Synthesis of long-wavelength chlorins by chemical modification for methyl pyropheophorbide-a and their in vitro cell viabilities

Article abstract of DOI:10.1142/S1088424611004403

A series of novel chlorophyll-a homologs with long wavelength absorption were synthesized via modification of methyl pyropheophorbide-a used as starting material. For introducing electron-withdrawing group methylenemalononitrile moiety was established on

Full text of DOI:10.1142/S1088424611004403

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 122–129
DOI: 10.1142/S1088424611004403
Published at http://www.worldscinet.com/jpp/
Synthesis of long-wavelength chlorins by chemical
modification for methyl pyropheophorbide-a and
their in vitro cell viabilities
Jin Jun Wang*a, Jia Zhu Lib, Judit Jakusc andYoung Key Shimb◊
a College of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, China
b PDT Research Institute, School of Nano Engineering, Inje University, Gimhae 621-749, Korea
c Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest 1025,
Hungary
Received 28 August 2011
Accepted 28 September 2011
ABSTRACT: A series of novel chlorophyll-a homologs with long wavelength absorption were
synthesized via modification of methyl pyropheophorbide-a used as starting material. For introducing
electron-withdrawing group methylenemalononitrile moiety was established on the periphery of modified
chlorin by Knoevenagel reaction of malononitrile with formyl group at 3-, 15-position and 131-carbonyl
group on the exocyclic ring. All of chlorins containing the methylenemalononitrile structure show
Qy-absorption at more than 700 nm. Moreover, we have examined a preliminary in vitro photodynamic
anticancer effect of these new derivatives on mouse sarcoma S-180 cell line.
KEYWORDS: chlorin, methyl pyropheophorbide-a, chemical modification, visible absorption.
such as stable chlorins are needed, because they should
INTRODUCTION
also be able to localize in relatively high concentration at
Long wavelength absorbing photosensitizers (>700
the tumor site relative to normal tissues. Chlorophyll-a is
nm) have been described as potential candidates for
a natural chlorin pigment and his degradation products
achieving maximum tissue penetration in photodynamic
stand in marked contrast to symmetric porphyrin
therapy (PDT) [1–5]. Among such compounds with
pigments due to substantially stabilized S1 energies, a
long-wavelength absorption, some naturally occurring
strong Qy absorption band, and unique redox reactivates.
bacteriochlorins have been reported as effective
Therefore the synthesis of novel photosensitizers,
photosensitizers in preliminary in vitro and in vivo
possessed the basic skeleton of chlorophyll-a, have
studies [6, 7].However, most of these naturally occurring
become the focus of research in photodynamic therapy
bacteriochlorins are extremely sensitive to oxidation,
[9]. For chlorophyll-a compounds, the Qy bands as a
which result in rapid transformation into relative stable
longest absorption band were strongly affected by the
chlorin state which has an absorption maximum at or
substitutents on the Qy axis (N21–N23, see Scheme 1)
below 670 nm [4, 8]. Furthermore, if a laser is used to
[10]. Considering that introducing strong electron-
excite the bacteriochlorin in vivo, oxidation may result in
withdrawing group on the chromophore or expanding the
the formation of a new chromophore absorbing outside the
conjugation system of the macrocycle can bring about
laser window, which reduces the photodynamic efficacy.
obvious red shift of its long wavelength absorption,
In order to render PDT more generally applicable to tumor
therapy, long wavelength absorbing photosensitizers,
the modifications of methyl pyropheophorbide-a 1
(MPPa), which derived from methyl pheophorbide a
(MPa), the initial degradative product extracted from
Spirulina pacfica alga, were carried out for synthesis
SPP full member in good standing
of long-wavelength photosensitizers with stable chlorin
structure in our studies. Here we report the synthesis
*Correspondence to: Jin Jun Wang, email: wjj1955@163.com,
fax: +86 535-6902078
Copyright © 2012 World Scientific Publishing Company

SYNTHESIS OF LONG-WAVELENGTH CHLORINS BY CHEMICAL MODIFICATION
123
of chlorophyll a homologs, which possess absorption
maximum around longer than 700 nm, by modification
of methyl pyropheophorbide-a.
same condition for preparing chlorin 2 to also give 5a
and 5b (Scheme 1). It is worth noting that E-ketone and
C3-formyl group exhibited different activities, thereby in
the reaction of malononitrile with C3-formyl group, TEA
was used as a moderate catalyst, while its condensation
with E-ketone need a stronger base (NaOEt).
RESULTS AND DISCUSSION
In order to introduce methylenemalononitrile moiety
at meso-position, purpurin-5 dimethyl esters 11 and
132-oxopyropheophoride-a 12, obtained from the
allomerization of MPPa 1 [13], were used as reaction
precursors on account of the high reactivities of their
C15-carbonyl groups. The former (11) reacted with
malononitrile smoothly to form expected product 13
in 67% yield under the catalysis of TEA in THF. The
corresponding reaction of the latter (12) did not gave
designed tetracyanochlorin 15, but diacyanochlorin 14 in
52% instead. The further reaction of 14 with malononitrile
using sodium ethoxide as catalyst by refluxing in EtOH
also not generated 15, but rearranged into purpurin-18 16
(Scheme 2).
Synthesis and characterization
Malononitrile exhibits high activity and its
introduction will extend greatly the Qy peak of chlorin
comparing with other active methylene compounds such
as dimethyl malonate and acetylacetone [11, 12], on the
other hand, in our preliminary experiment, the reaction of
dimethyl malonate or acetylacetone with E-ketone gave
very poor yield in same condition or no react. Therefore
in this study we choose malononitrile as main building
block to prepare the stable long-wavelength chlorins.
The reaction of starting material 1 with malononitrile in
the presence of sodium ethoxide in EtOH by refluxing to
give 131-dicyanomethylene chlorin 2 in 75% yield which
shows Qy-absorption at 704 nm. The exocyclic carbon–
carbon double bonds at 3-position of 2 was oxidized with
OsO4 in dichloromethane in the presence of pyridine
followed by glycol cleavage, using sodium periodate to
form chlorin 3 and methyl pyropheophorbide-d 4 (MPPd)
whose Qy peaks were observed at 727 and 694 nm,
respectively. To expand conjugated p-system of chlorin
chromophore the C3-formyl of 3 reacted continuously
with Ph3PCH2Ph in dichloromethane in the presence of
aqueous NaOH solution at room temperature for 30 min
to give a C3-phenyl substituted chlorin mixture composed
of pyropheophorbide 5a as cis-isomer (19%) and 5b
as trans-isomer (59%), which were easily separated by
chromatography and showed their Qy bands at 703 and
710 nm, respectively. The Knoevenagel reaction of 3 was
performed readily in THF in the presence of TEA to give
tetracyano-substituted chlorin 6 in 70% yield, whose
Qy-absorption attained to 734 nm. By the same way
MPP-d 4 was converted into dicyano-substituted chlorin
7 (Qy = 706 nm) from which chlorin 6 was also obtained
by means of refluxing in THF with malononitrile. The
chlorination for MPPa 1 produced 20-chlorochlorin 9
whosep-systemwasfurtherextendedthroughits20-meso-
position. The subsequent Knoevenagel reaction of 9 with
malononitrile smoothly gave 131-dicyanomethylene-
substituted chlorochlorin 10 whose Qy peak appeared at
714 nm. Although this compound also could be obtained
from 2 by same chlorination with NCS, the speed and
yield of the reaction were relatively low in comparison
with that of MPPa 1 due to the absorb-electron effect of
the dicyanomethylene group at 131-position. The Wittig
reaction of MPPd 4 with benzyltriphenylphosphonium
bromide generated a pair of cis-trans isomers 8a
(Qy = 667 nm) and 8b (Qy = 673 nm) in 20% and 58%
yields, respectively [11]. The condensations of these
isomers with malononitrile were carried out under the
All constructions for dicyanomethylene moiety were
based on Knoevenagel reaction. Instead of forming
expected product, only the condensation of C132-carbonyl
of 14 with malononitrile was rearranged to known
purpurin-18 ester 16, whose possible formation process
was outlined in Fig. 1. we postulated that in ethanol
containing sodium ethoxide the resulting malononitrile
carbonion failed to attack to the carbonyl at 132-position,
or carbon-carbon double bond at 131-position either due
to its steric hindrance, but the smaller ethyioxide ion as
catalyst attacked the carbon atom at 132-position to form
Michael adduct A. Subsequent nucleophilic substitution
at the carbon atom of C131-ethoxyl group brought
about leaving malononitrile carbonion to rearrange
to diketochlorin 12 which was readily converted into
purpurin-18 eater 16 under strong alkaline condition [13].
The visible spectra of chlorins show that the introduction
of dicyanomethylene moiety on the tetrapyrrole macrocycles
can cause various degree of bathochromic shift in their Qy
peaks (Fig. 2). Compared the differences of the longest
absorption bands in their visible spectra between chlorins
before and after Knoevenagel reaction, each change from
C=O on the exocyclic E-ring to C=C(CN)2, such as 1→2
(DQy = 36 nm) or 2→6 (DQy = 30 nm), increases their
Qy wavelengths more than 30 nm. The transform from
formyl group to dicyanomethylene structure at 3-position or
15-position, such as 3→6 (DQy = 7 nm), 4→7 (DQy = 12
nm) and 11→13 (DQy = 12 nm), bring about completely
different red-shift distance. Besides structure modification,
these results showed that the changes of Qy wavelengths of
chlorins also related to the existing substituted groups in the
opposite direction of introduced dicyanomethylene along
N21–N23 axis. The lmax of trans-phenyl-substituted isomer
5b was red shifted compared to those of unsubstituted
vinyl compound 2 because the C3b-phenyl conjugated
with the chlorin chromophore through the 3a–3b double
bond. However, the corresponding Qy band of cis-isomer
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 123–129

124
J. J. WANG ET AL.
NC
NC
NC
NC
NH
N
N
NH
N
N
NH
N
N
NH
N
N
(a)
(a)
Cl
Cl
HN
HN
HN
HN
CN
CN
O
O
7
9
10
6
O
NC
O
NC
O
O
OCH3
OCH3
OCH3
OCH3
(d)
(e)
(e)
(d)
32
31
O
O
5
NH
N
N
NH
N
N
NH
N
N
NH
N
N
1
20
(a)
(b)
+
10
HN
HN
HN
HN
18
17
131
O
CN
CN
132
O
2
3
4
NC
O
O
O
O
1
NC
OCH3
OCH3
OCH3
OCH3
(c)
(c)
NH
N
N
NH
N
NH
N
N
NH
N
N
(a)
+
+
HN
N
HN
HN
HN
CN
CN
O
O
5b
OCH3
8a
OCH3
5a
OCH3
8b
OCH3
O
NC
O
O
O
NC
Scheme 1. Synthesis of dicyanomethylene bearing long-wavelength chlorins. Reagents and conditions: (a) CH2(CN)2/NaOEt/EtOH;
(b) Py, OsO4/THF/NaIO4/SiO2; (c) Ph3PCH2PhCl/NaOH; (d) CH2(CN)2/CH2Cl2/N(Et)3; (e) NCS/CH2Cl2
NH
N
N
NH
N
N
NH
N
N
NH
N
N
(a)
(a)
+
HN
HN
HN
HN
OCH3
O
O
CN
O
O
O
O
11
12
14
1
O
O
O
O
NC
(c)
OCH3
OCH3
OCH3
OCH3
(b)
NH
N
N
NH
N
NH
N
HN
N
HN
N
HN
CO2CH3
CN
CN
O
O
O
NC
O
O
O
NC
15
NC
OCH3
CN
OCH3
OCH3
13
16
Scheme 2. Synthesis of 15-meso dicyanomethylene bearing purpurin-5 13, and attempts at synthesizing tetracyano bearing chlorin
15. Reagents and conditions: (a) LiOH/THF; (b) CH2(CN)2/CH2Cl2/N(Et)3; (c) NaOEt/EtOH
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS OF LONG-WAVELENGTH CHLORINS BY CHEMICAL MODIFICATION
125
- CH(CN)2
N
HN
N
HN
N
HN
N
HN
O
C2H5
O
OC2H5
O
C2H5
CN
O
O
CN
O
O
O
O
O
O
O
O
CN
CN
NC
16
A
12
OCH3
OCH3
OCH3
OCH3
NC
14
Fig. 1. Possible conversion process from chlorin 14 to purpurin-18 methyl ester 16
Table 1. Absorption properties of dicyanomethylene bearing
long-wavelength chlorins
Compound
Absorption lmax, nm (relative intensity)
Soret
DSoret*
Qy
DQy*
1
414 (1.00)
452 (1.00)
393 (1.00)
452 (1.00)
455 (1.00)
422 (1.00)
380 (1.00)
456 (1.00)
432 (1.00)
407 (1.00)
0
38
-21
38
41
8
668 (0.38)
704 (1.11)
726 (0.86)
703 (1.01)
710 (0.97)
734 (0.93)
706 (0.54)
711 (1.00)
700 (0.29)
722 (0.91)
0
2
36
58
35
42
66
38
43
32
54
3
5a
5b
6
7
-34
42
18
-7
10
13
14
Fig. 2. UV-vis spectra in dichloromethane of dicyano- and
tetracyano-substituted chlorin 2 and 6
*DSoret and DQy represent the change of the Soret band and Qy
band between the dicyano-methylene bearing long-wavelength
chlorin and their starting material MPPa 1.
5a appeared at 703 nm, which was almost the same as that
of 2 (704 nm). This difference was ascribable to that cis-
isomer has less conjugation of the C3-double bond with the
chlorin chromophore because of steric repulsion between
the phenyl group and C2-methyl group. The Soret/Qy
absorbance ratios of chlorins including 2, 5–7, 10 and 14,
which are in the range of 0.88–1.16, were reduced after
linking with electro-withdrawing groups at 131-position.
The corresponding ratios of chlorins 3 and 13, which beared
b, b-dicyanomethylene at 3- and 15-position respectively,
were not much difference in comparison with that of their
precursors 4 and 11. It implied that rigid dicyanomethylene
structure on the E-ring, except chlorin 13, improved the
absorbing intensity of Qy band more effectively than that on
C3 position (Table 1).
explained due to introduction of b, b-dicyanomethylene to
reduce ring current density and (de)shielding action.
In vitro photosensitizing efficacy
In the present study, viability of the cell using
the methodology reported by Ahn et al. [14] was
determined by comparison with that of MPPa 1 for new
photosensitizers on mouse sarcoma S-180 cell line at
0.01, 0.05, 0.1, 0.5 and 1 mM after PDT (Fig. 3). For PDT
treatment, it was observed that all compounds showed
an improved effect for cell death or cell viability as the
concentration of the photosensitizer increased. And all
compounds obtained showed better effect than that of
MPPa. Among all the dicyano- or tetracyano-substituted
chlorins we obtained, compound 10 showed highest
effect when compared with the others.
Table2showstheIC50 valuesofthesenewphotosensitizers
on mouse sarcoma S-180 cell line after PDT. The reference
compound MPPa showed a relative low effect after PDT
(IC50 > 1.0 mM), all the dicyano- or tetracyano-substituted
chlorins showed lower IC50 value than that of MPPa.
Compound 10 among the tested photosensitizers showed
relatively high PDT effect (IC50 = 0.060 mM). Furthermore,
we are aiming to explore, in greater depth, the other
biological effects of these compounds for PDT.
The 1H NMR spectrum of chlorins introduced
dicyanomethylene structure at 131-position, including
2, 3, 10 and 14, clearly showed the chemical shifts of
all sorts of the proton signals corresponding to these
of their precursor. While for the chlorins introduced
dicyanomethylene structure at 3-position, after the
conversion from formyl group to dicyanomethylene a 1H
singlet proton signal corresponding to the proton attached
to dicyanomethylene was discovered at about 8.00 ppm,
at the same time the original signal for formyl group
disappeared. By contrast, allmost all proton absorption
signalsmovedtoupfieldexceptthatofonehydrogenlinked
with central nitrogen. These regular migrations could be
Copyright © 2012 World Scientific Publishing Company
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126
J. J. WANG ET AL.
1
are given in nanometers and relative intensity. The H
NMR spectra were obtained using a Varian spectrometer
(400 MHz). The chemical shifts (d) are given in parts per
million (ppm) relative to tetramethylsilane (TMS, 0 ppm),
unless otherwise indicated. Elemental analyses were
carried out using a Perkin Elmer 240-C microanalyzer.
Materials obtained from commercial suppliers were used
without further purification. Methyl pyropheophorbide a
1 was prepared according to the procedures described in
the literature [15].
Fig. 3. Cell viabiliy results of photosensitizers on mouse
sarcoma S-180 cell line. Compounds were tested at 0.01, 0.05,
0.1, 0.5 and 1 mM concentrations in triplicate. MPPa was used
as reference compound. Mouse sarcoma S-180 cell line at 80%
confluency in 96-well plates was used for in vitro PDT tests.
Cells were illuminated with a lamp in a wavelength range of
640–700 nm, with a peak at 660 nm, for 20 min. Total light dose
was 8.4 J. Statistical analyses were performed using unpaired
Student’s t test
Synthesis
Preparation of 131-b,b-dicyanomethylene-131-
deoxopyropheophorbide-a methyl ester (2). MPPa 1
(120 mg, 0.219 mmol) was dissolved in 30 mL EtOH,
to which malononitrile (100 mg, 1.515 mmol) and
sodium ethoxide (25 mg) was added under stirring. The
solution was refluxed under N2 and decrease of 1 was
monitored by TLC. After disappearance of 1 the reaction
mixture was poured into ice water and extracted with
dichloromethane (2 × 50 mL). The combined extract was
washed with water, dried over anhydrous Na2SO4. After
evaporating the residue was chromatographed on silica
gel (eluent: hexane/ethyl acetate, 3:1) to afford 98 mg
of 2 (0.164 mmol, 75%). UV-vis (CH2Cl2): lmax, nm (rel.
intensity log e) 704 (1.11), 644 (0.32), 574 (0.21), 535
(0.16), 452 (1.00), 430 (0.97), 384 (0.98), 350 (0.78). 1H
NMR (400 MHz; CDCl3): d, ppm 9.09, 8.97, 8.32 (each
s, each 1H, meso-H), 7.82 (dd, J = 17.8, 12.3 Hz, 1H,
31-H), 6.20 (dd, J = 17.8, 1.5 Hz, 1H, 32-trans-H), 6.09
(dd, J = 12.3, 1.5 Hz, 1H, 32 -cis-H), 5.31 (d, J = 20.0
Hz, 1H, 132-H), 5.26 (d, J = 20.0 Hz, 1H, 132-H), 4.06–
4.27 (m, 2H, 17 + 18-H), 3.58, 3.35, 3.27, 3.06 (each s,
each 3H, CH3 + OCH3), 3.50 (q, J = 7.8 Hz, 2H, 81-H),
2.39–2.58, 2.11–2.30 (m, 4H, 171 + 172-H), 1.70 (d,
J = 7.2 Hz, 3H, 18-CH3), 1.53 (t, J = 7.8 Hz, 3H, 82-H),
0.78 (br s, 1H, NH), -1.18 (br s, 1H, NH). IR (KBr): n,
cm-1 3469 (N–H), 2962, 2869 (C–H), 1737 (C=O), 1664
(C=C), 1564, 1444, 1240, 1172, 1074, 908, 796 (chlorin
skeleton). Anal. calcd. for C37H36N6O2: C 74.47, H 6.08,
N 14.08; found C 74.59, H 5.89, N 14.28.
Table 2. IC50 results of photosensitizers on mouse sarcoma
S-180 cell line
Compound
1(MPPa)
2
3
5a
0.559 0.394 0.194 0.123
10 13 14
0.735 0.060 0.853 0.241
5b
IC50, mM
Compound
IC50, mM
>1.0
6
7
0.312
In conclusion, we have developed a new approach for
the preparation of some pyropheophorbide derivatives
possessing Qy-absorption at more than 700 nm by
constructing two or four electron-withdrawing cyano-
group on the perphery of chlorin chromophore such
as at 3-positon or 131-position. These long wavelength
absorbing chlorins with basic skeleton of chlorophyll-a
possess relative stable tetrapyrrole macrocyclic structure.
Their unique optical and photochemical properties and
polyfunctional groups may be valuable for the synthesis
of new generation photosensitisers using in PDT. Among
all the compounds we obtained, compound 10 showed
relatively high PDT effect (IC50 = 0.060 mM).
Preparationof3-formyl-131-b,b-dicyanomethylene-
3-devinyl-131-deoxopyropheophorbide-a methyl ester
(3) and methyl pyeopheophorbide-d (MPPd, 4).
Chlorin 2 (70 mg, 0.117 mmol) was dissolved in a mixed
solution of THF (20 mL) and pyridine (0.3 mL) at 0 °C
and stirred at same temperature for 30 min after adding
51 mg (0.200 mmol) osmium(VIII) oxide in THF (2 mL),
and then stirred at room temperature for an additional 1
h. An excess of a solution of sodium hydrogensulfite (15
g) in a 50% mixture of methanol in water was added.
The mixture was stirred violently for 20 min. After
filtrating the brown osmium(VI) oxide precipitate,
dichloromethane was added to the mixture. The organic
layer was separated and dried over anhydrous sodium
sulfate. The solvent was removed to give solid material
EXPERIMENTAL
General
All of the reactions were monitored by thin layer
chromatography (TLC) using 0.20-mm silica gel plates
with or without a UV indicator (60F-254). Silica gel 60
(70–230 or 230–400 mesh, Merck) was used for flash
columnchromatography.Themeltingpoints(uncorrected)
were measured using an Electrothermal IA9000 Series
digital melting point apparatus. The electronic absorption
spectra were measured using a SCINCO S-3100 UV-vis
spectrophotometer. The absorption maxima (lmax) values
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SYNTHESIS OF LONG-WAVELENGTH CHLORINS BY CHEMICAL MODIFICATION
127
that was suspended in a mixture of THF (15 mL) and
(C=C), 1528, 1400, 1173, 1071, 1040, 968, 728 (chlorin
skeleton). Anal. calcd. for C43H40N6O2: C 76.76, H 5.99,
N 12.49; found C 76.60, H 5.81, N 12.24. 5b. UV-vis
(CH2Cl2): lmax, nm (rel. intensity log e) 710 (0.97), 649
(0.27), 577 (0.12), 496 (0.08), 455 (1.00), 434 (0.89), 390
(0.84), 353 (0.52). 1H NMR (400 MHz; CDCl3): d, ppm
9.12, 9.10, 8.36 (each s, each 1H, meso-H), 8.13 (d, J =
16.3 Hz, 1H, 31-H), 7.78 (d, J = 7.2 Hz, 2H, Ph-H), 7.55
(d, J = 16.3 Hz, 1H, 32-H), 7.44–7.57 (m, 3H, Ph-H), 5.54
(d, J = 21 Hz, 1H, 132-H), 5.40 (d, J = 21 Hz, 1H, 132-
H), 4.24–4.40, 4.08–4.17 (m, each 1H, 17 + 18-H), 3.63,
3.34, 3.32, 3.09 (each s, each 3H, CH3 + OCH3), 3.60 (q,
J = 7.8 Hz, 2H, 81-H), 2.40–2.63, 2.17–2.36 (m, each 2H,
171 + 172-H), 1.75 (d, J = 7.2 Hz, 1H, 18-CH3), 1.61 (t,
J = 7.4 Hz, 3H, 82-H), 0.06 (br s, 1H, NH), -0.98 (br s, 1H,
NH). IR (KBr): n, cm-1 3449 (N–H), 2924 (C-H), 1736
(C=O), 1686 (C=C), 1560, 1459, 1341, 1086, 1040, 976,
669 (chlorin skeleton). Anal. calcd. for C43H40N6O2: C
76.76, H 5.99, N 12.49; found C 76.89, H 6.13, N 12.67.
Preparation of 32,32-dicyano-131-b,b-dicyanomethy-
lene-131-deoxopyropheophorbide-a methyl ester (6).
Formyl chlorin 3 (40 mg, 0.067 mmol) was dissolved in 15
mL dichloromethane containing TEA (1.5 mL), to which
malononitrile (70 mg, 1.060 mmol) was added under
stirring. The solution was stirred under N2 for 2 h. The
reaction mixture was poured into ice water and extracted
with dichloromethane (2 × 15 mL). The combined
extract was washed with water, dried over anhydrous
Na2SO4. The combined organic phases were evaporated
to druness and the residue was chromatographed on silica
gel (eluent: hexane/ethyl acetate, 3:1) to afford 20 mg
6 (0.0031 mmol, 46%). UV-vis (CH2Cl2): lmax, nm (rel.
intensity log e) 734 (0.93), 590 (0.31), 545 (0.24), 422
(1.00), 353 (0.83). 1H NMR (400 MHz; CDCl3): d, ppm
9.46, 9.12, 8.69 (each s, each 1H, meso-H), 9.25 (s, 1H,
31-H), 5.71 (d, J = 21.0 Hz, 1H, 132-H), 5.55 (d, J = 21.0
Hz, 1H, 132-H), 4.38–4.56 (m, 1H, 18-H), 4.21–4.43
(m, 1H, 17-H), 3.76, 3.62, 3.48, 3.26 (each s, each 3H,
CH3 + OCH3), 3.69 (q, J = 7.5 Hz, 2H, 81-H), 2.51–2.73,
2.16–2.40 (m, each 2H, 171 + 172-H), 1.81 (d, J = 7.2 Hz,
3H, 18-CH3), 1.68 (t, J = 7.5Hz, 3H, 81-CH3), 0.79 (br s,
1H, NH), -1.35 (br s, 1H, NH). IR (KBr): n, cm-1 3450
(N–H), 2978 (C–H), 1735 (C=O), 1637 (C=C), 1561,
1475, 1225, 1084, 982, 722 (chlorin skeleton). Anal.
calcd. for C39H34N8O2: C 72.43, H 5.30, N 17.33; found
C 72.59, H 5.17, N 17.50.
silica gel (2.5 g). After addition of a solution of sodium
metaperiodate (1 g) in water (15 mL), the color of the
solution changed from green to bronze within 30 min.
After adding dichloromethane (20 mL), the mixture was
filtered through cotton wool and then the resultant crude
material was chromatographed on silica gel (eluent:
hexane/ethyl acetate, 3:1) to give 54 mg chlorin 3 (0.090
mmol, 77%) and 8 mg MPPd 4 (0.014 mmol, 12%),
respectively. 3. UV-vis (CH2Cl2): lmax, nm (rel. intensity
log e) 726 (0.86), 663 (0.22), 583 (0.21), 544 (0.15),
1
412 (0.99), 393 (1.00), 384 (0.86). H NMR (400 MHz;
CDCl3): d, ppm 11.36 (s, 1H, CHO), 10.06, 9.28, 8.62 (s,
each 1H, meso-H), 5.61, 5.55 (each d, J = 19.8 Hz, each
1H, 132-H), 4.23–4.53, 4.13–4.20 (m, each 1H, 17, 18-H),
3.68 (q, J = 7.8 Hz, 2H, 81-H), 3.67, 3.63, 3.57, 3.15 (each
s, each 3H, CH3 + OCH3), 2.49–2.69, 2.15–2.39 (m, 4H,
171 + 172-H), 1.74 (d, J = 7.4 Hz, 3H, 18-CH3), 1.58 (t, J
= 7.8 Hz, 3H, 82-H), 0.80 (br s, 1H, NH), -1.50 (br s, 1H,
NH). IR (KBr): n, cm-1 3443 (N–H), 2962, 2926 (C–H),
1740, 1735 (C=O), 1654 (C=C), 1564, 1438, 1400, 1256,
1169, 1083, 940, 722 (chlorin skeleton). Anal. calcd. for
C36H34N6O3: C 72.22, H 5.72, N 14.04; found C 72.40, H
5.56, N 14.19. Analytical data of MPPd 4 in line with the
literature values [16].
Preparation of cis-32-phenyl-131-b,b-dicyanome-
thylene-131-deoxopyropheophorbide-a methyl ester
(5a) and trans-32-phenyl-131-b,b-dicyanomethylene-
131-deoxopyropheophorbide-a methyl ester (5b).
Formylchlorin3(65mg,0.109mmol)andbenzyltriphenyl-
phosphonium chloride (93 mg, 0.240 mmol) was
dissolved in 10 mL dichloromethane and a solution of
NaOH (70 mg) in H2O (3 mL) was added with stirring.
The solution was stirred at room temperature under N2
and the decrease of 3 was monitored by TLC. After
disappearance of 3, the reaction mixture was poured into
ice water and dichloromethane. The aqueous phase was
extracted with several portions of dichloromethane and
the combined organic phases were washed with aqueous
aq. 2% HCl, aq. 4% NaHCO3 and water and evaporated
in vacuo to dryness. The residue was chromatographed
on silica gel (eluent: hexane/ethyl acetate, 4:1) to give 14
mg chlorin 5a (0.021 mmol, 19%) and 43 mg chlorin 5b
(0.064 mmol, 59%). 5a. UV-vis (CH2Cl2): lmax, nm (rel.
intensity log e) 703 (1.01), 644 (0.34), 574 (0.23), 533
(0.19), 495 (0.16), 452 (1.00), 434 (0.94), 386 (0.88), 350
1
(0.72). H NMR (400 MHz; CDCl3): d, ppm 9.10, 9.02,
Preparation of 32,32-dicyanopyropheophorbide-a
methyl ester (7). MPPd 4 (42 mg, 0.076 mmol)
was dissolved in 20 mL dichloromethane, to which
malononitrile (70 mg, 1.060 mmol) and TEA (1.5 mL)
was added under stirring. The solution was stirred under
N2 for 2 h. The reaction mixture was poured into ice
water and extracted with dichloro-methane (2 × 15 mL).
The combined extracts was washed with water, dried
over anhydrous Na2SO4. After evaporating the solution
the residue was chromatographed on silica gel (eluent:
hexane/ethyl acetate, 3:1) to afford 34 mg 7 (0.057 mmol,
8.27 (each s, each 1H, meso-H), 7.70 (m, 2H, Ph-H), 7.54
(d, J = 11.4 Hz, 1H, 31-H), 7.52 (m, 1H, Ph-H), 7.35 (d,
J = 11.4 Hz, 1H, 32-H), 6.94–7.25 (m, 3H, Ph-H), 5.52,
5.38 (d, J = 20 Hz, each 1H, 132-H), 4.18–4.35, 4.06–4.15
(m, 2H, 17 + 18-H), 3.53 (q, J = 7.8 Hz, 2H, 81-H), 3.64,
3.53, 3.00, 2.90 (each s, each 3H, CH3 + OCH3), 2.46–
2.64, 2.20–2.35 (m, each 1H, 171 + 172-H), 1.75 (d, J =
7.2 Hz, 3H, 18-CH3), 1.58 (t, J = 7.8 Hz, 3H, 82-H), 0.78
(br s, 1H, NH), -0.86 (br s, 1H, NH). IR (KBr): n, cm-1
3448, 3180 (N–H), 2929, 2866 (C–H), 1741 (C=O), 1638
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 127–129

128
J. J. WANG ET AL.
76%). UV-vis (CH2Cl2): lmax, nm (rel. intensity log e) 706
(0.54), 638 (0.08), 590 (0.11), 522 (0.11), 434 (0.54), 431
(0.49), 380 (1.00). 1H NMR (400 MHz; CDCl3): d, ppm
9.58, 9.19, 8.79 (s, each 1H, meso-H), 9.23 (s, 1H, 31-H),
5.26 (d, J = 20.0 Hz, 1H, 132-H), 5.19 (d, J = 20.0 Hz, 1H,
132-H), 4.34 (q, J = 7.3 Hz, 1H, 18-H), 4.12 (d, J = 7.2
Hz, 1H, 17-H), 3.67, 3.62, 3.51, 3.28 (each s, each 3H,
CH3 + OCH3), 3.70 (q, J = 7.6Hz, 2H, 81-H), 2.52–2.70,
2.20–2.43 (m, each 2H, 171 + 172-H), 1.84 (d, J = 7.2 Hz,
3H, 18-CH3), 1.71 (t, J = 7.6 Hz, 3H, 81-CH3), -0.16 (br
s, 1H, NH), -2.08 (br s, 1H, NH). IR (KBr): n, cm-1 3449,
3167 (N–H), 2963 (C–H), 1740 (C=O), 1672 (C=C),
1542, 1510, 1400, 1223, 1075, 1021, 980, 799 (chlorin
skeleton). Anal. calcd. for C36H34N6O3: C 72.22, H 5.72,
N 14.04; found C 72.06, H 5.60, N 13.91.
NH). IR (KBr): n, cm-1 3448, 3145 (N–H), 2956 (C–H),
1736 (C=O), 1665 (C=C), 1619, 1437, 1401, 1257, 1170,
1010, 976, 710, 708, 597 (chlorin skeleton). Anal. calcd.
for C37H35ClN6O2: C 70.41, H 5.59, N 13.32; found C
70.26, H 5.72, N 13.25.
Preparationof15-b,b-dicyanomethylenepurpurin-5
methyl ester (13). This compound as a red solid
was obtained from compound 11 by reacting with
malononitrile in the yield of 71% according to the
method for preparing compound 6. UV-vis (CHCl3):
l
max, nm (rel. intensity log e) 700 (0.29), 620 (0.07),
1
564 (0.20), 502 (0.08), 432 (1.00), 358 (0.67) nm. H
NMR (400 MHz; CDCl3): d, ppm 9.59 (s, 1H, 151-H),
8.45, 9.34, 9.62 (each s, each 1H, meso-H), 7.88 (dd,
J = 17.7, 11.5 Hz, 1H, 31-H), 6.28 (d, J = 17.7 Hz, 1H,
trans-32-H), 6.16 (d, J = 11.5 Hz, 1H, cis-32-H), 4.50 (d,
J = 10.7 Hz, 1H, 17-H), 4.36 (q, J = 7.3 Hz, 1H, 18-H),
3.66 (q, J = 7.6 Hz, 2H, 81-H), 4.25, 3.37, 3.51, 3.32, 3.18
(each s, each 3H, CH3 + OCH3), 1.95–2.08, 2.38–2.41,
2.58–2.69 (each m, 4H, 171 + 172-H), 1.78 (d, J = 6.6
Hz, 3H, 18-CH3), 1.67 (t, J = 7.6 Hz, 3H, 82-CH3), 0.20
(br s, 1H, NH), -0.20 (br s, 1H, NH). IR (KBr): n, cm-1
3423, 3163 (N–H), 2958 (C–H), 1740, 1735 (C=O), 1672
(C=C), 1606, 1400, 1256, 1084, 924 (chlorin skeleton).
Anal. calcd. for C38H38N6O4: C 71.01, H 5.96, N 13.08;
found C 71.24, H 5.79, N 13.20.
Preparation of 20-chloropyropheophorbide-a methyl
ester (9). 150 mg of NCS was partialy added to a solution
of MPPa 1 (200 mg, 0.330 mmol) in methylene chloride
(50 mL), and the reaction mixture was stirred for 5 h
under nitrogen in the dark. The resulting solution was
then poured into 200 mL of iced water and extracted with
methylene chloride (3 × 100 mL). The combined extract
was washed with 10% aqueous NaHCO3, water and dried
over Na2SO4 and evaporated to dryness. The residue
was chromatographed on silica gel (eluent: hexane/ethyl
acetate, 2:1) to afford 237 mg chlorin 9 as dark-red solid
in 80% yield. mp 210–214 °C. UV-vis (CHCl3): lmax, nm
(rel. intensity log e) 676 (0.41), 620 (0.07), 550 (0.12),
Preparation of 131-b,b-dicyanomethylene-132-oxo-
131-deoxopyropheophorbide-a methyl ester (14). This
compound as a red solid was obtained from compound
12 by reacting with malononitrile in the yield of 68%
according to the method for preparing compound 6.
UV-vis (CHCl3): lmax, nm (rel. intensity log e) 722 (0.91),
618 (0.08), 512 (0.28), 472 (0.36), 425 (0.35), 407 (1.00).
1H NMR (400 MHz; CDCl3): d, ppm 8.94, 9.50, 9.80
(each s, each 1H, meso-H), 8.08 (dd, J = 17.8, 11.5 Hz,
31-H), 6.36 (dd, J = 17.8, 1.0 Hz, 1H, trans-32-H), 6.30
(dd, J = 11.5, 1.0 Hz, 1H, cis-32-H), 5.11 (d, J = 8.0 Hz,
1H, 17-H), 4.63 (q, J = 7.5 Hz, 1H, 18-H), 3.75 (q, J =
7.6 Hz, 2H, 81-H), 3.62, 3.51, 3.50, 3.35 (each s, each 3H,
CH3 + OCH3), 1.95–2.07, 2.18–2.43, 2.67–2.80 (each m,
4H, 171 + 172-H), 1.88 (d, J = 7.3 Hz, 3H, 18-CH3), 1.70
(t, J = 7.6 Hz, 3H, 8-CH3), 0.51 (br s, 1H, NH), -1.98 (br
s, 1H, NH). IR (KBr): n, cm-1 3449 (N–H), 2989 (C–H),
1741, 1735 (C=O), 1638 (C=C), 1542, 1528, 1308, 1080,
726 (chlorin skeleton). Anal. calcd. for C37H34N6O3: C
72.77, H 5.61, N 13.76; found C 72.60, H 5.40, N 13.91.
1
518 (0.09), 416 (1.00). H NMR (400 MHz; CDCl3): d,
ppm 9.52, 9.55 (each s, each 1H, meso-H), 7.92 (dd, J =
17.8, 11.5 Hz, 1H, 31-H), 6.27 (dd, J = 11.5, 1.5 Hz, 1H,
32-H), 6.14 (dd, J = 17.8, 1.5 Hz, 1H, 32-H), 5.24 (d, J =
3.2 Hz, 2H, 132-H), 4.80 (q, J = 7.0 Hz, 1H, 18-H), 4.24
(dd, J = 9.0, 2.8 Hz, 1H, 17-H), 3.68 (q, J = 7.6 Hz, 2H,
81-CH2), 3.67, 3.60, 3.59, 3.24 (each s, each 3H, OCH3 +
CH3), 2.05–2.70 (m, 4H, 171 + 172-H), 1.70 (t, J = 7.6 Hz,
3H, 82-CH3), 1.64 (d, J = 7.0 Hz, 3H, 18-CH3), 0.48 (br s,
1H, NH), -1.94 (br s, 1H, NH). Other analytical data are
consistent with the literature data [17].
Preparation of 20-chloro-131-b,b-dicyanomethy-
lene-131-deoxopyropheophorbide-a methyl ester (10).
This compound as a red solid was obtained from
compound 9 by reacting with malononitrile in the yield of
59% according to the method for preparing compound 2.
UV-vis (CHCl3): lmax, nm (rel. intensity log e) 711 (1.00),
651 (0.17), 585 (0.17), 545 (0.08), 456 (1.00), 385 (0.79).
1H NMR (400 MHz; CDCl3): d, ppm 9.17, 9.35 (each
s, each 1H, meso-H), 7.85 (dd, J = 17.8, 11.5 Hz, 1H,
31-H), 6.28 (dd, J = 11.5, 1.4 Hz, 1H, cis-32-H), 6.07 (dd,
J = 17.8, 1.4 Hz, 1H, trans-32-H), 5.44 (d, J = 21.0 Hz,
1H, 132-H), 5.28 (d, J = 21.0 Hz, 1H, 132-H), 4.66 (q,
J = 7.1 Hz, 1H, 18-H), 4.04 (dd, J = 9.2, 2.4 Hz, 1H,
17-H), 3.62 (q, J = 7.6 Hz, 2H, 81-H), 3.63, 3.54, 3.50,
3.19 (each s, each 3H, CH3 + OCH3), 2.53–2.61, 2.15–
2.25, 1.85–1.93 (each m, 4H, 171 + 172-H), 1.64 (t, J =
7.6 Hz, 3H, 82-CH3), 1.55 (d, J = 7.1 Hz, 3H, 18-CH3),
-1.32 (each br s, each 1H, NH), -1.51 (each br s, each 1H,
MTT assay for in vitro photosensitizing efficacy
The photosensitizing activities of long-wavelength
dicyanomethylene bearing compounds (2, 3, 5a, 5b,
6, 7, 10, 13, 14) and MPPa 1 were determined in the
mouse sarcoma S-180 cell line. The cells were grown
in a-MEM with 10% fetal calf serum, L-glutamine,
penicillin, and streptomycin. Cells were maintained in
5% CO2, 95% air, and 100% humidity. Cells were plated
in 96-well plates at a density of 5 × 103 cells per well
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS OF LONG-WAVELENGTH CHLORINS BY CHEMICAL MODIFICATION
129
in complete medium. After an overnight incubation
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at 37 °C, the photosensitizers were added at varying
concentrations and incubated at 37 °C for 3 h in the
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drug-free complete medium. Cells were then illuminated
with a lamp in a wavelength range of 640–700 nm, with
a peak at 660 nm, for 20 min. Total light dose was 8.4
J. After PDT, the cells were incubated for 48 h at 37 °C
in the dark. Following the 48 h incubation, 10 mL of
4.0 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazoliumbromide (MTT) (Sigma) dissolved
in PBS was added to each well. After a 4 h incubation at
37 °C, unreacted MTT and medium were removed and
100 mL DMSO was added to solubilize the formazan
crystals. The 96-well plate was read on a microtiter plate
reader (ELISA-reader, BioTek, Synergy HT, USA) at an
absorbance of 570 nm. The results were plotted as percent
survival of the corresponding dark (drug no light) control
for each compound tested after subtracting medium only
control absorbance. Each data point represents the mean
from 3 separate experiments with 6 replicates at each
dose, and the standard errors were less than 10%.
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Acknowledgements
This work was supported by the Natural Science
Foundation of Shandong Province of China (No.
Y2008B49) and by the open project of state key laboratory
breeding base of green chemistry-synthesis technology
(Zhejiang University of Technology), as well as by the
Framework of the Intergovernmental Sino-Hungarian
Scientific and Technological Cooperation (CHN-30/04).
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J. Porphyrins Phthalocyanines 2012; 16: 129–129