Synthesis of functionalized chlorins sterically-prevented from self-aggregation

Article abstract of DOI:10.1016/j.dyepig.2013.05.024

The synthesis of six new regioisomeric chlorin derivatives sterically-prevented from self-aggregation is described. The compounds were prepared by the Diels-Alder reaction between protoporphyrin IX dimethyl ester, and N-[p-(1,3-dithiolan)phenyl]maleimide and N-(p-formylphenyl)maleimide. The protopophyrin IX dimethyl ester was synthesized in 2 steps from natural hematoporphyrin using a modified procedure from literature, and the substituted maleimides were conveniently synthesized, aiming at producing formyl-chlorins for subsequent functionalization with amphiphilic groups. The Diels-Alder reactions were systematically studied in order to establish optimized conditions for the cycloadditions. The regioisomers were fully characterized and the aggregation studies were performed by NMR, UV-Vis spectroscopy, and also HRMS (ESI-TOF and MALDI-TOF). Preliminary evaluations on the photosensitizing activities and amphiphilicity were carried out indicating that these new compounds are potential candidates, to be studied in more advanced tests of Photodynamic Therapy (PDT). This work represents on advance on our previous study, with respect to these new structures, their photophysical properties and amphiphilicity.

Full text of DOI:10.1016/j.dyepig.2013.05.024

Accepted Manuscript
Synthesis of Functionalized Chlorins Sterically-Prevented from Self-Aggregation
Fabiane A.B. dos Santos, Adjaci F. Uchoa, Mauricio S. Baptista, Yassuko Iamamoto,
Osvaldo A. Serra, Timothy J. Brocksom, Kleber T. de Oliveira
PII:
S0143-7208(13)00195-2
10.1016/j.dyepig.2013.05.024
DYPI 3961
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 19 March 2013
Revised Date: 24 April 2013
Accepted Date: 23 May 2013
Please cite this article as: dos Santos FAB, Uchoa AF, Baptista MS, Iamamoto Y, Serra OA, Brocksom
TJ, de Oliveira KT, Synthesis of Functionalized Chlorins Sterically-Prevented from Self-Aggregation,
Dyes and Pigments (2013), doi: 10.1016/j.dyepig.2013.05.024.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of Functionalized Chlorins Sterically-Prevented from
Self-Aggregation
a
b,c
c
Fabiane A. B. dos Santos, Adjaci F. Uchoa, Mauricio S. Baptista, Yassuko
b
b
a
a,
Iamamoto, Osvaldo A. Serra, Timothy J. Brocksom, Kleber T. de Oliveira *
a. Departamento de Química, Universidade Federal de São Carlos - UFSCar,
São Carlos - SP – Brazil, 13565-905.
b. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de
Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900,
14040-901, Ribeirão Preto-SP, Brazil.
c. Departamento de Bioquímica, Instituto de Química, Universidade de São
Paulo, Avenida Prof. Lineu Prestes 748, Cidade Universitária, 05508-000, São
Paulo-SP, Brazil
*
Corresponding author: Kleber Thiago de Oliveira
Departamento de Química - UFSCar
Universidade Federal de São Carlos
Rodovia Washington Luiz, km 235, São Carlos, Brazil
CEP: 13565-905
Phone: +55 - 16 – 3351 8083 Fax: +55 – 16 – 3351 8350
*
kleber.oliveira@ufscar.br
1

ACCEPTED MANUSCRIPT
Abstract
The synthesis of six new regioisomeric chlorin derivatives sterically-prevented
from self-aggregation is described. The compounds were prepared by the Diels-
Alder reaction between protoporphyrin IX dimethyl ester, and N-[p-(1,3-
dithiolan)phenyl]maleimide
and
N-(p-formylphenyl)maleimide.
The
protopophyrin IX dimethyl ester was synthesized in 2 steps from natural
hematoporphyrin using a modified procedure from literature, and the substituted
maleimides were conveniently synthesized, aiming at producing formyl-chlorins
for subsequent functionalization with amphiphilic groups. The Diels-Alder
reactions were systematically studied in order to establish optimized conditions
for the cycloadditions.
The regioisomers were fully characterized and the aggregation studies were
performed by NMR, UV-Vis spectroscopy, and also HRMS (ESI-TOF and
MALDI-TOF). Preliminary evaluations on the photosensitizing activities and
amphiphilicity were carried out indicating that these new compounds are
potential candidates, to be studied in more advanced tests of Photodynamic
Therapy (PDT). This work represents on advance on our previous study, with
respect to these new structures, their photophysical properties and
amphiphilicity.
2

ACCEPTED MANUSCRIPT
Keywords: Protoporphyrin IX, chlorin derivatives, Diels-Alder, non-aggregation,
photosensitizers, PDT and amphiphilicity.
Highlights:
-
-
-
-
Synthesis of six chlorins sterically self-prevented from aggregation is described
A vinyl porphyrin and p-substituted maleimides were used as building blocks.
Amphiphilic chlorins were obtained after functionalizations
These new chlorins have proven to be good candidates for PDT studies.
3

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Introduction
Protoporphyrin IX and chlorophylls can be considered as the dyes of life
since animal and vegetal existence on earth depends on the activities of these
molecules.[1] For animals, protoporphyrin IX is essential in cellular breathing,
and for plants the chlorophylls are essential to transfer light energy through the
photosynthetic process. Therefore, there is no surprise that these
biosynthetically-related pigments can act as photosensitizers, and some
modified structures present improved ability to transfer light energy.[2] It is well
known that when light excites a photosensitizer this may provide a type-II redox
reaction in cells through singlet oxygen formation, which is an essential
component of photodynamic therapy (PDT).[3]
In this context, the chemistry of protopophyrin IX has attracted attention
since the discovery of the photosensitizing ability of this compound, and many
papers have been published recently.[2,4,5,6] In general, protoporphyrin IX
exhibits an exceptional combination of advantages, such as optimal
photophysical properties, low systemic toxicity, and a great affinity for
hyperproliferating tissues.[2] In addition, several of the successful commercial
photosensitizers, such as Visudyne® and Lemuteporfin® are an excellent
example of chlorin derivatives synthesized from protoporphyrin IX, and present
remarkable photodynamic activities.[2]
However, a general limitation for the use of chlorin derivatives in PDT is
their strong tendency for aggregation in solution and their low chemical stability.
Stability of chlorin derivatives has been achieved by using other methodologies,
4

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highlighting those based on C-C bond formation at the β-position by using
cycloaddition reactions.[4,5,6]
In conclusion, methodologies to produce stable and non-aggregating
photosensitizers are the aim of our research, since the aggregation
phenomenon has also been decisive for low singlet oxygen generation and for
decreased ability to act as a photosensitizer in PDT treatments.
In previous work we have demonstrated that chlorin derivatives, which were
synthesized through the Diels-Alder reaction between protoporphyrin IX
dimethyl ester and maleimides, presented a special “L-shape” structure and
were sterically prevented from self-aggregation.[6] We now describe the
synthesis and preliminary evaluation of some functionalized chlorins prepared
from different substituted maleimides, which were designed to produce non-
aggregating and amphiphilic photosensitizers.
1. Materials and Methods
1.1. Reagents and measurements
®
All reagents were of analytical grade and were purchased from Aldrich or
some national suppliers. If necessary, solvents and reagents were only used
after purification according to standard procedures.[7] Protoporphyrin IX was
supplied by Frontier (Texas, USA). Ultrasound irradiation was employed for
1
13
deoxygenation of the toluene used in the Diels-Alder reactions. H and C NMR
spectra were recorded on a Bruker Avance 400 spectrometer at 400.13 MHz
and 100.13 MHz, respectively, using CDCl
3
as the solvent and TMS as the
5

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1
13
internal reference. Unequivocal H and C assignments were carried out by
1
1
1
13
1
13
using 2D gCOSY ( H/ H), gHSQC ( H/ C), gHMBC ( H/ C) and NOESY
spectra (mixing time of 800 ms). The chemical shifts are expressed in δ (ppm)
and the coupling constants (J) are given in Hertz (Hz). HRMS were obtained
using the ESI-TOF (Max Impact Bruker) and MALDI-TOF (AutoFlex Speed
Bruker, laser 355 nm, 50 Hz, spectrometer, α-cyano-4-hydroxycinnamic acid as
matrix, and Flex analysis as processing software). The UV-Vis absorption
spectra were recorded on a Perkin Elmer Lambda 25 spectrophotometer.
Column chromatography was carried out using silica gel 200-400 mesh
from Aldrich, and the preparative thin layer chromatography (TLC) was
conducted on Aluminum sheets (1 mm thick) – Merck TLC Silica gel 60 F254
.
1.2. Synthesis of 2-(4-nitrophenyl)-1,3-dithiolane (2):
To a solution of 4-nitrobenzaldehyde (1) (5.10 g, 34.0 mmol) in anhydrous
dichloromethane (150 mL), 1,2-ethanedithiol (14.8 mL, 17.6 mmol) and
BF
h. After that, the reaction was washed with 5% NaOH (100 mL), water (100
mL) and brine (100 mL). The organic layer was dried over anhydrous Na SO
3 2
.OEt (0.8 mL) were added, maintaining the stirring at room temperature for
6
2
4
and the solvent was distilled off under reduced pressure. Compound 2 was
purified by flash chromatography in silica gel using hexane:ethyl acetate (8:2)
o
1
as eluent. Yield: 6.90 g; 30.4 mmol; 89%. mp: 76-78 C. H NMR (CDCl
3
, 400
MHz) δ (ppm): 3.36-3.56 (m, 4H); 5.65 (s, 1H); 7.67 (d, 2H, J = 8.6 Hz); 8.16 (d,
1
3
2
H, J = 8.6 Hz). C NMR (CDCl
3
, 100 MHz) δ (ppm): 148.6 (1C); 147.4 (1C);
-
1
128.8 (2CH); 123.7 (2CH); 54.9 (1CH); 40.5 (2CH
2
). IR (νmax., KBr, cm ): 3115,
6

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3099, 3078, 2920, 2853,1108, 1605, 1593, 1517, 1349, 866, 727 , 715. GC-MS
m/z: 227, 199, 182, 166, 152, 121, 77.
1.3. Synthesis of 4-(1,3-dithiolan-2-yl)aniline (3):
2 2
To a solution of compound 2 (5.08 g, 22.0 mmol) in 95% ethanol, SnCl .2H O
o
(
25.3 g, 0.11 mol) was added. The reaction was heated at 70 C for 3h. The
reaction mixture was then poured onto crushed ice (100 g) and the pH was
adjusted with aqueous NaHCO 5% to about 8. The mixture was extracted with
ethyl acetate (300 mL) and the organic layer was washed with brine (100 mL)
and dried over Na CO . The solvent was distilled off under reduced pressure
and compound 3 was obtained without further purification. Yield: 4.30 g; 21.7
3
2
3
o
1
mmol; 93%. mp: 56-58 C. H NMR (CDCl
3
, 400MHz) δ (ppm): 3.28-3.36 (m,
2
7
H); 3.45-3.52 (m, 2H); 3.69 (br.s, 2H); 5.61 (s, 1H); 6.61 (d, 2H, J = 8.4 Hz);
1
3
3
.31 (d, 2H, J = 8.4 Hz). C NMR (CDCl , 100MHz) δ (ppm): 146.3 (1C); 129.1
-
1
(1C); 129.0 (2CH); 114.9 (2CH); 56.4 (1CH); 40.1 (2CH
2
). IR (νmax., KBr, cm ):
3
5
423, 3383, 3308, 3032, 3007, 2916, 1624, 1607, 1513, 1279, 1174, 750, 685,
25.
1.4. Synthesis of 1-[4-(1,3-dithiolan-2-yl)phenyl]-1H-pyrrole-2,5-dione (4):
o
To a solution of compound 3 (3.73 g, 18.9 mmol) in CH Cl (20 mL) at 0 C,
2
2
maleic anhydride (1.85 g, 18.9 mmol) dissolved in CH
2
Cl
2
(20 mL) was slowly
added. After 1h, the solid formed was filtered and washed with small portions of
7

ACCEPTED MANUSCRIPT
cooled CH
2
Cl
2
. The solid amide-acid was dried under vacuum and used without
o
1
further purification. Yield: 4.98 g; 16.9 mmol; 84%. mp: 181-183 C. H NMR
DMSO-d , 400 MHz) δ (ppm): 3.30-3.55 (m, 4H); 5.72 (s, 1H); 6.31 (d, 1H, J =
2.1Hz); 6.47 (d,1H, J = 12.1 Hz); 7.46-7.48 (m, 2H); 7.56-7.58 (m, 2H); 10.43
(
6
1
1
3
(
(
(
s,1H); 12.97 (s,1H). C NMR (DMSO-d
6 2
, 100 MHz) δ (ppm): 39.7 (2CH ); 54.7
1CH); 119.4 (1CH); 128.4 (1CH); 130.5 (2CH); 131.5 (2CH); 136.0 (1C); 138.1
-
1
1C); 163.2 (1C); 166.9 (1C). IR (νmax., KBr, cm ): 3092; 1920; 1857; 1794;
1698; 1524; 1504; 1323; 852.
To the amide-acid obtained previously (2.50 g, 8.46 mmol), anhydrous sodium
acetate (1.21 g, 8.46 mmol) and freshly purified acetic anhydride (100 mL) were
added at room temperature. After 1h stirring under a N atmosphere, the
2
o
reaction was heated at 90 C for 1h. Cold water (200 mL) was added and the
reaction was stirred up to room temperature (~ 30 min). The reaction was
extracted with ethyl acetate (2 x 100 mL) and the organic layer washed with
saturated NaHCO solution (2 x 100 mL), brine (100 mL) and dried over
3
anhydrous Na SO . Compound 4 was obtained without further purification, after
2
4
solvent evaporation under reduced pressure. Yield: 2.14 g; 7.75 mmol; 92%.
o
1
mp: 168-170 C. H NMR (CDCl
3
, 400 MHz) δ (ppm): 3.34-3.40 (m, 2H); 3.47-
3
=
.54 (m, 2H); 5.65 (s, 1H); 6.85 (s, 2H); 7.31 (d, 2H, J = 8.5 Hz); 7.63 (d, 2H, J
1
3
8.5 Hz). C NMR (CDCl
3
, 100 MHz) δ (ppm): 169.4 (2C); 140.3 (1C);134.2
(
2CH); 130.7 (1C); 128.8 (2CH); 125.9 (2CH); 55.6(1CH); 40.2 (2CH
2
). IR
-
1
(
ν
max., KBr, cm ): 3097, 2920,1513, 1400, 1711, 1151, 832, 710. HRMS (ESI-
+
TOF): m/z calculated for [M+H] 278.0304; found 278.0301.
8

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1.5. Synthesis of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzaldehyde (5):
o
To a solution of compound 4 (2.00 g, 7.21 mmol) in acetone (80 mL) at 0 C,
NBS (5.13 g, 28.8 mmol) dissolved in acetone:water 9:1 (80 mL) was added,
and the stirring was maintained for 1h. Water (100 mL) was added and the
reaction was extracted with CH
2 2
Cl (3 x 100 mL). The organic layer was dried
over anhydrous Na SO and the solvent distilled off under reduced pressure.
2
4
Compound 5 was purified by flash chromatography in silica gel using
toluene:ethyl acetate (9:1) as eluent. Yield: 1.26 g; 6.26 mmol; 87%. mp: 129-
o
1
1
2
1
31 C. H NMR (CDCl , 400 MHz) δ (ppm): 10.04 (s, 1H); 6.91 (s, 2H); 7.63 (d,
3
1
3
H, J = 8.6 Hz); 7.99 (d, 2H, J = 8.6 Hz). C NMR (CDCl
3
, 100 MHz) δ (ppm):
25.7 (2CH); 130.4 (2CH); 134.5 (2CH); 135.0 (C); 136.6 (C); 168.8 (2C); 191.1
-
1
(
1CH). IR (νmax., KBr, cm ): 2854, 1776, 1718, 1701, 1603, 1395, 837, 827.
+
HRMS (ESI-TOF): m/z calculated for [M+H] 202.0499; found 202.0504.
1.6. Synthesis of protoporphyrin IX dimethyl ester (7):
To a solution of 2.00 g of hematophorphyrin (6) (3.34 mmol) in chlorobenzene
(1L), dried p-toluenesulfonic acid (5.0 g, 30 mmol) was added and the reaction
o
was heated at 140 C under a N
2
atmosphere for 3h. After that, the
chlorobenzene was distilled off under reduced pressure, and the reaction was
treated with a 5% methanolic solution of H SO (670 mL) for 12h at room
2
4
temperature and with protection from light. The reaction was then poured onto
00 g of crushed ice and neutralized with concentrated NH OH solution to a pH
8
4
of about 9. The reaction was extracted with CH
2
Cl
2
(3 x 100 mL) and the
9

ACCEPTED MANUSCRIPT
2 4
organic layer was dried over Na SO . Compound 7 was obtained after solvent
evaporation and crystallization from CHCl :MeOH (1:3). Yield: 1.40 g, 2.40
3
1
mmol, 72%. H NMR (CDCl
3
, 400MHz)
δ
ppm: –3.69 (s, 2H); 3.28 (t, 4H, J = 7.5
Hz); 3.62 (s, 3H); 3.63 (s, 3H); 3.66 (s, 6H); 3.70 (s, 3H); 3.71 (s, 3H); 4.40 (t,
4
H, J = 7.5 Hz); 6.17 (d, 2H, J = 11.6 Hz); 6.37 (d, 2H, J = 17.7 Hz), 8.27 (dd,
H, J = 17.7 Hz, J = 11.6 Hz); 10.03 (s, 3H); 10.08 (s, 1H); 10.16 (s, 3H); 10.21
2
(s, 3H).
1.7. General procedure for the Diels-Alder reactions between 7 and
maleimides 4 and 5.
To a solution of protoporphyrin dimethyl ester (7) (60 mg, 0.1 mmol) in
degassed and dry toluene (5 mL), maleimides 4 or 5 were added in different
molar proportions (Tables 1 and 2). The reactions were performed in glass
®
pressure tubes (Ace pressure tubes from Aldrich ) at different temperatures and
reaction times. The reactions were monitored by TLC and UV-Vis spectroscopy
in order to verify the end of the starting material. The reaction products were
purified by flash column chromatography in silica gel using toluene:ethyl acetate
2 2
8.5:1.5 as eluent, and the chlorin derivatives were crystallized from CH Cl and
hexanes. Compounds 8a-b and 9a-b were obtained in different yields as
described in Tables 1 and 2.
1
2
2
,2 [N,N-dicarbonyl-N-4-(1,3-dithiolan-2-yl)phenyl]-13,17-bis[2-
1
2
3
(
methoxycarbonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,2 ,2 ,2 -
1
0

ACCEPTED MANUSCRIPT
1
tetrahydrobenzo[b]porphyrin (8a): H NMR (CDCl
3
, 400 MHz) δ (ppm): -2.40
5
and -2.48 (br.s, 2H, H-22 e H-24); 2.08 (s, 3H, CH -H2 ); 3.05-3.11 and 3.14-
3
2
2
15
16
1
3
2
1
2
.22 (m, 6H e 2H, H-13 , H-17 , H2 , H2 ); 3.40 (s, 3H, H-12 ); 3.45-3.47 (m,
3
α
3β
1
1
H, H-2 e 2 ); 3.49 (s, 3H, H-18 ); 3.56 (s, 3H, H-7 ); 3.65 and 3.68 (s, 6H; H-
4
4
2
1
3 and H-17 ); 3.90-3.94 (m, 1H, H-2 ); 4.18 (t, 2H, J = 7.8 Hz, H-13 ); 4.31 (t,
1
1
H, J = 6.6 Hz, H-17 ), 4.65 (d, 1H, J = 8.6 Hz, H-2 ); 6.15 (dd, 1H, J = 1.3 Hz,
2
α
2β
J = 11.6 Hz, H-8 ); 6.35 (dd, H, J = 1.3 Hz and J = 17.8 Hz, H-8 ); 6.64 (d, 2H,
9
13
10
12
J = 8.6 Hz, H-2 and H-2 ); 7.10 (d, 2H, J = 8.6 Hz, H-2 and H-2 ); 7.41 (t,
4
1
1
H, J = 5.3 Hz, H-2 ); 8.19 (dd, 1H, J = 17.8 Hz and 11.6 Hz, H-8 ); 9.09 (s, 1H,
1
3
H-20); 9.32 (s, 1H, H-5); 9.67 (s, 1H, H-15), 9.85 (s, 1H, H-10). C NMR
1
1
1
1
(
CDCl
3
, 100 MHz) δ (ppm): 11.4 (C-18 ); 11.6 (C-12 ); 12.3 (C-7 ); 21.5 (C-17 );
1
3
5
2
2
2
2
3
2
1
1.9 (C-13 ); 25.6 (C-2 ); 26.5 (C-2 ); 36.6 (C-17 ); 37.0 (C-13 ); 38.5 (C-2 );
1
5
16
1
4
4
9,8 and 39,9 (C2 and C2 ); 50.1 (C-2 ); 51.7 (C-13 ); 51.8 (C-17 ); 52.3 (C-
1
4
4
); 55.3 (C-2 ); 90.4 (C-5); 93.4 (C-20); 97.9(C-15); 99.8 (C-10); 115.6 (C-2 );
2
9
13
10
12
21.4 (C-8 ); 125.9 (C-2 and C-2 ); 128.2 (C-2 and C-2 ); 129.2 (C-7); 129.8
1
8
(
C-8 ); 130.8 (C-2 ); 130.9 (C-18); 132.6 (C-9); 133.7 (C-8); 133.9 (C-16); 136.2
11
(
C-17); 136.5 (C-6);138.3 (C-12 e C-19);139.6 (C-13); 140.5 (C-2 ); 149.5 (C-
3
3
); 151.0 (C-14); 151.3 (C-11); 152.2 (C-4); 165.9 (C-1); 173,4 (C-17 ); 173.8
3
6
7
(
C-13 );174.8 (C-2 ); 178.5 (C-2 ). HRMS (MALDI-TOF): m/z calculated for
+
[
M+H] 868.3197; found 868.3207. UV-Vis (CH
2
Cl
2
) λmax nm (log ε) 407 (5.26),
505 (4.08), 540 (4.10), 610 (3.59), 667 (4.58).
1
2
2
,2 [N,N-dicarbonyl-N-4-(formylphenyl)]-13,17-bis[2-(methoxycarbonyl)ethyl]-
1
2
3
1
2
,7,12,18-tetramethyl-18-vinyl-2,2 ,2 ,2 -tetrahydrobenzo[b]porphyrin (9a):
H
1
1

ACCEPTED MANUSCRIPT
NMR (CDCl
3
, 400 MHz) δ (ppm): -2.47 and -2.39 (br.s, 2H, H-22 e H-24); 2.07
5
2
2
(
s, 3H, H-2 ); 3.15 (t, 2H, J = 7.8 Hz, H-13 ); 3.20 (t, 2H, J = 7.8 Hz, H-17 );
1
3α
3β
1
3
.40 (s, 3H, H-12 ); 3.44-3.47 (m, 2H, H-2 and 2 ); 3.50 (s, 3H, H-18 ); 3.55
1
4
4
2
(
s, 3H, H-7 ); 3.65 and 3.68 (s, 6H; H-13 and H-17 ); 3.89-3.93 (m, 1H, H-2 );
1
1
4
.18 (t, 2H, J = 7.8 Hz, H-13 ); 4.31 (t, 2H, J = 7.6 Hz, H-17 ), 4.64 (d, 1H, J =
1
2α
8
.6 Hz, H-2 ); 6.15 (dd, 1H, J = 1.5 Hz, J = 11.5 Hz, H-8 ); 6.34 (dd, H, J = 1.5
2
β
9
13
11
Hz, J = 17.8 Hz, H-8 ); 6.91-6.94 (m, 2H, H-2 and 2 );7.44-7.47 (m, 2H, H-2
1
2
4
and H-2 ); 7.41 (t, 1H, J = 5.4 Hz, H-2 ); 8.17 (dd, 1H, J = 17.8 and 11.5 Hz, H-
1
8
9
1
1
2
1
1
1
1
1
); 9.07 (s, 1H, H-20); 9.33 (s, 1H, H-5); 9.69 (s, 1H, H-15), 9.86 (s, 1H, H-10);
1
4
13
1
3
.64 (s, 1H, H-2 ). C NMR (CDCl , 100 MHz) δ (ppm): 11.4 (C-18 ); 11.6 (C-
1
1
1
1
3
5
2 ); 12.3 (C-7 ); 21.5 (C-17 ); 21.9 (C-13 ); 25.6 (C-2 ); 26.4 (C-2 ); 36.6 (C-
2
2
2
1
4
4
7 ); 37.0 (C-13 ); 38.5 (C-2 ); 50.2 (C-2 ); 51.7 (C-13 ); 51.8 (C-17 ); 52.3 (C-
4
2
); 90.4 (C-5); 93.2 (C-20); 98.1(C-15); 99.9 (C-10); 115.6 (C-2 ); 121.4 (C-8 );
9
13
10
12
1
26.3 (C-2 and C-2 ); 129.7 (C-2 and C-2 ); 129.2 (C-7); 129.8 (C-8 );
1
1
30.9 (C-18); 132.6 (C-9); 133.8 (C-8); 133.9 (C-16); 135.2 (C-2 ); 136.3 (C-
8
7); 136.5 (C-6 and C-2 );138.3 (C-19); 138.4 (C-12); 139.7 (C-13); 149.6 (C-3);
3
51.1 (C- 14); 151.5 (C-11); 152.0 (C-4); 165.7 (C-1); 173.4 (C-17 ); 173.8 (C-
3
6
7
14
3 ); 174.3 (C-2 ); 178.1 (C-2 ); 190.8 (C-2 ). HRMS (MALDI-TOF): m/z
+
calculated for [M+H] 792.3392; found 792.3420. UV-Vis (CH Cl ) λ
(log ε)
2
2
max nm
407 (5.25), 505 (4.11), 540 (4.11), 610 (3.65), 667 (4.65).
1
2
2
,2 [N,N-dicarbonyl-N-4-(1,3-dithiolan-2-yl)phenyl]-8,12-bis[2-
1
2
3
(
methoxycarbonyl)ethyl]-2,7,13,17-tetramethyl-8-vinyl-2,2 ,2 ,2 -
1
tetrahydrobenzo[b]porphyrin (8b): H NMR (CDCl
3
, 400 MHz) δ (ppm):-2.44
1
2

ACCEPTED MANUSCRIPT
5
(
br.s, 2H, H-21 and H-23); 2.06 (s, 3H, H-2 ); 3.15-3.22 and 3.05–3.12 (m, 8H,
2
2
15
16
1
3α
3β
H-12 , H-8 , H-2 , H-2 ); 3.42 (s, 3H, H-13 ); 3.44-3.45 (m, 2H, H-2 and 2 );
1
1
4
4
3
.47 (s, 3H, H-7 ); 3.61 (s, 3H, H-17 ); 3.65 and 3.66 (s, 6H; H-12 and H-8 );
2
1
3
.89-3.93 (m, 1H, H-2 ); 4.18 (t, 2H, J = 7.7 Hz, H-12 ); 4.32 (t, 2H, J = 7.7 Hz,
1
1
H-8 ), 4.63 (d, 1H, J = 8.6 Hz, H-2 ); 6.11 (dd, 1H, J = 1.4 Hz, J = 11.5 Hz, H-
2
α
2β
9
1
2
1
8 ); 6.33 (dd, H, J = 1.4 Hz, J = 17.8 Hz, H-18 ); 6,62-6,64 (m, 2H, H-2 and
1
3
10
12
4
); 7,08-7,10 (m, 2H, H-2 and H-2 ); 7.41 (t, 1H, J = 5.3 Hz, H-2 ); 8.13 (dd,
1
H, J = 17.8 Hz and 11.5 Hz, H-18 ); 9.25 (s, 1H, H-5); 9.27 (s, 1H, H-20), 9.68
1
3
(
3
s, 1H, H-10), 9.75 (s, 1H, H-15). C NMR (CDCl , 100 MHz) δ (ppm): 11.2 (C-
1
1
1
1
1
3
7
2
); 11.7 (C-13 ); 12.4 (C-17 ); 21.6 (C-8 ); 21.9 (C-12 ); 25.6 (C-2 ); 26.6 (C-
5
2
2
2
15
16
); 36.7 (C-8 ); 37.1 (C-12 ); 38.6 (C-2 ); 39.8 and 39.9 (C2 and C2 ); 50.1
1
4
4
14
(
C-2 ); 51.7 (C-12 ); 51.8 (C-8 ); 52.2 (C-2); 55.3 (C2 ); 89.9 (C-5); 94.1 (C-
4
2
9
2
2
1
1
1
0); 98.4 (C-10); 99.4 (C-15); 116.0 (C-2 ); 120.9 (C-18 ); 125.8 (C-2 and C-
1
3
10
12
1
8
); 128.2 (C-2 and C-2 ); 129.9 (C-18 ); 130,4 (C-7); 130,8 (C-2 ); 131.2 (C-
8); 133.3 (C-9 and C-17); 133.8 (C-16); 135.9 (C-8); 136.8 (C-6); 137.3 (C-19);
1
1
38.0 (C-13); 139.7 (C-12); 140.4 (C-2 ); 149.6 (C-3); 150.6 (C-11); 151.4 (C-
3
3
6
4); 152.8 (C-4); 165.6 (C-1); 173.4 (C-8 ); 173.8 (C-12 ); 174.7 (C-2 ); 178.5
7
+
(
C-2 ). HRMS (MALDI-TOF): m/z calculated for [M+H] 868.3197; found
8
68.3192. UV-Vis (CH Cl ) λ
(log ε) 407 (5.22), 505 (4.07), 539 (4.10),
max nm
2
2
574 (3.63), 667 (4.64).
1
2
2
,2 [N,N-dicarbonyl-N-4-(formylphenyl)]-8,12-bis[2-(methoxycarbonyl)ethyl]-
1
2
3
1
2
,7,13,17-tetramethyl-18-vinyl-2,2 ,2 ,2 -tetrahydrobenzo[b]porphyrin (9b):
H
3
NMR (CDCl , 400 MHz) δ (ppm): -2.43 (br.s, 2H, H-21 and H-23); 2.07 (s, 3H,
5
2
2
1
H-2 ); 3.14-3.17 (m, 2H, H-12 ); 3.18-3.21 (m, 2H, H-8 ); 3.41 (s, 3H, H-13 );
1
3

ACCEPTED MANUSCRIPT
3
α
3β
1
1
3
.44-3.45 (m, 2H, H-2 and H-2 ); 3.46 (s, 3H, H-7 ); 3.61 (s, 3H, H-17 ); 3.64
4
4
2
and 3.66 (s, 6H; H-12 and H-8 ); 3.89-3.93 (m, 1H, H-2 ); 4.18 (t, 2H, J = 7.8
1
1
1
Hz, H-12 ); 4.31 (t, 2H, J = 7.7 Hz, H-8 ), 4.64 (d, 1H, J = 11.8 Hz, H-2 ); 6.11
2
α
(
dd, 1H, J = 1.6 Hz, J = 11.6 Hz, H-18 ); 6.33 (dd, H, J = 1.6 Hz, J = 17.8 Hz,
2β
9
13
10
12
H-18 ); 6.91-6.93 (m, 2H, H-2 and H-2 ); 7.44-7.46 (m, 2H, H-2 and H-2 );
4
1
7
.40 (t, 1H, J = 5.4 Hz, H-2 ); 8.11 (dd, 1H, J = 17.8 Hz and 11.6 Hz, H-18 );
9.26 (s, 2H, H-5 and H-20), 9.70 (s, 1H, H-10), 9.75 (s, 1H, H-15); 9.64 (s, 1H,
1
4
13
1
1
3
H-2 ). C NMR (CDCl , 100 MHz) δ (ppm): 11.2 (C-7 ); 11.7 (C-13 ); 12.4 (C-
1
1
1
3
5
2
1
1
9
7 ); 21.5 (C-8 ); 21.9 (C-12 ); 25.6 (C-2 ); 26.5 (C-2 ); 36.7 (C-8 ); 37.0 (C-
2
2
1
4
4
2 ); 38.6 (C-2 ); 50.1 (C-2 ); 51.7 (C-12 ); 51.8 (C-8 ); 52.2 (C-2); 89,9 (C-5);
4
2
9
3.9 (C-20); 98.5 (C-10); 99.5 (C-15); 115.8 (C-2 ); 120.9 (C-18 ); 126.3 (C-2
13
10
12
1
and C-2 ); 129.7 (C-2 and C-2 ); 129.9 (C-18 ); 130.4 (C-7); 131.2 (C-18);
1
1
1
33.4 (C-9); 133.4 (C-17); 133.8 (C-16); 135.2 (C-2 ); 136.0 (C-8); 136.5 (C-
8
2
);136,8 (C-19); 137.3 (C-6); 138.1 (C-13); 139.8 (C-12); 149,7 (C-3); 150.7
3
3
(
C-11); 151.5 (C-14); 152.7(C-4); 165.4 (C-1); 173.3 (C-8 ); 173.8 (C-12 );
6
7
14
1
74.2 (C-2 ); 178.1 (C-2 ); 190.8 (C-2 ). HRMS (MALDI-TOF): m/z calculated
+
for [M+H] 792.3392; found 792.3356. UV-Vis (CH
2
Cl
2
) λmax nm (log ε) 407
(5.25), 505 (4.09), 539 (4.10), 574 (3.62), 667 (4.65).
2.8. General procedure for the reaction between 9a-b and 2-(aminooxy)ethanol:
To a solution of 10 mg (0.012 mmol) of compounds 9a or 9b in anhydrous and
deoxygenated THF, 2-(aminooxy)ethanol (1.5 mg, 0.02 mmol) and PTSA (2
mg) were added. The reaction was performed in a glass pressure tube (Ace
pressure tube from Aldrich®), purged with argon and stirred at room
1
4

ACCEPTED MANUSCRIPT
temperature for 6 h. The consumption of starting material 9a-b was monitored
by TLC and UV-Vis spectroscopy. The solvent was removed under reduced
pressure when no starting material was detected. The purification was carried
out
dichloromethane:methanol 99:1 as eluent, and the chlorin derivatives were
crystallized from CH Cl /hexane.
by
flash
column
chromatography
in
silica
gel
using
2
2
1
2
2
,2 [N,N-dicarbonyl-N-4-(O-2-hydroxyethyloxime)phenyl]-13,17-bis[2-
1
2
3
(
methoxycarbonyl)ethyl]-2,7,12,18-tetramethyl-18-vinyl-2,2 ,2 ,2 -
tetrahydrobenzo[b]porphyrin (10a): Yield: 7.5 mg; 8.0 µmol; 70%. H NMR
CDCl , 400 MHz) (ppm): -2.47 e -2.39 (sl, 2H); 2.09 (s, 3H); 3.16 (t, 2H, J =
.9 Hz); 3.21 (t, 2H, J = 8.1 Hz); 3.41 (s, 3H); 3.46-3.48 (m, 2H); 3.50 (s, 3H);
.56 (s, 3H); 3.65 and 3.68 (2s, 3H and 3H); 3.73 (t, 2H, J = 4.4Hz) 3.93-3.97
1
(
δ
3
7
3
(m, 1H); 4.09-4.07 (m, 2H); 4.18 (t, 2H, J = 7.8 Hz); 4.31 (t, 2H, J = 7.5 Hz),
4.68 (d, 1H, J = 8.7 Hz); 4.72 (br.s, 1H); 6.15 (dd, 1H, J = 1.3 Hz and 11.5 Hz,
2
α
H-8 ); 6.35 (dd, H, J = 1.3 Hz and 17.9 Hz); 6.77-6.74 (m, 2H); 7.17-7.15 (m,
2H,); 7.42 (t, 1H, J = 5.3 Hz); 7.76 (s, 1H,); 8.19 (dd, 1H, J = 17.9 and
13
=
11.5Hz); 9.09 (s, 1H, H-20); 9.33 (s, 1H, H-5); 9.68 (s, 1H,), 9.86 (s, 1H,). C
(ppm): 11.4 (1CH ); 11.6 (1CH ); 12.3 (1CH ); 21.5
NMR (CDCl , 100 MHz)
δ
3
3
3
3
(
(
(
(
(
(
1CH
1CH); 50.1 (1CH); 51.7 (1CH
1CH ); 90.4 (1CH); 93.2 (1CH); 98.0 (1CH); 99.8 (1CH); 115.5 (1CH); 121.3
2
); 21.9 (1CH
2
); 25.6 (1CH
2
); 26.4 (1CH
3
); 36.6 (1CH
2
); 37.0 (1CH
2
); 38.5
3
); 51.8 (1CH
3
); 52.3 (1C); 62.0 (1CH
2
); 75.1
2
2
1CH ); 126.2 (2CH); 127.1 (2CH); 129.2 (1C); 129.8 (1CH); 130.9 (1C); 131.5
1C); 132.57 (1C); 132.64 (1C); 133,8 (1C); 133.9 (1C); 136.3 (1C); 136.5
1C);138.3 (1C); 138.4 (1C); 139.7 (1C); 148.1 (1CH); 149,6 (1C); 151.1 (1C);
1
5

ACCEPTED MANUSCRIPT
151.4 (1C); 152.1 (1C); 165.8 (1C); 173.4 (1C); 173.8 (1C); 174.6 (1C); 178.4
+
(
1C). HRMS (ESI-TOF): m/z calculated for [M ] 850.3685; found 850.3684.
2 2
UV-Vis (CH Cl ) λmax (log ε) 407 (5.14), 503 (4.00), 540 (4.00), 610 (3.57),
668 (4.50).
1
2
2
,2 [N,N-dicarbonyl-N-4-(O-2-hydroxyethyloxime)]-8,12-bis[2-
1
2
3
(
methoxycarbonyl)ethyl]-2,7,13,17-tetramethyl-18-vinyl-2,2 ,2 ,2 -
tetrahydrobenzo[b]porphyrin (10b): Yield: 8.0 mg; 9.0 µmol; 74%. H NMR
CDCl , 400 MHz) (ppm): -2.43 (br.s, 2H); 2.07 (s, 3H); 3,15-3,18(m, 2H);
,18-3,22 (m, 2H); 3.42 (s, 3H); 3.45-3.46 (m, 2H); 3.47 (s, 3H); 3.62 (s, 3H);
.85 and 3.86 (2s, 3H and 3H); 3.72-3.74(m, 2H); 3,92-3,96 (m, 1H); 4.07-4.09
1
(
3
δ
3
3
(m, 2H) 4.19 (t, 2H, J = 7.8 Hz); 4.32 (t, 2H, J = 7.8 Hz), 4.65 (d, 1H, J = 8.7
Hz); 6.11 (dd, 1H, J = 1.1 Hz and 11.6 Hz); 6.33 (dd, H, J = 1.1 Hz and 17.9
Hz); 6.74 (d, 2H, J = 8.7Hz); 7.15 (d, 2H, J = 8.7Hz); 7.42 (t, 1H, J = 5.3 Hz);
7
9
1
3
.76 (s, 1H); 8.13 (dd, 1H, J = 11.6 and 17.9 Hz); 9.27 (s, 2H), 9.69 (s, 1H),
13
3 3 3
.76 (s, 1H). C NMR (CDCl , 100 MHz) δ (ppm): 11.2 (1CH ); 11.7 (1CH );
2.4 (1CH ); 21.5 (1CH ); 21.9 (1CH ); 25.6 (1CH ); 26.5 (1CH ); 36.7 (1CH );
3
2
2
2
3
2
7.1 (1CH
); 75.1 (1CH
1CH); 120.9 (2CH
2
); 38.6 (1CH); 50.1 (1CH); 51.7 (1CH
3
3
); 51.8 (1CH ); 52.2 (1C); 62.0
(
1CH
2
2
); 89,9 (1CH); 94.0 (1CH); 98.4 (1CH); 99.4 (1CH); 115.9
); 126.2 (2CH); 127.0 (2CH); 129.9 (1CH); 130.4 (1C);
31.2 (1C); 131.4 (1C); 132.7 (1C); 133.35 (1C); 133.41 (1C); 133,8 (1C);
36.0 (1C); 136.9 (1C); 137.4 (1C); 138.0 (1C); 139.8 (1C); 148.1 (1CH); 149.7
(
2
1
1
(1C); 150.6 (1C); 151.4 (1C); 152.8 (1C); 165.6 (1C); 173.4 (1C); 173.8 (1C);
+
1
74.5 (1C); 178.4 (1C). HRMS (ESI-TOF): m/z calculated for [M ] 850.3685;
1
6

ACCEPTED MANUSCRIPT
found 850.3692. UV-Vis (CH
2 2
Cl ) λmax (log ε) 407 (5.20), 502 (4.07), 539
(4.05), 611 (3.56), 668 (4.55) nm.
2.9. Aggregation studies
The self-aggregation on porphyrins and chlorins is a natural tendency due
to the strong attractive interactions between π-systems of the polyaromatic
compounds. As it is usual in any aggregation equilibrium, the amount of
aggregates should increase with the total solution concentration of the
photosensitizers, unless there are sterically-inhibiting functions on molecules as
those present in 8a-b, 9a-b, 10a-b. To evaluate this phenomenon, NMR and
UV-Vis analyses give clear evidences about the presence or absence of the
aggregates in solution.
1
Thus, the H NMR spectra were acquired in different concentrations in order
to evaluate the phenomenon. Chemical shift variations for each sample were
the analysed parameters. The same was performed for the UV-Vis analyses at
different concentrations and different solvents (CH
2 2
Cl and water). Changes in
the λmax from the bands in UV-Vis spectra were monitored.
2.10. Singlet oxygen measurements
The singlet oxygen quantum yield was determined using 1,3-
-
diphenylisobenzofuran (DPBF) as singlet oxygen quencher.[8] A 1.01x10
4
-1
mol.L solution of DPBF in ethanol was prepared. Solutions of the new chlorins
8a-b, 9a-b, 10a-b) were prepared in the same solvent, with an absorbance of
(
1
7

ACCEPTED MANUSCRIPT
0.4 at the Soret band. The cuvette was filled with 1.5 mL of the chlorin solution
and 1.5 mL of the DPBF solution. The absorbance was recorded and then the
solution was irradiated for 3 second periods, using a red laser operating at 50
mW and 661 nm. Absorbance was recorded again and twelve more irradiations
were made, each one followed by an absorbance measurement. Experiments
were done in triplicate. The same was performed with standard methylene blue
(
MB). The singlet oxygen quantum yields were obtained from Equation (1).[9]
Equation (1)
In this equation, (Φ
∆
) represents the singlet oxygen quantum yield of the
Std
Std
sample and (Φ
∆
) is the standard singlet oxygen quantum yield. R and R are
the rate constants for DPBF consumption in the presence of the chlorins (8a-b,
a-b, 10a-b) and MB, respectively. Since it is a first order reaction, these
9
parameters were obtained from a plot of the natural logarithm (ln) of DPBF
Std
absorbance versus the irradiation time. Iabs and Iabs
are the intensity of
absorbed light for the sample and the standard, respectively.
2.11. Photobleaching studies
2 2
A solution of the new chlorins (8a-b, 9a-b, 10a-b) in CH Cl with an
absorbance value around 1 was prepared, and irradiated for 1 min using 50 mW
as laser potency. The absorbance spectra were recorded always after the
irradiations (10 times), in order to observe the possible photobleaching by
reduction of the photosensitizer concentration.[5]
2.12. Partition coefficients in n-octanol/water (log P)
1
8

ACCEPTED MANUSCRIPT
The partition coefficients were determined at room temperature in n-octanol
water (log POW) according to the shake-flask method. Distilled water and n-
/
butanol were mixed vigorously for 24 h at room temperature, to promote solvent
saturation in both phases. The chlorins (10a-b) were dissolved in the organic
phase (absorbance∼0.5 at the Soret band). Then, 4 mL of each, water and
stock chlorin solution were individually taken, and the vessel was vigorously
stirred for 10 min to allow the partitioning between the aqueous and organic
3
phase. The mixture was centrifuged at 3×10 g for 10 min, and the solvents
o
were separated and kept for equilibration at the test temperature (25 C) for 2 h
before the analysis. The UV–Vis absorption spectra were measured for organic
phases, before and after partitioning and the differences were measured at the
Soret band. The value of log POW can be easily determined by Equation (2):
Equation (2)
Where A
W
is the absorbance of water, given by difference between the
is the absorbance of n-
stock solution before and after partitioning and A
O
octanol, given by stock solution after partitioning. Both absorbances were
measured at the Soret band. VW and V are the volumes of aqueous and n-
B
octanol phases, respectively.[10]
1
9

ACCEPTED MANUSCRIPT
2. Results and Discussion
First, we have performed the syntheses of the new dienophiles 4 and 5
(Scheme 1) which were designed to avoid aggregation in the Diels-Alder
adducts with protopophyrin IX dimethyl ester (7). For this purpose, p-
nitrobenzaldehyde (1) was protected with ethanedithiol under catalytic acid
conditions (yield 89%).[11] Then, compound 2 was reduced with SnCl
2 2
.H O in
CH Cl yielding the amine 3 (93%).[11] Maleimide 4 was synthesized in two
2
2
steps: the first using an addition of 3 to maleic anhydride (84%) [6]; after that,
maleimide 4 was prepared with the mixed anhydride of Ac O/NaOAc (92%).[12]
2
The imide 5 was obtained from the reaction between 4 and NBS/acetone-water
(87%).[13]
Scheme 1
Aiming at the chlorin syntheses, we first prepared protoporphyrin IX
dimethyl ester (7) starting from hematoporphyrin (6) through a modified
literature procedure.[14] Hematoporphyrin (6) was dehydrated in chlorobenzene
under reflux and acid catalysis (PTSA). The solvent was distilled off and the
crude product was immediately esterified in methanol:H SO4 (5%) with
2
protection from light, yielding 7 in 72% (2 steps) after crystallization.
Scheme 2.
2
0

ACCEPTED MANUSCRIPT
After the preparation of compounds 4, 5 and 7, we then performed a
systematic study of the Diels-Alder reaction between 4 and 7, as presented in
Table 1.
Table 1
In our hands, the best condition was obtained by using toluene as solvent,
catalytic amounts of BHT (10 mg) to avoid polymerizations and 20 equiv of
o
maleimide 4, maintaining the temperature at 125 C (glass pressure tube) for
24h. In these conditions, the regioisomeric chlorins 8a and 8b were each
obtained in 37% yield (74% overall yield) after purifications.
Cycloaddition studies were also performed by using maleimide 5 as
presented in Table 2. In all entries, chlorins 9a and 9b were obtained in a
shorter time and by using a reduced amount of equivalents of dienophile 5. The
o
best result was obtained by using 8 equiv of maleimide 5 for 14h, at 125 C
(glass pressure tube) in toluene. In these conditions, chlorins 9a and 9b were
obtained in 36% and 35%, respectively (71% overall yield) after purification.
Table 2
Attempts to deprotect chlorins 8a and 8b were performed in order to have
convergent syntheses of chlorins 9a and 9b, but there was no success. Thus,
the desired functionalized chlorins 9a and 9b were only successfully obtained
2
1

ACCEPTED MANUSCRIPT
from the reaction between 7 and 5. The amphiphilic derivatives 10a and 10b
were then prepared from 9a or 9b by the reaction with 2-(hydroxyamino)ethanol
in THF under PTSA catalysis. The oxime derivatives 10a and 10b were
obtained after purifications in 70% and 74% yield, respectively.
3. Characterizations using 1D and 2D NMR.
1
13
Compounds 8-10 were characterized using 1D ( H, C and DEPT 135) and
2D (gHMBC, gHSQC, gCOSY and gNOESY) NMR analyses, MALDI-TOF or
ESI-TOF HRMS and UV-Vis. Particularly, the 2D NOESY analyses were
decisive to define the regiochemistry of the Diels–Alder reaction (reaction in ring
A or B of porphyrin 7) and the stereochemistry (cis-endo) of the products. For
example, Figure 1 depicts the main NOE correlations for compounds 9a and 9b
to exemplify the characterizations. First, we started from the principle that
1
chlorins 9a and 9b presented a doublet in the H NMRspectrum near to 4.65
1
ppm, and this signal corresponds to H-2 in both cases (Figure 1). For both
f
isolated products (higher or lower R ) we observed a NOE correlation between
1
H-2 and one meso-hydrogen (H-20) in both regioisomers 9a and 9b. For the
f
product with higher R , the meso-hydrogen H-20 presented a NOE correlation
with one vinylic hydrogen (double doublet at 8.11 ppm, J = 17.8 Hz and J = 11.6
Hz) giving us the first evidence that it could be the isomer 9b (Figure 1b).
Looking at the structures of compounds 9a and 9b, only 9b should present a
1
NOE correlation between H-20 and the vinylic hydrogen H-18 . In addition, H-
1
1
8 presents a very elucidative multiplicity (double doublet at 8.11 ppm, J = 17.8
Hz and J = 11.6 Hz) as expected for this hydrogen in the compound 9b. Other
2
2

ACCEPTED MANUSCRIPT
correlations were decisive to conclude about the regiochemistry and the
stereochemistry of 9b and can be seen in Figure 1b. Remarkably, the NOE
1
correlation between H-2 (Figure 1b) and the easily distinguished methyl group
5
H-2 (at 2.06 ppm) was decisive to assign the cis-endo structure of chlorin 9b.
1
2
Also, the NOE correlation between H-2 and H-2 gave us support to conclude
about the endo-stereoselectivity.
Figure 1
The compound with lower Rf, proposed to be 9a, was also deduced from
1
the 2D NOESY spectrum. We observed NOE correlations between H-2 and H-
1
5
2
0 and between H-2 and H-2 . However, in this case we observed a NOE
4
effect between the easily distinguished exocyclic vinylic hydrogen H-2 (triplet at
7.41 ppm and J = 5.4 Hz) and another meso-hydrogen, apparently H-5.
Considering other NOE correlations, we have observed that the proposed H-5
1
presents a NOE correlation with the methyl group H-7 , and these hydrogen
atoms present a NOE correlation with a vinylic hydrogen, with an unambiguous
multiplicity (double doublet at 6.34 ppm, J = 17.8 Hz and J = 1.5 Hz); this last
2β
hydrogen has to be assigned as H-8 , and all the previous correlations gave us
a confirmation about the regiochemistry of this more polar isomer 9a. Similarly,
the cis-endo structure of chlorin 9a was finally deduced from the NOE
1
5
1
2
correlations between H-2 and H-2 and between H-2 and H2 (Figure 1a). As
depicted in Figure 1a, other NOE correlations were decisive for the complete
1
13
assignment of structure 9a. The complete assignments of the H and C of
2
3

ACCEPTED MANUSCRIPT
chlorins 9a and 9b are described in the experimental section, and all the 1D and
2D NMR analyses are included in the supporting information.
The chlorins 8a and 8b were completely elucidated by using 1D and 2D
NMR as carried out for 9a and 9b. Since compounds 10a and 10b were
1
13
synthesized from 9a and 9b respectively, only the H and C NMR and HRMS
analyses were performed and were thus conclusive to assign the structures of
compounds 10a and 10b.
1
4
. Aggregation studies by H NMR spectroscopy.
Aggregation is
a
very undesirable phenomenon presented by
photosensitizers in solutions, and this behavior makes such photosensitizers
less efficient because the singlet oxygen production is strongly affected. In
order to evaluate the aggregation behavior of compounds 8-10 in solution, we
1
decided to carry out a large number of H NMR analyses at different
3
concentrations. First, we prepared 8 solutions of porphyrin 7 in CDCl and the
1
H NMR spectra were recorded with 64 scans. As observed in Figure 2 there
was a remarkable variation in the chemical shift of meso-hydrogen starting from
-
2
-1
-2
-1
0
.2x10 mol.L to 2.1x10 mol.L . These results indicate that the porphyrin 7
presents a strong aggregation in solution, as described in our previous
literature.[6] We have shown that the magnetic anisotropy is strongly affected
1
by aggregation, resulting in a change of the chemical shift in H NMR (around
0.1-0.5 ppm) in more concentrated solutions.
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4

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After that, solutions of compounds 8a, 8b, 9a, 9b, 10a and 10b were
1
individually prepared in CDCl and the H NMR analyses were recorded (64
3
scans) using the same concentrations as for the compound 7 analysis. As
shown in Figure 3 and Figures S1-S7 (Supporting information) all the new
chlorin derivatives were completely prevented from aggregation due to the “L
shape” of these potential photosensitizers. It is important to mention that all the
chlorin concentrations used in these studies were larger than the concentrations
used in common PDT treatments.
Figure 3
5. Aggregation studies by UV-Vis spectroscopy
Aggregation phenomena were also measured by UV-Vis spectroscopy
since this technique is very sensitive, and the concentrations used here are in
the same scale of PDT treatments. The measurements were performed in
2 2
CH Cl and water (containing 4% of DMSO) (Figures 4-5 and Figures S8-S13 -
supporting information). Note that there is no shift at the maximum absorbance
wavelengths. Also, there is a linear increase in the absorption with
concentration confirming the very low tendency for aggregation of the new
chlorins 8a, 8b, 9a, 9b, 10a and 10b. In addition, the solubilization of
compounds 10a-b in aqueous solution, attest their amphiphilic character, which
may have good impact in the PDT efficiency
Figure 4
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5

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Figure 5
6. Singlet oxygen measurements (Φ∆)
In order to verify the ability of these new chlorins to produce singlet oxygen,
we measured the singlet oxygen quantum yield as described in the
literature.[15] For the quantifications, methylene blue (MB) was used as
standard and 1,3-diphenylisobenzofuran (DPBF) as scavenger reagent. The
measurements were performed in ethanol (Figures 6 and 7), detecting the
consumption of DPBF after successive irradiations in the presence of MB or
compounds 8a, 8b, 9a, 9b, 10a and 10b, respectively, giving the rate of singlet
oxygen production (first order kinetics).
Figure 6
Figure 7
∆
Comparing these rates and using the Equation (1) the Φ values for the
series were 8a, Φ = 0.41; 8b, Φ = 0.38; 9a, Φ = 0.35; 9b, Φ = 0.39; 10a, Φ =
∆
∆
∆
∆
∆
0.37; 10b, Φ
∆
=0.36, when compared with methylene blue (Φ
∆
= 0.52). These
singlet oxygen quantum yields showed that the compounds are good
candidates for PDT treatments.
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8. Photobleaching studies
Photobleaching is the photodegradation of photosensitizers after irradiation
with light where singlet oxygen reacts with the proper photosensitizer promoting
its consumption. This is an important parameter to be determined because
stability under light irradiation represents a fundamental characteristic for a
good photosensitizer.[16] All the studies described here were performed in
dichloromethane, by selective irradiation with a red laser at 661 nm and 50 mW.
After 10 irradiation periods of 1 min. each, we obtained the curves shown in
Figure 8 and Figures S14-S19 (supporting information). All the new chlorin
derivatives showed an absorbance decrease around 0.1-0.2 as exemplified for
chlorin 10a in Figure 8. Only the chlorin 8a presented a strong photobleaching
effect with an absorbance decrease around 0.4.
Figure 8
9. Preliminary Amphiphilicity Measurements (Log P)
Another essential property of a dye for PDT treatments is its ability to
incorporate into the cell. This is especially crucial in the case of hydrophobic
compounds, in which passive partitioning into the cytoplasm membrane
appears to be a pre-requisite for their cellular absorption. For such molecules, a
partition coefficient (logP) between an organic solvent phase and water is one
factor that is used to predict the ability of a molecule to diffuse into
biomembranes. In quantitative terms, the hydrophilic character is associated
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with poor distribution of chlorins in n-octanol, with log P <0, and lipophilic
compounds present log P> 1.5. Amphiphilic substances have intermediate
values of log P.[17]
The procedure employed for determining log P was the shake-flask method.
We measured the log P for chlorins 10a and 10b obtaining the same value for
both (log P= 1.3). Based on these results we can suggest that the designed
compounds 10a and 10b can be considered amphiphilic with potential for future
PDT treatments.[18]
7. Conclusions
In this paper we have described a study on the synthesis of six new chlorin
derivatives, using protoporphyrin IX dimethyl ester and p-substituted
phenylmaleimides as building blocks, thus obtaining the chlorin derivations in
1
13
very good yields. All the compounds were fully characterized by H and
C
NMR (1D and 2D), HRMS and UV-Vis. Preliminary photophysical and
photochemical evaluations were performed, showing that the new
photosensitizers are self-prevented from aggregation in solution, and good
candidates for PDT studies. This work was inspired by results that we have
described previously, but here we have achieved advances in improving the
structural features of more amphiphilic photosensitizers for PDT studies.
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8