C NMR (CDCl3)
d
15.09, 56.09, 55.42, 87.69, 88.14, 115.97, 135.38, 138.10, 138.68,
153.15, 154.48, 155.66; MS (ESI): Mꢁ1þ found: 760.74 calcd for
C21H20BF2I3N2O2 761.92; HPLC: retention time¼1500400 (99.6%).
4.2.4. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(3-methoxyphenyl)-4,40-di-
fluoroboradiazaindacene (3a). 2,4-Dimethyl-3-ethylpyrrole (3.3
mmol) and 3-methoxybenzaldehyde (1.5 mmol) were made to react
as described for compound 1a. The raw material was purified by
column chromatography (SiO2, petroleum ether/CH2Cl2, 6:4)
affording 236 mg (yield: 38%) of 3a as orange needles, mp 184 ꢀC;
EA: calcd for C24H29BF2N2O C, 70.25; H, 7.12; N, 6.83; found: C, 70.48;
found: C, 53.47; H, 5.38; N, 4.92; UV ꢁ vis
: 528 nm
ðCH2Cl2Þ
(
3
¼92,600); IR (KBr, cmꢁ1) 2934 (CH), 2862 (CH), 1526, 1172, 988,
H, 7.13; N, 6.81; UV ꢁ vis
: 524 nm (
3
¼84,300); IR (KBr, cmꢁ1
)
ðCH2Cl2Þ
524 (CI); 1H NMR (CDCl3)
2ꢂ CH3), 2.32 (q, 4H, J¼8 Hz, 2ꢂ CH2), 2.55 (s, 6H, 2ꢂ CH3), 3.76 (s,
d
1.00 (t, 6H, J¼8 Hz, 2ꢂ CH3), 1.35 (s, 6H,
2976 (CH), 2922 (CH), 2854 (CH), 1528, 1179, 992, 754; 1H NMR
(CDCl3)
d
1.01 (t, 6H, J¼7.5 Hz, 2ꢂ CH3), 1.37 (s, 6H, 2ꢂ CH3), 2.33 (q,
3H, OCH3), 3.84 (s, 3H, OCH3), 6.75 (br s, 1H), 7.25 (br s, 1H); 13C
4H, J¼7.5 Hz, 2ꢂ CH2), 2.55 (s, 6H, 2ꢂ CH3), 3.84 (s, 3H, OCH3), 6.85
NMR (CDCl3)
d 12.12, 12.50, 15.23, 17.39, 56.03, 55.21, 86.10, 117.22,
(m, 1H), 6.89 (d, J¼8 Hz, 1H), 7.02 (dd, J¼8, 3 Hz, 1H), 7.41 (t, J¼8 Hz,
128.53, 133.88, 138.00, 139.21, 153.76, 154.35, 155.72; MS (ESI):
MNaþ, found: 588.98 calcd for C25H30BF2IN2O2 566.23; HPLC: re-
tention time¼1002700 (98.8%).
1H); 13C NMR (CDCl3)
d 12.13,12.74,15.18,17.25, 56.04, 116.19,128.01,
128.59,131.43,133.02,138.59,139.69,152.99,155.15; MS (ESI): Mꢁ1þ
found: 409.11 calcd for C24H29BF2N2O 410.31; HPLC: retention
time¼905000 (98.4%).
Compound 13a, mp 204 ꢀC; EA: calcd for C17H23BF2N2: C, 67.12;
H, 7.62; N, 9.21; found: C, 67.54; H, 7.65; N, 9.30; UV ꢁ vis
:
ðCH2Cl2Þ
526 nm (
3
¼133,000); IR (KBr, cmꢁ1) 2956 (CH), 2924 (CH), 2839
4.2.5. 1,3,5,7-Tetramethyl-8-(3-methoxyphenyl)-4,40-difluoroboradi-
azaindacene (3b). 3-Methoxybenzaldehyde (1.5 mmol) and 2,4-
dimethylpyrrole (3.3 mmol) were made to react as described for
compound 1a. The raw material was purified by column chroma-
tography (SiO2, petroleum ethereCH2Cl2, 1:1) affording 131 mg
(yield: 25%) of 3b as orange needles, mp 180 ꢀC; EA: calcd for
C20H21BF2N2O C, 67.82; H, 5.98; N, 7.91; found: C, 67.99; H, 6.00; N,
(CH),1526,1172, 988; 1H NMR (CDCl3)
d
1.00 (t, 6H, J¼8 Hz, 2ꢂ CH3),
2.18 (s, 6H, 2ꢂ CH3), 2.39 (q, 4H, J¼8 Hz, 2ꢂ CH2), 2.54 (s, 6H, 2ꢂ
CH3), 6.96 (s, 1H); 13C NMR (CDCl3)
d 12.02, 12.61, 14.88, 16.99,
131.29, 134.00, 137.13, 139.52, 153.21; MS (ESI): MNaþ, found:
327.00 calcd for C17H23BF2N2 304.19; HPLC: retention time¼801900
(99.58%).
7.91; UV ꢁ vis
:
500 nm (
3
¼62,200); IR (KBr, cmꢁ1) 2928
ðCH2Cl2Þ
4.2.2. 1,3,5,7-Tetramethyl-8-(3-iodo-4,5-dimethoxyphenyl)-4,40-di-
fluoroboradiazaindacene (1b). 3-Iodo-4,5-dimethoxybenzaldehyde
(1.5 mmol) and 2,4-dimethylpyrrole (3.3 mmol) were reacted as
described for compound 1a. The raw material was chromato-
graphed (SiO2, petroleum ether/CH2Cl2, 1:1) affording 140 mg
(yield: 18%) of 1b as orange needles, mp 218 ꢀC; EA: calcd for
C21H22BF2IN2O2 C, 49.44; H, 4.35; N, 5.49; found: C, 49.08; H, 4.36;
(CH), 2841 (CH), 1526, 1183, 998, 752; 1H NMR (CDCl3)
d 1.47 (s, 6H,
2ꢂ CH3), 2.57 (s, 6H, 2ꢂ CH3), 3.84 (s, 3H, OCH3), 6.00 (s, 2H), 6.85
(br s, 1H), 6.89 (d, J¼8 Hz, 1H), 7.02 (dd, J¼8, 2 Hz, 1H), 7.41 (t,
J¼8 Hz, 1H); 13C NMR (CDCl3)
d 14.79, 55.55, 114.75, 121.08, 121.36,
127.85, 129.48, 141.81, 143.39, 155.66, 158.64; MS (ESI): Mꢁ1þ
found: 353.28 calcd for C20H21BF2N2O 354.20; HPLC: retention
time¼702800 (97.2%).
N, 5.53; UV ꢁ vis
: 504 nm (
3
¼109,800); IR (KBr, cmꢁ1) 2946
ðCH2Cl2Þ
(CH), 2854 (CH), 1534, 1184, 990, 524 (CI); 1H NMR (CDCl3)
d
1.43 (s,
4.2.6. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(3-methoxyphenyl)-4,40-di-
fluoroboradiazaindacene (3c). Compound 3b (0.127 mmol), I2
(0.254 mmol), and HIO3 (0.254 mmol) were made to react as de-
scribed for compound 1c. The crude product was purified by col-
umn chromatography (SiO2, petroleum ethereCH2Cl2, 1:1)
6H, 2ꢂ CH3), 2.48 (s, 6H, 2ꢂ CH3), 3.76 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 5.92 (s, 2H), 6.75 (d, J¼3 Hz, 1H), 7.23 (d, J¼3 Hz, 1H); 13C
NMR (CDCl3)
d 14.78, 56.03, 55.38, 87.29, 116.32, 130.22, 135.22,
138.10, 138.98, 153.26, 154.61, 155.29; MS (ESI): MNaþ, found:

S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
4853
affording 33 mg (yield: 43%) of 3c as red needles, mp 209 ꢀC; EA:
described for compound 1a. At the end of the reaction the organic
layer was treated as above. The raw material was chromatographed
on silica gel (petroleum ethereCH2Cl2, 1:1) affording 270 mg (yield:
41%) of 6b as orange needles, mp 252 ꢀC; EA: calcd for
C24H23BF2N2O C, 71.30; H, 5.73; N, 6.93; found: C, 70.97; H, 5.68; N,
calcd for C20H19BF2I2N2O C, 39.64; H, 3.16; N, 4.62; found: C, 40.01;
H, 3.18; N, 4.65; UV ꢁ vis
: 534 nm (
3
¼61,500); IR (KBr, cmꢁ1
)
ðCH2Cl2Þ
2922 (CH), 2850 (CH), 1533, 1177, 991, 753; 524 (CI); 1H NMR
(CDCl3)
d
1.38 (s, 6H, 2ꢂ CH3), 2.57 (s, 6H, 2ꢂ CH3), 3.74 (s, 3H,
OCH3), 6.71 (m, 1H), 6.76 (d, J¼4 Hz, 1H), 6.97 (dd, J¼8, 2 Hz, 1H),
6.89; UV ꢁ vis
: 504 nm (
3
¼103,300); IR (KBr, cmꢁ1) 2956
ðCH2Cl2Þ
7.35 (t, J¼8 Hz, 1H); 13C NMR (CDCl3)
d
15.23, 55.67, 86.35, 115.01,
(CH), 2852 (CH), 1537, 1506, 1187, 1153, 977; 1H NMR (CDCl3)
d 1.11
122.12, 122.39, 127.36, 141.02, 143.25, 155.99, 158.72; MS (ESI):
Mꢁ1ꢁ found: 604.86 calcd for C20H19BF2I2N2O 605.99; HPLC: re-
tention time¼1402700 (99.1%).
(s, 6H, 2ꢂ CH3), 2.59 (s, 6H, 2ꢂ CH3), 4.07 (s, 3H, OCH3), 5.91 (s, 2H),
6.90 (d, J¼9 Hz,1H), 7.25 (d, J¼9 Hz,1H), 7.42 (td, J¼9, 2 Hz,1H), 7.49
(td, J¼9, 2 Hz,1H), 7.70 (d, J¼9 Hz,1H), 8.31 (d, J¼9 Hz,1H); 13C NMR
(CDCl3)
d 11.61, 13.11, 55.82, 102.32, 103.57, 116.39, 117.91, 120.36,
4.2.7. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(4-hydroxy-3,5-diiodophenyl)-
4,40-difluoroboradiaza indacene (4c). Compound 4b (0.088 mmol), I2
(0.166 mmol), and HIO3 (0.166 mmol) were made to react as de-
scribed for compound 1c. The crude product was purified by column
chromatography (SiO2, petroleum ethereCH2Cl2, 4:6) affording
45 mg (yield: 61%) of 4c as red needles, mp>300 ꢀC; EA: calcd for
C19H15BF2I4N2O C, 27.05; H,1.79; N, 3.32; found: C, 26.93; H,1.78%; N,
124.41, 124.27, 127.61, 128.32, 132.56, 134.51, 138.66, 138.94, 153.68,
155.98; MS (ESI): Mꢁ1þ found: 403.17 calcd for C24H23BF2N2O
404.26; HPLC: retention time¼805900 (94.0%).
4.2.11. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(4-methoxynaphthalene)-
4,40-difluoroboradiaza indacene (6c). Compound 6b (0.074 mmol),
I2 (0.148 mmol), and HIO3 (0.148 mmol) were made to react as
described for compound 1c. The crude product was recovered by
filtration then crystallized out of MeOH affording 30 mg (yield:
62%) of 6c as red needles, mp>300 ꢀC; EA: C, 43.94; H, 3.23; N, 4.27;
3.30; UV ꢁ vis
: 532 nm (
3
¼73,700); IR (KBr, cmꢁ1) 3450 (OH),
ðCH2Cl2Þ
2922 (CH), 1535, 1305, 1172, 993, 524 (CI); 1H NMR (CDCl3)
d 1.54 (s,
6H, 2ꢂ CH3), 2.63 (s, 6H, 2ꢂ CH3), 7.61 (s, 2H); 13C NMR (CDCl3)
d
15.02, 81.66, 85.02, 121.33, 127.31, 129.66, 141.99, 142.88, 155.71,
found: C, 43.90; H, 3.22; N, 4.28; UV ꢁ vis
: 538 nm
ðCH2Cl2Þ
156.32; MS (ESI): Mꢁ1ꢁ found: 842.52 calcd for C19H15BF2I4N2O
(
3
¼72,000); IR (KBr, cmꢁ1) 2956 (CH), 2852 (CH), 1529, 1305, 1172,
843.76; HPLC: retention time¼1305000 (100.0%).
994; 769, 524 (CI); 1H NMR (CDCl3)
d
1.07 (s, 6H, 2ꢂ CH3), 2.60 (s,
6H, 2ꢂ CH3), 4.01 (s, 3H, OCH3), 6.83 (d, J¼8 Hz, 1H), 7.17 (d, J¼8 Hz,
4.2.8. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(3-hydroxy-4-iodophenyl)-
4,40-difluoroboradiaza indacene (5c). Compound 5b (0.088 mmol),
I2 (0.166 mmol), and HIO3 (0.166 mmol) were made to react as
described for compound 1c. The crude product was purified by
column chromatography (SiO2, petroleum ethereCH2Cl2, 6:4)
affording 40 mg (yield: 63%) of 5c as red needles, mp>300 ꢀC; EA:
calcd for C19H16BF2I3N2O C, 31.79; H, 2.25; N, 3.90; found: C, 32.00;
1H), 7.38 (td, J¼8, 1 Hz, 1H), 7.42 (td, J¼8, 1 Hz, 1H), 7.55 (d, J¼8 Hz,
1H), 8.27 (d, J¼8 Hz, 1H); 13C NMR (CDCl3)
d 11.79, 14.16, 55.61,
84.98, 103.14, 104.01, 116.32, 117.80, 121.21, 124.56, 124.95, 127.32,
132.11, 134.63, 138.79, 138.38, 153.57, 155.26; MS (ESI): Mꢁ1þ
found: 654.93 calcd for C24H21BF2I2N2O 656.05; HPLC: retention
time¼2004400 (99.7%).
H, 2.27; N, 3.92; UV ꢁ vis
:
536 nm (
cmꢁ1) 3481 (OH), 2961 (CH), 2927 (CH), 2869 (CH),1538,1474,1190,
977, 526 (CI); 1H NMR (CDCl3)
1.42 (s, 6H, 2ꢂ CH3), 2.56 (s, 6H, 2ꢂ
3
¼67,700); IR (KBr,
4.2.12. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(4,7-dimethoxynaphthale-
ðCH2Cl2Þ
ne)-4,40-difluoroboradiaza
indacene
(7a). 4,7-Dimethoxy-1-
d
naphthaldehyde (1.5 mmol) and 2,4-dimethyl-3-ethylpyrrole
(3.3 mmol) were made to react as described for compound 1a. At
the end of the reaction the organic layer was treated as above. The
CH3), 5.54 (s, 1H, OH), 6.52 (dd, J¼8, 2 Hz, 1H), 6.82 (d, J¼2 Hz, 1H);
7.74 (d, J¼8 Hz, 1H); 13C NMR (CDCl3)
d 14.78, 81.98, 85.74, 115.33,
124.78, 130.96, 136.35, 139.77, 143.09, 153.71, 156.32; MS (ESI):
Mꢁ1ꢁ found: 716.67 calcd for C19H16BF2I3N2O 717.86; HPLC: re-
tention time¼1202100 (97.2%).
raw
material
was
chromatographed
(SiO2,
petroleum
ethereCH2Cl2, 7:3) affording 124 mg (yield: 17%) of 7a as orange
needles, mp 280 ꢀC; EA: calcd for C29H33BF2N2O2 C, 71.03; H, 6.78;
N, 5.71; found: C 71.37; H, 6.83, N, 5.73; UV ꢁ vis
: 528 nm
ðCH2Cl2Þ
4.2.9. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(4-methoxynaphthalene)-
(
3
¼83,100); IR (KBr, cmꢁ1) 2962 (CH), 2926 (CH), 2869 (CH), 1536,
4,40-difluoroboradiaza
indacene
(6a). 4-Methoxy-1-naphthal-
1316, 1187, 976; 1H NMR (CDCl3)
d
0.87 (t, 6H, J¼8 Hz, 2ꢂ CH3), 1.02
dehyde (1.5 mmol) and 2,4-dimethyl-3-ethylpyrrole (3.3 mmol)
were made to react as described for compound 1a. After stirring for
12 h, the organic layer containing the crude product was washed
with a solution of HCl (10%), two times with water and one time
with NaCl saturated solution (Brine); the organic solution was dried
over Na2SO4 and evaporated to dryness. The raw material was
chromatographed on silica gel (petroleum ethereCH2Cl2, 6:4)
affording 280 mg (yield: 41%) of 6a as orange needles, mp 241 ꢀC;
EA: calcd for C28H31BF2N2O C, 73.05; H, 6.79; N, 6.09; found: C,
(s, 6H, 2ꢂ CH3), 2.19 (q, 4H, J¼8 Hz, 2ꢂ CH2), 2.50 (s, 6H, 2ꢂ CH3),
3.66 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 6.70 (d, J¼8 Hz, 1H), 6.91 (d,
J¼3 Hz, 1H), 7.06 (dd, J¼9, 3 Hz, 1H), 7.13 (d, J¼8 Hz, 1H), 8.15 (d,
J¼9 Hz, 1H); 13C NMR (CDCl3)
d 11.41, 12.70, 14.79, 17.18, 55.68,
55.71, 102.21, 103.88, 117.71, 120.88, 124.08, 124.40, 127.35, 131.85,
132.62, 134.54, 138.46, 139.17, 153.64, 156.41, 159.16; MS (ESI):
MNaþ found: 513.12 calcd for C29H33BF2N2O2 490.39; HPLC: re-
tention time¼1102400 (98.6%).
72.88; H, 6.80; N, 6.10; UV ꢁ vis
:
528 nm (
(KBr, cmꢁ1) 2964 (CH), 2854 (CH), 1538, 1502, 1189, 977; 1H NMR
(CDCl3)
3
¼80,500); IR
4.2.13. 1,3,5,7-Tetramethyl-8-(4,7-dimethoxynaphthalene)-4,40-di-
fluoroboradiazaindacene (7b). 4,7-Dimethoxy-1-naphthaldehyde
(1.5 mmol) and 2,4-dimethyl-3-ethylpyrrole (3.3 mmol) were
made to react as described for compound 1a. At the end of the
reaction the organic layer was treated as above. The raw material
was purified by column chromatography (SiO2, petroleum ether/
CH2Cl2, 4:6) affording 124 mg (yield: 19%) of 7b as orange needles,
mp 234 ꢀC; EA: calcd for C25H25BF2N2O2 C, 69.14; H, 5.80; N, 6.45;
ðCH2Cl2Þ
d
0.87 (t, 6H, J¼8 Hz, 2ꢂ CH3), 0.93 (s, 6H, 2ꢂ CH3), 2.18 (q,
4H, J¼8 Hz, 2ꢂ CH2), 2.48 (s, 6H, 2ꢂ CH3), 4.01 (s, 3H, OCH3), 6.82 (d,
J¼9 Hz, 1H), 7.19 (t, J¼9 Hz, 1H), 7.35 (t, J¼9, 2 Hz, 1H), 7.41 (t, J¼9,
2 Hz, 1H), 7.62 (d, J¼9 Hz, 1H), 8.23 (d, J¼9 Hz, 1H); 13C NMR (CDCl3)
d
11.56, 13.01, 14.96, 16.85, 55.70, 101.93, 103.76, 116.21, 117.88,
120.52, 124.29, 124.02, 127.66, 131.32, 132.47, 134.45, 138.79, 139.22,
153.63, 156.09; MS (ESI): Mꢁ1ꢁ found: 459.20 calcd for
C28H31BF2N2O 460.37; HPLC: retention time¼1201200 (93.5%).
found:
C
68.99; H, 5.79; N, 6.48; UV ꢁ vis
:
504 nm
ðCH2Cl2Þ
(
3
¼94,300); IR (KBr, cmꢁ1) 2921 (CH), 2851 (CH), 1548, 1199, 1078,
976, 838; 1H NMR (CDCl3)
d
1.11 (s, 6H, 2ꢂ CH3), 2.52 (s, 6H, 2ꢂ
4.2.10. 1,3,5,7-Tetramethyl-8-(4-methoxynaphthalene)-4,40-difluoro-
boradiazaindacene (6b). 4-Methoxy-1-naphthaldehyde (1.5 mmol)
and 2,4-dimethylpyrrole (3.3 mmol) were made to react as
CH3), 3.66 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 5.85 (s, 2H), 6.70 (d,
J¼8 Hz, 1H), 6.89 (d, J¼3 Hz, 1H), 7.07 (dd, J¼9, 3 Hz, 1H), 7.12 (d,
J¼8 Hz, 1H), 8.16 (d, J¼9 Hz, 1H); 13C NMR (CDCl3)
d 11.39, 12.76,

4854
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
55.70, 55.76, 102.21, 103.79, 117.52, 120.81, 124.09, 124.78, 127.50,
128.85, 129.61, 134.39, 138.51, 139.46, 153.92, 156.16, 158.62; MS
(ESI): Mꢁ1þ found: 433.19 calcd for C25H25BF2N2O2 434.29; HPLC:
retention time¼801600 (99.4%).
Mꢁ1þ found: 642.79 calcd for C19H15BCl2F2I2N2 644.86; HPLC: re-
tention time¼503300 (92.5%).
4.2.18. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(2,3,4,5,6-pentafluorophe-
nyl)-4,40-difluoroboradiaza indacene (9a). 2,4-Dimethyl-3-ethyl-
pyrrole (3.3 mmol) and 2,3,4,5,6-pentafluorobenzaldehyde
(1.5 mmol) were made to react as described for compound 1a. The
raw material was chromatographed (SiO2, petroleum ethereCH2Cl2,
1:1), the fractions were collected to dryness and the product was
treated with cold methanol affording 112 mg (yield: 16%) of 9a as
orange needles, mp 206 ꢀC; EA: calcd for C23H22BF7N2 C, 58.75; H,
4.2.14. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(4,7-dimethoxynaphtha-
lene)-4,40-difluoroboradiaza
indacene
(7c). Compound
7b
(0.069 mmol), I2 (0.138 mmol), and HIO3 (0.138 mmol) were made
to react as described for compound 1c. The crude product was re-
covered by filtration then crystallized out of MeOH affording 30 mg
(yield: 63%) of 7c as red needles, mp>300 ꢀC; EA: calcd for
C25H23BF2I2N2O2 C, 43.77; H, 3.38; N, 4.08; found: C, 43.98; H, 3.41;
4.72; N, 5.96; found: C, 59.03; H, 4.76; N, 6.00. UV ꢁ vis
:
ðCH2Cl2Þ
534 nm (
3
¼56,800); IR (KBr, cmꢁ1) 2956 (CH), 2932 (CH), 2850 (CH),
N, 4.12; UV ꢁ vis
: 538 nm (
3
¼97,800); IR (KBr, cmꢁ1) 2965
ðCH2Cl2Þ
1548, 1468, 1180, 970; 1H NMR (CDCl3)
1.51 (s, 6H, 2ꢂ CH3), 2.33 (q, 4H, J¼8 Hz, 2ꢂ CH2), 2.54 (s, 6H, 2ꢂ CH3);
d
1.02 (t, 6H, J¼8 Hz, 2ꢂ CH3),
(CH), 2840 (CH), 1532, 1189, 984, 836, 524 (CI); 1H NMR (CDCl3)
d
1.11 (s, 6H, 2ꢂ CH3), 2.59 (s, 6H, 2ꢂ CH3), 3.64 (s, 3H, OCH3), 3.99
13C NMR (CDCl3)
d
14.78, 15.32, 16.59, 17.21, 131.25, 132.36, 134.54,
(s, 3H, OCH3), 6.71 (d, J¼8 Hz, 1H), 6.80 (d, J¼3 Hz, 1H), 7.09 (d,
140.26, 142.16 (dd, J¼248.9, 13.9 Hz), 143.39 (dd, J¼249.9, 15.1 Hz),
147.52 (dd, J¼241.3, 14.9 Hz), 155.11; MS (ESI): Mꢁ1þ found: 469.17
calcd for C23H22BF7N2 470.23; HPLC: retention time¼804100 (96.0%).
J¼3 Hz, 1H), 7.11 (d, J¼3 Hz, 1H), 8.18 (d, J¼8 Hz, 1H); 13C NMR
(CDCl3)
d 11.93, 12.62, 55.68, 55.75, 86.11, 102.60, 103.09, 117.42,
120.57, 124.11, 124.62, 127.31, 129.19, 134.62, 138.38, 139.45, 154.31,
155.81, 158.20; MS (ESI): Mꢁ1þ found: 684.95 calcd for
C25H23BF2I2N2O2 686.08; HPLC: retention time¼1703200 (99.4%).
4.2.19. 1,3,5,7-Tetramethyl-8-(2,3,4,5,6-pentafluorophenyl)-4,40-di-
fluoroboradiazaindacene (9b). 2,3,4,5,6-Pentafluorobenzaldehyde
(1.5 mmol) and 2,4-dimethylpyrrole (3.3 mmol) were made to re-
act as described for compound 1a. The raw material was purified by
column chromatography (SiO2, petroleum ether/CH2Cl2, 7:3), the
fractions were collected to dryness and the product precipitated
from methanol affording 92 mg (yield: 15%) of 9b as green needles,
mp 220 ꢀC; EA: calcd for C19H14BF7N2 C, 55.10; H, 3.41; N, 6.76;
4.2.15. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(2,6-dichlorophenyl)-4,40-
difluoroboradiazaindacene (8a). 2,4-Dimethyl-3-ethylpyrrole (3.3
mmol) and 2,6-dichlorobenzaldehyde (1.5 mmol) were made to
react as described for compound 1a. The raw material was chro-
matographed (SiO2, petroleum ether/CH2Cl2, 1:1) affording 230 mg
(yield: 34%) of 8a as brown needles, mp 255 ꢀC; EA: calcd for
C23H25BCl2F2N2 C, 61.50; H, 5.61; N, 6.24; found: C 61.79; H, 5.64; N,
found: C, 54.87; H, 3.43; N, 6.70; UV ꢁ vis
:
518 nm
ðCH2Cl2Þ
(
3
¼61,500); IR (KBr, cmꢁ1) 2948 (CH), 2836 (CH), 1549, 1478, 1184,
¼72,100); IR (KBr, cmꢁ1) 2960
961; 1H NMR (CDCl3)
d
1.53 (s, 6H, 2ꢂ CH3), 2.49 (s, 6H, 2ꢂ CH3),
6.18; UV ꢁ vis
:
536 nm (
3
ðCH2Cl2Þ
(CH), 2924 (CH), 1545, 1186, 973, 788; 1H NMR (CDCl3)
d 0.93 (t, 6H,
5.98 (s, 2H); 13C NMR (CDCl3)
d 14.45, 16.67, 129.13, 132.22, 135.63,
J¼8 Hz, 2ꢂ CH3), 1.31 (s, 6H, 2ꢂ CH3), 2.25 (q, 4H, J¼8 Hz, 2ꢂ CH2),
139.13, 141.13 (dd, J¼249.6, 12.8 Hz), 144.13 (dd, J¼250.0, 15.2 Hz),
147.63 (dd, J¼243.4, 11.9 Hz), 155.02; MS (ESI): Mꢁ1þ found: 413.02
calcd for C19H14BF7N2 414.13; HPLC: retention time¼604800 (99.5%).
2.47 (s, 6H, 2ꢂ CH3), 7.29 (dd, J¼9, 7 Hz, 1H), 7.32-7.42 (m, 2H); 13
C
NMR (CDCl3)
d 14.95, 15.58, 16.35, 17.42, 128.41, 129.98, 130.12,
131.64, 132.21, 134.42, 144.26, 157.18; MS (ESI): Mꢁ1þ found: 447.09
calcd for C23H25BCl2F2N2 449.17; HPLC: retention time¼401700
(96.3%).
4.2.20. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(2,3,4,5,6-pentafluorophe-
nyl)-4,40-difluoroboradiaza indacene (9c). Iodine (0.170 mmol),
iodic acid (0.170 mmol), and compound 9b (0.085 mmol) were
made to react as reported for compound 1c. The crude product was
purified by column chromatography (SiO2, petroleum
ethereCH2Cl2, 1:1) affording 54 mg (yield: 96%) of 9c as red nee-
dles, mp 250 ꢀC; EA: calcd for C19H12BF7I2N2 C, 34.27; H, 1.82; N,
4.2.16. 1,3,5,7-Tetramethyl-8-(2,6-dichlorophenyl)-4,40-difluorobora-
diazaindacene (8b). 2,4-Dimethylpyrrole (3.3 mmol) and 2,6-
dichlorobenzaldehyde (1.5 mmol) were made to react as de-
scribed for compound 1a. The raw material was chromatographed
(SiO2, petroleum ethereCH2Cl2, 1:1) affording 257 mg (yield: 44%)
of 8b as brown needles, mp 255 ꢀC; EA: calcd for C19H17BCl2F2N2 C,
4.21; found: C, 34.08; H, 1.81; N, 4.18; UV ꢁ vis
: 548 nm
ðCH2Cl2Þ
(
3
¼50,500); IR (KBr, cmꢁ1) 2948 (CH), 2836 (CH), 1549, 1478, 1184,
961. 1H NMR (CDCl3)
d
1.56 (s, 6H, 2ꢂ CH3), 2.59 (s, 6H, 2ꢂ CH3); 13
C
58.06; H, 4.36; N, 7.13; found:
C 57.89; H, 4.38; N, 7.08;
UV ꢁ vis
: 512 nm (
3
¼91,100); IR (KBr, cmꢁ1) 2956 (CH), 2924
NMR (CDCl3) d 15.53, 16.12, 85.01, 131.62, 135.21, 138.11, 140.29 (dd,
ðCH2Cl2Þ
(CH), 2854 (CH), 1551, 1465, 1192, 976; 1H NMR (CDCl3)
d 1.41 (s, 6H,
J¼241.2, 11.2 Hz), 143.26 (dd, J¼251.2, 13.9 Hz), 146.32 (dd, J¼243.2,
12.8 Hz), 154.66; MS (ESI): Mꢁ1ꢁ found: 664.7 calcd for
C19H12BF7I2N2 665.92; HPLC: retention time¼1200800 (94.9%).
2ꢂ CH3), 2.50 (s, 6H, 2ꢂ CH3), 5.93 (s, 2H), 7.28 (d, J¼9, 7 Hz, 1H),
7.35e7.41 (m, 2H); 13C NMR (CDCl3)
d 15.69, 16.73, 127.32, 129.09,
130.58, 131.60, 132.72, 134.48, 144.36, 156.95; MS (ESI): Mꢁ1þ
found: 391.08 calcd for C19H17BCl2F2N2 393.07; HPLC: retention
time¼400800 (99.6%).
4.2.21. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(2,3,5,6-tetrafluoro-3-[(4-
methoxybenzyl)sulfanyl]phenyl)-4,40-difluoroboradiazaindacene
(10a). Compound 9a (0.128 mmol) was dissolved in anhydrous THF
(8 mL), and the solution was stirred under N2 for 5 min. After this
period K2CO3 (354 mg, 2.56 mmol) was added and the mixture was
4.2.17. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(2,6-dichlorophenyl)-4,40-
difluoroboradiazaindacene (8c). Compound 8b (0.237 mmol), I2
(0.474 mmol), and HIO3 (0.474 mmol) were made to react as de-
scribed for compound 1c. The crude product was purified by col-
umn chromatography (SiO2, petroleum ether/CH2Cl2, 1:1) affording
101 mg (yield: 66%) of 8c as red needles mp 210 ꢀC; EA: calcd for
C19H15BCl2F2I2N2 C, 35.39; H, 2.34; N, 4.34; found: C, 35.40; H, 2.34;
brought to reflux. A solution of 4-methoxy-a-toluenethiol (1 mL,
2.56 mmol), dissolved in anhydrous THF (9 mL), was then added
dropwise over 15 min and the reaction was kept under reflux for
30 min. After cooling, the mixture was evaporated under vacuum.
The crude product was purified by column chromatography (SiO2,
petroleum ether/CH2Cl2, 1:1), precipitated from methanol affording
52 mg (yield 67%) of 10a as red-purple needles, mp 170 ꢀC; EA:
calcd for C31H31BF6N2OS C, 61.60; H, 5.17; N, 4.63; found: C, 62.11;
N, 4.31; UV ꢁ vis
: 548 nm (
3
¼76,400); IR (KBr, cmꢁ1) 2965
ðCH2Cl2Þ
(CH), 2942 (CH), 2848 (CH), 1548, 1462, 1188, 970, 528 (CI); 1H NMR
(CDCl3)
1H), 7.37e7.45 (m, 2H); 13C NMR (CDCl3)
128.98, 130.40, 131.68, 132.95, 134.79, 144.15, 157.94; MS (ESI):
d
1.42 (s, 6H, 2ꢂ CH3), 2.58 (s, 6H, 2ꢂ CH3), 7.34 (d, J¼9, 7 Hz,
d
15.84, 16.37, 86.06,
H, 5.21; N, 4.66; UV ꢁ vis
:
532 nm (
3
¼60,200); IR (KBr,
ðCH2Cl2Þ
cmꢁ1) 2957 (CH), 2924 (CH), 2852 (CH), 1549, 1471, 1186, 969, 829;

S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
4855
1H NMR (CDCl3)
d
0.93 (t, 6H, J¼8 Hz, 2ꢂ CH3), 1.25 (s, 6H, 2ꢂ CH3),
C22H26BF2N3 C, 69.30; H, 6.87; N, 11.02; found: C 69.53; H, 6.88; N,
2.24 (q, 4H, J¼8 Hz, 2ꢂ CH2), 2.45 (s, 6H, 2ꢂ CH3), 3.70 (s, 3H, OCH3),
11.05; UV ꢁ vis
: 524 nm (
3
¼57,000); IR (KBr, cmꢁ1) 3032
ðCH2Cl2Þ
4.10 (s, 2H, SCH2), 6.98 (d, J¼9 Hz, 2H), 7.11 (d, J¼9 Hz, 2H); 13C NMR
(CH), 2963 (CH), 2920 (CH), 2848 (CH), 1577, 1486, 1161, 972; 1H
(CDCl3)
d
13.95, 14.72, 15.63, 16.89, 38.06, 55.45, 114.13, 116.12 (t,
NMR (CDCl3)
d
0.91 (t, 6H, J¼8 Hz, 2ꢂ CH3),1.21 (s, 6H, 2ꢂ CH3), 2.23
J¼21.1 Hz), 124.20, 127.89, 128.35, 130.68, 131.46, 140.56, 143.62 (dd,
J¼251.2, 15.9 Hz), 147.71 (dd, J¼248.0, 13.9 Hz), 157.23, 158.86; MS
(ESI): Mꢁ1þ found: 603.15 calcd for C31H31BF6N2OS 604.46; HPLC:
retention time¼1001400 (99.0%).
(q, 4H, J¼8 Hz, 2ꢂ CH2), 2.46 (s, 6H, 2ꢂ CH3), 7.48 (dd, J¼8, 5 Hz,1H),
7.60 (dt, J¼8, 2 Hz, 1H), 8.39 (d, J¼2 Hz, 1H), 8.71 (dd, J¼5, 2 Hz, 1H);
13C NMR (CDCl3)
d 14.76, 15.52, 16.01, 17.26, 123.12, 131.00, 131.65,
134.15, 138.89, 145.26, 150.96, 151.32, 156.35; MS (ESI): Mꢁ1þ
found: 380.04 calcd for C22H26BF2N3 381.27; HPLC: retention
time¼802700 (98.0%).
4.2.22. 1,3,5,7-Tetramethyl-8-(2,3,5,6-tetrafluoro-3-[(4-methoxyben-
zyl)sulfanyl]phenyl)-4,40-difluoroboradiazaindacene (10b). 4-Metho-
xy-
a
-toluenethiol (3 mmol) and 9b (0.15 mmol) were made to react
4.2.26. 1,3,5,7-Tetramethyl-8-(3-pyridyl)-4,40-difluoroboradiazainda-
cene (12b). 3-Pyridylcarbaldehyde (3 mmol) and 2,4-dimethyl-
pyrrole (6 mmol) were made to react as described for compound
1a. The raw material was purified by column chromatography
(SiO2, CH2Cl2), affording 75 mg (yield: 8%) of 11b as green needles,
mp 190 ꢀC; EA: calcd for C18H18BF2N3 C, 66.49; H, 5.58; N, 12.92;
as described for compound 10a. The crude product was purified by
column chromatography (SiO2, petroleum ether/CH2Cl2, 6:4),
crystallized out of methanol affording 63 mg (yield 76%) of 10b as
yellow needles, mp 188 ꢀC; EA: calcd for C27H23BF6N2OS C, 59.14; H,
4.23; N, 5.11; found: C, 59.49; H, 4.26; N, 5.12. UV ꢁ vis
:
ðCH2Cl2Þ
516 nm (
3
¼92,800); IR (KBr, cmꢁ1) 2978 (CH), 2934 (CH), 2850 (CH),
found: C, 66.38; H, 5.54; N, 13.05; UV ꢁ vis
: 504 nm
ðCH2Cl2Þ
1552,1465,1178, 971; 1H NMR (CDCl3)
d
1.45 (s, 6H, 2ꢂ CH3), 2.56 (s,
(
3
¼69,800); IR (KBr, cmꢁ1) 3028 (CH), 2969 (CH), 2840 (CH), 1571,
6H, 2ꢂ CH3), 3.78 (s, 3H, OCH3), 4.18 (s, 2H, SCH2), 6.03 (s, 2H), 6.76
1488, 1167, 984; 1H NMR (CDCl3)
d
: 1.31 (s, 6H, 2ꢂ CH3), 2.49 (s, 6H,
(d, J¼9 Hz, 2H), 7.18 (d, J¼9 Hz, 2H); 13C NMR (CDCl3)
d
13.59, 14.87,
2ꢂ CH3), 5.94 (s, 2H), 7.38 (dd, J¼8, 5 Hz,1H), 7.57 (dt, J¼8, 2 Hz,1H),
38.01, 55.44, 114.09, 115.83 (t, J¼20.4 Hz), 122.20, 124.08, 128.23,
130.08, 130.98, 141.84, 143.51 (dd, J¼250.4, 16.1 Hz), 147.63 (dd,
J¼247.9, 13.8 Hz), 157.62, 159.27; MS (ESI): Mꢁ1þ found: 547.10
calcd for C27H23BF6N2OS 548.35; HPLC: retention time¼705000
(98.3%).
8.50 (d, J¼2 Hz, 1H), 8.68 (dd, J¼5, 2 Hz, 1H); 13C NMR (CDCl3)
d
14.96, 15.78, 122.85, 129.63, 131.93, 133.68, 137.32, 146.24, 150.21,
150.74, 156.21; MS (ESI): Mꢁ1þ found: 324.05 calcd for
C18H18BF2N3 325.16; HPLC: retention time¼604000 (99.7%).
4.2.27. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(3-pyridyl)-4,40-difluoro-
boradiazaindacene (12c). Iodine (0.435 mmol), iodic acid
(0.435 mmol), and compound 12b (0.217 mmol) were made to react
as reported for compound 1c. The crude product was purified by
column chromatography (SiO2, CHCl3) affording 100 mg (yield:
79%) of 12c as purple needles, mp>300 ꢀC; EA: calcd for
C18H16BF2I2N3 C, 37.47; H, 2.80; N, 7.28 found: C, 37.56; H, 2.82; N,
4.2.23. 2,6-Diiodo-1,3,5,7-tetramethyl-8-(2,3,5,6-tetrafluoro-3-[(4-
methoxybenzyl)sulfanyl]phenyl)-4,40-difluoroboradiazaindacene
(10c). 4-Methoxy-a-toluenethiol (0.6 mmol) and 9c (0.03 mmol)
were made to react as described for compound 10a. The crude
product was purified by column chromatography (SiO2, petroleum
ethereCH2Cl2, 1:1) affording 15 mg (yield 63%) of 6 as red needles,
mp 135 ꢀC; EA: calcd for C27H21BF6I2N2OS C, 40.53; H, 2.65; N, 3.50;
7.32. UV ꢁ vis
: 538 nm (
3
¼67,100); IR (KBr, cmꢁ1) 3021 (CH),
ðCH2Cl2Þ
found: C, 40.59; H, 2.65; N, 3.51; UV ꢁ vis
:
546 nm
2962 (CH), 2839 (CH), 1581, 1498, 1176, 978, 528; 1H NMR (CDCl3)
ðCH2Cl2Þ
(
3
¼48,300); IR (KBr, cmꢁ1) 2962 (CH), 2842 (CH), 1559, 1480, 1161,
d
1.56 (s, 6H, 2ꢂ CH3), 2.59 (s, 6H, 2ꢂ CH3), 7.52 (dd, J¼8, 5 Hz, 1H),
970, 524 (CI); 1H NMR (CDCl3)
d
1.48 (s, 6H, 2ꢂ CH3), 2.56 (s, 6H, 2ꢂ
7.65 (dt, J¼8, 2 Hz, 1H), 8.58 (d, J¼2 Hz, 1H), 8.67 (dd, J¼5, 2 Hz, 1H);
CH3), 3.51 (s, 2H, SCH2), 3.72 (s, 3H, OCH3), 6.77 (d, J¼9 Hz, 2H), 7.10
13C NMR (CDCl3)
d 15.63, 16.23, 87.25, 123.95, 132.24, 133.59, 139.14,
(d, J¼9 Hz, 2H); 13C NMR (CDCl3)
d
13.69, 15.26, 38.10, 55.67, 87.11,
146.52, 151.58, 151.99, 155.21; MS (ESI): Mꢁ1þ found: 575.21 calcd
114.26, 115.71 (t, J¼20.1 Hz), 124.28, 128.01, 129.82, 130.53, 141.11
143.36 (dd, J¼249.3, 15.6 Hz), 147.14 (dd, J¼247.0, 12.9 Hz), 157.50,
159.15; MS (ESI): Mꢁ1þ found: 799.06 calcd for C27H21BF6I2N2OS
800.14; HPLC: retention time¼1502700 (92.3%).
for C18H16BF2I2N3 576.96; HPLC: retention time¼1100700 (99.1%).
4.3. Fluorescence properties and quantum yield of
fluorescence
4.2.24. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(4-pyridyl)-4,40-difluorobo-
Steady-state fluorescence spectroscopic studies were performed
on a Jasco FP-750 spectrofluorimeter. The slit width was 2.5 nm for
both excitation and emission. Values of absorbance, necessary for
the equation described below, were obtained for the same solutions
used in the fluorescence measurements on a Varian Cary 50 Scan
instrument. The relative quantum efficiencies of fluorescence of
each BODIPY derivative were obtained comparing the emission
spectra of the test sample with that of a 0.1 N NaOH solution of
fluorescein excited at 490 nm, whose quantum efficiency literature
radiazaindacene (11a). 2,4-Dimethyl-3-ethylpyrrole (620
mL,
6 mmol) and 4-pyridylcarbaldehyde (283 L, 3 mmol) were made to
m
react as described for compound 1a. The raw material was purified
by column chromatography (SiO2, CH2Cl2), affording 120 mg (yield:
17%) of 11a as red needles, mp 196 ꢀC; EA: calcd for C22H26BF2N3 C,
69.30; H, 6.87; N, 11.02; found: C 69.52; H, 6.91; N, 11.11;
UV ꢁ vis
: 524 nm (
3
¼71,400); IR (KBr, cmꢁ1) 3025 (CH), 2978
ðCH2Cl2Þ
1
(CH), 2922 (CH), 2856 (CH), 1575, 1490, 1170, 981; H NMR (CDCl3)
value is 0.85.20 The quantum efficiencies of fluorescence (
Ffluo) of
d
0.91 (t, 6H, J¼8 Hz, 2ꢂ CH3), 1.23 (s, 6H, 2ꢂ CH3), 2.22 (q, 4H,
J¼8 Hz, 2ꢂ CH2), 2.46 (s, 6H, 2ꢂ CH3), 7.23 (dd, J¼6, 1 Hz, 2H), 8.69
the BODIPYs were calculated according to the following equation
(Ifluo is the fluorescence intensity at the specific excitation wave-
length, Abs denotes the absorbance at the excitation wavelength)
(dd, J¼6, 1 Hz, 2H); 13C NMR (CDCl3)
d 15.21, 15.89, 16.45, 17.01,
124.13,130.39,131.52,132.12,138.42,144.26,151.12,156.81; MS (ESI):
Mꢁ1þ found: 380.15 calcd for C22H26BF2N3 381.27; HPLC: retention
time¼805200 (98.5%).
ꢀ
ꢁ
sample
fluo
standard
fluo
sample
standard
=I
fluo
fluo
F
¼
F
ꢂ
I
ꢀ
ꢁ
4.2.25. 2,6-Diethyl-1,3,5,7-tetramethyl-8-(3-pyridyl)-4,40-di-
ꢂ
Absstandard=Abssample
fluoroboradiazaindacene
(12a). 2,4-Dimethyl-3-ethylpyrrole
L, 3 mmol)
(620 L, 6 mmol) and 3-pyridylcarbaldehyde (283
m
m
4.4. Comparative singlet-oxygen generation measurements
were made to react as described for compound 1a. The raw material
was purified by column chromatography (SiO2, CH2Cl2), affording
120 mg (yield: 17%) of 12a as red needles, mp 196 ꢀC; EA: calcd for
A solution of aerated isopropanol, containing 50
mM of 1,3-
diphenylisobenzofuran (DPBF) and 2.5 M of photosensitizers
m

4856
S. Banfi et al. / Tetrahedron 69 (2013) 4845e4856
was prepared in a glass flask and kept in the dark; 2 mL of this
solution was transferred into a cuvette, placed in the UVevis in-
strument and irradiated, at rt, from the open top side with the
green LED source for the time required to observe the chemical
event (maximum 20 min). The DPBF absorbance decrease was
measured at 410 nm and at fixed time intervals, as defined for each
experiment. The rate of singlet oxygen production was determined
measuring the decrease in the intensity of DPBF absorbance
recorded over time. Irradiation of the DPBF-isopropanol solution in
the absence of photosensitizer (negative control) and a solution
Possible intrinsic (i.e., non photodynamic) cytotoxic effects of
the BODIPY were assessed on control treated as described above,
with BODIPY concentrations tenfold higher than those used for PDT
experiments, omitting cell irradiation.
IC50 (i.e., the concentration affecting 50% of cells) values were
obtained by nonlinear regression analysis, using the GraphPad
PRISM 3.03 software (GraphPad Software Inc., San Diego CA).
References and notes
1. Treibs, A.; Kreuzer, F. H. Justus Liebigs Ann. Chem. 1968, 718, 208e223.
2. Ulrich, G.; Ziessel, R.; Harriman, A. Angew Chem., Int. Ed. 2008, 47, 1184e1201.
3. Shah, M.; Thangaraj, K.; Soong, M. L.; Wolford, L.; Boyer, J. H.; Politzer, I. R.;
Pavlopoulos, T. G. Heteroat. Chem. 1990, 1, 389e399.
4. (a) Rurack, K.; Kollmannsberger, M.; Daub, J. Angew Chem., Int. Ed. 2001, 40,
385e387; (b) Boens, N.; Leen, V.; Dehaen, W. Chem. Soc. Rev. 2012, 41,
1130e1172.
5. Golovkova, T. A.; Kozlov, D. V.; Neckers, D. C. J. Org. Chem. 2005, 70, 5545e5549.
6. Monsma, F. J.; Barton, A. C.; Kang, H. C.; Brassard, D. L.; Haugland, R. P.; Sibley,
D. R. J. Neurochem. 1989, 52, 1641e1644.
7. Ziessel, R.; Ulrich, G.; Harriman, A. New J. Chem. 2007, 31, 496e501.
8. (a) Ventura, B.; Marconi, G.; Broering, M.; Kruger, R.; Flamigni, L. New J. Chem.
2009, 33, 428e438; (b) Guzow, K.; Kornowska, K.; Wiczk, W. Tetrahedron Lett.
2009, 50, 2908e2910; (c) Fron, E.; Coutino-Gonzalez, E.; Pandey, L.; Sliwa, M.;
Van der Auweraer, M.; De Schryver, F. C.; Thomas, J.; Dong, Z.; Leen, V.; Smet,
M.; Dehaen, W.; Vosch, T. New J. Chem. 2009, 33, 1490e1496.
containing 2.5 mM of Rose Bengal as a comparative control were
also carried out. The relative singlet-oxygen generation rate of each
photosensitizer was determined by using Rose Bengal as
standard.23
4.5. Photodegradation of the BODIPYs
A 10 mM Phosphate Buffer Solution (PBS) of photosensitizer was
prepared in a flask. This solution was irradiated, at rt, for 2 h with
the green LED and a 1 mL sample was collected every 15 min and
analyzed with the spectrophotometer. The photodegradation rate
was determined from the plot of the decrease in the intensity of
absorbance versus time. The curves were fitted with an exponential
equation from which the half-degradation time could be calculated.
9. (a) Li, L.; Nguyen, B.; Burgess, K. Bioorg. Med. Chem. Lett. 2008, 18, 3112e3116;
(b) Sobenina, L. N.; Vasil’tsov, A. M.; Petrova, O. V.; Petrushenko, K. B.; Ushakov,
I. A.; Clavier, G.; Meallet-Renault, R.; Mikhaleva, A. I.; Trofimov, B. A. Org. Lett.
2011, 13, 2524e2527.
4.6. Computational methods
10. (a) Kee, H. L.; Kirmaier, C.; Yu, L.; Thamyongkit, P.; Youngblood, W. S.; Calder,
M.; Ramos, L.; Noll, B. C.; Bocian, D. F.; Scheidt, W. R.; Birge, R. R.; Lindsey, J. S.;
Holten, D. J. Phys. Chem. B 2005, 109, 20433e20443; (b) Lager, E.; Liu, J.; Aguilar-
Aguilar, A.; Tang, B. Z.; Pena-Cabrera, E. J. Org. Chem. 2009, 74, 2053e2058.
11. (a) Kolemen, S.; Bozdemir, O. A.; Cakmak, Y.; Barin, G.; Erten-Ela, S.; Marszalek,
M.; Yum, J.; Zakeeruddin, S. M.; Nazeeruddin, M. K.; Gratzel, M.; Akkaya, E. U.
Chem. Sci. 2011, 2, 949e954; (b) Cakmak, Y.; Kolemen, S.; Duman, S.; Dede, Y.;
Dolen, Y.; Kilic, B.; Kostereli, Z.; Yildirim, L. T.; Dogan, A. L.; Guc, D.; Akkaya, E. U.
Angew. Chem., Int. Ed. 2011, 50, 1e6.
The structures corresponding to the aromatic moieties tethered
on the BODIPY meso position were drawn and minimized to their
lowest energy conformation using a semi-empirical PM3 method in
HYPERCHEM software. With the same software the HOMO energy
of the structures was calculated.
12. (a) Yogo, T.; Urano, Y.; Ishitsuka, Y.; Maniwa, F.; Nagano, T. J. Am. Chem. Soc.
2005, 127, 12162e12163; (b) Banfi, S.; Caruso, E.; Zaza, S.; Mancini, M.; Gar-
iboldi, M.; Monti, E. J. Photochem. Photobiol., B 2012, 114, 52e60.
13. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.;
Moan, J.; Perez, O. J. Natl. Cancer Inst. 1998, 90, 889e905.
14. (a) De Rosa, M. C.; Crutchley, R. J. Coord. Chem. Rev. 2002, 233e234, 351e371;
(b) Sharman, W. M.; Allen, C. M.; van Lier, J. E. Methods in Enzymol. 2000, 319,
376e400.
4.7. Cytotoxicity studies
Human ovarian carcinoma SKOV3 cells were obtained from the
American Type Culture Collection (Rockville, MD, USA) and main-
tained in Dulbecco’s modified Eagle medium-F12 (DMEM-F12,
Mascia-Brunelli, Milano, Italy) supplemented with 10% fetal bovine
serum, 1% glutamine, and 0.5% antibiotic mixture (penicillin,
streptomycin, and neomycin), were used for this study. The cell
lines were maintained in standard culture conditions at 37 ꢀC in
a humidified 5% CO2 atmosphere. The antiproliferative effect of the
different PS was assessed using the MTT assay.26 Briefly,
5ꢂ104 cells/mL were seeded onto 96-well plates and allowed to
grow for 48 h prior to treatment with different BODIPY concen-
trations. After 24 h, the BODIPY-containing medium was replaced
by PBS, and cells were irradiated under visible light for 2 h using
a green LED (fluence 33 J/cm2). At the end of this time, cells were
incubated for 24 h at 37 ꢀC in drug-free medium; MTT was then
added to each well (final concentration 0.4 mg/mL) for 3 h at 37 ꢀC
and formazan crystals formed through MTT metabolism by viable
cells were dissolved in DMSO. Optical densities were measured at
570 nm using a Universal Microplate Reader EL800 (Bio-Tek
Instruments).
15. Turro, N. J. Modern Molecular Photochemistry; University Science Books: Sau-
salito, CA, 1991191e195.
16. Yuster, P.; Weissman, S. I. J. Chem. Phys. 1949, 17, 1182e1188.
17. (a) Koziar, J. C.; Cowan, D. O. Acc. Chem. Res. 1978, 334e341; (b) McGlynn, S. P.;
Reynolds, M. J.; Daigre, G. W.; Christodoyleas, N. D. J. Phys. Chem. 1962, 66,
2499e2505.
18. (a) Akkaya, E. U.; Dost, Z.; Atilgan, S. Tetrahedron 2006, 62, 8484e8488; (b) Liu,
J. Y.; Yeund, H. S.; Xu, W.; Li, X.; Ng, D. K. P. Org. Lett. 2008, 10, 5421e5424.
19. Lim, S. H.; Thivierge, C.; Nowak-Sliwinska, P.; Han, J.; Van den Bergh, H.;
ꢀ
Wagnieres, G.; Burgess, K.; Lee, H. B. J. Med. Chem. 2010, 53 (7), 2865e2874.
20. Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587e600.
21. Rurack, K.; Kollmannsberger, M.; Daub, J. New J. Chem. 2001, 25, 289e292.
22. (a) Yamada, K.; Toyota, T.; Takakura, K.; Ishimura, M.; Sugawara, T. New J. Chem.
2001, 25, 667e669; (b) Prieto, J. B.; Arbeloa, F. L.; Martinez, V. M.; Lopez, T. A.;
Amat-Guerri, F.; Liras, M.; Arbeloa, I. L. Chem. Phys. Lett. 2004, 385, 29e35.
23. Kochevar, I. E.; Redmond, R. W. Methods Enzymol. 2000, 319, 20e28.
24. Lissi, E. A.; Encinas, M. V.; Lemp, E.; Rubio, M. A. Chem. Rev. 1993, 93, 699e723.
25. Caruso, E.; Banfi, S.; Barbieri, P.; Leva, B.; Orlandi, V. T. J. Photochem. Photobiol., B
2012, 114, 44e51.
26. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D.
L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48,
589e601.