Water-soluble, core-modified porphyrins as novel, longer-wavelength-absorbing sensitizers for photodynamic therapy. II. Effects of core heteroatoms and meso-substituents on biological activity

Article abstract of DOI:10.1021/jm0103662

Water-soluble, core-modified porphyrins were prepared and evaluated as sensitizers for photodynamic therapy (PDT). The addition of an aromatic aldehyde to 2,5-dilithiothiophene or -selenophene gave diol 3 as a nearly equimolar mixture of meso and d,1 dias

Full text of DOI:10.1021/jm0103662

J . Med. Chem. 2002, 45, 449-461
449
Wa ter -Solu ble, Cor e-Mod ified P or p h yr in s a s Novel,
Lon ger -Wa velen gth -Absor bin g Sen sitizer s for P h otod yn a m ic Th er a p y. II.
Effects of Cor e Heter oa tom s a n d Meso-Su bstitu en ts on Biologica l Activity
David G. Hilmey,† Masako Abe,† Marina I. Nelen,† Corey E. Stilts,† Gary A. Baker,† Sheila N. Baker,†
Frank V. Bright,† Sherry R. Davies,‡ Sandra O. Gollnick,‡ Allan R. Oseroff,‡ Scott L. Gibson,§ Russell Hilf,§ and
Michael R. Detty*,†
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000,
PDT Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263, and
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue,
Box 607, Rochester, New York 14642
Received August 3, 2001
Water-soluble, core-modified porphyrins were prepared and evaluated as sensitizers for
photodynamic therapy (PDT). The addition of an aromatic aldehyde to 2,5-dilithiothiophene
or -selenophene gave diol 3 as a nearly equimolar mixture of meso and d,l diastereomers, which
gave a single diastereomer following careful recrystallization. The condensation of pyrrole with
a diol 3 using catalytic BF3-etherate gave bispyrrolochalcogenophenes (4). Condensation of a
diol 3 with 4 in the presence BF3-etherate gave 21,23-dichalcogenaporphyrins (5). 21-
Thiaporphyrins (6) were prepared by condensation of a diol 3 with excess pyrrole and
benzaldehyde in the presence of tetrachlorobenzoquinone and catalytic BF3-etherate. Sulfonation
of 5 and 6 with concentrated sulfuric acid at 100 °C gave sulfonated derivatives 7-15. Bis-4-
methoxy-21,23-dithiaporphyrins 5h and 5l were demethylated with BBr3, and the resulting
phenols were alkylated with ethyl bromoacetate. Saponification gave 21,23-dithiaporphyrin
dicarboxylate salts 16 and 17. The 21,23-core-modified porphyrins gave band I absorption
maxima (λmax of 689-717 nm) at longer wavelengths than band I for the corresponding 21-
core-modified porphyrins, but both classes had band I maxima at longer wavelengths than
either TPPS4 or Photofrin (λmax of 630 nm for both). The core heteroatoms had little effect on
either absorption maxima or quantum yields of singlet oxygen generation in 7-17. The meso
substituents had a greater impact on absorption maxima. Compounds 7-17 were evaluated
for phototoxicity against Colo-26 cells in culture using 4 J cm-2 of 570-800 nm light. Compounds
8-12, 14, 16, and 17 gave a 50% cell kill in vitro at a lower concentration than Photofrin [5.7
mg (9 µmol)/kg]. Compounds 14, 16, and 17 gave a 50% cell kill with 4 J cm-2 of light and
submicromolar concentrations of sensitizer. Sensitizers 8 and 11 showed no toxicity or side
effects in BALB/c mice observed for 90 days following a single intravenous injection of 10 mg/
kg of sensitizer. Distribution studies show that sensitizer 8 accumulates in the tumors of BALB/c
mice. PDT with 8 at 0.125 mg (0.13 µmol)/kg or 11 at 2.5 mg (2.5 µmol)/kg and 135 J cm-2 of
694 nm light was comparable to PDT with Photofrin at 2.5 mg (4 µmol)/kg and 135 J cm-2 of
630 nm light against Colo-26 tumors in BALB/c mice.
In tr od u ction
using Photofrin as a photosensitizer.1,3-5 PDT with
Photofrin is also being evaluated as a protocol for
treating cancers of the head and neck region6 and for
treating pancreatic cancer7 as well as a possible therapy
against Karposi’s sarcoma and cancers of the brain,
breast (both primary and metastatic), skin, and abdo-
men.8
In the treatment of cancer, one protocol that can
better differentiate cancerous and normal tissue is
photodynamic therapy (PDT).1,2 PDT combines light and
endogenous oxygen with a photosensitizer localized in
or around the tumor. Irradiation of the sensitizer
produces a cascade of biochemical events that inactivate
cancer cells either directly through attack at specific
cellular sites or indirectly through the induction of
vascular damage to blood vessels feeding the tumor.1,2
PDT has regulatory approval in the USA, Canada, The
Netherlands, France, Germany, and J apan for cancers
of the lung, digestive tract, and genitourinary tract
Although Photofrin has been shown to be effective
against a number of malignancies, it is not the “ideal”
photosensitizer. Photofrin and most other porphyrin-
related sensitizers have a weak absorbance in the red
region of the spectrum (g630 nm where the penetration
of light in tissue is optimal) and induce long-lasting skin
photosensitivity (2-3 months) due to the retention of
porphyrin moieties in cutaneous tissue.9 Because Photo-
frin is not a well-defined, single agent,10 it is difficult
to modify it chemically for the investigation of struc-
ture-activity relationships. These limitations have
* To whom correspondence should be addressed. Tel: 716-645-6800.
Fax: 716-645-6963. E-mail: mdetty@acsu.buffalo.edu.
† The State University of New York.
‡ Roswell Park Cancer Institute.
§ University of Rochester Medical Center.
10.1021/jm0103662 CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/19/2001

450 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
Ch a r t 1
Sch em e 1
more, the 21-selena analogue showed no skin photosen-
sitization in animals irradiated 24 h postinjection.16
In view of our promising results with 111 and the
reported results with the 21-core-modified porphyrins,16
we were interested in examining 21- and 21,23-core-
modified porphyrins more thoroughly as sensitizers for
PDT. Herein, we describe a general synthetic strategy
to 21- and 21,23-core-modified porphyrins that permits
the regiospecific incorporation of S, Se, and N-H groups
in the 21- and 23-positions and the incorporation of
different pairs of meso substituents at the 5- and 10-
positions or the 15- and 20-positions. We also describe
the effects of heteroatom substitution and meso group
substitution on physical and photophysical properties
and on biological properties of these new core-modified
porphyrins.
stimulated research efforts toward the development of
second and third generation sensitizers for PDT that
better fit the definition of an ideal sensitizer.8
We have recently described the synthesis and proper-
ties of 21,23-dithia (1) and 21,23-diselena (2) core-mod-
ified derivatives11 of 5,10,15,20-tetrakis(4-sulfonato-
phenyl)porphyrin (TPPS4, Chart 1). TPPS4 once was a
promising photosensitizer displaying excellent mem-
brane permeability and lysosomal accumulation in cells
and is one of the more selective porphyrins for ac-
cumulation in tumors with demonstrated efficacy both
in vitro and in vivo.12 The development of TPPS4 as a
potential photosensitizer in the clinic has been hindered
by its reported neurotoxicity in mice at high concentra-
tions.13
Ch em istr y
Syn th esis. A. Wa ter -Solu ble Su lfon a ted Der iva -
tives. Analogues of TPPS4 have been prepared and a
limited quantitative structure-activity relationship
(QSAR) has been developed,12b,c,17 in which the number
and placement of solubilizing groups markedly affect
biodistribution, localization, and efficacy. In the series
of porphyrin analogues of TPPS4, molecules with two
identical sulfonatoaryl groups at the 5- and 10-positions
of the porphyrin core12b,c,17 and with other substituents
at the 15- and 20-positions have optimal properties for
uptake and distribution. Our general synthetic approach
to 21- and 21,23-core-modified porphyrins is shown in
Scheme 1 and allows the various combinations of
heteroatoms to be placed regiospecifically. The synthetic
route also allows the 5- and 10-positions to be identical
to or different from the 15- and 20-positions.
The 2,5-dilithiation of thiophene or selenophene was
accomplished with excess BuLi-TMEDA (N,N,N,N-
tetramethylethylenediamine) at -78 °C in hexanes.14a
The addition of various aromatic aldehydes to the 2,5-
dilithiochalcogenophenes gave the 2,5-bis(arylhydroxy-
methyl)chalcogenophenes (3) shown in Chart 2 in 37-
89% isolated yield as a single, crystalline diastereomer
(by 1H and 13C nuclear magnetic resonance (NMR))
following recrystallization of a mixture of meso and d,l
diastereomers.11 The condensation of pyrrole with 2,5-
bis(arylhydroxymethyl)chalcogenophenes 3a -c and 3h
was catalyzed by BF3-etherate to give bis(arylpyrrolo-
methyl)chalcogenophenes 4a -d in 46-92% isolated
yields (Chart 2).
Structurally, the chalcogen heteroatoms of the
21,23-core-modified porphyrins fill the core and prevent
binding to metals,14 which is a striking difference from
the normal porphyrin core and which might be expected
to impart different biological properties to core-modified
porphyrins in comparison to the normal tetrapyrrolic
porphyrins. The 21,23-dithiaporphyrin 1 was found to
be more potent than TPPS4 both in vitro and in vivo
although compound 1 generates singlet oxygen less
efficiently than TPPS4.11 Both 1 and 2 absorb light of
significantly longer wavelengths (λmax of 695 nm for 1
and 2 in water) than either TPPS4 or Photofrin (both
with λmax of 630 nm in water).11
Sulfonated 21-thia and 21-selena core-modified por-
phyrins have been prepared and evaluated as sensitiz-
ers for PDT. The 21-thia and 21-selena derivatives
absorb light of longer wavelengths than either TPPS4
or Photofrin.15 Sulfonated 21-core-modified porphyrins
were reported to be comparable to chlorin e6 for efficacy
in vivo with BFS1 sarcoma-bearing mice.16 Further-

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 451
Ch a r t 2
Compounds 5 and 6 were sulfonated with concen-
trated sulfuric acid at 100 °C11 to give the 4-sul-
fonatophenyl-substituted derivatives 7-14 in 14-56%
yields (Chart 1). The core-modified porphyrins were
isolated as the disodium salts following neutralization
of the sulfuric acid with NaOH and reverse phase
chromatography. Under the reaction conditions for the
preparation of 14, the dimethylanilino group of 5f is
protonated, thus deactivating the aniline ring to sul-
fonation. The deactivating fluoro and trifluoromethyl
groups allow selective sulfonation of the phenyl sub-
stituents of 5a -e, respectively.
Sulfonation of 5g gave a tetrasulfonated product. The
tolyl and phenyl groups of 5g are close enough in
reactivity that sulfonation gave a single tetrasulfonated
material, which we have assigned structure 15 in Chart
1. MALDI time-of-flight (TOF) mass spectrometry con-
firmed the tetrasulfonation product (m/z 1085 for M +
1) and the 20 signals observed in the 13C NMR spectrum
are consistent with sulfonation of the phenyl rings at
the 4-positions and monosulfonation of each tolyl group
at either the 2-position or the 3-position (21 signals
expected for either).
Dithiaporphyrin 5h was unstable to the sulfonation
conditions. No methoxy singlets were observed in the
1H NMR spectrum of the product mixture following
workup. Sulfonation of 5h was not investigated further.
B. Wa ter -Solu ble Ca r boxyla te Der iva tives. Com-
pounds 8-14 each have two sulfonato substituents to
provide water solubility. For comparison purposes, we
prepared the biscarboxylates 16 and 17, whose pKa
values should be nearer physiological pH. Compound
16 was prepared via initial demethylation of dimethoxy
porphyrin 5h with BBr3 to give bisphenol 5i in 57%
yield, which was alkylated with ethyl bromoacetate to
give 5j in 50% isolated yield. Saponification gave bis-
carboxylic acid 5k in 91% yield, which was then
converted to the disodium salt 16 in 70% yield.
Biscarboxylate 17 was prepared from porphyrin 5l via
a similar route. The condensation of pyrrole with either
2,5-bis(4-fluorophenylhydroxymethyl)thiophene (3c) or
2,5-bis(4-methoxyphenylhydroxymethyl)thiophene (3h )
was catalyzed by BF3-etherate in the presence of
TCBQ15 to give bis(4-fluorophenylpyrrolomethyl)thio-
phene (4c) in 80% isolated yield or bis(4-methoxyphen-
ylpyrrolomethyl)thiophene (4d ) in 92% isolated yield,
respectively (Chart 2). Equimolar amounts of 3h and
4c in dichloromethane were condensed with BF3-ether-
ate in the presence of TCBQ15 to give dithiaporphyrin
5l (Chart 2) in 29% yield. Demethylation with BBr3 gave
bisphenol 5m in 57% isolated yield. The bisphenol 5m
was treated with ethyl bromoacetate and sodium car-
bonate to give diester 5n in 76% yield. The diester 5n
was saponified to the corresponding acid with NaOH
in aqueous tetrahydrofuran (THF) to give carboxylic
acid 5o in 93% yield, which was then converted to the
sodium salt 17 in 91% yield.
As we previously described for the preparation of
3a ,b,11 the diols 3 can exist as either meso or d,l
diastereomers, which should be comparable energeti-
cally and spectroscopically. The meso and d,l forms can
interconvert in the presence of trace amounts of acid.11
1
The crude diol 3 displayed H NMR signals consistent
with both diastereomers. However, slow recrystalliza-
tion of the crude diol 3 gave crystalline materials that
were isolated as a single diastereomer, although we
cannot assign the meso or d,l stereochemistry with
certainty. For 3c and 3h , the reported literature melting
points are 103-104 and 154 °C,14a respectively, which
are significantly different than the melting points of
77-79 °C for 3c and 125-127 °C for 3h that we observe.
By 1H and 13C NMR, compounds 3c and 3h that we have
isolated each appear to be a single, pure diastereomer,
which may be the alternate diastereomer to those
reported previously.14a
Compounds 3 and 4 are the building blocks for the
regiospecific preparation of 21,23-core-modified por-
phyrins (5) and 21-core-modified porphyrins (6). Equimo-
lar amounts of 3 and 4 in dichloromethane were
condensed with catalytic BF3-etherate in the presence
of tetrachlorobenzoquinone (TCBQ)15 to give 21,23-
core-modified porphyrins (5) in isolated yields of 2-29%
(Chart 2). Diols 3a and 3c were each condensed with
pyrrole and benzaldehyde in the presence of TCBQ
using BF3-etherate as a catalyst15 to give 21-thiapor-
phyrins 6a 18 and 6b in 15 and 8% yields, respectively
(Chart 2).
Ch a r a cter iza tion of Wa ter -Solu ble, Cor e-Mod i-
fied P or p h yr in s. All of the sulfonated materials 7-15
and the dicarboxylates 16 and 17 gave parent ions by
mass spectrometry [fast atom bombardment (FAB(+)),
MALDI TOF, or Q-TOF electrospray] corresponding to
the di- or tetrasodium salts. The elemental analyses in
this series were consistent with several waters of

452 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
Ta ble 1. UV-Vis Band Maxima and Extinction Coefficients
crystallization per sulfonate or carboxylate group. In
compounds 7-14, sulfonation at the 4-position was
indicated by the appearance of a new AA′BB′ pattern
for TPPS4, 1, 2, and 7-17 in Water, λmax nm (∈ × 10-3 M-1
cm-1
compd
TPPS4 411 (464) 513 (15.5) 549 (7.0)
)
1
soret
band IV
band III
band II
band I
in the H NMR spectrum and appropriate symmetry in
the 13C NMR spectrum for each sample.
577 6.5) 630 (3.9)
633 (2.0) 695 (4.0)
631 (2.2) 695 (4.5)
607 (4.0) 666 (4.2)
633 (2.3) 701 (4.6)
1
434 (190) 513 (19.3) 546 (5.5)
434 (221) 513 (22.4) 546 (6.2)
425 (445) 515 (22.4) 550 (7.2)
431 (476) 518 (15.7) 560 (8.3)
From the 13C NMR spectrum of 15, the tolyl groups
are sulfonated either at the 2-position adjacent to the
dithiaporphyrin ring or at the 3-position adjacent to the
methyl substituents. The 1H NMR spectrum is more
complicated (Figure S1a, Supporting Information). The
thiophene protons would be expected to display two two-
proton singlets reflecting the mirror plane perpendicular
to the two rings. At ambient temperature, four broad-
ened yet distinct one-proton singlets are observed at δ
9.68, 9.66, 9.64, and 9.63. Similarly, the four pyrrole
protons would be expected to display two coupled, two-
proton doublets reflecting the two sets of chemically
different protons. At ambient temperature, four one-
proton doublets are observed (J ) 7.5 Hz for each)
centered at δ 8.11, 8.02, 7.69, and 7.64. These data are
consistent with two scenarios: (a) two isomeric mol-
ecules, one with a plane of symmetry through the
thiophene rings and the other with a C2 axis through
the thiophene rings, or (b) a single molecule with two
4-sulfonatophenyl substituents, one 4-methyl-3-sul-
fonatophenyl and one 4-methyl-2-sulfonato substituent.
The 13C NMR spectrum of 15 displayed 20 distinct
signals, one short of the 21 expected for the first scenario
but far short of the 42 signals expected for the second.
2
7
8
9
437 (443) 515 (34.9) 549 (10.8) 634 (2.6) 698 (7.1)
435 (243) 529 (20.4) 569 (10.5) 635 (3.6) 702 (6.3)
10
11
12
13
14
439 (65)
538 (10.3) 580 (5.1)
634 (3.0) 703 (3.8)
627 (2.5) 691 (3.2)
637 (2.1) 701 (4.2)
432 (224) 527 (12.6) 566 (5.4)
440 (270) 524 (19.7) 559 (7.0)
428 (79.7 515 (18.3) 577 (16.0) 635 (3.1) 716 (7.5)
447 (81.5)
15
16
17
433 (266) 515 (19.2) 549 (7.1)
434 (234) 514 (21.6) 549 (9.9)
442 (127) 533 (14.0) 573 (10.8) 645 (2.5) 717 (4.8)
627 (2.4) 689 (4.3)
634 (1.7) 699 (5.6)
modified porphyrins 1, 2, 8-11, and 13-17 in water
are summarized in Table 1. A typical absorption spec-
trum is shown in Figure S2 (Supporting Information)
for dithiaporphyrin 8. The absorption maxima of band
I, the long-wavelength band excited during PDT, are
all at significantly longer wavelengths (689-717 nm)
when compared to the band I maximum for either
Photofrin or TPPS4 (630 nm, ∈ ) 3000 M-1 cm-1 for
Photofrin and ∈ ) 3900 M-1 cm-1 for TPPS4) and have
significantly stronger absorbances (Table 1). The spec-
tral data for 21-thiaporphyrins 7 and 12 are also
compiled in Table 1. The absorption maxima of band I
for 7 (666 nm) and 12 (691 nm) are intermediate
between the Photofrin and the 21,23-core-modified
porphyrins.
The wavelength of the band I absorption maximum
in the 21,23-core-modified porphyrins is little affected
by the identity of the chalcogen atom. 21,23-Dithiapor-
phyrin (1) and 21,23-diselenaporphyrin (2) with four
sulfonatophenyl substituents have identical band I
maxima at 695 nm. Compounds 8-11 with 5,10-bis-4-
fluorophenyl and 15,20-bis-4-sulfonatophenyl substitu-
ents and all combinations of sulfur and selenium atoms
at the 21- and 23-positions have band I maxima that
fall between 698 and 703 nm.
Qu a n tu m Yield s for F lu or escen ce. In vivo fluo-
rescence techniques have had a significant impact on
the development of PDT with respect to the pharmaco-
kinetics of sensitizers and with respect to PDT dosim-
etry.19 Photofrin has been used in fluorescent diagnos-
tics,20 and the fluorescence of TPPS4 has been extensively
studied.21 Several of the 21,23-core-modified porphyrins
of this study were emissive although quantum yields
for fluorescence (φF) were low (Table 2). A small (e10
nm) Stokes shift was observed for the core-modified
porphyrins.
The quantum yield standard used in these studies
was a freshly prepared solution of TPPS4 (φF ) 0.12)21a
dissolved in water. Compounds 1, 8, and 13 have values
of φF of just under 0.01, and compound 17 has a value
of φF of 0.003. A representative absorption/emission
spectrum is shown for compound 8 in Figure S2 (Sup-
porting Information). While these values of φF are low,
they are sufficient to facilitate measurements of tissue
distribution as described in the biological results below.
The dimethylanilino analogue 14 was the only dithia-
porphyrin examined that was nonemissive.
1
When the H NMR spectrum of 15 is acquired at 60
°C (Figure S1b, Supporting Information), the two sets
of thiophene singlets collapse to two, two-proton singlets
at δ 9.98 and 9.95, and the four pyrrole doublets collapse
to an exchange-broadened singlet at δ 8.39 and a
doublet at δ 7.98 with J ) 7.5 Hz (Figure S1b, Sup-
porting Information). At ambient temperature, the
protons of the 4-sulfonatophenyl and 4-methylsul-
fonatophenyl groups are unresolved (Figure S1a, Sup-
porting Information). At 60 °C, the 4-sulfonatophenyl
protons resolve into an AA′BB′ pattern centered at δ
8.59 and 8.53 while the sulfonatotolyl groups resolve
into a poorly resolved doublet at δ 9.08 with a coupling
constant of ≈1 Hz, a broadened doublet at δ 8.93 with
a coupling constant of 4.4 Hz (and presumably longer-
range coupling to the proton at δ 9.08), and a well-
resolved doublet at δ 8.88 with a coupling constant of
4.4 Hz. These results are only consistent with the first
scenario involving two isomers with a symmetry ele-
ment through the thiophene rings. If sulfonation of the
tolyl groups was at the 2-position, hindered rotation of
the 4-phenyl-2-sulfonatophenyl rings would create two
isomers: one with both 2-sulfonato groups cis relative
to the plane of the dithiaporphyrin ring (mirror plane
of symmetry through the thiophenes) and one with the
2-sulfonato groups trans (C2 axis through the thiophenes).
In contrast, 3-sulfonatoaryl substituents would not
experience the same hindered rotation. As a conse-
quence, we have assigned the structure of 15 to have
the 4-methyl-2-sulfonatophenyl substituents as shown
in Chart 1. We have been unable to isolate or enrich
one isomer relative to the other either through careful
crystallization or through chromatographic separation.
Electr on ic Absor p tion Sp ectr a of Cor e-Mod ified
P or p h yr in s. The absorption spectra of 21,23-core-

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 453
Ta ble 2. Effective Concentrations To Give 50% Cell-Kill with 4 J cm-2 of Light (EC50), Quantum Yields for the Generation of Singlet
Oxygen [φ(1O2)], Rates of Photobleaching (kbleach), Flouresence Emission Maxima (λF), and Quantum Yields for Fluoresence (φF) for
Sensitizers 1, 2, and 7-17 as Compared to Photofrin and TPPS4
compd
E
E′
X
Araˆ
EC50 (µM)
φ(1O2)
kbleach × 104 s-1 λF (nm)
1.27 ( 0.01
φF ((σ)
Photofrin
TPPS4
1
9.0
125
30
>100
>100
1.6
NH NH SO3Na 4-C6H4SO3Na
S
Se
S
S
0.71b
0.12c
S
Se
SO3Na 4-C6H4SO3Na
SO3Na 4-C6H4SO3Na
0.50d
0.84 ( 0.02
700
700
8.6 ((0.4) × 10-3
2
7
8
9
0.17d
8.9 (( 0.4) × 10-4
NH SO3Na 4-C6H4SO3Na
S
0.80 ( 0.02
0.74 ( 0.03
0.65 ( 0.01
0.64 ( 0.01
0.55 ( 0.02
0.78 ( 0.05
F
F
F
F
4-C6H4SO3Na
4-C6H4SO3Na
4-C6H4SO3Na
4-C6H4SO3Na
4-C6H4SO3Na
4-C6H4SO3Na
4-C6H4SO3Na
0.85 ( 0.03
703
700
6.9((1.1) × 10-3
1.4 ((0.1) × 10-4
S
Se
S
Se
NH
S
S
S
S
S
2.1
1.2
7.9
2.1
10
11
12
13
14
15
16
17
Se
Se
S
S
S
S
S
S
1.44 ( 0.03
2.4 ( 0.2
< 10-4
F
CF3
NMe2
>100
0.64
12
0.13 ( 0.02
0.77 (0.04
697
703
8.8 ((0.8) × 10-3
<10-4
0.60 ( 0.02
SO3Na C6H4(CH3)SO3Nae
H
F
4-C6H4OCH2CO2Na
4-C6H4OCH2CO2Na
0.15
0.43
0.18 ( 0.02
0.16 ( 0.02
0.71 ( 0.02
3.2((0.1) × 10-3
a
b
d
4-Substituted aryl. Ref 23. c Ref 22a. Ref 11. e 4-Methyl, 2-sulfonato.
The introduction of selenium into the porphyrin core
greatly reduces the quantum yield for fluorescence due
to heavy atom effects.22 Diselenaporphyrin 2 is 10% as
emissive in comparison to its dithiaporphyrin analogue
1. Thiaselenaporphyrin 9 is 2% as emissive in compari-
son to its dithiaporphyrin analogue 8. Diselenaporphy-
rin 11 gave no detectable emission.
21-thiaporphyrin 12 occurs more rapidly than photo-
bleaching of Photofrin while photobleaching of 21,23-
diselenaporphyrin 11 occurs at a rate comparable to the
photobleaching of Photofrin. 21,23-Dithiaporphyrins 1,
8, and 14 are less susceptible to photobleaching in
comparison to Photofrin while dithiaporphyrins 13, 16,
and 17 are nearly an order of magnitude more photo-
stable in comparison to Photofrin.
Qu a n tu m Yield s for Sin glet Oxygen Gen er a tion .
The generation of singlet oxygen appears to be impor-
tant in the phototoxicity of the core-modified porphy-
rins.11 The quantum yield for singlet oxygen generation
[φ(1O2)] by TPPS4 has been reported to be 0.71,23 while
we have reported values of φ(1O2) for 1 and 2 to be 0.50
and 0.17, respectively (Table 2).11 Values of φ(1O2) were
measured for compounds 7-12, 14, and 17 and are
compiled in Table 2. Values of φ(1O2) are uniformly
higher for the 21,23-core-modified porphyrins with two
4-sulfonatonphenyl substituents relative to 1 and 2 with
four 4-sulfonato groups. Values of φ(1O2) appear to be
higher for the 21-thiaporphyrins relative to 21,23-core-
modified porphyrins with identical substituents. Values
of φ(1O2) for selenium-containing 21,23-core-modified
porphyrins 9-11 are lower than for dithiaporphyrin 8,
which is somewhat surprising since spin-orbit effects
from the heavy atom(s) would be more pronounced for
9-11 relative to 8.22 As we previously noted in compar-
ing 1 and 2,11 one possible explanation is that the short,
intramolecular Se‚‚‚S contacts in 9 and 10 and the
Se‚‚‚Se contacts in 11 disrupt the planarity of the
π-framework much more than the S‚‚‚S contacts of the
dithiaporphyrins. Such disruption may lead to less
efficient intersystem crossing relative to the various
photophysical routes to return to the ground state.
Other substituent changes in the meso positions have
little impact on values of φ(1O2) as represented by 14
[φ(1O2) ) 0.60] and 17 [φ(1O2) ) 0.71].
Solu bility Ch a r a cter istics of Cor e-Mod ified P or -
p h yr in s. In comparing a series of compounds in order
to establish structure-activity relationships, one must
be concerned with the bioavailability of each of the
individual compounds in the series. Of particular con-
cern are the varying solubilities of different photosen-
sitizer candidates, which can skew the comparison of
results from studies in vitro and in vivo. The tetrasul-
fonato, disulfonato, and dicarboxylate derivatives of this
study displayed varying degrees of aqueous solubility.
The tetrasulfonato derivatives 1, 2, 7, and 15 as well
as disulfonato derivatives 8-13 were all soluble at 10
mg of sensitizer per milliliter of saline (≈10-2
M
solutions), and the resulting solutions remained as such
even upon storage at 5 °C. Dimethylanilino derivative
14 and dicarboxylate derivatives 16 and 17 were not
soluble at 10 mg/mL in saline and gave stable solutions
at 5 °C at concentrations of 1 mg of sensitizer per
milliliter of saline (≈10-3 M).
Su m m a r y of Ch em ica l a n d P h otop h ysica l P r op -
er ties of Cor e-Mod ified P or p h yr in s. The 21- and
21,23-core-modified porphyrins 1, 2, and 7-17 of this
study have chemical and photophysical properties that
offer advantages as sensitizers for PDT. Synthetically,
both the 21- and the 21,23-core-modified porphyrins can
be prepared in pure form with a variety of aryl substit-
uents and solubilizing groups as well as different
combinations of heteroatoms at the 21- and 23-positions.
The band I absorption maxima of all of these compounds
are at longer wavelengths than the band I absorption
maximum of either Photofrin or TPPS4. The new core-
modified porphyrins of this study (compounds 7-17)
produce singlet oxygen efficiently upon irradiation of
band I. The meso substituents have little impact on
values of φ(1O2). It should be noted that the 21,23-
diselaporphyrins have lower values of φ(1O2) than other
analogues perhaps reflecting the nonplanarity of the
diselenaporphyrins. The 21,23-dithiaporphyrins exam-
P h otoblea ch in g of 21,23-Cor e-Mod ified P or p h y-
r in s. One of the limitations of PDT dosimetry is the rate
of bleaching of the photosensitizer.1,2,10 Rates of photo-
bleaching of core-modified porphyrins 1, 8, 11-14, 16,
and 17 in phosphate-buffered saline (PBS) under condi-
tions of constant initial absorbance between 570 and 750
nm using 570-750 nm light at 500 mW cm-2 are
compiled in Table 2 and can be compared to that for
Photofrin as shown in Figure S3 (Supporting Informa-
tion) for several of these compounds. Photobleaching of

454 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
ined for photobleaching in this study are all more
photostable in comparison to Photofrin, and many of
these species exhibit fluorescence quantum yields that
are sufficient to assess cellular uptake and tissue
distribution. Finally, all of the compounds 1, 2, and
7-17 are sufficiently water-soluble for the comparison
of biological properties in vitro.
Biology
In Vit r o St u d ies. Da r k a n d P h ot ot oxicit ies
Aga in st Colo-26 Cells in Cu ltu r e. The core-modified
porphyrins of this study and Photofrin were evaluated
in culture for dark- and light-induced toxicities toward
Colo-26 cells, a murine colon carcinoma cell line. Cell
cultures were incubated for 24 h in the dark with
various concentrations of sensitizer and were then
washed prior to treatment with filtered 590-800 nm
light for a total light dose of 4 J cm-2. Light-treated cells
and dark controls were incubated for 24 h, and cell
survival was determined. Results are compiled in Table
2 as the effective concentration of sensitizer to give 50%
cell kill with 4 J cm-2 of 590-800 nm light (EC50).
Values of EC50 were determined from a plot of the
surviving fraction of cells vs concentration of sensitizer
(Figures S4-S16 for 1, 2, and 7-17, respectively, and
Figure S17 for Photofrin, Supporting Information). None
of the core-modified porphyrins displayed significant
dark toxicity at concentrations e100 µM (Figures
S4-S16, Supporting Information).
Against Colo-26 cells, all of the tetrasulfonated ma-
terials [dithiaporphyrin 1 (EC50 of 30 µM), diselenapor-
phyrin 2 (EC50 of >100 µM), thiaporphyrin 7 (EC50 of
>100 µM), and dithiaporphyrin 15 (EC50 of 12 µM)] were
less effective in comparison to Photofrin [EC50 of 5.7 mg/
L, 9.0 µM (assuming a monomer)]. In analogy to the
QSAR developed for porphyrin derivatives of TPPS4,12a,b,17
replacing the sulfonato groups on adjacent meso posi-
tions in the core-modified porphyrins should improve
the relative phototoxicity. Replacing the 5- and 10-
sulfonato substituents with fluoro substituents (com-
pounds 8-12) gave markedly lower values of EC50.
Diselenaporphyrin 11 with an EC50 value of 7.9 µM was
comparable to Photofrin while compounds 8-10 and 12
with at least one sulfur heteroatom in the core were
more effective than Photofrin with EC50 values of
1.2-2.1 µM. A comparison of Photofrin at several
concentrations [2.5-10 µg/mL (4-16 µM)] with com-
pound 8 at 3 µg/mL (3 µM) is shown in Figure 1.
Other substituent changes gave submicromolar val-
ues of EC50. Replacing the sulfonato water-solubilizing
groups of 8 with the carboxylate groups of 16 and 17
gave values of EC50 of 0.43 and 0.15 µM, respectively.
Replacing the fluoro substituents of 8 with the di-
methylamino groups of 14 gave a value of EC50 of 0.64
µM. In contrast, replacing the fluoro substituents of 8
with trifluoromethyl substituents in 13 gave greatly
reduced phototoxicity with an EC50 of >100 µM.
F igu r e 1. Comparison of dithiaporphyrin 8 and Photofrin
photosensitization on the cell viability of cultured Colo-26
tumor cells. Data are expressed as the surviving fraction of
viable cells for cells treated with various concentrations of
Photofrin [2.5-10.0 µg/mL (4-16 µM)] and 630 nm light (75
mW cm-2) or dithiaporphyrin 8 [3 µg/mL (3 µM)] and 694 nm
light (75 mW cm-2). Each data point represents the mean
surviving fraction of viable cells calculated from at least three
separate experiments performed in duplicate; bars are the
SEM.
and cell survival was determined. The surviving fraction
24 h postirradiation is shown graphically in Figure S18
(Supporting Information). As observed with Colo-26 cells
(Table 2), 8, 11, 14, and 17 were all more phototoxic
than Photofrin against R3230AC cells. Furthermore,
compounds 14 and 17 are more phototoxic than 8 and
11 toward R3230AC cells, which is also analogous to
their behavior relative to 8 and 11 against Colo-26 cells
(Table 2).
Although compounds 14, 16, and 17 were the most
phototoxic materials against Colo-26 cells in vitro, these
materials were not the most water-soluble in the series.
For ease of formulation, initial in vivo studies were
conducted with dithiaporphyrin 8 and diselenaporphy-
rin 11, which readily dissolved in saline or 5% aqueous
dextrose solutions.
In Vivo Stu d ies. A. In itia l Da r k Toxicities. With
porphyrin-related photosensitizers, the therapeutic dose
for rodents is typically 1-5 mg/kg.1,2 We chose a starting
dose of 10 mg/kg to evaluate the core-modified porphy-
rins for dark toxicity. In groups of five BALB/c mice
given a single intravenous injection of 10 mg (10 µmol)/
kg of dithiaporphyrin 8 or 10 mg (10 µmol)/kg of
diselenaporphyrin 11 as a saline solution, neither
toxicity nor morbidity was observed. The animals were
followed for 90 days postinjection under normal vi-
varium conditions (daily cycle of 12 h of fluorescent
light/12 h of darkness). After the animals were sacri-
ficed, no gross abnormalities were noted in the organs
and tissues of sacrificed animals.
B. Distr ibu tion Stu d ies w ith Dith ia p or p h yr in 8.
Initial distribution studies were done at higher than
therapeutic doses to facilitate the tracking of dithiapor-
phyrin 8 in various tissues. In BALB/c mice bearing
Colo-26 tumors, the presence of 8 was measured by
fluorescence spectroscopy at 1, 4, and 24 h postinjection
of 5 mg (5 µmol)/kg of 8 (Figure 2). Fluorescence
quenching in diselenaporphyrin 11 made similar studies
impractical with this sensitizer. From these three time
Similar results were obtained with R3230AC rat
mammary adenocarcinoma cells in vitro. Cell cultures
were incubated for 24 h in the dark with 10 µM
concentrations of Photofrin, 8, 11, 14, or 17 and were
then washed prior to treatment with filtered 570-800
nm light for a total light dose of 0.9 J cm-2. Light-
treated cells and dark controls were incubated for 24 h,

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 455
F igu r e 2. Tissue distribution of dithiaporphyrin 8 in BALB/c
mice bearing Colo-26 tumors. Animals were given 5 mg (5
µmol)/kg of sensitizer and were sacrificed at the indicated time
points (three animals per point). Tissues were cut, homog-
enized, and taken up in Solvable. Bars are the SEM.
F igu r e 3. PDT with dithiaporphyrins 1 (open squares), 8
(filled triangles), and diselenaporphyrin 11 (open circles)
against implanted Colo-26 tumors in BALB/c mice. Animals
received 10 mg (10 µmol)/kg of 1, 0.125 mg (0.13 µmol)/kg of
8, or 2.5 mg (2.5 µmol)/kg of 11 and 135 J cm-2 of 694 nm
light 4 h postinjection. Times were measured posttreatment
until tumor volumes reached 400 mm3. These data can be
compared to a control group receiving neither drug nor light
(filled circles) and to a group treated with 2.5 mg (4 µmol)/kg
of Photofrin and 135 J cm-2 of 630 nm light (open triangles)
24 h postinjection.
points, the highest concentration of 8 in the tumor was
found at 4 h, which suggested that the maximum
concentration of 8 in tumors occurred between 1 and
24 h postinjection. In contrast, the concentration of 8
showed apparent decreases from the 1 h peak in the
liver, kidney, lung, heart, and spleen. At 24 h, the
concentration of 8 was higher in the tumor than in the
liver, kidney, lung, and heart.
The uptake of dithiaporphyrin 8 in tumor was much
greater than the uptake of dithiaporphyrin 1. At a dose
of 5 mg (5 µmol)/kg, a concentration of 19 ( 7 ng of 1/g
of protein was measured in tumors of BALB/c mice
bearing Colo-26 tumors 24 h postinjection. In contrast,
a concentration of 494 ( 90 ng of 8/g of protein was
measured in the tumors of animals 24 h postinjection
of 5 mg (5 µmol) of 8/kg (Figure 2), which represents a
greater than 25-fold increase in photosensitizer uptake.
C. P DT w ith Dith ia p or p h yr in 8 a n d Diselen a -
p or p h yr in 11. On the basis of the EC50 values in Table
2, diselenaporphyrin 11 [EC50 of 7.9 µg/mL (7.9 µM)]
should be comparable to Photofrin (EC50 of 9.0 µM) with
respect to treatment dose while dithiaporphyrin 1 (EC50
> 100 µM) should require a significantly higher dose.
In contrast, dithiaporphyrin 8 with an EC50 of 1.6 µM
should require a significantly smaller treatment dose
than either 11 or Photofrin.
BALB/c mice bearing Colo-26 tumors were given 10
mg (10 µmol)/kg of dithiaporphyrin 1, 0.125 mg (0.13
µmol)/kg of dithiaporphyrin 8, or 2.5 mg (2.5 µmol)/kg
of diselenaporphyrin 11 as a 5% dextrose in water
solution via tail vein injection. The animals were
irradiated 4 h postinjection with 135 J cm-2 of 694 nm
red light from a dye laser (75 mW cm-2 for 30 min). The
time in days to 400 mm3 tumor volume was noted for
each animal in each group, and the results are pre-
sented as a Kaplan-Meyer plot in Figure 3. Of the five
animals treated with dithiaporphyrin 8 and light, one
cure was obtained, and the other four animals gave a
mean time to 400 mm3 of 18 ( 4 days, which is a 300%
increase relative to untreated controls (6 ( 2 days, P <
0.001). Of the 10 animals treated with diselenaporphy-
rin 11 and light, three cures were obtained and the other
seven animals gave a mean time to 400 mm3 of 17 ( 3
days, which again is statistically different from un-
treated controls (P < 0.001). Of the four animals treated
with dithiaporphyrin 1 and light, a mean time to 400
mm3 of 13 ( 6 days was obtained, which represents a
significant response relative to untreated controls (P <
0.014). However, no cures were obtained with 1 and the
delay in tumor regrowth was significantly smaller than
for 8 and 11.
The results for PDT with 8 and 11 can be compared
to those with Photofrin in the same animal model
(Figure 3). BALB/c mice bearing Colo-26 tumors were
given 2.5 mg (4.0 µmol)/kg of Photofrin as a 5% aqueous
dextrose solution and were irradiated 24 h later with
135 J cm-2 of 630 nm laser light at a power of 75 mW
cm-2. The dose, interval between administration and
irradiation, fluence, and fluence rate are published
conditions for PDT with Photofrin.2,8,9 Of the eight
animals treated in this group, four cures were obtained
and the other four animals gave a mean time to 400
mm3 of 21 ( 4 days, which is not significantly different
than the results with either 8 at a dose of 0.125 mg/kg
or 11 at a dose of 2.5 mg/kg and irradiation 4 h after
sensitizer injection (P > 0.59).
It should be noted that no aspect of PDT with either
8 or 11 has yet been studied to achieve optimization (i.e.,
the sensitizer dose, the delivery vehicle, the interval
between sensitizer administration and irradiation, light
dose, fluence, and application of fractionated irradia-
tion). Despite this, PDT with either 8 or 11 clearly gives
efficacy comparable to that of Photofrin using published
conditions.2,8,9
D. Clea r a n ce Stu d ies Usin g Ea r -Sw ellin g Re-
sp on se a s a n En d P oin t. With Photofrin and related
porphyrin sensitizers, long-term acute cutaneous pho-
tosensitivity is a significant side effect in their clinical
use.9 The time course of acute cutaneous photosensitiv-
ity following administration of 0.125 mg (0.13 µmol)/kg

456 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
from GIBCO (Grand Island, NY). Fetal bovine serum (FBS)
was obtained from Atlanta Biologicals (Atlanta, GA). Concen-
tration in vacuo was performed on a Bu¨chi rotary evaporator.
NMR spectra were recorded at 30.0 °C on a Varian Gemini-
300, Inova 400, or Inova 500 instrument with residual solvent
signal as the internal standard: CDCl3 (δ 7.26 for proton, δ
77.0 for carbon). Infrared spectra were recorded on a Perkin-
Elmer FT-IR instrument. UV-visible near-IR spectra were
recorded on a Perkin-Elmer Lambda 12 spectrophotometer or
on a Sequential DX17 MV Stopped-Flow Spectrometer (Ap-
plied Photophysics, Leatherhead, U.K.). Both were equipped
with a circulating constant-temperature bath for the sample
chambers. Elemental analyses were conducted by Atlantic
Microanalytical, Inc. Electrospray and MALDI mass spectrom-
etry were conducted by the Campus Chemical Instrumentation
Center of The Ohio State University (Columbus, OH). Com-
pounds 1-3a ,b were prepared as described in ref 11.
of 8 or 5.0 mg (5 µmol)/kg of 11 was examined using
the established murine ear-swelling response (ESR).24
The data for 8 and 11 can be compared to the ESR
observed in Photofrin-treated [5 mg (8 µmol)/kg] mice
as reported in ref 24. The absolute ESR at day 1 for 8
at 0.125 mg (0.13 µmol)/kg or for 11 at 5.0 mg (5 µmol)/
kg was on the order of 0.11-0.13 mm of swelling above
the normal untreated ear thickness of 0.25-0.30 mm.
The absolute swelling observed with Photofrin-treated
animals on day 1 was on the order of 0.20-0.25 mm.24
The ESR disappeared between days 3 and 4 with 8 at
0.125 mg/kg and between days 5 and 7 with 11 at 5.0
mg/kg. Even at 31 days, there remained a positive ESR
in Photofrin-treated animals.24 Importantly, concentra-
tions of 8 or 11 that gave comparable results to Photo-
frin for PDT efficacy gave significantly reduced duration
of cutaneous photosensitivity.
Su m m a r y of Biologica l P r op er ties. The sulfonated
and carboxylated core-modified porphyrins examined in
this study are all sufficiently soluble at the concentra-
tions examined for the PDT studies in vitro to allow a
comparison of structure and activity. A comparison of
sulfonates 1, 8, and 11 with Photofrin demonstrated
that relative phototoxicity in vitro (against Colo-26 and
R3230AC cells) roughly translates to relative effective-
ness for PDT in vivo. It should be possible to optimize
sensitizer performance through structure-activity stud-
ies in vitro in the sulfonato series. The dicarboxylate
derivatives 16 and 17 were even more effective than
sulfonato derivatives 1, 2, and 7-15 as PDT sensitizers
in vitro although the dicarboxylates were less water-
soluble. We are evaluating delivery vehicles for dicar-
boxylates 16 and 17 (as well as other carboxylate
derivatives) and for dimethylanilino derivative 14 in
order to evaluate their effectiveness as PDT sensitizers
in vivo.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2,5-Bis-
(a r ylh yd r oxym eth yl)th iop h en es a n d Selen op h en es.
P r ep a r a tion of 2,5-Bis[(4-flu or op h en yl)h yd r oxym eth -
yl]th iop h en e (3c). Thiophene (8.4 g, 0.10 mol) was added to
a solution of n-butyllithium (177 mL, 1.6 M in hexanes, 0.30
mol) and TMEDA (45 mL, 0.31 mol) in 500 mL of hexanes,
under an argon atmosphere. The reaction mixture was heated
at reflux for 2 h, cooled to ambient temperature, and trans-
ferred via a cannula to a pressure-equalizing addition funnel.
This dilithiothiophene suspension was then added dropwise
to a 0 °C solution of 4-fluorobenzaldehyde (37.2 g, 0.30 mol)
in THF (500 mL), which had been dried over basic alumina
and degassed with argon for 15 min. After the addition was
complete, the mixture was allowed to warm to room temper-
ature, 750 mL of a 1 M solution of ammonium chloride was
added, and the organic phase was separated. The aqueous
phase was extracted with ether (3 × 400 mL). The combined
organic extracts were washed with water (3 × 500 mL) and
brine (500 mL), dried over MgSO4, and concentrated to a
yellow oil. The crude product was recrystallized from toluene
to give 14.3 g (43%) of white crystalline product of one
diasteromer (meso or d,l); mp 77-79 °C (literature mp14a
103-104 °C). IR (KBr): 3402 br, 1663, 1516, 1225, 1015 cm-1
.
FAB(+) MS: m/z 331 (C18H14F2O2S + H, M + 1). Anal. C, H.
If one sulfur atom is in the core, the second hetero-
atom at the 21- or 23-position can be selected from NH,
S, or Se with little effect on phototoxicity in vitro.
However, one structural feature that appears to be im-
portant for increased phototoxicity in vitro in the sul-
fonato series is the presence of hydrogen bond-ac-
cepting groups on two of the aromatic rings. The
fluorophenyl substituents of 8-12 and the dimethyl-
anilino substituents of 14 contribute to lower values of
EC50 relative to the (trifluoromethyl)phenyl substituents
of 13 or the tolyl substituents of 15.
Studies in vivo with dithiaporphyrin 8 and diselena-
porphyrin 11 suggest that effective core-modified por-
phyrin sensitizers can be developed that (i) can be
prepared with well-defined structure and purity; (ii) are
amenable to structure-activity studies to identify better
materials; (iii) absorb light of longer wavelengths than
Photofrin, which increases the effective depth of pen-
etration of light; (iv) have minimal dark toxicity; (v)
localize in the tumor; and (vi) have limited cutaneous
photosensitivity, which limits PDT with Photofrin. We
are currently optimizing PDT with our lead candidates
8, 11, 14, 16, and 17 as well as examining new
derivatives.
P r ep a r a tion of 2,5-Bis[(4-flu or op h en yl)h yd r oxym eth -
yl]selen op h en e (3d ). Selenophene (7.5 g, 0.06 mol) was
treated with n-butyllithium (89 mL, 1.6 M in hexanes, 0.15
mol) and TMEDA (23 mL, 0.15 mol) in 250 mL of hexanes as
described. The dilithioselenophene suspension was then slowly
added dropwise to a solution of 4-fluorobenzaldehyde (37.2 g,
0.3 mol) in THF (300 mL) as described. Following workup, the
crude product was recrystallized from toluene to give 8.42 g
(37%) of 3d as a white crystalline product of one diasteromer
(meso or d,l); mp 95-95.5 °C. IR (KBr): 3246, 1675, 1613,
1512, 1225 cm-1. FAB(+) MS: m/z 381 (C18H14F2O280Se + H,
M + 1). Anal. C, H.
P r ep a r a tion of 2,5-Bis[(4-tr iflu or om eth ylp h en yl)h y-
d r oxym eth yl]th iop h en e (3e). Thiophene (1.68 g, 0.020 mol)
was treated with n-butyllithium (31 mL, 1.6 M in hexanes,
0.050 mol) and TMEDA (10 mL, 0.07 mol) in 100 mL of
hexanes as described. The dilithiothiophene suspension was
added dropwise to a solution of 4-trifluorotolualdehyde (7.66
g, 0.044 mol) in THF (125 mL) as described. Following workup,
the crude product was recrystallized from toluene to give 4.17
g (52%) of 3e as a white crystalline solid; mp 160-162 °C. IR
(KBr): 3377 (br), 1338, 1118, 1072 cm-1. High-resolution
Q-TOF MS: m/z 455.0524 (calcd for C20H14F6O2S + Na,
455.0516). Anal. C, H.
P r ep a r a tion of 2,5-Bis[(4-N,N-d im eth yla m in op h en yl)-
h yd r oxym eth yl]th iop h en e (3f). Thiophene (4.2 g, 0.050 mol)
was treated with n-butyllithium (78 mL, 1.6 M in hexanes,
0.13 mol) and TMEDA (19 mL, 0.13 mol) in 200 mL of hexanes
as described. The dilithiothiophene suspension was added
dropwise to a solution of 4-dimethylaminobenzaldehyde (19.4
g, 0.13 mol) in THF (250 mL) as described. Following workup,
the crude product was recrystallized from toluene to give 12.34
Exp er im en ta l Section
Gen er a l Meth od s. Solvents and reagents were used as
received from Sigma-Aldrich Chemical Co. (St. Louis, MO)
unless otherwise noted. Cell culture medium was purchased

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 457
g (65%) of 3f as a white crystalline solid; mp 101-103 °C.
FAB(+) MS: m/z 383 (C22H26N2O2S + H, M + 1). Anal.
C, H, N.
been degassed under a stream of Ar bubbles for 20 min. TCBQ
(4.52 g) was added, and the reaction vessel was covered with
aluminum foil, followed by purging of the system with argon.
Boron trifluoride etherate (0.2 mL) was added, and the
resulting mixture was heated at reflux for 1 h. The reaction
mixture was cooled and concentrated, and the residue was
redissolved in minimal CH2Cl2. The resulting solution was
purified via chromatography on basic alumina eluted with
CH2Cl2. The first red band was collected, and the product was
washed with acetone. The crude product was then recrystal-
lized from CH2Cl2/MeOH to give 0.252 g (11%) of 5a as a purple
solid, which was recrystallized from toluene to give purple
P r ep a r a tion of 2,5-Bis[(4-tolyl)h yd r oxym eth yl]th io-
p h en e (3g). Thiophene (4.2 g, 0.050 mol) was treated with
n-butyllithium (78 mL, 1.6 M in hexanes, 0.13 mol) and
TMEDA (19 mL, 0.13 mol) in 200 mL of hexanes as described.
The dilithiothiophene suspension was added dropwise to a
solution of 4-methylbenzaldehyde (15.6 g, 0.13 mol) in THF
(250 mL) as described. Following workup, the crude product
was recrystallized from toluene to give 13.2 g (82%) of 3g as a
white crystalline product; mp 92-94 °C. IR (KBr): 3283 (br),
2920, 2877, 1451, 1009 cm-1. High-resolution Q-TOF MS: m/z
347.1079 (calcd for C20H20O2S + Na, 347.1082). Anal. C, H.
P r ep a r a tion of 2,5-Bis[(4-m eth oxyp h en yl)h yd r oxy-
m eth yl]th iop h en e (3h ). Thiophene (5.12 g, 0.060 mol) was
treated with n-butyllithium (78 mL, 1.6 M in hexanes, 0.13
mol) and TMEDA (19 mL, 0.13 mol) in 200 mL of hexanes as
described. The dilithiothiophene suspension was added drop-
wise to a solution of 4-methoxybenzaldehyde (17.0 g, 0.125 mol)
in THF (250 mL) as described. Following workup, the crude
product was recrystallized from toluene to give 8.75 g (41%)
of 3h as a white crystalline product; mp 125-127 °C (literature
mp14a 154 °C). FAB(+) MS: m/z 357 (C20H20O4S + 1, M + 1).
Anal. C, H.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2,5-Bis(1-
a r yl-1-p yr r olom eth yl)ch a lcogen op h en es. P r ep a r a tion of
2,5-Bis(1-p h en yl-1-p yr r olom eth yl)th iop h en e (4a ). Com-
pound 3a 11 (1.3 g, 4.0 mmol) was dissolved in excess pyrrole
(10.6 mL), and argon was bubbled through it. Boron trifluoride
etherate was added (0.1 mL), and the resulting mixture was
allowed to stir for 1 h. The reaction was stopped by the addition
of CH2Cl2 (100 mL) followed by 40% NaOH (25 mL). The
organic layer was separated, washed with water (3 × 100 mL)
and brine (100 mL), dried over MgSO4, and concentrated. The
excess pyrrole was removed via vacuum distilled at ambient
temperature. The residual oil was purified via chromatography
on silica gel eluted with 75:25 hexanes/ethyl acetate. The
yellow band was collected to give a yellow oil, which was
recrystallized from ethyl acetate/hexanes to give 0.74 g (46%)
of 4a as a white solid; mp 98-100 °C. IR (KBr): 3510, 2978,
1716, 1521, 1363, 1222 cm-1. FAB(+) MS: m/z 395 (C26H22N2S
+ H, M+ + 1). Anal. C, H, N.
P r ep a r a tion of 2,5-Bis(1-p h en yl-1-p yr r olom eth yl)se-
len op h en e (4b). 2,5-Bis-(phenylhydroxymethyl)selenophene11
(1.21 g, 3.5 mmol) and pyrrole (10.8 mL) were treated with
boron trifluoride etherate (0.1 mL) as described. The resid-
ual oil was purified via chromatography on silica gel eluted
with 75:25 hexanes/ethyl acetate and was recrystallized from
ethyl acetate/hexanes to give 0.70 g (51%) of 4b as a white
solid; mp 109-111 °C. IR (KBr): 3426, 1700, 1628, 1457
cm-1. FAB(+) MS: m/z 443 (C26H22N280Se + H, M + 1). Anal.
C, H, N.
P r ep a r a tion of 2,5-Bis[1-(4-flu or op h en yl-1-p yr r olo-
m eth yl]th iop h en e (4c). Compound 3c14a (0.40 g, 1.5 mmol)
and pyrrole (4 mL) were treated with boron trifluoride etherate
(0.080 mL) as described. The residual oil was purified via
chromatography on silica gel eluted with 80:20 hexanes/ethyl
acetate to give 0.40 g (78%) of 4c as a colorless oil that was
used without further purification. HRMS: m/z 430.1305 (calcd
for C26H20F2N2S, 430.1315). Anal. C, H, N.
needles; mp >300 °C. IR (KBr): 3446, 1679, 1628, 1472 cm-1
.
FAB(+) MS: m/z 685 (C44H26F2N2S2 + H, M + 1). Anal. C, H,
N.
P r ep a r a tion of 5,10-Bis-4-flu or op h en yl-15,20-d ip h en -
yl-21,23-d iselen a p or p h yr in (5b). Compounds 4b (1.48 g, 3.4
mmol) and 3d (1.27 g, 3.4 mmol) in 1 L of CH2Cl2 were treated
with TCBQ (4.52 g) and then boron trifluoride etherate (0.2
mL) as described. The crude product was recrystallized from
CH2Cl2/MeOH to give 0.257 g (12%) of 5b as a purple solid;
mp >300 °C. FAB(+) MS: m/z 781 (C44H26F2N280Se2 + H, M
+ 1). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-flu or op h en yl-15,20-d ip h en -
yl-21-th ia -23-selen a p or p h yr in (5c). Compounds 4b (1.59 g,
3.6 mmol) and 3c (1.20 g, 3.6 mmol) in 1 L of CH2Cl2 were
treated with TCBQ (4.5 g) and then boron trifluoride etherate
(0.2 mL) as described. The crude product was recrystallized
from CH2Cl2/MeOH to give 0.42 g (16%) of 5c as a purple solid;
mp >300 °C. High-resolution Q-TOF MS: m/z 733.1034 (calcd
for C44H26F2N2S80Se + H, 733.1027). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-flu or op h en yl-15,20-d ip h en -
yl-21-selen a -23-th ia p or p h yr in (5d ). Compounds 4a (1.69 g,
4.3 mmol) and 3d (1.62 g, 4.3 mmol) in 1 L of CH2Cl2 were
treated with TCBQ (4.41 g) and then boron trifluoride etherate
(0.2 mL) as described. The crude product was recrystallized
from CH2Cl2/MeOH to give 0.19 g (6%) of 5d as a purple solid;
mp >300 °C. High-resolution Q-TOF MS: m/z 733.1034 (calcd
for C44H26F2N2S80Se + H, 733.1027). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-(tr iflu or om eth yl)p h en yl-15,-
20-d ip h en yl-21,23-d ith ia p or p h yr in (5e). Compounds 4a
(1.70 g, 4.3 mmol) and 3e (1.73 g, 4.3 mmol) in 1 L of CH2Cl2
were treated with TCBQ (4.5 g) and boron trifluoride etherate
(0.2 mL) as described. The crude product was recrystallized
from MeOH/CH2Cl2 to give 0.075 g (2%) of 5e, which was
recrystallized from toluene to give purple needles; mp >300
°C. FAB(+) MS: m/z 785 (C46H26F6N2S2 + H, M + 1). Anal. C,
H, N.
P r ep a r a tion of 5,10-Bis-4-(N,N-d im eth yla m in o)p h en -
yl-15,20-d ip h en yl-21,23-d ith ia p or p h yr in (5f). Compounds
4a (0.71 g, 1.8 mmol) and 3f (0.69 g, 1.8 mmol) in 1 L of
CH2Cl2 were treated with TCBQ (2.20 g) and boron trifluoride
etherate (0.1 mL) as described. The crude product was purified
via chromatography on basic alumina eluted with CH2Cl2. The
crude product was recrystallized from CH2Cl2/MeOH to give
0.225 g (17%) of 5f as a purple solid; mp >300 °C. High-
resolution Q-TOF MS: m/z 735.2645 (calcd for C48H38N4S2
H, 735.2616). Anal. C, H, N.
+
P r ep a r a tion of 5,10-Bis-4-tolyl-15,20-d ip h en yl-21,23-
d ith ia p or p h yr in (5g). Compounds 4a (1.42 g, 3.6 mmol) and
3g (1.17 g, 3.6 mmol) in 1 L of CH2Cl2 were treated with TCBQ
(4.52 g) and boron trifluoride etherate (0.2 mL) as described.
The crude product was recrystallized from CH2Cl2/MeOH to
give 0.52 g (22%) of 5 g as a purple solid; mp >300 °C. High-
P r ep a r a tion of 2,5-Bis[1-(4-m eth oxyp h en yl-1-p yr r o-
lom eth yl]th iop h en e (4d ). Compound 3h 14a(0.41 g, 1.15
mmol) and pyrrole (4 mL) were treated with boron trifluoride
etherate (0.080 mL) as described. The residual oil was purified
via chromatography on silica gel eluted with 80:20 hexanes/
ethyl acetate to give 0.48 g (92%) of 4d as a colorless oil that
was used without further purification. HRMS: m/z 454.1694
(calcd for C28H26N2O2S, 454.1715). Anal. C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5,10,15,20-
Tetr a (a r yl)-21,23-cor e-m od ified P or p h yr in s. P r ep a r a -
t ion of 5,10-Bis-4-flu or op h en yl-15,20-d ip h en yl-21,23-
d ith ia p or p h yr in (5a ). Compounds 4a (1.33 g, 3.6 mmol) and
3c (1.12 g, 3.6 mmol) were dissolved in 1 L of CH2Cl2 that had
resolution Q-TOF MS: m/z 677.2094 (calcd for C46H32N2S2
H, 677.2085). Anal. C, H, N.
+
P r ep a r a tion of 5,10-Bis-4-m eth oxyp h en yl-15,20-d i-
p h en yl-21,23-d ith ia p or p h yr in (5h ). Compounds 4a (1.42 g,
3.6 mmol) and 3h (1.28 g, 3.6 mmol) in 1 L of CH2Cl2 were
treated with TCBQ (4.40 g) and boron trifluoride etherate (0.2
mL) as described. The crude product was recrystallized from
CH2Cl2/MeOH to give 0.71 g (29%) of 5h as a purple solid; mp
>300 °C. High-resolution Q-TOF MS: m/z 677.2094 (calcd for
C
46H32N2S2 + H, 677.2085). Anal. C, H, N.

458 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
P r ep a r a t ion of 5,10-Bis-4-flu or op h en yl-15,20-b is-4-
m eth oxyp h en yl-21,23-d ith ia p or p h yr in (5l). Compounds 4c
(1.00 g, 2.33 mmol) and 3i (0.83 g, 2.33 mmol) in 750 mL of
CH2Cl2 were treated with TCBQ (2.2 g, 8.95 mmol) and boron
trifluoride etherate (0.15 mL, 1.2 mmol) as described. The
crude product was recrystallized from MeOH/CH2Cl2 to give
0.50 g (29%) of 5l as a purple solid; mp >300 °C. High-
resolution Q-TOF MS: m/z 745.1793 (calcd for C46H30F2N2O2S2
+ H, 745.1795). Anal. C, H, N.
P r ep a r a tion of 5,10,15,20-Tetr a p h en yl-21-th ia p or p h y-
r in (6a ).18 Pyrrole (1 mL, 0.015 mol), benzaldehyde (1 mL,
0.01 mol), and 3a (1.48 g, 5.00 mmol) in 1 L of CH2Cl2 were
treated as described to give 0.38 g (12%) of 6a as dark purple
crystals; mp > 300 °C (literature mp18 >300 °C). IR (KBr):
3010, 2977, 1717, 1512, 1370, 1225 cm-1. FAB(+) MS: m/z
632 (C44H29N3S + H, M + 1).
washed with several portions of THF, and dried to give 41 mg
(70%) of porphyrin 16; mp >300 °C. 1H NMR (300 MHz,
CD3OD): δ 9.72 (s, 2H), 9.60 (s, 2H), 8.61 (d, 2H, J ) 4.5 Hz),
8.53 (d, 2H, J ) 4.5 Hz), 8.10-8.17 (m, 4H), 8.06 (d, 4H, J )
8.1 Hz), 7.74-7.80 (m, 6H), 7.37 (d, 4H, J ) 8.1 Hz), 4.62 (s,
4H). 13C NMR (75 MHz, CD3OD): δ 176.51, 160.72, 158.13,
157.76, 149.46, 148.81, 142.39, 136.87, 136.46, 135.82, 135.64,
135.19, 135.10, 134.56, 129.37, 128.69, 115.13, 68.78. High-
resolution Q-TOF MS: m/z 841.1427 (calcd for C48H30N2-
Na2O6S2 + H, 841.1419). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-flu or op h en yl-15,20-bis-4-h y-
d r oxyp h en yl-21,23-d ith ia p or p h yr in (5m ). A solution of
porphyrin 5l (312 mg, 0.44 mmol) in 100 mL of 1,2-dichloro-
ethane was degassed with a stream of argon. Boron tribromide
methyl sulfide complex (1 M in CH2Cl2, 8 mL, 8 mmol) was
added, and the resulting solution was heated at reflux for 15
h. The reaction mixture was cooled to ambient temperature,
and 50 mL of MeOH was added. After 15 min, the reaction
mixture was concentrated, and 200 mL of brine and 200 mL
of ethyl acetate were added. The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate (3 ×
200 mL). The combined organic layers were dried over MgSO4
and concentrated. The crude solid was washed with several
portions of MeOH to give 170 mg (57%) of the phenolic
porphyrin 5m , which was recrystallized from CH2Cl2/toluene
to give purple needles; mp >300 °C. IR (KBr): 3400 (br), 1603,
1508, 1223, 1157 cm-1. High-resolution Q-TOF MS: m/z
717.1475 (calcd for C44H26F2N2O2S2 + H, 717.1482). Anal. C,
H, N.
P r ep a r a t ion of 5,10-Bis-4-flu or op h en yl-15,20-b is(4-
eth yl p h en oxya ceta te)-21,23-d ith ia p or p h yr in (5n ). Por-
phyrin 5m (185 mg, 0.26 mmol), 1.5 of K2CO3, and 2 mL of
ethyl bromoacetate in 50 mL of acetone were heated at reflux
for 15 h until 5m was consumed. The reaction mixture was
cooled to ambient temperature, and the K2CO3 was removed
by filtration. The filter cake was washed with THF until the
filtrate was colorless. The combined filtrates were concen-
trated. The crude product was washed with MeOH to give 175
mg (76%) of 5n as a purple solid; mp 255-257 °C. IR (KBr):
1757, 1603, 1511, 1223, 1195, 1180 cm-1. High-resolution
Q-TOF MS: m/z 889.2198 (calcd for C52H38F2N2O6S2 + H,
889.2217). Anal. C, H, N.
P r ep a r a t ion of 5,10-Bis-4-flu or op h en yl-15,20-b is(4-
p h en oxya cetic a cid )-21,23-d ith ia p or p h yr in (5o). Porphy-
rin 5n (190 mg, 0.21 mmol) was dissolved in 30 mL of THF,
and 200 mg (2.5 mmol) of NaOH in 10 mL of H2O was added.
The resulting solution was stirred at ambient temperature for
15 h. After the solution was acidified with 1 mL of acetic acid,
100 mL of H2O and 100 mL of ethyl acetate were added. The
organic layer was separated, and the aqueous layer was
extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were dried over MgSO4 and concentrated. The
crude product was washed with several portions of Et2O to
give 164 mg (93%) of 5o as a purple solid; mp >300 °C. IR
(KBr): 3420 (br), 1730, 1602, 1512, 1223 cm-1. High-resolution
Q-TOF MS: m/z 833.1589 (calcd for C48H30F2N2O6S2 + H,
883.1591). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-flu or op h en yl-15,20-d ip h en -
yl-21-th ia p or p h yr in (6b). Pyrrole (1 mL, 0.015 mol), ben-
zaldehyde (1 mL, 0.01 mol), and 3c (1.66 g, 5.0 mmol) in 1 L
of CH2Cl2 were treated with TCBQ (4.52 g) and boron trifluo-
ride etherate (0.2 mL) as described. The crude product was
recrystallized from CH2Cl2/hexanes to give 0.25 g (8%) of 6b
as
a purple solid; mp >300 °C. FAB(+) MS: m/z 668
(C44H27F2N3S + H, M + 1). Anal. C, H, N.
P r ep a r a tion of 5,10-Dip h en yl-15,20-bis-4-h yd r oxy-
p h en yl-21,23-d ith ia p or p h yr in (5i). A solution of porphyrin
5h (312 mg, 0.44 mmol) in 100 mL of 1,2-dichloroethane was
degassed with a stream of argon. Boron tribromide methyl
sulfide complex (1 M in CH2Cl2, 8 mL, 8 mmol) was added,
and the resulting solution was heated at reflux for 15 h. The
reaction mixture was cooled to ambient temperature, and 50
mL of MeOH was added. After 15 min, the reaction mixture
was concentrated, and 200 mL of brine and 200 mL of ethyl
acetate were added. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (3 × 200 mL).
The combined organic layers were dried over MgSO4 and
concentrated. The crude solid was washed with several por-
tions of MeOH to give 170 mg (57%) of the phenolic porphyrin
5i, which was recrystallized from CH2Cl2/toluene to give purple
needles; mp >300 °C. High-resolution Q-TOF MS: m/z 717.1475
(calcd for C44H28N2O2S2 + H, 717.1482). Anal. C, H, N.
P r ep a r a tion of 5,10-Dip h en yl-15,20-bis-(4-eth yl p h e-
n oxya ceta te)-21,23-d ith ia p or p h yr in (5j). Porphyrin 5i (126
mg, 0.185 mmol), 1.5 g of K2CO3, and 2 mL of ethyl bromo-
acetate in 50 mL of acetone were heated at reflux for 15 h
until 5i was consumed. The reaction mixture was cooled to
ambient temperature, and the K2CO3 was removed by filtra-
tion. The filter cake was washed with THF until the filtrate
was colorless. The combined filtrates were concentrated. The
crude product was washed with MeOH to give 79 mg (50%) of
5j as a purple solid; mp 255-257 °C. High-resolution Q-TOF
MS: m/z 751.2084 (calcd for C52H40N2O6S2 + H, 751.2089).
Anal. C, H, N.
P r ep a r a tion of 5,10-Dip h en yl-15,20-bis-(4-p h en oxy-
a cetic a cid )-21,23-d ith ia p or p h yr in (5k ). Porphyrin 5j (75
mg, 0.088 mmol) was dissolved in 30 mL of THF and 285 mg
(0.14 mmol) of NaOH in 13 mL of H2O was added. The
resulting solution was stirred at ambient temperature for 15
h. After the solution was acidified with 3 mL of acetic acid, 50
mL of H2O and 100 mL of ethyl acetate were added. The
organic layer was separated, and the aqueous layer was
extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were dried over MgSO4 and concentrated. The
crude product was washed with several portions of Et2O to
give 63.5 mg (91%) of 5k , which was recrystallized from EtOAc
to give purple needles; mp >300 °C. High-resolution Q-TOF
MS: m/z 797.1778 (calcd for C48H32N2O6S2 + H, 797.1780).
Anal. C, H, N.
P r ep a r a t ion of 5,10-Bis-4-flu or op h en yl-15,20-b is(4-
p h en oxya cetic a cid )-21,23-d ith ia p or p h yr in Disod iu m
Sa lt (17). Porphyrin 5o (140 mg, 0.17 mmol) was dissolved in
30 mL of THF, and 1.4 mL (0.34 mmol) of 0.24 M aqueous
NaOH was added. The solution was stirred for 15 h at ambient
temperature. The purple precipitate was collected by filtration,
washed with several portions of THF, and dried to give 140
1
mg (91%) of porphyrin 17; mp >300 °C. H NMR (300 MHz,
CD3OD): δ 9.79 (s, 2H), 9.63 (s, 2H), 8.68 (d, 2H, J ) 4.5 Hz),
8.58 (d, 2H, J ) 4.5 Hz), 8.07-8.24 (m, 8H), 7.56 (t, 4H, J )
8.4 Hz), 7.44 (d, 4H, J ) 8.7 Hz), 4.70 (s, 4H). 13C NMR (75
MHz, CD3OD): δ 176.51, 164.61 (d, J ) 245 Hz), 160.74,
158.18, 157.76, 149.59, 148.83, 138.46 (d, J ) 3 Hz), 136.98,
136.78 (d, J ) 8 Hz), 136.49, 136.33, 135.99, 135.83, 134.92,
134.49, 133.82, 115.70, 115.28 (d, J ) 20 Hz), 68.78. High-
resolution Q-TOF MS: m/z 877.1262 (calcd for C48H28F2N2-
Na2O6S2 + H, 877.1230). Anal. C, H, N.
P r ep a r a tion of 5,10-Dip h en yl-15,20-bis-(4-p h en oxy-
a cetic a cid )-21,23-d ith ia p or p h yr in Disod iu m Sa lt (16).
Porphyrin 5k (55.3 mg, 0.0694 mmol) was dissolved in 30 mL
of THF, and 1.46 mL (0.146 mmol) of 0.10 M aqueous NaOH
was added. The solution was stirred for 15 h at ambient
temperature. The purple precipitate was collected by filtration,

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 459
Gen er a l P r oced u r e for th e Su lfon a tion of Cor e-
Mod ified P or p h yr in s. P r ep a r a t ion of 5,10-Bis-4-su l-
fon a t op h en yl-15,20-bis-4-flu or op h en yl-21,23-d it h ia p or -
p h yr in Disod iu m Sa lt (8). Compound 5a (0.53 g, 0.77 mmol)
was dissolved in excess H2SO4 (26 mL) and allowed to stir at
100 °C overnight. The acid was slowly neutralized with
concentrated NaOH until the solution was slightly basic. An
equal volume of MeOH was added, and the solid Na2SO4 was
removed by filtration. The filtrate was concentrated, and the
residue was dissolved in acetone. The resulting solution was
chilled precipitating more Na2SO4, which was removed via
filtration. The acetone solution was concentrated, and the
residue was dissolved in a minimal amount of water. The
resulting solution was subjected to reverse phase chromatog-
raphy eluting with MeOH. The purple band was collected, and
the residue was recrystallized from 10% aqueous MeOH to give
146.37, 143.63, 138.10, 137.93, 137.63, 137.54, 137.05 (d, J )
8 Hz), 135.22, 135.09, 126.40, 115.71 (d, J ) 22 Hz). IR (KBr):
3432 (br), 2365, 2345, 1126, 1040 cm-1. MALDI TOF MS: m/z
984 (C44H24F2N2Na2O6S280Se2 + H, M + 1), 962 (C44H25F2N2-
NaO6S280Se2 + H), 940 (C44H26F2N2O6S280Se2 + H). Anal. C,
H, N.
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
flu or op h en yl-21-th ia p or p h yr in Disod iu m Sa lt (12). 5,10-
Bis-4-fluorophenyl-15,20-bisphenyl-21-thiaporphyrin (0.25 g,
0.37 mmol) was treated as described to give 0.15 g (46%) of
1
12 as a metallic purple solid; mp >300 °C. H NMR (CD3OD,
500 MHz): δ 9.56 (s, 2H), 8.89 (s, 2H), 8.49 (d, 2H, J ) 5 Hz),
8.44 (d, 2H, J ) 5 Hz), 8.18 (AA′, 4H, J ) 8 Hz), 8.13 (BB′,
4H, J ) 8 Hz), 7.99 (m, 4H), 7.40 (t, 4H, J ) 8 Hz). 13C NMR
(CD3OD, 75 MHz): δ 157.26 (d, J ) 240 Hz, C-F), 148.60,
146.40, 145.31, 139.80, 138.01, 136.80, 136.69, 136.39, 135.52
(d, J ) 7 Hz), 135.25, 134.15, 131.81, 130.14, 125.53, 124.11,
115.61 (d, J ) 22 Hz, C-C-F). IR (KBr): 3448 (br), 2366,
1221, 1124, 1039 cm-1. High-resolution Q-TOF MS: m/z
872.0767 (calcd for C44H25F2N3Na2O6S3 + H, 872.0747). Anal.
C, H, N.
1
0.34 g (49%) of 8 as a metallic purple solid; mp >300 °C. H
NMR (CD3OD, 500 MHz): δ 9.71 (s, 2H), 9.67 (s, 2H), 8.62 (s,
4H), 8.32 (AA′, 4H, J ) 8 Hz), 8.26 (BB′, 4H, J ) 8 Hz), 8.18
(m, 4H), 7.57 (t, 4H, J ) 9 Hz). 13C NMR (CD3OD, 125 MHz):
δ 164.63 (d, J ) 247 Hz), 157.81, 157.61, 149.24, 148.96,
146.46, 143.99, 138.23, 136.85 (d, J ) 8 Hz), 136.84, 136.68,
135.53, 135.43, 135.05, 134.50, 126.33, 115.60 (d, J ) 22 Hz).
IR (KBr): 3453, 1696, 1653, 1482, 1358, 1184 cm-1. MALDI
TOF MS: m/z 888 (C44H24F2N2Na2O6S4 + H, M + 1), 866
(C44H25F2N2NaO6S4 + H), 844 (C44H26F2N2O6S4 + H). Anal. C,
H, N.
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
(tr iflu or om eth yl)p h en yl-21,23-d ith ia p or p h yr in Disod i-
u m Sa lt (13). Compound 5e (0.056 g, 0.71 mmol) was treated
as described to give 0.040 g (56%) of 13 as a metallic purple
solid; mp >300 °C. 1H NMR (CD3OD, 300 MHz): δ 9.68 (d,
4H, J ) 9 Hz), 8.60 (m, 4H), 8.37 (AA′, 4 H, J ) 8 Hz), 8.25 (s,
8H), 8.18 (BB′, 4H, J ) 8 Hz). 13C NMR (CD3OD, 75 MHz): δ
180.42, 175.22, 157.78, 157.62, 149.21, 148.94, 146.47, 144.14,
143.89, 139.26, 136.97, 136.71, 135.75, 135.69, 135.40 (q, J )
8 HZ), 134.9 (q, J ) 21 Hz), 129.50, 127.9 (q, J ) 135 Hz),
126.36. IR (KBr): 3448 (br), 2367, 1578, 1396 cm-1. MALDI
TOF MS: m/z 889 (C46H24F6N2Na2O6S4 + H, M + 1), 867
(C46H25F6N2NaO6S4 + H), 845 (C46H26F6N2O6S4 + H). Anal. C,
H, N.
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
N,N-(d im eth yla m in o)p h en yl)-21,23-d ith ia p or p h yr in Di-
sod iu m Sa lt (14). Compound 5f (0.28 g, 0.38 mmol) was
treated as described to give 0.15 g (42%) of 14 as a dark purple
solid; mp >300 °C. 1H NMR (CD3OD, 300 MHz): δ 9.73 (s,
2H), 9.68 (s, 2H), 8.66 (d, 4H, J ) 4 Hz), 8.59 (d, 4H, J ) 4
Hz), 8.24 (AA′, 4H, J ) Hz), 8.16 (BB′, 4H, J ) 8 Hz), 7.98
(AA′, 4H, J ) 9 Hz), 7.05 (BB′, 4H, J ) 9 Hz), 3.07 (s, 12H).
13C NMR (CD3OD, 75 MHz): δ 157.87, 157.12, 151.43, 149.69,
148.14, 146.21, 144.39, 137.16, 136.78, 136.60, 136.06, 135.90,
134.99, 134.18, 133.12, 129.64, 126.30, 112.33, 40.38. High-
resolution Q-TOF MS: m/z 939.1433 (calcd for C48H36N4-
Na2O6S4 + H, 939.1391). Anal. C, H, N.
P r ep a r a tion of 5,10-bis-4-su lfon a top h en yl-15,20-bis(2-
su lfon a t o-4-m et h ylp h en yl)-21,23-d it h ia p or p h yr in (15).
5,10-Bis(4-methylphenyl)-15,20-bis-phenyl-21,23-dithioporphy-
rin (0.46 g, 0.68 mmol) was treated as described and recrystal-
lized from MeOH/CH2Cl2 to give 0.40 g (57%) of 15 as a
metallic purple solid; mp >300 °C. 1H NMR (CD3OD, 300
MHz): δ 9.68 (s, 1H), 9.66 (s, 1H), 9.64 (s, 1H), 9.63 (s, 1H),
8.73 (d, 2H, J ) 4 Hz), 8.58 (m, 4H), 8.25 (collapsed AA”BB”,
8H), 8.11 (d, 1H, J ) 7.5 Hz), 8.02 (d, 1H, J ) 7.5 Hz), 7.69 (d,
1H, J ) 8 Hz), 7.64 (d, 1H, J ) 7.5 Hz), 2.94 (s, 6H). 13C NMR
(CD3OD, 75 MHz): δ 157.89, 157.68, 149.35, 148.97, 146.48,
144.14, 144.08, 139.31, 138.19, 136.93, 136.84, 136.78, 135.74,
135.37, 135.06, 134.47, 133.71, 132.13, 126.36, 20.76. IR
(KBr): 3458 (br), 2365, 2343, 1183, 1032. MALDI TOF MS:
m/z 1085 (C46H28N2Na4O12S6 + H, M + 1), 1063 (C46H29N2-
Na3O12S6 + H), 1041 (C46H31N2Na2O12S6 + H), 1019 (C46H32N2-
NaO12S6 + H), 997 (C46H32N2O12S6 + H). Anal. C, H, N.
P r ep a r a tion of Tetr a -4-su lfon a top h en yl-21-th ia p or -
p h yr in Tetr a sod iu m Sa lt (7). Compound 6a (0.20 g, 0.30
mmol) was treated as described to give 0.27 g (84%) of 7 as
dark purple crystals; mp > 300 °C. 1H NMR (CD3OD, 300
MHz): δ 9.97 (s, 2H), 9.02 (s, 2H), 8.69 (d, 2H, J ) 5 Hz), 8.61
(d, 2H, J ) 5 Hz), 8.34 (AA′, 8H), 8.28 (BB′, 8H). 13C NMR
(CD3OD, 75 MHz): δ 157.73, 154.75, 147.53, 145.31, 144.36,
142.91, 138.89, 135.59, 134.84, 134.31, 134.11, 133.27, 131.11,
129.24, 125.47, 124.56, 123.36. IR (KBr): 3442, 1700, 1507,
1217, 1038 cm-1. MALDI TOF MS: m/z 1040 (C44H25N3-
Na4O12S5 + H, M + 1), 1018 (C44H26N3Na3O12S5 + H), 996
(C44H27N3Na2O12S5 + H), 974 (C44H28N3NaO12S5 + H), 952
(C44H29N3O12S5 + H). Anal. C, H, N, S.
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
flu or op h en yl-21-selen a -23- th ia p or p h yr in Disod iu m Sa lt
(9). Compound 5c (0.21 g, 0.28 mmol) was treated as described
to give 0.040 g (14%) of 9 as a metallic purple solid; mp >300
°C. 1H NMR (DMSO-d6, 300 MHz): δ 10.24 (s, 2H), 9.88 (s,
2H), 8.91 (m, 4H), 8.51 (br s, 4H), 8.43 (AA′, J ) 7 Hz), 8.34
(BB′, 4H, J ) 7 Hz), 7.93 (t, 4H, J ) 8 Hz). 13C NMR (DMSO-
d6, 75 MHz): δ 162.63 (d, J ) 246 Hz, C-F), 157.12, 154.40,
151.93, 147.97, 144.44, 139.78, 138.31, 136.57, 135.70 (d, 8 Hz,
C-C2-F), 135.51, 135.28, 134.10, 133.51, 125.21, 114.76 (d, J
) 22 Hz, C-C-F). IR (KBr): 3448 (br), 2364, 2345, 1223, 1187
cm-1. High-resolution Q-TOF MS: m/z 936.9770 (calcd for
C
44H24F2N2Na2O6S380Se + H, 936.9801). Anal. C, H, N.
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
flu or op h en yl-21-th ia -23-selen a p or p h yr in Disod iu m Sa lt
(10). Compound 5d (0.50 g, 0.69 mmol) was treated as
described to give 0.40 g (67%) of 10 as a metallic purple solid;
1
mp >300 °C. H NMR (DMSO-d6, 300 MHz): δ 10.20 (s, 2H),
9.90 (s, 2H), 8.91 (d, 4H, J ) 4 Hz), 8.47 (m, 8H), 8.35 (d, 4H,
J ) 8 Hz), 7.93 (d, 4H, J ) 8 Hz). 13C NMR (DMSO-d6, 75
MHz): δ 162.54 (d, J ) 246 Hz, C-F), 157.19, 154.29, 151.91,
148.17, 144.40, 140.35, 138.07, 136.07, 136.03, 135.70 (d, J )
7 Hz, C-C2-F), 135.31, 134.37, 134.04, 133.49, 124.98, 115.00
(d, J ) 22 Hz, C-C-F). IR (KBr): 3448 (br), 2364, 2345, 1223,
1187 cm-1. High-resolution Q-TOF MS: m/z 936.9770 (calcd
for C44H24F2N2Na2O6S380Se + H, 936.9801). Anal. C, H, N.
Qu a n tu m Yield Deter m in a tion s. Quantum yields for
singlet oxygen were measured in 0.01 M PBS at pH 7.4 with
1.6% NaCl at 25 °C using methods we have previously
described.22,24
Cells a n d Cu ltu r e Con d ition s. Colo-26, a murine colon
carcinoma cell line, was maintained in RPMI 1640 supple-
mented with 10% fetal calf serum and antibiotics (all compo-
nents purchased from GIBCO Laboratories, Grand Island, NY)
at 37 °C, 5% CO2. R3230AC, a rat mammary adenocarcinoma
P r ep a r a tion of 5,10-Bis-4-su lfon a top h en yl-15,20-bis-4-
flu or oph en yl-21,23-diselen apor ph yr in Disodiu m Salt (11).
Compound 5b (0.27 g, 0.35 mmol) was treated as described to
give 0.15 g (43%) of 11 as a metallic purple solid; mp >300
1
°C. H NMR (CD3OD, 300 MHz): δ 9.60 (s, 2H), 9.57 (s, 2H),
8.40 (s, 4H), 8.22 (AA′, 4H, J ) 7 Hz), 7.99 (BB′, 4H, J ) 7
Hz), 7.91 (br s, 4H), 7.46 (t, 4H). 13C NMR (CD3OD, 75 MHz):
δ 163.02 (d, J ) 247 Hz), 157.73, 157.61, 151.05, 150.67,

460 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Hilmey et al.
(2) Henderson, B. W.; Dougherty, T. J . How Does Photodynamic
Therapy Work? Photochem. Photobiol. 1992, 55, 145-157.
(3) Kato, H. Photodynamic Therapy for Lung CancersA Review of
19 Years’ Experience. J . Photochem. Photobiol., B 1998, 42, 96-
99.
(4) Guillemin, F.; Feintrenie, X.; Lignon, D. Photodynamic Therapy
of Bronchial Cancers. Rev. Pneumologie Clin. 1992, 48, 111-
114.
(5) Puolakkainen, P.; Schroder, T. Photodynamic Therapy of Gas-
trointestinal Tumors: A Review. Dig. Dis. Sci. 1992, 10, 53-
60.
(6) Noske, D. P.; Wolbers, J . G.; Sterenborg, H. J . Photodynamic
Therapy of Malignant Glioma. A Review of Literature. Clin.
Neurol. Neurosurg. 1991, 93, 293-307.
(7) Moesta, K. T.; Schlag, P.; Douglass, H. O., J r.; Mang, T. S.
Evaluating the Role of Photodynamic Therapy in the Manage-
ment of Pancreatic Cancer. Lasers Surg. Med. 1995, 16, 84-92.
(8) Sharman, W. M.; Allen, C. M.; van Lier, J . E. Photodynamic
Therapeutics: Basic Principles and Clinical Applications. Drug
Discovery Today 1999, 4, 507-517.
(9) Bellnier, D. A.; Dougherty, T. J . A Preliminary Pharmacokinetic
Study of Intravenous Photofrin in Patients. J . Clin. Laser Med.
Surg. 1996, 14, 311-314.
(10) Dougherty, T. J .; Marcus, S. L. Photodynamic Therapy. Eur. J .
Cancer 1992, 28A, 1734-1742.
(11) Stilts, C. E.; Nelen, M. I.; Hilmey, D. G.; Davies, S. R.; Gollnick,
S. O.; Oseroff, A. R.; Gibson, S. L.; Hilf, R.; Detty, M. R. Water-
Soluble, Core-Modified Porphyrins as Novel, Longer-Wavelength-
Absorbing Sensitizers for Photodynamic Therapy. J . Med. Chem.
2000, 43, 2403-2410.
(12) (a) Berg, K.; Prydz, K.; Moan, J . Photochemical Treatment with
the Lysosomally Localized Dye Tetra(4-sulfonatophenyl)por-
phine Results in Lysosomal Release of the Dye but not of â-N-
Acetyl-D-glucosamidase Activity. Biochim. Biophys. Acta 1993,
1158, 300-306. (b) Kessel, D.; Thompson, P.; Saatio, K.; Nantwi,
K. D. Tumor Localization and Photosensitization by Sulfonated
Derivatives of Tetraphenylporphine. Photochem. Photobiol. 1987,
45, 787-790. (c) Kessel, D.; Woodburn, K. Biodistribution of
Photosensitizing Agents. Int. J . Biochem. 1993, 25, 1377-1383.
(d) Moan, J .; Iani, V.; Ma, L. W. In Vivo Fluorescence of
Phthalocyanines During Light Exposure. J . Photochem. Photo-
biol., B 1998, 42, 100-103.
cell line, was maintained in minimum essential media supple-
mented with 10% FBS (Atlanta Biologicals, Atlanta, GA), 50
units/mL of penicillin G, 50 mg/mL of streptomycin, and 1.0
mg/mL of fungizone (MEM).
In Vitr o P h ototoxicity Mea su r em en ts. Cells were plated
at 5 × 103 cells/well of a 96 well tissue culture plate the
evening before the assay. The day of the assay, the cells were
washed twice with PBS, and 100 µL of HBSS containing
various concentrations of photosensitizer was added to each
well. The sensitizer and cells were incubated for 24 h at 37 °C
followed by a wash with PBS and the addition of 100 µL of
PBS. The plates were irradiated with filtered 570-800 nm
light for a total light dose of 4 J cm-2. Following irradiation,
100 µL of growth media was added and the plates were
incubated for 24 h at 37 °C, 5% CO2. Cell survival was
monitored using the MTT assay as described in Mosmann.25
An im a ls. All animals were cared for under the guidelines
of the Roswell Park Cancer Institute Committee on Animal
Resources.
P h otosen sitizer Ad m in istr a tion . For animal experimen-
tation, the sensitizers were dissolved in saline or 5% dextrose
in water (D5W). Injection was intravenous via the tail vein.
F lu or escen ce Mea su r em en t of Tissu e Distr ibu tion of
Sen sitizer s (Solva ble Assa y.) Tissue samples and tumors
were harvested at various times posttreatment with 1 or 8 [5
mg (5 µmol)/kg] and flash frozen at -70 °C. Samples were
thawed on ice, and 1 mL of Solvable (Packard Instrument
Company, Meriden, CT) was added prior to incubation at 55
°C for 18-24 h. Samples were allowed to cool to ambient
temperature, and the fluorescence per sample was determined
and compared to a standard curve generated by the dilution
of pure photosensitizer. Results are presented as total dye
concentration per milligram of total protein. Total protein is
measured using the BioRad protein assay (BioRad Laborato-
ries).
P DT w ith 1, 8, 11, a n d P h otofr in . Colo-26 tumors were
implanted in BALB/c mice via the sterile trochar method.
Sensitizer was administered 96 h following tumoring of the
animals. Four hours following administration of sensitizer 1
[10 mg (10 µmol)/kg], 8 [0.125 mg (0.13 µmol)/kg], 11 [2.5 mg
(2.5 µmol)/kg], or Photofrin [2.5 mg (4 µmol)/kg], the tumors
were irradiated with red light at 630 nm at 75 mW cm-2 for
30 min for Photofrin or 694 nm at 75 mW cm-2 for 30 min for
1, 8, and 11 for a total light dose of 135 J cm-2. Treated
animals were followed until a tumor volume of 400 mm3 was
reached. An untreated control group received neither light nor
drug.
(13) (a) Sema, A. A. F.; Kennedy, J . C.; Blakeslee, D.; Robertson, D.
M. Experimental Porphyric Neuropathy: A Preliminary Report.
Can. J . Neurol. Sci. 1981, 8, 105-114. (b) Winkelman, J . W.;
Collins, G. H. Neurotoxicity of Tetraphenylporphinesulfonate
TPPS4 and Its Relation to Photodynamic Therapy. Photochem.
Photobiol. 1987, 46, 801-807.
(14) (a) Ulman, A.; Manassen, J . Synthesis of Tetraphenylporphyrin
Molecules Containing Heteroatoms Other than Nitrogen. Part
4. Symmetrically and Unsymmetrically Substituted Tetraphen-
yl-21, 23-dithiaporphyrins. J . Chem. Soc., Perkin Trans. 1 1979,
4, 1066-1069. (b) Ulman, A.; Manassen, J .; Frolow, F.; Rabinov-
ich, D. Synthesis of New Tetraphenylporphyrin Molecules
Containing Heteroatoms Other than Nitrogen: II. Tetraphenyl-
21-selena-23-thiaporphyrin and tetraphenyl-21, 23-diselenapor-
phyrin. Tetrahedron Lett. 1978, 167-170. (c) Ulman, A.; Ma-
nassen, J .; Frolow, F.; Rabinovich, D. Synthesis of New
Tetraphenylporphyrin Molecules Containing Heteroatoms other
than Nitrogen. III. Tetraphenyl-21-tellura-23-thiaporphyrin: An
Internally-Bridged Porphyrin. Tetrahedron Lett. 1978, 1885-
1886. (d) J eyaprakash Narayanan, S.; Sridevi, B.; Chandrashek-
ar, T. K.; Vij, A.; Roy, R. Novel Core-Modified Expanded
Porphyrins with meso-Aryl Substituents; Synthesis, Spectral and
Structural Characterization. J . Am. Chem. Soc. 1999, 121,
9053-9068.
Sta tistica l An a lyses. All statistical analyses were per-
formed using the Student’s t-test for pairwise comparisons. A
P value of < 0.05 was considered significant.
Ack n ow led gm en t. The authors thank 3-Dimen-
sional Pharmaceuticals and Merck Pharmaceuticals for
partial support of this work. The authors also thank the
staff of the Campus Chemical Instrumentation Center
of The Ohio State University for timely high-resolution
mass spectral analyses of molecules difficult to purify
free of solvents of crystallization.
(15) (a) Latos-Grazynski, L.; Lisowski, J .; Olmstead, M. M.; Balch,
A. L. Five-Coordinate Complexes of 21-Thiaporphyrin. The
Preparation, Spectra, and Structures of Iron(II), Nickel(II), and
Copper(II) Complexes. Inorg. Chem. 1989, 28, 1183-1188. (b)
Latos-Grazynski, L.; Pacholska, E.; Chmielewski, P. J .; Olm-
stead, M. M.; Balch, A. L. Synthesis of 5,20-Diphenyl-10, 15-
bis(p-tolyl)-21-selenaporphyrin (SeDPDTPH). Inorg. Chem. 1996,
35, 566-573.
Su p p or tin g In for m a tion Ava ila ble: NMR data for com-
pounds 3-6, Figure S1 for ambient and 60 °C 1H NMR spectra
of 15, Figure S2 for the absorbance/emission spectrum of 8,
Figure S3 for the photobleaching of various sensitizers, Figures
S4-S17 for the dark and phototoxicity of 1, 2, 7-17, and
Photofrin against the murine colon carcinoma cell line Colo-
26, and Figure S18 for the phototoxicity of 8, 9, 11, 14, 17,
and Photofrin against R3230AC rat mammary adenocarci-
noma cells. This information is available free of charge via the
Internet at http://pubs.acs.org.
(16) (a) Ziolkowski, P.; Milach, J .; Symonowicz, K.; Chmielewski, P.;
Latos-Grazynski, L.; Marcinkowska, E. 5,20-Bis(4-sulphophen-
yl)-10,15-bis(2-methoxy-4-sulfophenyl)-21-thiaporphyrin as
a
New Potent Sensitizer in Photodynamic Therapy. Tumori 1995,
81, 364-369. (b) Marcinkowska, E.; Ziolkowski, P.; Pacholska,
E.; Latos-Grazynski, L.; Chmielewski, P.; Radzikowski, C. Z. The
New Sensitizing Agents for Photodynamic Therapy: 21-Selena-
porphyrin and 21-Thiaporphyrin. Anticancer Res. 1997, 17,
3313-3320.
Refer en ces
(17) Meng, G. G.; J ames, B. R.; Skov, K. A.; Korbelik, M. Porphyrin
Chemistry Pertaining to the Design of Anti-cancer Drugs; Part
2, the Synthesis and in Vitro Tests of Water-soluble Porphyrins
(1) Dougherty, T. J .; Gomer, C. J .; Henderson, B. W.; J ori, G.; Kessel,
D.; Korbelik, M.; Moan, J .; Peng, Q. Photodynamic Therapy. J .
Natl. Cancer Inst. 1998, 90, 889-905.

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 461
Containing in the meso Positions, the Functional Groups:
4-Methylpyridinium, 4-Sulfonatophenyl, in Combination with
Phenyl, 4-Pyridyl, 4-Nitrophenyl, or 4-Aminophenyl. Can. J .
Chem. 1994, 71, 2447-2457.
Structural and Photophysical Properties of a Water-soluble
Porphyrin Associated with Polycations in Solution and Electro-
statically assembles Ultrathin Films. J . Phys. Chem. B 2000,
104, 5986-5992.
(22) Detty, M. R.; Merkel, P. B. Chalcogenopyrylium Dyes as
Potential Photochemotherapeutic Agents. III. Solution Studies
of Heavy Atome Effects on Triplet Yields, Quantum Efficiencies
of Singlet Oxygen Generation, Rates of Reaction with Singlet
Oxygen, and Emission Quantum Yields. J . Am. Chem. Soc. 1990,
112, 3845-3851.
(23) Wilkinson, F.; Helman, W. P.; Ross, A. B. Quantum Yields for
the Photosensitized Formation of the Lowest Electronically
Excited Singlet State of Molecular Oxygen in Solution. J . Phys.
Chem. Ref. Data 1993, 22, 113-262.
(24) (a) Hawkins, C. W.; Bickers, D. R.; Mukhtar, H.; Elmets, C. A.
Cutaneous Porphyrin Photosensitization: Murine Ear Swelling
as a Marker of the Acute Response. J . Invest. Dermatol. 1986,
86, 638-642. (b) Bellnier, D. A.; Dougherty, T. J . The Time
Course of Cutaneous Porphyrin Photosensitization of the Murine
Ear. Photochem. Photobiol. 1989, 49, 369-372.
(25) Leonard, K. A.; Nelen, M. I.; Anderson, L. T.; Gibson, S. L.; Hilf,
R.; Detty, M. R. 2,4,6-Triarylchalcogenopyrylium Dyes Related
in Structure to the Antitumor Agent AA1 as in Vitro Sensitizers
for the Photodynamic Therapy of Cancer. J . Med. Chem. 1999,
42, 3942-3952.
(26) Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and
Survival: Application to Proliferation and Cytotoxicity Assays.
J . Immunol. Methods 1983, 65, 55-63.
(18) Pandian, R.; Chandrashekar, T. K. Water-soluble Thiaporphy-
rins and Metallothiaporphyrins: Synthesis, Characterization,
Ground- and Excited-state Properties. J . Chem. Soc., Dalton
Trans. 1 1993, 119-125.
(19) Wagnie`res, G. A.; Star, W. M.; Wilson, B. C. In Vivo Fluorescence
Spectroscopy and Imaging for Oncological Applications. Photo-
chem. Photobiol. 1998, 68, 603-632.
(20) (a) Braichotte, D. G.; Wagnie`res, R.; Bays, P.; Monnier, P.; van
den Bergh, H. Clinical Pharmacokinetic Studies of Photofrin by
Fluorescence Spectroscopy in the Oral Cavity, the Esophagus,
and the Bronchi. Cancer 1995, 75, 2768-2778. (b) Wagnie`res,
R.; Studzinski, A. P.; Braichotte, D. R.; Monnier, P.; Depeursinge,
C.; Chaˆtelain, A.; van den Bergh, H. E. Clinical Imaging
Fluorescence Apparatus for the Endoscopic Photodetection of
Early Cancers by Use of Photofrin-II. Appl. Opt. 1997, 36, 5608-
5620.
(21) (a) Knyukshto, V. N.; Solovyov, K. N.; Egorova, G. D. Lumines-
cence and Structure of the Protonated Forms of meso-Tet-
raarylporphyrins in Solution. Biospectroscopy 1998, 4, 121-133.
(b) Micali, N.; Mallamace, F.; Romeo, A.; Purrello, R.; Monsu`
Scolaro, L. Mesoscopic Structure of meso-Tetrakis(4-sulfonato-
phenyl)porphine J -Aggregates J . Phys. Chem. B 2000, 104,
5897-5904. (c) Van Patten, P. G.; Shreve, A. P.; Donohoe, R. J .
J M0103662