Water-soluble, core-modified porphyrins as novel, longer-wavelength- absorbing sensitizers for photodynamic therapy

Article abstract of DOI:10.1021/jm000044i

Water-soluble, core-modified 5,10,15,20-tetrakis(4-sulfonatophenyl)- 21,23-dithiaporphyrin (1) and 5,10,15,20-tetrakis(4-sulfonatophenyl)-21,23- diselenaporphyrin (2) were prepared as the tetrasodium salts by the sulfonation of 5,10,15,20-tetraphenyl-21,2

Full text of DOI:10.1021/jm000044i

J . Med. Chem. 2000, 43, 2403-2410
2403
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
Corey E. Stilts,† Marina I. Nelen,† David G. Hilmey,† Sherry R. Davies,‡ Sandra O. Gollnick,‡ Allan R. Oseroff,‡
Scott L. Gibson,§ Russell Hilf,§ and Michael R. Detty*,†
Departments of Chemistry and Medicinal Chemistry, State University of New York at Buffalo, Buffalo, New York 14260;
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 607,
Rochester, New York 14642; and Department of Dermatology, Roswell Park Cancer Institute, Elm and Carlton Streets,
Buffalo, New York 14263
Received February 1, 2000
Water-soluble, core-modified 5,10,15,20-tetrakis(4-sulfonatophenyl)-21,23-dithiaporphyrin (1)
and 5,10,15,20-tetrakis(4-sulfonatophenyl)-21,23-diselenaporphyrin (2) were prepared as the
tetrasodium salts by the sulfonation of 5,10,15,20-tetraphenyl-21,23-dithiaporphyrin (3) and
-21,23-diselenaporphyrin (4), respectively, with sulfuric acid. Compounds 3 and 4 were prepared
by the condensation of pyrrole with either 2,5-bis(phenylhydroxymethyl)thiophene (5) or 2,5-
bis(phenylhydroxymethyl)selenophene (6) in propionic acid. The addition of benzaldehyde to
2,5-dilithiothiophene or 2,5-dilithioselenophene gives 5 or 6, respectively, as a nearly equimolar
mixture of meso- and d,l-diastereomers. Careful crystallization of 5 gives a single diastereomer
by removing the crystalline product from the equilibrating mixture of diastereomers in solution.
Photodynamic therapy (PDT) with 1 has an LD50 of less than 25 µg/mL against Colo-26 cells
in culture and exhibits a lethal dose for 90% or more at concentrations greater than 50 µg/mL.
In contrast, PDT with 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) requires
concentrations of greater than 100 µg/mL to achieve LD50. Neither 1 nor TPPS4 shows significant
photoactivity against the murine T-cell line, MOLT-4, above the dark toxicity. Sensitizer 1
shows no toxicity or side effects in BALB/c mice observed for 30 days following a single
intravenous injection of 10 mg (9.1 µmol)/kg. Distribution studies show that sensitizer 1
accumulates in the tumors of BALB/c mice bearing Colo-26 or EMT-6 tumors with sensitizer
concentration roughly doubling as the dosage of 1 increased from 5 to 10 mg/kg. In vivo studies
show that PDT with sensitizer 1 at both 3.25 and 10 mg/kg with 135 J cm-2 of 694-nm light is
effective against Colo-26 tumors in BALB/c mice.
Ch a r t 1
Photodynamic therapy (PDT) has been developed as
a cancer therapy over the last 25 years and has
regulatory approval in many countries for cancers of the
lung, digestive tract, and genitourinary tract using
Photofrin as a photosensitizer.1-4 PDT with Photofrin
is also being evaluated as a protocol for treating cancers
of the head and neck region5 and for treating pancreatic
cancer.6 The ideal sensitizer would absorb light strongly
in the red region of the spectrum (600-900 nm), where
light has greater penetration into tissue,7 would have
highly efficient photochemistry for killing tumor cells,
would localize in or around the tumor and not in normal
tissues, and would rapidly clear the system following
treatment. Although Photofrin (a mixture of materials
containing as one component dimeric porphyrin ethers)
has received regulatory approval, Photofrin and many
other porphyrin sensitizers have weak absorbance at the
shorter end of the red region of the spectrum (maxima
at approximately 630 nm) and induce long-lasting skin
photosensitivity. These drawbacks have resulted in the
preparation and evaluation of many other synthetic,
porphyrin-related molecules.8-10
Part of the challenge to chemists trying to replace
Photofrin with other porphyrin-like molecules is the
synthesis of water-soluble porphyrins and related ma-
terials with suitable solubility and bioavailability for use
in PDT and with minimal dark toxicity. Some ap-
proaches to increased water solubility and bioavailabil-
ity include the incorporation of sugar residues around
the porphyrin perimeter to enhance aqueous solubility11
as well as the incorporation of ionic groups such as
pyridinium, sulfonato, or carboxylato groups.8,12
5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrin
(TPPS4, Chart 1) and less highly sulfonated derivatives
of tetraphenylporphyrin have been prepared and stud-
ied as photosensitizers.13-15 Although the sulfonated
derivatives are soluble in water and have shown mem-
* To whom correspondence should be addressed.
† State University of New York at Buffalo.
‡ Roswell Park Cancer Institute.
§ University of Rochester Medical Center.
10.1021/jm000044i CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/26/2000

2404 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Stilts et al.
Ch a r t 2
densed with pyrrole in propionic acid. The direct
conversion of 6 to 4 has been described, although 6 was
prepared by the addition of selenium reagents to 1,6-
diphenylhexa-2,4-diyne-1,6-diol.17 Both 3 and 4 have
been prepared in trace amounts from the conversion of
5 and 6 to dipyrroles 7 and 8, respectively, followed by
treatment with trifluoroacetic acid and chloranil.18
Ch a r a cter iza tion a n d Sp ectr a l P r op er ties. The
1
structures of 1 and 2 followed directly from the H and
13C NMR spectra. In comparing 1 to 3 and 2 to 4, the
chemical shifts for the singlets corresponding to the four
pyrrole and four chalcogenophene protons remain un-
changed and integrate to four protons for each signal.
These data suggest that sulfonation has not occurred
on either heterocyclic ring. The aromatic signals for 1
and 2 have collapsed to slightly broadened 16-proton
singlets (ν1/2 ≈ 8 Hz), which do not allow unequivocal
assignment of substitution patterns. However, the 13C
NMR spectra are only consistent with para-substitution
of the phenyl groups. In each case, nine lines were
observed, corresponding to a 4-fold symmetric molecule
with four aromatic signals from each of the identical
para-substituted phenyl substituents, one signal from
the four identical meso-carbons, and two signals each
from the two pyrrole and thiophene rings that are each
2-fold symmetric.
brane permeability, TPPS4 and derivatives still absorb
weakly near the 630-nm maxima. A greater concern
with TPPS4 is its reported neurotoxicity.14 21,23-Core-
modified porphyrins containing the chalcogen atoms
sulfur, selenium, and tellurium have been described16,17
and are characterized by significantly longer wave-
lengths of absorption in band I. The 21,23-core-
modified porphyrins do not bind divalent metals and the
intramolecular 21,23-heteroatom contacts become sig-
nificantly less than the sum of van der Waals’ radii as
the heteroatoms become larger (2.85 Å for Se‚‚‚Se
distances and 2.65 Å for Te‚‚‚S distances). These fea-
tures suggested that water-soluble derivatives might be
interesting new sensitizer candidates for PDT with
optical and photophysical properties and perhaps bio-
chemical properties different than tetraarylporphyrin
derivatives.
In this paper, we describe the synthesis of core-
modified derivatives of TPPS4, 21,23-dithia derivative
1, and 21,23-diselena derivative 2 (Chart 1), with water
solubility and with longer wavelength absorption maxima
(λmax 695 nm) than TPPS4. Dithia derivative 1 was
evaluated as the first member of a new class of photo-
sensitizers and was found to display in vitro and in vivo
phototoxicity. Importantly, no toxicity was observed in
animals given a single dosage of 10 mg/kg of 5,10,15,20-
tetrakis(4-sulfonatophenyl)-21,23-dithiaporphyrin (1).
The absorption spectra of compounds 1 and 2 in water
as a solvent (Table 1) are nearly identical to the
absorption spectra of compounds 3 and 4 in dichloro-
methane as solvent. Importantly, the absorption maxima
of band I, the long-wavelength band, are little affected
by the sulfonation or by the change in solvent. The
absorption maxima are at 695 nm, which are signifi-
cantly longer than the Photofrin maximum at 630 nm
in water as well as the TPPS4 maximum at 630 nm in
water.
Dia ster eoselectivity in th e F or m a tion of Diols
5 a n d 6. One intriguing feature in the syntheses of diols
516 and 618 is that only a single diastereomer has been
described for the addition of benzaldehyde to either 2,5-
dilithiothiophene or 2,5-dilithioselenophene. The diols
5 and 6 can exist as either meso- or d,l-diastereomers
and, energetically, both diastereomers should be com-
parable and, spectroscopically, would be expected to
have similar but not necessarily identical spectral
properties. As described in the literature examples,16
recrystallization of the crude product mixture for the
preparation of 5 in our hands gave a single, crystalline
Ch em istr y
Syn th esis. 5,10,15,20-Tetrakis(4-sulfonatophenyl)-
21,23-dithiaporphyrin (1) and 5,10,15,20-tetrakis(4-
sulfonatophenyl)-21,23-diselenaporphyrin (2) were each
prepared as the tetrasodium salts by sulfonation of the
corresponding 5,10,15,20-tetraphenylporphyrin 3 or 4
(Chart 1) with sulfuric acid followed by treatment with
NaOH, a procedure that has worked well for the
sulfonation of other porphyrins.13 The water solubility
of 1 and 2 made purification somewhat difficult, but
Soxhlet extraction followed by a final recrystallization
gave 1 and 2 as the crystalline hexahydrates. Both 1
and 2 were isolated in 85% yield from the sulfonation,
which suggests that the chalcogenophene rings survive
the functionalization.
5,10,15,20-Tetraphenyl-21,23-dithiaporphyrin (3) was
prepared in 5% overall yield by the condensation of
pyrrole with 2,5-bis(phenylhydroxymethyl)thiophene
(5), which in turn was prepared by the addition of
benzaldehyde to 2,5-dilithiothiophene.16 5,10,15,20-
Tetraphenyl-21,23-diselenaporphyrin (4) was prepared
in 2% overall yield by the reaction of 2,5-dilithioseleno-
phene with benzaldehyde to give 2,5-bis(phenylhydroxy-
methyl)selenophene (6, Chart 2), which was then con-
1
diastereomer in up to 89% isolated yield. The H NMR
spectrum of the crystalline product displayed two two-
proton singlets corresponding to the 2,5-disubstituted
thiophene ring and the R-protons on the hydroxymethyl
substitutents in addition to the expected patterns for
the phenyl substituents. The seven lines observed in the
13C NMR spectrum (six aromatic, one alkyl) are also
consistent with a single diastereomer.
However, examination of the crude product mixture
(from a preparation of 5 prior to recrystallization) by
1H and 13C NMR spectroscopy indicated that both
diastereomers were present in nearly equal amounts.
The 500-MHz 1H NMR spectrum of crude 5 showed
superimposable resonances for the meso- and d,l-phenyl
substituents but two distinct singlets for the thiophene
protons (δ 6.67 and 6.64) and two distinct singlets for

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2405
Ta ble 1. UV-Visible Band Maxima and Extinction Coefficients for TPPS4, 1, and 2 in Water
λ
max, nm (ꢀ × 10-3 cm-1 mol-1 L) in water
compd
Soret
Band IV
Band III
Band II
Band I
Φ(1O2)b
0.71c
0.50 ( 0.01
0.17 ( 0.01
a
TPPS4
411 (464)
434 (190)
434 (221)
513 (15.5)
513 (19.3)
513 (22.4)
549 (7.0)
546 (5.5)
546 (6.2)
577 (6.5)
633 (2.0)
631 (2.2)
630 (3.9)
695 (4.0)
695 (4.5)
1
2
a
b
Reference 16. In 0.01 M PBS at pH 7.4, 1.6% by weight NaCl at 25 °C. c Reference 19.
the R-protons of the hydroxymethyl substituents (δ 5.83
and 5.82). The two sets of signals are consistent with a
mixture of both the meso- and d,l-diastereomers. The
13C NMR spectrum of the crude product mixture dis-
played, in addition to the seven signals for the crystal-
line diastereomer of 5 (δ 149.37, 144.97, 128.09, 127.06,
125.99, 122.91, and 70.75), six new signals (δ 149.18,
144.44, 127.03, 126.06, 122.8, and 70.55) corresponding
to the second diastereomer (with a 128.09-ppm signal
common to both diastereomers). If the crystalline dia-
stereomer is exposed to trifluoroacetic acid in dimethyl
sulfoxide at ambient temperature, the mixture of dia-
stereomers is reestablished.
Qu a n tu m Yield s for Sin glet Oxygen Gen er a tion
for 1 a n d 2. The quantum yield for singlet oxygen
generation [Φ(1O2)] by TPPS4 has been determined to
be 0.71.19 Using rose bengal as a standard,20 we con-
firmerd a value of 0.71 for Φ(1O2) for TPPS4 and
measured values of Φ(1O2) for 1 and 2 to be 0.50 ( 0.01
and 0.17 ( 0.01, respectively (Table 1). The lower value
of Φ(1O2) for 2 relative to 1 was somewhat surprising
since spin-orbit effects from the heavy atom would be
more pronounced for 2 relative to 1.21 One possible
explanation is that the close intramolecular Se‚‚‚Se
contacts17 disrupt the planarity of the π-framework and
lead to less efficient intersystem crossing relative to the
various photophysical routes to return to the ground
state.
The crystallization of a single diastereomer from the
preparation of 5 is an excellent illustration of Le
Chaˆtelier’s principle. As the single diastereomer crystal-
lizes and is removed from the solution equilibrium of
meso- and d,l-diastereomers, equilibration and further
crystallization produce a single diastereomer. The equili-
bration of doubly benzylic alcohols is not surprising and
could readily be catalyzed by trace amounts of acid.
Biology
In Vitr o Stu dies. P h otodyn am ic Th er apy again st
Cells in Cu ltu r e. Core-modified porphyrin 1 and
TPPS4 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 2
h in the dark with various concentrations of 1 or TPPS4
and were then washed prior to treatment with red light
at either 694 nm for 1 or 630 nm for TPPS4 for a total
light dose of 5 J cm-2. Light-treated cells and dark
controls were incubated for 24 h, and cell survival was
determined. Results are shown in Figure 1. Compound
1 and TPPS4 displayed comparable dark toxicity with
dark toxicity becoming significant at concentrations
>100 µg/mL. PDT with core-modified porphyrin 1 gave
an LD50 of less than 25 µg/mL and exhibits lethality
(LD90 or better) at doses greater than 50 µg/mL. In
contrast, TPPS4 requires concentrations of greater than
100 µg/mL to achieve LD50.
Compound 1 and TPPS4 were also evaluated in
culture for dark and light toxicities toward MOLT-4, a
murine T-cell line. As shown in Figure S1 (Supporting
Information), neither 1 nor TPPS4 showed significant
photoactivity above the dark toxicity achieved at higher
doses (>50 µg/mL).
In h ibition of Cytoch r om e c Oxid a se in Isola ted
Mitoch on d r ia l Su sp en sion s. TPPS4 and other sul-
fonated photosensitizers accumulate in the lysosomes,
where they are released to the cytoplasm upon irradia-
tion.15 The mitochondria are one possible subcellular
target23 for the sulfonated sensitizers following release.
To evaluate the ability of compound 1 to target mito-
chondria during PDT, mitochondria, isolated from
R3230AC rat mammary adenocarcinoma, were treated
as suspensions with 10-5 M solutions of 1 for 5 min.
The mixtures were centrifuged and the pellets were
resuspended. The cytochrome c oxidase activity in
sensitizer-treated mitochondrial suspensions was mea-
sured over time and compared to samples kept in the
dark for comparable periods of time as shown in Figure
The addition of benzaldehyde to 2,5-dilithioseleno-
phene has been reported and the diol has been iso-
lated.18 In our hands, the reaction gave a nearly one-
to-one mixture of both meso- and d,l-diastereomers in
63% yield as an oil. Crystallization/recrystallization of
the diastereomeric mixture gave the same mixture of
both diastereomers in 42% isolated yield. The 500-MHz
1H NMR spectra in DMSO-d6 for both crude and
recrystallized 6 showed nearly superimposable reso-
nances for the meso- and d,l-phenyl substituents (∆δ of
approximately 0.005 ppm) but two distinct singlets for
the selenophene protons (δ 6.81 and 6.77) and two
distinct singlets for the R-protons of the hydroxymethyl
substituents (δ 5.79 and 5.78). The two sets of signals
are consistent with a mixture of both the meso- and d,l-
diastereomers. The literature report of the 1H NMR
spectrum of 6 prepared in this manner describes only
one singlet at δ 6.77 for the selenophene protons and
another singlet at δ 5.85 for the phenylhydroxymethyl
substituents (in CDCl3), again illustrating that LeChaˆt-
elier’s principle can give a single diastereomer under
proper crystallization conditions.18
The 13C NMR spectrum of either the crude or recrys-
tallized product mixture displayed signals in seven
different chemical shift regions. Five of these signals
were doubled (δ 157.04/156.84, 145.30/145.24, 128.10/
128.02, 127.06/127.02, 125.98/125.92) and two peaks
were isolated singlets (δ 124.38, 72.40), which again is
consistent with a diastereomeric mixture of diols 6.
The crude diol mixtures of 5 and 6 gave 3 and 4 in
5% and 2% isolated yields, respectively, when treated
with pyrrole as described above. The use of the crude
diol mixtures gives somewhat higher overall yields for
the preparation of 3 and 4.

2406 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Stilts et al.
F igu r e 3. Accumulation of sensitizer 1 in Colo-26 and EMT-6
tumors in Balb/c mice determined by total fluorescence and
expressed as nanograms of 1 per milligram of protein. Tumors
were excised 24 h postinjection of 5% sodium bicarbonate
solutions of 1 at either 5 mg/kg or 10 mg/kg. Details of
experimental conditions are described in the Experimental
Section; bars are the SEM.
enzyme or to other sites preceding it in the respiration
chain.23
In Vivo Stu d ies. Toxicity a n d Distr ibu tion . In
applications of PDT with porphyrin-related sensitizers,
the therapeutic dose for rodents is typically on the order
of 1-5 mg/kg.1,9 Sensitizer 1 was evaluated for dark
toxicity in BALB/c mice given a single tail-vein injection
of 10 mg (9.1 µmol)/kg of 1 in saline. No toxicity,
morbidity, or side effects were observed in a group of
five animals followed for 30 days postinjection.
In BALB/c mice bearing Colo-26 tumors or EMT-6
tumors, a murine mammary tumor, the presence of
sensitizer 1 in the tumors was followed by fluorescence
spectroscopy. Twenty-four hours following intravenous
injection of a saline solution of 1, tissues were excised,
minced, and homogenized. The homogenate was taken
up in Solvable and the emission from sensitizer 1 was
quantified in terms of nanograms of 1/milligrams of
protein in tissue. Fluorescence from 1 that was above
background levels was found only in the tumor and the
fluorescence level was roughly linear with respect to
dosage, as shown in Figure 3.
F igu r e 1. Effect of (a) sensitizer 1 and (b) TPPS4 photosen-
sitization on the cell viability of cultured Colo-26 tumor cells.
Details of experimental conditions are described in the Ex-
perimental Section. Data are expressed as percent viable cells,
compared to untreated cells, for cells treated with sensitizer
in the dark (b) or for cells treated with sensitizer and 5.0 J
cm-2 irradiation (O). Each datum point represents the mean
percent viable cells calculated from at least three separate
experiments performed in duplicate; bars are the SEM.
P DT in Vivo. BALB/c mice bearing Colo-26 tumors
were given 3.25 or 10 mg/kg of sensitizer 1 as an
aqueous solution in 5% sodium bicarbonate via tail-vein
injection. Four hours postinjection, the animals were
treated with 135 J cm-2 of laser light at 694 nm (75
mW cm-2 for 30 min). The times to 400 mm3 tumor
volume were noted, and the results are presented as a
Kaplan-Meyer plot in Figure 4. Animals treated with
sensitizer 1 and light gave a significant response at both
3.25 mg/kg (198 ( 19 h, P < 0.01) and 10 mg/kg (276 (
133 h, P < 0.014) relative to control animals (104 ( 25
h) receiving neither drug nor light.
F igu r e 2. Effect of sensitizer 1 (10 µM) on mitochondrial
cytochrome c oxidase activity in the dark (b) and with
photosensitization (O). Details of experimental conditions are
described in the Experimental Section. Data are expressed as
the percent of initial preirradiation cytochrome c oxidase
activity in mitochondrial suspensions. Each datum point
represents the mean of two separate experiments performed
in duplicate; bars are the individual values of the separate
runs.
Su m m a r y a n d Con clu sion s
Core-modified 5,10,15,20-tetraaryl-21,23-dichalcoge-
naporphyrins can be prepared and sulfonated without
destruction of the chalcogenophene rings. The sul-
fonated dithia derivative 1 and diselena derivative 2
2. Cytochrome c oxidase is the last enzyme in the
mitochondrial respiration chain and the loss of activity
shown in Figure 2 is due to direct photodamage to this

Modified Porphyrins for Photodynamic Therapy
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2407
signal of CDCl3 as internal standard (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 (Applied
Photophysics, Leatherhead, UK). Both were equipped with a
circulating constant-temperature bath for the sample cham-
bers. Elemental analyses were conducted by Atlantic Micro-
analytical, Inc.
P r ep a r a tion of Tetr a sod iu m 5,10,15,20-Tetr a k is(4-su l-
fon a t op h en yl)-21,23-d it h ia p or p h yr in (1). 5,10,15,20-
Tetraphenyl-21,23-dithiaporphyrin (3, 0.18 g, 0.28 mmol) was
dissolved in 6 mL of sulfuric acid and the resulting solution
was heated at 100 °C for 18 h. The solution was cooled to
ambient temperature and was added slowly to 50 mL of ice
water. A 30% sodium hydroxide solution was added dropwise
to raise the pH of the solution to 8. Ice was continually added
to keep the solution cold. After the pH adjustment, the reaction
mixture was concentrated in vacuo to give crude product,
which was collected by filtration. The crude crystals were
washed with several small portions of methanol (5 × 30 mL),
the filtrate was combined with the aqueous filtrate, and more
product precipitated that was again collected by filtration and
washed with several small portions of methanol. The crude
product was purified via Soxhlet extraction with 90% ethanol
to give 0.25 g (85%) of 1 as a purple crystalline solid, mp >
300 °C: 1H NMR (CD3OD) δ 9.77 (s, 4 H, thiophene H’s), 8.69
(s, 4 H, pyrrole H’s), 8.34 (s, 16 H, Ar); 13C NMR (CD3OD)
δ 157.5, 148.9, 146.3, 143.9, 136.7, 135.4, 134.9, 134.5,
126.2; IR (KBr) 3413 (br), 1635, 1185, 1130 cm-1. Anal.
(C44H24N2Na4O12S6‚6H2O) C, H, N.
P r ep a r a tion of Tetr a sod iu m 5,10,15,20-Tetr a k is(4-su l-
fon a top h en yl)-21,23-d iselen a p or p h yr in (2). 5,10,15,20-
Tetraphenyl-21,23-diselenaporphyrin (4, 0.030 g, 0.040 mmol)
was dissolved in 2 mL of sulfuric acid and the resulting
solution was heated at 100 °C for 18 h. The solution was cooled
to ambient temperature and was added slowly to 25 mL of ice
water. A 30% sodium hydroxide solution was added dropwise
to raise the pH of the solution to 8. Ice was continually added
to keep the solution cold. After the pH adjustment, the reaction
mixture was concentrated in vacuo to give crude product,
which was collected by filtration. The crude crystals were
washed with several small portions of methanol (5 × 5 mL) to
remove the soluble porphyrin from the less soluble sodium
sulfate. The crude product was purified via Soxhlet extraction
with 90% ethanol to give 0.040 g (85%) of 2 as a purple
crystalline solid, mp > 300 °C: 1H NMR (CD3OD) δ 9.77 (s, 4
H, selenophene H’s), 8.69 (s, 4 H, pyrrole H’s), 8.34 (s, 16 H,
Ar); 13C NMR (CD3OD) δ 170.67, 150.27, 145.23, 144.86,
138.79, 138.13, 136.92, 135.64, 127.71; IR (KBr) 3448 (br),
2955, 1558, 1418, 1247 cm-1. Anal. (C44H24N2Na4O12Se2S4‚
6H2O) C, H, N.
F igu r e 4. PDT with sensitizer 1 at 3.25 mg/kg (O, n ) 5
animals, mean ) 198 ( 19 h) and 10 mg/kg (b, n ) 3 animals,
mean ) 276 ( 133 h) against implanted Colo-26 tumors in
BALB/c mice. These results can be compared with a control
group (2, n ) 11 animals, mean ) 104 ( 25 h) that received
neither light nor drug. Animals received 135 J cm-2 of 694-
nm light delivered at 75 mW cm-2 for 30 min. Times were
measured posttreatment till tumor volumes reached 400 mm3.
have absorption maxima at 695 nm in water, which is
65 nm longer than that of TPPS4. The dithia derivative
1 is an effective photosensitizer in vitro against Colo-
26 cells and is more effective than TPPS4 at comparable
concentrations and light dose. In vivo studies indicated
that 1 is not toxic at an intravenous dosage of 10 mg
(9.1 µmol)/kg with no signs of toxicity, morbidity, or side
effects in animals followed for 30 days postinjection. In
animals bearing Colo-26 or EMT-6 tumors, sensitizer 1
is found in the tumor 24 h postinjection as determined
by fluorescence spectroscopy with levels corresponding
to dosage. In animals bearing Colo-26 tumors, PDT with
694-nm light was effective at both 3.25 and 10 mg/kg
of sensitizer 1 relative to untreated controls.
Although TPPS4 is one of the more selective porphy-
rins for accumulation in tumors, its development as a
sensitizer has been hindered by its reported neurotox-
icity.14 Sensitizer 1 appears to be as effective as TPPS4
as a sensitizer for PDT and also appears to localize in
the tumor. Hopefully, the changes to the core in 1 and
2 and related molecules will avoid the neurotoxicity
associated with TPPS4. We are currently optimizing
appropriate in vivo protocols for PDT with 1 and 2 with
the Colo-26 tumor line. Encouraged by this preliminary
survey of biological results, we are also preparing other
core-modified porphyrin derivatives for functionalization
with water-solubilizing groups and evaluation as sen-
sitizers for PDT.
P r epa r a tion of 5,10,15,20-Tetr a p h en yl-21,23-d ith ia p or -
p h yr in (3). 2,5-Bis(phenylhydroxymethyl)thiophene (5, 8.50
g, 0.0297 mol) and acetic anhydride (30 mL) were dissolved in
1.5 L of propionic acid. The pyrrole (2.0 mL, 0.029 mol) was
then added and the resulting solution was stirred for 1 h at
reflux. The reaction mixture was cooled to ambient tempera-
ture and was then added slowly to a mixture of 400 mL of a
NH4OH solution and 800 mL of ice water. The products were
extracted with CH2Cl2 (3 × 300 mL) and the combined extracts
were washed with more of the NH4OH-ice water solution. The
organic extracts were dried over MgSO4 and concentrated. The
crude product was purified via chromatography on SiO2 eluted
with 1:1 CHCl3/toluene. The initial red band was collected and
concentrated to give 3, which was recrystallized from CH2Cl2/
MeOH to give 0.56 g (6.0%) of metallic purple crystal, mp >
300 °C:16 1H NMR (CDCl3) δ 9.69 (s, 4 H, thiophene H’s), 8.69
(s, 4 H, pyrrole H’s), 8.25 (d, 8 H, J ) 7 Hz, Ph), 7.81 (m, 12
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 media was purchased from
GIBCO (Grand Island, NY). Fetal bovine serum (FBS) was
obtained from Atlanta Biologicals (Atlanta, GA). Concentration
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 instruments with the residual solvent
H, Ph); IR (KBr) 3454, 2915, 2850, 1595, 1458, 1358 cm-1
;
FAB(+)MS, m/z 649 (C44H28N2S2 + H, M+ + 1). Anal.
(C44H28N2S2) C, H, N.

2408 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Stilts et al.
P r ep a r a tion of 5,10,15,20-Tetr a p h en yl-21,23-d iselen a -
p or p h yr in (4). 2,5-Bis(phenylhydroxymethyl)selenophene (6,
2.32 g, 6.76 mmol) and acetic anhydride (10 mL) were dissolved
in 500 mL of propionic acid. The pyrrole (0.50 mL, 0.029 mol)
was then added and the resulting solution was stirred for 1 h
at reflux. The reaction mixture was cooled to ambient tem-
perature and was then added slowly to a mixture of 200 mL
of a NH4OH solution and 400 mL of ice water. The products
were extracted with CH2Cl2 (3 × 300 mL) and the combined
extracts were washed with more of the NH4OH-ice water
solution. The organic extracts were dried over MgSO4 and
concentrated. The crude product was purified via chromatog-
raphy on SiO2 eluted with 1:1 CHCl3/toluene. The initial red
band was collected and concentrated to give 4, which was
recrystallized from CH2Cl2/MeOH to give 0.056 g (2.2%) of
metallic purple crystal, mp > 300 °C:17 1H NMR (CDCl3) δ
9.725 (s, 4 H, selenophene H’s), 8.72 (s, 4 H, pyrrole H’s), 8.29
(m, 8 H, Ph), 7.85 (m, 12 H, Ph); IR (KBr) 3454, 2915, 2850,
1595, 1458, 1358 cm-1; FAB(+)MS, m/z 745 (C44H28N280Se2 +
H, M+ + 1). Anal. (C44H28N2Se2) C, H, N.
P r ep a r a t ion of 2,5-Bis(p h en ylh yd r oxym et h yl)t h io-
p h en e (5). Thiophene (20.3 mL, 21.0 g, 0.250 mol) was added
slowly via syringe to a solution of n-BuLi (390 mL of a 1.6 M
solution in hexanes, 0.62 mol) and tetramethylethylenedi-
amine (75 mL) in 1 L of dry hexanes under an argon
atmosphere. The resulting solution was warmed to reflux for
1 h, cooled to ambient temperature, and transferred via
cannula to a pressure-equalizing addition funnel. The dilithio-
thiophene solution was added slowly to a solution of benz-
aldehyde (61 g, 0.58 mol) in 1 L of dry THF (distilled from
sodium benzophenone ketyl) cooled to 0 °C in an ice bath. The
ice bath was removed after addition was complete and the
reaction was warmed to ambient temperature. A cold, satu-
rated NH4Cl solution was added (2 L) and the organic phase
was separated. The aqueous phase was extracted with ether
(3 × 500 mL). The combined organic phases were washed with
water (3 × 1 L) and brine (1 L), dried over Na2SO4, and
concentrated. The crude product was recrystallized from
chloroform to give 65.9 g (89%) of crystalline 5 as one
diastereomer (meso- or d,l-), mp 137-138 °C (lit.19 mp 137-
138 °C): 1H NMR (DMSO-d6) δ 7.38 (d, 4 H, J ) 7 Hz, Ph),
7.31 (t, 4 H, J ) 7 Hz, Ph), 7.22 (t, 2 H, J ) 7 Hz, Ph), 6.67 (s,
2 H, thiophene H’s), 6.11 (br s, 2 H, -CHOH), 5.83 (s, 2 H,
-CH-OH); 13C NMR (DMSO-d6) δ 149.37, 144.97, 128.09,
127.06, 125.99, 122.91, 70.75; IR (KBr) 3403 (br), 3075, 2874,
1453 cm-1; FAB(+)MS m/z 297 (C18H16O2S + H, M + 1). Anal.
(C18H16O2S) C, H.
of the crystals and 140-144 (dec) °C for the other half (lit.18
mp 143-144 °C): 1H NMR (CD3OD) δ 7.49 (d, 4 H, J ) 7 Hz,
Ph), 7.36 (t, 4 H, J ) 7 Hz, Ph), 7.26 (t, 2 H, J ) 7 Hz, Ph),
6.81/6.77 (s, 2 H, selenophene H’s), 5.79/5.78 (s, 2 H, -CHOH),
2.88 (br s, 2 H, -CHOH); 13C NMR (CD3OD) δ 157.04/156.84,
145.30/145.24, 128.10/128.02, 127.06/127.02, 125.98/125.92,
124.38, 72.40; IR (KBr) 3437 (br), 3062, 3029, 2867, 1493 cm-1
;
FAB(+)MS m/z 345 (C18H16O280Se + H, M + 1). Anal.
(C18H16O2Se) C, H.
Qu a n tu m Yield Deter m in a tion s. Quantum yields for
singlet oxygen were measured in 0.01 M phosphate-buffered
saline (PBS) at pH 7.4 with 1.6% NaCl at 25 °C using methods
we have previously described.20,21
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 (FCS) and antibiotics (all
components purchased from GIBCO Laboratories, Grand
Island, NY) at 37 °C, 5% CO2. EMT-6, a murine mammary
carcinoma cell line, was maintained in MEM supplemented
with 15% fetal calf serum and antibiotics. MOLT-4, a murine
T cell leukemia cell line, was maintained in RPMI 1640, 5%
FCS and antibiotics at 37 °C, 5% CO2.
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 either 1 or TPPS4 was added to each
well. The sensitizer and cells were incubated for 2 h at 37 °C
followed by a wash with PBS and the addition of 100 µL of
PBS. The plates were irradiated with red light at either 694
or 630 nm for a total light dose of 5 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.24
An im a ls. All animals were cared for under the guidelines
of the Roswell Park Cancer Institute Committee on Animal
Resources or the University Committee on Animal Resources
at the University of Rochester.
P r ep a r a t ion of Mit och on d r ia l Su sp en sion s. The
R3230AC mammary adenocarcinoma was transplanted sub-
cutaneously in the axillary region of 80-100g female Fischer
344 rats using the sterile trochar method.25 Two to three weeks
after transplantation, when tumors had grown to 2-3 cm in
diameter, the animals were sacrificed. The tumors were
excised and placed in 0.9% sodium chloride on ice. The tissue
was finely minced with scissors and homogenized on ice at a
ratio of 1 g of tumor tissue to 5 mL of buffer containing 0.33
M sucrose, 1 mM dithiothreitol, 1 mM ethylene glycol bis(â-
aminoethyl ether)-N,N,N′,N′-tetraaetic acid, 0.03% bovine
serum albumin, and 0.1 M potassiom chloride (pH 7.4), using
30-s bursts with a Polytron PCU-2110 homogenizer at a setting
of 6 (Brinkmann Ind., Westbury, NY). Preparation of isolated
mitochondria from the homogenized tumor tissue followed a
method described earlier.23 Mitochondrial suspensions were
divided into 0.5-mL aliquots (6-10 mg of mitochondrial
protein/mL) and stored at -86 °C until used for in vitro
experiments.
Exp osu r e of Tu m or Mitoch on d r ia to Sen sitizer 1 a n d
TP P S4 in Vitr o. Mitochondrial suspensions were removed
from storage and thawed on ice. Solutions of sensitizer 1 and
TPPS4 were prepared by dissolving 2.5 mg of dye in 5.0 mL of
mitochondrial preparation buffer, which approximated a 1.0
mM solution for each of the three dyes studied. Final concen-
trations of the sensitizers were determined using their absor-
bance. Ten microliters of the sensitizer/buffer solution was
transferred to 1.0 mL mitochondrial preparation buffer and
the absorbance determined using a diode array spectropho-
tometer (HP8452A, Hewlett-Packard, Palo Alto, CA). The
sensitizers in mitochondrial preparation buffer, at a final
concentration that gave an OD of 0.2, were then added to
mitochondrial suspensions (1.0 mL) and allowed to incubate
in the dark on ice for 15 min. The sensitizer/mitochondrial
suspension was then centrifuged at 8000g for 3 min using an
Eppendorf microcentrifuge (Model 3200, Brinkmann Ind.,
The crude diol prior to crystallization displayed approxi-
mately a 1:1 mixture of both diastereomers. For the second
diastereomer of 5: 1H NMR (DMSO-d6) δ 7.38 (d, 4 H, J ) 7
Hz, Ph), 7.31 (t, 4 H, J ) 7 Hz, Ph), 7.22 (t, 2 H, J ) 7 Hz,
Ph), 6.64 (s, 2 H, thiophene H’s), 6.11 (br s, 2 H, -CH-OH),
5.82 (s, 2 H, -CHOH); 13C NMR (DMSO-d6) δ 149.18, 144.44,
128.09, 127.03, 126.06, 122.80, 70.65.
P r ep a r a tion of 2,5-Bis(p h en ylh yd r oxym eth yl)selen o-
p h en e (6). Selenophene (10.0 g, 0.0760 mol) was added slowly
via syringe to a solution of n-BuLi (100 mL of a 1.6 M solution
in hexanes, 0.16 mol) and tetramethylethylenediamine (25 mL)
in 350 mL of dry hexanes under an argon atmosphere. The
resulting solution was warmed to reflux for 1 h, cooled to
ambient temperature, and transferred via cannula to a pres-
sure-equalizing addition funnel. The dilithiothiophene solution
was added slowly to a solution of benzaldehyde (17.0 g, 0.160
mol) in 350 mL of dry THF (distilled from sodium benzophe-
none ketyl) cooled to 0 °C in an ice bath. The ice bath was
removed after addition was complete and the reaction was
warmed to ambient temperature. A cold, saturated NH4Cl
solution was added (1 L) and the organic phase was separated.
The aqueous phase was extracted with ether (3 × 200 mL).
The combined organic phases were washed with water (3 ×
300 mL) and brine (300 mL), dried over Na2SO4, and concen-
trated. The crude product was recrystallized from chloroform
to give 11.1 g (42%) of crystalline 6 as one-to-one mixture of
diastereomers (meso- and d,l-), mp 92-93 °C for roughly half

Modified Porphyrins for Photodynamic Therapy
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Westbuty, NY), the supernatant was aspirated with a Pasteur
pipet, and the pellet was resuspended in 1.0 mL of mitochon-
drial preparation buffer. The suspension was then transferred
to a 3.0-mL quartz cuvette which was positioned in a focused,
1.0-cm diameter, filtered (530 to 750 nm) light beam emitted
from a 750-W tungsten source. The intensity of the beam was
uniform over the wavelength band used and adjusted to a
fluence rate of 100 mW cm-2 using neutral density filters.
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P h otosen sitizer Ad m in istr a tion . For animal experimen-
tation, the sensitizers were dissolved in 5% sodium bicarbonate
solutions. Injection was intravenous via the tail vein.
F lu or escen ce Mea su r em en ts (Solva ble Assa y.) Tissue
samples and tumors were harvested at various times post-
treatment 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 dilution of pure 1. Results
are presented as total dye concentration per milligram of total
protein. Total protein is measured using the Bio-Rad protein
assay (Bio-Rad Laboratories).
P DT w ith 1. Colo-26 tumors were implanted in BALB/c
mice via the sterile trochar method. Sensitizer at 3.25 or 10
mg/kg was administered 96 h following tumoring of the
animals. Four hours following administration of sensitizer 1,
the tumors were irradiated with red light at 694 nm at 75 mW
cm-2 for 30 min 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.
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Sta tistica l An a lyses. All statistical analyses were per-
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Ack n ow led gm en t. The authors thank the National
Institutes of Health (Grant CA69155 to M.R.D., Grant
CA55791 to A.R.O., and Grant CA36856 to R.H.) and
the shared resources of the Roswell Park Cancer Center
support grant (P30 CA16056) for partial support of this
work.
Su p p or tin g In for m a tion Ava ila ble: Figure S1 showing
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T-cell line MOLT-4. This information is available free of charge
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