Synthesis and evaluation of chalcogenopyrylium dyes as potential sensitizers for the photodynamic therapy of cancer

Article abstract of DOI:10.1021/jm990245q

A series of thiopyrylium (2), selenopyrylium (3), and telluropyrylium dyes (4) was prepared via the addition of Grignard reagents to either 2,6- di(4-dimethylamino)phenylchalcogenopyran-4-ones (5a) or 2-[4- (dimethylamino)phenyl]-6-phenylchalcogenopyran-4

Full text of DOI:10.1021/jm990245q

J . Med. Chem. 1999, 42, 3953-3964
3953
Syn th esis a n d Eva lu a tion of Ch a lcogen op yr yliu m Dyes a s P oten tia l Sen sitizer s
for th e P h otod yn a m ic Th er a p y of Ca n cer
Kristi A. Leonard,† Marina I. Nelen,† Todd P. Simard,† Sherry R. Davies,§ Sandra O. Gollnick,§ Allan R. Oseroff,§
Scott L. Gibson,‡ Russell Hilf,‡ Lan Bo Chen,| and Michael R. Detty*,†
Departments of Chemistry and Medicinal Chemistry, State University of New York at Buffalo, Buffalo, New York 14260,
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 607,
Rochester, New York 14642, Department of Dermatology and PDT Center, Roswell Park Cancer Institute, Elm and Carlton
Streets, Buffalo, New York 14263, and Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street,
Boston, Massachusetts 02115
Received May 17, 1999
A series of thiopyrylium (2), selenopyrylium (3), and telluropyrylium dyes (4) was prepared
via the addition of Grignard reagents to either 2,6-di(4-dimethylamino)phenylchalcogenopyran-
4-ones (5a ) or 2-[4-(dimethylamino)phenyl]-6-phenylchalcogenopyran-4-ones (5b) followed by
elimination and ion exchange to give the chloride salts. The absorption spectra and quantum
yields for singlet oxygen generation of these dyes suggested that the dyes would have utility
as sensitizers for PDT. Selenopyrylium dyes 3a and 3d with quantum yields for singlet oxygen
generation of 0.040 and 0.045, respectively, were phototoxic to Colo-26 cells in culture. The
toxicity of the dyes 2-4 was evaluated in clonogenic assays of human carcinoma cell lines.
Importantly, the presence of a sulfur, selenium, or tellurium heteroatom in the molecules had
no predictable impact on the toxicity of any particular dye set. Substituents at the 2-, 4-, and
6-positions of the dye had a much greater impact on cytotoxicity. The IC50 values determined
in the clonogenic assays did not correlate with chemical properties in the dye molecules such
as reduction potential or lipophilicity. Initial in vivo toxicity studies showed no toxicity for
these dyes at dosages between 7.2 and 38 µmol/kg in BALB/c mice.
In tr od u ction
The cationic sensitizers for PDT share structural
features with several classes of π-electron-delocalized,
lipophilic, cationic compounds (DLCs) that have been
evaluated as selective anticarcinoma agents. DLCs such
as rhodamine 123 (Rh-123),8 dequalinium chloride,9
thiacarbocyanines,10 Victoria blue BO (also a sensitizer
for PDT),2a tetraphenylphosphonium,11 rhodacyanines,12,13
and the thiopyrylium dye AA114 show selective toxicity
toward carcinoma cells relative to normal epithelial
cells. Like the cationic sensitizers for PDT, many of the
DLCs are targeted to the mitochondria of cells presum-
ably in response to the high negative charge in the
mitochondria. It is hypothesized that the difference in
transmitochondrial membrane potentials between nor-
mal epithelial cells and carcinoma cells may be respon-
sible for the increased uptake and prolonged retention
of cationic dyes and other DLCs in carcinoma cells.8,15
Photodynamic therapy (PDT) is a relatively recent
development in cancer therapy that has recently won
regulatory approval in the United States, Canada, The
Netherlands, France, Germany, and J apan for cancers
of the lung, digestive tract, and genitourinary tract.1 As
a therapy, PDT uses a light-activated sensitizer to
produce a cytotoxic reagent or a cytotoxic reaction in
the tumor cell, typically via generation of singlet oxygen
or superoxide from molecular oxygen.1a Photofrin, a
mixture of porphyrins derived from hematoporphyrin,
has received regulatory approval for use in PDT, but
the Photofrin mixture is not an ideal sensitizer. Photo-
frin and other porphyrin derivatives are only weakly
absorbing at wavelengths of light longer than 600 nm,
where penetration of light in tissue is optimal.
Cationic dyes have been explored as sensitizers for
PDT, and the classes explored include the Victoria blue
dyes,2 methylene blue3 and the related nile blue dyes,4
cyanine dyes,5 rhodacyanine dyes,6 and telluropyrylium
dyes.7 These classes of dyes have absorption maxima of
greater than 600 nm and have molar extinction co-
efficients of g104 M-1 cm-1, which makes them more
effective at harvesting light during PDT. Many of the
cationic dyes accumulate selectively in transformed cells
relative to normal cells with the mitochondria as
important cellular targets.2,3,5-7
Even though they accumulate selectively in tumors
in vivo, the chalcogenopyrylium dyes Rh-1238 and AA114
are poor sensitizers for PDT presumably due to their
low quantum yields for singlet oxygen generation.
Earlier studies with the chalcogenopyrylium dye series
1 (Chart 1) demonstrated that the heteroatom impacts
the rate of intersystem crossing to the triplet and,
consequently, the quantum yield for singlet oxygen
generation.2a,7 Those analogues incorporating at least
one selenium or tellurium atom are useful as PDT
sensitizers. By analogy, analogues of Rh-123 and AA1
incorporating the heavier chalcogen atoms should be
more efficient sensitizers for PDT. We have recently
described the synthesis of thiopyrylium dye 2a , sele-
nopyrylium dye 3a , and telluropyrylium dye 4a (Chart
* To whom correspondence should be addressed.
† State University of New York at Buffalo.
‡ University of Rochester Medical Center.
§ Roswell Park Cancer Institute.
| Dana-Farber Cancer Institute.
10.1021/jm990245q CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/03/1999

3954 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Leonard et al.
Ch a r t 1
Sch em e 2
variety of different groups at the 2-, 4-, and 6-posi-
tions.17,18 Thiopyranones, selenopyranones, and telluro-
pyranones 5 (X ) S, Se, Te) can be prepared from 1,4-
pentadiyn-3-ones 6 as a common intermediate through
the formal addition of hydrogen sulfide, selenide, and
telluride, respectively.18 Unsymmetrical diynones 6 (R1
* R2) are prepared by the formal coupling of a terminal
acetylene with an alkynyl aldehyde,19 while symmetrical
diynones 6 (R1 ) R2) are prepared by the formal
coupling of 2 equiv of a terminal acetylene and formal-
dehyde.17,20
Sch em e 1
2,6-Di[4-(dimethylamino)phenyl]chalcogenopyran-4-
ones 5a were prepared by the addition of disodium
chalcogenides to 1,5-di[4-(dimethylamino)phenyl]-1,4-
pentadiyn-3-one (6a ).16 Dyes 2a -4a were prepared by
the addition of 4-(dimethylamino)phenylmagnesium
bromide to chalcogenopyranones 5a (Scheme 2).16 The
intermediate alcohols were dehydrated with cold 10%
HPF6, and the hexafluorophosphate salts were ex-
changed for chloride on an ion-exchange resin. Following
the same sequence with methylmagnesium bromide
gave dyes 2b-4b in 68-74% isolated yields; with
phenylmagnesium bromide, dyes 2c-4c in 51-80%
isolated yields; with mesitylmagnesium bromide, dyes
2d -4d in 45-75% isolated yields; with 2-thienylmag-
nesium bromide, dye 4e in 67% isolated yield; with 3,5-
dimethylphenylmagnesium bromide, dyes 3f and 4f in
62% and 67% isolated yields, respectively; and with 3,5-
di(trifluoromethyl)phenylmagnesium bromide, dyes 3g
and 4g in 74% and 51% isolated yields, respectively.
Unsymmetrical dyes 2h -4h and 2i-4i were prepared
as shown in Scheme 3 from 1-[4-(dimethylamino)-
phenyl]-5-phenyl-1,4-pentadiyn-3-one (6b) as a common
intermediate. The addition of lithium 4-(dimethylami-
no)phenylacetylide20 to phenylpropargyl aldehyde fol-
lowed by oxidation of the intermediate diynol with
manganese dioxide gave diynone 6b in 67% overall
yield. 2-[4-(Dimethylamino)phenyl]-6-phenylchalcogeno-
pyran-4-ones 5b were prepared in two steps from 6b.
The initial step converted 6b to a mixture of enol ethers
through the addition of ethanol across one triple bond
in 0.2 N NaOEt in ethanol.16 The enol ether mixture
was then added to solutions of disodium chalcogenides
to give chalcogenopyranones 5b (X ) S, Se, Te) in 67-
73% isolated yields. The addition of 4-(dimethylamino)-
1) as analogues of the AA1 structure and have demon-
strated that all three dyes are photosensitizers in
vitro.16 The synthetic routes to 2a -4a also permit the
incorporation of a variety of different substituents
around the π-framework in addition to the heteroatom
changes in the chalcogenopyrylium ring.
In this article, we describe the synthesis of a series
of dyes related in structure to AA1 with varied substit-
uents at the 2-, 4-, and 6-positions of the thiopyrylium
(2), selenopyrylium (3), and telluropyrylium (4) nucleus.
The effects of structure on chemical and photophysical
properties important to PDT sensitizers (absorption
maxima, reduction potentials, quantum yields for sin-
glet oxygen generation) were evaluated. The effects of
heteroatom substitution in the chalcogenopyrylium ring
on toxicity were evaluated in vitro with a clonogenic
assay and in vivo with BALB/c mice.
Ch em ica l Resu lts a n d Discu ssion
Syn th esis. The thio-, seleno-, and telluropyrylium
dyes related to AA1 were prepared by a common
synthetic approach as shown in the retrosynthesis of
Scheme 1. The addition of Grignard reagents or orga-
nolithium compounds to the carbonyl carbon of chalco-
genopyranones 5 followed by acid-induced elimination
of water leads to chalcogenopyrylium dyes 2-4 with a

Chalcogenopyrylium Dyes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3955
Sch em e 3
Ch a r t 2
Earlier studies have shown that heteroatom substitu-
tion affects absorption maxima (λmax), with λmax increas-
ing in the following order: pyrylium < thiopyrylium <
selenopyrylium < telluropyrylium dyes.7,21 The thiopy-
rylium dyes of this study have values of λmax between
592 nm for 2j and 675 nm for 2c. The corresponding
selenopyrylium analogues 3 absorb at longer wave-
lengths (620-723 nm) and the telluropyrylium ana-
logues 4 at yet longer wavelengths (653-771 nm), which
is consistent with the earlier studies. All of the dyes 2-4
prepared in this study absorb light of appropriate
wavelengths useful for PDT.
The chalcogenopyrylium nucleus when drawn as an
alternating Hu¨ckel π-system has starred carbons at the
2-, 4-, and 6-positions as shown in structure I of Chart
2. Following empirical rules, electron-donating substit-
uents attached to the starred carbons should give
shorter wavelengths of absorption while electron-
withdrawing substituents should give longer absorption
maxima.21 The dyes 2a -2d , 3a -3g, and 4a -4g have
two 4-(dimethylamino)phenyl substituents at the 2- and
6-positions but vary the substituent in the 4-position.
In comparisons of λmax for these dyes, dyes with a
4-substituent more electron-donating than phenyl have
shorter values of λmax [4-(dimethylamino)phenyl, methyl,
mesityl, 3,5-dimethylphenyl] than dyes with a 4-sub-
stituent more electron-withdrawing than phenyl [2-
thienyl, 3,5-di(trifluoromethyl)phenyl].
While the 4-(dimethylamino)phenyl substituents are
electron-donating relative to a phenyl substituent, the
nitrogen atoms help delocalize the positive charge and
give cyanine character to the dye chromophores. Two
4-(dimethylamino)phenyl substituents can be placed at
the 2,4-positions or at the 2,6-positions as shown in
structures II and III, respectively, of Chart 2. The 2,6-
placement of these groups gives longer wavelengths of
absorption if one compares values of λmax for dyes 2c-
4c with dyes 2h -4h (Table 1). Dyes 2a -4a with three
4-(dimethylamino)phenyl substituents have absorption
maxima closer to values of λmax for 2h -4h with 2,4-di-
4-(dimethylamino)phenyl substituents relative to values
of λmax for 2c-4c with 2,6-di-4-(dimethylamino)phenyl
substituents. This observation suggests that resonance
contributions from structure II are dominant in the
chromophore of 2a -4a . With one 4-(dimethylamino)-
phenyl substituent, placement at the 2-position (dyes
2i-4i) gives longer wavelengths of absorption than
placement at the 4-position (dyes 2j-4j).
phenylmagnesium bromide and phenylmagnesium bro-
mide, respectively, to chalcogenopyranones 5b followed
by dehydration with 10% HPF6 and ion exchange gave
dyes 2h -4h and 2i-4i.
The presence of a nitrogen-containing functionality
appears to be important in cationic PDT sensitizers and
in DLCs that show either selective uptake in carcinoma
cells or selective toxicity toward carcinoma cells. Both
AA114 and the Victoria blue dyes2 have three nitrogen-
containing functional groups while Rh-123,8 methylene
blue3 and the related nile blue dyes,4 cyanine dyes,5
thiacarbocyanines,10 rhodacyanine dyes,6 and dequal-
inium salts9 have two. The dye series 2-4 incorporates
some systematic changes in the number and placement
of the nitrogen-containing functionality. Dyes 2a -4a
place three 4-(dimethylamino)phenyl substituents at the
2-, 4-, and 6-positions of the chalcogenopyrylium nucleus.
Dyes 2c-4c and 2h -4h place two 4-(dimethylamino)-
phenyl substituents at the 2- and 6-positions and at the
2- and 4-positions, respectively. Dyes 2i-4i and 2j-4j21
(Chart 1) place one 4-(dimethylamino)phenyl substitu-
ent at either the 2- or 4-position, respectively. Dyes 2b-
4b through dyes 2g-4g have two 4-(dimethylamino)-
phenyl substituents at the 2- and 6-positions as a
common structural feature while varying the substitu-
ents attached to the 4-position, which include alkyl, aryl,
and heteroaryl groups.
Absor p tion Sp ectr a of Dyes 2-4. For the dyes 2-4
to be useful as sensitizers for PDT, they must absorb
light of greater than 600 nm and produce a cytotoxic
species such as singlet oxygen upon irradiation.22 The
depth of nonthermal penetration of light in tissue is
greatest between 600 and 900 nm. At longer wave-
lengths, absorption of light by the infrared harmonics
of water both generates heat during excitation and
reduces the fraction of light reaching the sensitizer,
while at shorter wavelengths, absorption by biological
chromophores produces similar effects.23
Red u ction P oten tia ls of Dyes 2-4. One concern
with respect to the use of mitochondrial-specific, cationic
photosensitizers for PDT is toxicity derived from disrup-

3956 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Leonard et al.
Ta ble 1. Selected Physical and Photophysical Properties for
Chalcogenopyrylium Dyes 2-4
Ch a r t 3
a
dye
λmax
,
nm
E°, Vb (vs Fc/Fc+)
Φ(1O2) ( 2σ
2a
2b
2c
2d
2h
2i
594
655
675
665
627
624
592
631
669
704
695
689
723
657
654
620
672
703
738
727
746
729
771
689
693
653
-0.76
-0.71
-0.62
-0.67
-0.63
-0.45
-0.33
-0.70
-0.63
-0.55
-0.60
-0.55
-0.39
-0.55
-0.39
-0.31
-0.62
-0.58
-0.49
-0.50
-0.45
-0.49
-0.35
-0.47
-0.33
-0.24
0.011 ( 0.002
0.004 ( 0.001
0.025 ( 0.002
0.005 ( 0.001
0.004 ( 0.001
2j
3a
3b
3c
3d
3f
3g
3h
3i
0.040 ( 0.004
analogues 2-4 in this study would be expected to follow
a similar mechanism. The quantum yields for singlet
oxygen generation [Φ(1O2)] for many of the dyes 2-4
were measured and are compiled in Table 1.
0.015 ( 0.001
0.045 ( 0.002
Several generalizations can be made with respect to
the effects of substituents on values of Φ(1O2) for dyes
2-4. For a given set of substituents, the selenopyrylium
dye has a higher value of Φ(1O2) than the telluro-
pyrylium dye, which in turn has a higher value of
Φ(1O2) than the corresponding thiopyrylium dye. Pre-
sumably, the selenopyrylium nucleus is the best com-
promise for maximizing spin-orbit effects that promote
singlet oxygen generation while minimizing geometry
distortions as the larger chalcogens disrupt planarity
in the π-framework.16
The excited state of chalcogenopyrylium dyes 2-4 can
return to ground state via many processes including
intersystem crossing to the triplet, which can then
interact with ground-state (triplet) oxygen to produce
singlet oxygen and ground-state dye. One process that
returns excited state to ground state is the rotational
release of energy by the substituents. Compounds
bearing substituents that hinder free rotation around
the C-C bond joining the substituents to the ring have
higher values of Φ(1O2) than compounds with substit-
uents that do not hinder rotation.
2,4,6-Triphenylselenopyrylium hexafluorophosphate
has a value of Φ(1O2) of 0.003.24 In the excited state, a
phenyl substituent is free to rotate as shown illustrated
in structure IV of Chart 3. The addition of a dimethyl-
amino group on the phenyl ring produces higher values
of Φ(1O2) as found in dye 3i with a value of Φ(1O2) of
0.007 (Table 1) or dye 3j with a value of Φ(1O2) of
0.005.24 As shown in structure V of Chart 3, delocal-
ization of the positive charge to nitrogen would create
partial double-bond character to the C-C bond joining
the substituent to the ring. The hindered rotation about
the C-C bond increases the relative amount of inter-
system crossing to the triplet and the value of Φ(1O2).
Dyes 3c and 3h with two 4-(dimethylmino)phenyl
substituents have higher values of Φ(1O2) of 0.015.
Selenopyrylium dye 3a bearing three 4-(dimethyl-
amino)phenyl substituents has an even higher value of
Φ(1O2) of 0.040 (Table 1).
Replacing the phenyl group of 3c with a mesityl group
in dye 3d increases the value of Φ(1O2) to 0.045. The
mesityl group also hinders rotation about the C-C bond
joining the substituent to the ring through the steric
interactions of the methyl groups as shown in structure
VI of Chart 3. The steric hindrance to rotation leads to
a 3-fold increase in Φ(1O2) for 3d relative to 3c.
The same trends are observed with thiopyrylium dyes
2 and telluropyrylium dyes 4. However, both of these
heterocyclic systems have lower values of Φ(1O2) relative
to the corresponding selenopyrylium dye.
0.015 ( 0.001
0.007 ( 0.001
0.005 ( 0.002c
0.018 ( 0.003
3j
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.007 ( 0.002
0.027 ( 0.002
0.006 ( 0.002
0.010 ( 0.001
0.004 ( 0.001
0.004 ( 0.001c
4j
a
b
In CH2Cl2. In CH2Cl2 at a Pt disk electrode with a scan rate
of 0.1 V s-1 with 0.2 M Bu4NBF4 as supporting electrolyte.
c Reference 23.
tion of the redox cascade in the mitochondria. Typically,
cationic dyes are good electron acceptors, and electron
transfer within the mitochondria should be possible. The
reduction potentials of dyes 2-4 were determined by
cyclic voltammetry, and values of E° for the cation/
neutral radical couple are compiled in Table 1. As is
observed with other chalcogenopyrylium dyes, replacing
a heavier chalcogen atom with a lighter chalcogen atom
in the ring leads to a cathodic (negative) shift in
reduction potential for all substitution patterns shown
in Table 1.21 Electron-donating groups give cathodic
shifts in reduction potentials (more negative values of
E°), while electron-withdrawing groups give anodic
shifts. The dye series 2-4 covers a 0.52-V range in
values of E° from -0.24 V for 4j to -0.76 V for 2a [vs
ferrocene/ferricinium (Fc/Fc+) at +0.40 V].
n -Octa n ol/Wa ter P a r tition Coefficien ts for Dyes
2-4. The dyes 2-4 all had similar values of log P, where
P is the n-octanol/water partition coefficient. The range
of values was 2.1-2.4 with the exception of dyes 3g and
4g with 3,5-di(trifluoromethyl)phenyl substituents at
the 4-position where log P was 2.9 for both. These values
can be compared to values of log P of 1.5 for Rh-123,
1.9 for AA1, and 2.4 for dye 1 (X ) Te, Y ) Se).
Qu a n tu m Yield s for Sin glet Oxygen Gen er a tion .
Many of the cationic photosensitizers for PDT such as
the Victoria blues,2 methylene blues3 and the related
nile blues,4 cyanine dyes,5 rhodacyanine dyes,6 and
chalcogenopyrylium dyes 17 generate singlet oxygen
upon irradiation in aerated solvents. Singlet oxygen
production upon irradiation is thought to be responsible
for part if not all of the biological damage that is
observed from PDT with these sensitizers both in vitro
and in vivo. We have recently shown that the photo-
damage produced in vitro from dyes 2a -4a is derived
from the production of singlet oxygen.16 The other

Chalcogenopyrylium Dyes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3957
Although values of Φ(1O2) are small for dyes 2-4
relative to solution values of Φ(1O2) for other photosen-
sitizers for PDT, the effects of rigidization in biological
membranes on values of Φ(1O2) have not been deter-
mined.25 Rotational degrees of freedom in rigid, planar
molecules such as the porphyrins, methylene blues, and
nile blues are few, and the photophysics of these
sensitizers will be little affected when the sensitizers
reside in biological membranes. If the dyes 2-4 were
immobilized in a biological membrane, rotational de-
grees of freedom would be greatly reduced and effective
values of Φ(1O2) would be higher than solution values.
Several of the chalcogenopyrylium dyes 1 with solution
values of Φ(1O2) < 0.01 have performed as efficient
photosensitizers in vitro.2a,7a
Biologica l Resu lts a n d Discu ssion
In Vitr o Clon ogen ic Assa y. The toxicity of the
individual chalcogenopyrylium dyes 2-4 will impact
selection of sensitizer candidates for PDT. In particular,
little is known about the relationship of the heteroatom
in the chalcogenopyrylium dyes with the acute and
chronic toxicities of these materials. As a first step in
developing the comparative toxicity of the chalcogeno-
pyrylium dyes, dyes 2-4 were evaluated for their
inhibitory effects on the growth of several human
carcinoma cell lines: colon carcinoma (CX-1), prostate
carcinoma (PC-3), and breast carcinoma (MDA-435). In
each case, the cells were incubated with one of the dyes
2-4 for 24 h and then in dye-free medium for 2 weeks.
The number of colonies was measured by a crystal
violet-staining method, and the results are expressed
as the IC50 value and are compiled in Table 2 (Support-
ing Information). The magnitude of the IC50 value for
each dye is a direct measure of toxicity.
For any particular substitution pattern at the 2-, 4-,
and 6-positions, thiopyrylium dye 2, selenopyrylium dye
3, and telluropyrylium dye 4 all had similar IC50 values
(within a factor of 3) toward any particular cell line as
shown. The one exception to this generalization was the
series 2i-4i in which the IC50 values for thiopyrylium
analogue 2i indicated 3-9-fold greater toxicity than that
found for the telluropyrylium analogue 4i for all three
cell lines. In general, however, the heteroatom had no
predictable impact on toxicity in vitro.
The selection of substituents at the 2-, 4-, and 6-posi-
tions of dyes 2-4 has more of an impact on toxicity than
the heteroatom. Substituent changes in the seleno-
pyrylium dyes 3 and telluropyrylium dyes 4 gave a
greater than 200-fold range of IC50 values among the
cell lines of Table 2. In general, the 4-methylchalco-
genopyrylium salts 3b and 4b displayed the least
toxicity against all three cell lines relative to the other
dyes, while the 2,6-di[4-(dimethylamino)phenyl]-4-
phenylchalcogenopyrylium salts 2c-4c displayed the
most.
Electr och em ica l Red u ction P oten tia ls a n d in
Vitr o Toxicity. In the search for selective anticarci-
noma agents, specific interactions of some structural
feature in the drug with the particular carcinoma cell
line can give greater specificity than toxicity derived
from an intrinsic chemical property such as reduction
potential or the n-octanol/water partition coefficient.
There are only small differences in lipophilicity among
F igu r e 1. Plot of log IC50 values (µM in dye) for dyes 2-4
against CX-1 cells as determined by clonogenic assay as a
function of the electrochemical reduction potential E° (V vs
Fc/Fc+) for dyes 2-4.
the dyes 2-4 (with the exception of 3g and 4g).
However, reduction potentials vary by 0.52 V among the
dyes. With cationic dyes and other DLCs targeted to the
mitochondria, electron transfer to the dye or DLC can
disrupt mitochondrial processes and give cell death.
Figure 1 shows a plot of IC50 values for dyes 2-4 against
the CX-1 cell line as a function of reduction potential
(E°). The lack of an apparent correlation suggests that
differences in IC50 values can be attributed to specific
drug-cell interactions in the clonogenic assays.
In itia l in Vivo Toxicity Stu d ies. The mitochondrial
accumulation of cationic sensitizers for PDT or DLCs
as selective anticarcinoma agents raises concerns that
such materials will have high dark or inherent toxicity.
In addition, cationic dyes are typically good electron
acceptors and might be expected to interfere with
electron transport at high concentrations. Nonlethal
doses of 5-10 mg (13-26 µmol)/kg/day of Rh-123 have
been administered for 10 consecutive days.8c Useful
dosages of cationic sensitizers for use in PDT are
typically much smaller and are delivered as a single
dosage in the 5-10 µmol/kg range.1,22
With selenopyrylium dyes 3 and telluropyrylium dyes
4, the toxicity associated with the heteroatom is an
added concern. Selenium is an essential trace element
and has been the subject of numerous toxicity stud-
ies.26,27 One is tempted to assume that since tellurium
is below selenium in the periodic table, it must be more
metallic and consequently more toxic. Toxicity with
respect to tellurium metal, which is in commercial use
in the semiconductor and reprographic industries, has
been reviewed, and in general, tellurium has been found
to be less toxic than selenium.26,27 Daily ingestion of
tellurium from air, food, and fluids is estimated to be
as high as 0.6 mg (4-5 µmol)/day in some areas for a
70-kg adult.26 Other tellurium-containing compounds
have emerged in recent years as potential drugs and
have shown acceptable toxicity in animal studies.28
Ammonium trichloro(dioxoethylene-O,O′)tellurate (AS-
101), an inorganic tellurate complex, was shown to have

3958 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Leonard et al.
Su m m a r y a n d Con clu sion s
A series of thiopyrylium, selenopyrylium, and telluro-
pyrylium dyes (2-4, respectively) with diverse substit-
uents was prepared. The absorption spectra and quan-
tum yields for singlet oxygen generation of these dyes
suggested that the dyes would have utility as sensitizers
for PDT. Dyes 3a and 3d with the highest quantum
yields for singlet oxygen generation were found to be
photosensitizers in vitro against Colo-26 cells. The dark
toxicity of the dyes 2-4 was evaluated in clonogenic
assays of human carcinoma cell lines and normal
monkey epithelial cells in order to compare the relative
toxicity of thiopyrylium, selenopyrylium, and telluro-
pyrylium analogues with identical carbon π-frameworks.
Importantly, the presence of a sulfur, selenium, or
tellurium heteroatom in the molecules had no predict-
able impact on the dark toxicity of any particular dye
set. Substituents at the 2-, 4-, and 6-positions of the dye
had a much greater impact on cytotoxicity. The IC50
values determined in the clonogenic assays did not
correlate with chemical properties in the dye molecules
such as reduction potential or lipophilicity. Initial in
vivo toxicity studies showed little toxicity at 7.2-38
µmol/kg dosages in BALB/c mice.
F igu r e 2. Effects of selenopyrylium dye 3a or 3d photosen-
sitization on the cell viability of cultured Colo-26 tumor cells.
Details of experimental conditions are described in the Ex-
perimental Section. Data are expressed as the fraction of viable
cells compared to untreated cells (O) and for cells treated with
1 µM solutions of dye 3a (b) or dye 3d (9) and light. Each
datum point represents the mean percent viable cells calcu-
lated from at least 3 separate experiments; bars are the
standard deviation.
Exp er im en ta l Section
Gen er a l Meth od s. Solvents and reagents were used as
received from Sigma-Aldrich Chemical Co. (St. Louis, MO)
unless otherwise noted. Cell culture media was purchased from
GIBCO (Grand Island, NY). Fetal bovine serum (FBS) was
obtained from Atlanta Biologicals (Atlanta, GA). Concentration
in vacuo was performed on a Bu¨chi rotary evaporator. NMR
spectra were recorded on a Varian Gemini-300, Inova 400, or
Inova 500 instrument with residual solvent signal as internal
standard: CDCl3 (δ 7.26 for proton, δ 77.0 for carbon). Infrared
spectra were recorded on a Perkin-Elmer FT-IR instrument.
UV-visible near-IR spectra were recorded on a Perkin-Elmer
Lambda 12 spectrophotometer or on a Sequential DX17 MV
stopped-flow spectrometer (Applied Photophysics, Leather-
head, U.K.). Both were equipped with a circulating constant-
temperature bath for the sample chambers. Elemental analy-
ses were conducted by Atlantic Microanalytical, Inc. Dyes were
analyzed as the hexafluorophosphate salts since the chloride
salts were hygroscopic.
both antitumor and immunomodulatory activity in
vivo.28
Dyes 2-4 were evaluated for dark toxicity in BALB/c
mice given a single tail-vein injection of dye dissolved
in 1% Tween 80 in saline. Generally, groups of five mice
were tested with escalating concentrations of dye and
followed for 60 days after injection. The maximum dye
dose was determined by its solubility in the 1% Tween
80/saline vehicle. For selenopyrylium dye 3h , no toxicity
was observed with 7.7 mg (19 µmol) of dye/kg of animal.
For dyes 2a -4a , no toxicity was observed following
injection of 4.0 mg (8.3 µmol) of thiopyrylium dye 2a /
kg, 20 mg (38 µmol) of selenopyrylium dye 3a /kg, and
7.0 mg (12 µmol) of telluropyrylium dye 4a /kg. However,
a group of three animals given a dosage of 27 mg (50.3
µmol) of selenopyrylium dye 3a /kg had no surviving
animals after 48 h. For dyes 2d -4d , no toxicity was
observed following injection of 4.4 mg (9.2 µmol) of
thiopyrylium dye 2d /kg (10 animals), 8.6 mg (16 µmol)
of selenopyrylium dye 3d /kg, and 11.0 mg (19 µmol) of
telluropyrylium dye 4d /kg. For telluropyrylium dye 4g,
no toxicity was observed at 5.0 mg (7.2 µmol) of dye/kg.
P h otod yn a m ic Th er a p y a ga in st Colo-26 Cells in
Cu ltu r e. Dyes 3a and 3d had the two highest quantum
yields for singlet oxygen generation (0.040 and 0.045,
respectively) in the series of dyes 2-4 (Table 1) and
were not dark toxic in vivo at 10 µmol of dye/kg, a typical
therapeutic dosage for PDT. The dyes were tested as
PDT photosensitizers in vitro against murine Colo-26
cells. Cell cultures incubated for 2 h with 1 µM concen-
trations of either dye 3a or 3d displayed essentially no
dark toxicity associated with the dye. As shown in
Figure 2, irradiation of dye-treated cells resulted in a
light-dose-dependent reduction of cell viability that was
significantly greater than that observed for irradiation
of nontreated cells (P < 0.001 at 30 J cm-2).
2,6-Di[4-(d im eth yla m in o)p h en yl]-4-m eth ylth iop yr yli-
u m Ch lor id e (2b). Methylmagnesium bromide (1.4 M in THF,
1.0 mL) was added dropwise to a solution of thiopyranone 5a
(0.150 g, 0.428 mmol) in THF (10 mL) under an argon
atmosphere. The resulting mixture was heated at reflux for 3
h, then cooled. The reaction mixture was concentrated. The
residue was dissolved in acetic acid (10 mL) and added
dropwise to 10% HPF6 (10 mL) at 0 °C. The product was
collected by filtration and washed with water (4 × 10 mL) and
ether (4 × 10 mL). The solid was redissolved in a small amount
of acetone, added to H2O (200 mL), and recollected by filtration.
The crude product was dissolved in 10 mL of methanol and
0.3 g of Amberlite IRA-400 Cl ion-exchange resin was added.
The resulting mixture was stirred 0.5 h and the exchange resin
was removed via filtration. The process was repeated with two
additional 0.3-g aliquots of the ion-exchange resin. The product
was recrystallized from CH2Cl2/ether to give 0.122 g (74%) of
1
2b: mp 228-231 °C; H NMR (CD2Cl2) δ 7.83 (m, 6 H), 6.85
(“doublet” AA′BB′, 4 H, J ) 8.7 Hz), 3.16 (s, 12 H), 2.70 (s, 3
H); 13C NMR (CD2Cl2) δ 161.1, 155.5, 151.8, 135.5, 129.5, 127.3,
127.0, 125.7, 120.1, 118.1, 110.2, 37.6; λmax (CH2Cl2) 655 nm
[ꢀ (5.5 ( 0.2) × 104 M-1 cm-1]. Anal. as the hexafluorophos-
phate salt (C22H25F6N2PS) C, H, N.
2,6-Di[4-(d im eth yla m in o)p h en yl]-4-m eth ylselen op yr y-
liu m Ch lor id e (3b). Methylmagnesium bromide (1.4 M in
THF, 1.0 mL) was added dropwise to a solution of selenopy-

Chalcogenopyrylium Dyes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3959
ranone 5a (0.150 g, 0.378 mmol) in THF (10 mL) under an
argon atmosphere. The resulting mixture was treated as
described above for 2b. The product was recrystallized from
CH2Cl2/ether to give 0.111 g (68%) of 3b: mp 229-231 °C; 1H
NMR (CD2Cl2) δ 7.77 (m, 6 H), 6.84 (“doublet” AA′BB′, 4 H, J
) 9.0 Hz), 3.15 (s, 12 H), 2.63 (s, 3 H); λmax (CH2Cl2) 669 nm
[ꢀ (7.5 ( 0.2) × 104 M-1 cm-1]. Anal. as the hexafluorophos-
phate salt (C22H25F6N2PSe) C, H, N.
2,6-Di[4-(d im e t h yla m in o)p h e n yl]-4-m e t h ylt e llu r o-
p yr yliu m Ch lor id e (4b). Methylmagnesium bromide (1.4 M
in THF, 1.0 mL) was added dropwise to a solution of telluro-
pyranone 5a (0.100 g, 0.224 mmol) in THF (10 mL) under an
argon atmosphere. The resulting mixture was treated as
described above for 2b. The product was recrystallized from
CH2Cl2/ether to give 0.075 g (70%) of 4b: mp 238-240 °C; 1H
NMR (CD2Cl2) δ 7.74 (s, 2 H), 7.66 (“doublet” AA′BB′, 4 H, J
) 9.0 Hz), 6.81 (“doublet” AA′BB′, 4 H, J ) 9.0 Hz), 3.13 (s,
12 H), 2.53 (s, 3 H); 13C NMR (CD2Cl2) δ 171.9, 158.6, 152.4,
127.4, 126.2, 124.7, 110.4, 37.6, 26.4; λmax (CH2Cl2) 703 nm [ꢀ
(7.1 ( 0.2) × 104 M-1 cm-1]. Anal. as the hexafluorophosphate
salt (C22H25F6N2PTe) C, H, N.
[ꢀ (5.79 ( 0.02) × 104 M-1 cm-1]. Anal. as the hexafluorophos-
phate salt (C27H27F6N2PTe) C, H, N.
2,6-Di[4-(d im et h yla m in o)p h en yl]-4-(2,4,6-t r im et h yl-
p h en yl)th iop yr yliu m Ch lor id e (2d ). A mixture of bro-
momesitylene (0.24 g, 0.180 mL, 1.2 mmol) and magnesium
turnings (0.060 g, 2.5 mg-at) was heated at reflux in anhydrous
THF (50 mL) for 3 h. A solution of thiopyranone 5a (0.20 g,
0.62 mmol) in 5 mL of anhydrous THF was added dropwise to
the reaction mixture and the resulting solution was heated at
reflux for an additional hour. The reaction mixture was
concentrated. The residue was dissolved in acetic acid (10 mL)
and added dropwise to 10% HPF6 (10 mL) at 0 °C. The product
was collected by filtration and washed with water (4 × 10 mL)
and ether (4 × 10 mL). The solid was redissolved in a small
amount of acetone, added to H2O (200 mL), and recollected by
filtration. The crude product was dissolved in 10 mL of
methanol and 0.3 g of Amberlite IRA-400 Cl ion-exchange resin
was added. The resulting mixture was stirred 0.5 h and the
exchange resin was removed via filtration. The process was
repeated with two additional 0.3-g aliquots of the ion-exchange
resin. The product was recrystallized from CH3CN to give 0.24
g (66%) of 2d : mp 209-211 °C; 1H NMR (CD2Cl2) δ 7.85
(“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 7.69 (s, 2 H), 7.03 (s, 2
H), 6.87 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 3.17 (s, 12 H),
2.36 (s, 3 H), 2.17 (s, 6 H); 13C NMR (CD2Cl2) δ 164.5, 160.8,
154.9, 140.0, 136.6, 134.8, 130.0, 129.5, 125.0, 120.7, 113.3,
40.6, 21.4, 20.1; λmax (CH2Cl2) 665 nm [ꢀ (7.2 ( 0.1) × 104 M-1
cm-1]. Anal. as the hexafluorophosphate salt (C30H33F6N2PS-
H2O) C, H, N.
2,6-Di[4-(d im eth yla m in o)p h en yl]-4-p h en ylth iop yr yli-
u m Ch lor id e (2c). A mixture of bromobenzene (0.060 mL,
0.57 mmol) and magnesium turnings (0.027 g, 1.1 mg-at) was
heated at reflux in THF (10 mL) for 3 h. A solution of
thiopyranone 5a (0.100 g, 0.285 mmol) in 5 mL of anhydrous
THF was added dropwise to the reaction mixture and the
resulting solution was heated at reflux for an additional hour.
The reaction mixture was concentrated. The residue was
dissolved in acetic acid (10 mL) and added dropwise to 10%
HPF6 (10 mL) at 0 °C. The product was collected by filtration
and washed with water (4 × 10 mL) and ether (4 × 10 mL).
The solid was redissolved in a small amount of acetone, added
to H2O (200 mL), and recollected by filtration. The crude
product was dissolved in 10 mL of methanol and 0.3 g of
Amberlite IRA-400 Cl ion-exchange resin was added. The
resulting mixture was stirred 0.5 h and the exchange resin
was removed via filtration. The process was repeated with two
additional 0.3-g aliquots of the ion-exchange resin. The product
was recrystallized from CH3CN to give 0.102 g (80%) of 2c:
2,6-Di[4-(d im et h yla m in o)p h en yl]-4-(2,4,6-t r im et h yl-
p h en yl)selen op yr yliu m Ch lor id e (3d ). A mixture of bro-
momesitylene (0.33 g, 0.29 mL, 1.5 mmol) and magnesium
turnings (0.080 g, 3.0 mg-at) was heated at reflux in anhydrous
THF (50 mL) for 5 h. A solution of selenopyranone 5a (0.33 g,
0.83 mmol) in 30 mL of anhydrous THF was added dropwise
to the reaction mixture and the resulting solution was treated
as described above for 2d . The product was recrystallized from
1
CH3CN to give 0.24 g (45%) of 3d : mp 209-211 °C; H NMR
(CD2Cl2) δ 7.87 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 7.57 (s, 2
H), 7.02 (s, 2 H), 6.85 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz),
3.15 (s, 12 H), 2.36 (s, 3 H), 2.15 (s, 6 H); 13C NMR (CD2Cl2) δ
167.0, 156.1, 150.3, 134.9, 133.2, 129.9, 125.4, 124.65, 120.35,
118.5, 108.7, 35.9, 16.65, 16.1; λmax (CH2Cl2) 695 nm [ꢀ (6.3 (
0.1) × 104 M-1 cm-1]. Anal. as the hexafluorophosphate salt
(C30H33F6N2PSe) C, H, N.
1
mp 238-240 °C; H NMR (CD2Cl2) δ 8.15 (s, 2 H), 7.86 (m, 6
H), 7.66 (m, 3 H), 6.88 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz),
3.18 (s, 12 H); 13C NMR (CD2Cl2) δ 161.1, 155.5, 151.8, 135.5,
129.5, 127.3, 127.0, 125.7, 120.1, 118.1, 110.2, 37.6; λmax (CH2-
Cl2) 675 nm [ꢀ (4.8 ( 0.1) × 104 M-1 cm-1]. Anal. as the
hexafluorophosphate salt (C27H27F6N2PS) C, H, N.
2,6-Di[4-(d im et h yla m in o)p h en yl]-4-(2,4,6-t r im et h yl-
p h en yl)tellu r op yr yliu m Ch lor id e (4d ). A mixture of bro-
momesitylene (0.29 g, 0.22 mL, 1.0 mmol) and magnesium
turnings (0.060 g, 2.5 mg-at) was heated at reflux in anhydrous
THF (50 mL) for 4 h. A solution of telluropyranone 5a (0.33 g,
0.74 mmol) in 10 mL of anhydrous THF was added dropwise
to the reaction mixture and the resulting solution was treated
as described above for 2d . The product was recrystallized from
2,6-Di[4-(d im eth yla m in o)p h en yl]-4-p h en ylselen op yr y-
liu m Ch lor id e (3c). A mixture of bromobenzene (0.060 mL,
0.57 mmol) and magnesium turnings (0.036 g, 1.5 mg-at) was
heated at reflux in THF (10 mL) for 3 h. A solution of
selenopyranone 5a (0.150 g, 0.378 mmol) in 5 mL of anhydrous
THF was added dropwise to the reaction mixture and the
resulting solution was treated as described above for 2c. The
product was recrystallized from CH3CN to give 0.095 g (51%)
of 3c: mp >260 °C; 1H NMR (CD2Cl2) δ 8.03 (s, 2 H), 7.81 (m,
5 H), 7.64 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 6.85 (“doublet”
AA′BB′, 4 H, J ) 9.2 Hz), 3.16 (s, 12 H); 13C NMR (CD2Cl2) δ
167.6, 155.2, 151.8, 136.9, 128.9, 127.2, 127.1, 125.7, 120.4,
119.9, 110.3, 37.6; λmax (CH2Cl2) 704 nm [ꢀ (6.8 ( 0.2) × 104
M-1 cm-1]. Anal. as the hexafluorophosphate salt (C27H27F6N2-
PSe) C, H, N.
1
CH3CN to give 0.38 g (75%) of 4d : mp 238-240 °C; H NMR
(CD2Cl2) δ 7.67 (“doublet” AA′BB′, 4 H, J ) 9 Hz), 7.54 (s, 2
H), 7.01 (s, 2 H), 6.78 (“doublet” AA′BB′, 4 H, J ) 9 Hz), 3.12
(s, 12 H), 2.36 (s, 3 H), 2.13 (s, 6 H); 13C NMR (CD2Cl2) δ 169.9,
156.9, 150.4, 135.2, 129.6, 125.8, 124.6, 123.6, 123.1, 108.8,
35.95, 16.4, 16.0; λmax (CH2Cl2) 727 nm [ꢀ (6.9 ( 0.1) × 104
M-1 cm-1]. Anal. as the hexafluorophosphate salt (C30H33F6N2-
PTe) C, H, N.
2,6-Di[4-(d im e t h yla m in o)p h e n yl]-4-p h e n ylt e llu r o-
p yr yliu m Ch lor id e (4c). A mixture of bromobenzene (0.14
g, 0.89 mmol) and magnesium turnings (0.044 g, 1.8 mg-at)
was heated at reflux in THF (10 mL) for 3 h. A solution of
telluropyranone 5a (0.200 g, 0.448 mmol) in 5 mL of anhydrous
THF was added dropwise to the reaction mixture and the
resulting solution was treated as described above for 2c. The
product was recrystallized from CH3CN to give 0.144 g (59%)
of 4c: mp 250-252 °C; 1H NMR (CD2Cl2) δ 7.98 (s, 2 H), 7.78
(m, 3 H), 7.69 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 7.62 (m, 2
H), 6.83 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 3.14 (s, 12 H);
13C NMR (CD2Cl2) δ 171.5, 157.6, 152.2, 140.0, 128.3, 127.6,
127.2, 125.4, 125.3, 124.9, 110.5, 37.7; λmax (CH2Cl2) 738 nm
2,6-Di[4-(d im eth yla m in o)p h en yl]-4-(2-th ien yl)tellu r o-
p yr yliu m Ch lor id e (4e). A mixture of 2-bromothiophene
(0.274 g, 1.68 mmol) and magnesium turnings (0.082 g) was
heated at reflux in anhydrous THF (30 mL) under argon
atmosphere for 3 h. A solution of telluropyranone 5a (0.300 g,
0.673 mmol) in THF (20 mL) was added dropwise to the
reaction mixture and the resulting mixture was heated at
reflux for an additional 1 h. The reaction mixture was
concentrated. The residue was dissolved in acetic acid (10 mL)
and added dropwise to 10% HPF6 (10 mL) at 0 °C. The product
was collected by filtration and washed with water (4 × 10 mL)
and ether (4 × 10 mL). The solid was redissolved in a small
amount of acetone, added to H2O (200 mL), and recollected by

3960 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Leonard et al.
filtration. The crude product was dissolved in 10 mL of
methanol and 0.3 g of Amberlite IRA-400 Cl ion-exchange resin
was added. The resulting mixture was stirred 0.5 h and the
exchange resin was removed via filtration. The process was
repeated with two additional 0.3-g aliquots of the ion-exchange
resin. The product was recrystallized from CH3CN to give
0.248 g (67%) of 4e: mp 244-246 °C; 1H NMR (CD2Cl2) δ 8.10
(s, 2 H), 8.03 (“doublet” AA′BB′, 4 H, J ) 9 Hz), 7.82 (“doublet”
AA′BB′, 4 H, J ) 9 Hz), 7.65 (“doublet” AA′BB′, 4 H, J ) 8.7
Hz), 7.31 (t, 1 H, J ) 9 Hz), 6.81 (“doublet” AA′BB′, 4 H, J )
8.7 Hz), 3.13 (s, 12 H); 13C NMR (CD2Cl2) δ 173.9, 156.8, 153.0,
147.8, 134.5, 133.5, 132.8, 132.5, 129.9, 126.1, 115.3, 42.7; λmax
(CH2Cl2) 746 nm [ꢀ (9.4 ( 0.7) × 104 M-1 cm-1]. Anal. as the
hexafluorophosphate salt (C25H25F6N2PSTe‚1/4CH3CN) C, H,
N.
250 °C; 1H NMR (CD2Cl2) δ 8.39 (s, 2 H), 8.24 (s, 1 H), 8.01 (s,
2 H), 7.85 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 6.85 (“doublet”
AA′BB′, 4 H, J ) 9 Hz), 3.13 (s, 12 H); 13C NMR (CD2Cl2) δ
165.7, 149.7, 149.3, 137.0, 126.5 (q), 124.8, 123.9, 119.1, 118.0,
117.8, 116.3, 107.7, 34.5; λmax (CH2Cl2) 723 nm [ꢀ (4.5 ( 0.1)
× 104 M-1 cm-1]. Anal. as the hexafluorophosphate salt
(C29H25F12N2PSe) C, H, N.
2,6-Di[4-(d im et h yla m in o)p h en yl]-4-[3,5-d i(t r iflu or o-
m eth yl)p h en yl]tellu r op yr yliu m Ch lor id e (4g). A mixture
of bromo-3,5-di(trifluoromethyl)benzene (0.50 g, 1.7 mmol) and
magnesium turnings (0.080 g, 3.3 mg-at) was heated at reflux
in anhydrous THF (20 mL) for 5 h. A solution of telluropyra-
none 5a (0.33 g, 0.83 mmol) in 30 mL of anhydrous THF was
added dropwise to the reaction mixture and the resulting
solution was treated as described above for 3g. The product
was recrystallized from CH3CN to give 0.30 g (51%) of 4 g:
2,6-Di[4-(d im e t h yla m in o )p h e n y l]-4-(3,5-d im e t h yl-
p h en yl)selen op yr yliu m Ch lor id e (3f). A mixture of bromo-
3,5-dimethylbenzene (0.37 g, 2.0 mmol) and magnesium turn-
ings (0.10 g, 4.4 mg-at) was heated at reflux in anhydrous THF
(50 mL) for 5 h. A solution of selenopyranone 5a (0.33 g, 0.83
mmol) in 30 mL of anhydrous THF was added dropwise to the
reaction mixture and the resulting solution was heated at
reflux for an additional hour. The reaction mixture was
concentrated. The residue was dissolved in acetic acid (10 mL)
and added dropwise to 10% HPF6 (10 mL) at 0 °C. The product
was collected by filtration and washed with water (4 × 10 mL)
and ether (4 × 10 mL). The solid was redissolved in a small
amount of acetone, added to H2O (200 mL), and recollected by
filtration. The crude product was dissolved in 10 mL of
methanol and 0.3 g of Amberlite IRA-400 Cl ion-exchange resin
was added. The resulting mixture was stirred 0.5 h and the
exchange resin was removed via filtration. The process was
repeated with two additional 0.3-g aliquots of the ion-exchange
resin. The product was recrystallized from CH3CN to give 0.43
1
mp g 240 °C; H NMR (CD2Cl2) δ 8.19 (s, 2 H), 8.18 (s, 1 H),
7.83 (s, 2 H), 7.75 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 6.90
(“doublet” AA′BB′, 4 H, J ) 9 Hz), 3.20 (s, 12 H); 13C NMR
(CD2Cl2) δ 169.6, 151.3, 150.7, 140.3, 128.4 (q), 127.6, 123.3,
121.7, 120.3, 119.2, 116.7, 108.7, 35.8; λmax (CH2Cl2) 771 nm
[ꢀ (7.6 ( 0.1) × 104 M-1 cm-1]. Anal. as the hexafluorophos-
phate salt (C29H25F12N2PTe) C, H, N.
P r ep a r a tion of 1-[4-(Dim eth yla m in o)p h en yl]-5-p h en -
yl-1,4-p en ta d iyn -3-on e (6b). Butyllithium (40 mL of a 2.5
M solution in hexanes) was added to a solution of 4-(dimethy-
lamino)phenylacetylene (15.8 g, 0.100 mol) in THF (200 mL)
at -78 °C. The resulting solution was stirred for 1 h at -78
°C. Phenylpropargyl aldehyde (11.8 g, 0.100 mol) was added
and the resulting mixture was stirred for 3 h. Brine (100 mL)
was added and the product was extracted with CH2Cl2 (3 ×
100 mL). The combined organic extracts were dried over
MgSO4 and concentrated. The residue was purified via column
chromatography on SiO2 eluted with CH2Cl2/hexanes to give
1-[4-(dimethylamino)phenyl]-5-phenyl-1,4-pentadiyn-3-ol (19.3
g, 70%) as an orange solid: mp 116-118 °C; 1H NMR (CDCl3)
δ 7.50 (m, 2 H), 7.34 (m, 5 H), 6.62 (“doublet” AA′BB′, 2 H, J
) 9 Hz), 5.57 (d, 1 H, J ) 6 Hz), 2.97 (s, 6 H), 2.37 (d, 1 H, J
) 6 Hz); 13C NMR (CDCl3) δ 150.4, 133.0, 131.9, 128.7, 128.2,
122.1, 111.7, 108.5, 86.5, 85.9, 84.1, 83.9, 53.3, 40.1; IR (KBr)
3262 (s), 2923 (s), 2224 (s) cm-1; FDMS m/z 277 (C19H19NO,
277). Anal. Calcd for C19H19NO: C, 82.29; H, 6.22; N, 5.09.
Found: C, 82.66; H, 6.14; N, 5.11.
1
g (62%) of 3f: mp 209-211 °C; H NMR (CD2Cl2) δ 8.02 (s, 2
H), 7.87 (“doublet” AA′BB′, 4 H, J ) 9.2 Hz), 7.48 (s, 2 H),
7.27 (s, 1 H), 6.81 (“doublet” AA′BB′, 4 H, J ) 9 Hz), 3.09 (s,
12 H), 2.42 (s, 6 H); λmax (CH2Cl2) 689 nm [ꢀ (6.3 ( 0.1) × 104
M-1 cm-1]. Anal. as the hexafluorophosphate salt (C29H31F6N2-
PSe) C, H, N.
2,6-Di[4-(d im e t h y la m in o )p h e n y l]-4-(3,5-d im e t h y l-
p h en yl)tellu r op yr yliu m Ch lor id e (4f). A mixture of bromo-
3,5-dimethylbenzene (0.28 g, 1.5 mmol) and magnesium turn-
ings (0.070 g, 2.9 mg-at) was heated at reflux in anhydrous
THF (50 mL) for 5 h. A solution of telluropyranone 5a (0.33 g,
0.74 mmol) in 30 mL of anhydrous THF was added dropwise
to the reaction mixture and the resulting solution was treated
as described above for 3f. The product was recrystallized from
1-[4-(N,N-Dimethylamino)phenyl]-5-phenyl-1,4-pentadiyn-
3-ol (9.60 g, 34.9 mmol) was dissolved in CH2Cl2 (200 mL).
Manganese dioxide (15.2 g, 0.175 mol) was added and the
resulting mixture was stirred at ambient temperature for 1
h. The reaction mixture was filtered through Celite and the
filtrate was concentrated to give 9.12 g (95%) of diynone 6b:
1
CH3CN to give 0.34 g (67%) of 4f: mp 209-211 °C; H NMR
1
(CD2Cl2) δ 8.01 (s, 2 H), 7.73 (“doublet” AA′BB′, 4 H, J ) 9.2
Hz), 7.48 (s, 2 H), 7.27 (s, 1 H), 6.81 (“doublet” AA′BB′, 4 H, J
) 9 Hz), 3.09 (s, 12 H), 2.42 (s, 6 H); λmax (CH2Cl2) 729 nm [ꢀ
(6.3 ( 0.1) × 104 M-1 cm-1]. Anal. as the hexafluorophosphate
salt (C29H31F6N2PTe) C, H, N.
mp 133-135 °C; H NMR (CDCl3) δ 7.63 (m, 2 H), 7.54 (m, 2
H), 7.39 (m, 3 H), 6.63 (dd, 2 H), 3.04 (s, 6 H); 13C NMR (CDCl3)
δ 152.1, 135.6, 133.1, 130.8, 128.6, 119.9, 111.5, 104.6, 97.1,
90.1, 89.5, 39.9; IR (KBr) 2904 (b), 2145 (s), 1598 (s) cm-1
;
FDMS m/z 275 (C19H19NO, 275). Anal. Calcd for C19H17NO:
C, 83.49; H, 5.53; N, 5.13. Found: C, 83.31; H, 5.46; N, 5.10.
2,6-Di[4-(d im et h yla m in o)p h en yl]-4-[3,5-d i(t r iflu or o-
m eth yl)p h en yl]selen op yr yliu m Ch lor id e (3g). A mixture
of bromo-3,5-di(trifluoromethyl)benzene (0.50 g, 1.7 mmol) and
magnesium turnings (0.080 g, 3.3 mg-at) was heated at reflux
in anhydrous THF (20 mL) for 5 h. A solution of selenopyra-
none 5a (0.33 g, 0.83 mmol) in 30 mL of anhydrous THF was
added dropwise to the reaction mixture and the resulting
solution was heated at reflux for an additional hour. The
reaction mixture was concentrated. The residue was dissolved
in acetic acid (10 mL) and added dropwise to 10% HPF6 (10
mL) at 0 °C. The product was collected by filtration and
washed with water (4 × 10 mL) and ether (4 × 10 mL). The
solid was redissolved in a small amount of acetone, added to
H2O (200 mL), and recollected by filtration. The crude product
was dissolved in 10 mL of methanol and 0.3 g of Amberlite
IRA-400 Cl ion-exchange resin was added. The resulting
mixture was stirred 0.5 h and the exchange resin was removed
via filtration. The process was repeated with two additional
0.3-g aliquots of the ion-exchange resin. The product was
recrystallized from CH3CN to give 0.45 g (74%) of 3g: mp 249-
∆-4H-2-[4-(Dim eth ylam in o)ph en yl]-6-ph en ylth iopyr an -
4-on e [5b (X ) S)]. A mixture of sulfur (0.282 g, 8.78 mmol),
sodium borohydride (0.415 g, 11.0 mmol), and 0.25 M sodium
ethoxide in ethanol (75 mL) was heated at reflux for 3 h until
the reaction turned clear. 1-[4-(Dimethylamino)phenyl]-5-
phenyl-1,4-pentadiyn-3-one (6b) (2.00 g, 7.32 mmol) was
dissolved in THF (10 mL) and the resulting solution was added
to 0.25 M sodium ethoxide in ethanol (10 mL) and stirred for
1 h until the diynone was converted to a mixture of enol ethers.
The solution of enol ethers was added to the reaction mixture
and stirring was continued for 1 h. Water (200 mL) was added
and the product was extracted with CH2Cl2 (4 × 100 mL). The
combined organic extracts were dried over MgSO4, filtered
through Celite, and concentrated. The residue was purified
via column chromatography on SiO2 eluted with CH2Cl2 to give
1.65 g (73%) of thiopyranone 5b as a brown solid: mp 154-
1
155 °C; H NMR (CDCl3) δ 7.66 (m, 2 H), 7.56 (m, 2H) 7.50
(m, 3H), 6.75 (“doublet” AA′BB′, 2 H, J ) 9 Hz), 3.04 (s, 6 H);
13C NMR (CDCl3) δ 182.6, 153.7, 152.5, 151.9, 136.3, 130.4,

Chalcogenopyrylium Dyes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3961
129.2, 127.6, 126.8, 126.7, 123.5, 122.6, 111.9, 40.0; IR (KBr)
3447, 3005, 1714 cm-1; EIMS m/z 307 (C19H17NOS). Anal.
(C19H17NOS) C, H, N.
aniline (0.452 g, 2.26 mmol) and magnesium (0.054 g, 2.3 mg-
at) was heated at reflux in THF (20 mL) for 3 h. A solution of
selenopyranone 5b (0.200 g, 0.564 mmol) in 10 mL of anhy-
drous THF was added dropwise to the reaction mixture and
the resulting solution was treated as described for the prepa-
ration of 2h . The product was recrystallized from CH3CN to
give 0.148 g (67%) of 3h : mp >260 °C; 1H NMR (CD2Cl2) δ
8.34 (s, 1 H), 8.26 (s, 1 H), 8.00 (“doublet” AA′BB′, 2 H, J ) 9
Hz), 7.83-7.65 (m, 5 H), 7.62 (m, 2 H), 6.93-6.84 (m, 4 H),
3.19 (s, 6 H), 3.16 (s, 6 H); λmax (CH2Cl2) 657 nm [ꢀ (5.5 ( 0.2)
× 104 M-1 cm-1]. Anal. as the hexafluorophosphate salt
(C27H27F6N2PSe) C, H, N.
2,4-Di[4-(d im e t h yla m in o)p h e n yl]-6-p h e n ylt e llu r o-
p yr yliu m Ch lor id e (4h ). A mixture of 4-bromo-N,N-di-
methylaniline (0.396 g, 1.98 mmol) and magnesium (0.024 g,
0.99 mg-at) was heated at reflux in THF (20 mL) for 3 h. A
solution of telluropyranone 5b (0.200 g, 0.496 mmol) in 10 mL
of anhydrous THF was added dropwise to the reaction mixture
and the resulting solution was treated as described for 2h . The
product was recrystallized from CH3CN to give 0.234 g (89%)
of 4h : mp 250-252 °C; 1H NMR (CDCl3) δ 8.32 (s, 2 H), 7.98
(“doublet” AA′BB′, 2 H, J ) 9 Hz), 7.68-7.66 (m, 7 H), 6.92
(“doublet” AA′BB′, 2 H, J ) 9 Hz), 6.85 (“doublet” AA′BB′, 2
H, J ) 9 Hz), 3.16 (s, 6 H), 3.15 (s, 6 H); λmax (CH2Cl2) 689 nm
[ꢀ (5.4 ( 0.02) × 104 M-1 cm-1]. Anal. as the hexafluorophos-
phate salt (C27H27F6N2PTe) C, H, N.
∆-4H-2-[4-(Dim eth yla m in o)p h en yl]-6-p h en ylselen op y-
r a n -4-on e [5b (X ) Se)]. A mixture of selenium (0.693 g, 8.78
mmol), sodium borohydride (0.415 g, 11.0 mmol), and 0.25 M
sodium ethoxide in ethanol (75 mL) was heated at reflux for 3
h until the reaction turned clear. 1-[4-(Dimethylamino)phenyl]-
5-phenyl-1,4-pentadiyn-3-one (6b) (2.00 g, 7.32 mmol) was
dissolved in THF (10 mL) and the resulting solution was added
to 0.25 M sodium ethoxide in ethanol (10 mL) and stirred for
1 h until the diynone was converted to a mixture of enol ethers.
The solution of enol ethers was added to the reaction mixture
and stirring was continued for 1 h. Water (200 mL) was added
and the product was extracted with CH2Cl2 (4 × 100 mL). The
combined organic extracts were dried over MgSO4, filtered
through Celite, and concentrated. The residue was purified
via column chromatography on SiO2 eluted with CH2Cl2 to give
1.77 g (68%) of selenopyranone 5b as a brown solid: mp 155-
1
156 °C; H NMR (CDCl3) δ 7.59-7.47 (m, 7 H), 7.25 (s, 2 H),
6.73 (“doublet” AA′BB′, 2 H, J ) 9 Hz), 3.03 (s, 6 H); 13C NMR
(CDCl3) δ 184.8, 156.2, 154.7, 152.0, 138.0, 130.4, 129.3, 128.0,
127.8, 126.8, 124.6, 124.5, 112.1, 40.1; IR (KBr) 3420, 2895,
1596 cm-1; EIMS m/z 355 (C19H17NO80Se). Anal. (C19H17NOSe)
C, H, N.
∆-4H -2-[4-(Dim et h yla m in o)p h en yl]-6-p h en ylt ellu r o-
p yr a n -4-on e [5b (X ) Te)]. A mixture of tellurium granules
(1.12 g, 8.78 mmol), sodium borohydride (0.415 g, 11.0 mmol),
and 0.25 M sodium ethoxide in ethanol (75 mL) was heated
at reflux for several hours until the reaction turned clear. 1-[4-
(Dimethylamino)phenyl]-5-phenyl-1,4-pentadiyn-3-one (6b) (2.00
g, 7.32 mmol) was dissolved in THF (10 mL) and the resulting
solution was added to 0.25 M sodium ethoxide in ethanol (10
mL). The resulting mixture was stirred for several hours until
the diynone was converted to the enol ethers. The solution of
enol ethers was added to the reaction mixture and the
resulting mixture was stirred for an additional hour. Water
(200 mL) was added and the product was extracted with CH2-
Cl2 (4 × 100 mL). The combined extracts were dried over
MgSO4, filtered through Celite, and concentrated. The residue
was purified by column chromatography on SiO2 eluted with
CH2Cl2 to give 1.98 g (67%) of the telluropyranone 5b as a
2-[4-(Dim eth yla m in o)p h en yl]-4,6-d ip h en ylth iop yr yli-
u m Ch lor id e (2i). Phenylmagnesium bromide (1.09 mL of a
3.0 M solution in ether) was added dropwise to a solution of
thiopyranone 5b (0.200 g, 0.651 mmol) in 20 mL of anhydrous
THF (10 mL) for 3 h. The resulting solution was heated at
reflux for an additional hour. The reaction mixture was
concentrated. The residue was dissolved in acetic acid (10 mL)
and added dropwise to 10% HPF6 (10 mL) at 0 °C. The product
was collected by filtration and washed with water (4 × 10 mL)
and ether (4 × 10 mL). The solid was redissolved in a small
amount of acetone, added to H2O (200 mL), and recollected by
filtration. The crude product was dissolved in 10 mL of
methanol and 0.3 g of Amberlite IRA-400 Cl ion-exchange resin
was added. The resulting mixture was stirred for 0.5 h and
the exchange resin was removed via filtration. The process was
repeated with two additional 0.3-g aliquots of the ion-exchange
resin. The product was recrystallized from CH3CN to give
0.102 g (84%) of 2i: mp 218-220 °C; 1H NMR (CD2Cl2) δ 8.47
(s, 1 H), 8.26 (s, 1 H), 8.02-7.86 (m, 5 H), 7.69 (m, 7 H), 6.92
(“doublet” AA′BB′, 2 H, J ) 9 Hz), 3.24 (s, 6 H); λmax (CH2Cl2)
624 nm [ꢀ (2.5 ( 0.1) × 104 M-1 cm-1]. Anal. as the hexafluo-
rophosphate salt (C25H22F6NPS) C, H, N.
1
brown solid: mp 130-131 °C; H NMR (CDCl3) δ 7.51-7.40
(m, 7 H), 7.31 (s, 2 H), 6.72 (“doublet” AA′BB′, 2 H, J ) 9 Hz),
3.02 (s, 6 H); 13C NMR (CDCl3) δ 188.3, 151.9, 150.1, 148.0,
141.1, 132.7, 130.0, 129.4, 128.7, 128.1, 127.7, 126.8, 112.2,
40.1; IR (KBr) 3447, 2894, 1586; EIMS m/z 405 (C19H17NO130
-
Te). Anal. (C19H17NOTe) C, H, N.
2-[4-(Dim eth yla m in o)p h en yl]-4,6-d ip h en ylselen op yr y-
liu m Ch lor id e (3i). Phenylmagnesium bromide (0.94 mL of
a 3.0 M solution in ether) was added dropwise to a solution of
selenopyranone 5b (0.200 g, 0.564 mmol) in 10 mL of anhy-
drous THF. The resulting solution was treated as described
for the preparation of 2i. The product was recrystallized from
CH3CN to give 0.170 g (67%) of 3i: mp 243-245 °C; 1H NMR
(CDCl3) δ 8. 33 (s, 1 H), 8.19 (s, 1 H), 7.97-7.66 (m, 10 H),
6.93 (“doublet” AA′BB′, 2 H, J ) 9 Hz) 3.24 (s, 6 H); λmax (CH2-
Cl2) 654 nm [ꢀ (3.4 ( 0.2) × 104 M-1 cm-1]. Anal. as the
hexafluorophosphate salt (C25H22F6NPSe) C, H, N.
2-[4-(Dim e t h yla m in o)p h e n yl]-4,6-d ip h e n ylt e llu r o-
p yr yliu m Ch lor id e (4i). Phenylmagnesium bromide (0.83 mL
of a 3.0 M solution in ether) was added to a solution of
telluropyranone 5b (0.200 g, 0.496 mmol) in 10 mL of
anhydrous THF. The resulting solution was treated as de-
scribed for the preparation of 2i. The product was recrystal-
lized from CH3CN to give 0.220 g (73%) of 4i: mp 243-244
°C; 1H NMR [500 MHz] (CDCl3) δ 8.22 (d, 2 H), 7.83 (dd, 4 H),
7.71-7.62 (m, 8 H), 6.91 (dd, 2 H) 3.20 (s, 6 H); λmax (CH2Cl2)
693 nm [ꢀ (3.5 ( 0.1) × 104 M-1 cm-1]. Anal. as the hexafluo-
rophosphate salt (C25H22F6NPTe) C, H, N.
2,4-Di[4-(d im eth yla m in o)p h en yl]-6-p h en ylth iop yr yli-
u m Ch lor id e (2h ). A mixture of 4-bromo-N,N-dimethylaniline
(0.520 g, 2.60 mmol) and magnesium (0.032 g, 1.30 mg-at) was
heated at reflux in THF (20 mL) for 3 h. A solution of
thiopyranone 5b (0.200 g, 0.651 mmol) in 10 mL of anhydrous
THF was added dropwise to the reaction mixture and the
resulting solution was heated at reflux for an additional hour.
The reaction mixture was concentrated. The residue was
dissolved in acetic acid (10 mL) and added dropwise to 10%
HPF6 (10 mL) at 0 °C. The product was collected by filtration
and washed with water (4 × 10 mL) and ether (4 × 10 mL).
The solid was redissolved in a small amount of acetone, added
to H2O (200 mL), and recollected by filtration. The crude
product was dissolved in 10 mL of methanol and 0.3 g of
Amberlite IRA-400 Cl ion-exchange resin was added. The
resulting mixture was stirred 0.5 h and the exchange resin
was removed via filtration. The process was repeated with two
additional 0.3-g aliquots of the ion-exchange resin. The product
was recrystallized from CH3CN to give 0.203 g (90%) of 2h :
1
mp 238-240 °C; H NMR (CD2Cl2) δ 8.38 (s, 1 H), 8.26 (s, 1
H), 8.01 (d, 2 H), 7.98-7.64 (m, 7 H), 6.91-6.84 (m, 4 H), 3.20
(s, 6 H), 3.17 (s, 6 H); λmax (CH2Cl2) 627 nm [ꢀ (8.7 ( 0.3) ×
104 M-1 cm-1]. Anal. as the hexafluorophosphate salt (C27H27
F6N2PS) C, H, N.
-
Electr och em ica l P r oced u r es. A Princeton Applied Re-
search Model 173 potentiostat/galvanostat and a model 175
universal programmer were used for the electrochemical
measurements. The working electrode for cyclic voltammetry
2,4-Di[4-(d im eth yla m in o)p h en yl]-6-p h en ylselen op yr y-
liu m Ch lor id e (3h ). A mixture of 4-bromo-N,N-dimethyl-

3962 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Leonard et al.
was a platinum disk electrode (diameter, 1 mm) obtained from
Princeton Applied Research. The auxiliary electrode was a
platinum wire. The reference for cyclic voltammetry was the
Fc/Fc+ couple at +0.40 V at a scan rate of 0.1 V s-1. All samples
were run in J .T. Baker HPLC-grade dichloromethane that had
been stored over 3A molecular sieves. Electrometric-grade
tetrabutylammonium fluoroborate (Southwestern Analytical
Chemicals, Inc.) was recrystallized from ethyl acetate-ether
and then dried overnight at 80 °C before it was used as
supporting electrolyte at 0.2 M. Argon was used for sample
deaeration.
comparisons to rose bengal, which is identical to the value
obtained with methylene blue.
Deter m in a tion of P a r tition Coefficien ts. The octanol/
water partition coefficients were all measured at pH 7.4 in
phosphate-buffered saline using UV-visible spectrophotom-
etry. The measurements were done using a “shake flask” direct
measurement.34 Three to five minutes of mixing was followed
by 1 h of settling time. Equilibration and measurements were
made at 23 °C using a Perkin-Elmer Lambda 12 spectropho-
tometer. HPLC grade 1-octanol was obtained from Sigma-
Aldrich.
Qu a n tu m Yield s for Sin glet Oxygen Gen er a tion . The
singlet oxygen acceptor 1,3-diphenylisobenzofuran (DPBF),
HPLC-grade methanol, and certified rose bengal and methyl-
ene blue were used as received from Aldrich Chemical Co.
Quantum yields for singlet oxygen generation in air-saturated
methanol were determined by monitoring the dye-sensitized
photooxidation of DPBF. DPBF is a convenient acceptor since
Cells a n d Cu ltu r e Con d ition s. CX-1, PC-3, and MDA-
435 cell lines were grown in a 50:50 mix of Dulbecco’s modified
Eagle’s medium (DMEM) and RMPI 1640 medium (GIBCO
Laboratories, Grand Island, NY) supplemented with 10% calf
serum (Hyclone Laboratories, Inc., Logan, UT) and antibiotics
at 37 °C under 5% CO2, 95% air, and 100% humidity. PC-3
(human prostate carcinoma) and MDA-435 (human breast
carcinoma) cells were obtained from Dr. L. Nadler (Dana-
Farber Cancer Institute), and CX-1 (human colon carcinoma)
was from Dr. M. Wolpert (National Cancer Institute).
Colo-26 cells were maintained in RPMI 1640 supplemented
with 10% fetal calf serum and antibiotics. The cells were
incubated at 37 °C, 5% CO2 and were split every 2-3 days.
In Vitr o Clon ogen ic Assa y. Cells were seeded at 1500
cells/well for CX-1, PC-3, and MDA-435 cells in 96-well plates
(Becton Dickinson Labware, Lincoln Park, NJ ). The assay was
performed in duplicate. Dyes 2-4 were first dissolved in
dimethyl sulfoxide, to prepare 10 mg/mL stock solutions. The
final drug solution was made by mixing 100 µL of this stock
solution with 10 mL of 5% CS DME media solution. On the
following day, cells were treated with test compounds at varied
concentrations and cultured precisely for 24 h in the media.
After rinsing, cells were incubated in drug-free medium for 2
weeks. Colonies were stained with 2% crystal violet in 70%
ethanol and counted by an automated colony counter (Artek
counter model 880, Dynatech Laboratories, Inc., Chantilly,
VA).
In Vitr o P h ototoxicity Mea su r em en ts. Colo-26 cells
were plated at 2.5 × 104 cells/well of a 96-well tissue culture
plate the evening before the assay. The day of the assay, the
cells were washed twice with PBS, and 100 µL of HBSS,
containing 1 µM 3a or 3d , 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. The plates were
irradiated with filtered 590-800-nm light at 30 mW cm-2 with
various light doses, and 100 µL of growth media was added.
Cells were then incubated overnight at 37 °C, 5% CO2. Cell
survival was monitored using the MTT assay as described in
Mosmann.35
it absorbs in
a region of dye transparency and rapidly
scavenges singlet oxygen to give colorless products. This
reaction occurs with little or no physical quenching.29 The
indirect studies of quantum yields of singlet oxygen generation
were carried out in a stopped-flow spectrophotometer (Applied
Photophysics, Ltd.; ×18). The output beam of a 150-W Xenon
arc lamp was passed through a monochromator (0.30-mm slits)
to separate the 410-nm radiation and to direct it to the mixing
chamber (approximate volume of 100 µL) via a flexible optical
fiber to provide the excitation for DPBF. The excitation beam
entered the chamber through a port that provides a 2-mm path
length. The fluorescence of DPBF was monitored at a 90° angle
to the excitation beam via a PMT (Hamamatsu) close-coupled
to the chamber assembly. The fluorescence was passed through
a 460-nm interference filter (Oriel) placed in front of the PMT
window. The photolysis radiation was supplied by a 100-W
QTH lamp (Oriel). The output of the lamp was passed through
590-nm long-pass and 660-nm interference filters (both from
Oriel) and focused onto an input window of a flexible optical
fiber. The photolysis light via the fiber reached the mixing
chamber by entering through the 10-mm path length port. The
solution of the sensitizer and the solution of DPBF were
prepared separately to prevent sample degradation and were
injected into the mixing chamber that was kept at (25.0 ( 0.1)
°C via a circulating water bath. The decay of fluorescence of
DPBF was then observed on a 200- or 2000-s time scale. The
fluorescence decays were analyzed using the routines provided
with the stopped-flow instrument software package by fitting
them to a first-order exponential decay function with a floating
endpoint. A total of three or more measurements were recorded
for each sample and the results averaged to obtain a mean
value for the decay rate.
On the basis of our earlier work,6a methylene blue has a
singlet oxygen quantum yield [Φ(1O2)] of 0.50 in methanol,
which is close to the quantum yield of 0.52 determined for
methylene blue in both ethanol and water.30 The quantum
yield for singlet oxygen generation by dyes 2-4 may be
calculated by comparing the rates of the decay for the dye of
interest and methylene blue (MB) at the same DPBF concen-
tration and optical density at 660 nm:
Sta tistica l An a lyses. All statistical analyses were per-
formed using the Student’s t-test for pairwise comparisons. A
P value < 0.05 was considered significant.
An im a ls. All animals were cared for under the guidelines
of the Roswell Park Cancer Institute Committee on Animal
Resources. Stock solutions of dyes 2-4 in 1% Tween 80/0.9%
NaCl were prepared by dissolving the dye in Tween 80 and
diluting to the appropriate volume with 0.9% NaCl. Animals
were give 100-µL aliquots of the stock solutions via tail-vein
injection.
Φ(1O2)(dye) ) [Φox,DPBF(dye) × Φ(1O2)(MB)]/Φox,DPBF(MB)
Ack n ow led gm en t. The authors thank the National
Institutes of Health (Grant CA69155 to M.R.D. and
Grant CA36856 to R.H.) for support of this work.
The rate of disappearance of DPBF can be expressed as follows:
-d[DPBF]/dt ) {Ia × Φ(1O2) × kr[DPBF]}/{kr[DPBF] +
kd}
Su p p or tin g In for m a tion Ava ila ble: Table 2 for IC50
values against several human carcinoma cell lines. This
material is available free of charge via the Internet at http://
pubs.acs.org.
where kr is the rate of reaction of DPBF with singlet oxygen,
kd is the rate of decay of singlet oxygen, and Ia is the absorbed
light.31 At low concentrations of DPBF, where kr[DPBF] , kd
Refer en ces
(DPBF] < 3 µM, kd ) 1.0 × 10-5 s-1 32
, first-order decay kinetics
(1) (a) Dougherty, T. J .; Gomer, C. J .; Henderson, B. W.; J ori, G.;
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Moan, J .; Ma, L. W.; Nesland, J . M. Uptake, Localization, and
were observed.
The value of Φ(1O2) for 2 was confirmed with rose bengal
where Φ(1O2) ) 0.76.33 Dye 2 gave Φ(1O2) of 0.020 ( 0.004 in

Chalcogenopyrylium Dyes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3963
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