New π-Extended Watersoluble Squaraines as Singlet Oxygen ...

New π-Extended Watersoluble Squaraines as
Singlet Oxygen Generators
Luca Beverina, Alessandro Abbotto, Mirko Landenna, Michele Cerminara, Riccardo Tubino,
Francesco Meinardi and Giorgio A. Pagani*
Experimental
Supporting Information
1 H and 13 C NMR spectra were recorded using a Bruker AMX-500 spectrometer operating at
500 and 125.70 MHz, respectively. The spectral parameters and calibrations have been previously
reported. 1 Coupling constants are presented in Hz. Absorption spectrometry was performed using a
jasco V570 spectrophotometer. Anhydrous N,N-dimethylformamide (DMF) was supplied by
FLUKA and stored over molecular sieves. Anhydrous acetonitrile was supplied by Aldrich and
stored under nitrogen. Dry solvents were dried overnight on Na 2 SO 4 and filtered immediately
before use. All other commercial chemicals were supplied by Aldrich and used without purification.
Melting points are uncorrected.
1-(3,6,9-trioxadecyl)-pyrrole-2-carbaldehyde (5b). A solution of pyrrole-2-carbaldehyde (10.00
g, 105.15 mmol) in anhydrous DMF (100 ml) was added dropwise under nitrogen atmosphere and
at 0°C to a suspension of NaH (4.40 g of a 60 % mineral oil suspension, 110.00 mmol). The
resulting white suspension was stirred for 30 min. A solution of 3,6,9-trioxadecyl-4-
toluenesulfonate 2 (44.75 g, 106.00 mmol) in anhydrous DMF (50 ml) was added dropwise and the
reaction mixture was stirred at ambient temperature over a week end. The mixture was poured into
500 ml of icy brine to give a brown suspension that was washed with hexane (100 ml) and
extracted with AcOEt (4 x 200 ml). The combined organic phase was washed with brine (4 x 200
ml), dried over Na 2 SO 4 and evaporated to give a yellow, viscous oil that was further purified by
chromatography (SiO 2 , AcOEt) yielding the pure product as a light yellow oil (21.00 g, 66.0 mmol,
83.0 %). 1 H NMR (CDCl 3 ) δ 9.51 (1H, s), 7.07 (1H, m, broad), 6.94 (1H, m, broad), 6.21 (1H, m,
broad), 4.50 (2H, t, J = 6.1 Hz), 3.75 (2H, t, J = 6.1 Hz), 3.52-3.55 (8H, m), 3.37 (3H, s). Anal.
Calcd for C 12 H 19 NO 4 : C, 59.73; H, 7.94; N, 5.81; Found: C, 59.38; H, 7.91; N, 6.23.
1

2-(N,N-diphenylhydrazonomethyl)-1-methyl-pyrrole (6a). A mixture of N,N-diphenylhydrazine
hydrochloride (4.19 g, 19.00 mmol) and N-methylpyrrole-2-carboxaldehyde 5a (2.03 g, 18.62
mmol) was suspended in degassed THF (80 ml) and refluxed under nitrogen atmosphere for 1h. The
Color turned gradually from yellow to deep green and a dark precipitate was formed. The
suspension was cooled and filtered, the solution was evaporated to give a blue sticky solid that was
taken up with a CH 2 Cl 2 and filtered through a pad of florisil. Evaporation of the solvent gave a
waxy off-white solid that was readily air oxidized turning dark and that was used directly without
further purification (3.35 g, 12.17 mmol, 65.3 %). 1 H NMR (DMSO-d 6 ) δ 7.45 (4H, t, J = 7.9 Hz),
7.18 (2H, t, J = 7.3 Hz), 7.14 (1H, s), 7.09 (4H, d, J = 7.2 Hz), 6.87 (1H, t, J = 2.1 Hz), 6.18 (1H,
dd, J = 3.6, 1.52 Hz), 6.00 (2H, t, J = 3.5 Hz), 3.92 (3H, s).
2-(N,N-diphenylhydrazonomethyl)-1-(3,6,9-trioxadecyl)-pyrrole (6b). A mixture of N,Ndiphenylhydrazine
hydrochloride (1.24 g, 5.60 mmol) and aldehyde 5b (1.35 g, 5.60 mmol) was
suspended in degassed THF (50 ml) and refluxed under nitrogen atmosphere for 1h. The color
turned gradually from yellow to black. The suspension was cooled and the solvent evaporated to
give a dark oil that was taken up with a AcOEt and filtered through a pad of Florisil. Evaporation of
the solvent gave the product as a brown oil that was used directly without further purification (1.56
g, 3.80 mmol, 68.0 %). 1 H NMR (DMSO-d 6 ) δ 7.44 (4H, t, J = 7.9 Hz), 7.18 (2H, t, J = 7.3 Hz),
7.14 (1H, s), 7.09 (4H, d, J = 7.7 Hz), 6.92 (1H, m, broad), 6.19 (1H, m, broad), 6.02 (2H, t, J = 3.3
Hz), 4.49 (2H, t, J = 6.1 Hz), 3.77 (2H, t, J = 6.1 Hz), 3.52-3.55 (8H, m), 3.23 (3H, s). 13 C (acetoned
6 ) δ 145.9, 132.7, 131.7, 129.9, 128.4, 126.0, 124.2, 115.7, 109.7, 73.7, 72.5, 72.3 (2C), 72.1, 59.8,
50.2.
2-(N,N-dimethylhydrazonomethyl)-1-(3,6,9-trioxadecyl)-1H-pyrrole (6c). A mixture of N,Ndimethylhydrazine
(1.27 g, 21.13 mmol) p-toluenesulphonic acid (3.29 g, 21.13 mmol) and
aldehyde 5b (5.13 g, 21.13 mmol) was suspended in degassed THF (100 ml) and refluxed under
nitrogen atmosphere for 30 min. A second aliquot of N,N-dimethylhydrazine (1.27 g, 21.13 mmol)
was added and the reflux maintained for an additional 30 min. The solvent was removed and the
residue taken up with AcOEt and purified by filtration through a short silica gel column.
Evaporation of the solvent afforded a yellow-brown viscous oil that was used directly without
further purification (3.90 g, 13.8 mmol, 65.3 %).
2

2-(N-(4-bromophenylhydrazonomethyl)-1-methylpyrrole (6d). A mixture of 4-
bromophenylhydrazine hydrochloride (3.00 g, 13.42 mmol) and 5a (1.47 g, 13.42 mmol) was
suspended in degassed THF (50 ml) and stirred at room temperature for 6 h under nitrogen
atmosphere. The color turns from light yellow to dark blue. The solvent was removed and the black
sticky residue purified by chromatography (SiO 2, CH 2 Cl 2 ) to give the pure compound as a yellow
powder (1.02 g, 3.67 mmol, 33 %). 1 H NMR (CDCl 3 ) δ 7.68 (1H, s), 7.34 (2H, d, J = 8.8 Hz), 6.88
(2H, d, J = 8.8 Hz), 6.71 (1H, t, J = 2.2 Hz), 6.34 (1H, dd, J = 3.7, 1.8 Hz), 6.14 (1H, dd, J = 3.6,
2.6), 3.96 (3 H, s).
1-[4-pyridyl]-2-(N-(3,6,9-trioxadecyl)-2-pyrrolyl)ethylene (7a). tBuOK (2.56 g, 22.80 mmol)
was added portionwise under nitrogen atmosphere to a solution of 4-picoline (1.93 g, 20.70 mmol)
and 5b (5.00 g, 20.74 mmol) in anhydrous DMF (20 ml). The resulting red solution was stirred at
80 °C for 1 h and at room temperature overnight. The reaction mixture was poured into 200 ml of
crushed ice to give a yellow suspension that was extracted with AcOEt (3 x 200 ml). The combined
organic phase was washed with brine (4 x 200 ml), dried over Na 2 SO 4 and evaporated affording the
product as a dark yellow oil that was used directly without further purification in the subsequent
step (5.00 g, 15.80 mmol, 76.3 %). 1 H NMR (DMSO-d 6 ) δ 8.49 (2H, d, J = 5.5 Hz), 7.27 (2H, d, J =
5.5 Hz), 7.24 (1H, d, J = 16.1 Hz), 6.77 (1H, s, broad), 6.73 (1H, d, J = 16.1 Hz), 6.58 (1H, d, J =
3.5 Hz), 6.17 (1H, t, J = 3.2 Hz), 4.16 (2H, t, J = 5.6 Hz), 3.72 (2H, t, J = 5.6 Hz), 3.59-3.53 (6H,
m), 3.47 (2H, m), 3.23 (3H, s).
1-[4-quinolyl]-2-(N-(3,6,9-trioxadecyl)-2-pyrrolyl)ethylene (7b). tBuOK (1.12 g, 10.01 mmol)
was added portionwise under nitrogen atmosphere to a solution of 4-methylquinoline (1.30 g, 9.10
mmol) and 5b ( 2.20 g, 9.12 mmol) in anhydrous DMF (15 ml). The resulting red fluorescent
solution was stirred at 80 °C for 1 h and at room temperature overnight. The reaction mixture was
poured into 200 ml of crushed ice to give a red-orange suspension that was extracted with AcOEt (3
x 200 ml). The combined organic phase was washed with brine (4 x 200 ml), dried over Na 2 SO 4 and
evaporated affording a red oil that was purified by chromatography (SiO 2 AcOEt) to give the
product as an orange oil (1.92 g, 5.50 mmol, 60.0 %). 1 H NMR (CDCl 3 ) δ 8.85 (1H, d, J = 4.7 Hz),
8.21 (1H, d, J = 8.3 Hz), 7.72 (1H, t, J = 8.5 Hz), 7.55 (1H, d, J = 5.1 Hz), 7.57 (1H, d, J = 8.5 Hz),
7.58 (1H, d, J = 15.8 Hz), 7.31 (1H, d, J = 15.8 Hz), 6.83 (1H, dd, J = 2.5, 1.7 Hz), 6.72 (1H, dd,
3

3.8, 1.5 Hz), 6.24 (1H, t, J = 3.5 Hz), 4.22 (2H, t, J = 5.7 Hz), 3.77 (2H, t, J = 5.7 Hz), 3.75-3.35
(8H, m), 3.23 (3H, s).
Preparation of squaraine 1a. Squaric acid (0,510 g, 4.50 mmol) was added as a solid to a
refluxing solution of compound 6a (2,600 g, 9.44 mmol) in absolute ethanol (150 ml). The colour
immediately turned deep green. After 1 h at the reflux temperature, the green suspension was
cooled at 0°C and the precipitate filtered at reduced pressure. The crude product was washed with
hot EtOH (80 ml) and crystallized from AcOH to give the pure compound as a powder with golden
luster (0,800 g, 1.27 mmol, 28.3 %). M.p. > 300 °C (dec.) 1 H NMR (CD 2 Cl 2 ) δ 7.66 (2H, d, J = 4.4
Hz), 7.41 (8H, t, J = 7.7 Hz), 7.22 (4H, t, J = 7.3 Hz), 7.15 (8H, d, J = 7.9 Hz), 7.07 (2H, s), 6.69
(2H, dd, J = 4.5 Hz), 4.26 (6H, s); 13 C (CDCl 3 ) 168.3, 168.2, 143.8, 142.4, 131.9, 130.1, 125.8,
125.0, 123.8, 122.4, 117.1, 36.0. Anal. Calcd for C 40 H 32 N 6 O 2 : C, 76.41; H, 5.13; N, 13.37; Calcd
for C 40 H 32 N 6 O 2 x ½ H 2 O: C, 75.33; H, 5.22; N, 13.18; Found: C, 75.33; H, 5.19; N, 12.88.
Preparation of squaraine 1b. A suspension of squaric acid (0.140 g, 1.22 mmol) and hydrazone
6b (1.000 g, 2.45 mmol) in a 1:1 BuOH/toluene mixture (120 ml) was refluxed in a Dean-Stark
apparatus for 3 h. The solvent was removed and the deep green oily residue was washed with water
(50 ml) and dissolved in boiling isopropanol (60 ml). The solvent evaporation afforded a golden,
shiny solid that was taken up with Et 2 O and filtered at reduced pressure. (0.530 g, 0.59 mmol, 48.6
%). M.p. [EtOH] 127-130 °C. 1 H NMR (DMSO-d 6 ) δ 7.62 (2H, d, J = 4.6 Hz), 7.52 (8H, t, J = 7.8
Hz), 7.31 (4H, t, J = 7.4 Hz), 7.26 (2H, s), 7.23 (8H, d, J = 7.8 Hz), 7.06 (2H, d, J = 4.6 Hz), 4.79
(4H, t, J = 4.8 Hz), 3.61 (4H, t, J = 5.2 Hz), 3.43-3.31 (16H, m), 3.15 (6H, s). 13 C (DMSO-d 6 ) 166.8,
166.7, 144.5, 141.9, 130.1, 130.0, 125.6, 125.0, 122.6, 122.1, 116.0, 71.1, 70.5, 69.8, 69.7, 69.5,
57.9, 46.7. Anal. Calcd for C 52 H 56 N 6 O 8 : C, 69.94; H, 6.32; N, 9.41; Found: C, 70.18; H, 6.45; N,
9.32.
Preparation of squaraine 1c. A suspension of squaric acid (0.370 g, 3.25 mmol) and hydrazone 6c
(1.850 g, 6.54 mmol) in a 1:1 BuOH/toluene mixture (120 ml) was refluxed in a Dean-Stark
apparatus for 3 h. The solvent was removed and the deep green oily residue was taken up with
deionised water (100 ml) and extracted with AcOEt (3 x 150 ml). The combined organic phase was
dried over Na 2 SO 4 and evaporated to give a dark oil. Addition of 10 ml of toluene started the
4

crystallization of a golden solid that was filtered and further crystallized from EtOH (0.420 g, 0.65
mmol, 20.0 %). M.p. [EtOH] 121-122 °C. 1 H NMR (DMSO-d 6 ) δ 7.52 (2H, d, J = 4.5 Hz), 7.26
(2H, s), 6.75 (2H, d, J = 4.5 Hz), 4.94 (4H, t, J = 5.0 Hz), 3.67 (4H, t, J = 5.2 Hz), 3.47-3.32 (16H,
m), 3.19 (6H, s), 3.11 (12H, s). 13 C (DMSO-d 6 ) 163.4, 157.9, 146.0, 128.9, 122.2, 120.1, 113.5,
71.1, 70.7, 70.0, 69.7, 69.5, 57.9, 46.1, 42.4. Anal. Calcd for C 32 H 48 N 6 O 8 : C, 59.61; H, 7.50; N,
13.03; Found: C, 59.76; H, 7.59; N, 12.99.
Preparation of squaraine 1d. A suspension of hydrazone 6d (0.500 g, 1.80 mmol) and squaric
acid (0.103 g, 0.90 mmol) in absolute ethanol (80 ml) was refluxed for 3 h. Upon cooling a dark
green precipitate was formed and filtered at reduced pressure. Crystallization form AcOH gave the
pure compound as a green powder (0.306 g, 0.48 mmol, 53.6 %). M.p. > 300 °C (dec.). 1 H NMR
(DMSO-d 6 ) δ 7.93 (2H, s), 7.61 (2H, d, J = 4.6 Hz), 7.44 (4H, d, J = 8.8 Hz), 7.09 (4H, d, J = 8.8
Hz), 7.0 (2H, d, J = 4.6 Hz), 4.30 (6H, s). The 13 C NMR could not be recorded because of
insufficient solubility. Anal. Calcd for C 28 H 22 Br 2 N 6 O 2 : C, 53.02; H, 3.50; N, 13.25; Found: C,
53.54; H, 3.79; N, 13.23.
Preparation of squaraine 2a. A suspension of squaric acid (0.230 g, 2.01 mmol), pyridine (0.320
g, 4.03 mmol) and chromophore 7a (1.400 g, 4.42 mmol) in a 1:1 toluene/BuOH mixture (40 ml)
was refluxed in a Dean-Stark apparatus for 1.5 h. The resulting deep green solution was chilled at
0°C overnight. A golden precipitate was formed and filtered at reduced pressure. Crystallization
from EtOH/H 2 O gave the pure compound as a golden green powder (0.600 mg, 0.84 mmol, 20 %).
M.p. 172-173 °C. 1 H NMR (DMSO-d 6 ) δ 8.72 (4H, d, J = 6.4 Hz), 7.90 (4H, d, J = 6.4 Hz), 7.87
(2H, d, J = 16.1 Hz), 7.74 (2H, d, J = 4.6 Hz), 7.60 (2H, d, J = 16.1 Hz), 7.33 (2H, d, J = 4.7 Hz),
4.60 (4H, t, J = 4.4 Hz), 3.70 (4H, t, J = 4.7 Hz), 3.44 (4H, m), 3.34 (4 H, m), 3.29 (4H, m), 3.23
(4H, m), 3.12 (6 H, s); 13 C (DMSO-d 6 ) 193.2, 170.4, 147.5, 146.7, 146.4, 130.7, 130.6, 123.5,123.4,
122.1, 116.0, 71.0, 70.9, 70.1, 70.0, 69.5, 57.8, 46.3. Anal. Calcd for C 40 H 46 N 4 O 8 : C, 67.59; H,
6.52; N, 7.88; Calcd for C 40 H 46 N 4 O 8 x 2/3 H 2 O: C, 65.11; H, 6.69; N, 7.59; Found: C, 65.26; H,
6.80; N, 8.13.
Preparation of squaraine 2b. A suspension of squaric acid (0.078 g, 0.68 mmol), pyridine (0.108
g, 1.36 mmol) and chromophore 7b (0.500 g, 1.36 mmol) in a 1:1 toluene/BuOH mixture (40 ml)
5

was refluxed in a Dean-Stark apparatus for 4 h. The resulting deep green solution was chilled at 0°C
overnight and the formed golden precipitate collected by suction filtration. Crystallization from
EtOH gave the pure compound as a golden green powder (0.100 mg, 0.12 mmol, 18.1 %). M.p.
169-170 °C. 1 H NMR (DMSO-d 6 ) δ 8.94 (2H, J = 4.7 Hz), 8.57 (2H, d, J = 8.4 Hz), 8.34 (2H, d, J =
15.8 Hz), 8.06 (2H, d, J = 8.3 Hz), 7.98 (2H, d, J = 4.7 Hz), 7.81 (2H, d, J = 15.8 Hz), 7.79 (2H, t, J
= 8.3 Hz), 7.77 (2H, d, J = 4.7 Hz), 7.69 (2H, t, J = 7.9 Hz), 7.62 (2H, d, J = 4.6 Hz), 5.12 (4H, t, J =
4.8 Hz), 3.74 (4H, t, J = 4.8 Hz), 3.47 (4H, m), 3.35 (4H, m), 3.27 (4H, m), 3.16 (4H, m), 3.05 (6H,
s). Anal. Calcd for C 48 H 50 N 4 O 8 : C, 71.09; H, 6.21; N, 6.91; Found: C, 70.64; H, 6.19; N, 6.83.
Preparation of squaraine 3. A suspension of 2-methylindolizine 3 (1.680 g, 12.81 mmol) and
squaric acid (0.730 g, 6.40 mmol) in a 1:1 mixture of BuOH/toluene (75 ml) was refluxed for 2h in
a Dean-Stark apparatus. Upon cooling a dark green precipitate was formed and collected by suction
filtration. Crystallization from CH 3 CN afforded the pure compound as a green powder (0.101 g,
0.25 mmol, 26 %). M.p. > 300 °C (dec). 1 H NMR (CD 2 Cl 2 ) δ 10.27 (2H, d, J = 6.9 Hz), 7.26 (2H,
d, J = 8.5 Hz), 7.11 (2H, t, J = 8.0 Hz), 6.81 (2H, t, J = 6.6 Hz), 6.46 (2H, s), 2.76 (6H, s); 13 C
(CDCl 3 ) 176.3, 166.5,143.0, 139.2, 135.0, 127.5, 122.5, 117.7, 113.8, 111.9, 15.4. Anal. Calcd for
C 22 H 16 N 2 O 2 : C, 77.63; H, 4.74; N, 8.23; Anal. Calcd for C 22 H 16 N 2 O 2 x ¼ H 2 O: C, 76.62; H, 4.82;
N, 8.12; Found: C, 76.90; H, 4.72; N, 7.63.
N
O
O
H 3 C
N
CH 3
3
Squaraine 3 structure was assigned on the basis of its 1 H NMR spectrum. 2-methylindolizine 1 H
NMR spectrum (DMSO-d 6 ) in fact shows two singlets in the high field aromatic region at 7.31 ppm
and 6.18 ppm corresponding respectively to the H 3 and H 1 protons. Squaraine 3 1 H NMR spectrum
(CD 2 Cl 2 ) only displays one singlet at 6.46 ppm. According to the electron withdrawing nature of the
squaraine core, and so with the expected low field shift of the indolizine signals in the squaraine,
the 6.46 ppm singlet can be unambiguously assigned to the position 1 of a 3 substituted 2-
methylindolizine, hence the assigned condensation regiochemistry.
Indolizine numbering:
6

7
6
8
N
4
5
1
3
2
DPBF degradations.
ABS
1,2
1,0
0,8
0,6
0,4
0,2
0 min
3 min
6 min
9 min
12 min
15 min
20 min
25 min
30 min
35 min
40 min
46 min
50 min
55 min
60 min
0,0
300 400 500 600 700 800 900
Wavelength (nm)
Figure S1. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 2b (5 µM) CH 2 Cl 2
solution.
7

1,2
1,0
0,8
415
727,5
0 min
15 min
25 min
40 min
55 min
70 min
85 min
100 min
ABS
0,6
0,4
0,2
400 500 600 700 800
Wavelength [nm]
Figure S2. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 1b (5 µM) CH 2 Cl 2
solution.
ABS
1,0
0,8
0,6
0 min
15 min
30 min
45 min
60 min
75 min
90 min
120 min
150 min
180 min
210 min
0,4
0,2
0,0
400 500 600 700 800
Wavelength [nm]
Figure S3. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 1c (5 µM) CH 2 Cl 2
solution.
8

ABS
1,2
1,0
0,8
0,6
DPBF
1d
0 min
3 min
6 min
9 min
12 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
0,4
0,2
0,0
300 400 500 600 700 800 900
Wavelength (nm)
Figure S4. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 1d (5 µM) CH 2 Cl 2
solution.
ABS
1,2
1,0
0,8
0,6
0,4
0,2
00 min
03 min
06 min
09 min
12 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
0,0
300 400 500 600 700 800 900
Wavelength (nm)
9

Figure S5. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 2a (5 µM) CH 2 Cl 2
solution.
ABS
1,0
0,8
0,6
0,4
0,2
00 min
03 min
06 min
09 min
12 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
0,0
300 400 500 600 700 800
Wavelength (nm)
Figure S6. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and 3 (5 µM) CH 2 Cl 2
solution.
10

ABS
1,2
1,0
0,8
0,6
0 min 14 min
1 min 16 min
2 min 18 min
3 min 20 min
4 min 26 min
5 min 30 min
6 min 34 min
8 min 38 min
10 min 42 min
12 min
0,4
0,2
0,0
350 400 450 500
Wavelenght [nm]
Figure S7. Time evolution of the UV-Vis spectrum of a DPBF (50 µM) and TPP (5 µM) CH 2 Cl 2
solution. The TPP spectrum has been subtracted from all the spectra.
References
1 Abbotto, A.; Alanzo, V.; Bradamante, S.; Pagani, G. A. J. Chem. Soc., Perkin Trans. 2 1991, 481.
2 Lottner, C.; Bart, K.; Bernhardt, G.; Brunner, H. J. Med. Chem. 2002, 45, 2079
3 Chichibabin, A. E. Chem. Ber. 1927, 60, 1607.
11