Water soluble, core-modified porphyrins. 3. Synthesis, photophysical properties, and in vitro studies of photosensitization, uptake, and localization with carboxylic acid-substituted derivatives

Article abstract of DOI:10.1021/jm030136i

Water soluble, core-modified porphyrins 1-5 bearing 1-4 carboxylic acid groups were prepared and evaluated in vitro as photosensitizers for photodynamic therapy. The 21,23-core-modified porphyrins 1-5 gave band I absorption maxima with λ_max of

Full text of DOI:10.1021/jm030136i

3734
J . Med. Chem. 2003, 46, 3734-3747
Wa ter Solu ble, Cor e-Mod ified P or p h yr in s. 3. Syn th esis, P h otop h ysica l
P r op er ties, a n d in Vitr o Stu d ies of P h otosen sitiza tion , Up ta k e, a n d Loca liza tion
w ith Ca r boxylic Acid -Su bstitu ted Der iva tives
Youngjae You,† Scott L. Gibson,‡ Russell Hilf,‡ Sherry R. Davies,§ Allan R. Oseroff,§ Indrajit Roy,†
Tymish Y. Ohulchanskyy,† Earl J . Bergey,† and Michael R. Detty*,†
Institute for Lasers, Photonics, and Biophotonics, Department of Chemistry, University at Buffalo, The State University of New
York, Buffalo, New York 14260-3000, Department of Biochemistry and Biophysics, University of Rochester Medical Center,
601 Elmwood Avenue, Box 607, Rochester, New York 14642, and PDT Center, Roswell Park Cancer Institute,
Elm and Carlton Streets, Buffalo, New York 14263
Received March 26, 2003
Water soluble, core-modified porphyrins 1-5 bearing 1-4 carboxylic acid groups were prepared
and evaluated in vitro as photosensitizers for photodynamic therapy. The 21,23-core-modified
porphyrins 1-5 gave band I absorption maxima with λmax of 695-701 nm. The number of
carboxylic acid groups in the dithiaporphyrins 1-4 had little effect on either absorption maxima
(λmax of 696-701 nm for band I) or quantum yields of singlet oxygen generation [φ(1O2) of 0.74-
0.80]. Substituting two Se atoms for S gave a shorter band I absorption maximum (λmax of 695
nm) and a smaller value for the quantum yield for generation of singlet oxygen [φ(1O2) of 0.30].
The phototoxicity of 1-5 was evaluated against R3230AC cells. The phototoxicities of
dithiaporphyrin 2, sulfonated thiaporphyrin 30, HPPH, and Photofrin were also evaluated
against Colo-26 cells in culture using 4 J cm-2 of 570-800 nm light. Compound 2 was
significantly more phototoxic than sulfonated dithiaporphyrin 30, HPPH, or Photofrin. Cellular
uptake was much greater for compounds 1, 2, and 5 relative to compounds 3 and 4. Confocal
scanning laser microscopy and double labeling experiments with rhodamine 123 suggested
that the mitochondria were an important target for dithiaporphyrins 1 and 2. Inhibition of
mitochondrial cytochrome c oxidase activity in whole R3230AC cells was observed in the dark
with compounds 1 and 30 and both in the dark and in the light with core-modified porphyrin
2.
In tr od u ction
meso-substituents have been incorporated in the por-
phyrin molecule to give longer wavelength absorption
maxima. Other variations of porphyrins and porphyrin-
related molecules (chlorins and bacteriochlorins (Chart
2), texaphyrins, phthalocyanines) have been prepared
and evaluated as photosensitizers for PDT.3 Chlorin and
bacteriochlorin derivatives are the products of reduction
of one or two pyrrole carbon-carbon double bonds,
respectively. Texaphyrin derivatives incorporate five
nitrogen atoms in the central core.
Photodynamic therapy (PDT) has gained acceptance
as a front line cancer therapy for a variety of malignan-
cies.1-3 Regulatory agencies in the U.S.A., J apan, The
Netherlands, Germany, France, and Great Britain have
approved this treatment regimen for cancers of the lung,
esophagus, genitourinary tract, and head and neck as
well as other disease conditions such as actinic kero-
tinitis and macular degeneration.3-8 The majority of the
clinical trials and PDT protocols that have received
approval utilized Photofrin, a complex derivative of
hematoporphyrin, as the photosensitizer. While effec-
tive, Photofrin has several drawbacks that limit its
general clinical use including minimal absorption in the
red region of the visible spectrum where light penetra-
tion into tissue is maximal, long-term skin photosensi-
tivity,9 loss of absorbance during irradiation due to rapid
photobleaching, structural composition that does not
easily allow chemical modification,10 and a lack of
specificity for either target malignancies or intracellular
sites.
We have been investigating the photosensitizing
properties of 5,10,15,20-tetraaryl-21,23-dithia, 21-sel-
ena-23-thia, and 21,23-diselenaporphyrins as a porphy-
rin variation with several desirable features.12 These
core-modified porphyrins can be synthesized in a pure
form, have longer wavelengths of absorption than the
corresponding porphyrin, and generate singlet oxygen
with high quantum yields. Unlike any of the porphyrin,
chlorin, bacteriochlorin, texaphyrin, or phthalocyanine
derivatives, the 21,23-heteroatom contacts in the core-
modified porphyrins are less than the sum of van der
Waals’ radii13 and the 21,23-core-modified porphyrins
are no longer capable of binding a metal ion within the
core and can only bind to metals that approach the
heteroatoms from above or below the porphyrin core.14
This aspect of the core-modified porphyrins represents
a significant difference from other porphyrin-related
photosensitizers and should lead to novel pharmacoki-
netics and related biological properties.
The drawbacks associated with Photofrin have pro-
vided the impetus for the search for new photosensitiz-
ers with greater specificity and efficacy.3,11 Various
* 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.
‡ University of Rochester Medical Center.
§ Roswell Park Cancer Institute.
10.1021/jm030136i CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/16/2003

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3735
Ch a r t 1
Sch em e 1. Synthesis of Core-Modified Porphyrins 2, 4,
and 5a
Our initial biological studies demonstrated that the
core-modified porphyrins were potent photosensitizers
with limited dark toxicity and minimal cutaneous
phototoxicity.12 Core-modified porphyrins with two 4-sul-
fonatophenyl substituents at the 5- and 10-meso-posi-
tions of the ring were more effective photosensitizers
both in vitro and in vivo than core-modified porphyrins
with four 4-sulfonatoaryl substituents at the 5-, 10-, 15-,
and 20-positions of the porphyrin ring. Several core-
modified porphyrins with two 4-sulfonatophenyl sub-
stituents were significantly more effective than Photo-
frin in vitro and, at 20-fold lower concentrations than
Photofrin, were comparable to Photofrin in efficacy in
vivo.12b
The pKa values of the sulfonato substituents described
above are sufficiently low to keep the core-modified
porphyrins anionic over the range of physiological pH.
Replacing the sulfonato groups with carboxylic acid
groups gives core-modified porphyrins that achieve both
neutral and anionic states at physiological pH. For the
study of carboxylate derivatives of the core-modified
porphyrins, we have prepared 21,23-dithiaporphyrins
1-4 with 1-4 4-phenoxyacetic acid residues, respec-
tively, in the meso-positions and 21,23-diselenaporphy-
rin 5 with two 4-phenoxyacetic acid residues at the 5,10-
meso-positions (Chart 1). Several physical properties of
these compounds were investigated, including electronic
absorption, quantum yields for the generation of singlet
oxygen and for fluorescence, photobleaching, and lipo-
philicity. In general, the core modification of heteroat-
oms alters the photophysical properties significantly,
while changes in the meso-groups affect the lipophilicity.
To evaluate further the potential development of these
core-modified porphyrins as therapeutics, a series of
studies were performed in vitro to evaluate dark and
phototoxicities, cellular uptake, and localization.
a
Key: (a) 2.5 equiv of BuLi, TMEDA, hexanes, 23 °C to reflux.
(b) PhCHdO or 4-MeOC6H4CHdO. (c) BF3, pyrrole. (d) 7 of 8,
TCBQ, TsOH, CH2Cl2. (e) BBr3, CH2Cl2. (f) BrCH2CO2Et, K2CO3,
acetone. (g) NaOH, aqueous THF.
29% isolated yield (Scheme 1). Similarly, diol 8a and
10 gave dithiaporphyrin 13 in 20% isolated yield and
diol 7b and selenophene 11 gave 21,23-diselenaporphy-
rin 14 in 13% isolated yield. The demethylation of 12-
14 with BBr3 in CH2Cl2 gave phenolic core-modified
porphyrins 15-17, which were alkylated with ethyl
bromoacetate and K2CO3 in acetone to give 18-20. The
ester functional groups of 18-20 were saponified with
NaOH in aqueous tetrahydrofuran (THF) to give dicar-
boxylic acid derivatives 2 and 5 and tetracarboxylic acid
derivative 4.
Syn th esis of Mon oca r boxylic Acid 1 a n d Tr ica r -
boxylic Acid 3. The critical step in the synthesis of
core-modified porphyrins with an odd number of phe-
noxyacetic acid residues is breaking the symmetry of
the 2,5-di(arylhydroxymethyl)thiophenes as shown in
Scheme 2. Treating thiophene with a limiting amount
of BuLi gives 2-lithiothiophene, which reacts with
benzaldehyde to give 2-(1-phenyl-1-hydroxymethyl)-
thiophene (21) in 68% isolated yield.15 Compound 21
was then protected as the TBS ether to give 22, which
was then lithiated with BuLi. The resulting 5-lithio-
thiophene was treated with p-methoxybenzaldehyde to
give 23, which is a common intermediate for the
synthesis of 1 and 3, following removal of the TBS
protecting group.
The condensation of thiophene 912b and diol 23 with
TsOH and TCBQ in CH2Cl2 gave methoxyphenyl dithia-
porphyrin 24 in 22% isolated yield. Demethylation with
BBr3 gave 25, which was alkylated with ethyl bromoace-
tate to give ester 26. Saponification with NaOH in
aqueous THF gave monocarboxylic acid 1. Similarly,
condensation of thiophene 1012b and diol 23 gave tris-
(methoxyphenyl)dithiaporphyrin 27 in 22% isolated
yield, which was then demethylated with BBr3 to give
trisphenol 28. Compound 28 was then alkylated with
ethyl bromoacetate to give triester 29, which was
saponified with NaOH in aqueous THF to give tricar-
boxylic acid 3.
Resu lts a n d Discu ssion
Ch em istr y. Syn th esis of Dica r boxylic Acid s 2
a n d 5 a n d Tetr a ca r boxylic Acid 4. The synthesis of
compounds 2, 4, and 5 with an even number of carbox-
ylic acid groups is shown in Scheme 1 and follows our
previous synthetic strategy.12 2,5-Dilithiochalcogeno-
phenes 613 were added to benzaldehyde to give 2,5-di-
(phenylhydroxymethyl)chalcogenophenes 7a ,b12 and p-
methoxybenzaldehyde to give chalcogenophenes 8a 12b
and 8b. Diols 7a and 8 were converted to the corre-
sponding 2,5-di(arylpyrrolomethyl)chalcogenophenes
9-11 with pyrrole and BF3-etherate. Diol 7a was
condensed with thiophene 10 in the presence of tetra-
chlorobenzoquinone (TCBQ) and p-toluenesulfonic acid
(TsOH) in CH2Cl2 to give 21,23-dithiaporphyrin 12 in
Electr on ic Absor p tion Sp ectr a of Cor e-Mod ified
P or p h yr in s 1-5. 21- and 21,23-Core-modified porphy-
rins absorb light of longer wavelengths than the corre-

3736 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You et al.
Ta ble 1. UV-Vis Band Maxima and Molar Absorptivities for
Sch em e 2. Synthesis of Core-Modified Porphyrins 1
and 3a
1-5 and 30 in MeOH, λmax nm (∈ × 10-3 M-1 cm-1
)
compd
soret
band IV
band III
band II
band I
1
2
433 (241) 512 (18.7)
434 (234) 514 (21.6)
546 (6.2) 633 (1.8) 696 (3.7)
549 (9.9) 634 (1.7) 699 (5.6)
3
436 (275) 515 (22.9) 550 (10.2) 635 (1.8) 700 (5.7)
4
5
30
437 (178) 516 (12.0)
448 (147) 527 (17.2)
431 (476) 518 (15.7)
552 (7.1) 636 (2.2) 701 (4.2)
564 (3.4) 631 (1.7) 695 (2.8)
560 (8.3) 633 (2.3) 701 (4.6)
tical meso-substituents have similar values of φ(1O2)
while 21,23-dithiaporphyrins have slightly smaller val-
ues of φ(1O2).12 21,23-Dithiaporphyrin 30 is an effective
photosensitizer both in vitro and in vivo with φ(1O2) )
0.74 in MeOH at 25 °C.12b
Values of φ(1O2) for dithiaporphyrins 1-5 were de-
termined by direct methods16 using dithiaporphyrin
3012b as a standard. As shown in Figure S6 of the
Supporting Information, the luminescence intensities
from singlet oxygen generated by irradiation of MeOH
solutions of 1-4 and 30 at ambient temperature with
identical absorbance at 532 nm (Figure S7, Supporting
Information) were essentially identical from all five
solutions. Values of φ(1O2) for 1-4 were between 0.74
and 0.80 with φ(1O2) for 30 of 0.74 (Table 2). The
diselenaporphyrin 5 was less efficient at generating 1O2
(Figure S8, Supporting Information) with φ(1O2) of 0.30.
P h otoblea ch in g of 21,23-Cor e-Mod ified P or p h y-
r in s 1-5. Although core-modified porphyrins 1-5 and
30 appeared to be photostable to 532 nm irradiation in
air-saturated MeOH for at least 20-30 min, broadband
irradiation in pH 7.4 phosphate-buffered saline (PBS)
gave some photobleaching. Rates of photobleaching of
core-modified porphyrins 1-5 and 30 as well as the rate
of photobleaching of Photofrin in 0.05 M, pH 7.4, PBS
were determined under irradiation with 200 mW cm-2
of broadband 350-750 nm white light emitted from a
focused and filtered 750 W tungsten source. Concentra-
tions (≈5 × 10-6 M) were adjusted such that the initial
absorbance of the soret band was ≈1.0 in a 1 cm cell.
21,23-Diselenaporphyrin 5 was the most photostable of
the five compounds examined with a rate of loss of <3
× 10-6 s-1 under these conditions (Table 2). Compounds
1, 2, and 30 have comparable photostability with a rate
of loss of 1.26 × 10-4 s-1 for 2, 1.60 × 10-4 s-1 for 1,
and 1.90 × 10-4 s-1 for 30 (Table 2). Compound 2 was
a factor of 4.5 times more stable than Photofrin, which
had a rate of loss of 5.69 × 10-4 s-1. 21,23-Dithiapor-
phyrins 3 and 4 were the least photostable compounds
of the series with rates of loss of 6.77 × 10-4 and 1.00
× 10-3 s-1, respectively (Table 2).
a
Key: (a) 1 equiv of BuLi, TMEDA, hexanes, 0 °C. (b) PhCHdO.
(c) TBSCl, Et3N. (d) (i) 4-MeOC6H4CHdO; (ii) aqueous HCl. (e) 9,
TsOH, CH2Cl2. (f) BBr3, CH2Cl2. (g) BrCH2CO2Et, K2CO3, acetone.
(h) NaOH, aqueous THF. (i) 10, TsOH, CH2Cl2.
Ch a r t 2
sponding porphyrins. For core-modified porphyrins 1-5
and bis-4-sulfonatophenyl-21,23-dithiaporphyrin 30
(Chart 2), which is an effective photosensitizer both in
vitro and in vivo,12b substituents on the aryl groups at
the meso-positions appear to have little effect on the
absorption maxima of the core-modified porphyrins.12
Individual absorption spectra for 1-5 in MeOH are
shown in Figures S1-S5 in the Supporting Information.
For 1-4, the band maxima for all bands move to slightly
longer wavelengths as the number of carboxylic acid
groups increases.
Replacing the core sulfur atoms of 2 with selenium
atoms had a more pronounced effect than varying the
number of carboxylic acid groups or other meso-substit-
uents. The absorption maxima for bands III and IV and
the soret band are at 13-15 nm longer wavelengths for
21,23-diselenaporphyrin 5 relative to 21,23-dithiapor-
phyrin 2 while maxima for bands I and II are at 3-4
nm shorter wavelengths relative to 2.
n -Octa n ol/Wa ter P a r tition Coefficien ts. The oc-
tanol/water partition coefficients for 1-5 and 30 were
all measured with pH 7.4 PBS (0.05 M) as the aqueous
phase using UV-visible spectrometry (Table 2). The
measurements were done using a “shake flask” direct
measurement, and results are the average of three
independent measurements.17 Compound 1 with one
carboxylic acid was the most lipophilic with log P > 3.5.
Log P values for dithiaporphyrin 2 and diselenaporphy-
rin 5 with two carboxylic acids were determined to be
0.036 and -0.67, respectively. Replacing the two S
atoms of 2 with the two Se atoms of 5 gave a -0.7
change in log P, which is consistent with our observa-
tions in chalcogenopyrylium dyes where replacing one
Sin glet Oxygen Gen er a tion . The photochemical
production of singlet oxygen is thought to be the primary
mechanism for cytotoxicity for PDT with many photosen-
sitizers.1-3,10 One measure of a photosensitizer’s poten-
tial is the efficiency of singlet oxygen generation as
measured by φ(1O2). It has been observed that 5,10,15,-
20-tetraarylporphyrins and 21-thiaporphyrins with iden-

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3737
Ta ble 2. Quantum Yields for the Generation of Singlet Oxygen [φ(1O2)], Rates of Photobleaching (kPB) with Broadband Light,
n-Octanol/Water Partition Coefficients (log P), Emission Maxima (λEM), and Quantum Yields for Fluorescence (φF) for Core-Modified
Porphyrins 1-5 and 30
kPB × 104
λEM
c
compda
φ(1O2)
(s-1
)
log P
>3.5
0.036 ( 0.005
-1.49 ( 0.05
-0.95 ( 0.08
-0.67 ( 0.04
-1.9 ( 0.1
(nm)c
φF
1
2
3
4
5
30
0.78 ( 0.03
0.80 ( 0.03
0.74 ( 0.03
0.78 ( 0.03
0.30 ( 0.03
0.74b
1.26 ( 0.09
1.60 ( 0.05
6.77 ( 0.08
10.0 ( 0.4
<3 × 10-2
1.90 ( 0.04
710
710
710
712
0.004 ( 0.001
0.007 ( 0.001
0.008 ( 0.001
0.008 ( 0.001
<0.001
710
0.004 ( 0.001
a
b
Value ( standard deviation. Ref 12b. c In MeOH.
S atom with one Se atom led to a 0.1-0.3 more negative
value of log P.18 Compounds 3 and 4 were much more
hydrophilic with values of log P of -1.49 ( 0.05 and
-0.95 ( 0.08, respectively. Compound 30 with two
sulfonate groups was the most hydrophilic compound
with a log P of -1.9 ( 0.1.
Qu a n tu m Yield s for F lu or escen ce. Dithiaporphy-
rins 1-4 were emissive although quantum yields for
fluorescence (φF) were low (Table 2). The quantum yield
standard used in these studies was a freshly prepared
solution of Rhodamine 6G (φF ) 1.0) dissolved in MeOH.
A small (e15 nm) Stokes shift was observed for the core-
modified porphyrins 1-4, and values of φF were between
0.003 and 0.008. Selenium-containing core-modified
porphyrin 5 was nonemissive.
Su m m a r y of P h ysica l P r op er ties. Substituent
changes in the meso-aryl groups of core-modified por-
phyrins have little impact on absorption spectra and
quantum yields for the generation of singlet oxygen and
fluorescence. Compounds 1-4 and 30 all have band I
maxima near 700 nm and values of φ(1O2) between 0.74
and 0.80. Heteroatom changes have a much greater
impact. The substitution of two Se atoms for the two S
atoms of 2 gives compound 5. The two Se atoms are
much less than van der Waals’ radii apart distorting
the planarity of the core-modified porphyrin ring,12
which leads to slight spectral differences from 1-4 and
to smaller values of φ(1O2) and φF. Compound 5 has
greater photostability than compounds 1-4 and 30, and
compounds 1, 2, 5, and 30 all have significantly greater
photostability than Photofrin with respect to broadband
irradiation. The number and identity of the water-
solubilizing functionality on meso-aryl groups have its
greatest impact on values of log P.
Biology. While a successful photosensitizer will have
certain desirable physical and photophysical properties,
the viability of a photosensitizer as a functioning drug
will depend on adsorption, distribution, metabolism, and
excretion (ADME) properties.19 As a starting point to
evaluate biological properties, we examined compounds
1-5 in vitro using R3230AC rat mammary adenocar-
cinoma cells to establish dark and phototoxicity, cellular
uptake, and sites of localization.
F igu r e 1. Cell viability of cultured R3230AC cells after
photosensitization in the presence of core-modified porphyrins
1-5. Data are expressed as the percent survival of viable cells
relative to untreated controls following treatment with various
concentrations of 1-5 (1 × 10-8 to 1 × 10-6 M) and broadband
light (350-750 nm) delivered at 1.4 mW cm-2 from a filtered
750 W tungsten source for a total of 5.0 J 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.
None of the core-modified porphyrins 1-5 displayed any
significant dark toxicity at concentrations e1 × 10-6
M
(Figures S9-S13, Supporting Information). In addition,
dithiaporphyrins 3 and 4 with three and four carboxylic
acid residues, respectively, showed little phototoxicity
upon irradiation (Figure 1). In contrast, dithiaporphyrin
1 (1 × 10-6 M) showed significant cell phototoxicity (P
< 0.005) as compared to cells maintained in the dark
for the same 24 h period with 1 × 10-6 M 1. Irradiation
of cultures in the presence of 1 × 10-7 or 1 × 10-8 M 1
did not result in any significant decrease in cell viability
as compared to nonirradiated controls or cells exposed
to the same concentrations of 1 in the dark over the
same time period.
The data presented in Figure 1 demonstrate that
dithiaporphyrin 2 was significantly more phototoxic
than 1. At 1 × 10-7 M, 2 combined with irradiation
resulted in a 53% loss in cell viability while 1 under
the same conditions induced no significant reduction in
cell numbers, a significant difference of P < 0.001 when
the phototoxic effects of the two core-modified porphy-
rins were compared. At 1 × 10-6 M, dithiaporphyrin 1
reduced cell viability by 41% 24 h after irradiation, but
Da r k a n d P h ototoxicities In Vitr o. One very
desirable property for new photosensitizers is high
phototoxicity with minimal or no dark toxicity. Cultured
R3230AC cells were incubated for 24 h in the dark with
various concentrations of photosensitizer and were then
washed and irradiated with broadband light (350-750
nm) delivered at 1.4 mW cm-2 for 1 h (5 J cm-2). Light-
treated cells and dark controls were subsequently
incubated for 24 h, and cell survival was determined.

3738 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You et al.
F igu r e 2. Cell viability of cultured Colo-26 cells after pho-
tosensitization in the presence of core-modified porphyrins 2
and 30, HPPH, and Photofrin. Data are expressed as the
surviving fraction of viable cells relative to untreated controls
following treatment with various concentrations of 2, 30,
HPPH, and Photofrin and filtered 590-800 nm light at 37 °C
F igu r e 3. Cellular uptake of core-modified porphyrins 1-5
in cultured R3230AC rat mammary adenocarcinoma cells.
Cells were incubated with 1 × 10-6, 5 × 10-6, and 1 × 10-5
M
concentrations of 1-5 for 3 h. After the incubation period, cells
were washed, detached, and dissolved in 25% Scintigest. The
intracellular fluorescence for 1-4 or the absorbance for 5 was
compared to standard curves, and the data are presented as
femtomoles of 1-5 per cell.
with 14 mW cm-2 for a total light dose of 4 J cm-2
.
irradiation of cells treated with core-modified porphyrin
2 gave a 96% reduction in cell viability, resulting in a
significant difference in phototoxicity of P < 0.001.
Cells treated with diselenaporphyrin 5 and light
showed some phototoxicity as compared to that observed
in dark controls when 5 was present in cell cultures at
1 × 10-8 to 1 × 10-6 M. However, the phototoxicity of 5
was significantly less than that observed for core-
modified porphyrin 2 (P < 0.001, Figure 1).
A Com p a r ison of Dith ia p or p h yr in 2 w ith 30,
HP P H, a n d P h otofr in . Compound 2 was significantly
more phototoxic than compounds 1 or 3-5. Comparison
of the phototoxicity of 2 with 30, Photofrin, and HPPH,
a pyropheophorbide derivative currently in clinical
trials,11a is presented in Figure 2. These experiments
were carried out with cultured Colo-26 cells, a murine
colon carcinoma cell line. Cells 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 delivered at 14 mW cm-2 for a total light
dose of 4 J cm-2. Light-treated cells and dark controls
were then incubated for 24 h, and cell survival was
determined. Results are compiled in Figure 2. No
significant dark toxicity was observed at concentrations
<10-4 M. The concentration of Photofrin that gave a
50% cell kill under these conditions was approximately
10 µM, 1 µM for compound 30 and HPPH, and 0.1 µM
for compound 2. In this comparison, compound 2 was
significantly (P < 0.001) more phototoxic than 30,
HPPH, or Photofrin.
digested in 25% Scintigest. The fluorescence or absor-
bance values from the resulting solutions were com-
pared to measurements from known concentrations of
1-5. For compounds 1-4, the fluorescence peak at 712
nm was measured following excitation at 440 nm. Spin-
orbit effects from the two Se atoms of 5 effectively
eliminated fluorescence in solutions of this molecule,
and cellular uptake was determined by the absorbance
at 440 nm. The data in Figure 3 depict the amount of
core-modified porphyrins 1-5, expressed as fmol/cell,
measured intracellularly 3 h after exposure of R3230AC
cells in culture to various concentrations of 1-5. Core-
modified porphyrin uptake reached a plateau after 3 h,
and intracellular concentrations remained fairly con-
stant over 24 h.
Cellular uptake was significantly greater for core-
modified porphyrins 1, 2, and 5 relative to core-modified
porphyrins 3 and 4. Following incubation in media
containing 5 × 10-6 M core-modified porphyrin, the
intracellular concentrations of 1, 2, and 5 are 12 ( 0.8,
15 ( 1, and 8.9 ( 1.5 fmol/cell, respectively, at 3 h after
exposure. In contrast, the intracellular concentration of
3 was 2.5 ( 2.0 fmol/cell 3 h following incubation with
5 × 10-6 M 3. Core-modified porphyrin 4 was not
detectable in cells at concentrations e5 × 10-5 M (<0.3
fmol/cell), and at 1 × 10-4 M, 4 was measured at 1.0 (
0.5 fmol/cell.
Up ta k e of Cor e-Mod ified P or p h yr in s 1-5 in to
Cu ltu r ed R3230AC Cells. As shown in Figure 1, the
number of carboxylic acid residues and the 21,23-
heteroatoms has an impact on the efficacy of 1-5. Two
likely consequences of structural changes are changes
in cellular uptake or cellular localization. The uptake
of core-modified porphyrins 1-5 was determined by
fluorescence for compounds 1-4 and by absorbance for
compound 5. Following the incubation periods in the
presence of 1-5, cells were washed, detached, and
Su bcellu la r Ta r gets. Mitoch on d r ia l Cytoch r om e
c Oxid a se Activity in Wh ole Cells. The subcellular
localization of a photosensitizer can impact both dark
and phototoxicity. For lipophilic porphyrin and porphy-
rin-related structures, accumulation of the photosensi-
tizer occurs in the cellular membranes of organelles such
as the mitochondria and lysosomes.20 We examined the
impact of carboxylated photosensitizers 1 and 2 and
sulfonated dithiaporphyrin 30 on the mitochondria of
R3230AC cells in culture both in the dark and in the

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3739
F igu r e 5. Spectrofluorometry of the emission from R3230AC
cells treated with 2 µM dithiaporphyrins 2 and a solution of
compound 2 in MeOH.
F igu r e 4. Effect of 1, 2, and 30 on mitochondrial cytochrome
c oxidase activity in cultured whole R3230AC tumor cells in
the dark and following irradiation. Cell culture and light
exposure conditions are detailed in the Experimental Section.
Columns represent the change in optical density (mOD) units
per minute for 1 × 105 cells that were exposed to 1, 2, or 30 at
indicated concentrations in the dark (solid columns) or 24 h
after light exposure (striped columns). Each datum point
represents the mean of at least three separate experiments
performed in duplicate; the error bars are the SEM.
1.0 µM photosensitizer. Either compounds 1 and 30 are
much less toxic toward mitochondria than 2 (i.e., not
localized near a sensitive intramitochondrial site) or
these photosensitizers do not localize initially in the
mitochondria, but the photosensitizer may relocalize to
the mitochondria during irradiation.22
In tr a cellu la r Loca liza tion of Cor e-Mod ified P or -
p h yr in s 1, 2, a n d 30 via Con foca l La ser Sca n n in g
Micr oscopy. Confocal laser scanning microscopy (CLSM)
is a powerful technique for visualizing a fluorescent
photosensitizer within a cell, and when an organelle
specific dye is employed along with the photosensitizer,
dual label confocal microscopy can be used to image both
the organelle specific dye and the photosensitizer in the
same cell.23 We examined the localization of dithiapor-
phyrins 1-4 and 30 in cells and used rhodamine 123
(Rh-123) to identify mitochondria intracellularly.24
light by measuring the activity of cytochrome c oxidase.
Cytochrome c oxidase is the terminal enzyme in the
mitochondrial respiratory chain, and dark or photodam-
age either to this enzyme or to any preceding it in the
respiration chain can lead to inhibition of cytochrome c
oxidase activity.21
R3230AC cells were incubated with a 0.01-1.0 µM
solution of 1, 2, or 30 for 24 h. Cell cultures were then
washed and irradiated for 1 h with 350-750 nm light
delivered at 1.4 mW cm-2 (5.0 J cm-2). Both irradiated
cells and photosensitizer-treated dark controls were
then incubated for an additional 24 h in the dark at
which time the activity of cytochrome c oxidase was
determined (Figure 4). As shown in Figure 4, 1.0 µM
solutions of 1 or 30 (as well as lower concentrations)
had no impact on cytochrome c oxidase activity in
nonirradiated R3230AC cells. In contrast, treating
R3230AC cells with either 0.1 or 1.0 µM concentrations
of 2 inhibited cytochrome c oxidase activity by 35 and
65%, respectively, in nonirradiated cells. Irradiation of
the cells gave 55% inhibition of cytochrome c oxidase
activity following treatment with 1.0 µM 1, 70% inhibi-
tion following treatment with 0.1 µM 2, 90% inhibition
following treatment with 1.0 µM 2, and 90% inhibition
following treatment with 1.0 µM 30.
Untreated R3230AC cells, as well as cells treated with
2-10 µM 3 or 4 for 3 h, displayed no detectable
fluorescence. Although the fluorescence from 3 or 4 in
solution is weak (Table 2), the lack of any cellular
fluorescence is consistent with the low uptake of dithia-
porphyrins 3 and 4 (Figure 3). In contrast, emission was
apparent from R3230AC cells treated with 2 µM 1 or 2
for 3 h. At lower concentrations of 1 or 2, no cellular
fluorescence was observed, which is most likely due to
the low values of φF for 1 and 2 (Table 2). Localized
spectrofluorometry indicated that the emission from the
R3230AC cells was identical to emission from MeOH
solutions of 1 (Figure S14, Supporting Information) or
2 (Figure 5).
Fluorescence micrographs of R3230AC cells treated
with 2 µM dithiaporphyrin 1, 2, or sulfonated dithiapor-
phyrin 30 are shown in Figure 6. Three different
patterns of staining were observed. In each case, cells
were treated with a 2 µM solution of photosensitizer in
culture media and were then washed and covered with
fresh media. For cells treated with 1, the emission was
granular and observed throughout the cell except in the
nucleus (Figure 6a). For cells treated with 2, the
emission was very diffuse and was observed throughout
the entire cell except in the nucleus (Figure 6b). For cells
As shown in Figure 4, mitochondrial cytochrome c
oxidase activity in whole cells treated with 2 was
inhibited both in the dark and following irradiation
relative to control cells. These data are consistent with
initial localization in the mitochondria of R3230AC cells
in the absence of light and with further mitochondrial
damage upon irradiation of photosensitizer-treated cells.
In contrast, no significant inhibition of cytochrome c
oxidase activity was observed in either 1- or 30-treated
dark controls while 55 and 90% inhibition, respectively,
were observed following irradiation of cells treated with

3740 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You et al.
F igu r e 7. CSLM fluorescence images of R3230AC cells
treated with (A) 1 µM Rh-123 for 20 min and (B) 2 µM 2 for 3
h followed by costaining with 1 µM Rh-123 for 20 min. Cell
culture conditions and experimental details are detailed in the
Experimental Section.
treated with sulfonated dithiaporphyrin 30, the emis-
sion was localized near the nuclear membrane (Figure
6c).
The emission from cells treated with dithiaporphyrins
1, 2, and 30 can be compared with the emission from
cells treated with Rh-123, a known mitochondrial stain,
in Figure 7.24 Emission from cells treated with a 1 µM
solution of Rh-123 for 20 min was highly granular and
similar to the emission observed from cells treated with
dithiaporphyrin 1 (Figure 7a).
F igu r e 6. CSLM fluorescence images of R3230AC cells
treated with (A) 2 µM 1, (B) 2 µM 2, and (C) 2 µM 30 for 3 h.
Cell culture conditions and experimental details are detailed
in the Experimental Section.
One problem with dual-labeling experiments with the
dithiaporphyrins and Rh-123 is the difference in mag-

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3741
Ch a r t 3
nitude of quantum yields for fluorescence for Rh-123
(λem ) 529 nm, φF ) 1.0) and dithiaporphyrins 1, 2, and
30 (λem ) 710-712 nm, φF ) 0.004-0.007). The strong
emission from Rh-123 results in overlap of the tail of
the emission of Rh-123 with the emission from the
dithiaporphyrins. However, the primary emission from
Rh-123 can be separated from the emission from the
dithiaporphyrins using a 640 nm short-pass dichroic
mirror. Most of the emission from Rh-123 is transmitted
through the mirror while the emission from the core-
modified porphyrins as well as the tail of the emission
from Rh-123 is reflected orthogonally.
A fluorescence micrograph from emission collected
through the dichroic mirror for R3230AC cells treated
first with 2 µM 2 for 3 h, washed, treated with 1 µM
Rh-123 for 20 min, washed, and covered with fresh
media is shown in Figure 7b. In stark contrast to the
granular fluorescence observed in cells treated only with
Rh-123 in Figure 7a, the Rh-123 emission from cells
treated with 2 and Rh-123 is diffuse throughout the
cytoplasm. These data are consistent with disruption
of the mitochondrial membrane potential by 2 or with
exclusion of Rh-123 from the mitochondria in the
presence of 2.
hydrophilic end as shown in the examples of Chart 3.
Porphyrin derivatives with two polar groups on adjacent
meso-positions provide more active photosensitizers
than those with one, three, or four as determined by
cell viability.25 Sulfonated dithiaporphyrin 30 has two
4-sulfonatophenyl groups at the 5- and 10-positions with
4-fluorophenyl substituents at the 15- and 20-positions.
Among the core-modified porphyrins 1-5, the enhanced
phototoxicity of compounds 1, 2, and 5 with hydrophobic
and hydrophilic ends is reflected in increased cellular
uptake of these core-modified porphyrins relative to
compounds 3 and 4.
The CLSM images from R3230AC cells treated with
dithiaporphyrins 1 and 2 as well as the effects of
compounds 1 and 2 on mitochondrial function strongly
suggest that the mitochondria are an important target
for these core-modified porphyrins. Many of the photo-
sensitizers of Chart 2 also have the mitochondria as a
significant target, and the similarities in structure
suggest a common binding site. While a common binding
site has yet to be ascertained, some possibilities include
cytochrome c oxidase or an earlier enzyme in the
respiratory chain, the peripheral benzodiazepine recep-
tor (PBR) for which protoporphyrin IX and other por-
phyrins are natural ligands,26 the antiapoptotic protein
Bcl-2,27 or some part of the complex that includes the
PBR, Bcl-2, the voltage-dependent anion channel, the
adenine nucleotide transporter, and the mitochondrial
pore transition. The importance of binding to the PBR
has been challenged in several studies.11a,28 One of these
studies correlates increased phototoxicity of pyropheo-
phorbide derivatives with enhanced lipophilicity rather
than with PBR binding.11a In contrast to those observa-
tions, the phototoxicity of dithiaporphyrin 2, with log P
) 0.036, is much greater than dithiaporphyrin 1, with
log P > 3.5.
Su m m a r y a n d Con clu sion s
Sequential lithiation of thiophenes and selenophenes
permits the preparation of 2,5-di-(1-hydroxymethyl)-
chalcogenophenes with two different substituents on the
hydroxymethyl groups. When these derivatives are
combined with symmetrical 2,5-di-(1-aryl-1-hydroxy-
methyl)chalcogenophenes, synthetic routes are now
available to core-modified porphyrins with (i) one pair
of identical, adjoining meso-substituents and two dif-
ferent meso-substituents, (ii) two adjoining pairs of
identical meso-substituents, (iii) three identical meso-
substituents, and (iv) four identical meso-substituents.
The chalcogen atoms can be systematically varied as
well to provide two S atoms, one S and one Se atom, or
two Se atoms.12b Systematic variation of structure is
now possible with the core-modified porphyrins.
Photophysical properties are relatively insensitive to
the number of carboxylic acid groups. However, replac-
ing S atoms in the core with Se atoms, as in diselena-
porphyrin 5, results in diminished quantum yields for
the generation of singlet oxygen and for fluorescence.
In contrast, phototoxicity and cellular uptake were quite
sensitive to the number of carboxylic acid residues in
the meso-positions of compounds 1-5. Compounds 3 and
4 display essentially no phototoxicity, which correlates
with greatly reduced cellular uptake relative to 1, 2, and
5. Of the three remaining compounds, dithiaporphyrin
2 with two carboxylic acid groups displays the greatest
phototoxicity followed by dithiaporphyrin 1 with one
carboxylic acid group and diselenaporphyrin 5 with two
carboxylic acid groups. In a comparison of dithiapor-
phyrin 2 with sulfonated dithiaporphyrin 30,12b HPPH
(Chart 3),11a and Photofrin, dithiaporphyrin 2 displayed
significantly greater phototoxicity (Figure 2). While dark
toxicity as measured by cell viability did not appear to
be an issue with compounds 1-5, changes in the meso-
substituents appear to impact site of localization (Figure
6) as well as mitochondrial function (Figures 4 and 7).
Most of the effective porphyrin-related photosen-
sitizers3b have a distinct hydrophobic end and a distinct
At the molecular level, we are able to separate
photophysical properties from lipophilicity through our
choice of meso and core substituents. We have also
demonstrated a correlation of uptake with phototoxicity
as well as a correlation of uptake with lipophilicity. We
are actively examining the correlation of structural
features with subcellular localization.
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

3742 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You 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 23 °C on a Varian Gemini-300,
Inova 400, or Inova 500 instrument with residual solvent
signal as the internal standard: CDCl3 (7.26 for proton, 77.16
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 equipped
with a circulating constant temperature bath for the sample
chambers. Elemental analyses were conducted by Atlantic
Microlabs, Inc. Q-TOF 2 electrospray mass spectrometry was
conducted by the Campus Chemical Instrumentation Center
of The Ohio State University (Columbus, OH). Compounds 13
and 20 were prepared as previously described in ref 12b.
colorless oil. 1H NMR (400 MHz, CDCl3): δ -0.02 (s, 3H), 0.05
(s, 3H), 0.93 (s, 9H), 5.97 (s, 1H), 6.74 (d, 1H, J ) 3 Hz), 6.87
(dd, 1H, J ) 5, 3 Hz), 7.18 (d, 1H, J ) 5 Hz), 7.24 (dd, 1H, J
) 7, 6 Hz), 7.32 (dd, 2H, J ) 7, 7 Hz), 7.41 (d, 2H, J ) 7 Hz).
13C NMR (75 MHz, CDCl3): δ -4.89, -4.68, 18.45, 25.92, 73.31,
123.69, 124.70, 126.33, 126.47, 127.57, 128.38, 144.54, 150.58.
High-resolution Q-TOF MS: m/z 327.1191 (calcd for C17H24
-
OSSi + Na+, 327.1215).
P r ep a r a tion of 2-[1-(4-Meth oxyp h en yl)-1-h yd r oxym e-
th yl]-5-(1-p h en yl-1-h yd r oxym eth yl)th iop h en e (23). Com-
pound 22 (10.7 g, 35 mmol) was added to a solution of
n-butyllithium (23 mL, 1.6 M in hexanes, 37 mmol) and
TMEDA (5.8 mL, 38 mmol) in 150 mL of hexanes under an
Ar atmosphere. The reaction mixture was stirred at ambient
temperature for 30 min and was transferred via cannula to a
pressure-equalizing addition funnel. The suspension of 2 lithio-
22 was then added dropwise to a 0 °C solution of 4-methoxy-
benzaldehyde (5.1 mL, 33 mmol) in anhydrous THF (150 mL),
which had been degassed with Ar for 15 min. After addition
was complete, the mixture was allowed to warm to ambient
temperature, 300 mL of a 1 M solution of NH4Cl was added,
and the organic phase was separated. The aqueous phase was
extracted with ether (3 × 300 mL). The combined organic
extracts were washed with water (3 × 300 mL) and brine (300
mL), dried over MgSO4, and concentrated to give a yellow oil.
The oil was dissolved in a 1 M solution of Bu4NF in THF (95
mL, 95 mmol) and stirred at ambient temperature for 1 h at
which point 100 mL of saturated aqueous NH4Cl was added.
The resulting mixture was extracted with ether (4 × 100 mL).
The combined organic extracts were washed with water (3 ×
400 mL) and brine (400 mL), dried over MgSO4, and concen-
trated. The crude diol was purified by column chromatography
on SiO2 eluted with 25% EtOAc/hexanes to give 7.8 g (68%) of
predominantly one diastereomer of 23 as a yellow solid; mp
73-75 °C. IR (KBr): 3397 (br), 1511, 1161 cm-1. 1H NMR (400
MHz, CDCl3): δ 2.62 (br s, 2H), 3.79 (s, 3H), 5.87 (s, 1H), 5.92
(s, 1H), 6.67 (s, 2H), 6.86 (d, 2H, J ) 8.4 Hz), 7.32 (m, 5H),
7.40 (d, 2H, J ) 7.6 Hz). 13C NMR (75 MHz,CDCl3): δ 55.40,
72.29, 72.62, 114.00, 124.36, 124.58, 126.37, 126.40, 127.73,
127.78, 128.08, 128.62, 135.33, 143.01, 148.08, 148.67, 159.44.
High-resolution Q-TOF MS: m/z 349.0874 (calcd for C19H18O3S
+ Na+, 349.0875).
P r epar ation of 2,5-Bis[1-(4-m eth oxyph en yl)h ydr oxym e-
th yl]selen op h en e (8b). Selenophene (5.7 g, 43 mmol) was
added to a solution of n-butyllithium (60 mL, 1.6 M in hexanes,
95 mmol) and N,N,N,N-tetramethylenediamine (TMEDA, 15
mL, 100 mmol) in 200 mL of hexanes, under an Ar atmosphere.
The reaction mixture was heated at reflux for 2 h, cooled to
ambient temperature, and transferred via a cannula to a
pressure-equalizing addition funnel. This dilithioselenophene
suspension was then added dropwise to a 0 °C solution of
4-methoxybenzaldehyde (11.2 g, 82 mmol) in anhydrous THF
(200 mL), which had been degassed with Ar for 15 min. After
the addition was complete, the mixture was warmed to
ambient temperature, 300 mL of a 1 M solution of NH4Cl was
added, and the organic phase was separated. The aqueous
phase was extracted with ether (3 × 300 mL). The combined
organic extracts were washed with water (3 × 300 mL) and
brine (300 mL), dried over MgSO4, and concentrated to give a
yellow oil. The crude product was precipitated by the slow
addition of hexanes to an ether solution of 8b to give 13.0 g
(74%) of 8b as a white solid; mp 90-92 °C. IR (KBr): 3400
(br), 2953, 1612, 1512 cm-1. 1H NMR (400 MHz, CDCl3 + CD3-
OD): δ 3.78 (s, 6H), 4.44 (s, 2H), 5.31 (s, 2H), 6.76 (d, 2H, J )
4 Hz), 6.85 (d, 2H), 6.87 (d, 2H, J ) 3 Hz), 7.27 (d, 2H, J ) 3
Hz), 7.29 (d, 2H, J ) 3 Hz). 13C NMR (75 MHz, 1:1 CDCl3/
CD3OD): δ 54.95, 82.70, 113.64, 126.18, 127.97, 132.76, 153.64,
159.14. High-resolution Q-TOF MS: m/z 427.0429 (calcd for
C
20H20O480Se + Na+, 427.0426).
P r ep a r a tion of 2,5-Bis[1-(4-m eth oxyp h en yl)-1-p yr r o-
Gen er a l P r oced u r e for th e P r ep a r a tion of P or p h yr in s
12-14, 24, a n d 27. P r ep a r a tion of 5-(4-Meth oxyp h en yl)-
10,15,20-tr ip h en yl-21,23-d ith ia p or p h yr in (24). Compound
23 (3.0 g, 10 mmol), 9 (4.3 g, 10 mmol), and TCBQ (9.8 g, 40
mmol) were dissolved in 600 mL of CH2Cl2 that had been
degassed under a stream of Ar bubbles for 20 min. TsOH
monohydrate (1.9 g, 10 mmol) was added, and the reaction
mixture was stirred for 0.5 h at ambient temperature. The
reaction vessel was covered with aluminum foil, and the
reaction mixture was heated at reflux for 1 h. The reaction
mixture was concentrated, and the residue was redissolved
in minimal CH2Cl2. The crude product was purified via
chromatography on basic alumina eluted with CH2Cl2. Por-
phyrin 24 was isolated as the first red band. The crude product
was washed with acetone and was then recrystallized from
CH2Cl2/MeOH to give 1.5 g (22%) of 24 as a purple solid; mp
lom eth yl]selen op h en e (11). Compound 8b (5.1 g, 13 mmol)
was dissolved in excess pyrrole (35 mL), and the resulting
solution was degassed with Ar. Boron trifluoride etherate was
added (0.32 mL), and the resulting mixture was allowed to
stir for 1 h at ambient temperature. The reaction was stopped
by addition of CH2Cl2 (200 mL) followed by 40% NaOH (50
mL). The organic layer was separated, washed with water (3
× 200 mL) and brine (200 mL), dried over MgSO4, and
concentrated. The excess pyrrole was removed at reduced
pressure at ambient temperature. The residual oil was purified
via chromatography on SiO2 eluted with 25% EtOAc/hexanes
to give 4.5 g (71%) of 11 as a yellow oil. IR (neat): 3400 (br),
2957, 1610, 1510 cm-1. 1H NMR (500 MHz, CDCl3): δ 3.82 (s,
6H), 5.57 (s, 2H), 5.99 (s, 2H), 6.18 (d, 2H, J ) 2 Hz), 6.70 (s,
2H), 6.82 (s, 2H), 6.88 (AA′, 4H, J ) 8 Hz), 7.22 (BB′, 4H, J )
8 Hz), 7.96 (br s, 2H). 13C NMR (75 MHz, CDCl3): δ 47.41,
55.36, 107.40, 108.38, 114.04, 117.21, 127.34, 129.47, 133.82,
135.53, 154.09. High-resolution Q-TOF MS: m/z 525.1062
(calcd for C28H26N2O280Se + Na+, 525.1059).
P r ep a r a tion of 2-[1-P h en yl-1-(ter t-bu tyld im eth ylsilyl-
oxy)m eth yl]th iop h en e (22). A mixture of 2-(1-phenyl-1-
hydroxymethyl)thiophene, (4 g, 0.021 mol), which was pre-
pared according to ref 15, TBSCl (9.5 g, 0.063 mol), DMAP
(2.6 g, 0.021 mol), and Et3N (3.7 mL, 0.063 mol) in 100 mL of
dry CH2Cl2 was stirred at 0 °C for 2 h and was then warmed
to ambient temperature for 24 h. The reaction mixture was
partitioned between 400 mL of ether and 400 mL of saturated
aqueous NaHCO3. The organic layer was washed with water
(3 × 400 mL) and brine (400 mL), dried over MgSO4, and
concentrated to give a yellow oil. Chromatography on SiO2
eluted with 5% EtOAc/hexanes gave 5.5 g (86%) of 22 as a
1
> 300 °C. H NMR (400 MHz, CDCl3): δ 4.11 (s, 3H), 7.36 (d,
2H, J ) 8.4 Hz), 7.81 (s, 9H), 8.18 (d, 2H, J ) 8.4 Hz), 8.26 (d,
6H, J ) 5.6 Hz), 8.69 (s, 3H, s), 8.72 (d, 1H, J ) 4.4 Hz), 9.69
(s, 3H), 9.73 (d, 1H, J ) 4.8 Hz). 13C NMR (75 MHz, CDCl3):
δ 55.75, 113.21, 127.56, 128.16, 133.81, 134.00, 134.10, 134.17,
134.27, 134.58, 134.61, 134.70, 135.48, 135.57, 135.65, 141.44,
147.83, 147.99, 148.03, 148.25, 156.48, 156.63, 156.89, 159.92.
High-resolution Q-TOF MS: m/z 679.186 (calcd for C45H30N2-
OS2 + H, 679.1878).
P r epar ation of 5,10-Bis-4-m eth oxyph en yl-15,20-diph en -
yl-21,23-d ith ia p or p h yr in (12). 2,5- Bis(1-phenyl-1-pyrro-
lomethyl)thiophene12 (9, 1.42 g, 3.6 mmol) and 2,5-bis[1-(4-
methoxyphenyl)-1-hydroxymethyl]thiophene12b (8a , 1.28 g, 3.6
mmol) in 1 L of CH2Cl2 were treated with TCBQ (4.40 g, 18
mmol) and TsOH (0.76 g, 4.0 mmol) as described. The crude
product was recrystallized from CH2Cl2/MeOH to give 0.75 g

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3743
1
(29%) of 12 as a purple solid; mp > 300 °C. H NMR (CDCl3,
300 MHz): δ 4.07 (s, 6 H), 7.34 (BB′, 4 H, J ) 8.1 Hz), 7.79
(m, 6 H), 8.17 (AA′, 4 H, J ) 8.1 Hz), 8.24 (m, 4 H), 8.67 (d, 2
H, J ) 4.5 Hz), 8.71 (d, 2 H, J ) 4.5 Hz), 9.67 (s, 2 H), 9.72 (s,
2 H). 13C NMR (CDCl3, 75 MHz): δ 55.56, 113.03, 127.40,
127.97, 133.69, 133.79, 134.00, 134.16, 134.35, 134.56, 135.31,
135.40, 141.30, 147.68, 148.15, 156.38, 156.65, 159.75. High-
147.67, 147.95, 156.16, 156.38, 156.76, 157.24. High-resolution
Q-TOF MS: m/z 665.1708 (calcd for C44H28N2OS2 + H,
665.1721).
P r epar ation of5,10-Bis-(4-h ydr oxyph en yl)-15,20-Diph en -
yl-21,23-d ith ia p or p h yr in (15). Dithiaporphyrin 12 (312 mg,
0.44 mmol) was treated with BBr3 (0.78 mL, 8.0 mmol) as
described to give 170 mg (57%) of the phenolic dithiaporphyrin
15, which was recrystallized from CH2Cl2/toluene to give
resolution Q-TOF MS: m/z 677.2094 (calcd for C46H32N2S2
+
1
H, 677.2085).
purple needles, mp > 300 °C. H NMR (300 MHz, DMSO-d6):
P r epar ation of 5,10-Bis-4-m eth oxyph en yl-15,20-diph en -
yl-21,23-d ith ia p or p h yr in (14). 2,5- Bis[1-(4-methoxy)phenyl-
1-pyrrolomethyl]selenophene (11, 3.8 g, 7.6 mmol) and diol 7b12
(2.6 g, 7.6 mmol) in 1 L of CH2Cl2 were treated with TCBQ
(7.3 g, 30 mmol) and TsOH (1.52 g, 8.0 mmol) as described to
give 0.80 g (13%) of 14 as a purple solid; mp > 300 °C. 1H
NMR (500 MHz, CDCl3): δ 4.11 (s, 6H), 7.40 (AA′, 4H, J ) 8
Hz), 7.86 (m, 6H), 8.25 (BB′, 4H, J ) 8 Hz), 8.32 (d, 4H, J )
5 Hz), 8.86 (s, 2H), 8.89 (d, 2H, J ) 2 Hz), 9.91 (s, 2H), 9.95 (s,
2H). 13C NMR (75 MHz, CDCl3): δ 55.73, 113.35, 127.68,
128.12, 133.64, 134.50, 134.70, 135.85, 137.05, 137.33, 137.63,
141.17, 149.66, 150.35, 156.84, 156.94, 159.96. High-resolution
Q-TOF MS: m/z 805.0907 (calcd for C46H32N2O280Se2 + H,
805.0878).
P r epar ation of 5,10,15-Tr i-(4-m eth oxyph en yl)-20-ph en -
yl-21,23-d ith ia p or p h yr in (27). Compounds 1012b (2.39 g, 5.3
mmol) and 23 (1.7 g, 5.2 mmol) were treated with TCBQ (5.4
g, 22 mmol) and TsOH (0.76 g, 4.0 mmol) as described to give
0.72 g (19%) of 27 as a purple solid; mp > 300 °C. 1H NMR
(400 MHz, CDCl3): δ 4.11 (s, 9H), 7.36 (d, 6H, J ) 8.0 Hz),
7.81 (m, 3H), 8.18 (d, 6H, J ) 7.2 Hz), 8.25 (m, 2H), 8.67 (d,
1H, J ) 4.4 Hz), 8.70 (s, 3H), 9.66 (d, 1H, J ) 5.2 Hz), 9.71 (s,
3H). 13C NMR (75 MHz, CDCl3): δ 55.74, 113.18, 127.54,
128.11, 133.90, 134.32, 134.53, 134.63, 135.34, 135.55, 141.51,
147.88, 148.14, 148.29, 156.43, 156.71, 156.85, 159.88. High-
resolution Q-TOF MS: m/z 739.2038 (calcd for C47H34N2O3S2
+ H, 739.2089).
δ 10.14 (br s, 2 H), 9.78 (s, 2 H), 9.66 (s, 2 H), 8.67 (d, 2 H, J
) 4.4 Hz), 8.57 (d, 2 H, J ) 4.4 Hz), 8.22 (m, 4 H), 8.06 (AA′,
4 H, J ) 8.2 Hz,), 7.86 (m, 6 H), 7.28 (BB′, 4 H, J ) 8.2 Hz).
13C NMR (75 MHz, DMSO-d6): δ 157.89, 155.82, 155.45,
147.38, 146.60, 140.34, 136.02, 135.67, 135.53, 134.84, 134.62,
134.24, 133.86, 133.40, 130.93, 128.33, 127.76. High-resolution
Q-TOF MS: m/z 717.1475 (calcd for C44H28N2O2S2 + H,
717.1482).
P r epar ation of5,10-Bis-(4-h ydr oxyph en yl)-15,20-Diph en -
yl-21,23-diselen apor ph yr in (17). Diselenaporphyrin 14 (0.40
g, 0.50 mmol) was treated with BBr3 (0.49 mL, 5.1 mmol) as
described to give 0.37 g (96%) of 17 as a purple solid; mp >
300 °C. 1H NMR (500 MHz, 1:1:1 CDCl3/CD3OD/DMSO-d6):
δ 7.24 (AA′, 4H, J ) 8 Hz), 7.75 (m, 6H), 8.02 (d, 4H, J ) 6
Hz), 8.15 (BB′, 4H, J ) 8 Hz), 8.68 (d, 2H, J ) 4 Hz), 8.76 (d,
2H, J ) 4 Hz), 9.77 (s, 2H), 9.87 (s, 2H). 13C NMR (126 MHz,
1:1:1 CDCl3/CD3OD/DMSO-d6): δ 115.04, 127.84, 128.34,
131.93, 134.26, 134.38, 134.84, 136.11, 137.12, 137.17, 137.37,
138.37, 140.74, 149.12, 150.20, 156.61, 156.74, 157.98. High-
resolution Q-TOF MS: m/z 777.0553 (calcd for C44H28N2-
O280Se2 + H, 777.0565).
P r epar ation of 5,10,15-Tr is-(4-h ydr oxyph en yl)-20-P h en -
yl-21,23-d ith ia p or p h yr in (28). Core-modified porphyrin 27
(0.49 g, 0.66 mmol) was treated with BBr3 (0.49 g, 5.1 mmol)
as described to give 0.38 g (82%) of 28 as a purple solid; mp >
1
300 °C. H NMR (400 MHz, 1:1 CD3OD/CDCl3): δ 7.07 (AA′,
6H, J ) 7.6 Hz), 8.09 (m, 3H), 7.83 (BB′, 6H, J ) 7.6 Hz), 7.98
(m, 2H), 8.40 (d, 1H, J ) 4.4 Hz), 8.44 (s, 2H), 8.47 (d, 1H, J
) 4.4 Hz), 9.42 (d, 1H, J ) 5.2 Hz), 9.51 (d, 3H, J ) 5.2 Hz).
13C NMR (75 MHz, 1:1 CD3OD/CDCl3): δ 114.33, 127.20,
127.84, 132.15, 133.29, 133.87, 134.07, 135.00, 135.43, 140.83,
147.23, 147.52, 147.66, 147.85, 155.89, 156.45, 157.20. High-
resolution Q-TOF MS: m/z 697.1624 (calcd for C44H28N2O3S2
+ H, 697.1619).
Gen er a l P r oced u r e for Dem eth yla tion of Cor e-Mod i-
fied P or p h yr in s 12-14, 24, a n d 27. P r ep a r a tion of 5,10,-
15,20-Tetr a-(4-h ydr oxyph en yl)-21,23-dith iapor ph yr in (16).
Compounds 8a 12b (1.75 g, 4.9 mmol) and 10 (2.25 g, 5.0 mmol)
were treated with TCBQ (2.25 g, 22 mmol) and TsOH (0.95 g,
5.0 mmol) as described. The porphyrin product displayed
minimal solubility. The crude product was washed with
acetone to give 0.74 g (20%) of 13 as a purple solid; mp 280-
282 °C. High-resolution Q-TOF MS: m/z 769.2180 (calcd for
Gen er a l P r oced u r e for th e P r ep a r a tion of Eth yl Ca r -
b oxyla t om et h oxy Cor e-Mod ified P or p h yr in s. P r ep a -
r a tion of Eth yl 5,10,15-Tr ip h en yl-20-(4-ca r boxyla tom eth -
oxy)p h en yl-21,23-d ith ia p or p h yr in (26). Core-modified por-
phyrin 25 (0.25 g, 0.38 mmol), 2.6 g of K2CO3, and 2.1 mL of
ethyl bromoacetate in 60 mL of acetone were heated at reflux
for 15 h. The reaction mixture was cooled to ambient temper-
ature, and the K2CO3 was removed by filtration. The filter cake
was washed with acetone until the filtrate was colorless. The
combined filtrates were concentrated. The crude product was
washed with MeOH to give 0.22 g (78%) of 26 as a purple solid;
C
48H36N2O4S2 + H, 769.2195). This particular porphyrin was
too insoluble for NMR studies and was characterized following
demethylation in the next step.
Core-modified porphyrin 13 (0.77 g, 1.0 mmol) was dissolved
in CH2Cl2 (100 mL), and BBr3 (0.49 mL, 5.1 mmol) was added
at 0 °C. The resulting solution was stirred for 5 h at ambient
temperature. The reaction mixture was added to 200 mL of
EtOAc and 200 mL of saturated NaHCO3. The organic layer
was separated and washed three times with brine, dried over
MgSO4, and concentrated. The crude solid was washed with
3/1 EtOAc/hexanes several times to give 0.60 g (84%) of 16 as
1
mp > 300 °C. H NMR (500 MHz, CDCl3): δ 1.40 (t, 3H, J )
7.0 Hz), 4.42 q, (2H, J ) 7.0 Hz), 4.92 s, (2H), 7.36 (AA′, 2H,
J ) 8.5 Hz), 7.81 (br s, 9H), 8.18 (BB′, 2H, J ) 8.5 Hz), 8.26
(m, 6H), 8.69 (m, 4H), 9.69 (m, 4H). 13C NMR (75 MHz,
CDCl3): δ 14.43, 61.73, 65.92, 113.94, 127.57, 128.18, 133.79,
134.32, 134.65, 134.89, 135.53, 141.41, 147.92, 148.02, 148.17,
156.54, 156.64, 156.79, 158.24, 169.13. High-resolution Q-TOF
MS: m/z 751.2065 (calcd for C48H34N2O3S2 + H, 751.2089).
1
a dark blue solid; mp > 300 °C. H NMR (400 MHz, DMSO-
d6): δ 7.26 (AA′, 8H, J ) 7.6 Hz), 7.95 (BB′, 8H, J ) 8.0 Hz),
8.47 (s, 4H), 9.65 (s, 4H). 13C NMR (75 MHz, DMSO-d6): δ
115.44, 131.48, 134.38, 134.57, 136.14, 147.47, 155.81, 158.23.
High-resolution Q-TOF MS: m/z 713.1542 (calcd for C44H28
N2O4S2 + H, 713.1569).
-
P r ep a r a tion of 5-(4-Hyd r oxyp h en yl)-10,15,20-tr ip h en -
yl-21,23-d ith ia p or p h yr in (25). Core-modified porphyrin 24
(0.35 g, 0.52 mmol) in 50 mL of CH2Cl2 was treated with BBr3
(0.49 mL, 5.1 mmol) as described for the preparation of 16 to
give 0.30 g (87%) of 25 as a purple solid; mp > 300 °C. 1H
NMR (400 MHz, 1:1 CD3OD/CDCl3): δ 7.11 (AA′, 2H, d, J )
8.4 Hz, 2 × HOCCH), 7.63 (m, 9H), 7.91 (BB′, 2H, J ) 8.4
Hz), 8.07 (m, 6H), 8.49 (d, 3H, J ) 4.4 Hz), 8.56 (d, 1H, J )
4.4 Hz), 9.52 (d, 3H, J ) 6.8 Hz), 9.61 (d, 1H, J ) 4.8 Hz). 13C
NMR (75 MHz, 1:1 CD3OD/CDCl3): δ 114.40, 127.28, 127.94,
132.24, 133.68, 133.85, 133.95, 134.10, 134.21, 134.33, 134.46,
134.70, 135.24, 135.36, 135.46, 135.64, 140.94, 148.34, 147.57,
P r ep a r a tion of Dieth yl 5,10-Dip h en yl-15,20-bis-(4-ca r -
boxylatom eth oxy)ph en yl-21,23-dith iapor ph yr in (18). Core-
modified porphyrin 15 (126 mg, 0.185 mmol), 1.5 g of K2CO3,
and 2 mL of ethyl bromoacetate in 50 mL of acetone were
treated as described for the preparation of 29 to give 94 mg
1
(60%) of 18 as a purple solid; mp 255-257 °C. H NMR (300
MHz, CDCl3): δ 9.70 (s, 2 H), 9.67 (s, 2 H), 8.68 (m, 4 H), 8.63
(d, 2 H, J ) 4.5 Hz), 8.22-8.27 (m, 4 H), 8.18 (AA′, 4 H, J )
8.4 Hz), 7.77-7.85 (m, 6 H), 7.37 (BB′, 4 H, J ) 8.4 Hz), 4.93
(s, 4 H), 4.42 (q, 4 H, J ) 7.2 Hz), 1.42 (t, 6 H, J ) 7.2 Hz). 13
C
NMR (75 MHz, CDCl3): δ 168.99, 158.08, 156.57, 156.42,
148.03, 147.76, 141.25, 135.38, 134.74, 134.48, 134.16, 133.93,

3744 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You et al.
133.57, 128.01, 127.41, 113.78, 65.77, 61.59, 14.29. High-
resolution Q-TOF MS: m/z 751.2084 (calcd for C52H40N2O6S2
+ H, 751.2089).
resolution Q-TOF MS: m/z 871.1750 (calcd for C50H34N2O9S2
+ H, 871.1784). Anal. C, H, N.
P r ep a r a t ion of 5,10,15,20-Tet r a -(4-ca r boxyla t om et h -
oxy)p h en yl-21,23-d ith ia p or p h yr in (4). Core-modified por-
phyrin 19 (0.18 g, 0.17 mmol) in 20 mL of THF was treated
with 2 mL (2 mmol) of 1 M NaOH as described to give 0.11 g
P r ep a r a tion of Tr ieth yl 5-P h en yl-10,15,20-Tr is-(4-ca r -
boxylatom eth oxy)ph en yl-21,23-dith iapor ph yr in (29). Core-
modified porphyrin 28 (0.30 g, 0.43 mmol) was treated with
2.0 mL of ethyl bromoacetate as described to give 0.38 g (92%)
1
(68%) of core-modified porphyrin 4; mp 212-214 °C. H NMR
1
(500 MHz, 1:1 CD3OD/CDCl3): δ 4.92 (s, 8 H), 7.40 (AA′, 8 H,
J ) 8.0 Hz), 8.14 (BB′, 8 H, J ) 8.0 Hz), 8.64 (s, 4 H), 9.70 (s,
4 H). 13C NMR (126 MHz, 1:1 CD3OD/CDCl3): δ 64.15, 67.93,
112.82, 126.60, 132.62, 133.19, 133.24, 134.11, 134.31, 147.19,
148.24, 155.57, 157.70, 167.07, 168.82. High-resolution Q-TOF
MS: m/z 945.1807 (calcd for C52H36N2O12S2 + H, 945.1788).
Anal. C, H, N.
of 29 as a purple solid; mp 182-184 °C. H NMR (500 MHz,
CDCl3): δ 1.43 (d, 9H, J ) 7.0 H), 4.43 (q, 6H, J ) 7.0 Hz),
4.92 (s, 6H), 7.36 (d, 6H, J ) 8.0 Hz), 7.82 (m, 3H), 8.18 (d,
6H, J ) 8.0 Hz), 8.25 (m, 2H), 8.69 (m, 4H), 9.69 (m, 4H). 13C
NMR (75 MHz, CDCl3): δ 14.40, 61.69, 65.84, 113.90, 127.54,
128.14, 133.61, 133.65, 133.71, 134.04, 134.29, 134.61, 134.84,
135.49, 141.37, 147.92, 148.07, 148.18, 156.51, 156.66, 156.76,
158.17, 169.11. High-resolution Q-TOF MS: m/z 955.2726
(calcd for C56H46N2O9S2 + H, 955.2723).
P r epa r a tion of 5,10-Diph en yl-15,20-Bis-(4-ca r boxyla to-
m eth oxy)p h en yl-21,23-d iselen a p or p h yr in (5). Core-modi-
fied porphyrin 20 (0.28 g, 0.30 mmol) in 30 mL of THF was
treated with 3 mL (3 mmol) of 1.0 M aqueous NaOH as
described to give 0.25 g (95%) of porphyrin 5; mp 255-257
P r epar ation of Tetr aeth yl 5,10,15,20-Tetr a-(4-car boxyl-
a t om et h oxy)p h en yl-21,23-d it h ia p or p h yr in (19). Core-
modified porphyrin 16 (0.35 g, 0.49 mmol) was treated with 2
mL of ethyl bromoacetate as described to give 0.30 g (58%) of
19 as a purple solid; mp 146-148 °C. 1H NMR (500 MHz,
CDCl3): δ 1.43 (t, 12 H, J ) 7.5 Hz), 4.43 (q, 8 H, J ) 7.5 Hz),
4.92 (s, 8 H), 7.36 (AA′, 8 H, J ) 8.5 Hz), 8.18 (BB′, 8 H, J )
8.5 Hz) 8.69 (s, 4 H), 9.69 (s, 4 H). 13C NMR (75 MHz, CDCl3):
δ 14.43, 61.85, 65.95, 113.99, 133.69, 134.70, 134.95, 135.58,
135.64, 148.17, 156.78, 158.25, 169.24. High-resolution Q-TOF
MS: m/z 1057.3035 (calcd for C60H52N2O12S2 + H, 1057.3040).
1
°C. H NMR (500 MHz, 1:1 CDCl3/DMSO-d6): δ 4.90 (s, 4H),
7.40 (AA′, 4H, J ) 8 Hz), 7.81 (m, 6H), 8.19 (BB′, 4H, J ) 8
Hz), 8.24 (m, 4H), 8.79 (s, 4H), 9.86 (s, 2H), 9.88 (s, 2H). 13C
NMR (75 MHz, 1:1 CDCl3/DMSO-d6): δ 65.40, 113.94, 127.56,
128.06, 134.35, 134.48, 135.68, 137.03, 137.15, 137.36, 140.90,
149.59, 150.05, 156.73, 156.73, 156.78, 158.18, 171.19. High-
resolution Q-TOF MS: m/z 893.0654 (calcd for C48H32N2-
O680Se2 + H, 893.0676). Anal. C, H, N.
Qu a n tu m Yield Deter m in a tion s for th e Gen er a tion of
Sin glet Oxygen . The quantum yields for singlet oxygen
generation with 21,23-dithiaproprphyrin 1-4 were measured
by direct methods in MeOH.16 A SPEX 270M spectrometer
(J obin Yvon) equipped with InGaAs photodetector (Electroop-
tical Systems Inc., U.S.A.) was used for recording singlet
oxygen emission spectra. A diode-pumped solid state laser
(Millenia X, Spectra-Physics) at 532 nm was the excitation
source. The sample solution in a quartz cuvette was placed
directly in front of the entrance slit of the spectrometer, and
the emission signal was collected at 90° relative to the exciting
laser beam. An additional long-pass filter (850LP) was used
to attenuate the excitation laser and the fluorescence from the
photosensitizer.
Deter m in a tion of P a r tition Coefficien ts. The octanol/
water partition coefficients were all measured at pH 7.4 (PBS)
using UV-visible spectrophotometry. The measurements were
done using a shake flask direct measurement.18 Mixing for 3-5
min was followed by 1 h of settling time. Equilibration and
measurements were made at 23 °C using a Perkin-Elmer
Lambda 12 spectrophotometer. High-performance liquid chro-
matography grade 1-octanol was obtained from Sigma-Aldrich.
Cells a n d Cu ltu r e Con d ition s. R3230AC Cells. Cells
cultured from the rodent mammary adenocarcinoma (R3230AC)
were used for these studies. The R3230AC tumors were
maintained by transplantation in the abdominal region of
100-120 g Fischer female rats, using the sterile trochar
technique described by Hilf et al.29 R3230AC cells were
cultured from tumor homogenates using the method described
earlier.30 All cell lines were maintained in passage culture on
100 mm diameter polystyrene dishes (Becton Dickinson,
Franklin Lakes, NJ ) in 10 mL of minimum essential medium
(R-MEM) supplemented with 10% FBS, 50 units/mL penicillin
G, 50 ug/mL streptomycin, and 1.0 ug/mL Fungizone growth
media. Only cells from passages 1-10 were used for experi-
ments. Cells, passages 1-4, that were maintained at -86 °C
were used to initiate the experimental cultures. Cultures were
maintained at 37 °C in a 5% CO2 humidified atmosphere
(Forma Scientific, Marietta, OH). Passage was accomplished
by removing the culture medium, and then adding a 1 mL
solution containing 0.25% trypsin, incubating at 37 °C for 2-5
min to remove the cells from the surface followed by seeding
new culture dishes with an appropriate number of cells in 10
mL of R-MEM. Cell counts were performed using a particle
counter (model ZM, Coulter Electronics, Hialeah, FL).
Gen er a l P r oced u r e for Sa p on ifica tion of Eth yl Ester s
of Cor e-Mod ified P or p h yr in s. P r ep a r a tion of 5,10,15-Tr i-
p h en yl-20-(4-ca r b oxyla t om et h oxy)p h en yl-21,23-d it h ia -
p or p h yr in (1). Core-modified porphyrin 26 (0.18 g, 0.24
mmol) was dissolved in 50 mL of THF, and 1 M NaOH (20
mL, 20 mmol) was added. The resulting solution was stirred
at ambient temperature for 15 h. The solution was acidified
by the addition of 8 mL of acetic acid. The reaction mixture
was diluted with 100 mL of H2O, and the products were
extracted with EtOAc (3 × 100 mL). The combined organic
extracts were dried over MgSO4 and concentrated. The crude
product was washed with several portions of hexanes/MeOH
to give 0.16 g (92%) of 1 as a purple solid; mp 265-267 °C. 1H
NMR (500 MHz, CDCl3): δ 5.00 (s, 2H), 7.40 (AA′, 2H, J )
8.0 Hz), 7.81 (m, 9H), 8.20 (BB′, 2H, J ) 8.0 Hz), 8.24 (m, 6H),
8.68 (s, 4H), 9.68 (d, 4H, J ) 5.0 Hz). 13C NMR (75 MHz,
CDCl3): δ 65.35, 114.00, 127.60, 128.23, 133.56, 134.20, 134.24,
134.36, 134.52, 134.63, 134.71, 135.21, 135.55, 135.64, 135.68,
141.31, 147.93, 147.97, 148.14, 156.37, 156.52, 156.56, 156.60,
157.83, 172.71. High-resolution Q-TOF MS: m/z 723.1761
(calcd for C46H30N2O3S2 + H, 723.1776). Anal. C, H, N.
P r ep a r a t ion of 5,10-Dip h en yl-15,20-Bis-(4-ca r b oxyl-
a tom eth oxy)p h en yl-21,23-d ith ia p or p h yr in (2). Core-modi-
fied porphyrin 18 (55.3 mg, 0.0694 mmol) in 30 mL of THF
was treated with 1.46 mL (0.146 mmol) of 0.10 M aqueous
NaOH as described to give 52 mg (91%) of core-modified
porphyrin 2; mp > 300 °C. 1H NMR (300 MHz, CD3OD): δ
9.72 (s, 2 H), 9.60 (s, 2 H), 8.61 (d, 2 H, J ) 4.5 Hz), 8.53 (d,
2 H, J ) 4.5 Hz), 8.10-8.17 (m, 4 H), 8.06 (d, 4 H, J ) 8.1
Hz), 7.74-7.80 (m, 6 H), 7.37 (d, 4 H, J ) 8.1 Hz), 4.62 (s, 4
H). 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-P h en yl-10,15,20-Tr is-(4-ca r boxyla to-
m eth oxy)p h en yl-21,23-d ith ia p or p h yr in (3). Core-modified
porphyrin 29 (0.30 g, 0.31 mmol) in 30 mL of THF was treated
with 3 mL (3 mmol) of 1.0 M aqueous NaOH as described to
give 0.25 g (91%) of core-modified porphyrin 3 as a purple solid;
mp 210-212 °C. 1H NMR (400 MHz, 1:1 CD3OD/CDCl3): δ
4.96 (s, 6H), 7.44 (AA′, 4H, J ) 7.6 Hz), 7.42 (AA′, 2 H, J )
7.6 Hz), 7.85 (m, 3H), 8.21 (BB′, 6H, J ) 7.6 Hz), 8.23 (BB′, 2
H, J ) 7.6 Hz)), 8.26 (m, 2 H), 8.71 (m, 4H), 9.74 (m, 4H). 13C
NMR (75 MHz, 1:1 CD3OD/CDCl3): δ 65.21, 114.37, 128.28,
133.39, 134.15, 134.31, 135.10, 135.70, 136.31, 140.58, 147.25,
147.45, 147.60, 155.92, 156.14, 156.28, 158.42, 170.85. High-
In cu ba tion w ith Cor e-Mod ified P or p h yr in s 1-5. For
experiments designed to determine cell viability in the pres-

Water Soluble, Core-Modified Porphyrins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3745
ence of individual core-modified porphyrins (1-5) in the dark
and after light exposure, R3230AC cells were removed from
the 100 mm dishes via trypsinization, see above, and seeded
on 12 well plates at 0.5-0.8 × 105 cells/well in 1.0 mL of
growth media. Cultures were incubated for 24 h, as above,
after which the appropriate concentrations of 1-5 were added
directly to the wells in the growth medium. Cells were then
incubated with the core-modified porphyrins for selected times.
For experiments designed to determine the amount of
intracellular porphyrin after 3 or 24 h incubation periods in
the presence of core-modified porphyrins, cells were seeded on
12 well plates as above, and 24 h after seeding, compounds
1-5 were added at the appropriate concentrations in the
growth. Cells were incubated at 37 °C in the dark for either 3
or 24 h when the intracellular porphyrin content was deter-
mined.
the evening before the assay. The day of the assay, the cells
were washed twice with PBS and 100 µL of Hank’s basic salt
solution (HBSS) with 1% FCS containing various concentra-
tions of dithiaporphyrins 2 and 30, HPPH, and Photofrin was
added to each well. The dye and cells were incubated for 2 h
at 37 °C followed by a wash with PBS and the addition of 100
µL of PBS supplemented with 1% FCS. The plates were
irradiated with filtered 590-800 nm light at 14 mW cm-2 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. Control cells (no dye + light; dye + no
light) were treated using the same conditions. Cell survival
was monitored using the MTT assay as described by Mos-
mann.31
P h otoblea ch in g Con d ition s. Studies of the rates of
photobleaching of core-modified porphyrins 1-5 were carried
out in PBS (0.05 M, pH 7.4). Stock solutions of 1-5 and
Photofrin (1 × 10-3 M) in clear medium were diluted in PBS.
Concentrations (≈5 × 10-6 M) were adjusted such that the
initial absorbance of the soret band was ≈1.0 in a 1 cm cell.
Initial absorption was measured using a diode array spectro-
photometer (HP8452, Hewlett-Packard, Palo Alto, CA). Ir-
radiation of 1-5 or Photofrin was accomplished by exposing
1.0 mL of 1-5 or Photofrin to 200 mW/cm2 broadband 350-
750 nm white light emitted from a focused and filtered 750 W
tungsten source. Absorption spectra were acquired at selected
intervals, and rates of photobleaching were determined by the
reduction in the absorption maximum of each compound over
time of irradiation.
Mea su r em en t of P or p h yr in Con ten t in Cu ltu r ed Cells.
Determination of the levels of core-modified porphyrins in
cultured cells was performed in 12 well plates using fluores-
cence. Following the incubation periods in the presence of 1-5,
the growth medium was removed, and the cell monolayers
were washed once with 1.0 mL of 0.9% saline. Cells in selected
wells were detached using a 0.25% trypsin solution, and the
number of cells per well was determined using a Coulter
counter as described above. The saline wash was removed, and
1.0 mL of Scintigest was added. Porphyrin fluorescence was
determined using a multiwell fluorescence plate reader (Mo-
lecular Devices, Sunnyvale, CA). Excitation was at 440 nm,
and the major fluorescent peak at 725 nm was used to
calculate the porphyrin concentration, which was obtained by
comparing the fluorescence values (or absorbance values for
5) with measurements from known concentrations of 1-5 in
Scintigest. Data are expressed as femtomoles of core-modified
porphyrin/cell.
Ir r a d ia tion of Cu ltu r ed R3230AC Cells. Following in-
cubation for 3 or 24 h with porphyrins on 12 well plates, the
growth medium containing the porphyrins was removed from
cell monolayers, cells were washed once with 1.0 mL of 0.9%
NaCl and 1.0 mL of medium minus FBS, and phenol red (clear
medium) was added. Plates, with lids removed, were positioned
on a orbital shaker (LabLine, Melrose Park, IL) and then
exposed for various times to broadband light (350-750 nm)
delivered at 1.4 mW cm-2 from a filtered 750 W tungsten
source defocused to encompass the whole 12 well plate. The
culture plates were gently orbited on the shaker in order to
ensure uniform illumination of all of the wells on the plate.
The medium was them removed, and 1.0 mL of fresh growth
medium was added and cultures were incubated at 37 °C for
24 h in the dark. Plates were also maintained in the dark and
underwent the same changes in cell culture medium as those
that were irradiated. These were used to determine the dark
toxicity of 1-5 at 24 and 48 h after addition of 1-5 to the
monolayers. Cell counts, using the Coulter counter as above,
were made on control nonirradiated cells and on irradiated
cells or cells maintained in the dark. Previously, when we used
trypan blue dye exclusion to determine cell viability, we found
that all of the cells that had detached from the surface and
were floating in the medium stained with trypan blue, i.e.,
nonviable, while those cells that remained attached to the
surface excluded trypan blue. Culturing of the cells that
detached was unsuccessful while passing those cells that
remained attached resulted in a plating efficiency similar to
that of untreated cells. Therefore, we counted the cells that
remained attached to the plate after treatment and used those
numbers to determine viability. Data for cell viability are
expressed as the percent of control cell counts, cells that were
not exposed to either porphyrins or irradiation.
CLSM Stu d ies. R3230AC cells were grown in a tissue
culture flask containing R-MEM medium with phenol red
(GIBCO, Rockville, MD) and 10% FBS (GIBCO). Cells were
trypsinized from the culture flask and were resuspended in
medium at a concentration of 7.5 × 105 cells/mL. Then, in 60
mm culture plates, 5 mL of R-MEM medium was combined
with 0.10 mL of cell suspension. The plates were then placed
overnight in an incubator at 37 °C with 5% CO2 (VWR
Scientific model 2400, Bridgeport, NJ ). The next day, the cells
(with about 70% confluence) were rinsed with PBS (GIBCO),
and 5 mL of R-MEM medium (without phenol red) containing
1 µM Rh-123 or 2 µM 1 or 2 was replaced in each plate. The
plates were returned to the incubator (37 °C, 5% CO2) for a
duration of 3 h for 1 and 2 and 20 min for Rh-123. After
incubation, the plates were rinsed with sterile PBS, and fresh
media (without phenol red) was replaced at a volume of 5 mL/
plate. The plates were then observed directly under the
confocal microscope. For costaining experiments, cells were
treated as described with 1 or 2 for 3 h, were rinsed once with
sterile PBS, and were then treated with 1 µM Rh-123 for 20
min as described.
CLSM images were obtained using a commercial CLSM
system, model MRC-1024 (Bio-Rad, Richmond, CA), which was
attached onto an upright microscope (Nikon, model Eclipse
E800). A water immersion objective lens (Nikon, Fluor-60X,
NA ) 1.0) was used for cell imaging. A solid state diode-
pumped laser (Verdi, Coherent) was used as a source of
excitation (532 nm). An additional long-pass filter (585LP) was
introduced in front of the photomultiplier to minimize leakage
of the excitation light. To confirm that the observed fluores-
cence was from photosensitizer permeated to cells, we used
localized spectrofluorometry. The fluorescence signal was
collected from the upper port of the confocal microscope using
a multimode optical fiber of core diameter 1 mm and was
delivered to a spectrometer (Holospec from Kaiser Optical
Systems, Inc.) equipped with a cooled charge coupled device
camera (Princeton Instruments) as detector. A comparison of
the fluorescence spectra from cells and the fluorescence spectra
of the photosensitizers confirmed that the origin of the
fluorescence observed in the image channel was from the
photosensitizer. Control cells showed no fluorescence under
the same imaging conditions in the absence of photosensitizer.
A 640 nm short-pass dichroic mirror (Chroma) mounted at a
45° angle to the fluorescence beam was employed in costaining
experiments.
Cells a n d Cu ltu r e Con d ition s. Colo-26 Cells. Colo-26,
a murine colon carcinoma cell line, was maintained in a growth
medium of RPMI 1640 supplemented with 10% fetal calf serum
(FCS) and antibiotics (all components purchased from GIBCO
Laboratories) at 37 °C, 5% CO2.
Ir r a d ia tion of Cu ltu r ed Colo-26Cells. Colo-26 cells were
plated at 5 × 103 cells/well of a 96 well tissue culture plate

3746 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
You et al.
Clearance to the In Vivo Quantitative Structure-Activity Re-
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Photochem. Photobiol. 1999, 70, 781-788. (b) Sessler, J . L.;
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Cytoch r om e c Oxid a se Mea su r em en ts in Cu ltu r ed
R3230AC Tu m or Cells. To determine whether core-modified
porphyrin 2 affected mitochondrial cytochrome c oxidase
activity, R3230AC cells were plated on 12 well culture plates
as described until they reached 4-7 × 105 cells/well, a number
when the cells were still in log phase growth. The complete
medium was removed, and fresh clear medium with core-
modified porphyrin 2 at 1 × 10-8, 1 × 10-7, and 1 × 10-6 final
concentration or without photosensitizer for control cells was
added. Cells were then incubated for 24 h in the dark at 37
°C with 5% CO2, the medium was removed, and the cells were
washed once with 0.9% NaCl, and 1.0 mL of clear medium
was added. Monolayers were then exposed to filtered, 350-
750 nm light from a 750 W tungsten source (1.3 mW cm-2) for
1 h as described above (4.7 J cm-2). Immediately following
irradiation, the medium was removed from the substrate and
replaced with complete medium. Both irradiated cells and dye-
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the activity of cytochrome c oxidase was determined according
to the method described earlier.28 Data are expressed as ∆m
OD/min/105 cells at 550 nm and compared to that determined
from cells not exposed to dye or light. Cytochrome c oxidase
activity was also determined in cells exposed to dye alone or
light alone, drug and light controls, respectively.
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.
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Ack n ow led gm en t. We thank Dr. Haridas Pudavar
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confocal microscopy images. We also thank the National
Institutes of Health (Grant CA69155 to M.R.D. and
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Su p p or tin g In for m a tion Ava ila ble: Figures S1-S5
showing absorption spectra for compounds 1-5, Figure S6
showing singlet oxygen luminescence following irradiation of
1-4 and 30, Figure S7 showing absorbance at 532 nm for 1-5
and 30, Figure S8 showing singlet oxygen luminescence
following irradiation of 5, Figures S9-S13 showing dark
toxicity for 1-5, and Figure S14 showing localized spectro-
fluorometry from R3230AC cells treated with dithiaporphyrin
1. This information is available free of charge via the Internet
at http://pubs.acs.org.
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