Fluorinated photosensitizers: Synthesis, photophysical, electrochemical, intracellular localization, in vitro photosensitizing efficacy and determination of tumor-uptake by 19F in vivo NMR spectroscopy

Article abstract of DOI:10.1016/j.tet.2003.10.016

For in vivo NMR studies, starting from pyrroles, a series of fluorinated porphyrins were synthesized by following the MacDonald reaction conditions. Upon reaction with osmium tetroxide, a fluorinated porphyrin containing four trifluoromethyl groups (12 fl

Full text of DOI:10.1016/j.tet.2003.10.016

Tetrahedron 59 (2003) 10059–10073
Fluorinated photosensitizers: synthesis, photophysical,
electrochemical, intracellular localization, in vitro
photosensitizing efficacy and determination of tumor-uptake
by 19F in vivo NMR spectroscopy
Suresh K. Pandey,a Amy L. Gryshuk,a Andrew Graham,b Kei Ohkubo,c Shunichi Fukuzumi,c
,
*
†
Mahabeer P. Dobhal,a Gang Zheng,a Zhongping Ou,d Riqiang Zhan,d Karl M. Kadish,d
,
,
*
a,f
Allan Oseroff,b S. Ramaprasade and Ravindra K. Pandey
,
,
*
*
aPhotodynamic Therapy Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
bDepartment of Dermatology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
cDepartment of Material and Life Science, Graduate School of Engineering, Osaka University, CREST,
Japan Science and Technology Agency (JST), Yamada-oka, Suita, Osaka 565-0871, Japan
dDepartment of Chemistry, University of Houston, Houston, TX 77204-5003, USA
eDepartment of Radiology, University of Nebraska, Omaha, Nebraska, USA
fDepartment of Nuclear Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Received 4 September 2003; revised 6 October 2003; accepted 6 October 2003
Abstract—For in vivo NMR studies, starting from pyrroles, a series of fluorinated porphyrins were synthesized by following the MacDonald
reaction conditions. Upon reaction with osmium tetroxide, a fluorinated porphyrin containing four trifluoromethyl groups (12 fluorine units)
was converted into the related chlorin and bacteriochlorin which exhibited long-wavelength absorptions at 652 and 720 nm, respectively. All
compounds produced good singlet oxygen production efficiency. A comparative study of nine porphyrins with and without fluorine
substituents indicated no adverse effects of the presence of fluorinated groups in the photophysical properties of the porphyrins, chlorins or
bacteriochlorins. The first and second one-electron reduction potentials (vs SCE) of the investigated compounds range between 21.29 and
21.49 V and between 21.66 and 21.84 V in PhCN containing 0.1 M TBAP. UV–visible spectroelectrochemical data suggested the
formation of p-anion and p-cation radicals upon the first reduction and first oxidation. The in vivo 19F MR study of a representative fluorine
labeled compound with twelve equivalent fluorines confirmed the presence of the fluorine labeled sensitizer in mouse (C3H/HeJ) implanted
with RIF tumors on mouse foot dorsum by inoculating 2£105 cells (the studies were repeated on four tumored mice to confirm the feasibility
and reproducibility). All fluorinated compounds were found to be quite effective in vitro. In a comparative intracellular localization study
with Rhodamine-123 in RIF tumor cells, the most soluble porphyrin containing two propionic ester side chains was found to localize in
mitochondria as well as the related chlorin and bacteriochlorin.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
PDT has emerged as one of the promising strategies in
cancer treatment. In this therapy, patients are given
intravenous injections of a drug that accumulates in cancer
cells in much higher concentrations than in normal cells.
Laser light with an appropriate wavelength delivered by
fiber optics to these tumor sites produces highly reactive
oxygen species (e.g. 1O2) that destroy the tumor cells.2
Therefore, for a drug to be effective, it is necessary that the
compound be in high concentration in tumor cells. PDT is
most beneficial when laser light is delivered at a time point
when the photosensitizer’s concentration is greater in the
tumor than in the surrounding tissue. Thus, a comprehensive
knowledge of the extent of localization and the rate
of accumulation is of immense value. While the
photo sensitizer’s concentration in tissue may be determined
by chemical extraction techniques, these methods are
Photodynamic therapy (PDT) is now a well recognized
modality that has been used both independently and in
conjunction with other cancer treatments.1 Combining the
use of a light-sensitive drug, lasers and fiber-optic probes,
Keywords: photodynamic therapy; photosensitizers; porphyrins; chlorins;
bacteriochlorins; mitochondria.
*
Corresponding authors. Address: Photodynamic Therapy Center, Roswell
Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Tel.: þ1-7168453203; fax: þ1-7168458920;
e-mail: ravindra.pandey@roswellpark.org;
fukuzumi@ap.chem.eng.osaka-u.ac.jp; kkadish@uh.edu;
sramaprasad@unmc.edu
†
On leave from the Department of Chemistry, University of Rajasthan,
Jaipur 302004, India.
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.016

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S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
Chart 1.
invasive, time consuming, and clinically non-feasible. In
contrast, in vivo NMR is minimally invasive, considered
safe, and the therapy can be monitored over time in a single
living system.3
of the drug in tumors, it is necessary to have photosensi-
tizers containing multiple fluorine units. It would be
advantageous to have a photosensitizer with equivalent
fluorine substituents that will render signal addition of all
equivalent nuclei.
In vivo MR spectroscopy has been used increasingly to
monitor metabolism and disease states in humans. Both
magnetic resonance imaging and spectroscopy have evolved
into sophisticated diagnostic techniques. In addition to the
proton, nuclei such as fluorine4 can be studied by in vivo
spectroscopy and imaging. Fluorine-19 (19F) NMR is a
technique with significant potential because of the relatively
high sensitivity and low endogenous background. Due in
part to its high MR sensitivity, fluorine has received
considerable attention as an MR nucleus. Fluorine-19 MR
has been used to study metabolism, tumor growth, and blood
flow.5 More recently, in vivo 19F MR has been used to
measure tumor integrity and vasculature in subcutaneously
implanted tumors in rats.6 MR of fluorinated compounds is
particularly attractive for in vivo studies of human and
animal models. The 19F isotope has a 100% natural
abundance, a spin of 1/2, and a MR sensitivity that is 83%
then that of hydrogen.7
In this manuscript we report the synthesis of a series of
fluorinated and the corresponding non-fluorinated por-
phyrin-based compounds, the effect of the substituents on
their photophysical and electrochemical characteristics,
their intracellular localization and their in vitro photo-
sensitizing efficacy.
2. Results and discussion
2.1. Chemistry
For the synthesis of porphyrin-based fluorinated photo-
sensitizers, our synthetic strategy was divided into two
parts. These were the (a) synthesis of porphyrins 1 and
2 containing symmetrical trifluoromethyl groups (total
fluorine: 6) introduced at the meta-or para-position(s) of
the phenyl ring and (b) porphyrins 3 and 4 bearing
symmetrical trifluoromethyl groups introduced at the 3
and 5-positions (total fluorine: 12) of the phenyl ring
present at the meso-position of the porphyrin ring system
(see Chart 1).
To date, most of the fluorinated porphyrin-based analogs
synthesized for in vivo 19F NMR studies have been
unsymmetrical,8 and thus lead to signal dispersion. In
order to have a strong fluorine signal at a low concentration

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10061
Scheme 1.
Porphyrins 1–4 were synthesized from pyrroles 5,9 1510 or
1911 by following the well established ‘2þ2’ reaction
approach12 (see0 Scheme 1). In brief, reactions of pyrrole 5
with p-, m- or 3 ,50-dimethoxyphenyl aldehyde under acidic
reaction conditions produced the corresponding dipyrro-
methanes 6, 7 and 8 in 60–80% yield, which on refluxing
with ethylene glycol/KOH gave the related a-free dipyrro-
methanes 9, 11 and 13, respectively in .85% yield. Further
reaction of these dipyrromethanes with POCl3/DMF under
Vilsmeier’s reaction conditions13 produced the correspond-
ing diformyl dipyrromethanes 10, 12 and 14 in excellent
yields (.75%). For the preparation of dipyrromethane 17,
pyrrole 15 was first converted into the acetoxy derivative 16,
which on treating with K-10 clay14 in dichloromethane at
room temperature gave the desired dipyrromethane in
70% overall yield. Reaction of pyrrole 19 with bromine/
methanol15 gave the corresponding dipyrromethane 20 in
72% yield. Hydrogenation of dipyrromethane 17 and 20 in
presence of Pd/C at room temperature produced the
corresponding carboxylic acids 18 and 21 in quantitative
yields.
porphyrins 22–24 were isolated in modest yield. By
following a similar approach, pyrromethane 14 was also
reacted with pyrromethane dicarboxylic acid 21, and
porphyrin 25 was obtained in 38% yield. For the preparation
of the fluorinated analogs, porphyrins 22–25 containing
methoxy groups in the phenyl rings were reacted with boron
tribromide and the related hydroxyphenyl porphyrins 26–29
were obtained in 70–77% yields. Further reaction of these
porphyrins with 3,5-trifluoromethylbenzyl bromide pro-
duced the desired fluorinated porphyrins 1–4 in modest
yield (Scheme 2). Reaction of 29 with 3,5-dimethylbenzyl
bromide produced the non-fluorinated porphyrin 4a in
good yield. The structures of the intermediates and the
final products were confirmed by 1H, 19F NMR, mass
spectrometry and elemental analyses.
Among the fluorinated porphyrins 1–4, compound 4,
containing two propionic ester functionalities at the bottom
half of the molecule showed enhanced solubility compared
to those containing alkyl groups in 1% Tween 80/water
formulation as well as improved in vitro photosensitizing
efficacy in comparison to the other analogues. Therefore, for
investigating the photosensitizing effect(s) of fluorinated
groups in long-wavelength absorbing compounds, por-
phyrin 4 was reacted with osmium tetroxide (OsO4) and
Diformyldipyrromethanes 10, 12 and 14 were then indi-
vidually reacted with dipyrromethane dicarboxylic acid 18
under McDonald reaction conditions.12 After purification

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S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
Scheme 2.
Scheme 3.
the corresponding vic-dihydroxy chlorin 30 (lmax: 648 nm)
and tetra-hydroxy-bacteriochlorin 31 (as an isomeric
mixture, lmax: 716 nm) were synthesized. The relative
yields of 31 and 32 were found to depend on the amount of
OsO4 used (Scheme 3).
peaks with equal intensity at 12.98 and 12.99 ppm,
respectively.
2.2. In vivo 19F MR spectral characteristics
A typical 19F MR spectrum of the fluorinated photo-
sensitizer recorded on a 7T animal imager 8.0 h post IP
administration of the compound (100 mM kg21) is shown in
Figure 1. The tumor volume was 0.18 mL and spectra were
recorded over 30 min. The ability to observe the photo-
sensitizer by 19F MR non-invasively is an important step
towards understanding its pharmacokinetic characteristics
and relating these to its in vivo photosensitizing efficacy.
These details will be published elsewhere.
As expected, the 19F NMR spectra of all the fluorinated
porphyrins 1–4 exhibited a single peak at d 13.0 ppm,
whereas chlorin 30 and bacteriochlorin 31 showed two
2.3. Photophysical properties
A typical fluorescence spectrum of fluorinated porphyrins in
PhCN is shown in Figure 2. For the case of 3, virtually the
same fluorescence maxima at 628 and 693 nm was observed
as for non-fluorinated porphyrins. The absorption and
fluorescence maxima of investigated porphyrins 1–4,
Figure 1. In vivo F-19 NMR spectrum of compound 4 obtained from a RIF
tumor implanted in a C3H/HeJ mouse foot dorsum in one of the hind legs.

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10063
Figure 2. Fluorescence spectrum of 3 (7.3£1026 M) in deaerated PhCN at
298 K by excitation at 535 nm.
26–29, chlorin 30 and bacteriochlorin 31 are listed in Table
1. The absorption and the fluorescence maxima are red-
shifted in the order: porphyrin, chlorin and bacteriochlorin,
but they are not affected by the fluorine substituents.
Figure 3. Fluorescence decay (lem¼628 nm) of 3 (7.3£1026 M) in
deaerated PhCN at 298 K by excitation at 535 nm. The instrument response
(gray line), decay data (solid line), and single-exponential fitted line are
plotted in the bottom frame. The residuals of the fit for a lifetime of
t¼18.5 ns are plotted in the top frame.
The fluorescence decay of 3 is well fitted by a single
exponential line with lifetime of 18.5 ns as shown in Figure
3. The fluorescence lifetimes are also listed in Table 1. The
fluorescence lifetimes (t) are also unaffected by fluorinated
substituents. The t values of the nine porphyrins in this table
are all similar in the range of 16.1–18.6 ns irrespective of
substituents, but they are significantly longer than those of
chlorin 30 (3.8 ns) and bacteriochlorin 31 (3.3 ns).
order kinetics as shown in Figure 5. This indicates that there
is no contribution of the triplet–triplet annihilation under
the present experimental conditions.
The T–T absorption maxima and the triplet lifetimes are
summarized in Table 2. The triplet lifetimes of porphyrins
are in the range of 8.3–25 ms, which are shorter than the
lifetimes of chlorin 30 (185 ms) and the bacteriochlorin 31
(77 ms).
Phosphorescence spectra were observed in deaerated frozen
2-MeTHF at 77 K. The phosphorescence maxima are also
summarized in Table 1. Again the phosphorescence maxima
of porphyrins (822–823 nm) are not affected by the
fluorinated substituents. The triplet excited states of the
porphyrins were detected from the transient absorption
spectra measured 4.0 ns after laser excitation at 355 nm. A
typical example is shown in Figure 4 for the case of 3.
The negative absorption at 410 nm in Figure 3 is due to
bleaching of the ground-state absorption bands. The positive
absorption at 450 nm in Figure 4 is due to the triplet–triplet
(T–T) transition. The T–T absorption decay obeys first-
The decay of the T–T absorption in oxygen-saturated PhCN
is enhanced significantly over that observed in deaerated
PhCN. The decay rate obeys first-order kinetics and the
decay rate constant increases with increasing oxygen
concentration. Thus, an efficient energy transfer from the
triplet excited state of 3 to oxygen occurs to produce singlet
oxygen. The rate constants of the energy transfer (kEN) were
Table 1. Fluorescence emission maxima and fluorescence lifetimes in deaerated PhCN at 298 K, and phosphorescence emission maxima in deaerated
2-MeTHF at 77 K
l
max(fluorescence) (nm)
693
t (fluorescence)a (nm)
l
max(phosphorescence) (nm)
26
27
28
29
1
2
3
4
628
18.5
17.9
17.3
17.6
18.6
16.1
18.5
17.7
18.2
3.8
822
822
822
823
823
823
822
822
822
842
806
627
628
628
628
627
628
628
629
652
720
693
693
692
693
692
693
691
4a
30
31
693
b
b
3.3
a
The experimental errors are within ^5%.
Shoulder peak.
b

10064
S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
determined from the dependence of the decay rate constants
on oxygen concentration as listed in Table 2. The kEN values
are in the range of 8.9£108–1.1£109 M21 s21. There is no
specific effects of the fluorinated substituents on the kEN
values which are smaller than the diffusion-limited value in
PhCN (5.6£109 M21 s21).16
Irradiation of an oxygen-saturated benzene solution of 3
results in formation of singlet oxygen which was detected
1
by the O2 emission at 1270 nm (see Section 3). Quantum
1
yields (F) of O2 generation were determined from the
emission intensity which was compared to the intensity
obtained using a C60 reference compound.17 Relatively high
F values are obtained for all investigated compounds as
summarized in Table 2. The highest F value is obtained as
0.81 for the tetrafluorinated compound 3.
2.4. Electrochemical studies
Figure 4. T–T absorption spectrum of 3 (7.3£1026 M) obtained by the
laser flash photolysis in deaerated PhCN at 4.0 ms after laser excitation
(355 nm) at 298 K.
The electrochemically investigated compounds can be
divided into three groups based on the macrocycle and
substitutents. The first group includes the four fluorinated
porphyrins 1, 2, 3 and 4 (Chart 1, Scheme 2), the second, the
four non-fluorinated porphyrins 26, 27, 28 and 29 (Scheme
2) and the third, the one fluorinated chlorin 30 and the one
fluorinated bacteriochlorin 31 (Scheme 3). The electro-
chemistry of these ten complexes was carried out in PhCN
containing 0.1 M TBAP as supporting electrolyte. The
half-wave or peak potential for each reduction is listed in
Table 3.
In all but one case (compound 29), the first reduction was
reversible and the second irreversible due to a chemical
reaction following formation of the dianion. This led to
cyclic voltammograms of the type illustrated in Figure 6.
A chemical reaction also followed the first oxidation of each
porphyrin and this led to irreversible or quasi-reversible
oxidations as graphically shown in Figure 7 for the case of
compounds 3 and 4. The UV–visible spectral changes
obtained upon the first reduction were recorded in PhCN
containing 0.2 M TBAP. An example of the thin-layer
spectral changes are shown in Figure 8 for compounds 3 and
30 and a summary of the spectral data for the eight singly
reduced porphyrins is given in Table 3. The neutral
compounds have a Soret band at 407–412 nm and four
visible bands between 503 and 626 nm (see Table 4). Upon
the first reduction, the Soret and visible bands decrease in
intensity while a new broad visible band appears at
799–813 nm (see Table 3). These results are consistent
with a one-electron addition to the porphyrin p-ring system
and indicate the formation of a porphyrin p-anion radical.18
The cyclic voltammograms illustrating the stepwise oxi-
dation of compounds 3, 4 and 30 are shown in Figure 7. The
same spectral patterns are observed for the eight porphyrins
in Table 3 and these data may be contrasted to what is
observed for reduction of the chlorin 30 (Fig. 8(b)) and
bacteriochlorin 31 whose p-anion radical absorption bands
are also summarized in this table. The spectral changes
obtained upon oxidation of the porphyrin 3 and the chlorin
30 are illustrated in Figure 8. The reaction of the porphyrin
is quasi-reversible while that of the chlorin is reversible on
the cyclic voltammetry timescale (see cyclic voltamogram
Figure 7). Thus only the spectrum of the chlorin 30
Figure 5. Kinetic trace for the T–T absorption of 3 (7.3£1026 M) at
450 nm in deaerated PhCN after laser excitation (355 nm) at 298 K. Inset:
First-order plot.
Table 2. T–T absorption maxima (lmax(T–T)), triplet lifetimes (t(T–T))
in the deaerated PhCN at 298 K, quenching rate constants of the triplet
excited states by oxygen in PhCN, and quantum yields of singlet oxygen
in C6D6
l
max(T–T) (nm)
t(T–T) (s)a
kEN (M21 s21 a
)
F(1O2)a
26
27
28
29
1
2
3
4
450
450
450
450
450
450
450
450
450
440
440
1.0£1025
1.1£1025
1.1£1025
1.1£1025
8.3£1026
1.4£1025
1.9£1025
2.5£1025
2.3£1025
1.9£1024
7.7£1025
9.4£108
1.1£109
1.0£109
8.9£108
9.9£108
9.0£108
1.0£109
1.0£109
9.7£108
1.1£109
1.1£109
0.26
0.57
0.68
0.33
0.61
0.75
0.81
0.36
0.27
0.30
0.31
4a
30
31
a
The experimental errors are within ^5%.

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10065
Table 3. Reduction potentials of investigated complexes and UV–visible spectral data of singly reduced compounds in PhCN containing 0.1 M TBAP
Group
Compound
Potential (V vs. SCE)
Singly reduced complexes
E1/2
Ep
l
max, nm (1£1024, M21 cm21
)
I
1
2
3
4
21.43
21.38
21.40
21.29
21.78
21.80
21.80
21.66
409 (6.1)
408 (5.7)
407 (4.3)
412 (7.1)
432 (4.9)sh
–
–
–
–
806 (0.7)
813 (1.0)
801 (0.9)
801 (1.1)
435 (3.7)sh
–
–
II
26
27
28
29
21.44
21.44
21.49
21.46a
21.68
21.62
21.66
21.66
410 (6.8)
409 (5.1)
409 (5.7)
408 (5.2)
434 (6.3)
437 (4.1)
439 (2.8)
439 (2.5)sh
–
–
–
–
799 (1.8)
800 (1.9)
800 (1.0)
800 (1.2)
III
30
31
21.30
21.46b
21.74
21.84
402 (3.5)
406 (6.8)
–
–
535 (0.7)
750 (0.8)
735 (1.2)
827 (0.8)
a
Ep at a scan rate of 0.1 V/s. Two small peaks at Ep¼20.52 and 20.94 V also can be seen on first scan.
A peak at Ep¼21.29 V also can be seen. sh¼shoulder peak.
b
corresponds to a true measurement of the first electro-
sensitizers 1–4 was determined in radiation induced
fibrosarcoma (RIF) tumor cell lines.19 For determining the
drug dose, one of the fluorinated porphyins 4 was initially
tested at two different concentrations (0.5 and 1.0 mM). The
drug concentration at 1.0 mM, together with a light dose at
generated oxidation product which should be a p-cation
radical. The oxidations of other compounds were not
chemically or electrochemically reversible and thus no
meaningful spectra could be obtained (Fig. 9).
2.5. In vitro photosensitizing efficacy
The in vitro photosensitizing activity of fluorinated photo-
Figure 6. Cyclic voltammograms illustrating the stepwise reductions of
compounds (a) 3, (b) 4 and (c) 30 in PhCN containing 0.1 M TBAP.
Figure 7. Cyclic voltammograms illustrating the stepwise oxidation of
compounds (a) 3, (b) 4 and (c) 30 in PhCN containing 0.1 M TBAP.

10066
S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
Figure 9. Thin-layer UV–visible spectral changes of (a) compound 3 and
(b) compound 30 upon the first oxidation at 1.10 and 0.90 V in PhCN
containing 0.2 M TBAP.
Figure 8. Thin-layer UV–visible spectral changes of (a) compound 3 and
(b) compound 30 upon the first reduction at 21.50 V in PhCN containing
0.2 M TBAP.
singlet oxygen efficiency with photosensitizing activity,
these compounds were also evaluated for their photo-
physical characteristics. Although the presence and position
of the fluorinated substituents in the porphyrin macrocycle
produced a remarkable difference in singlet oxygen
producing efficiency, no direct correlation was observed
between singlet oxygen yield and in vitro photodynamic
activity. For example, the singlet oxygen yields of
porphyrins 1–3, are quite similar (0.61–0.81) but these
compounds showed a significant difference in photo-
sensitizing activity. In contrast, similar photosensitizing
results were obtained among the tetrakis-trifluorinated
4.0 J/cm2, produced a significant phototoxicity without any
dark toxicity. Other photosensitizers were then evaluated
under similar drug concentration. From the results sum-
marized in Figure 10 it can be seen that all the fluorinated
porphyrins, the chlorin and the bacteriochlorin produced
significant in vitro efficacy. However, the compounds in the
porphyrin series containing bis-trifluoromethyl groups were
found to be less effective than those containing tetrakis-
trifluoromethyl groups. The vic-dihydroxy chlorin 30 and
the related tetrahydroxy bacteriochlorin 31 were quite
effective with similar efficacy. In order to correlate the
Table 4. UV–visible data of investigated compounds in PhCN containing 0.2 M TBAP
Group
Compound
l
max, nm (1£1024, M21 cm21
)
Soret band
Visible bands
I
4
2
3
1
408 (12.71)
409 (11.23)
407 (7.25)
407 (13.12)
503 (1.13)
503 (0.74)
503 (0.65)
503 (1.29)
536 (0.51)
538 (0.60)
535 (0.30)
535 (0.62)
572 (0.47)
572 (0.74)
571 (0.27)
572 (0.58)
626 (0.19)
624 (0.29)
625 (0.10)
625 (0.28)
II
26
27
29
28
407 (16.20)
407 (12.47)
412 (12.70)
407 (9.58)
504 (1.24)
503 (0.48)
504 (0.57)
503 (0.88)
536 (0.27)
535 (0.20)
538 (0.74)
535 (0.40)
573 (0.23)
572 (0.16)
570 (0.95)
572 (0.37)
624 (0.09)
624 (0.10)
615 (0.21)
624 (0.15)
III
30
31
402 (8.28)
399 (8.90)
501 (0.79)
506 (1.28)
548 (0.17)
593 (0.35)
593 (0.26)
645 (2.02)
648 (2.12)
715 (1.70)

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10067
philicity of the molecule which has proven to be an
important molecular descriptor that often is well correlated
with the bioactivity of drugs. The lipophilicity is indicated
by lipophilic indice such as the logarithm of a partition
coefficient, log P, which reflects the equilibrium partitioning
of a molecule between a non-polar and a polar phase, such
as n-octanol/water system. Partition coefficients can be
measured either experimentally by following a simple
‘shake flask’ approach or by using a currently available
computer program (PALLAS system).19c We calculated the
log P values of the fluorinated porphyrins 1–4, the chlorin
30 and the bacteriochlorin 31 and these were in the range
of 12.76–19.75 [1 and 2: 15.58, 3: 19.75, 4: 18.43,
30:15.60 and 31:12.76]. For investigating a correlation
between singlet oxygen yields and PDT efficacy, the in vivo
studies of fluorinated photosensitizers at different drug/ight
doses are currently in progress. These results along with
the in vivo 19F tumor imaging data will be reported
elsewhere.
Figure 10. The in vitro photosensitizing activity of various fluorinated
porphyrins 1–4, chlorin 30 and bacteriochlorin 31 (1.0 mM) in RIF tumor
cells at 4 h incubation. Control: Cell exposed to light without photo-
sensitizer and cells with photosensitizer but no light exposure.
2.6. Intracellular localization
In general, porphyrin-based compounds have shown very
diverse patterns of localization, based on structure,
lipophilicity and charge.20 Localization in the lysosomes
and mitochondria are reported to be predominant. However,
in a QSAR study of certain photosensitizers the compounds
that localize in mitochondria are generally found to be more
effective. Therefore, the site of localization of the
fluorinated porphyrin 4 and the related chlorin 30 and
bacteriochlorin 31 were compared with Rhodamine-123,
which is known to target mitochondria. Images of the
analogs (3 and 4) in spite of a significant difference in
singlet oxygen yields (0.81 and 0.36, respectively).
On the basis of in vitro results, it is difficult to predict the in
vivo photosensitizing efficacy of these photosensitizers
because the pharmacokinetic and pharmacodynamic pro-
files as well as photobleaching characteristics play impor-
tant roles in drug localization and clearance. These
properties could also be influenced by the overall lipo-
Figure 11. Comparative intracellular localization of Rhodamine-123 (mitochondrial probe) and fluorinated chlorin 30 in RIF cells at 24 h post-incubation.
Similar patterns were observed from fluorinated porphyrin 4 and the corresponding bacteriochlorin 31 (only a representative example is shown). The overlay
picture clearly indicates that both Rhodamine-123 and chlorin 30 localize in mitochondria.

10068
S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
photosensitizers and Rhodamine-123 were taken in rapid
succession. The resulting images clearly indicate that these
photosensitizers localize to the same cellular region as
Rhodamine-123, suggesting that these compounds localize
in mitochondria, a more sensitive site for cell damage by
PDT (see Figure 11 for a representative example).
and the corresponding dipyrromethane 7 and 8 were
synthesized.
3.2.2. 3,9-Diethyl-6-(m-methoxyphenyl)-4,8-dimethyl-
2,10-diethoxycarbonyl dipyrromethane (7). Yield 70%;
1
mp 122–1258C; H NMR: d 8.25 (brs, 2H, 2£NH), 7.23–
7.27 (m, 1H, ArH), 6.82 (dd, J¼2.2, 7.8 Hz, 1H, ArH), 6.68
(d, J¼7.4 Hz, 1H, ArH), 6.63 (s, 1H, ArH), 5.45 (s, 1H, CH),
4.25 (q, J¼7.3 Hz, 4H, 2£OCH2CH3), 3.76 (s, 3H, OCH3),
2.73 (q, J¼7.6 Hz, 4H, 2£CH2CH3), 1.79 (s, 6H, 2£CH3),
1.30 (t, J¼7.1 Hz, 6H, 2£OCH2CH3), 1.11 (t, J¼7.3 Hz,
6H, 2£CH2CH3). Anal. calcd for C28H36N2O5: C, 69.98; H,
7.55; N, 5.83. Found: C, 70.08; H, 7.60; N, 5.82.
3. Experimental
3.1. Chemistry
All chemicals were of reagent grade and used as received.
Solvents were dried using standard methods unless stated
otherwise. Reactions were carried out under a N2 atmos-
phere and were monitored by analytical precoated
(0.20 mm) silica TLC plates (POLYGRAMw SIL N-HR).
Melting points were determined on electrically heated
melting point apparatus and are uncorrected. UV–vis
spectra were recorded on a Varian (Cary-50 Bio) spectro-
3.2.3. 3,9-Diethyl-6-(30,50-dimethoxyphenyl)4,8-dimethyl-
2,10-diethoxycarbonyldipyrromethane (8). Yield 60%;
viscous oil; 1H NMR: d 8.25 (brs, 2H, 2£NH), 6.38 (s, 1H,
ArH), 6.24 (s, 2H, ArH), 5.39 (s, 1H, CH), 4.26 (q, J¼
7.3 Hz, 4H, 2£OCH2CH3), 3.74 (s, 6H, 2£OCH3), 2.70–
2.78 (m, 4H, 2£CH2CH3), 1.79 (s, 6H, 2£CH3), 1.32
(t, J¼7.2 Hz, 6H, 2£OCH2CH3), 1.10–1.16 (m, 6H,
2£CH2CH3).
1
photometer. H and 19F NMR spectra were recorded on a
Brucker AMX 400 and 376.5 MHz NMR spectrometer,
respectively at 3038K in CDCl3 containing tetramethyl-
silane (TMS) as an internal standard. Proton chemical shifts
(d) are reported in parts per million (ppm) relative to TMS
(0.00 ppm) or CDCl3 (7.26 ppm) while fluorine chemical
shifts are reported in ppm relative to trifluoroacetic acid
(0.00 ppm). Coupling constants (J) are reported in Hertz
(Hz) and s, d, t, q, m, dd and br refer to singlet, doublet,
triplet, quartet, multiplet, doublet of doublet and broad
respectively. Mass spectral data (FAB) were obtained from
the University of Michigan, East Lansing, MI (the matrix is
usually nitrobenzyl alcohol) and from the Biopolymer
Facility of Basic Studies Center, Roswell Park Cancer
Institute, Buffalo, NY. Elemental analysis data were
obtained from Midwest Microlab, LLC, Indianapolis, IN.
3.3. General method for the preparation of pyrro-
methanes 9, 11, 13
The diethoxycarbonyl dipyrromethanes 6, 7 or 8 (for
example 11.6 g of 6) were individually dissolved in ethylene
glycol (250 mL). Sodium hydroxide (12 g, crushed powder)
was added, and the reaction mixture was refluxed for 1 h. It
was then cooled, diluted with dichloromethane (250 mL)
and washed with water (2£200 mL). The dichloro-
methane layer was dried over anhydrous sodium sulfate.
The residue obtained after evaporating the solvent was
chromatographed over silica column eluted with chloro-
form. The appropriate eluates were combined, the solvent
was evaporated, and the desired dipyrromethane 9 was
obtained in 95% yield.
3.2. General method for the synthesis of dipyrromethane
6, 7 and 8
3.3.1. 3,9-Diethyl-6-(p-methoxyphenyl)4,8-dimethyl-
dipyrromethane (9). Mp 64–668C; lit.21 1H NMR: d 7.30
(brs, 2H, 2£NH), 7.06 (d, J¼8.8 Hz, 2H, ArH), 6.85 (d,
J¼8.8 Hz, 2H, ArH), 6.38 (s, 2H, 2£Pyrrolic CH), 5.45
(s, 1H, CH), 3.81 (s, 3H, OCH3), 2.44 (q, J¼7.2 Hz, 4H,
2£CH2CH3), 1.81 (s, 6H, 2£CH3), 1.19 (t, J¼7.9 Hz, 6H,
2£CH2CH3).
Pyrrole 5 (10.0 g, 0.055 mol) and p-methoxy benzaldehyde
(3.74 g, 0.0275 mol) were dissolved in ethanol (70 mL) and
p-toluenesulfonic acid (200 mg) was added. The reaction
mixture was refluxed for 2 h under nitrogen. Analytical TLC
at frequent intervals was used to monitor the completion of
the reaction. It was then cooled, the solid was filtered, the
product was washed with cold water and dried under
vacuum at room temperature. The desired pyrromethane
6 [3,9-diethyl-6-(p-methoxyphenyl)-4,8-dimethyl-2,10-
diethoxycarbonyl dipyrromethane] was isolated as a white
powder in 80% (21.2 g) yield.
3.3.2. 3,9-Diethyl-6-(m-methoxyphenyl)-4,8-dimethyl-
dipyrromethane (11). Yield 88%; mp 202–2048C;
1H NMR: d 9.49 (s, 2H, 2£CHO), 9.08 (brs, 2H, 2£NH),
7.23–7.27 (m, 1H, ArH), 6.83 (dd, J¼2.5, 8.0 Hz, 1H,
ArH), 6.66 (d, J¼7.6 Hz, 1H, ArH), 6.62 (s, 1H, ArH),
5.53 (s, 1H, CH), 3.75 (s, 3H, OCH3), 2.71 (q, J¼7.6 Hz,
4H, 2£CH2CH3), 1.86 (s, 6H, 2£CH3), 1.20 (t, J¼7.8 Hz,
6H, 2£CH2CH3). Anal. calcd for C22H28N2O.2H2O:
C, 70.94; H, 8.66; N, 7.52. Found: C, 71.29; H, 7.17; N,
7.24.
3.2.1. 3,9-Diethyl-6-(p-methoxyphenyl)-4,8-dimethyl-
2,10-diethoxycarbonyl dipyrromethane (6). Mp 105–
1
1088C; H NMR: d 8.20 (brs, 2H, 2£NH), 7.01 (d, J¼
8.9 Hz, 2H, ArH), 6.87 (d, J¼8.9 Hz, 2H, ArH), 5.43 (s, 1H,
CH), 4.26 (q, J¼7.3 Hz, 4H, 2£OCH2CH3), 3.81 (s, 3H,
OCH3), 2.74 (q, J¼7.5 Hz, 4H, 2£CH2CH3), 1.78 (s, 6H,
2£CH3), 1.31 (t, J¼7.1 Hz, 6H, 2£OCH2CH3), 1.12 (t, J¼
7.7 Hz, 6H, 2£CH2CH3). Anal. calcd for C28H36N2O4:
C, 69.98; H, 7.55; N, 5.83. Found: C, 70.15; H, 7.37;
N, 5.93. By following a similar approach, pyrrole 5 was
reacted with m-methoxy or 3, 5-dimethoxy benzaldehyde
3.3.3. 3,9-Diethyl-6-(30,50-dimethoxyphenyl)-4,8-dimethyl-
dipyrromethane (13). Yield 97%; mp 119–1218C; 1H
NMR: d 7.34 (brs, 2H, 2£NH), 6.33–6.36 (m, 3H, ArH),
6.31 (s, 2H, 2£pyrrolic CH), 5.40 (s, 1H, CH), 3.74 (s, 6H,
2£OCH3), 2.40–2.45 (m, 4H, 2£CH2CH3), 1.81 (s, 6H,

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10069
2£CH3), 1.14–1.19 (m, 6H, 2£CH2CH3). Anal. calcd for
C23H30N2O2.5H2O: C, 60.50; H, 8.83; N, 6.40. Found: C,
60.00; H, 8.82; N, 5.91.
reaction was stirred for another 12 h. It was then diluted
with dichloromethane, washed with water and the organic
layer was dried over anhydrous sodium sulfate. Evaporation
of the solvent gave a residue which was dissolved in
trifluoroacetic acid (30 mL) and stirred at room temperature
for 30 min. The Zn-free porphyrin thus obtained after
standard work-up was chromatographed over a short
Grade III Alumina column and eluted with dichloro-
methane. The major band was collected and the solvent
evaporated. The residue was crystallized from dichloro-
methane/hexane and porphyrin 22 was isolated in 17%
(640 mg) yield.
3.4. General method for the preparation of diformyl-
dipyrromethanes 10, 12 and 14
The pyrromethane(s) [e.g. 9 (7.1 g)] was dissolved in
Vilsmeier reagent, prepared by reaction POCl3 (10.5 mL)
and DMF (45 mL) was added and the reaction mixture was
stirred at room temperature overnight. The reaction mixture
was poured into ice cold water (250 mL) and aqueous
sodium hydroxide (50%, 35 mL) was then added slowly; the
pH was adjusted to 10–12 and stirred overnight before
extracting with dichloromethane (3£200 mL). The organic
layer was separated, washed with water (2£200 mL) until
neutral and dried over anhydrous sodium sulfate. Evapo-
ration of the solvent gave a residue which was chromato-
graphed over a silica column, eluting with 1:1 ethyl acetate/
cyclohexane. The major band was separated, and the
product obtained after evaporating the solvent was crystal-
lized with methanol and compound 10 was isolated in
80% (6.80 g) yield.
3.5.1. 2,8,13,17-Tetraethyl-5-(p-methoxyphenyl)-3,7,12,
18-tetramethylporphyrin (22). Mp 280–2828C; UV–vis
[CH2Cl2, nm (1, M21 cm21)] 403 (3.82£105), 503 (3.15£
1
104), 535 (1.39£104), 571 (1.35£104), 623 (4.82£103); H
NMR: d 10.13 (s, 2H, 2£mesoH), 9.92 (s, 1H, mesoH), 7.86
(d, J¼9.0 Hz, 2H, ArH), 7.16 (d, J¼9.0 Hz, 2H, ArH),
4.00–4.06 (m, 8H, 4£CH2CH3), 3.99 (s, 3H, OCH3), 3.62
(s, 6H, 2£CH3), 2.47 (s, 6H, 2£CH3), 1.87 (t, J¼7.7 Hz, 6H,
2£CH2CH3), 1.75 (t, J¼7.6 Hz, 6H, 2£CH2CH3), 23.19
(brs, 1H, NH), 23.27 (brs, 1H, NH). Anal. calcd for
C39H44N4O: C, 80.10; H, 7.58; N, 9.58. Found: C, 80.14; H,
7.54; N, 9.62.
3.4.1. 3,9-Diethyl-2,10-diformyl-6-(p-methoxyphenyl)-
4,8-dimethyldipyrromethane (10). Mp 168–1698C;
1H NMR: d 9.50 (s, 2H, 2£CHO), 8.70 (brs, 2H, 2£NH),
6.98 (d, J¼8.7 Hz, 2H, ArH), 6.86 (d, J¼8.7 Hz, 2H, ArH),
5.47 (s, 1H, CH), 3.80 (s, 3H, OCH3), 2.70 (q, J¼7.5 Hz,
4H, 2£CH2CH3), 1.81 (s, 6H, 2£CH3), 1.20 (t, J¼7.5 Hz,
6H, 2£CH2CH3). Anal. calcd for C24H28N2O3.1/2H2O:
C, 71.80; H, 7.28; N, 6.98. Found: C, 71.69; H, 6.74; N,
6.76.
3.5.2. 2,8,13,17-Tetraethyl-5-(m-methoxyphenyl)-3,7,12,
18-tetramethylporphyrin (23). Yield 30%; mp 270–
1
2728C; H NMR: d10.16 (s, 2H, 2£mesoH), 9.96 (s, 1H,
meso H), 7.67 (d, J¼7.2 Hz, 2H, ArH), 7.59 (t, J¼
7.8 Hz, 1H, ArH), 7.50 (s, 1H, ArH), 4.02–10 (m,
8H, 4£CH2CH3), 4.00 (s, 3H, OCH3), 3.65 (s, 6H,
2£CH3), 2.60 (s, 6H, 2£CH3), 1.90 (t, J¼7.7 Hz, 6H,
2£CH2CH3), 1.78 (t, J¼ 7.94 Hz, 6H, 2£CH2CH3), 23.20
(brs, 1H, NH), 23.35 (brs, 1H, NH). Anal. calcd for
C39H44N4O: C, 80.10; H, 7.58; N, 9.58. Found: C, 80.76; H,
7.60; N, 9.54.
3.4.2. 3,9-Diethyl-2,10-diformyl-6-(m-methoxyphenyl)-
4,8-dimethyldipyrromethane (12). Yield 79%; mp 202–
1
2048C; H NMR: d 9.49 (s, 2H, 2£CHO), 9.08 (brs, 2H,
2£NH), 7.23–7.27 (m, 1H, ArH), 6.83 (dd, J¼2.5, 8.0 Hz,
1H, ArH), 6.66 (d, J¼7.6 Hz, 1H, ArH), 6.62 (s, 1H, ArH),
5.53 (s, 1H, CH), 3.75 (s, 3H, OCH3), 2.71 (q, J¼7.6 Hz,
4H, 2£CH2CH3), 1.86 (s, 6H, 2£CH3), 1.20 (t, J¼7.8 Hz,
6H, 2£CH2CH3). Anal. calcd for C24H28N2O3: C, 73.44; H,
7.19; N, 7.14. Found: C, 73.65; H, 7.29; N, 7.06.
3.5.3. 2,8-Diethyl-5-(30,50-dimethoxyphenyl)-2, 8-diethyl-
13,17-bis-(2-methoxycarbonylethyl)-3,7,12,18-tetra-
methylporphyrin (25). The title compound was prepared
by reacting diformyldipyrromethane 14 with dipyrro-
methane dicarboxylic acid 21 by following the procedure
reported by Chen et al.22
3.4.3. 3,9-Diethyl-2,10-diformyl-6-(3,5-di-methoxyphenyl)-
4,8-dimethyldipyrromethane (14). Yield 78%; mp 202–
2048C; 1H NMR (CDCl3, 400 MHz) d 9.52 (s, 2H, 2£CHO),
8.56 (brs, 2H, 2£NH), 6.40 (s, 1H, ArH), 6.20 (s, 2H, ArH),
5.42 (s, 1H, CH), 3.74 (s, 6H, 2£OCH3), 2.70 (q, J¼7.7 Hz,
4H, 2£CH2CH3), 1.81 (s, 6H, 2£CH3), 1.20 (t, J¼7.8 Hz,
6H, 2£CH2CH3). Anal. calcd for C25H30N2O4.1/2H2O: C,
69.58; H, 7.24; N, 6.49. Found: C, 69.52; H, 7.00; N, 6.16.
3.6. General method for the synthesis of porphyrins
26–29
Reaction of porphyrins 22–25 (100 mg each) with ethereal
boron tribromide solution afforded porphyrin 26–29 in
70–77%.
3.6.1. 2,8,13,17-Tetraethyl-5-(p-hydroxyphenyl)-3,7,12,
18-tetramethylporphyrin (26). Yield 77%; mp .3008C;
UV–vis [CH2Cl2, nm (1, M21 cm21)] 404 (3.79£105), 503
3.5. General method for the synthesis of porphyrins
22–25
1
(2.91£104), 536 (1.19£104), 570 (1.14£104); H NMR: d
The diformyl dipyrromethane (e.g. 10, 2.52 g, 6.43 mmol),
and dipyrromethane 18 (2.04 g, 6.42 mmol) were dissolved
in dichloromethane (500 mL). p-Toluenesulfonic acid
(6.0 g) dissolved in methanol (100 mL) was added, and
the reaction mixture was stirred at room temperature
overnight under nitrogen atmosphere. A saturated solution
of zinc acetate/methanol (125 mL) was added, and the
10.15 (s, 2H, 2£meso H), 9.94 (s, 1H, meso H), 7.89 (d,
J¼9.2 Hz, 2H, ArH), 7.18 (d, J¼8.7 Hz, 2H, ArH), 4.00–
4.08 (m, 8H, 4£CH2CH3), 3.64 (s, 6H, 2£CH3), 2.53 (s, 6H,
2£CH3), 1.88 (t, J¼7.7 Hz, 6H, 2£CH2CH3), 1.76 (t, J¼
7.5 Hz, 6H, 2£CH2CH3), 23.75 (brs, 2H, 2£NH). Anal.
calcd for C38H42N4O: C, 79.66; H, 7.42; N, 9.82. Found: C,
79.75; H, 7.45; N, 9.70.

10070
S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
3.6.2. 2,8,13,17-Tetraethyl-5-(m-hydroxyphenyl)-3,7,12,
18-tetramethylporphyrin (27). Yield 72%; mp .3008C;
UV–vis [CH2Cl2, nm (1, M21 cm21)] 402 (5.00£105),
502 (4.03£104), 536 (1.76£104), 570 (1.61£104), 623
8H, 4£CH2CH3), 3.70 (s, 6H, 2£CH3), 2.52 (s, 6H, 2£CH3),
1.94 (t, J¼7.7 Hz, 6H, 2£CH2CH3), 1.82 (t, J¼7.5 Hz, 6H,
2£CH2CH3). 19F NMR: d 13.12 (s, 6F, 2£CF3). Anal. calcd
for C47H46N4F6O.H2O: C, 69.27; H, 5.94; N, 6.87. Found:
C, 69.37; H, 6.19; N, 6.33.
1
(4.60£103); H NMR: d 10.18 (s, 2H, 2£meso H), 9.98
(s, 1H, mesoH), 7.69 (d, J¼7.0 Hz, 2H, ArH), 7.61 (t, J¼
7.7 Hz, 1H, ArH), 7.51 (s, 1H, ArH), 4.04–4.12 (m, 8H,
4£CH2CH3), 3.66 (s, 6H, 2£CH3), 2.58 (s, 6H, 2£CH3),
1.91 (t, J¼7.7 Hz, 6H, 2£CH2CH3), 1.79 (t, J¼7.94 Hz, 6H,
2£CH2CH3), 23.85 to 23.75 (each brs, 1H, 2£NH). Anal.
calcd for C38H42N4O: C, 79.66; H, 7.42; N, 9.82. Found: C,
80.02; H, 7.37; N, 9.83.
3.7.2. 5-[3-Bis{3,5-trifluromethyl)benzyloxy}phenyl]-
2,8,13,17-tetraethyl-3,7,12,18-tetramethylporphyrin (2).
Yield 96%; mp 109–1108C; UV–vis[CH2Cl2, nm (1, M21
cm21)] 404 (9.49£104), 501 (6.83£103), 537 (4.27£ 103),
570 (4.98£103); 1H NMR: d 10.17 (s, 2H, 2£meso CH), 9.96
(s, 1H, meso CH), 7.94 (s, 2H, ArH), 7.75–7.85 (m, 3H,
ArH), 7.62 (t, J¼7.8 Hz, 1H, ArH), 7.40 (dd, J¼2.6, 7.8 Hz,
1H, ArH), 5.32 (s, 2H, OCH2), 4.00–4.10 (m, 8H,
4£CH2CH3), 3.65 (s, 6H, 2£CH3), 2.53 (s, 6H, 2£CH3),
1.89 (t, J¼7.8 Hz, 6H, 2£CH2CH3), 1.77 (t, J¼7.6 Hz, 6H,
2£CH2CH3), 23.10 and 23.30 (each brs, 1H, 2£NH). 19F
NMR: d 13.00 (s, 6F, 2£CF3). Mass (FAB) calcd for
C47H46N4F6O: 796.35. Found: 797.36 (Mþ1).
3.7.3. 5-[30,50-Bis{300,500-trifluromethyl)benzyloxy}phenyl]-
2,8,13,17-tetraethyl-3,7,12,18-tetra-methyl porphyrin
(3). Yield 45%; mp 214–2168C; UV–vis[CH2Cl2, nm (1,
M21 cm21)] 403 (2.29£105), 502 (1.93£104), 536 (9.27£
103), 570 (8.53£103), 623 (3.34£103); 1H NMR: d 10.20 (s,
2H, 2£meso CH), 9.98 (s, 1H, meso CH), 7.96 (s, 4H, ArH),
7.87 (s, 2H, ArH), 7.42 (d, J¼2.9 Hz, 2H, ArH), 7.12–7.16
(m, 1H, ArH), 5.37 (s, 4H, 2£OCH2), 4.03–4.10 (m, 8H,
4£CH2CH3), 3.66 (s, 6H, 2£CH3), 2.60 (s, 6H, 2£CH3),
1.91 (t, J¼7.5 Hz, 6H, 2£CH2CH3), 1.78 (t, J¼7.5 Hz, 6H,
2£CH2CH3). 19F NMR: d 12.98 (s, 12F, 4£CF3). Anal.
calcd for C56H50N4F12O2: C, 64.74; H, 4.85; N, 5.39.
Found: C, 64.20; H, 4.94; N, 5.00.
3.6.3. 2,8,13,17-Tetraethyl-5-(30,50-di-hydroxyphenyl)-
3,7,12,18-tetramethylporphyrin (28). Yield 75%; mp
.3008C; UV–vis [CH2Cl2, nm (1, M21 cm21)] 402
(4.16£105), 502 (3.45£104), 536 (1.62£104), 569 (1.49£
104), 623 (6.50£103); 1H NMR (CDCl3, 400 MHz) d 10.18
(s, 2H, 2£mesoH), 9.98 (s, 1H, meso H), 7.05 (s, 2H, ArH),
6.69 (s, 1H, ArH), 4.04–4.12 (m, 8H, 4£CH2CH3), 3.66 (s,
6H, 2£CH3), 2.67 (s, 6H, 2£CH3), 1.90 (t, J¼7.7 Hz, 6H,
2£CH2CH3), 1.80 (t, J¼7.5 Hz, 6H, 2£CH2CH3). Anal.
calcd for C38H42N4O2.H2O: C, 75.47; H, 7.33; N, 9.26.
Found: C, 75.31; H, 7.38; N, 9.14.
3.6.4. 2,8-Diethyl-5-(30,50-dimethoxyphenyl)-13,17-bis-
(2-methoxycarbonylethyl)-3,7,12,18-tetramethylporphy-
rin (29). Yield 70%; mp .3008C; UV–vis [CH2Cl2, nm
(1, M21 cm21)] 403 (4.64£105), 502 (5.21£104), 536
(3.39£104), 571 (4.00£104); 1H NMR: d 10.18 (s, 2H,
2£meso H), 9.95 (s, 1H, meso H), 7.10 (s, 2H, ArH), 6.70
(s, 1H, ArH), 4.30–4.40 (m, 4H, 2£CH2CH2CO2CH3),
3.98–4.08 (m, 4H, 2£CH2CH3), 3.70 (s, 6H, 2£OCH3),
3.65 (s, 6H, 2£CH3), 3.30–3.40 (m, 4H, 2£CH2CH2CO2
CH3), 2.66 (s, 6H, 2£CH3), 1.80–1.90 (m, 6H, 2£CH2CH3).
Anal. calcd for C42H46N4O6.H2O: C, 63.69; H, 6.11; N,
7.08. Found: C, 63.32; H, 6.07; N, 6.41.
3.7.4. 5-[30,50-Bis{300,500-trifluromethyl)benzyloxy}phe-
nyl]-2,8-diethyl-13,17-bis(2-methoxycarbonylethyl)-3,7-
dimethylporphyrin (4). Yield 75%; mp 72–758C; UV–vis
[CH2Cl2, nm (1, M21 cm21)] 404 (3.81£105), 502
(3.02£104), 536 (1.41£104), 570 (1.32£104), 624 (4.82£
3.7. General method for the synthesis of porphyrins 1 to 4
1
103), 653 (2.59£103); H NMR: d 10.16 (s, 2H, 2£meso
3,5-Bis(trifluromethyl)benzylbromide (40 mL, 0.22 mmol)
and anhydrous K2CO3 (250 mg) were added to a stirred
solution of 5-(4-hydoxyphenyl)tetraethylporphyrin 26
(85 mg, 0.15 mmol) in dry acetonitrile (10 mL) and the
reaction mixture was refluxed overnight under nitrogen.
Solvent was evaporated under reduced pressure; water
(20 mL) was poured and extracted with CH2Cl2 (2£20 mL).
The combined organic extracts were washed with water
(2£20 mL) and organic fraction was dried over Na2SO4.
Removal of organic solvent in vacuo gave a crude
solid residue, which was chromatographed over silica
column using CH2Cl2 as an eluant to yield 77 mg (65%)
of 5-[4-{3,5-Bis(trifluromethyl)benzyloxy}phenyl]tetra-
ethylporphyrin 1 as purple plates.
CH), 9.97 (s, 1H, meso CH), 7.92 (s, 3H, ArH), 7.81–7.84
(m, 3H, ArH), 7.37 (d, J¼2.0 Hz, 2H, ArH), 7.11 (d,
J¼2.4 Hz, 1H, ArH), 5.35 (s, 4H, 2£OCH2), 4.40 (t,
J¼7.6 Hz, 4H, 2£CH2CH2CO2CH3), 4.00 (q, J¼7.8 Hz,
4H, 2£CH2CH3), 3.67 (s, 6H, 2£OCH3), 3.66 (s, 6H,
2£CH3), 3.30 (t, J¼7.6 Hz, 4H, 2£CH2CH2CO2CH3), 2.56
(s, 6H, 2£CH3), 1.74 (t, J¼7.4 Hz, 6H, 2£CH2CH3), 23.27
(brs, 1H, NH), 23.33 (brs, 1H, NH). 19F NMR: d 12.96
(s,12F, 4£CF3). Anal. calcd for C60H54N4F12O6.H20: C,
61.43; H, 4.81; N, 4.77. Found: C, 61.56; H, 4.71; N, 4.71.
3.7.5. 5-{30,50-Bis(300,500-dimethylbenzyloxy)}phenyl-2,8-
diethyl-13,17-bis(2-methoxycarbonylethyl)-3,7-di-
methylporphyrin (4a). Yield 30%; mp 158–1608C; UV–
vis [CH2Cl2, nm (1, M21 cm21)] 404 (3.81£105), 502
(3.43£104), 536 (1.56£104), 570 (1.50£104), 623 (5.19£
3.7.1. 5-[4-{3,5-Bis(trifluromethyl)benzyloxy}phenyl]-2,
8,13,17-tetraethyl-3,7,12,18-tetramethylporphyrin (1).
Mp. 274–2768C; UV–vis [CH2Cl2, nm (1, M21 cm21)]
403 (2.52£105), 502 (2.08£104), 535 (9.25£103), 570
1
103); H NMR (CDCl3, 400 MHz) d 10.18 (s, 2H, 2£meso
CH), 9.98 (s, 1H, meso CH), 7.31–7.32 (m, 2H, ArH), 7.11
(brs, 1H, ArH), 7.08 (s, 3H, ArH), 6.99 (s, 1H, ArH), 6.96
(brs, 2H, ArH), 5.17 (s, 4H, 2£OCH2), 4.42 (t, J¼7.6 Hz, 4H,
2£CH2CH2CO2CH3), 4.04 (q, J¼7.0 Hz, 4H, 2£CH2CH3),
3.70 (s, 6H, 2£OCH3), 3.69 (s, 6H, 2£CH3), 3.32 (t, J¼
7.8 Hz, 4H, 2£CH2CH2CO2CH3), 2.58 (s, 6H, 2£CH3), 2.30
1
(8.82£ 103), 624 (3.13£103); H NMR: d 10.21 (s, 2H,
2£meso CH), 10.00 (s, 1H, meso CH), 8.14 (s, 2H, ArH),
7.98 (s, 1H, ArH), 7.96 (d, J¼4.5 Hz, 2H, ArH), 7.31 (d,
J¼4.5 Hz, 2H, ArH), 5.42 (s, 2H, OCH2), 4.06–4.13 (m,

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10071
(s, 12H, 4£PhCH3), 1.78 (t, J¼7.4 Hz, 6H, 2£CH2CH3),
23.25 (brs, 1H, NH), 23.28 (brs, 1H, NH). Mass (FAB)
Calcd for C60H66N4O6: 938.22. Found: 939.20 (Mþ1).
a-MEM with 10% fetal calf serum, penicillin and
streptomycin. Cells were maintained in 5% CO2, 95% air
and 100% humidity. For determining the PDT efficacy,
these cells were plated in 96-well plates at a density of
1.25£104 cells well in complete media. After an overnight
incubation at 378C, the photosensitizers were added at the
same concentration (1.0 mM), incubated at 378C for 4 h in
the dark. Cells were then illuminated with a 1000 W quartz
halogen lamp with IR and band pass dichroic filters to allow
light between 400 and 700 nm, at a dose rate of 16 mW/cm2.
After PDT, the cells were washed once and placed in
complete media and incubated for 48 h. Then 10 mL of
5.0 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazoliumbromide dissolved in PBS (Sigma, St.
Louis, MO) was added to each well. After a 4 h incubation
at 378C the MTT and media were removed and 100 mL
DMSO was added to solubilize the formazin crystals.
The 96-well plate was read on a microtiter plate reader
(Miles Inc. Titertek Multiscan Plus MK II) at 560 nm. The
results were plotted as percent survival of the corresponding
dark (drug no light) control for each compound tested.
Each data point is the average of 5 replicate wells and
the error bars are the standard deviation of a single
experiment.
3.8. Synthesis of chlorin 30 and bacteriochlorin 31 from
porphyrin 4
OsO4 (75 mg) dissolved in diethyl ether (5 mL) was added
to a stirred solution of 4 (100 mg) in dry CH2Cl2 (20 mL)
and pyridine (0.2 mL). The reaction mixture was stirred at
room temperature for 6 h. UV–vis spectrum showed two
peaks at 645 nm (chlorin 30, major, Rf¼0.6 in 5% MeOH in
CH2Cl2) and at 713 nm (bacteriochlorin 31, minor, Rf¼0.4
in 5% MeOH in CH2Cl2). The reaction was worked up by
passing a stream of H2S gas for one minute, diluted with
CH2Cl2 (50 mL) and then filtered through fluted filter paper.
The residue was washed with CH2Cl2 (3£50 mL), dried
over Na2SO4. The solvent was concentrated and the crude
mixture so obtained was purified by preparative TLC using
5% MeOH in CH2Cl2 as an eluant. Three bands were
isolated. The fast moving band (10 mg) was identified as
unreacted starting material 4, the middle band (25 mg, 40%)
was found to be chlorin 30 and the slowest moving band
isolated in 13 mg (20%) was characterized as bacterio-
chlorin 31.
3.10. Intracellular localization
3.8.1. 5-[3,5-Bis{3,5-bis(trifluromethyl)benzyloxy}phenyl]-
2,8-diethyl-7,8-dihydroxy-3,7,12,18-tetramethyl-13,17-
bis(2-methoxycarbonylethyl)porphyrin (30). Mp 172–
1748C; UV–vis [CH2Cl2, nm (1, M21 cm21)] 397 (4.44£
In order to determine the subcellular localization of the
fluorinated porhyrin 4, chlorin 30 and bacteriochlorin 31, the
RIF cells were seeded on poly-L-lysine coated glass cover
slips at 1£105 in 6 well plates and cultered for 48 h to allow
for attachment and spreading. The cells were incubated at
378C in dark with the 1 mM concentration of photosensitizers
for 1, 4 and 24 h and then co-incubated with the 0.25 mM
concentration of the organelle-specific dyes Rhodamine-123
for mitochondria and Fluospheres for lysosomes. Immedi-
ately prior to microscopy (Zeiss Axiovert 35, Carl Zeiss, Inc.
Germany), the cells were gently rinsed with PBS. Cells were
illuminated with a mercury arc lamp with a filter cube
containing 530–585 nm excitation filter, a 600 nm dichoric
filter and a long pass emission filter to detect the
photosensitizer. Fluorescent images were recorded and
analyzed using a GenIISys intesifier coupled toa Dage MTI
CCD72 camera and digitally processed with Metamorph
software (Universal Imaging Corp., Downingtown, PA).
1
105), 500 (4.16£104), 648 (1.17£105); H NMR: d 9.88 (s,
1H, meso CH), 9.61 (s, 1H, meso CH), 9.21 (s, 1H, meso
CH), 7.97 (s, 2H, ArH), 7.91 (s, 2H, ArH), 7.88 (s, 1H,
ArH), 7.84 (s, 1H, ArH), 7.35 (s, 1H, ArH), 7.12 (s, 1H,
ArH), 7.06 (s, 1H, ArH), 5.40 (s, 2H, OCH2), 5.30 (s, 2H,
OCH2), 4.24 (t, J¼7.2 Hz, 2H, CH2CH2CO2CH3), 4.15
(t, J¼7.4 Hz, 2H, CH2CH2CO2CH3), 3.96 (q, J¼7.4 Hz, 2H,
CH2CH3), 3.69 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.47
(s, 3H, CH3), 3.44 (s, 3H, CH3), 3.17 (t, J¼7.6 Hz, 4H,
2£CH2CH2CO2CH3), 2.26 (s, 3H, CH3), 2.23–2.34 (m, 3H,
CH3), 1.70 (t, J¼7.8 Hz, 3H, CH3), 0.89–0.93 (m, 2H,
CH2CH3), 0.44 (t, J¼8.0 Hz, 3H, CH2CH3), 22.32 (brs, 1H,
NH), 19F NMR: d 12.98 (s, 6F, 2£CF3), 12.99 (s, 6F,
2£CF3). Mass (FAB) calcd. for C60H56N4F12O8: 1188.12.
Found: 1189.50 (Mþ1).
3.8.2. 5-[3,5-Bis{3,5-bis(trifluromethyl)benzyloxy}phenyl]-
2,8-diethyl-7,8,17,18-tetrahydroxy-3,7,12,18-tetramethyl-
13,17-bis(2-methoxycarbonylethyl)porphyrin (31). Mp
100–1038C; UV–vis (CH2Cl2) 395 (1.69£105), 504
(2.22£104), 644 (3.81£104), 716 (2.67£104). Note: Due to
mixture of isomers it was difficult to assign the resonances
3.11. Photophysical measurements
Absorption spectra were recorded on a Hewlett Packard
8453A diode array spectrophotometer. Time-resolved
fluorescence and phosphorescence spectra were measured
by a Photon Technology International GL-3300 with a
Photon Technology International GL-302, nitrogen laser/
pumped dye laser system, equipped with a four channel
digital delay/pulse generator (Stanford Research System
Inc. DG535) and a motor driver (Photon Technology
International MD-5020). Excitation wavelengths were
from 535 to 551 nm using coumarin 540A (Photon
Technology International, Canada) as a dye. Fluorescence
lifetimes were determined by a two-exponential curve fit
using a microcomputer. Nanosecond transient absorption
measurements were carried out using a Nd:YAG laser
(continuum, SLII-10, 4–6 ns fwhm) at 355 nm with the
1
for each proton in the H NMR spectrum. The 19F NMR
spectrum showed mainly two peaks at d 12.97 and
12.96 ppm. Mass (FAB) calcd for C60H58N4F12O10:
1223.13. Found: 1223.30 (Mþ1).
3.9. Method for in vitro biological studies
The in vitro photosensitizing activity of fluorinated photo-
sensitizers 1–4, chlorin 30 and bacteriochlorin 31 was
determined in the radiation induced fibrosarcoma (RIF)
tumor cell line.18 The RIF tumor cells were grown in

10072
S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
power of 10 mJ as an excitation source. Photoinduced
events were estimated by using a continuous Xe-lamp
(150 W) and an InGaAs-PIN photodiode (Hamamatsu
2949) as a probe light and a detector, respectively. The
output from the photodiodes and a photomultiplier tube was
recorded with a digitizing oscilloscope (Tektronix,
TDS3032, 300 MHz). The transient spectra were recorded
using fresh solutions in each laser excitation. All experi-
ments were performed at 298 K.
References
1. (a) Pandey, R. K.; Zheng, G. In The Porphyrin Handbook;
Smith, K. M., Kadish, K. M., Guilard, R., Eds.; Academic: San
Diego, 2000. (b) Dougherty, T. J.; Gomer, C.; Henderson,
B. W.; Jori, G.; Kessel, D.; Kobrelik, M. J.; Peng, Q. J. Natl.
Cancer Inst. 1998, 90, 889. (c) Bonnett, R. J. Heterocycl.
Chem. 2002, 39, 455. (d) Boyle, R. W.; Rousseau, J.;
Kudrevich, S. V.; Obochi, M. O. K.; van Lier, J. E. Br.
J. Cancer 1996, 73, 49.
1
For the O2 phosphorescence measurements, an O2-satu-
2. Sherman, W. M.; Allen, C. M.; van Lier, J. E. Methods
Enzymol. 2000, 319, 376.
rated C6D6 solution containing the sample in a quartz cell
(optical path length 10 mm) was excited at 532 nm using a
Cosmo System LVU-200S spectrometer. A photomultiplier
(Hamamatsu Photonics, R5509-72) was used to detect
emission in the near infrared region (band path 1 mm).
3. (a) Workman, P.; Maxwell, R. J.; Griffiths, J. R. NMR Biomed.
1992, 5, 270. (b) Blackstock, A. W.; Lightfoot, H.; Case, L. D.;
Tepper, J. E.; Mukherji, S. K.; Mitchell, B. S.; Swarts, S. G.;
Hess, S. M. Clin. Cancer Res. 2001, 7, 3263–3268.
(c) Martino, R.; Malet-Martino, M.; Gilard, V. Curr. Drug
Metab. 2000, 1, 271–303. (d) Hunjan, S.; Zhao, D.;
Constantinescu, A.; Hahn, E. W.; Antich, P. P.; Mason, R. P.
Int. J. Radiat. Oncol. Biol. Phys. 2001, 49, 1097–1108.
4. Bottomly, P. A. Radiology 1989, 170, 1.
3.12. Electrochemical and spectroelectrochemical
measurements
Cyclic voltammetry (CV) measurements were performed at
298 K on an EG&G Model 173 potentiostat coupled with an
EG&G Model 175 universal programmer in deaerated
benzonitrile solution containing 0.1 M TBAP as a support-
ing electrolyte. A three-electrode system was utilized and
consisted of a glassy carbon working electrode, a platinum
wire counter electrode and a saturated calomel reference
electrode (SCE). The reference electrode was separated
from the bulk of the solution by a fritted-glass bridge filled
with the solvent/supporting electrolyte mixture. Thin-layer
spectroelectrochemical measurements of the one-electron
oxidized and one-electron reduced bacteriochlorin
derivatives were carried out using an optically transparent
platinum thin-layer working electrode and a Hewlett–
Packard model 8453 diode array spectrophotometer coupled
with an EG&G Model 173 universal programmer.
5. Mason, R. P.; Shukla, H.; Antich, P. P. Magn. Reson. Med.
1993, 29, 296.
6. (a) Ceckler, T. L.; Gibson, S. L.; Hilf, R.; Bryant, R. G. Magn.
Reson. Med. 1990, 13, 416. (b) Thomas, C.; Counsell, C.;
Wood, P.; Adams, G. Bull. Cancer 1993, 80, 666.
7. Jameson, C. J. In Fluorine; Mason, J., Ed.; Plenum: New York,
1987; p 437 Chapter 16.
8. Omote, M.; Ando, A.; Takagi, T.; Koyama, M.; Kumadaki, I.
Tetrahedron 1996, 52, 13961.
9. Barton, D. H. R.; Zard, S. Z. J. Chem. Soc. Chem. Commun.
1985, 1098.
10. Caveleiro, J. A. S.; Gonsalves, J. A. S.; Kenner, A. M. d.’A. R.;
Smith, G. W. K. M. J. Chem. Soc. Perkin Trans. 1973, 240.
11. Jackson, A. H.; Smith, K. M. The Total Synthesis of Natural
Products; ApSimon, J., Ed.; Wiley: New York, 1973; Vol. 3,
p 144 1984, Vol. 6, p 23.
3.13. In vivo 19F measurements
12. Arsenault, G. P.; Bullock, E.; MacDonald, S. F. J. Am. Chem.
Soc. 1960, 82, 4384.
The radiation induced fibrosarcoma (RIF) cells were
maintained according to the protocol of Twentyman
et al.23 Tumors were grown on mouse foot dorsum by
inoculating 2£105 cells. The photosensitizer was adminis-
tered IP (,100 mM). 19F MR spectra were collected on a
Bruker 7T instrument using a home built surface coil. The
19F MR spectral parameters included a 908 pulse of 16 ms, a
spectral width of 20 KHz, 8 K data points, and a 2 s
repetition time for a total accumulation time of 30 min.
13. Clezy, P. S.; Fookes, C. J. R.; Liepa, A. J. Aust. J. Chem. 1977,
30, 2017.
14. (a) Pandey, R. K.; Jackson, A. H.; Smith, K. M. J. Chem. Soc.
Perkin Trans. 1 1991, 1211. (b) Smith, K. M.; Pandey, R. K.
Tetrahedron Lett. 1986, 27, 2717. (c) Jackson, A. H.; Pandey,
R. K.; Rao, K. R. N.; Roberts, E. Tetrahedron Lett. 1985, 793.
15. Smith, K. M. In The Porphyrins and Metalloporphyrins;
Smith, K. M., Ed.; Elesvier: Amsterdam, 1975.
16. Fukuzumi, S.; Suenobu, T.; Patz, M.; Hirasaka, T.; Itoh, S.;
Fujitsuka, M.; Ito, O. J. Am. Chem. Soc. 1998, 120, 8060.
17. Arbogast, J. W.; Darmanyan, A. P.; Chrostopher, P. D.; Foote,
S.; Rubin, Y.; Diederich, F. N.; Alvarez, M. M.; Anz, S. J.;
Whetten, R. L. J. Phys. Chem. 1991, 95, 11.
Acknowledgements
18. Felton, R. H. The Porphyrins; Dolphin, D., Ed.; Academic:
New York, 1978; Vol. 5. Chapter 3.
This research was supported by grants from DOD
(DMAD17-99-1-9065), NIH (CA 55792), the Robert
A. Welch Foundation (Grant E-680 to KMK), from the
Ministry of Education, Culture, Sports, Science and
Technology, Japan (13440216 and 13031059 to SF) and
the shared resources of the Roswell Park Cancer Center
Support Grant (P30CA16056). We thank Beverly
Chamberlin, Michigan State University, East Lansing for
mass spectrometry results. The elemental analyses were
performed at Midwest Micro lab, Indianapolis.
19. (a) Pandey, R. K.; Sumlin, A. B.; Potter, W. R.; Bellnier, D. A.;
Henderson, B. W.; Constantine, S.; Aoudia, M.; Rodgers,
M. A. J.; Smith, K. M.; Dougherty, T. J. Photochem.
Photobiol. 1996, 63, 194. (b) Henderson, B. W.; Bellnier,
D. A.; Graco, W. R.; Sharma, A.; Pandey, R. K.; Vaughan, L.;
Weishaupt, K. R.; Dougherty, T. J. Cancer Res. 1997, 57,
4000. (c) Zheng, G.; Potter, W. R.; Camacho, S. H.; Missert,
J. R. G.; Wang, G.; Bellnier, D. A.; Henderson, B. W.;

S. K. Pandey et al. / Tetrahedron 59 (2003) 10059–10073
10073
Rodgers, M. A. J.; Dougherty, T. J.; Pandey, R. K. J. Med.
Chem. 2001, 44, 1540. and references therein.
22. Chen, Y. C.; Medforth, C. J.; Smith, K. M.; Alderfer, J. L.;
Dougherty, T. J.; Smith, K. M. J. Org. Chem. 2001, 66, 3930.
23. Twentyman, P. R.; Brown, J. M.; Gray, J. W.; Franko, A. J.;
Scoles, M. A.; Kallman, R. F. J. Natl. Cancer Inst. 1980, 64,
595.
20. Pandey, R. K.; Potter, W. R.; Meunier, I.; Sumlin, A. B.;
Smith, K. M. Photochem. Photobiol. 1995, 62, 764.
21. Li, G.; Graham, A.; Potter, W. R.; Grossman, Z. D.; Oseroff,
A.; Dougherty, T. J.; Pandey, R. K. J. Org. Chem. 2001, 66,
1316.