Core-modified porphyrins. Part 5: Electronic effects on photophysical and biological properties in vitro

Article abstract of DOI:10.1016/j.bmc.2005.07.006

21,23-Dithiaporphyrins 2-11 were prepared as analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin 1 to examine the impact of electronic properties at the 5- and 20-meso-positions. The effects of the electronic properties

Full text of DOI:10.1016/j.bmc.2005.07.006

Bioorganic & Medicinal Chemistry 13 (2005) 5968–5980
Core-modified porphyrins. Part 5: Electronic effects
on photophysical and biological properties in vitro
Youngjae You,a,* Scott L. Gibsonb and Michael R. Dettya
aInstitute for Lasers, Photonics, and Biophotonics, Department of Chemistry, University at Buffalo,
The State University of New York, Buffalo, NY 14260, USA
bDepartment of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue,
Box 607, Rochester, NY 14642, USA
Received 27 June 2005; accepted 7 July 2005
Available online 9 August 2005
Abstract—21,23-Dithiaporphyrins 2–11 were prepared as analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-
21,23-dithiaporphyrin 1 to examine the impact of electronic properties at the 5- and 20-meso-positions. The effects of the electronic
properties at the meso-rings were not significant with respect to absorption spectra, quantum yields for the generation of singlet
oxygen and for fluorescence. While some differences were noted in the n-octanol/pH 7.4 buffer partition coefficient, log D7.4, among
the compounds, log D7.4 did not critically influence the cellular uptake or phototoxicity. None of the dithiaporphyrins 1–11 dis-
played dark toxicity at concentrations up to 1 · 10À5 M. Once irradiated with 5 J cmÀ2 of 350–750 nm light, five porphyrins 2, 3,
5, 6, and 8 killed over 80% of R3230AC rat mammary adenocarcinoma cells at 5 · 10À7 M photosensitizer. Among these five, com-
pound 3 bearing 5-phenyl and 20-(4-fluorophenyl) substituents was the most potent photosensitizer toward R3230AC cells showing
67% cell kill at 1 · 10À7 M 3. Bulky substituents at the 5- and 20-positions gave photosensitizers with minimal phototoxicity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Dithiaporphyrins have been extensively studied in our
laboratory as second-generation photosensitizers due
to several advantages. The various dithiaporphyrins
can be prepared in high purity through established syn-
thetic schemes, can absorb longer wavelengths of light
(690–710 nm) than natural tetra nitrogenic porphyrins
(ꢀ630 nm), and are more photostable than Photofrin
upon irradiation.12 Dithiaporphyrins with two carboxyl-
ic acids exhibited a higher phototoxic activity toward
R3230AC rat mammary adenocarcinoma cells than
derivatives with one, three, or four acid groups.13 Bulky
substituents at the meso-positions reduced phototoxici-
ty, while effects on physicochemical properties, such as
absorption spectra and quantum yields for the genera-
tion of singlet oxygen and fluorescence, were minimal.14
Of the dithiaporphyrins prepared in our previous
study, 5-phenyl-20-(2-thienyl)-10,15-bis-(4-carboxylato-
methoxyphenyl)-21,23-dithiaporphyrin (IY69) showed
the most potent phototoxic activity and irradiation of
cells treated with IY69 induced apoptotic cell death
along with the damage to mitochondrial function.14
Photodynamic therapy (PDT) is a promising new treat-
ment regimen for cancer that uses three components to
destroy the tumor: a photosensitizer, light, and oxy-
gen.1–9 The photosensitizer generates cytotoxic singlet
oxygen following absorption of a photon of appropriate
wavelength. Photofrin has gained regulatory approval in
many countries but it has several drawbacks, such as
long-term skin phototoxicity, chemical complexity, and
weak absorption at shorter wavelengths (ꢀ630 nm).10
Therefore, the development of more ‘‘ideal’’ photosensi-
tizers is a major emphasis in research on photodynamic
therapy, which should be chemically pure, generate
singlet oxygen efficiently, accumulate selectively in the
tumor, and observe longer wavelength light (650–
800 nm). Currently, several new photosensitizers are
approved or are under clinical study.11
Keywords: Core-modified porphyrin; Dithiaporphyrin; Photodynamic
therapy; Anticancer therapy; QSAR.
The phototoxicity of IY69 prompted us to develop a
more extensive study of substituent effects in the dithia-
porphyrin diacids. The structures of compounds in this
*
Corresponding author. Tel.: +1 716 645 6800x2197; fax: +1 716 645
6963; e-mail: yjyou@nsm.buffalo.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.006

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5969
work were designed to investigate the electronic effects
of substituents at the para-positions of meso-phenyl
rings on physicochemical and biological outcomes. Cel-
lular uptake and photo- and dark toxicity of the various
dithiaporphyrins were evaluated along with the impor-
tant physicochemical properties for a photosensitizer,
such as absorption spectra, quantum yields for the gen-
eration of singlet oxygen and for fluorescence, and the
oxyphenyl)-1-pyrrolomethyl]-thiophene 22 and oxidized
in the presence of 2,3,5,6-tetrachlorobenzoquinone
(TCBQ) and p-toluenesulfonic acid monohydrate
(TsOHÆH2O) or boron trifluoride etherate (BF3ÆOEt2)
in CH2Cl2 giving dimethoxy dithiaporphyrins 24–28 in
3–18% isolated yields (Scheme 2). Demethylation with
boron tribromide (BBr3) in CH2Cl2 gave diphenolic
dithiaporphyrins 31–35 (83–95%), which were then
alkylated in the presence of K2CO3 and ethyl bromoac-
etate in acetone to produce ester porphyrins 36–40. Fi-
nal diacid dithiaporphyrins 4, 5, 8, 10, and 11 were
prepared by hydrolysis of the esters with NaOH in a 1:1
solution of distilled water and tetrahydrofuran (THF).
n-octanol/water partition coefficient at pH 7.4, log D7.4
.
2. Results and discussion
2.1. Chemistry
In the preparation of di-4-methoxyphenyl- and di-
4-hydroxyphenyldithiaporphyrin 7 and 9, the upper
scheme could not be applied due to possible problems
in either the demethylation of 7 or the alkylation of 9.
Consequently, slightly modified routes were devised
(Scheme 2). To avoid any difficulty in selective demethyl-
ation of a tetramethoxy porphyrin in the preparation of
dimethoxy porphyrin 7, ester porphyrin 29 was prepared
directly by cyclization of 2,5-bis[1-(4-methoxyphenyl)-1-
2.1.1. Synthesis of symmetrical 21,23-dithiaporphyrins 4,
5, and 7–11. The synthesis of symmetric core-modified
porphyrins 4, 5, 8, 10, and 11 (Fig. 1) followed our pre-
vious method.12–14 As shown in Scheme 1, 2,5-bis[1-(4-
substituted-phenyl)-1-hydroxymethyl]thiophene
diols
12, 13, and 15–17 were synthesized from thiophene
and the corresponding aldehydes in 70–75% isolated
yields. The diols were condensed with 2,5-bis[1-(4-meth-
F
O
F
O
S
S
2
7
F
N
N
S
S
S
N
N
3
8
S
Cl
Cl
HO
OH
OH
HO
O
O
S
S
O
O
4
9
1
F C
CF
3
3
S
S
10
5
F C
3
S
S
11
6
Figure 1. Structures of 21,23-dithiaporphyrins with various group substitutions at the 5,20-meso-positions.
c
b
d
Ar
HO
Ph
OH
Ph
OH
Ph
S
S
S
S
S
OTBS
42
43
44, Ar = 4-FC6H4
45, Ar = 4-CF3C6H4
a
12: Ar = 4-ClC6H4
13: Ar = 4-CF3C6 H4
14: Ar = 4-OCH3C6H4
15: Ar = 4-N(CH3)2C6H4
16: Ar = 4-CH(CH3)2C6H4
17: Ar = 4-C6H5C6H4
18: Ar = 4-TIBSOC6H4
19: Ar = 4-DMIPSOC6H4
Ar
HO
Ar
OH
20: Ar = 4-CH3CH2OCOCH2OC6H4
Scheme 1. Reagents: (a) i. 2.5 equiv n-BuLi, ii. 2 equiv aryl aldehyde; (b) i. 1 equiv BuLi, ii. 1 equiv benzaldehyde; (c) TBSCl, DMAP, Et3N;
(d) i. 1 equiv n-BuLi, ii. 1 equiv aryl aldehyde, iii. aqueous HCl.

5970
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
Ar
Ar
Ar
HO
Ar
OH
a
S
S
NH
HN
21, Ar = 4-MeOC6H4
20, Ar = 4-CH3CH2OCOCH2OC6H4
22, Ar = 4-MeOC6H4
23, Ar = 4-CH3CH2OCOCH2OC6H4
31: Ar = 4-ClC6H4, R = H
32: Ar = 4-CF3C6 H4, R = H
33: Ar = 4-N(CH3)2C6H4, R = H
34: Ar = 4-CH(CH3)2C6H4, R = H
35: Ar = 4-C6H5C6H4, R = H
Ar
Ar
S
S
b
c
N
12-17 + 22
N
d
OR
RO
36: Ar = 4-ClC6H4, R = CH2CO2Et
37: Ar = 4-CF3C6H4 , R = CH2CO2Et
38: Ar = 4-N(CH3)2C6H4, R = CH2CO2Et
39: Ar = 4-CH(CH3)2C6H4, R = CH2CO2Et
40: Ar = 4-C6H5C6H4, R = CH2CO2Et
e
24: Ar = 4-ClC6H4, R = Me
25: Ar = 4-CF3C6 H4, R = Me
26: Ar = 4-N(CH3)2C6H4, R = Me
27: Ar = 4-CH(CH3)2C6H4, R = Me
28: Ar = 4-C6H5C6H4, R = Me
4,5
8,10,11
e
29: Ar = 4-OCH3C6H4, R = CH2CO2Et
41: Ar = 4-OHC6H4, R = CH2CO2Et
f
b
7,9
14,18 + 23
30: Ar = 4-TIBSOC6H4, R = CH2CO2Et
Scheme 2. Reagents: (a) pyrrole, BF3ÆOEt2; (b) TCBQ, TsOH, CH2Cl2; (c) BBr3, CH2Cl2; (d) BrCH2CO2Et, K2CO3, acetone; (e) NaOH, aqueous
THF; (f) TBAF.
Ph
Ar
2.2. Photophysical properties
S
S
c
48, Ar = 4-FC6H4, R = H
49, Ar = 4-CF3C6H4, R = H
b
44 or 45
+ 22
N
N
2.2.1. Absorption maxima. The absorption maximum
and molar extinction coefficient of band I of porphyrins
are important parameters in evaluating potential
photosensitizers for PDT. Longer wavelength light
(650–800 nm) reaches deeper into tissue to activate
photosensitizers.15 Photosensitizers with higher extinc-
tion coefficients harvest the irradiating light more effi-
ciently. So, photosensitizers having longer-wavelength
absorption maxima (ꢀ800 nm) and higher extinction
coefficient are highly desirable. Although most 4-aryl
substituents at the meso-positions have minimal impact
on the absorption maximum of band I, introduction of
fluoro 2 or dimethylamino 8 groups results in a signifi-
d
50, Ar = 4-FC6H4, R = CH2CO2Et
51, Ar = 4-CF3C6H4, R = CH2CO2Et
e
OR
RO
46, Ar = 4-FC6H4, R = Me
47, Ar = 4-CF3C6H4, R = Me
3 or 6
Scheme 3. Reagents: (a) pyrrole, BF3ÆOEt2; (b) TCBQ, TsOH,
CH2Cl2; (c) BBr3, CH2Cl2; (d) BrCH2CO2Et, K2CO3, acetone; (e)
NaOH, aqueous THF.
hydroxymethyl]thiophene 14 and diethyl 2,5-bis[1-(4-
carboxylatomethoxyphenyl)-1-pyrrolomethyl]thiophene
23. For the preparation of 9, protection of the phenolic
functionality was necessary. Protected diol, 2,5-bis[1-(4-
triisobutylsilyoxy)phenyl-1-hydroxymethyl]thiophene
18, was synthesized and condensed with 23 to give
hydroxyl-protected ester 30 in 32% isolated yield. Depro-
tection of 30 with tetrabutylammonium fluoride (TBAF)
in THF provided ester 41.
cant redshift from the diphenyl derivative 1 (Dkmax
+19 and +16 nm, respectively, Table 1). Also notewor-
thy is the extinction coefficient of band I for compound
=
Table 1. UV–vis–near-IR band maxima and molar absorptivities for
dithiaporphyrins 1–11 in methanola
Compound Soret
Band IV Band III Band II Band I
2.1.2. Synthesis of unsymmetrical dicarboxylic acid
derivatives 3 and 6. Reported methods to prepare
unsymmetrical core-modified porphyrins were used in
the preparation of dithiaporphyrins 3 and 6 with 4-flu-
orophenyl and 4-trifluoromethylphenyl substituents,
respectively (Scheme 3). Unsymmetric diols 44 and 45
were prepared from 2-lithiothiophene via the tert-
butylsilyl (TBS)-protected monohydroxythiophene 43.
Cyclization of 44 and 45 with 22 gave dimethoxydithia-
porphyrins 46 and 47 in 5% and 7% isolated yields,
respectively. Diacid porphyrins 3 and 6 were successfully
synthesized through demethylation, alkylation, and
hydrolysis, under the same conditions as those used
for the symmetrical analogues.
1
435 (314) 513 (27.7) 549 (10.7) 632 (3.2) 698 (7.0)
442 (127) 533 (14.0) 573 (10.8) 645 (2.5) 717 (4.8)
435 (407) 514 (26.9) 548 (9.9) 634 (1.8) 698 (5.3)
436 (296) 515 (21.4) 550 (8.8) 634 (1.7) 699 (4.6)
435 (297) 514 (24.3) 548 (9.2) 633 (1.8) 697 (4.6)
435 (327) 514 (25.4) 549 (11.3) 634 (2.1) 698 (5.6)
440 (176) 517 (14.6) 554 (10.3) 638 (1.2) 704 (4.1)
2b
3
4
5
6
7c
8c
9c
449 (100) 522 (17.7) 573 (19.4) —
714 (8.9)
440 (263) 518 (21.5) 555 (12.3) 639 (1.6) 704 (5.9)
436 (306) 515 (21.6) 551 (9.6) 635 (1.6) 700 (5.5)
439 (97.6) 518 (13.7) 553 (7.5) 637 (1.1) 702 (3.8)
10
11c
a kmax nm (e · 103 MÀ1 cmÀ1).
b Di-sodium salts and data from Ref. 12.
c In THF.

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5971
8 (8.9 · 103 MÀ1 cmÀ1), which is the highest among the
dithiaporphyrin derivatives.
Quantum yields of fluorescence (/F) were determined
for the compounds 1–11 compared to rhodamine 6G
(/F = 1.0). Interesting enough is the increase of /F of
compound 8 (0.029), which is theoretically consistent
with its low value of /(1O2), 0.32 (Table 2). In contrast,
compound 9 displayed low quantum yields for both the
generation of singlet oxygen [/(1O2) = 0.03] and for
fluorescence (/F = 0.002). All the other compounds have
poor fluorescence with values of /F < 0.012.
2.2.2. Quantum yields for the generation of singlet
oxygen. Singlet oxygen (1O2) is thought to be the toxic
species that damages tumor cells following irradiation
of the photosensitizer.16 Higher quantum yields for the
generation of singlet oxygen [/(1O2)] endow photosensi-
tizers with greater potential for biological activity, that
is, phototoxicity. Values of /(1O2) for 1–11 were deter-
mined by direct detection of singlet-oxygen lumines-
cence at 1270 nm, which was compared with the
standard, rose Bengal [/(1O2) = 0.80]. Excitation was
effected with a frequency-doubled neodinium–YAG la-
ser emitting at 532 nm. All the core-modified porphyrins
1–11 showed high values of /(1O2) (>0.73), with the
exception of two compounds, 4-dimethylaminophenyl
derivative 8 [/(1O2) = 0.32] and 4-hydroxyphenyl deriv-
ative 9 [/(1O2) = 0.03] (Table 2).
2.2.4. n-Octanol/water partition coefficients. The lipo-
philicity of a molecule is an important determinant of
biomembrane permeability. The partition between n-
octanol and pH 7.4 buffer was measured by a slightly
modified ꢀshake-flaskꢁ method.17 The change in substitu-
ents on the meso-aryl groups gave a range of values of
log D7.4 from À0.16 for 4-isopropylphenyl derivative
10 to 0.78 for 4-hydroxyphenyl derivative 9 (Table 2).
Interestingly, the effects of the hydroxyl and isopropyl
groups were contrary to our expectations. The isopropyl
group increases log D7.4 (Dlog D7.4 = +0.74) and
hydroxyl group decreases log D7.4(DlogD7.4 = À0.20)
from compound 1 (log D7.4 = 0.04, Table 2), even
though the hydrophobic constants (p) of isopropyl and
hydroxyl groups are 1.53 and À0.67, respectively.18 This
observation strongly suggests that log D7.4 might be
influenced by other factors, such as dimerization or
aggregation in solution.
2.2.3. Quantum yields for fluorescence. Molecular fluo-
rescence is a useful tool to study the localization and
pharmacokinetics of dyes both in vitro and in vivo.
Table 2. Quantum yields for the generation of singlet oxygen [/(1O2)],
quantum yields for fluorescence (/F), and n-octanol/water partition
coefficients in pH 7.4 phosphate buffer (log D7.4) for 1–11a
Compound
/(1O2)
/
log D7.4
F
2.3. Biology
1
2
0.80
0.71b
0.83
0.76
0.75
0.87
0.81
0.32
0.03
0.74
0.73
0.007
0.003b
0.010
0.011
0.010
0.009
0.012
0.029
0.002
0.010
0.012
0.04 0.02
NDc
2.3.1. Intracellular accumulation of core-modified por-
phyrins into cultured R3230AC cells. The cellular uptake
of core-modified porphyrins after 24 h incubation was
determined by fluorescence techniques (Fig. 2). Follow-
ing exposure to photosensitizers, cells were digested and
the porphyrins were solubilized with 25% Scintigest
(100% DMSO for compound 9). Owing to low values
of /F for porphyrins (Table 2), higher concentrations
of photosensitizer were employed, 5 · 10À6 and
1 · 10À5 M, in the uptake experiment, relative to the
phototoxicity experiments, although there was no signif-
icant dark toxicity with any of the compounds at
1 · 10À5 M.
3
À0.06 0.01
0.19 0.06
0.27 0.05
À0.08 0.01
À0.15 0.08
0.56 0.15
0.78 0.08
À0.16 0.06
0.12 0.14
4
5
6
7
8
9
10
11
a Detailed methods for measurements above are presented in Section 4.
b Ref.12.
c ND (not determined) indicates samples for which measurements were
not performed.
4
3
2
1
0
1
2
3
4
5
6
7
8
9
10
11
Dithiaporphyrin
Figure 2. Cellular uptake of 21,23-dithiaporphyrins in cultured R3230AC rat mammary adenocarcinoma cells. Each bar represents the mean
intracellular uptake of each compound incubated with R3230AC cells for 24 h at 5 · 10À6 M (black bars) or 1 · 10À5 M (white bars). Data are
expressed as femtomole porphyrin/cell and error bars are the SEM.

5972
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
Cellular uptake of the dithiaporphyrins at 5 · 10À6 M of
the photosensitizer covered a range from 0.1 fmol/cell
for 11 to 1.9 fmol/cell for 2. Cellular uptake was similar
for the symmetrically substituted dithiaporphyrins 1, 2,
5, 7, and 10 (1.8, 1.9, 1.7, 1.7, and 1.8 fmol/cell, respec-
tively) and was somewhat lower for 4-chlorophenyl
derivative 4 (0.8 fmol/cell) and 4-dimethylaminophenyl
derivative 8 (0.7 fmol/cell). Cellular uptake of the
unsymmetrically substituted derivatives 3 (0.7 fmol/cell)
and 6 (0.3 fmol/cell) was significantly less than the corre-
sponding symmetrical derivatives 1, 2, and 5 for these
substituents (P < 0.05 for all pairwise comparisons).
The lowest cellular uptake was observed with compound
11 (0.1 fmol/cell) with bulky biphenyl substituents. The
value of /F for 9 is much smaller than any of the other
derivatives and no fluorescence could be detected fol-
lowing cell digestion of cells treated with 5 · 10À6 M
photosensitizer. At 1 · 10À5 M photosensitizer, uptake
of compound 9 (0.2 fmol/cell) was comparable to the
For the dark controls, cell survival was >85% for all
dithiaporphyrins at concentrations up to 1 · 10À5 M.
Phototoxicity with the porphyrins at 1 · 10À7 and
5 · 10À7 M is given in Figure 3. Dithiaporphyrins 2–6
with the electron-withdrawing substituents –F and
–CF3, dithiaporphyrin 8 with dimethylamino substitu-
ents, and dithiaporphyrin 1 with phenyl substituents
showed comparable phototoxicity with <20% survival
at 5 · 10À7 M. However, compound 3 with meso-phenyl
and 4-fluorophenyl substituents gave only 33% cell
survival in R3230AC cells treated with 1 · 10À7 M 3.
Dithiaporphyrin 4 with chloro substituents and dith-
iaporphyrin 7 with methoxy substituents were not as
phototoxic with treated cells displaying 44% and 54%
cell survival, respectively, at 5 · 10À7 M. Dithiaporphy-
rin 9 with hydroxyl substituents and dithiaporphyrins 10
and 11 with bulky substituents showed essentially no
phototoxicity at 5 · 10À7 M. Even at 1 · 10À6 M, com-
pounds 10 and 11 showed 63% and 75% survival, respec-
tively, with 5 J cmÀ2 of 350–750 nm light in treated cells.
This is consistent with our earlier observations on
dithiaporphyrins with two bulky substituents.14
uptake of compounds
6
(0.4 fmol/cell) and 11
(0.6 fmol/cell), but the uptake of these compounds was
still less than for any of the others at either 5 · 10À6 M
or 1 · 10À5 M.
The uptake does not appear to be strictly a function of
liphophilicity. log D7.4 values for compounds 1, 5, 7, and
10 with the highest cellular uptake and compounds 6
and 11 with two of the lowest values of cellular uptake
are all near 0. Additionally, compound 9 with the high-
est value of log D7.4 at 0.78 showed the lowest uptake,
which might be explained by either loss of amphiphilic-
ity of the molecule or formation of aggregates in the
media.
Examination of the quantum yields for the generation of
singlet oxygen, cellular uptake, and phototoxicity of the
various dithiaporphyrins revealed some inconsistencies.
Compounds 1, 2, 6, and 8 were the most phototoxic
compounds in the series 1–11, yet /(1O2) was 0.80 for
1, 0.71 for 2, and 0.87 for 6 in solution but only 0.32
for 8. Furthermore, cellular uptake was 1.8 fmol/cell
for 1, 1.9 fmol/cell for 2, and only 0.3 and 0.7 fmol/cell
for 6 and 8, respectively, when the R3230AC cells were
exposed to 5 · 10À6 M photosensitizer. In contrast, the
photosensitizer 10 with /(1O2) of 0.73 and cellular up-
take of 1.3 fmol/cell in cells treated with 5 · 10À7 10
was one of the poorest photosensitizers in the series.
Clearly, efficacy of photosensitizers in the series 1–11
cannot be predicted by either values of /(1O2) or by cel-
lular uptake or by a combination of the two.
2.3.2. Dark toxicity and phototoxicity of core-modified
porphyrins toward cultured R3230AC cells. Dark toxicity
and phototoxicity of the core-modified porphyrins
toward R3230AC rat mammary adenocarcinoma cells
were determined using the MTT colorimetric assay.
Cells were incubated for 24 h with 5 · 10À8 to
1 · 10À6 M core-modified porphyrin. Treated cells were
then irradiated with 1.4 mW cmÀ2 broadband light
Values of log D7.4 also did not correlate with efficacy.
Compounds 1, 2, 6, and 10 had values of log D7.4 near
0, while compound 8 was more lipophilic with log D7.4
of 0.56. Compound 10 showed no phototoxicity in
(350–750 nm) for 1 h giving a total fluence of 5 J cmÀ2
.
Cell survival was then determined 24 h following irradi-
ation of treated cells and 24 h later for dark controls.
1.25
1.00
0.75
0.50
0.25
0.00
1
2
3
4
5
6
7
8
9
10
11
Dithiaporphyrin
Figure 3. Cell viability of cultured R3230AC cells after photosensitization with 5 J cmÀ2 of broadband light (350–750 nm) in the presence of 21,23-
dithiaporphyrins, 1 · 10À7 M (black bars) or 5 · 10À7 M (white bars). Each data point represents the mean of at least three separate experiments
performed in duplicate and error bars are the SEM. Data are expressed as the surviving fraction of viable cells relative to untreated controls.

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5973
marked contrast to compounds 1, 2, and 6 with similar
values of log D7.4, uptake, and /(1O2). Furthermore, the
more lipophilic compound 8 was similar in potency to
the amphiphilic molecules 1, 2, and 6. In the absence
of a single parameter to predict phototoxic behavior,
one needs to consider the possibilities of differences in
uptake and phototoxicity among monomeric and aggre-
gate species, as well as the possibility of specific cellular
sites of localization.
4-isopropylphenyl or 4-biphenyl substituents, respec-
tively, showed essentially no phototoxicity. The presence
of two bulky meso-substituents leads to an ineffective
photosensitizer. In studies of over thirty dithiaporphyrin
derivatives, no clear correlation of either physical or
photophysical properties has allowed prediction of effi-
cacy. While two derivatives appear to be superior in
studies in vitro, we have as yet to determine a molecular
target, although mitochondria are implicated as a gener-
al site of action.14 The efficacy of IY69 and compound 3
may be due to a specific molecular target or may be due
to some physical phenomenon peculiar to these deriva-
tives, relative to the others, such as aggregation/deaggre-
gation in the cell. Future studies will focus on
compounds IY69 and 3 to identify reasons for their
added efficacy.
3. Summary and conclusions
As an extension of our previous study of structure–
activity relationships that focused on the effects of steric
bulk and symmetry in dithiaporphyrins, ten new core-
modified porphyrins were prepared whose substituents
varied in electronic properties. These derivatives were
successfully prepared through the synthetic methods
developed in our previous work.13 However, the prepa-
ration of dimethoxy and dihydroxy compounds, 7 and 9,
respectively, required new synthetic approaches, as
shown in Scheme 2.
4. Experimental
4.1. General methods
Solvents and reagents were used as received from Sig-
ma–Aldrich Chemical (St. Louis, MO) unless otherwise
noted. Cell culture medium was purchased from GIBCO
(Grand Island, NY). Fetal bovine serum (FBS) was ob-
tained from Atlanta Biologicals (Atlanta, GA). Concen-
tration in vacuo was performed on a Buchi 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 (d 7.26 for proton, d 77.16 for carbon). Infrared
spectra were recorded on a Perkin-Elmer FT-IR instru-
ment. UV–vis–near-IR spectra were recorded on a Per-
kin-Elmer Lambda 12 spectrophotometer. Elemental
analyses were conducted by Atlantic Microlabs, Inc.
Q-TOF 2 electrospray and ESI mass spectrometry were
conducted at the Campus Chemical Instrumentation
Center of The Ohio State University (Columbus, OH)
and the Instrument Center of the Department of Chem-
istry at the University at Buffalo. Compounds 1, 12–15,
21–23, 45, and 46 were prepared, as previously de-
scribed12–14 and compound 2, prepared in our earlier
works,12 was used as the disodium salt. In biological
studies, core-modified porphyrins 1–11 were dissolved
in DMSO to make a stock solution at 2 · 10À3 M. The
stock solutions were then used after appropriate dilu-
tions with sterilized doubly distilled water.
The differences in electronic properties among the sub-
stituents had minimal impact on physical and photo-
physical features, such as absorption maxima (kmax),
quantum yields for the generation of singlet oxygen [/
(1O2)] and fluorescence (/F), and n-octanol/water parti-
tion coefficients (log D7.4). Compounds 2 and 8 with two
4-fluorophenyl or two 4-dimethylaminophenyl substitu-
ents in the meso-positions displayed a small bathochro-
mic shift in kmax, relative to the other derivatives,
compound 8 had a value of /(1O2) that was less than
half of any other derivative in the series, and compounds
8 and 9 were both more lipophilic than other derivatives
in the series based on the values of log D7.4
.
In the series 1–11, five of the dithiaporphyrins (com-
pounds 2, 3, 5, 6, and 8) were efficient photosensitizers,
giving less than 20% cell survival in R3230AC rat
mammary adenocarcinoma cells following treatment
with 5 · 10À7 M photosensitizer and 5 J cmÀ2 of 350–
750 nm light. Compound 3 with meso-phenyl and
4-fluorophenyl substituents was the most potent photo-
sensitizer among these five toward R3230AC cells, with
only 33% cell survival following treatment with 1 ·
10À7 M photosensitizer and 5 J cmÀ2 of 350–750 nm
light. Dark controls indicated that there was no signifi-
cant cellular toxicity in the absence of light.
4.2. Synthesis
Empirically, the substituent studies of this manuscript
and our previous work have identified two dithiapor-
phyrins with two 4-(carboxylatomethoxy)phenyl substit-
uents at the 10- and 15-positions, a phenyl substituent at
the 5-position, and either a 4-fluorophenyl substituent
(compound 3 of this study) or a 2-thienyl substituent
(IY69 of our previous study)14 at the 20-position as
superior photosensitizers with submicromolar EC50ꢁs
and 5 J cmÀ2 of 350–750 nm light. As was observed in
our earlier work with dithiaporphyrins with two 2,4,6-
trimethylphenyl substituents or two 4-tert-butylphenyl
substituents,14 compounds 10 and 11 with two bulky
4.2.1. General method for the preparation of 2,5-bis(aryl-
1-hydroxymethyl)thiophenes (16–18). Compounds 17
and 18 were prepared as described for the preparation
of 16.
4.2.2. 2,5-Bis[1-(4-isopropyl)phenyl-1-hydroxymethyl]thi-
ophene (16). Thiophene (4.2 g, 50 mmol) was added to a
solution of n-butyllithium (69 mL of a 1.6 M solution in
hexanes, 110 mmol) and TMEDA (17 mL, 115 mmol) in
200 mL hexanes under an Ar atmosphere. The reaction
mixture was heated at reflux for 1 h, cooled to ambient
temperature, and transferred via a cannula to a pres-

5974
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
sure-equalizing addition funnel. This dilithiothiophene
suspension was then added dropwise to a solution of
4-isopropylbenzaldehyde (14 g, 95 mmol) in 200 mL of
anhydrous THF cooled to 0 ꢁC, which had been de-
gassed with Ar for 15 min. After the addition was com-
plete, the mixture was warmed to ambient temperature,
300 mL of NH4Cl (aqueous 1 M solution) was added,
and the organic phase was separated. The aqueous
phase was extracted with ether (3· 300 mL). The com-
bined organic extracts were washed with water (3·
300 mL) and brine (300 mL), dried over MgSO4, and
concentrated. The crude product was purified by a silica
column with the mixture of hexanes and ethyl acetate to
give 14 g (73%) of 16 as a light yellow oil. IR (film):
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· 200 mL) and brine (200 mL), dried over
MgSO4, and concentrated to give the crude diol. The
crude diol was purified by column chromatography on
SiO2 eluted with a mixture of hexanes and ethyl acetate
to give 3.5 g (56%) of 44 as a light yellow oil. IR (film):
3406, 3063, 2874, 1700, 1604 cmÀ1; 1H NMR (300 MHz,
CDCl3): d 6.22 (2H, s), 6.97 (2H, s), 6.78 (1H, d,
J = 3.0 Hz), 6.85 (1H, t, J = 4.8 Hz), 6.98 (2H, t,
J = 8.4 Hz), 7.22 (1H, d, J = 4.8 Hz), 7.44 (2H, dd,
J1 = 8.3, J2 = 5.7); 13C NMR (75 MHz, CDCl3): d 71.92,
72.62, 115.47 (d, J = 22), 124.60, 126.41, 128.16 (d,
J = 8), 128.19, 128.69, 138.80, 142.94, 148.02, 148.38,
162.51 (d, J = 244), 164.14; High Resolution Q-TOF
MS: m/z 337.0693 (calcd for C18H15FN2O2S+Na,
337.0674).
3415, 3036, 2940, 2870, 1699 cmÀ1
;
1H NMR
(500 MHz, CDCl3): d 1.17 (12H, d, J = 7.0 Hz), 3.16–
3.20 (2H, m), 5.85 (2H, s), 6.62 (2H, s), 7.14 (4H, d,
J = 8.0 Hz), 7.27 (4H, d, J = 7.5 Hz); 13C NMR
(126 MHz, CDCl3): d 24.10, 33.96, 72.62, 124.42,
124.45, 126.42, 126.47, 126.73, 140.47, 148.26, 148.85;
High Resolution Q-TOF MS, m/z 403.1703 (calcd for
C24H28O2S+H, 403.1708).
4.2.6.
2-(1-Hydroxy-1-phenylmethyl)-5-[1-(4-trifluoro-
methylphenyl)-1-hydroxymethyl]-thiophene (45). Com-
pound 45 was prepared with the same method as that
used for the preparation of 44. Yield: 57%; IR (film):
3428, 3063, 2874, 1700, 1620 cmÀ1; 1H NMR (500 MHz,
DMSO-d6): d 5.82 (1 H, s), 5.89 (1H, s), 6.21 (1H, s),
6.42 (1H, s), 6.68 (1H, d, J = 10.4 Hz), 6.71 (1H, d,
J = 11.6 Hz), 7.18–7.25 (1H, m), 7.30 (2H, br s), 7.35
(2H, br s), 7.60 (2H, br s), 7.66 (2H, br s); 13C NMR
(75 MHz, DMSO-d6): d 70.50, 71.13, 123.54 (123.90),
125.48 (125.52), 126.31 (126.39), 127.02 (127.08),
127.59 (127.64), 128.56, 145.02 (145.08), 148.35
(148.52), 149.67, 149.91 (150.12); High Resolution Q-
TOF MS: m/z 387.0635 (calcd for C19H15F3N2O2S+Na,
387.0643).
4.2.3. 2,5-Bis(1-biphenyl-1-hydroxymethyl)thiophene (17).
1
Yield: 70%; IR (film): 3392, 3030, 1732, 1698 cmÀ1; H
NMR (500 MHz, CD3OD): d 5.44 (2H, s), 6.70 (2H, s),
7.28–7.35 (2H, m), 7.38–7.52 (8H, m), 7.54–7.62 (8H,
m); 13C NMR (126 MHz, 1:1 CDCl3/CD3OD): d 81.41,
81.45, 125.14, 127.09, 127.11, 127.15, 127.33, 127.39,
127.44, 127.47, 127.51, 128.88, 140.02, 140.06, 140.84,
141.08, 146.29, 146.33; High Resolution ESI MS: m/z
448.1481 (calcd for C30H24O2S, 448.1492).
4.2.4. 2,5-Bis[1-(4-triisobutylsilyoxy)phenyl-1-hydroxy-
methyl]thiophene (18). Yield: 58%; H NMR (300 MHz,
1
CDCl3): d 1.31 (18H, d, J = 6.9 Hz), 1.42–1.50 (6H, br
s), 6.13 (2H, s), 6.88 (2H, s), 7.07 (4H, d, J = 8.4 Hz),
7.48 (4H, d, J = 7.5 Hz); 13C NMR (75 MHz, CDCl3): d
.83, 18.06, 72.46, 120.03, 124.44, 127.73, 135.60, 148.50,
156.10. High Resolution ESI MS: m/z 663.3344 (calcd
for C36H56O4SSi2+Na, 663.3330).
4.2.7. Diethyl 2,5-bis[1-(4-carboxylatomethoxyphenyl)-1-
pyrrolomethyl]thiophene (23). Diol 20 (3.2 g, 5.8 mmol)
was dissolved in excess pyrrole (16 mL) and the resulting
solution was degassed with Ar. Boron trifluoride etherate
was added (0.10 mL, 1.1 mmol) and the resulting mixture
was stirred for 1 h at ambient temperature. The reaction
was stopped by the addition of CH2Cl2 (100 mL), fol-
lowed by 40% NaOH (50 mL). The organic layer was sep-
arated, washed with water (3· 150 mL) and brine
(150 mL), dried over MgSO4, and concentrated. The ex-
cess pyrrole was removed at reduced pressure at ambient
temperature. The residual oil was purified via chromato-
graphy on SiO2 eluted with the mixture of hexanes and
ethyl acetate to give 2.7 g (63%) of 23 as a yellow oil. 1H
NMR (500 MHz, CDCl3): d 1.10 (18H, d, J = 7.5 Hz),
1.40–1.48 (6H, s), 5.51 (2H, s), 5.90 (2H, s), 6.14 (2H, br
s), 6.58 (2H, s), 6.68 (2H, s), 6.82 (4H, d, J = 8.5 Hz),
7.07 (4H, d, J = 8.5 Hz), 7.89 (2H, br s); 13C NMR
(126 MHz, CDCl3): d 12.76, 18.05, 45.33, 107.45,
108.35, 117.20, 120.01, 125.17, 129.44, 133.62, 135.26,
146.46, 155.14; High Resolution ESI MS: m/z 761.3958
(calcd for C44H62N2O2SSi2+Na, 761.3963).
4.2.5. 2-(1-Hydroxy-1-phenylmethyl)-5-[1-(4-fluorophe-
nyl)-1-hydroxymethyl]-thiophene (44). 2-[1-(tert-Butyl-
dimethylsilyloxy)-1-phenylmethyl]thiophene14 (43, 6.0 g,
20 mmol) was added to a solution of n-butyllithium
(14 mL of 1.6 M in hexanes, 22 mmol) and TMEDA
(3.6 mL, 24 mmol) in 150 mL of hexanes under an Ar
atmosphere. The reaction mixture was stirred at ambient
temperature for 30 min and transferred via cannula to a
pressure-equalizing addition funnel. The suspension of
2-lithio 43 was then added dropwise to a solution of 4-flu-
orobenzaldehyde (2.1 mL, 20 mmol) in 150 mL of anhy-
drous THF at 0 ꢁC, which was 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· 200 mL). The combined organic extracts were
washed with water (3· 200 mL) and brine (200 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
4.2.8. General method for the preparation of 5,20-diaryl-
10,15-bis(4-methoxyphenyl)-21,23-dithiaporphyrins (24–
30, 46, 47). Compounds, 24–30, 46, and 47, were
prepared with similar methods described for the prepa-
ration of 24.

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5975
4.2.9. 5,20-Bis(4-chlorophenyl)-10,15-bis(4-methoxyphe-
nyl)-21,23-dithiaporphyrin (24). Diol 12 (3.0 g, 0.8 mmol),
2,5-bis[1-(4-chlorophenyl)-1-pyrrolomethyl]thiophene (22,
3.8 g, 0.8 mmol), and 2,3,5,6-tetrachloro-1,4-benzoqui-
none (TCBQ, 6.1 g, 25 mmol) were dissolved in 600 mL
CH2Cl2. Boron trifluoride etherate (0.57 mL, 0.45 mmol)
was added and the reaction mixture was stirred for 0.5 h in
the dark. The reaction mixture was concentrated and the
residue was redissolved in minimal CH2Cl2. The crude
product was purified via chromatography on basic alumi-
na eluted with CH2Cl2. Dithiaporphyrin 24 was isolated
as the first red band. The crude product was washed with
acetone to give 0.32 g (5%) of 24 as a purple solid. Mp:
J = 4.5 Hz), 9.73 (2H, s), 9.77 (2 H, s); High Resolution
ESI MS: m/z 861.2592 (calcd for C58H40N2O2S2+H,
861.2604).
4.2.14. Diethyl 5,20-bis(4-methoxyphenyl)-10,15-bis(4-
carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (29).
Yield: 18%; mp: 117–119 ꢁC; 1H NMR (500 MHz,
CDCl3): d 1.42 (6H, t, J = 7.0 Hz), 4.10 (6H, s), 4.42
(4H, q, J = 7.0 Hz), 4.92 (4H, s), 7.36 (8H, d,
J = 7.0 Hz), 8.17 (8H, d, J = 7.5 Hz), 8.67 (2H, d,
J = 5.0 Hz), 8.72 (2H, d, J = 4.5 Hz), 9.68 (2H, s), 9.70
(2H, s); High Resolution ESI MS: m/z 913.2645 (calcd
for C54H44N2O8S2+H, 913.2612).
1
>300 ꢁC; H NMR (400 MHz, CDCl3): d 4.11 (6H, s),
7.36 (4H, d, J = 8.4 Hz), 7.80 (4H, d, J = 8.0 Hz), 8.168
(4H, d, J = 8.4 Hz), 8.169 (4H, d, J = 8.0 Hz), 8.64 (2H,
d, J = 4.4 Hz), 8.72 (2H, d, J = 4.4 Hz), 9.63 (2H, s),
9.73 (2H, s); 13C NMR (75 MHz, 1:1 CDCl3/CD3OD): d
55.79, 113.29, 127.93, 132.36, 133.70, 134.22, 134.63,
134.80, 135.13, 135.21, 135.32, 135.63, 135.86, 139.86,
147.59, 148.60, 156.38, 156.96, 160.04; High Resolution
Q-TOF MS: m/z 777.1204 (calcd for C46H30Cl2N2
O2S2+H, 777.1204).
4.2.15. Diethyl 5,20-bis(4-triisobutylsilyoxyphenyl)-10,15-
bis(4-carboxylatomethoxyphenyl)-21,23-dithiaporphyrin
1
(30). Yield: 32%; mp: 195–198 ꢁC; H NMR (500 MHz,
CDCl3): d 1.29 (36H, d, J = 7.5 Hz), 1.42 (6H, t,
J = 7.0 Hz), 1.45–1.51 (6H, s), 4.42 (4H, q, J = 7.0 Hz),
4.93 (4H, s), 7.32–7.39 (8H, s), 8.09 (4H, d, J = 6.5 Hz),
8.18 (4H, d, J = 7.0 Hz), 8.66–8.70 (4H, s), 9.68 (2H, s),
9.71 (2H, s); High Resolution ESI MS: m/z 1197.4987
(calcd for C70H80N2O8S2Si2+H, 1197.4967).
4.2.10. 5,20-Bis(4-trifluoromethylphenyl)-10,15-bis(4-
methoxyphenyl)-21,23-dithiaporphyrin (25). Yield: 3%;
mp: >300 ꢁC; H NMR (400 MHz, CDCl3): d 4.10 (6H,
4.2.16. 5-Phenyl-20-(4-fluorophenyl)-10,15-bis(4-methoxy-
phenyl)-21,23-dithiaporphyrin (46). Yield: 5%; mp:
>300 ꢁC; H NMR (300 MHz, CDCl3): d 4.10 (6H, s),
1
1
s), 7.37 (4H, d, J = 7.6 Hz), 8.09 (4H, d, J = 7.2 Hz),
8.18 (4H, d, J = 8.0 Hz), 8.36 (4H, d, J = 7.2 Hz), 8.60
(2H, d, J = 4.4 Hz), 8.74 (2H, d, J = 4.0 Hz), 9.59 (2H,
s), 9.75 (2H, s); High Resolution Q-TOF MS: m/z
845.1735 (calcd for C48H30F6N2O2S2+H, 845.1731).
7.36 (4H, d, J = 8.1 Hz), 7.45 (2H, t, J = 8.1 Hz), 7.81
(3H, br s), 8.18 (4H, d, J = 8.1 Hz), 8.16–8.30 (4H, m),
8.60–8.76 (4H, m), 9.63 (1H, d, J = 4.8 Hz), 9.69 (1H, d,
J = 5.1 Hz), 9.73 (2H, br s); High Resolution Q-TOF
MS: m/z 727.1874 (calcd for C46H31FN2O2S2+H,
727.1889).
4.2.11.
5,20-Bis(4-dimethylaminophenyl)-10,15-bis(4-
methoxyphenyl)-21,23-dithiaporphyrin (26). Yield: 6.4%;
mp: >300 ꢁC; 1H NMR (400 MHz, CDCl3): d 3.25
(12H, s), 4.10 (6H, s), 7.17 (4H, d, J = 8.8 Hz), 7.35 (4H,
d, J = 8.8 Hz), 8.15 (4H, d, J = 5.2 Hz), 8.18 (2H, d,
J = 5.2 Hz), 8.66 (2H, d, J = 4.4 Hz), 8.75 (2H, d,
J = 4.8 Hz), 9.64 (2H, s), 9.76 (2H, s); High Resolution
Q-TOF MS: m/z 795.2834 (calcd for C40H42N4O2S2+H,
795.2827).
4.2.17. 5-Phenyl-20-(4-trifluoromethylphenyl)-10,15-
bis(4-methoxyphenyl)-21,23-dithiaporphyrin (47). Yield:
7%; mp: 125–127 ꢁC; H NMR (400 MHz, CDCl3): d
1
11 (6H, s), 7.37 (4H, d, J = 8.4 Hz), 7.79–7.85 (3H, m),
8.08 (2H, d, J = 8.0 Hz), 8.18 (4H, d, J = 8.0 Hz),
8.22–8.27 (2H, m), 8.37 (2H, d, J = 8.0 Hz), 8.60 (1H,
d, J = 4.4 Hz), 8.68 (1H, d, J = 4.4 Hz), 8.72 (1H, d,
J = 4.4 Hz), 8.74 (1H, d, J = 4.4 Hz), 9.58 (1H, d,
J = 5.2 Hz), 9.68 (1H, d, J = 5.2 Hz), 9.74 (2H, s);
4.2.12. 5,20-Bis(4-isobutylphenyl)-10,15-bis(4-methoxy-
phenyl)-21,23-dithiaporphyrin (27). Yield: 30%; mp:
13C NMR (75 MHz, CDCl3):
d
55.78, 111.07,
113.27, 124.57, 127.63, 128.25, 130.82, 131.53, 133.71,
133.99, 134.33, 134.62, 134.79, 134.95, 135.17, 135.61,
135.75, 135.85, 141.32, 145.20, 147.47, 147.70, 148.48,
148.67, 150.65, 155.96, 156.80, 160.01, 160.92; High
Resolution Q-TOF MS: m/z 777.1868 (calcd for
C47H31F3N2O2S2+H, 777.1857).
1
>300 ꢁC; H NMR (400 MHz, CDCl3): d 1.56 (12H, d,
J = 6.8 Hz), 3.26–3.30 (2H, m), 4.10 (6H, s), 7.35 (4H,
d, J = 8.4 Hz), 7.67 (4H, d, J = 7.6 Hz), 8.18 (8H, d,
J = 8.0 Hz), 8.45 (2H, d, J = 4.4 Hz), 8.73 (2H, d,
J = 4.4 Hz), 9.71 (2H, s), 9.72 (2H, s); 13C NMR
(75 MHz, CDCl3): d 24.40, 34.28, 55.72, 113.15, 125.67,
133.19, 133.95, 134.26, 134.53, 134.67, 135.55, 138.86,
148.02, 148.73, 156.62, 156.71, 159.84. High Resolution
Q-TOF MS: m/z 793.2938 (calcd for C52H44N2O2S2+H,
793.2922).
4.2.18. General method for the preparation of 5,20-diaryl-
10,15-bis(4-hydroxyphenyl)-21,23-dithiaporphyrins (31–
35, 48, 49). Compounds, 31–35, 48, and 49, were
prepared, as described for the preparation of 31.
4.2.13. 5,20-Di(biphenyl)-10,15-bis(4-methoxyphenyl)-
21,23-dithiaporphyrin (28). Yield: 18%; mp: 175–
177 ꢁC; H NMR (500 MHz, CDCl3): d 4.12 (6H, s),
7.37 (4H, d, J = 7.5 Hz), 7.48–7.52 (2H, m), 7.61 (4H,
t, J = 7.5 Hz), 7.93 (4H, d, J = 7.5 Hz), 8.06 (4H, d,
J = 7.5 Hz), 8.20 (4H, d, J = 7.5 Hz), 8.34 (4H, d,
J = 7.5 Hz), 8.73 (2H, d, J = 4.5 Hz), 8.76 (2H, d,
4.2.19. 5,20-Bis(4-chlorophenyl)-10,15-bis(4-hydroxyphe-
nyl)-21,23-dithiaporphyrin (31). Dithiaporphyrin 24
(0.27 g, 0.35 mmol) was dissolved in 50 mL CH2Cl2
and BBr3 (0.32 mL, 3.5 mmol) was added at 0 ꢁC. The
resulting solution was stirred for 5 h at ambient temper-
ature. The reaction mixture was added to 150 mL
EtOAc and 150 mL of saturated NaHCO3. The organic
1

5976
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
layer was separated and washed three times with 150 mL
of brine, dried over MgSO4, and concentrated. The
crude solid was washed with 25% EtOAc/hexanes sever-
al times to give 0.23 g (88%) of 31 as a dark blue solid.
Mp: >300 ꢁC; 1H NMR (400 MHz, 1:1 CDCl3/
CD3OD): d 7.18 (4H, d, J = 8.4 Hz), 7.69 (4H, d,
J = 8.0 Hz), 7.98 (4H, d, J = 8.4 Hz), 8.05 (4H, d,
J = 8.0 Hz), 8.52 (2H, d, J = 4.4 Hz), 8.64 (2H, d,
J = 4.4 Hz), 9.53 (2H, s), 9.67 (2H, s); 13C NMR
4.2.25. 5-Phenyl-20-(4-trifluoromethylphenyl)-10,15-bis(4-
hydroxyphenyl)-21,23-dithiaporphyrin (49). Yield: 93%;
mp: >300 ꢁC; 1H NMR (400 MHz, 1:1:1 CDCl3/
CD3OD/DMSO-d6) d 7.03 (4H, d, J = 8.4 Hz), 7.51–7.58
(3H, m), 7.78 (2H, d, J = 8.4 Hz), 7.81 (4H, d,
J = 8.0 Hz), 8.16–8.22 (2H, m), 8.08 (2H, d, J = 8.0 Hz),
8.30 (1H, d, J = 4.8 Hz), 8.36 (1H, d, J = 4.4 Hz), 8.44
(1H, d, J = 4.4 Hz), 8.46 (1H, d, J = 4.8 Hz), 9.30 (1H, d,
J = 5.2 Hz), 9.39 (1H, d, J = 5.2 Hz), 9.50 (2H, s); High
Resolution Q-TOF MS: m/z 749.1551 (calcd for
C45H27F3N2O2S2+H, 749.1544).
(75 MHz, 1:1 CDCl3/CD3OD):
d 114.53, 127.67,
132.00, 132.34, 133.79, 134.55, 134.98, 135.08, 135.60,
135.79, 139.55, 147.16, 148.40, 156.13, 156.81, 157.32;
High Resolution Q-TOF MS: m/z 749.0891 (calcd for
C44H26Cl2N2O2S2+H, 749.0888).
4.2.26. General method for the preparation of diethyl 5,20-
diaryl-10,15-bis(4-carboxylatomethoxyphenyl)-21,23-dithia-
porphyrins (36–40, 50, 51). Compounds, 36–40, 50, and 51,
were prepared as described for the preparation of 36.
4.2.20. 5,20-Bis(4-trifluoromethylphenyl)-10,15-bis(4-hydroxy-
phenyl)-21,23-dithiaporphyrin (32). Yield: 88%; mp:
1
>300 ꢁC; H NMR (400 MHz, 1:1 CDCl3/CD3OD): d
4.2.27. Diethyl 5,20-bis(4-chlorophenyl)-10,15-bis(4-carb-
oxylatomethoxyphenyl)-21,23-dithiaporphyrin (36). Dith-
iaporphyrin 31 (0.20 g, 0.27 mmol), K2CO3 (1.8 g,
13 mmol), and ethyl bromoacetate (2.96 mL, 27 mmol)
in 50 mL acetone were heated at reflux for 10 h. The reac-
tion mixture was cooled to ambient temperature and the
K2CO3 was removed by filtration. The filter cake was
washed with acetone until the filtrate became colorless.
The combined filtrates were concentrated. The crude
product was washed with MeOH to give 0.20 g (81%) of
36 as a purple solid. Mp: 212–214 ꢁC; 1H NMR
(400 MHz, CDCl3): d 1.45 (6H, t, J = 7.6 Hz), 4.44 (4H,
q, J = 6.8 Hz), 4.92 (4H, s), 7.36 (4H, d, J = 8.4 Hz),
7.80 (4H, d, J = 8.0 Hz), 8.17 (4H, d, J = 8.0 Hz), 8.17
(4H, d, J = 8.0 Hz), 8.66 (2H, d, J = 4.4 Hz), 8.72 (2H,
d, J = 4.8 Hz), 9.65 (2H, s), 9.73 (2H, s); 13C NMR
(75 MHz, CDCl3): d 14.43, 61.73, 65.86, 113.97, 127.88,
132.45, 134.17, 134.29, 134.66, 134.78, 134.88, 135.02,
134.27, 135.54, 135.79, 139.73, 147.62, 148.44, 156.37,
156.84, 158.28; High Resolution Q-TOF MS: m/z
921.1629 (calcd for C52H38Cl2N2O6S2+H, 921.1626).
7.23–7.33 (4H, m), 8.00–8.13 (8H, m), 8.31 (4H, d,
J = 7.6 Hz), 8.54 (2H, d, J = 4.4 Hz), 8.72 (2H, d,
J = 4.4 Hz), 9.56 (2H, d, J = 3.2 Hz), 9.75 (2H, d,
J = 2.4 Hz); High Resolution Q-TOF MS: m/z 817.1425
(calcd for C46H26F6N2O6S2+H, 817.1418).
4.2.21.
5,20-Bis(4-dimethylaminophenyl)-10,15-bis(4-
hydroxyphenyl)-21,23-dithiaporphyrin (33). Yield: 83%;
mp: >300 ꢁC; 1H NMR (400 MHz, 1:1 CDCl3/CD3OD):
d 3.21 (12H, s), 7.16 (4H, d, J = 7.6 Hz), 7.25 (4H, d,
J = 8.0 Hz), 8.02 (4H, d, J = 5.2 Hz), 8.06 (4H, d,
J = 5.2 Hz), 8.59 (2H, s), 8.62 (2H, s), 9.65 (2H, s), 9.69
(2H, s); HI ESI MS: 767.2521 (calcd for C48H38N4O2
S2+H, 767.2514).
4.2.22. 5,20-Bis(4-isobutylphenyl)-10,15-bis(4-hydroxyphe-
nyl)-21,23-dithiaporphyrin (34). Yield: 95%; mp: 234–
236 ꢁC; 1H NMR (500 MHz, acetone-d6): d 1.54 (12H, d,
J = 7.0 Hz), 3.21–3.34 (2H, m), 7.40 (4H, d, J = 8.0 Hz),
7.75 (4H, d, J = 7.5 Hz), 8.15 (4H, d, J = 8.5 Hz), 8.17
(4H, d, J = 8.0 Hz), 8.66 (2H, d, J = 4.0 Hz), 8.74 (2H, d,
J = 4.5 Hz), 9.73 (2H, s), 9.83 (2H, s); 13C NMR
(75 MHz, 1:1:1 CDCl3/CD3OD/DMSO-d6): d 24.33,
34.00, 115.00, 125.79, 131.70, 133.87, 134.34, 134.46,
134.61, 135.67, 138.31, 147.36, 147.86, 148.69, 156.10,
156.32, 158.10; High Resolution Q-TOF MS: m/z
765.2614 (calcd for C50H40N2O2S2+H, 765.2609).
4.2.28. Diethyl 5,20-bis(4-trifluoromethylphenyl)-10,15-bis(4-
carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (37).
Yield: 86%; mp: 133–135 ꢁC; 1H NMR (500 MHz,
CDCl3): d 1.38 (6H, t, J = 7.0 Hz), 4.38 (4H, q,
J = 7.0 Hz), 4.88 (4H, s), 7.40 (4 H, d, J = 9.0 Hz), 8.12
(4H, d, J = 7.5 Hz), 8.20 (4H, d, J = 8.0 Hz), 8.38 (4H,
d, J = 8.0 Hz), 8.63 (2H, d, J = 4.5 Hz), 8.75 (2H, d,
J = 4.0 Hz), 9.62 (2H, s), 9.76 (2H, s); High Resolution
Q-TOF MS: m/z 989.2190 (calcd for C54H38F6N2O6
S2+H, 989.2922).
4.2.23.
5,20-Di(biphenyl)-10,15-bis(4-hydroxyphenyl)-
21,23-dithiaporphyrin (35). Yield: 86%; mp: >300 ꢁC;
1H NMR (400 MHz, CD3OD): d 7.28 (4H, br s), 7.45
(2H, br s), 7.55 (4H, br s), 7.86 (4H, br s), 7.93–8.08
(8H, m), 8.18 (4H, br s), 8.62 (4H, br s), 9.68 (4H, br
s); High Resolution ESI MS: m/z 833.2274 (calcd for
C56H36N2O2S2+H, 833.2291).
4.2.29. Diethyl 5,20-bis(4-dimethylaminophenyl)-10,15-
bis(4-carboxylatomethoxyphenyl)-21,23-dithiaporphyrin
(38). Yield: 68%; mp: >300 ꢁC; 1H NMR (500 MHz,
CDCl3): d 1.42 (6H, t, J = 7.0 Hz), 3.25 (12H, s), 4.42
(4H, q, J = 7.5 Hz), 4.92 (4H, s), 7.18 (4H, d, J = 7.0 Hz),
7.35 (4H, d, J = 8.5 Hz), 8.16 (8H, t, J = 8.5 Hz), 8.63
(2H, d, J = 4.5 Hz), 8.74 (2H, d, J = 4.5 Hz), 9.62 (2H, s),
9.76 (2H, s); High Resolution ESI MS: m/z 939.3260
(calcd for C56H50N4O6S2+H, 939.3250).
4.2.24. 5-Phenyl-20-(4-fluorophenyl)-10,15-bis(4-hydroxy-
phenyl)-21,23-dithiaporphyrin (48). Yield: 93%; mp:
1
>300 ꢁC; H NMR (500 MHz, CDCl3): d 7.29 (4H, d,
J = 8.0 Hz), 7.49–7.57 (3H, m), 7.81 (2H, br s), 8.08
(4H, d, J = 8.5 Hz), 8.18–8.24 (4H, m), 8.63 (1H, d,
J = 5.0 Hz), 8.65 (1H, d, J = 4.5 Hz), 7.59 (2H, t,
J = 10.5 Hz), 9.65 (1H, d, J = 5.0 Hz), 9.69 (1H, d,
J = 5.0 Hz), 9.78 (2H, s); High Resolution Q-TOF MS:
m/z 699.1519 (calcd for C44H27FN2O2S2+H, 699.1498).
4.2.30. Diethyl 5,20-bis(4-isopropylphenyl)-10,15-bis(4-
carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (39).
Yield: 82%; mp: 180–182 ꢁC; 1H NMR (400 MHz,

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5977
CDCl3): d 1.43 (6H, t, J = 7.2 Hz), 1.56 (12H, d,
J = 6.8 Hz), 3.23–3.34 (2H, m), 4.43 (4H, q,
J = 6.8 Hz), 4.93 (4H, s), 7.36 (4H, d, J = 8.4 Hz), 7.67
(4H, d, J = 8.0 Hz), 8.17 (4H, d, J = 8.0 Hz), 8.19 (4H,
d, J = 8.4 Hz), 8.68 (2H, d, J = 4.4 Hz), 8.72 (2H, d,
J = 4.4 Hz), 9.68 (2H, s), 9.72 (2H, s); 13C NMR
(75 MHz, 1:1 CDCl3/DMSO-d6): d 14.44, 24.40, 34.29,
61.73, 65.93, 111.04, 113.91, 125.68, 133.44, 134.42,
134.54, 134.78, 135.00, 135.34, 135.52, 135.65, 138.82,
148.03, 148.12, 148.78, 156.67, 158.19, 169.16; High
Resolution Q-TOF MS: m/z 937.3388 (calcd for
C58H52N2O6S2+H, 937.3345).
d, J = 7.5 Hz), 8.60 (2H, s), 8.66 (2H, s), 9.70 (2H, s),
9.76 (2H, s), 10.08 (2H, s); High Resolution ESI MS:
m/z 885.2299 (calcd for C52H40N2O8S2+H, 885.2316).
4.2.35. General method for the preparation of diethyl 5,20-
diaryl-10,15-bis(4-carboxylatomethoxyphenyl)-21,23-dithia-
porphyrins (3–11). Compounds 3–11 were prepared, as
described for the preparation of 4.
4.2.36. 5,20-Bis(4-chlorophenyl)-10,15-bis(4-carboxylato-
methoxyphenyl)-21,23-dithiaporphyrin (4). Core-modi-
fied porphyrin 36 (0.13 g, 0.14 mmol) was dissolved in
20 mL THF and 10 mL of 1 M aqueous NaOH was add-
ed. The resulting solution was stirred at ambient temper-
ature for 15 h. The solution was acidified by the addition
of 4.3 mL acetic acid. The reaction mixture was diluted
with 100 mL 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 0.11 g (90%) of 4 as a purple solid. Mp:
4.2.31. Diethyl 5,20-di(biphenyl)-10,15-bis(4-carboxylato-
methoxyphenyl)-21,23-dithiaporphyrin (40). Yield: 87%;
1
mp: 122–124 ꢁC; H NMR (500 MHz, CDCl3): d 1.42
(6H, t, J = 7.0 Hz), 4.42 (4H, q, J = 7.0 Hz), 4.93 (4H,
s), 7.38 (4H, d, J = 7.5 Hz), 7.49 (2H, t, J = 7.5 Hz), 7.61
(4H, t, J = 7.5 Hz), 7.93 (4H, d, J = 7.0 Hz), 8.06 (4H, d,
J = 7.5 Hz), 8.20 (4H, d, J = 8.0 Hz), 8.33 (4H, d,
J = 7.5 Hz), 8.71 (2H, d, J = 4.5 Hz), 8.76 (2H, d,
J = 4.5 Hz), 9.71 (2H, s), 9.78 (2H, s); High Resolution
ESI MS: m/z 1005.3010 (calcd for C64H48N2O6S2+H,
1005.3027).
1
210–212 ꢁC: H NMR (400 MHz, 1:1 CDCl3/CD3OD):
d 4.93 (4H, s), 7.40 (4H, d, J = 8.4 Hz), 7.80 (4H, d,
J = 7.6 Hz), 8.12–8.21 (8H, m), 8.65 (2H, d,
J = 4.0 Hz), 8.71 (2H, d, J = 4.4 Hz), 9.66 (2H, s), 9.75
(2H, s); 13C NMR (75 MHz, DMSO-d6): 65.65, 114.17,
128.09, 132.74, 134.46, 134.54, 134.62, 135.04, 135.20,
135.48, 135.60, 135.78, 136.14, 139.80, 147.75, 148.59,
156.60, 157.10, 158.57, 171.57; High Resolution Q-
TOF MS: m/z 865.1021 (calcd for C48H30Cl2N2O6S2+H,
865.1000); Anal. Calcd for C48H30Cl2N2O6S2: C, 66.59;
H, 3.49; N, 3.24. Found: C, 66.99; H, 3.82; N, 2.94.
4.2.32. Diethyl 5-phenyl-20-(4-fluorophenyl)-10,15-bis(4-
carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (50).
Yield: 81%; mp: 145–147 ꢁC; 1H NMR (500 MHz,
CDCl3): d 1.43 (6H, t, J = 7.2 Hz), 4.43 (4H, q,
J = 6.8 Hz), 4.93 (4H, s), 7.37 (4H, d, J = 8.4 Hz), 7.52
(2H, t, J = 8.4 Hz), 7.79–7.86 (3H, m), 8.18 (4H, d,
J = 8.4 Hz), 8.18–8.28 (4H, m), 8.65 (1H, d, J = 4.4 Hz),
8.67–8.72 (3H, m), 9.63 (1H, d, J = 5.2 Hz), 9.68 (1H, d,
J = 5.2 Hz), 9.70 (2H, s); 13C NMR (75 MHz, CDCl3): d
14.37, 61.68, 64.46, 65.82, 110.97, 113.87, 114.59 (d,
J = 22), 127.52, 128.15, 133.83, 134.24, 134.73, 135.11,
135.47, 141.25, 147.81, 148.18, 156.70, 158.17, 162.97,
(d, J = 320), 169.06; High Resolution Q-TOF MS:
871.2320 (calcd for C52H39FN2O6S2+H, 871.2312).
4.2.37. 5,20-Bis(4-trifluoromethylphenyl)-10,15-bis(4-carb-
oxylatomethoxyphenyl)-21,23-dithiaporphyrin (5). Yield:
1
92%; mp: 220–222 ꢁC; H NMR (400 MHz, DMSO-d6):
d 4.90 (4H, s), 7.38 (4H, d, J = 8.4 Hz), 8.05 (8H, t,
J = 6.3 Hz), 8.36 (4H, d, J = 7.6 Hz), 8.58 (2H, d,
J = 4.4 Hz), 8.69 (2H, d, J = 4.4 Hz), 9.62 (2H, s), 9.76
(2 H, s); High Resolution Q-TOF MS: m/z 933.1529 (calcd
for C50H30F6N2O6S2+H, 933.1528); Anal. Calcd for
C50H30F6N2O6S2: C, 64.37; H, 3.24; N, 3.00. Found: C,
64.20; H, 3.02; N, 2.96.
4.2.33. Diethyl 5-phenyl-20-(4-trifluoromethylphenyl)-10,15-
bis(4-carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (51).
Yield: 87%; mp: 125–127 ꢁC; 1H NMR (400 MHz,
CDCl3): d 1.44 (6H, t, J = 6.8 Hz), 4.44 (4H, q, J =
6.8 Hz), 4.93 (4H, s), 7.38 (4H, d, J = 4.0 Hz), 7.79–7.89
(3H, m), 8.09 (2H, d, J = 8.0 Hz), 8.18 (4H, d, J =
8.0 Hz), 8.23–8.29 (2H, m), 8.37 (2H, d, J = 8.4 Hz),
8.62 (1H, d, J = 4.4 Hz), 8.68–8.76 (3H, m), 9.59 (1H, d,
J = 5.2 Hz), 9.70 (1H, d, J = 4.8 Hz), 9.73 (2H, s); 13C
NMR (75 MHz, CDCl3): d 14.43, 61.74, 65.88, 113.96,
124.55, 127.62, 128.27, 131.65, 134.02, 134.09, 134.31,
134.44, 134.73, 134.86, 135.08, 135.55, 135.71, 135.80,
135.91, 141.23, 145.10, 147.51, 147.76, 148.33, 148.52,
155.97, 156.82, 158.29, 169.10; High Resolution Q-TOF
MS: m/z 921.2308 (calcd for C53H39F3N2O6S2+H,
921.2280).
4.2.38. 5,20-Bis(4-methoxyphenyl)-10,15-bis(4-carboxy-
latomethoxyphenyl)-21,23-dithiaporphyrin (7). Yield:
80%; mp: >300 ꢁC; 1H NMR (400 MHz, 1:1 CDCl3/
CD3OD): d 4.06 (6H, s), 4.89 (4H, s), 7.33 (4H, d,
J = 8.4 Hz), 7.37 (4H, d, J = 8.4 Hz), 8.09 (4H, d,
J = 6.8 Hz), 8.11 (4H, d, J = 6.4 Hz), 8.62 (4H, s), 9.67
(4H, s); High Resolution ESI MS: m/z 857.1994 (calcd
for C50H36N2O8S2+H, 857.1986); Anal. Calcd for
C50H36N2O8S2: C, 70.08; H, 4.23; N, 3.27. Found: C,
70.23; H, 4.00; N, 3.24.
4.2.39. 5,20-Bis(4-dimethylaminophenyl)-10,15-bis(4-carb-
oxylatomethoxyphenyl)-21,23-dithiaporphyrin (8). Yield:
86%; mp: >300 ꢁC; 1H NMR (400 MHz, 1:1 CDCl3/
CD3OD): d 3.28 (12H, s), 4.91 (4H, s), 7.21 (4H, d,
J = 8.4 Hz), 7.41 (4H, d, J = 6.8 Hz), 8.10 (8H, t,
J = 7.6 Hz), 8.52 (2H, d, J = 4.0 Hz), 8.62 (2H, d,
J = 4.0 Hz), 9.57 (2H, s), 9.65 (2H, s); 13C NMR
(75 MHz, 1:1 CDCl3/CD3OD): d 29.02, 64.87, 110.95,
4.2.34. Diethyl 5,20-bis(4-hydroxyphenyl)-10,15-bis(4-
carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (41).
Yield: 84%; mp: 172–174 ꢁC; 1H NMR (500 MHz,
DMSO-d6): d 1.31 (6H, t, J = 6.5 Hz), 4.30 (4H, q,
J = 7.0 Hz), 5.09 (4H, s), 7.28 (4H, d, J = 7.5 Hz), 7.44
(4H, d, J = 7.5 Hz), 8.06 (4H, d, J = 8.0 Hz), 8.17 (4H,

5978
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
113.31, 128.40, 132.21, 133.33, 133.77, 134.17, 134.78,
135.05, 135.32, 146.75, 147.65, 155.56, 155.86, 170.26;
High Resolution ESI MS: m/z 883.2625 (calcd for
C52H42N4O6S2+H, 883.2624); Anal. Calcd for
C52H42N4O6S2: C, 70.73; H, 4.79; N, 6.34. Found: C,
70.62; H, 4.70; N, 6.28.
4.2.44. 5-Phenyl-20-(4-trifluoromethylphenyl)-10,15-
bis(4-carboxylatomethoxyphenyl)-21,23-dithiaporphyrin
(6). Yield: 90%; mp: 208–210 ꢁC; H NMR (400 MHz,
1
1:1 CDCl3/CD3OD): d 4.87 (4H, s), 7.34 (4H, d,
J = 8.4 Hz), 7.74–7.80 (3H, m), 8.02 (2H, d,
J = 8.0 Hz), 8.12 (4H, d, J = 8.4 Hz), 8.15–8.21 (2H,
m), 8.30 (2H, d, J = 7.6 Hz), 8.55 (1H, d, J = 4.4 Hz),
8.60–8.69 (3H, m), 9.54 (1H, d, J = 5.2 Hz), 9.65 (1H,
d, J = 4.8 Hz), 9.68 (2H, s); High Resolution Q-TOF
MS: m/z 887.1414 (calcd for C49H31F3N2O6S2+H,
887.1473); Anal. Calcd for C49H31F3N2O6S2: C, 68.04;
H, 3.61; N, 3.24. Found: C, 67.79; H, 3.37; N, 3.26.
4.2.40. 5,20-Bis(4-isopropylphenyl)-10,15-bis(4-carboxy-
latomethoxyphenyl)-21,23-dithiaporphyrin (10). Yield:
1
93%; mp: 220–222 ꢁC; H NMR (500 MHz, 1:1 CDCl3/
DMSO-d6): d 1.49 (12H, d, J = 7.0 Hz), 2.47–2.53 (2H,
m), 4.87 (4H, s), 7.36 (4H, d, J = 8.5 Hz), 7.65 (4H, d,
J = 7.5 Hz), 8.10 (4H, d, J = 8.0 Hz), 8.11 (4H, d,
J = 8.5 Hz), 8.12–8.21 (8H, m), 8.62 (2H, d, J = 4.5 Hz),
8.64 (2H, d, J = 4.5 Hz), 9.68 (2H, s), 9.68 (2H, s); 13C
NMR (75 MHz, 1:1 CDCl3/DMSO-d6): d 24.55, 34.20,
65.59, 114.27, 126.05, 133.90, 134.06, 134.44, 134.56,
134.78, 135.00, 135.63, 135.85, 135.98, 138.42, 147.76,
147.84, 148.94, 156.40, 156.46, 158.58, 170.79; High Res-
olution Q-TOF MS: m/z 881.2663 (calcd for
C54H44N2O6S2+H, 881.2719); Anal. Calcd for
C54H44N2O6S2: C, 73.61; H, 5.03; N, 3.18. Found: C,
73.34; H, 5.15; N, 3.08.
4.3. Photophysical properties
4.3.1. Determination of quantum yields for the generation
of singlet oxygen. The quantum yields for singlet oxygen
generation [/(1O2)] of 21,23-dithiaproprphyrins, 2–11,
were measured by direct methods in MeOH in a manner
identical to the determination of /(1O2) for 1.13,19
A
SPEX 270M spectrometer (Jobin Yvon) equipped with
InGaAs photodetector (Electrooptical 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 90ꢁ 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.
4.2.41. 5,20-Di(biphenyl)-10,15-bis(4-carboxylatometh-
oxyphenyl)-21,23-dithiaporphyrin (11). Yield: 85%; mp:
1
>300 ꢁC; H NMR (500 MHz, DMSO-d6): d 4.97 (4H,
s), 7.42 (4H, d, J = 8.0 Hz), 7.49 (2H, t, J = 7.0 Hz),
7.59 (4H, t, J = 7.0 Hz), 7.94 (4H, d, J = 7.0 Hz), 8.09
(4H, d, J = 7.5 Hz), 8.17 (4H, d, J = 8.0 Hz), 8.26 (4H,
d, J = 7.5 Hz), 8.65 (4H, s), 9.76 (4H, d, J = 4.5 Hz),
13.22 (2H, s); High Resolution Q-TOF MS: m/z
949.2401 (calcd for C60H40N2O6S2+H, 949.2397); Anal.
Calcd for C60H40N2O6S2: C, 75.93; H, 4.25; N, 2.95.
Found: C, 76.28; H, 4.24; N, 2.86.
4.3.2. Determination of quantum yields for fluorescence.
The quantum yields for fluorescence (/F) of the core-
modified porphyrins were measured in MeOH, as de-
scribed previously,20 and were compared to the standard
Rhodamine 6G (/F = 1.0). Steady-state fluorescence
spectra of the porphyrins were measured with excitation
at 532 nm using Fluorolog-3 spectrofluorometer (Jobin
Yvon).
4.2.42. 5,20-Bis(4-hydroxyphenyl)-10,15-bis(4-carboxyla-
tomethoxyphenyl)-21,23-dithiaporphyrin (9). Yield: 80%;
1
mp: >300 ꢁC; H NMR (500 MHz, DMSO-d6): d 4.98
(4H, s), 7.28 (4H, d, J = 8.0 Hz), 7.42 (4H, d,
J = 8.0 Hz), 8.06 (4H, d, J = 8.0 Hz), 8.17 (4H, d,
J = 7.5 Hz), 8.60 (2H, d, J = 4.0 Hz), 8.66 (2H, d,
J = 4.5 Hz), 9.71 (2H, s), 9.75 (2H, s), 10.07 (2H, s),
13.20 (2H, s); High Resolution Q-TOF MS: m/z
829.1673 (calcd for C48H32N2O8S2+H, 829.1678); Anal.
Calcd for C48H32N2O8S2: C, 69.55; H, 3.89; N, 3.38.
Found: C, 69.23; H, 3.83; N, 3.33.
4.3.3. Determination of n-octanol/water partition coeffi-
cients at pH 7.4. The n-octanol/water partition coeffi-
cients were determined at pH 7.4 using the absorbance
of the core-modified porphyrins. A ꢀshake-flaskꢁ direct
measurement17 with 3–5 min mixing, followed by a
4 h settling period, was used. Equilibration and mea-
surements were made at 23 ꢁC using a Perkin-Elmer
Lambda 12 spectrophotometer. Values are reported as
4.2.43. 5-Phenyl-20-(4-fluorophenyl)-10,15-bis(4-carboxy-
latomethoxyphenyl)-21,23-dithiaporphyrin (3). Yield:
log D7.4.
1
88%; mp: 218–220 ꢁC; H NMR (400 MHz, 1:1 CDCl3/
CD3OD): d 4.85 (4H, s), 7.33 (4H, d, J = 6.4 Hz), 7.43–
7.45 (2H, m), 7.73–7.79 (3H, m), 8.11 (4H, d,
J = 6.0 Hz), 8.13–8.20 (4H, m), 8.60–8.67 (3H, m), 9.57–
9.61 (1H, m), 9.62–9.66 (1H, m), 9.66–9.70 (2H, m); 13C
NMR (75 MHz, CDCl3): d 65.27, 113.73, 114.42 (d,
J = 22), 127.38, 128.06, 132.47, 133.78, 133.83, 134.04,
134.35, 134.48, 134.57, 135.33, 135.42, 135.50, 136.99,
140.95, 147.58, 147.99, 156.31, 156.44, 156.57, 158.08,
163.02 (d, J = 246), 171.11; High Resolution Q-TOF
MS: m/z 815.1666 (calcd for C48H31F1N2O6S2+H,
815.1686); Anal. Calcd for C48H31F1N2O6S2: C, 70.75;
H, 3.83; N, 3.44. Found: C, 71.13; H, 3.92; N, 3.46.
4.4. Biology
4.4.1. Cells and culture conditions. Cells cultured from
the rodent mammary adenocarcinoma (R3230AC) were
used for these studies. The R3230AC tumors were main-
tained by transplantation in the abdominal region of
100–120 g Fischer female rats, using the sterile trochar
technique described earlier by Hilf et al.21 R3230AC
cells were cultured from tumor homogenates using the
method described earlier.22 All cell lines were main-
tained in passage culture on 35 mm diameter polysty-
rene dishes (Becton Dickinson, Franklin Lakes, NJ) in

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
5979
3.0 mL of minimum essential medium (a-MEM) supple-
mented with 10% FBS, 50 units/mL penicillin G, 50 lg/
mL streptomycin, and 1.0 lg/mL Fungizone (complete
medium). Only cells from passages 1 to 10 were used
for experiments and cells from passages 1 to 4, stored
at À86 ꢁC, were used to initiate cultures. Cultures were
maintained at 37 ꢁC in a 5% CO2 humidified atmosphere
(Forma Scientific, Marietta, OH). Passage was accom-
plished by removing the culture medium, adding a
1.0 mL solution containing 0.25% trypsin, and incubat-
ing 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 3.0 mL a-MEM. Cell
counts were performed using a particle counter (model
ZM, Coulter Electronics, Hialeah, FL).
neither porphyrins nor light (control cells). Cell viabili-
ty, obtained for experimental samples, is expressed as
the percent of control cell counts.
4.4.4. Statistical analyses. All statistical analyses were
performed using Studentꢁs t-test for pairwise compari-
sons. A P value of <0.05 was considered significant.
Acknowledgments
We thank Dr. Tymish Y. Ohulchanskyy of the Institute
for Lasers, Photonics, and Biophotonics, The State Uni-
versity of New York at Buffalo, for helping in measuring
photophysical properties. This research was supported
by the Department of Defense [Breast Cancer Research
Program] under award number (W81XWH-04-1-0500).
Views and opinions of and endorsements by the
author(s) do not reflect those of the US Army or the
Department of Defense.
4.4.2. Incubation of cell cultures with dithiaporphyrins.
For experiments designed to determine the amount of
intracellular porphyrin after incubation with core-mod-
ified porphyrins, R3230AC cells were seeded on 96-well
plates as above. Compounds 1–11 were added at appro-
priate concentrations in complete medium 24 h after cell
seeding. Cells were incubated at 37 ꢁC in the dark for
various periods, the medium was removed, monolayers
were washed once with 0.9% NaCl, and 200 lL of a
25% solution of Scintigest (100% DMSO was used for
compound 9) was added to solubilize the cells. The
intracellular porphyrin content was determined using a
fluorescence multi-well plate reader (Molecular Devices,
Sunnyvale, CA) set at appropriate excitation and emis-
sion wavelengths. Intracellular dye concentration was
determined by comparing fluorescence values obtained
from solubilized cells with dye standards dissolved in
25% Scintigest (100% DMSO was used for compound
9). Data are expressed as femtomole porphyrin/cell.
References and notes
1. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori,
G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. J. Natl.
Cancer Inst. 1998, 90, 889.
2. Moan, J.; Peng, Q. Anticancer Res. 2003, 23, 3591.
3. Henderson, B. W.; Dougherty, T. J. Photochem. Photobiol.
1992, 55, 145.
4. Sharman, W. M.; Allen, C. M.; van Lier, J. E. Drug
Discovery Today 1999, 4, 507.
5. Detty, M. R. Expert Opin. Ther. Patents 2001, 11, 1849.
6. Levy, J. G.; Obochi, M. Photochem. Photobiol. 1996, 64,
737.
7. Pass, H. I. J. Natl. Cancer Inst. 1993, 85, 443.
8. Sternberg, E. D.; Dolphin, D.; Bruckner, C. Tetrahedron
1998, 54, 4151.
9. Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev.
Cancer 2003, 3, 380.
10. Bellnier, D. A.; Dougherty, T. J. J. Clin. Laser Med. Surg.
1996, 14, 311.
11. Detty, M. R.; Gibson, S. L.; Wagner, S. J. J. Med. Chem.
2004, 47, 3897.
12. Hilmey, D. G.; Abe, M.; Nelen, M. I.; Stilts, C. E.; Baker,
G. A.; Baker, S. N.; Bright, F. V.; Davies, S. R.; Gollnick,
S. O.; Oseroff, A. R.; Gibson, S. L.; Hilf, R.; Detty, M. R.
J. Med. Chem. 2002, 45, 449.
13. You, Y.; Gibson, S. L.; Hilf, R.; Davies, S. R.; Oseroff, A.
R.; Roy, I.; Ohulchanskyy, T. Y.; Bergey, E. J.; Detty, M.
R. J. Med. Chem. 2003, 46, 3734.
14. You, Y.; Gibson, S. L.; Hilf, R.; Ohulchanskyy, T. Y.;
Detty, M. R. Bioorg. Med. Chem. 2005, 13, 2235.
15. Shackley, D. C.; Whitehurst, C.; Moore, J. V.; George, N.
J.; Betts, C. D.; Clarke, N. W. BJU Int. 2000, 86, 638.
16. Weishaupt, K. R.; Gomer, C. J.; Dougherty, T. J. Cancer
Res. 1976, 36, 2326.
17. Sangster, J. In Octanol–Water Partition Coefficients:
Fundamentals and Physical Chemistry; Fogg, P. G. T.,
Ed.; John Wiley and Sons: New York, 1997.
18. Hansch, C.; Leo, A. Exploring QSAR: Fundamentals and
Applications in Chemistry and Biology; American Chemical
Society: Washington, DC, 1995.
For experiments designed to determine cell viability in
the presence of individual core-modified porphyrins 1–
11 in the dark or after light exposure, R3230AC cells
were seeded on 96-well plates at 1–1.5 · 104 cells/well
in complete medium. Cultures were then incubated for
24 h after which appropriate concentrations of 1–10
were added directly to the wells in complete medium.
4.4.3. Irradiation of cultured cells. Following incubation
of R3230AC cells with porphyrins, the medium was re-
moved, cells were washed once with 0.2 mL of 0.9%
NaCl and 0.2 mL of medium minus FBS, and phenol
red (clear medium) was added. Plates, with lids re-
moved, were positioned on an orbital shaker (LabLine,
Melrose Park, IL) and exposed for various times to
broadband visible light (350–750 nm) delivered at
1.4 mW cmÀ2 from a filtered 750 W halogen source
defocused to encompass the whole 96-well plate. The
culture plates were gently orbited on the shaker to en-
sure uniform illumination of all wells on the plate. The
clear medium was then removed, 0.2 mL of fresh com-
plete medium was added, and cultures were incubated
at 37 ꢁC for 24 h in the dark. Cell monolayers were also
maintained in the dark undergoing the same medium
changes and addition of dyes as those that were irradiat-
ed. Cell counts, as above, were performed on irradiated
cells, cells maintained in the dark or cells exposed to
19. Pandey, R. K.; Sumlin, A. B.; Constantine, S.; Aoudla,
M.; Potter, W. R.; Bellnier, D. A.; Henderson, B. W.;
Rodgers, M. A.; Smith, K. M.; Dougherty, T. J. Photo-
chem. Photobiol. 1996, 64, 194.

5980
Y. You et al. / Bioorg. Med. Chem. 13 (2005) 5968–5980
20. Baker, G. A.; Bright, F. V.; Detty, M. R.; Pandey, S.;
Stilts, C. E.; Yao, H. J. Porphyrins and Pthalocyanines
2000, 4, 669.
21. Hilf, R.; Michel, I.; Bell, C.; Freeman, J. J.; Borman, A.
Cancer Res. 1965, 25, 286.
22. Hissin, P. J.; Hilf, R. Cancer Res. 1978, 38, 3646.