Chlorin photosensitizers sterically designed to prevent self-aggregation

Article abstract of DOI:10.1021/jo201568n

The synthesis and photophysical evaluation of new chlorin derivatives are described. The Diels-Alder reaction between protoporphyrin IX dimethyl ester and substituted maleimides furnishes endo-adducts that completely prevent the self-aggregation of the chlorins. Fluorescence, resonant light scattering (RLS) and ¹H NMR experiments, as well as X-ray crystallographic have demonstrated that the configurational arrangement of the synthesized chlorins prevent π-stacking interactions between macrocycles, thus indicating that it is a nonaggregating photosensitizer with high singlet oxygen (Φ_Δ) and fluorescence (Φ_f) quantum yields. Our results show that this type of synthetic strategy may provide the lead to a new generation of PDT photosensitizers.

Full text of DOI:10.1021/jo201568n

ARTICLE
pubs.acs.org/joc
Chlorin Photosensitizers Sterically Designed To Prevent
Self-Aggregation
Adjaci F. Uchoa,†,§,z,* Kleber T. de Oliveira,‡ Mauricio S. Baptista,§ Adailton J. Bortoluzzi,^
Yassuko Iamamoto,z and Osvaldo A. Serraz,*
zDepartamento de Química, Faculdade de Filosofia, Ci^encias e Letras de Ribeir~ao Preto, Universidade de S~ao Paulo, Avenida
Bandeirantes 3900, 14040-901, Ribeir~ao Preto-SP, Brazil
§Departamento de Bioquímica, Instituto de Química, Universidade de S~ao Paulo, Avenida Prof. Lineu Prestes 748, Cidade Universitꢀaria,
05508-000, S~ao Paulo-SP, Brazil
^Departamento de Química, Centro de Ci^encias Físicas e Matemꢀaticas, Universidade Federal de Santa Catarina Trindade 88040-900—
Florianꢀopolis-SC, Brazil
‡Departamento de Química, Universidade Federal de S~ao Carlos, Rodovia Washington Luís, km 235 - SP-310, 13565-905,
S~ao Carlos-SP, Brazil
S Supporting Information
b
ABSTRACT: The synthesis and photophysical evaluation of
new chlorin derivatives are described. The DielsÀAlder reac-
tion between protoporphyrin IX dimethyl ester and substituted
maleimides furnishes endo-adducts that completely prevent the
self-aggregation of the chlorins. Fluorescence, resonant light
1
scattering (RLS) and H NMR experiments, as well as X-ray
crystallographic have demonstrated that the configurational
arrangement of the synthesized chlorins prevent π-stacking
interactions between macrocycles, thus indicating that it is a
nonaggregating photosensitizer with high singlet oxygen (ΦΔ) and fluorescence (Φf) quantum yields. Our results show that this
type of synthetic strategy may provide the lead to a new generation of PDT photosensitizers.
’ INTRODUCTION
Waals interactions, thus leading to self-aggregation in both the
solution and solid state.15À20 The interactions are affected mainly
by the solvent, sample concentration, temperature, and specific
interactions with biological structures.15À26
Administrated solutions used in PDT are developed to reduce
photosensitizer aggregation by using emulsion/polymer prepa-
rations.1À5 However, in the biological environment, it is difficult
to control aggregation with these strategies. For example, interac-
tion with membranes can concentrate photosensitizers, consider-
ably increasing local concentration and consequently shifting the
monomer/aggregate equilibrium to aggregation.24À26 Therefore,
the most relevant strategy would be to avoid aggregation in the
target tissue, which can only beobtained byusingmolecules whose
molecular structure avoids aggregation.1À5,16À25
In polar non-nucleophilic solvents such as chloroform, chlor-
ophyll a exists mainly in the dimeric form, whereas larger
aggregates are formed in nonpolar solvents like benzene or
octane.21À23 The same tendency has been observed for synthetic
chlorins.15À19 Such interactions are regarded as being due to
πÀπ and πÀσ interactions and are easily characterized using
NMR spectroscopy.27À31
Photodynamic therapy (PDT) is a consolidated technique for
the treatment of cancer and other diseases.1,2 It is based on the
damage of living tissues by visible light in the presence of a
photosensitizer and oxygen.3À5 However, the action mechanism
involved in PDT is still a matter of extensive study with respect to
photosensitizer incorporation in biological systems,6À9 to the
relationship between the structure of the photosensitizer and the
site of singlet oxygen generation,10 and to the action mechanism
of reactive oxygen species (ROS).11 There are several photo-
sensitizers in use today that are based on porphyrin, phenothia-
zinium, phthalocyanine, and chlorin derivatives. However, the
future of PDT is highly dependent on the development of more
efficient photosensitizers.12 In addition to the photochemical
yield, there are several factors that are important for PDT
efficiency, such as tissue/cell localization and membrane bind-
ing.13 Therefore, having a large photochemical yield is certainly a
necessary, though not the sole, characteristic required to develop
new PDT photosensitizers.14
A factor that limits the efficiency of most photosensitizers
is their tendency to self-aggregate via the strong attractive
interactions between π-systems of the polyaromatic macrocycles.
The geometries of π-conjugated systems induce strong van der
Received: July 28, 2011
Published: September 20, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of New Chlorins (13À15) through a Diels-Alder Reaction
Self-aggregated states reduce fluorescence quantum yields,
triplet states, and singlet oxygen generation, thereby diminishing
photosensitizing activity.32À34 In self-aggregated states, porphyr-
ins adopt cofacial arrangements with their centers offset in
both solution and solid state.27À29 With the aim of reducing
aggregation, several authors have proposed the use of bulky
peripheric groups intended to reduce soluteÀsolute and to
enhance soluteÀsolvent interactions.21,35,36 The tert-butyl group
has frequently been coupled to phthalocyanines and naphthalo-
cyanines.37 Picket fence porphyrins have also provided promis-
ing results.38,39 To achieve this same effect, the menthyl
group, which is reported to be bulkier and more efficient35 than
tert-butyl, has been used to synthesize new photosensitizers that
are less prone to aggregate in solution. Interestingly some
authors have shown strategies to actively manage aggregation
by adding substituent groups that favor aggregation and disag-
gregation in the same molecules, that is, by adding groups that
form hydrogen bonds and by avoiding planarity of the rings,
respectively.31,40À42 Additionally, the immobilization of photo-
sensitizers on nanoparticle carriers has also been shown to avoid
states of aggregation.36,43À45
All these strategies favor the presence of monomeric species of
the photosensitizer in solution. However, no strategy has proven
effective to the degree that only monomers are present even
in concentrated solutions or in the presence of biological systems
that favor aggregation. In general terms, all the aforemen-
tioned photosensitizer structures are somewhat planar, thus still
allowing electronic cloud overlap and aggregation at higher
concentrations.
From a synthetic viewpoint, the DielsÀAlder reaction, a
stereospecific reaction, has been efficiently employed to synthe-
size chlorin derivatives starting from protoporphyrin IX di-
methyl ester as diene and dimethyl acetylene dicarboxylate or
maleic anhydride as dienophiles.46À50 The former dienophile
led to the development of verteporfin, a potent photosensitizer
and the most relevant commercial success in the area of PDT.47
The latter dienophile is advantageous since it enables the
insertion of different amphiphilic groups in the anhydride
function.50
In this work, the DielsÀAlder reaction between protopor-
phyrin IX dimethyl ester and substituted maleimides is described.
Maleimide dienophiles51 have been utilized inorder toinduceendo
addition by secondary orbital stabilization. Compared to the other
antiaggregation strategies we have proposed,31 these new photo-
sensitizers have a bulkier substituent and a smaller photosensitizer
core, allowing the synthesis of new chlorin molecules that have a
nonplanar and rigid “L”-type shape, which completely prevents
aggregation, even at high concentrations (10À2 mol LÀ1).
’ RESULTS AND DISCUSSION
Synthesis and Characterization. Maleimides 8À10 were
synthesized according to the procedure described in the
literature.52 The DielsÀAlder reactions between maleimides
8À10 and protoporphyrin IX dimethyl ester or protoporphyrin
IX diethyl ester (11 or 12) were performed in a sealed tube by
using different concentrations of maleimide solutions (3À20,
equiv. to 8 or 9), in dry/deoxygenated toluene at 120 ꢀC
(Scheme 1).
Chlorins 13À15 (Scheme 1) were obtained by using different
concentrations of maleimides; however, the optimum concen-
trations of dienophiles were defined as 3 equiv of N-benzylma-
leimide or p-nitrophenyl maleimide (8 or 10) and 6 equiv of N-
octyl maleimide (9). The reactions were monitored by TLC and
UVÀvis at 666 nm, and the best results were achieved after 12 h
for dienophile 8 and 10 and after 20 h for 9. The products were
purified using column chromatography (silica) followed by
preparative TLC (silica), using a 25:1 mixture of CHCl3/AcOEt
as eluent, which furnished the pure isomers. Chlorins 13a and
13b have an Rf of 0.38 and 0.54, respectively; 14a and 14b have
an Rf of 0.52 and 0.67, respectively; and 15a and 15b have an
1
Rf of 0.28 and 0.42, respectively. An H NMR spectra con-
firmed that each spot corresponded to a single compound. The
amounts obtained and yields were as follows: 13a and 13b, 47 mg
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Figure 1. (a) Main NOE effects in compound 13a; (b) main NOE effects in compound 13b.
13b. For example, the NOE between H-21 (Figure 1b) and the
distinguished methyl group H-25 (at 2.07 ppm) was decisive to
assign the cis-endo structure of chlorin 13b. The NOE between
H-21 and H-22 was alsousedtoconfirmthe cis structureof 13b and
was in agreement to the DielsÀAlder reaction mechanism.
For the product with a lower Rf, that is, 13a, we also observed a
NOE between H-21 and H-20 and between H-21 and H-25;
however, a NOE was also observed between the distinguished
exocyclic vinylic hydrogen (triplet in 7.41 ppm and J = 4.9 Hz)
H-24 and another meso-hydrogen, supposedly H-5. This sup-
posed hydrogen H-5 presented a NOE with the methyl group
H-71, and the hydrogen H-71 presented a NOE with the vinylic
hydrogen H-82β (doublet at 6.34 and J = 17.6 Hz), confirming
the regiochemistry of this more polar isomer. The cis-endo
structure of 13a was also confirmed by a NOE between H-21
and H-25 and a NOE effect between H-21 and H22 (Figure 1a).
Other NOE signals were useful to complete assignment of
structure 13a. The HMBC shows several C/H correlations
(2JCH and 3JCH) that were decisive for the structural assignment
of these isomers. There is correlation between C-26 with H-21
(2JCH) and 22 (3JCH). The C-27 shows correlation with H-22
(2JCH) and H-23 (3JCH). The hydrogens were assigned unam-
biguously by gCOSY. The HMBC spectra and the other 2D
NMR analysis enabled a complete assignment of all protons and
carbons for the two isomeric compounds. The complete assign-
ments of spectroscopic data for chlorins are depicted in the
Experimental Section. Therefore, we can conclude that the more
polar compounds are a, and the less polar compounds are b. The
Figure 2. X-ray projections of compound 13b.
(0.062 mmol) with a 72% yield; 14a and 14b, 40 mg (0.050
mmol) and a 58% yield; and 15a and 15b, 49 mg (0.058 mmol)
with a 67% yield.
All compounds were fully characterized by 1D (1H, 13C and
DEPT-135) and 2D (HMBC, HSQC, COSY, NOESY) NMR
techniques, ESI-MS, UVÀvis, resonant light scattering and
photoluminescence. The regioselectivity of the DielsÀAlder
reaction (reaction in ring A or B of the porphyrin) and the
stereochemistry (cis-endo) were deduced from the NOESY
spectra. For example, Figure 1 details the main correlations for
compounds 13a and 13b. Furthermore, the structure identified
as 13b by 1D and 2D NMR analysis was confirmed using X-ray
crystallography (Figure 2).
Chlorins 13a and 13b presented an unequivocal signal in the
1H NMR spectra at 4.65 ppm (doublet), corresponding to H-21
in both structures (Figure 1). Also, both products presented a
nuclear overhauser effect (NOE) between H-21 and a meso-
hydrogen referred to as H-20 in both compounds 13a and 13b.
In the case of the product with a higher Rf, the meso-hydrogen
H-20 presented an NOE with one vinylic hydrogen (double
doublet at 8.12 ppm and J = 17.5 and 11.5 Hz), providing the first
piece of evidence that this is isomer 13b (Figure 1b). We
concluded that the unique possibility of an NOE between a
meso-hydrogen H-20 and a vinylic hydrogen (double doublet at
8.12 ppm and J = 17.5 and 11.5 Hz) occurs in compound 13b,
and the hydrogen with this chemical shift and these coupling
constants has to be H-181 (Figure 1b). Other correlations
observed by using NOE (Figure 1b) were also decisive in drawing
conclusions about the regiochemistry and the stereochemistry of
1
spectra 2D NMR for 13a and 13b and all H and 13C NMR
spectra are included in the Supporting Information.
UVÀVisible and Fluorescence Emission. The UVÀvis and
fluorescence spectra were obtained in methanolic solutions. The
new chlorins displayed typical UVÀvis spectra with Soret and Q
bands maximum absorption wavelength (λmax) in nm (log ε) of
402 (5.1) and 666 (4.5), respectively. The correlation between
Q/Soret bands was 0.22, with a Stokes shift of 5 nm. TPP, whose
fluorescence quantum yield (Φf) in methanol is 0.11, was used as
standard to quantify fluorescence yield. Φf values, which are
shown in the inset of Figure 3, are related to the group inserted
into the chlorin ring: R = phenyl, Φf = 0.16 for 13a, and Φf = 0.17
for 13b; R = octyl, Φf = 0.14 for 14a, and Φ f= 0.15 for 14b; R =
p-nitrophenyl, Φf = 0.12 for 15a, and Φf = 0.13 for 15b. All
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Figure 3. Absorption spectra in methanol (left) and fluorescence
emission spectra in methanol (right) both of compound 13a. Fluores-
cence quantum yield for the new chlorins. λexc = 500 nm; λemi
510À720 nm (insert).
=
Figure 5. Resonant light scattering (RLS) spectra of the compounds
13a, 13b, 14a, and 14b and protoporphyrin IX dimethyl ester (11) (A)
in CHCl3 and (B) in CH3OH/CHCl3 4:1, both at 10À4 mol LÀ1, in a
cuvette with 0.1 cm path length at room temperature.
respectively. The lower Φnr for compounds 13a and 13b is in
agreement with the larger rigidity of the phenyl group compared
to the other substituents.
Aggregation Studies of the New Chlorins. Self-aggregation
was investigated by means of several methods and using proto-
porphyrin IX dimethyl ester as reference, which was chosen
because it is the precursor of the new chlorins and because its
solubility and spectroscopic properties are similar to those of the
synthesized compounds.
Resonant light scattering (RLS) is a spectroscopic technique
whose signal intensity depends on both excitonic coupling
between light and the absorption cross section of the absorbing
species and the size of these species, making it ideal to characterize
aggregates of photosensitizer molecules.22À24 The spectrum of
protopophyrin IX dimethyl ester at 10À4 mol LÀ1 (11) displayed a
very intense RLS peak near 460 nm, which corresponds to theλmax
of the J-band aggregate (Figure 5A,B).15À19,21À23 None of the
synthesized chlorins (13a, 13b, 14a, and 14b) exhibited an RLS
signalunderthe sameexperimentalconditions, revealingthatthese
compounds are free of self-aggregation at this concentration
regime.15À19,21À23
Figure 4. Transient emission at 1270 nm of compound 13a in
CH3CN/H2O (9:1) solution. Inserts: (A) emission spectrum of singlet
oxygen of 13a; (B) singlet oxygen quantum yield (ΦΔ) in acetonitrile of
compounds 13a, 13b, 14a, 14b, 15a, 15b, and methylene blue (MB).
λexc = 532 nm; 10 Hz; 5 mJ/pulse.
compounds exhibit high Φf compared with related compounds,
such as other chlorins and porphyrins, thus indicating stronger
molecular rigidity.53,54
Singlet Oxygen Quantum Yield. Emission of singlet oxygen
was characterized and quantified by the direct method (NIR
phosphorescence emission) in a CH3CN/H2O 9:1 mixture,
using an instrument and technique described earlier.55 Figure 4
shows the transient at 1270 nm and spectra of singlet oxygen
emission after exciting compound 13a at 532 nm, as well as the
singlet oxygen quantum yields of the new chlorins (insert). 1O2
has a lifetime of 44 μs, which is shorter than its characteristic
lifetimeinpureacetonitrile (54.4( 1.3 μs),56 but itisin agreement
with the presence of water in the solvent mixture (CH3CN/H2O).
The ΦΔ values for the series were R = phenyl, ΦΔ = 0.66 for 13a
and ΦΔ = 0.71 for 13b; R = octyl, ΦΔ = 0.52 for 14a and ΦΔ =
0.54 for 14b; R = p-nitrophenyl, ΦΔ = 0.61 for 15a and 15b.
These results demonstrate that the new chlorins were able to
generate singlet oxygen with high quantum yields. Chlorins with
R = phenyl led to higher ΦΔ because the structures of these
chlorins (13a and 13b) seem to be more rigid and to present
fewer vibrational levels. Assuming that no other processes are
involved, the yield of nonradiative decay (Φnr) can be estimated
by adding ΦΔ and Φt and subtracting the result from 1. Φnr was
0.18 and 0.12 for compounds 13a and 13b; 0.34 and 0.31 for
compounds 14a and 14b; and 0.27 and 0.26 for 15a and 15b,
In addition, to determine the possible formation of self-
1
aggregates at high concentrations, the H NMR technique was
employed. It is known that the formation of aggregated states
affects the diamagnetic anisotropy of porphyrin and chlorin
rings.21À23 The 1H NMR spectra of protoporphyrin IX dimethyl
ester at different concentrations (between 0.4 Â 10À2 and 3.4
mol LÀ1, Figure 6A) were analyzed with respect to changes in the
chemical shifts as a function of concentration.21À23,57 Δδ-H
shifts in the case of the meso-H of protoporphyrin IX dimethyl
ester (Figure 6A, 10À8 ppm) demonstrated that aggregation is
well pronounced in this case due to the formation of partially
side-slipped face-to-face aggregates.21À23,58
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Figure 6. Changes in the chemical shifts of meso 1H and vinylic hydrogen in CDCl3 at 25 ꢀC as a function of concentration of protoporphyrin IX
dimethyl ester (A) and chlorin 13a (B).
The spectra of the new apolar chlorins (13, 14) were deter-
mined under the same experimental conditions. None of
the spectra presented Δδ-H shifts as a function of concentration.
The data for chlorin 13a are shown in Figure 6B and
for compounds 13b, 14a, and 14b are included in the
Supporting Information, indicating that the new chlorins do
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To test this hypothesis, two new “L”-type chlorins (15a, 15b)
having nitro substituents and increased aqueous solubility were
prepared. Experiments were performed in the presence of a
protein (BSA) because proteins are prone to induce photosensi-
tizer aggregation or disaggregation depending on the photosen-
sitizer/protein concentration ratio.59 We used hematoporphyrin
IX (HP IX) as reference because it has sufficient aqueous
solubility.
The fluorescence spectra of both compounds were deter-
mined in the presence of increasing amounts of BSA. Note that,
when BSA concentration is increased from 0 to 60 μg mLÀ1
,
there is a large increase (∼2.5 times) in the fluorescence emission
in the case of Hp IX and almost no change in the case of 15a
(Figure 8 insert). The increase in fluorescence intensity indicates
that BSA is able to dissociate the Hp IX aggregates that are
present in pure water solutions, and the fact that there was no
change in the emission profile of 15a indicates that this molecule
does not tend to aggregate in aqueous solution. Shifts in the
maxima wavelengths are in agreement with this explanation.
There were changes in the maxima of the emission (Figure 8)
and absorption spectra (from 377 to 400 nm, data not shown) in
the case of Hp IX with the increase of BSA concentration, which
can also be explained by the disaggregation of HP IX, and there
was no measurable change in the λmax of emission and absorption
of 15a, showing that the concept of stabilization of monomeric
species by the “L”-type scaffold is also valid in aqueous solution.
There are several works published in the literature showing
axial substituent groups, and that bulky groups disfavor
aggregation,31,35,40À42 although no-one has obtained results of
lack of aggregation in the high concentration regimes shown in
this work. We think that the conformation of molecules 13À15
have such a high efficiency to prevent the interaction between
π-systems, because of the extreme rigidity conferred by four sp3
carbons in the same ring, at positions 2, 21, 22, and 23.
Figure 7. Sum of the chemical shifts of meso-hydrogens (∑Δδ) for
compounds 11, 13a, 13b, 14a, and 14b as a function of the compound
concentration.
Figure 8. Fluorescence spectra of Hp IX (left) and 15a (right) as a
function of BSA concentration in μg mLÀ1 (a = 0, b = 10, c = 20, d = 30, e
= 60) in a water solution. Insert: Fluorescence integral versus BSA
concentration in μg mLÀ1
.
’ CONCLUSIONS
not undergo self-aggregation even in large concentration
solutions.
The new chlorins (13À15) have a bulky axial organic group
and a relatively small porphyrin core, forming an “L”-type shape
that hinders aggregation. These characteristics indicate that these
compounds are potentially useful for PDT applications, as
evidenced by the high singlet oxygen quantum yields and stability
of the monomeric species in aqueous and organic media. The
synthesis of these chlorins with substituted imides by DielsÀ
Alder reactions can be considered an excellent and versatile
method for the preparation of new chlorins that do not self-
aggregate.
To best view the changes in Δδ-meso-H as a function of
photosensitizer concentration, the sum of the chemical shifts of
meso-hydrogen (ΣΔδ-meso-H) as a function of concentration
was calculated and plotted in Figure 7. Note that ΣΔδ-meso-H is
directly proportional to the concentration of protopophyrin IX
dimethyl ester. In the case of the new compounds, there was no
observable shift. It is important to emphasize that chlorins usually
have a high tendency to form aggregates, often hindering the
acquisition of the 1H NMR spectra in CDCl3.35,50 The 1H NMR
data prove that the axial groups in chlorins 13a, 13b, 14a, and
14b definitely avoid self-aggregation.
’ EXPERIMENTAL SECTION
Obtaining sensitizers with low levels of aggregation was our
main goal.8,35,36 Indeed, chlorins 13a, 13b, 14a, and 14b are
completely free of aggregation, even at high concentrations.
These results can be considered a major breakthrough in the
development of photosensitizers, since the formation of aggre-
gated states deactivates the excited state of the molecules, reducing
the levelsof singlet oxygen generation. The new molecularfeatures
were attained by employing strategic dienophiles, which induced
the endo products and provided an “L”-type molecular scaffold that
does not allow aggregation. However, these compounds have low
aqueous solubility, and it is important to prove that this same
concept could be applied in water solutions.
General. Anhydrous toluene was purified according to a standard
procedure.50 Maleic anhydride was recrystallized in toluene, and other
commercially available reagents were used without further purification.
An ultrasound was employed for deoxygenation of the toluene. 1H NMR
and 13C RMN spectra were recorded at 500.13 and 125.77 MHz,
respectively, using CDCl3 as the solvent and TMS as the internal
1
reference. Unequivocal H assignments were carried out by using 2D
gCOSY (1H/1H) and NOESY spectra (mixing time of 800 ms). HRMS
were obtained using the ESI technique. The UVÀvis spectra were
registered using methanol as the solvent. Flash chromatography was
carried out by using silica gel 70À230 mesh, and the preparative thin
layer chromatography was conducted on 20 Â 20 cm glass plates coated
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with silica gel/gypsum 60 (1 mm thick). Analytical TLC was performed
on sheets precoated with silica gel (0.2 mm thick).
6.94À6.97 (m, 3H, H-210,H-211 and H-212); 7.41 (t, 1H, J = 4.9 Hz,
H-24); 8.18 (dd, 1H, J = 17.6 and 11.5 Hz, H-81); 9.10 (s, 1H, H-20);
9.33 (s, 1H, H-5); 9.67 (s, 1H, H-15), 9.85 (s, 1H, H-10). 13C NMR
(CDCl3, 125.77 MHz) δ (ppm): 11.4 (C-121); 11.6 (C-181); 12.3
(C-71); 21.5 (C-171); 21.9 (C-131); 25.6 (C-23); 26.5 (C-25); 36.6
(C-172); 37.0 (C-22); 38.5 (C-132); 50.1 (C-21); 51.6 (C-134); 51.8
(C-174); 52.3 (C-2); 90.5 (C-5); 93.4 (C-20); 97.9 (C-15); 99.8 (C-10);
115.6 (C-24); 121.3 (C-82); 126.0 (C-210 and C-212); 128.1 (C-211);
128.6 (C-29 and C-213); 129.2 (C-7); 129.8 (C-81); 131.0 (C-18); 131.3
(C-28); 132.6 (C-9); 133.8 (C-8); 133.9 (C-16); 136.3 (C-17); 136.5
(C-12); 138.3 (C-6); 138.4 (C-19); 139.6 (C-13); 149.6 (C-14); 152.3
(C-4); 166.1 (C-1); 173.4 (C-133); 173.8 (C-173); 174.9 (C-27); 178.6
Syntheses of N-Phenylmaleimide (8), N-Octylmaleimide
(9), and P-Nitrophenylmaleimide (10). To a solution of 0.196 g
(0.02 mol) of maleic anhydride in ethyl ether (4 mL) at 0 ꢀC, 0.02 mol of
the corresponding amine (2, 3, or 4) was added, and the reactional
mixture was stirred for 10 min. The white solid obtained was filtered off
and washed with ethyl ether (2 Â 10 mL). Then, intermediate 5, 6, or 7
was reacted in the presence of freshly distilled acetic anhydride (10 mL)
and anhydrous sodium acetate (0.1 g) at 60 ꢀC for 2 h. The reaction was
poured into a water/ice mixture (100 mL) and then extracted with
dichloromethane (2 Â 100 mL). Compounds 8, 9, and 10 were purified
by column chromatography on silica, using CH2Cl2/MeOH 9:1 as
eluent.
+
(C-26). ESI-MS-TOF, m/z 764.3443 calculated for C46H46N5O6
(MH+); found, 764.3415.
21,22[N,N-Dicarbonyl-N-phenyl]-8,12-bis[2-(methoxycar-
bonyl)ethyl]-2,7,13,17-tetramethyl-18-vinyl-2,21,22,23-tet-
rahydrobenzo[b]porphyrin (13b). 1H NMR (CDCl3, 500.13
MHz) δ (ppm): À2.43 (br s, 2H, H-21 and H-23); 2.07 (s, 3H, CH3-
25), 3.16 (t, 2H, J = 7.7 Hz, H-122), 3.20 (t, 2H, J = 7.7 Hz, H-82); 3.41
(s, 3H, CH3-131); 3.45À3.49 (m, 2H, H-23α and H-23β); 3.47 (s, 3H,
CH3-71); 3.60 (s, 3H, CH3-171), 3.64 and 3.65 (2s, 3H and 3H; OCH3-
124 and OCH3-84); 3.91À394 (m, 1H, H-22) 4.18 (t, 2H, J = 7.7 Hz,
H-121); 4.32 (t, 2H, J = 7.7 Hz, H-81), 4.65 (d, 1H, J = 8.6 Hz, H-21);
6.10 (d, 1H, J = 11.5 Hz, H-182α); 6.32 (d, 1H, J=17.5 Hz, H-182β)
6.65À6.72 (m, 2H, H-29 and 213); 6.92À6.99 (m, 3H, H-210, H-211 and
H-212); 7.42 (t, 1H, J = 5.0 Hz, H-24) 8.12 (dd, 1H, J = 17.5 and 11.5 Hz,
H-181); 9.27 and 9.28 (s, 2H, H-5 and, H-20); 9.70 (s, 1H, H-10), 9.75
(s, 1H, H-15). 13C NMR (CDCl3, 125.77 MHz) d (ppm): 11.2 (C-131);
11.7 (C-71); 12.4 (C-171); 21.6 (C-81); 21.9 (C-121); 25.6 (C-23); 26.5
(C-25); 36.7 (C-82); 37.0 (C-122); 38.6 (C-22); 50.1 (C-21); 51.6
(C-124); 51.7 (84); 52.2 (C-2); 90.0 (C-5); 94.2 (C-20); 98.4 (C-10);
99.4 (C-15); 116.0 (C-24); 120.9 (C-182); 126.0 (C-29 and 213); 128.1
(C-211); 128.5 (C-210 and 212); 129.9 (C-181); 131.3 (C-9); 131.4
(C-17); 133.4 (C-16); 133.9 (C-8); 136.1 (C-19); 137.5 (C-13); 137.9
(C-7); 139.1 (C-12); 139.7 (C-14); 149.7 (C-11); 151.2 (C-4); 163.9
(C-6); 165.9 (C-1); 173.4 (C-83); 173.8 (C-123); 174.7 (C-26); 178.6
N-Phenylmaleimide (8). Compound (8) was obtained in a global
74% yield and was characterized by 1H and 13C NMR analysis: 1H NMR
(CDCl3, 500 MHz), δ (ppm): 6.84 (br s, 2H, H-2 and H-3 vinyl group);
7.33À7.38 (m, H-7, H-8 and H-9); 7.45À7.48 (m, H-6 and H-10). 13C
NMR (CDCl3, 125.77 MHz), δ (ppm): 126.0 (2C, C-6 and C-10);
127.9 (C-8); 129.1 (2C, C-7 and C-9); 131.2 (C-5); 134.2 (2C, C-2 and
C-3); and 169.5 (2C, C-1 and C-4). ESI-MS-TOF, m/z 174.0550
calculated for C10H8NO2+ (MH+); found, 174.0582.
N-Octylmaleimide (9). Compound (9) was obtained in a global
1
1
52% yield and was characterized by H and 13C analysis: H NMR
(CDCl3, 500 MHz), δ (ppm): 0.87 (t, 3H, J = 6.5 Hz, CH3-12);
1.25À1.28 (m, 10H, H-7, H-8, H-9, H-10 and H-11); 1.56À1.58 (m,
2H, H-6); 3.50 (t, J = 7.0 Hz, 2H, H-5); 6.68 (br s, 2H, H-2 and H-3 vinyl
group). 13C NMR (CDCl3, 125.77 MHz) δ (ppm): 14.0 (C-12); 22.6
(C-11); 26.7 (C-10); 28.5 (C-9); 29.0 (C-8); 29.1 (C-7); 31.7 (C-6);
37.9 (C-5); 134.0 (C-2 and C-3); 170.9 (C-1 and C-4). ESI-MS-TOF,
m/z 210.1489 calculated for C12H20NO2+ (MH+); found, 210.1495.
P-Nitrophenylmaleimide (10). Compound (10) was obtained in
a global 72% yield and was characterized by 1H and 13C NMR analysis:
1H NMR (CDCl3, 500 MHz), δ (ppm): 6.93 (br s, 2H, H-2 and H-3
vinyl group); 7.69 (d, 2H, J = 10.0 H-6 and H-10); 8.34 (d, J = 10, H-7
and H-9). 13C NMR (CDCl3, 125.77 MHz), δ (ppm): 124.5 (2C, C-7
and C-9); 125.5 (2C, C-6 and C-10); 134.6 (2C, C-2 and C-3); 137.1
(C-5); 146.5 (C-8). ESI-MS-TOF, m/z 219.0400 calculated for
C10H7NO4+ (MH+); found, 219.0400.
(C-27). ESI-MS-TOF, m/z 764.3443 calculated for C46H46N5O6
+
(MH+); found, 764.3422.
21,22[N,N-Dicarbonyl-N-octhyl]-13,17-bis[2-(methoxycar-
bonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,21,22,23-tetra-
hydrobenzo[b]porphyrin (14a). 1H NMR (CDCl3, 500.13 MHz)
δ (ppm): À2.46 and À2.39 (br s, 2H, H-22 and H-24); 0.55À0.67
(m, 9H, H-215, 214, H-213 and H-212); 0.71À0.77 (m, 2H, H-211);
0.82À0.90 (m, 2H, H-210); 0.93À1.01 (m, 2H, H-29); 2.03 (s, 3H, CH3-
25); 3.00 (m, 2H, H-28); 3.16 (t, 2H, J = 7.6 Hz, H-132); 3.22 (t, 2H, J =
8.0 Hz, H-172); 3.35À3.39 (m, 2H, H-23α and H-23β); 3.40 (s, 3H, CH3-
71); 3.51 (s, 3H, CH3-181); 3.53 (s, 3H, CH3-121); 3.66 and 3.70 (2s, 3H
and 3H; OCH3-134 and OCH3À174); 3.73À3.76 (m, 1H, H-22); 4.18
(t, 2H, J = 8.0 Hz, H-131); 4.32 (t, 2H, J = 7.3 Hz, H-171); 4.46 (d, 1H,
J = 8.4 Hz, H-21); 6.14 (d, 1H, J = 11.5 Hz, H-82α); 6.34 (d, 1H, J = 17.7
Hz, H-82β); 7.34 (t, 1H, J = 5.0 Hz, H-24); 8.18 (dd, 1H, J = 17.7 and
11.5 Hz, H-81); 9.08 (s, 1H, H-20); 9.28 (s, 1H, H-5); 9.68 (s, 1H,
H-15), 9.84 (s, 1H, H-10). 13C NMR (CDCl3, 125.77 MHz) d (ppm):
11.4 (C-121); 11.6 (C-181); 12.2 (C-71); 13.8 (C-215); 21.5 (C-172);
21.9 (C-132); 22.2 (C-214); 25.5 (C-23); 26.1 (C-210); 26.5 (C-25); 27.1
(C-29); 28.6 (2C, C-212 and C-211); 31.4 (C-213); 36.6 (C-172); 37.1
(C-28); 38.3 (C-22); 38.7 (C-132); 49.9 (C-21); 51.6 (C-134); 51.8 (C-
174); 52.2 (C-2); 90.3 (C-5); 93.4 (C-20); 97.9 (C-15); 99.7 (C-10);
115.6 (C-24); 121.2 (C-82); 129.1 (C-7); 129.8 (C-81); 130.9 (C-18);
132.5 (C-9); 133.7 (C-8); 133.9 (C-16); 136.3 (C-17); 136.5 (C-9);
138.3 (C-6); 139.6 (C-19); 149.4 (C-14); 151.0 (C-16); 151.3 (C-9);
152.3 (C-4); 166.3 (C-1); 173.4 (C-133); 173.8 (C-173); 175.8 (C-27);
General Procedure for the DielsÀAlder Reaction. To a
solution of maleimides 8, 9, or 10 (3 equiv for 8 and 10 or 6 equiv for
9) in dry/deoxygenated toluene (5 mL), protoporphyrin IX diester (11
or 12) (42.3 mmol) was added. The reaction was performed in a sealed
tube at 120 ꢀC for 12 h in the case of dienophile 8 or 10, and 20 h for
dienophile 9. After that, the reaction mixtures were directly poured into a
column chromatograph (silica gel 70À230 mesh) and purified by using a
25:1 CHCl3/AcOEt mixture as eluent. This preliminary purification was
sufficient to isolate the isomeric mixture containing 13a/13b, 14a/14b,
and 15a/15b. The isomeric separations were carried out by preparative
thin layer chromatography on 20 Â 20 cm glass plates coated with silica
gel 60 and gypsum (1 mm thick), yielding 72% of 13a/13b isomers in a
1:1 ratio, 58% of 14a/14b, 66% of 15a/15b and isomers in the same
proportion for all reactions.
21,22[N,N-Dicarbonyl-N-phenyl]-13,17-bis[2-(methoxy-
carbonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,21,22,23-te-
trahydrobenzo[b]porphyrin (13a). 1H NMR (CDCl3, 500.13
MHz) δ (ppm): À2.47 and À2.38 (br s, 2H, H-22 and H-24); 2.07
(s, 3H, CH3-25); 3.15 (t, 2H, J = 7.8 Hz, H-132); 3.20 (t, 2H, J = 7.8 Hz,
H-172); 3.40 (s, 3H, CH3-121); 3.45À3.48 (m, 2H, H-23α and 23β); 3.49
(s, 3H, CH3-181); 3.56 (s, 3H, CH3-71); 3.64 and 3.68 (2s, 3H and 3H;
OCH3-134 and OCH3-174); 3.89À3.96 (m, 1H, H-22); 4.17 (t, 2H, J =
7.8 Hz, H-131); 4.29 and 4.30 (2dt,1H, J=7.8 and 4.4 Hz, H-171α and
171β), 4.65 (d, 1H, J = 8.6 Hz, H-21); 6.14 (d, 1H, J = 11.5 Hz, H-82α);
6.34 (d, 1H, J = 17.6 Hz, H-82β); 6.66À6.70 (m, 2H, H-29 and 213);
+
179.6 (C-26). ESI-MS-TOF, m/z 800.4382 calculated for C48H58N5O6
(MH+); found, 800.4389.
8830
dx.doi.org/10.1021/jo201568n |J. Org. Chem. 2011, 76, 8824–8832

The Journal of Organic Chemistry
ARTICLE
21,22[N,N-Dicarbonyl-N-octhyl]-8,12-bis[2-(methoxycar-
bonyl)ethyl]-2,7,13,17-tetramethyl-18-vinyl-2,21,22,23-tet-
rahydrobenzo[b]porphyrin (14b). 1H NMR (CDCl3, 500.13
MHz) δ (ppm): À2.43 (br s, 2H, H-21 and H-23), 0.58À0.64 (m,
9H, H-215, 214, H-213 and H-212) 0.76À0.89 (m, 4H, H-210 and H-211);
0.94À0.99 (m, 2H, H-29); 2.01 (s, 3H, CH3-25), 2.98 (t, 2H, J = 7.5 Hz,
H-28); 3.16 (t, 2H, J = 7.7 Hz, H-122), 3.17 (t, 2H, J = 7.7 Hz, H-82);
3.34À3.40 (m, 2H, H-23); 3.39 (s, 3H, CH3-131); 3.43 (s, 3H CH3-71);
3.59 (s, 3H, CH3-171); 3.65 and 3.66 (2s, 3H and 3H; OCH3-124 and
OCH3-84); 3.69À3.74 (m, 1H, H-22); 4.16 (t, 2H, J = 7.5 Hz, H-121);
4.29 (t, 2H, J = 7.5 Hz, H-81); 4.43 (d, 1H, J = 8.0 Hz, H-21); 6.11 (d, 1H,
J = 11.2 Hz, H-182α); 6.34 (d, 1H, J = 18.0 Hz, H-182β); 7.33 (t, 1H, J =
5.6 Hz, H-24), 8.14 (dd, 1H, J = 18.0 and 11.2 Hz, H-181); 9.20 (s, 1H,
H-5); 9.25 (s, 1H, H-20); 9.66 (s, 1H-10), 9.72 (s, 1H, H-15). 13C NMR
(CDCl3, 125.77 MHz) δ (ppm): 11.2 (C-131); 11.6 (C-71); 12.4
(C-171); 13.8 (C-215); 14.1 (CH3); 21.5 (C-81); 21.9 (C-121); 22.2
(C-214); 26.1 (C-23); 26.5 (C-25); 27.0 (C-29), 28,6 (2 signals C212 and
C-211); 29.7 (C-210); 31.4 (C-213); 36.7 (C-82); 37.1 (C-28); 38.3
(C-22); 38.7 (C-82); 49.9 (C-21); 51.6 (C-124); 51.7 (84); 52.1 (C-2);
89.8 (C-5); 94.1 (C-20); 98.3 (C-10); 99.3 (C-15); 115.9 (C-24); 120.7
(C-182); 130.0 (C-181); 131.1 (C-9); 133.3 (C-17); 133.4 (C-16);
133.8 (C-8); 135.9 (C-19); 136.8 (C-12); 137.3 (C-13); 137.9 (C-7);
139.7 (C-14); 149.5 (C-11); 150.5 (C-3); 151.4 (C-4); 153.0 (C-6);
165.9 (C-1); 173.4 (C-83); 173.8 (C-123); 175.6 (C-26); 179.6 (C-27).
H-15). 13C NMR (CDCl3, 125.77 MHz) δ (ppm): 11.3 (C-131); 11.7
(C-71); 12.4 (C-171); 14.1 (C-175); 14.2 (C-135); 21.5 (C-81); 21.8
(C-121); 25.6 (C-23); 26.6 (C-25); 36.8 (C-82); 37.2 (C-122); 38.7 (C-
22); 50.1 (C-21); 52.2 (C-2); 60.6 (C-124); 60.6 (84); 90.1 (C-5); 94.4
(C-20); 98.7 (C-10); 99.6 (C-15); 115.8 (C-24); 120.7 (C-182); 129.8
(C-28); 129.9 (C-181); 123.4 (C-210 and C-212); 126.5 (C-29 and
C-213); 133.6 (C-16); 133.9 (C-8); 136.8 (C-19); 146.5 (C-211);149.8
(C-4); 172.9 (C-83); 173.3 (C-123); 174.0 (C-26); 177.8 (C-27). ESI-
+
MS-TOF, m/z 837.3606 calculated for C48H49N6O8 (MH+); found,
837.3623.
Spectroscopic and Photophysical Studies. UVÀvis, fluores-
cence, and RLS spectra were obtained in conventional spectrophot-
ometer and spectrofluorometer equipments. Extinction coefficients
were obtained by measuring absorption spectra from 350 to 700 nm
and plotting them as a function of the chlorin concentration. Fluores-
cence spectra were obtained in a methanol solution, excited at 500 nm,
and detected from 530 to 800 nm. Fluorescence quantum yields (Φf)
were determined by measuring the area under the emission spectra
(530À800 nm range), using TPP in methanol (Φf = 0.11) as standard.
RLS were recorded using the spectrofluorometer operating in synchro-
nous scan mode, that is by simultaneously scanning the excitation and
emission monochromators (Δλ = 0 nm) of the spectrofluorometer from
350 to 700 nm in a cell with a path length of 0.1 cm. In all cases, the
absorbance values of the sample and reference solutions were kept below
0.1 at the excitation wavelength to minimize inner filter effects. For
studies of self-aggregation in water, Hp IX and 15a were dissolved in
DMSO. A 300 μL portion of this solution was added to a cuvette with a
1.0 cm path length containing 3 mL of water. The absorption and
fluorescence spectra were obtained before and after addition of increas-
ing amounts of the BSA solution of 3 mg mLÀ1, 10, 20, 30, and 60 μL.
The quantum yield of singlet oxygen production (ΦΔ) was calculated
by using a phosphorescence detection method. A Nd:YAG laser operat-
ing at 532 nm (5 ns, 10 Hz) was used as the excitation source. The
radiation emitted at 1270 nm was detected at a right angle by a liquid-
nitrogen cooled NIR photomultiplier.55 Three different concentrations
of chlorin in CH3CN/H2O 9:1 were tested. The absorbance of the
sample and standard (methylene blue)53 were kept the same. ΦΔ values
were calculated by comparing the emissions of samples and standards.
ESI-MS-TOF, m/z 800.4382 calculated for C48H58N5O6 (MH+);
+
found, 800.4377.
21,22[N,N-Dicarbonyl-N-(4-nitrophenyl)]-13,17-bis[2-(etho-
xycarbonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,21,22,23-
tetrahydrobenzo[b]porphyrin (15a). 1H NMR (CDCl3, 500
MHz),δ (ppm) À2.45 and À2.36 (br s, 2H, H-22 and H-24);1.17
and 1.18 (2t, 6H, CH3-135 and CH3-175); 2.11 (s, 3H, CH3-25); 3.14 (t,
2H, J = 7.5 Hz, H-132); 3.19 (t, 2H, J = 7.5 Hz, H-172); 3.40 (s, 3H, CH3-
121); 3.44À3.49 (m, 2H, H-23α and 23β); 3.50 (s, 3H, CH3-181); 3.55
(s, 3H, CH3-71); 3.97À4.01 (m, 1H, H-22); 4.12À4.18(m, 6H, H-131,
H-134 e H-174); 4.32 (m, 2H,J=7.0 Hz H-171) 4.71 (d, 1H, J = 8.5 Hz,
H-21); 6.16 (dd, 1H, J = 11.5 and 1.5 Hz, H-82α); 6.35 (dd, 1H, J = 17.7
and 1.5 Hz, H-82β); 6.99 (d, J = 9.5 2H, H-29 and 213); 7.42 (t, 1H, J = 3.5
Hz, H-24); 7.82 (d, J = 9.5 2H, H-210 and H-212); 8.18 (dd, 1H, J = 17.7
and 11.5 Hz, H-81); 9.08 (s, 1H, H-20); 9.33 (s, 1H, H-5); 9.71 (s, 1H,
H-15), 9.87 (s, 1H, H-10). 13C NMR (CDCl3, 125.77 MHz) δ (ppm):
11.4 (C-121); 11.7 (C-181); 12.3 (C-71); 14.1 (C-175); 14.2 (C-135),
19.9 (C-171), 21.5 (C-131) 21.8 (C-171); 22.7 (C-131); 25.7 (C-23);
26.4 (C-25); 36.8 (C-172); 37.2 (C-22); 38.6 (C-132); 50.2 (C-21); 52.3
(C-2); 60.4 (C-174); 60.6 (C-134); 90.4 (C-5); 93.1 (C-20); 98.2 (C-
15); 99.9 (C-10); 115.3 (C-24); 121.4 (C-82); 123.7 (C-210 and C-212);
126.5 (C-29 and C-213); 129.4 (C-7); 129.7 (C-81); 130.0 (C-18); 130.9
(C-28); 132.6 (C-9); 133.8 (C-8); 134.0 (C-16); 136.4 (C-17); 136.6
(C-12);136.8 (C-11);138.3 (C-6); 138.5 (C-19); 139.9 (C-13); 146.5
(C-211); 149.7 (C-14); 151.5 (C-4); 151.8 (C-3); 165.4 (C-1); 172.9
(C-133); 173.3 (C-173); 174.1 (C-27); 177.9 (C-26). ESI-MS-TOF, m/z
837.3606 calculated for C48H49N6O8+ (MH+); found, 837.3616.
21,22[N,N-Dicarbonyl-N-(4-nitrophenyl)]-8,12-bis[2-(meth-
oxycarbonyl)ethyl]-2,7,13,17-tetramethyl-18-vinyl-2,21,22,
23-tetrahydrobenzo[b]porphyrin (15b). 1H NMR (CDCl3, 500
MHz), δ (ppm): À2.45 (br s, 2H, H-21 and H-23); 1.15 and 1.17 (2t,
6H, CH3-125 and CH3-85); 2.10 (s, 3H, CH3-25), 3.15 (t, 2H, J = 8.0 Hz,
H-122), 3.19 (t, 2H, J = 8.0 Hz, H-82); 3.43 (s, 3H, CH3-131); 3.47 (s,
3H, CH3-71); 3.48À3.50 (m, 2H, H-23α and H-23β);3.62 (s, 3H, CH3-
171), 3.95À4.00 (m, 1H, H-22) 4.10À4.19(m, 6H, H-121, H-124 e
H-84); 4.32 (t, 2H, J = 8.0 Hz, H-81), 4.68 (d, 1H, J = 8.5 Hz, H-21); 6.12
(dd, 1H, J = 11.5 and 1.5 Hz, H-182α); 6.34 (dd, 1H, J = 17.5 and 1.5 Hz,
H-182β); 6.97 (d, J = 9.0 2H, H-29 and 213); 7.41À7.43 (m, 1H, H-24);
7.80 (d, 2H, J = 9.0, H-210 and H-212); 8.11 (dd, 1H, J = 17.5 and 11.5
Hz, H-181); 9.27 (s, 2H, H-5 and, H-20); 9.74 (s, 1H, H-10), 9.78 (s, 1H,
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR and 13C NMR spec-
S
b
tra of compounds 8, 9, 10, 13a, 13b, 14a, 14b; 15a, and 15b. The
X-ray crystallographic (CIF) file is available. Figures of 1H NMR
studies about aggregation for compounds 13b, 14a, and 14b have
also been included. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: adjaci@usp.br; osaserra@usp.br.
Present Addresses
†Centro de Engenharia Biomꢀedica, Universidade Camilo Castelo
Branco, Parque Tecnolꢀogico de S~ao Josꢀe dos Campos, Rodovia
Presidente Dutra, Km 138, 12247-004, S~ao Josꢀe dos Campos-SP,
Brazil.
’ ACKNOWLEDGMENT
The authors wish to thank the FAPESP, CNPq, and CAPES
for financial support of this research.
8831
dx.doi.org/10.1021/jo201568n |J. Org. Chem. 2011, 76, 8824–8832

The Journal of Organic Chemistry
ARTICLE
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