In vitro photodynamic properties of chalcogenopyrylium analogues of the thiopyrylium antitumor agent AA1

Article abstract of DOI:10.1021/jm020260m

Several series of chalcogenopyrylium dyes were prepared with one or two 4-anilino substituents at the 2- and 6-positions and with phenyl, 4-N,N-dimethylanilino, or 4-(N-morphilino)phenyl substituents at 2- and/or 4-positions. The dye series are all relate

Full text of DOI:10.1021/jm020260m

J . Med. Chem. 2002, 45, 5123-5135
5123
In Vitr o P h otod yn a m ic P r op er ties of Ch a lcogen op yr yliu m An a logu es of th e
Th iop yr yliu m An titu m or Agen t AA1
Nancy K. Brennan,† J onathan P. Hall,† Sherry R. Davies,§ Sandra O. Gollnick,§ Allan R. Oseroff,§
Scott L. Gibson,‡ Russell Hilf,‡ and Michael R. Detty†,*
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, Department of
Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 607, Rochester, New York
14642, and Department of Dermatology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263
Received J une 19, 2002
Several series of chalcogenopyrylium dyes were prepared with one or two 4-anilino substituents
at the 2- and 6-positions and with phenyl, 4-N,N-dimethylanilino, or 4-(N-morphilino)phenyl
substituents at 2- and/or 4-positions. The dye series are all related in structure to AA1, a
thiopyrylium dye that targets mitochondria. The chalcogenopyrylium nuclei included sulfur,
selenium, and tellurium at the 1-position. Key intermediates in the dye synthesis were the
corresponding ∆-4H-chalcogenopyran-4-ones. All of the dyes of this study were evaluated for
dark and phototoxicity toward Colo-26 cells in vitro. There was no correlation of dark toxicity
with either the reduction potential of the chalcogenopyrylium dye or the n-octanol/water
partition coefficient, log P. Several of the dyes of this study (thiopyrylium dyes 1-S and 13-S,
selenopyrylium dyes 1-Se, 2-Se, 3-Se, 4-Se, 13-Se, 14-Se, and 27-Se, and telluropyrylium
dye 13-Te) showed added phototoxicity upon irradiation. Dyes with the highest therapeutic
ratio as measured by dark toxicity/phototoxicity (15 J cm-2 of 360-800-nm light) had values
of log P of 1.0-1.2. Studies of cytochrome c oxidase activity in whole R3230AC cells suggested
that dyes 1-S and 3-Se, with values of log P of 2.2 and 1.7, respectively, were localized in the
mitochondria. Cytocrome c oxidase activity in whole cells was inhibited by 1-S and 3-Se in the
dark. Chalcogenopyrylium dyes 2-Se, 4-Se, 13-Te, and 14-Se inhibited whole-cell cytochrome
c oxidase activity only following irradiation, which suggests that these dyes relocalized to
mitochondria following irradiation.
Photodynamic therapy (PDT) as a protocol for treating
cancer, as well as other diseases such as age-related
macular degeneration, combines light and endogenous
oxygen with a photosensitizer localized in the target
tissue.1 The photosensitizer is a critical component in
PDT and a variety of molecular classes have been
evaluated including porphyrins and related molecules,
core-expanded porphyrins, core-modified porphyrins,
chlorins and bacteriochlorins, and a variety of cationic
dyes.1 A photosensitizer should absorb wavelengths of
light >600 nm, where penetration of light into tissue is
optimal and should have a high quantum yield for the
photochemical event that produces phototoxicity.1 As
with all drugs, adsorption, distribution, metabolism, and
excretion (ADME) properties2 of the photosensitizer are
critical to the success of the technique. A photosensitizer
with optimal ADME properties will have high specificity
for the tumor or other target tissue, will undergo rapid
distribution to the target tissue, will have minimal
toxicity in the absence of light, and will be excreted
following treatment to minimize any side effects such
as long-term light sensitivity.
that the cationic dyes appear to be bound intra-
cellularly.3-9 Uptake and retention of lipophilic cations
is greater in some carcinoma cells relative to normal
cells.3-9 Reports have demonstrated that the mito-
chondria3,5-9 and lysosomes4 are important targets for
lipophilic cations. A recent study has demonstrated that
lysosomal, nuclear, and mitochondrial localization of a
series of methylene blue analogues is directly related
to the n-octanol/water partition coefficient (log P).10
Initially, the methylene blue derivatives were found to
localize in lysosomes in vitro, but subsequently relocal-
ized upon irradiation as a function of log P.10 For
methylene blue (log P ) -1.0), relocalization to the
nucleus was observed. For the N,N-diethyl analogue (log
P ) 0.3), no relocalization was observed, and the dye
remained in the lysosome. For higher N,N-dialkyl
analogues (log P > 1.0), relocalization to the mitochon-
dria was observed upon irradiation.
The cationic thiopyrylium dye AA1 (Chart 1) inhibits
growth of the human colon carcinoma cell line CX-1 in
vitro incubated with the dye in the dark prolongs
survival of mice implanted with several different tumor
lines.9 Mitochondria appeared to be a target as deter-
mined by AA1-induced inhibition of mitochondrial AT-
Pase activity. Although AA1 appeared to be localized
in tumors, no additional toxicity was observed after irra-
diation of tumors borne on animals injected with AA1.
One concern with AA1 and other lipophilic cations is the
narrow therapeutic window between an effective treat-
ment dose and the dose where AA1 becomes lethal.9
One significant difference between porphyrin photo-
sensitizers and related molecules and cationic dyes is
* To whom correspondence should be addressed at: Department of
Chemistry, University at Buffalo, The State University of New York,
Buffalo, NY 14260, Phone: (716) 645-6800, ext 2200, FAX: (716) 645-
6963. e-mail: mdetty@acsu.buffalo.edu
† Department of Chemistry, University at Buffalo.
‡ Department of Biochemistry and Biophysics, University of Roch-
ester Medical Center.
§ Department of Dermatology, Roswell Park Cancer Institute.
10.1021/jm020260m CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/09/2002

5124 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
Ch a r t 1
We provide a general synthesis of selenopyrylium and
telluropyrylium analogues of AA1 with 4-anilino sub-
stituents at both the 2- and 6-positions as well as
various combinations of phenyl and aniline-related
substituents (4-anilino, 4-N-morpholinophenyl, and
4-N,N-dimethylanilino substituents).
Ch em ica l Resu lts a n d Discu ssion
Retr osyn th etic An a lysis a n d Syn th esis. A. Sele-
n op yr yliu m a n d Tellu r op yr yliu m An a logu es of
AA1. The chalcogenopyranones 5 are key intermediates
for the synthesis of AA1 and its selenopyrylium and
telluropyrylium analogues as shown in the retrosyn-
thesis of Scheme 1. The addition of Grignard reagents
to the pyranone carbonyl followed by acid-induced
dehydration will give the various chalcogenopyrylium
analogues of AA1.8c Formally, the chalcogenopyranones
5 can be prepared by the 1,4-addition of a hydrogen
chalcogenide to the triple bonds of 1,4-diynone 6, which
in turn can be prepared by addition of 2 equiv of an
acetylide derived from p-aminophenylethyne 7 to a
formate derivative followed by oxidation.
We have been interested in analogues of AA1 that
show enhanced phototoxicity upon irradiation and offer
promise as photosensitizers for PDT.8 The enhanced
phototoxicity of the cationic chalcogenopyrylium dyes
would allow for a lower effective dose relative to the
higher dose necessary for chemotherapy alone. The
lower effective dose in PDT would expand the thera-
peutic ratio relative to a lethal dose. In our pursuit of
AA1-like analogues, we have prepared and evaluated
chalcogenopyrylium dyes 18a and 28c (Chart 1) and
related compounds.8b Of these dyes, selenopyrylium dye
2-Se was found to be an effective sensitizer for PDT
against various carcinoma cell lines in culture and
against R3230AC mammary adenocarcinomas im-
planted in female Fisher rats in vivo, where a 300%
increase in tumor-doubling time was observed in treated
animals relative to untreated controls.8c For dye classes
1 and 2, the primary mechanism for phototoxicity
appears to be the photochemical generation of singlet
oxygen.8
From the work with chalcogenopyrylium dyes 1 and
2 and related structures, the following generalizations
can be made: (1) Analogues with the heavier chalcogen
atoms selenium and tellurium give longer wavelengths
of absorption and higher quantum yields for the pho-
tochemical generation of singlet oxygen than the cor-
responding sulfur analogues. (2) Two or three aniline-
related substituents at the 2-, 4-, and 6-positions are
required to give a photosensitizer. (3) The incorporation
of one (2-Se)8c or two unsubstituted aniline groups
(AA1)9 gives desirable biodistribution relative to dyes
1 with three N,N-dimethylanilino substituents. Given
these generalizations, we have investigated the effects
of chalcogen atom substitution at the 1-position and
substituent changes at the 2-, 4-, and 6-positions on
measurable physical properties such as log P and the
electrochemical reduction potential (E°) and correlated
these properties with the dark and phototoxicity of the
chalcogenopyrylium dyes toward cells in culture. Inhibi-
tion of cytochrome c oxidase activity in cultured whole
cells exposed to the chalcogenopyrylium dyes and light
suggests that the mitochondria are targets of localiza-
tion or relocalization for the chalcogenopyrylium dyes.
Sch em e 1
Although acetylene 7 is known,11 reactions of 7 with
Grignard reagents and alkyllithium compounds are
complicated by the free amino substituent. In view of
our successful synthesis of dyes 2, the amino group of 7
was protected with the bis-1,2-(dimethylsilyl)ethano
(Stabase) protecting group12 since this group is stable
to strong bases under aprotic conditions and is easily
hydrolyzed with either aqueous acid or base. The
Stabase-protected acetylene 8 was deprotonated with
n-BuLi in THF at -78 °C and 2 equiv of the resulting
acetylide were added to a solution of methyl formate to
give a mixture of 1,4-pentadiyn-3-ol 9 and 1,4-pen-
tadiyn-3-one 6 (Scheme 2). The Stabase protecting group
was lost during workup. Since the facile air oxidation
of 9 to 6 made purification of 9 problematic, the crude
product was oxidized directly with MnO2 to give 1,4-
pentadiyn-3-one 6 in 56% overall yield from 8.
The preparation of chalcogenopyranones 5 via the
formal addition of hydrogen chalcogenides to 6 was best
accomplished in two steps to minimize 5-exo-trig cy-
clization to give dihydrochalcogenophene products.13
Diynone 6 was stirred with 0.25 M NaOEt in EtOH for
2 h at ambient temperature to give enol ether 10 from
addition of ethanol across one triple bond of 6 (Scheme

Chalcogenopyrylium Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5125
Sch em e 2a
a
Key: (a) n-BuLi, THF, -78 °C; (b) HCO2Me; (c) MnO2, CH2Cl2 (56% overall); (d) 0.25 M NaOEt in EtOH; (e) S, Se, Te, NaBH4 (2.5
equiv), 0.1 M NaOEt in EtOH, add Na2E to 10; (f) i. Me2NC6H4MgBr, ii. HPF6; (g) Amberlite IRA-400 (Cl); (h) i. N-(4-bromophenyl)mor-
pholine, Mg°, THF, ii. HPF6, iii. Amberlite IRA-400 (Cl).
Ch a r t 2
Sch em e 3a
2). The addition of disodium chalcogenides to enol ether
10 gave chalcogenopyranones 5 in 21-57% isolated
yields.
AA1 and its heavier chalcogenopyrylium analogues
3-Se and 3-Te were prepared by the addition of 4-di-
methylaminophenylmagnesium bromide to chalcogeno-
pyranones 5 followed by dehydration with HPF6 (Scheme
2). Ion exchange with a chloride ion-exchange resin gave
the chloride salts of the dyes. Similarly, the N-mor-
pholinophenyl analogues 4 were prepared by the addi-
tion of the Grignard reagent derived from N-(4-bromo-
phenyl)morpholine14 followed by dehydration with HPF6
and ion exchange to give the chloride salts 4 (Scheme
2).
B. Ch a lcogen op yr yliu m An a logu es w ith On e
a
Key: (a) PdCl2(PPh3)2, CuI2, Et3N, HCtCCH2OH; (b) MnO2,
2-(4-An ilin o) Su bstitu en t. The key intermediates to
the preparation of dyes 11-14 (Chart 2), with one
4-anilino substituent, are the chalcogenopyranones 15
and 16, which were prepared as shown in Scheme 3.
Heck coupling of 4-iodo-N,N-dimethylaniline15 (17a ) or
N-(4-iodophenyl)morpholine16 (17b) with propargyl al-
CH2Cl2; (c) 8, LDA, THF, -78 °C; (d) i. 0.25 M NaOEt, EtOH; ii.
S, Se, or Te, NaBH4 (2.5 equiv), 0.25 M NaOEt, EtOH, add Na2E
to i.
cohol gave arylpropargyl alcohols 18a and 18b in 85%
and 99% isolated yields, respectively.17 The propargyl

5126 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
Ch a r t 3
followed by the addition of disodium selenide to give the
key intermediate, selenopyranone 26-Se, in 57% iso-
lated yield. The addition of the Grignard reagent derived
from N-(4-bromophenyl)morpholine17 to chalcogeno-
pyranone 26-Se followed by dehydration with HPF6
gave the hexafluorophosphate salt of dye 27-Se in 82%
isolated yield, which was then converted to the corre-
sponding chloride salt with an ion-exchange resin (Chart
2).
Sp ectr a l a n d P h ysica l P r op er ties. A. Absor p tion
Sp ectr a . Useful photosensitizers have some absorption
at wavelengths >600 nm, where penetration of light in
tissue is maximal.18 All of the dyes 3, 4, and 11-14 have
some absorption at wavelengths >600 nm (values of λmax
and log ꢀ are compiled in Table 1). For comparison
purposes, values of λmax and log ꢀ are also compiled in
Table 1 for dyes 18a and 2.8c As with other series of
chalcogenopyrylium dyes, the absorption maxima in-
crease in wavelength as the heteroatom increases in
size. Dye 11-Se and dye series 12, both with anilino
derivatives at the 2- and 6-positions and a phenyl
substituent at the 4-position, display large bathochromic
shifts relative to dyes 2 with anilino derivatives at the
2- and 4-positions and a phenyl substituent at the
6-position and relative to dyes 1, 13, 14, and 27-Se with
aniline related substituents at the 2-, 4-, and 6-positions.
Replacing an N,N-dimethylanilino substituent with a
4-N-morpholinophenyl substituent gives hypsochromic
shifts in absorption maxima as noted in comparisons of
dyes 3 and AA1 with dye series 4, 11-Se with 12-Se,
dye series 13 with 14, and 27-Se with 1-Se.
alcohols 18 were oxidized to the corresponding aryl-
propargyl aldehydes 19a 15 and 19b in 64% and 72%
isolated yields, respectively, with MnO2 in CH2Cl2.
The Stabase-protected12 acetylene 8 was deprotonated
with n-BuLi in THF at -78 °C, and 1 equiv of the
resulting acetylide was added to a solution of propargyl
aldehyde 19a to give a mixture of 1,4-pentadiyn-3-ol and
1,4-pentadiyn-3-one 20a (Scheme 3). The Stabase pro-
tecting group was lost during workup. The crude
product mixture was oxidized with MnO2 in CH2Cl2 to
give 1,4-pentadiynone 20a in 83% isolated yield. The
same procedure was applied to propargyl aldehyde 19b
to give 1,4-pentadiyn-3-one 20b in 31% overall yield.
As before, the preparation of chalcogenopyranones 15
and 16 via the formal addition of hydrogen chalco-
genides to 20 was best accomplished in two steps.
Diynone 20 was stirred with 0.25 M NaOEt in EtOH
for 2 h at ambient temperature followed by the addition
of disodium chalcogenides to give selenopyranone 15-
Se in 76% isolated yield and chalcogenopyranones 16
in 41-45% isolated yields.
The addition of phenylmagnesium bromide to sele-
nopyranone 15-Se and to chalcogenopyranones 16 fol-
lowed by dehydration with HPF6 gave the hexafluoro-
phosphate salts of selenopyrylium dye 11-Se and
chalcogenopyrylium dyes 12, respectively (Chart 2). The
addition of the Grignard reagent derived from N-(4-
bromophenyl)morpholine14 to chalcogenopyranones 16
followed by dehydration with HPF6 gave the hexafluo-
rophosphate salts of dyes 13 (Chart 2). The addition of
4-dimethylaminophenylmagnesium bromide to chalco-
genopyranones 16 followed by dehydration with HPF6
gave the hexafluorophosphate salts of chalcogenopyry-
lium dyes 14 (Chart 2). The hexafluorophosphate salts
of 11-14 were converted to the chloride salts using an
ion-exchange resin.
Dye 21-Se was prepared by the addition of the
Grignard reagent derived from N-(4-bromophenyl)-
morpholine14 to ∆-4H-2-(4-anilino)-6-phenylseleno-
pyran-4-one (22)8c followed by dehydration with HPF6
to give the hexafluorophosphate salt of dye 21-Se (Chart
2). As before, ion exchange gave the chloride salt of 21-
Se.
C. 2,4,6-Tr is-4-(N-m or p h olin o)p h en yl Selen o-
p yr yliu m Dye 27-Se. Propargyl aldehyde 19b was
treated with KOH to give N-ethynylmorpholine (23) in
97% isolated yield (Chart 3).17 Acetylene 23 was treated
with n-BuLi, and the resulting acetylide was added to
propargyl aldehyde 19b to give diynol 24 in 83% isolated
yield. Diynol 24 was oxidized to diynone 25 in 83%
isolated yield with MnO2. Diynone 25 was stirred with
0.25 M NaOEt in EtOH for 2 h at ambient temperature
Dyes AA1, 1-4, 11-14, 21-Se, and 27-Se all display
a large hypsochromic shift as the solvent is changed
from CH2Cl2 to water. Hydrogen-bond donation from the
anilino group(s) of dyes AA1, 3, 4, 11-14, and 21-Se to
water decreases the effective cationic charge on the
chromophore and the corresponding cyanine character.
Similarly, hydrogen bonding from water to the nitrogen
atoms of the various anilino groups of dyes AA1, 1-4,
11-14, 21-Se, and 27-Se decreases the availability of
the nitrogen lone-pair of electrons to the delocalized
π-system.
B. Electr och em ica l Red u ction P oten tia ls. With
the mitochondria as one possible target of the cationic
chalcogenopyrylium dyes, one must be concerned with
dark toxicity from disruption of the redox cascade in
mitochondrial respiration. The cationic chalcogenopy-
rylium dyes are electron acceptors and electron transfer
within the mitochondria to reduce the chalcogenopyry-
lium cation to the neutral chalcogenopyranyl radical is
possible along with the possibility of disruption of
mitochondrial function. The reduction potentials of AA1,
dyes 1-4, 11-14, 21-Se, and 27-Se were determined
by cyclic voltammetry and values of E° for the cation/
neutral radical couple are compiled in Table 1. Values
of E° for AA1, 1-4, 11-14, 21-Se, and 27-Se cover a
0.38-V range from -0.38 V for 12-Te to -0.76 V for 1-S
[vs the ferrocene/ferrocinium couple (Fc/Fc+) at +0.40
V]. Within each series of dyes, an anodic (positive) shift
in E° is observed as the chalcogen atom becomes
heavier.
C. n -Octa n ol/Wa ter P a r tition Coefficien ts. The
determination of n-octanol/water partition coefficients
for the chalcogenopyrylium dyes is complicated by the

Chalcogenopyrylium Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5127
Ta ble 1. Absorption Maxima in CH2Cl2 and Water, Electrochemical Reduction Potentials (E°), n-Octanol/Water Partition Coefficients
(log P), Dark Toxicity (LD50), and Phototoxicity (EC50) toward Colo-26 Cells in Culture, and Therapeutic Ratio (LD50/EC50) for
Chalcogenopyrylium Dyes AA1, 1-4, 11-14, 21-Se, and 27-Se
Colo-26 cells
compd
λmax, nm (log ꢀ) CH2Cl2
λ
max, nm (log ꢀ) H2O
E°, Va
log Pb
LD50 (µM)
EC50 (µM)
LD50/EC50
AA1
1-S
1-Se
2-S
2-Se
2-Te
3-Se
3-Te
4-S
4-Se
4-Te
11-Se
12-S
12-Se
12-Te
13-S
13-Se
13-Te
14-S
14-Se
14-Te
21-Se
27-Se
581 (4.74)
620 (4.76)
655 (4.50)
620 (4.49)
651 (4.47)
677 (4.50)
610 (4.84)
620 (4.60)
566 (4.66)
593 (4.65)
618 (4.73)
670 (4.71)
616 (4.64)
-645 (4.69)
674 (4.70)
578 (4.84)
608 (4.71)
638 (4.54)
589 (4.89)
616 (4.72)
646 (4.86)
611 (4.74)
622 (4.83)
541 (4.40)
595 (4.41)
627 (4.40)
573 (4.45)
615 (4.40)
640 (4.40)
554 (4.51)
581 (4.42)
535 (4.54)
553 (4.50)
582 (4.36)
635 (4.40)
597 (4.32)
626 (4.45)
653 (4.41)
546 (4.28)
566 (4.36)
584 (4.38)
553 (4.19)
557 (4.52)
583 (4.55)
580 (4.38)
582 (4.25)
-0.71
-0.76
-0.71
-0.60
-0.51
-0.44
-0.61
-0.55
-0.68
-0.57
-0.51
-0.46
-0.49
-0.42
-0.38
-0.58
-0.52
-0.41
-0.66
-0.58
-0.49
-0.47
-0.74
1.9
2.2
2.2
1.3
1.2
1.9
1.7
2.0
1.8
1.5
1.9
1.3
1.1
0.9
1.0
1.3
1.2
1.6
0.9
1.0
1.0
1.2
1.2
0.1
0.5
0.5
0.07
2.6
0.6
1.4
0.5
0.8
3.8
1.4
0.4
1.8
0.9
0.07
66
6.9
0.8
0.9
1.5
0.5
6.5
7.2
-
-
2.5
1.7
-
13
-
3.8
-
-
3.2
-
-
-
-
0.2
0.3
-
0.2
-
0.37
-
-
1.2
-
-
-
-
-
2.0
1.9
0.6
-
0.07
-
-
33
3.6
1.3
-
21
-
-
-
0.8
9.0
a
b
In CH2Cl2 with 0.2 N Bu4NBF4 as supporting electrolyte, V vs the ferrocene/ferricinium couple (E° ) +0.40 V). pH-6 Phosphate
buffer.
Sch em e 4
survival was determined. Results are reported in Table
1 for the LD50 for each dye with respect to dark toxicity
and, for those examples in which added phototoxicity
was observed, the effective concentration (EC50) to give
50% cell kill 24 h following irradiation with 15 J cm-2
of 360-800-nm light.
addition of H2O/HO- to the chalcogenopyrylium nucleus
at either the 2- or 4-position as shown in Scheme 4.8a,19
When the chalcogenopyrylium dye is partitioned be-
tween n-octanol and water, hydrolysis is more rapid in
the aqueous phase than in the organic phase. Apparent
values of log P are influenced by the rate of diffusion
between layers as well as the rate and extent of addition
of H2O/HO-. At physiological pH (7.4), apparent values
of log P can vary significantly ((0.7 units in log P). We
have found that minimal addition of H2O/HO- occurs
at pH 6 and that values of log P can be more reproduc-
ibly determined ((0.1 units of log P) using a mixture of
n-octanol and pH 6 phosphate buffer.
A. Da r k Toxicity tow a r d Colo-26 Cells in Cu l-
tu r e. With common substituents at the 2-, 4-, and
6-positions, the ring heteroatom had a significant impact
on the observed dark toxicity toward cultured Colo-26
cells in vitro (Table 1). For dye series 14, the dark
toxicity varied by a factor of 3 for the three chalcogen
atoms; for dye series 4, by a factor of 4.8; for the series
of AA1 and dyes 3, by a factor of 14; for dye series 12,
by a factor of 26; for dye series 2, by a factor of 37; and
for dye series 13, by a factor >50. For the series of AA1,
3-Se, and 3-Te and related series 2 and 4, the thiopy-
rylium analogues AA1, 2-S, and 4-S displayed the
greatest dark toxicity while the selenopyrylium ana-
logues 2-Se, 3-Se, and 4-Se displayed the least. In dye
series 12-14, the telluropyrylium analogues displayed
the greatest dark toxicity.
Values of log P were determined for AA1, 1-4, 11-
14, 21-Se, and 27-Se using a pH 6 phosphate buffer as
the aqueous phase via the “skake flask” direct measure-
ment.20 Values of log P are compiled in Table 1 and
cover a range of 0.9 to 2.2.
The substituents at the 2-, 4-, and 6-positions also
impacted the observed dark toxicity toward Colo-26 cells
in vitro. One general observation of the effect of sub-
stituents at the 2-, 4-, and 6-positions of the chalco-
genopyrylium dyes is that replacing a phenyl, 4-N,N-
dimethylanilino, or a 4-anilino substituent with a 4-N-
morpholinophenyl substituent resulted in a decrease in
dark toxicity (Table 1).
Biologica l Resu lts a n d Discu ssion
In Vitr o Stu d ies. Chalcogenopyrylium dyes AA1,
1-4, 11-14, 21-Se, and 27-Se were evaluated in culture
for dark or light-induced toxicity toward Colo-26 cells,
a murine colon carcinoma cell line. Cell cultures were
incubated for 24 h in the dark with various concentra-
tions of cationic dyes and were then washed prior to
treatment with filtered 360-800-nm light from a tung-
The changes in dark toxicity associated with changes
in either the ring heteroatom or in the 2-, 4-, and
6-substituents did not correlate with any systematic
changes in either reduction potential, E°, or n-octanol/
water partition coefficient, log P. As shown in Figure
S1 (Supporting Information), there is no correlation of
a 103 range in dark toxicity with a 0.38-V range in
sten-halogen source for a total light dose of 15 J cm-2
.
All of the chalcogenopyrylium dyes had absorption
bands with comparable extinction coefficients and band-
widths at half-height in this window. Light-treated cells
and dark controls were incubated for 24 h and cell

5128 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
F igu r e 2. Effect of 2-Se photosensitization on mitochondrial
cytochrome c oxidase activity in cultured R3230AC tumor cells.
Details of experimental conditions are described in the Ex-
perimental Section. Data are expressed as percentage of
control cytochrome c oxidase activity, obtained from cells not
exposed to either 2-Se or light (O) and in cells exposed to 1.0
× 10-8 M (b), 1.0 × 10-7 M (2) or 1.0 × 10-6 M (9) 2-Se and
light (0.5 mW/cm2 of 360-750-nm light). Each datum point
represents the mean of at least three separate experiments
performed in duplicate, the error bars are the SEM.
F igu r e 1. A plot of the therapeutic ratio between dark toxicity
and phototoxicity (LD50/EC50) as a function of the n-octanol/
water partition coefficient (log P) for dyes 1-S, 13-S, 1-Se, 2-Se,
3-Se, 4-Se, 13-Se, 13-Te, 14-Se, and 27-Se.
reduction potential. As shown in Figure S2 (Supporting
Information), there is no correlation of a 103 range in
dark toxicity with a range of values of log P from 0.9 to
2.2.
irradiation was followed by observing fluorescence from
the photosensitizer.10 Unfortunately, the dyes studied
in this present report are not fluorescent, which makes
detection within cultured cells difficult. Furthermore,
the heavy atoms present in the selenopyrylium and
telluropyrylium analogues can quench fluorescence,
which can complicate studies using fluorescent organelle
tags. However, the mitochondria were identified as sites
of localization or as sites of relocalization (following
irradiation) due to changes in mitochondrial cytochrome
c oxidase activity in whole cells treated with chalco-
genopyrylium dyes and light.8c
Cytochrome c oxidase is a multisubunit complex,
which spans the inner membrane of the mitochondria
and which contains four one-electron redox centers
including two copper ions and two heme units.21 It is
the last enzyme in the mitochondrial respiration chain,
and loss of enzymatic activity is due to direct damage
to this enzyme or to other sites preceding it in the
respiration chain.
Dye 2-Se has demonstrated efficacy in vivo8c and is
one of the more potent photosensitizers in this study
based on EC50 as well as the therapeutic ratio, LD50/
EC50 (Table 1). Dye 2-Se also has a value of log P of
1.2, which is in the optimal range suggested by Figure
1. R3230AC rat mammary adenocarcinoma cells were
incubated with 0.01-1.0 µM concentrations of 2-Se for
24 h, and cultures were washed once with 0.9% NaCl
and were then irradiated for various times up to 30 min
with 360-to-750-nm light from a tungsten-halogen
source delivered at 0.5 mW/cm2. Immediately following
irradiation, the cells were removed from the substrate
and the activity of cytochrome c oxidase was determined
in these samples. The data presented in Figure 2
demonstrate that mitochondrial cytochrome c oxidase
activity in whole cells treated with 2-Se and light is
inhibited in a concentration-and-light-dose-dependent
manner. Cells treated with 2-Se alone or light alone
showed no inhibition of cytochrome c oxidase compared
to untreated control cells under these conditions. Fur-
B. P h ototoxicity tow a r d Colo-26 Cells in Cu l-
tu r e. All of the chalcogenopyrylium dyes AA1, 1-4, 11-
14, 21-Se, and 27-Se were examined for phototoxicity
against cultured Colo-26 cells. Phototoxicity was ob-
served with thiopyrylium dyes 1-S and 13-S, with
selenopyrylium dyes 1-Se, 2-Se, 3-Se, 4-Se, 13-Se, 14-
Se, and 27-Se, and with telluropyrylium dye 13-Te.
Values of the effective concentration to give 50-% cell
kill (EC50) with 15 J cm-2 of 360-800-nm light are
compiled in Table 1. The range of EC50 values varied
from 0.07 µM for dye 14-Se to 2 µM for dye 13-S.
One trend observed in the phototoxicity studies was
that derivatives with a 4-phenyl substituent displayed
no increased toxicity upon irradiation compared to cells
exposed to dyes in the dark. Compound 2-Se proved to
be an efficient photosensitizer of Colo-26 cells in vitro.
If the 4-N,N-dimethylanilino and 6-phenyl substituents
are interchanged, one obtains 11-Se. Surprisingly,
selenopyrylium dye 11-Se displays no added photo-
toxicity. Similarly, none of the 4-phenyl-substituted
derivatives 12 displays any greater toxicity upon ir-
radiation than cells incubated with 12 in the dark.
The ratio of dark toxicity to phototoxicity as an
approximation of the therapeutic ratio for the photo-
sensitizers is perhaps a better measure of photosensi-
tizer effectiveness. Values of LD50/EC50 are compiled in
Table 1 and cover a range from 1.3 for 13-Te to 33 for
13-S. The series of photosensitizers 1-S, 1-Se, 2-Se,
3-Se, 4-Se, 13-S, 13-Se, 13-Te, 14-Se, and 27-Se cover
the 0.9-2.2 range of values of log P. In Figure 1, LD50/
EC50 is plotted as a function of log P. The data of Figure
1 suggest that the thiopyrylium and selenopyrylium
sensitizers with the largest therapeutic ratios are
clustered around log P values of 1.0-1.2.
C. Mitoch on d r ia l Loca liza tion or Reloca liza tion .
Cytoch r om e c Oxid a se Activity in Wh ole Cells. The
relocalization of a series of methylene blue photosensi-
tizers from lysosomes to mitochondria or DNA upon

Chalcogenopyrylium Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5129
Su m m a r y a n d Con clu sion s
General synthetic routes to selenopyrylium and tel-
luropyrylium analogues of AA1 and to chalcogenopyry-
lium analogues of a number of closely related structures
have been described. Several of these chalcogenopyry-
lium dyes are photosensitizers in vitro as determined
by phototoxicity studies in cultured Colo-26 cells and
whole cell mitochondrial cytochrome c oxidase studies
performed in cultured R3230AC cells. In general, the
selenopyrylium analogue in a series of chalcogenopyry-
lium dyes is most likely to show phototoxicity due to
the heavy atom effect increasing singlet oxygen yield,8
although all three chalcogen analogues of dye series 13
demonstrated some phototoxicity.
While phototoxicity was observed for the chalco-
genopyrylium dyes in this study for a range of log P
values from 1.0 to 2.2, the highest therapeutic ratio
(LD50/EC50) was found for dyes with log P values of
roughly from 1.0 to 1.2 (Figure 1). The chalcogena-
pyrylium dyes in this range include the more phototoxic
compounds (2-Se with EC50 of 0.2 µM, 14-Se with EC50
of 0.07 µM) as well as compounds that are phototoxic
at higher concentrations such as 13-S which has an
EC50 of 2.0 µM. Over the range of log P values from 0.9
to 2.2, there is no apparent correlation between dark
toxicity and log P (Figure S2, Supporting Information).
However, compounds with log P values ranging from
1.0 to 1.2 appear to be more effective photosensitizers
of cultured Colo 26 cells. This dependency on a range
of log P, hydrophobicity/hydrophilicity, for photosensi-
tizer efficacy has been reported for other dye classes
such as Nile blue derivatives,22 porphyrins with a
variety of side chain substituents,23 and methylene blue
analogues.10
At this time, we cannot define the site(s) of localiza-
tion of the chalcogenopyrylium dyes, but the mitochon-
dria appear to be one site of photodamage. On the basis
of studies with rhodamines,3 nile blue derivatives,4 and
methylene blue derivatives,10 it is likely that the chal-
cogenopyrylium dyes are concentrated initially in both
the lysosomes and/or the mitochondria. The lipophilicity
of the individual cations perhaps determines the relative
concentration in the two sites. For the dyes with values
of log P near the 1.0-1.2 range, initial localization may
be in the lysozomes. Irradiation may release dyes from
the lysosomes to concentrate in the mitochondria where
photodamage occurs, therefore these dyes may show less
dark toxicity intrinsically because damage may only
occur at the lysosomes prior to irradiation. Relocaliza-
tion of photosensitizers within the cell following irradia-
tion has been reported for Nile blue derivatives22 and
meso-tetraphenylporphyrins.23 Other chalcogenopyry-
lium dyes with higher values of log P may initially
concentrate to a higher degree in the mitochondria and
dark toxicity may be observed because of the probability
that the chalcogenapyrylium dyes may disrupt mito-
chondrial function as indicated below. Dyes 1-S and
3-Se, with values of log P of 2.2 and 1.7, respectively,
inhibit cytocrome c oxidase activity in whole cells in the
dark, which is consistent with mitochondrial localiza-
tion. Localization of lipophilic cationic dyes to the
mitochondria of whole cells may also be due to both the
plasma membrane potential and the mitochondrial
electrochemical gradient.9 These cellular properties
F igu r e 3. Effect of 1-S, 3-Se, 4-Se, 13-Te, or 14-Se photo-
sensitization on mitochondrial cytochrome c oxidase activity
in cultured R3230AC tumor cells. Data are cytochrome c
oxidase activity expressed as the change in optical density at
550 nm measured as mOD units/min/1 × 105 cells. A control
group of cells received neither dye nor light. Each experimental
group of cells was exposed to dye for 24 h in the dark and a
second group was irradiated for 1 h with 360-750-nm light
at 0.5 mW cm-2 (1.8 J cm-2) followed by incubation in the dark
for 24 h. Details of experimental conditions are described in
the Experimental Section. Each datum point represents the
mean of at least three separate experiments performed in
duplicate; the error bars are the standard error from the mean
(SEM).
thermore, inhibition of cytochrome c oxidase was ob-
served with as little as 0.01 µM 2-Se, which strongly
suggests that the mitochondria are sites of localization
or relocalization upon irradiation of cultured cells
treated with 2-Se.
The effects of dye and light treatment on whole cell
cytochrome c oxidase activity with dyes 1-S, 3-Se, 4-Se,
13-Te, and 14-Se was evaluated after a 24-hr incubation
period. These dyes were selected to cover all of the
chalcogen modifications, representative examples of
substituent changes, and a range of values of log P.
Cultured R3230AC rat mammary tumor cells were
incubated with a 1.0 µM solution of 1-S (log P 2.2), 3-Se
(log P 1.7), 4-Se (log P 1.5), 13-Te (log P 1.6), or 14-Se
(log P 1.0) for 24 hr. Cultures were washed once with
0.9% NaCl and irradiated for 1 h with 360-to-750-nm
light from a tungsten-halogen source delivered at 0.5
mW/cm2 (1.8 J cm-2). Immediately following irradiation,
the saline was removed from the substrate and replaced
with media (MEM + 10% FBS). Both irradiated dye-
treated cells and dye-treated dark controls were incu-
bated for 24 h in the dark, and the activity of cytochrome
c oxidase was determined in dye-treated irradiated cells
and dye-treated dark controls (Figure 3). The data
presented in Figure 3 demonstrate that mitochondrial
cytochrome c oxidase activity in whole cells treated with
1-S, 3-Se, 4-Se, 13-Te, or 14-Se and light is inhibited
relative to untreated control cells and control cells
treated with light only. Cells treated with 4-Se, 13-Te,
or 14-Se and no light did not inhibit mitochondrial
cytochrome c oxidase activity while dyes 1-S and 3-Se
displayed some inhibition of mitochondrial cytochrome
c oxidase in the dark. These data and the results with
2-Se suggest that mitochondria may be initial sites of
localization or relocalization of chalcogenopyrylium dyes
following irradiation. Upon irradiation, photodamage
occurs at the mitochondria as evidenced by the inhibi-
tion of cytochrome c oxidase.

5130 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
trile and a small amount of ether to give 0.012 g (75%) of AA1
as a dark blue solid; whose spectral properties were identical
to those of an authentic sample,9 mp >260 °C: 1H NMR [500
MHz, CD2Cl2] δ 8.28 (s, 2 H), 7.97 (AA′BB′, 2 H, J ) 9.5 Hz),
7.72 (AA′BB′, 4 H, J ) 8.5 Hz), 6.88 (m, 6 H), 4.53 (s, 4 H),
3.21 (s, 6 H); λmax (CH2Cl2) 581 nm, ꢀ ) 5.55 (( 0.05) × 104
appear to cause the lipophilic cationic dyes to concen-
trate intracellulary 100-10000-fold compared to the
concentration of the dye in the culture medium.9 In this
report, the mitochondria are damaged upon irradiation
of dye-treated cells as evidenced by the light-induced
inhibition of mitochondrial cytochrome c oxidase (Figure
3). Studies of the effects of the chalcogenapyrylium dyes
on this enzyme support earlier conclusions that lipo-
philic cationic dyes initially localize or relocalize upon
irradiation to the mitochondria of cultured cells.
The chalcogenopyrylium dyes are electron acceptors
as measured by E° for the cation/neutral radical redox
couple, but there is no overall correlation of E° with dark
toxicity in cultured cells. However, dyes 1-S and 3-Se,
with higher values of log P in this study (2.2 and 1.7,
respectively) show some inhibition of whole-cell, mito-
chondrial cytochrome c oxidase activity in the dark,
which may involve electron transfer to the chalco-
genopyrylium dye.
Whether damage to the mitochondria in the dark or
after irradiation in the presence of chalcogenapyrylium
dyes is the underlying cause for loss in cell viability is
not yet known. One aspect that is of interest is whether
members of this class of photosensitizers are cytotoxic
via induction of necrosis or through apoptosis. One could
surmise that compounds with a high affinity for mito-
chondria might act on this organelle which is the source
of cytochrome c, the factor, which when released from
the mitochondria activates the apoptotic machinery in
the cell leading to cell death. That this class of dyes may
be involved in apoptosis is the focus of future studies
in our laboratories.
M-1 cm-1
.
2,6-Bis(4-a n ilin o)-4-(4-N ,N -d im e t h yla n ilin o)se le n o-
p yr yliu m Ch lor id e (3-Se). A solution of ∆-4H-2,6-bis(4-
anilino)-selenopyran-4-one (5-Se, 0.050 g, 0.15 mmol) in THF
(5 mL) was added dropwise to the Grignard reagent prepared
from p-bromo-N,N-dimethylaniline, (0.29 g, 1.5 mmol) and Mg
turnings, (0.07 g, 2.9 mmol) in THF (2 mL). The resulting
mixture was heated at reflux for 0.5 h and was treated as
described for the preparation of AA1. The crude product was
recrystallized from CH3CN and a small amount of ether to give
0.024 g (28%) of the PF6 salt of 3-Se as a dark blue solid, mp
>260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.25 (s, 2 H), 7.98
(AA′BB′, 2 H, J ) 9.5 Hz), 7.67 (AA′BB′, 4 H, J ) 8.5 Hz),
6.87 (m, 6 H), 4.55 (s, 4 H), 3.19 (s, 6 H); λmax (CH2Cl2) 610
nm, ꢀ ) 6.90 (( 0.02) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.020 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.014 g (86%) of the product as a dark blue solid, mp
>260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.24 (s, 2 H), 7.97
(AA′BB′, 2 H, J ) 9.5 Hz), 7.67 (AA′BB′, 4 H, J ) 8.5 Hz),
6.88 (m, 6 H), 4.67 (s, 4 H), 3.18 (s, 6 H); λmax (H2O) 554 nm,
ꢀ ) 3.23 (( 0.01) × 104 M-1 cm-1. Anal. C, H, N.
2,6-Bis(4-a n ilin o)-4-(4-N,N-d im et h yla n ilin o)t ellu r o-
p yr yliu m Ch lor id e (3-Te). A solution of ∆-4H-2,6-(4-anilino)-
telluropyran-4-one (5-Te, 0.050 g, 0.13 mmol) in THF (5 mL)
was added dropwise to the Grignard reagent prepared from
p-bromo-N,N-dimethylaniline, (0.26 g, 1.3 mmol) and Mg
turnings, (0.06 g, 2.6 mmol) in THF (2 mL). The resulting
mixture was heated at reflux for 0.5 h and was treated as
described for the preparation of AA1. The crude product was
recrystallized from CH3CN and a small amount of ether to give
0.017 g (21%) of the PF6 salt of 3-Te as a dark blue solid, mp
>260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.29 (s, 2 H), 7.97
(AA′BB′, 2 H, J ) 8.4 Hz), 7.58 (AA′BB′, 4 H, J ) 8.5 Hz),
6.86 (m, 6 H), 4.56 (s, 4 H), 3.16 (s, 6 H); λmax (CH2Cl2) 620
nm, ꢀ ) 3.96 (( 0.02) × 104 M-1 cm-1. Anal. C, H, N.
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. Concentration in vacuo was performed
on a Bu¨chi rotary evaporator. Selenopyranone 22 was prepared
according to ref 8c. NMR spectra were recorded on a Varian
Gemini-300, Inova 400, or Inova 500 instruments 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
equipped with a circulating constant-temperature bath for the
sample chambers. Elemental analyses were conducted by
Atlantic Microanalytical, Inc.
2,6-(4-An ilin o)-4-(4-N,N-d im et h yla n ilin o)t h iop yr yli-
u m Ch lor id e (AA1).9 A solution of ∆-4H-2,6-bis(4-anilino)-
thiopyran-4-one (5-S, 0.050 g, 0.17 mmol) in THF (5 mL) was
added dropwise to the Grignard reagent prepared from p-
bromo-N,N-dimethylaniline, (0.34 g, 1.7 mmol) and Mg turn-
ings, (0.08 g, 3.4 mmol) in THF (2 mL). The resulting mixture
was heated at reflux for 0.5 h, allowed to cool to ambient
temperature and poured into CH3CO2H (3.0 mL). A 60%
weight solution of HPF6 in water was added dropwise until a
color change was observed. Water (10 mL) was added, and the
resulting solution was cooled to -10 °C. The precipitate was
collected by filtration, and the solid was washed with water
(10 mL) and ether (10 mL). The crude product was recrystal-
lized from CH3CN and a small amount of ether to give 0.032
g (34%) the PF6 salt of AA1 as a dark blue solid, mp > 260 °C.
The hexafluorophosphate salt (0.020 g) was dissolved in 10
mL of CH3CN, and 0.125 g of Amberlite IRA-400 Chloride ion-
exchange resin was added. The resulting mixture was stirred
0.5 h, followed by removal of the exchange resin via filtration.
The process was repeated with two additional 0.125-g aliquots
of the ion-exchange resin. Following the final ion exchange,
the filtrate was concentrated and recrystallized from acetoni-
The hexafluorophosphate salt (0.020 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.014 g (84%) of the product as a dark blue solid, mp
>260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.26 (s, 2 H), 7.96
(AA′BB′, 2 H, J ) 8.4 Hz), 7.57 (AA′BB′, 4 H, J ) 8.5 Hz),
6.85 (m, 6 H), 4.73 (s, 4 H), 3.16 (s, 6 H); λmax (H2O) 581 nm,
ꢀ ) 2.65 (( 0.08) × 104 M-1 cm-1. Anal. C, H, N.
2,6-Bis(4-a n ilin o)-4-(4-N-m or p h olin op h en yl)th iop yr y-
liu m Ch lor id e (4-S). A solution of ∆-4H-2,6-bis(4-anilino)-
thiopyran-4-one (0.050 g, 0.17 mmol) in THF (5 mL) was added
dropwise to the Grignard reagent prepared from N-(4-bro-
mophenyl)morpholine (0.41 g, 1.7 mmol) and Mg turnings,
(0.08 g, 3.4 mmol) in THF (2 mL). The resulting mixture was
heated at reflux for 0.5 h and was treated as described for AA1.
The crude product was recrystallized from CH3CN and a small
amount of ether to give 0.017 g (17%) of the product as a dark
blue solid, mp >260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.29
(s, 2 H), 7.95 (AA′BB′, 2 H, J ) 9.0 Hz), 7.75 (AA′BB′, 4 H, J
) 8.5 Hz), 7.07 (AA′BB′, 2 H, J ) 9.0 Hz), 6.87 (AA′BB′, 4 H,
J ) 8.5 Hz), 4.60 (s, 4 H), 3.86 (t, 4 H, J ) 4.8 Hz), 3.48 (t, 4
H, J ) 4.8 Hz); λmax (CH2Cl2) 566 nm, ꢀ ) 4.58 (( 0.02) × 104
M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.020 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.012 g (74%) of 4-S as a dark blue solid, mp >260 °C:
1H NMR [500 MHz, CD2Cl2] δ 8.28 (s, 2 H), 7.94 (AA′BB′, 2 H,

Chalcogenopyrylium Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5131
J ) 9.0 Hz), 7.75 (AA′BB′, 4 H, J ) 8.5 Hz), 7.07 (AA′BB′, 2
H, J ) 9.0 Hz), 6.87 (AA′BB′, 4 H, J ) 8.5 Hz), 4.60 (s, 4 H),
3.86 (t, 4 H, J ) 4.8 Hz), 3.48 (t, 4 H, J ) 4.8 Hz); λmax (H2O)
535 nm, ꢀ ) 3.5 (( 0.1) × 104 M-1 cm-1. Anal. C, H, N.
188 °C: 1H NMR [400 MHz, CD2Cl2] δ 8.24 (s, 1 H), 8.18 (s, 1
H), 7.96 (m, 2 H), 7.84 (AA′BB′, 2 H, J ) 9.2 Hz), 7.76 (AA′BB′,
2 H, J ) 8.8 Hz), 7.60 (m, 3 H), 7.04 (AA′BB′, 2 H, J ) 9.2
Hz), 6.88 (AA′BB′, 2 H, J ) 8.8 Hz), 4.93 (br s, 2 H), 3.18 (s,
6 H); λmax (CH2Cl2) 635 nm, ꢀ ) 2.5 (( 0.1) × 104 M-1 cm-1
.
2,6-Bis(4-a n ilin o)-4-(4-N-m or p h olin op h en yl)selen o-
p yr yliu m Ch lor id e (4-Se). A solution of ∆-4H-2,6-bis(4-
anilino)selenopyran-4-one (5-Se, 0.050 g, 0.15 mmol) in THF
(5 mL) was added dropwise to the Grignard reagent prepared
from N-(4-bromophenyl)morpholine (0.36 g, 1.5 mmol) and Mg
turnings, (0.08 g, 3.4 mmol) in THF (2 mL). The resulting
mixture was heated at reflux for 0.5 h and was treated as
described for AA1. The crude product was recrystallized from
CH3CN and a small amount of ether to give 0.017 g (17%) of
the product as a dark blue solid, mp >260 °C: 1H NMR [500
MHz, CD2Cl2] δ 8.25 (s, 2 H), 7.95 (AA′BB′, 2 H, J ) 9.2 Hz),
7.70 (AA′BB′, 4 H, J ) 8.8 Hz), 7.06 (AA′BB′, 2 H, J ) 9.2
Hz), 6.86 (AA′BB′, 4 H, J ) 8.8 Hz), 4.63 (s, 4 H), 3.87 (t, 4 H,
J ) 5.1 Hz), 3.46 (t, 4 H, J ) 5.1 Hz); λmax (CH2Cl2) 593 nm, ꢀ
) 4.48 (( 0.03) × 104 M-1 cm-1. Anal. C, H, N.
2-(4-An ilin o)-6-(4-N -m or p h olin op h e n yl)-4-p h e n yl-
th iop yr yliu m Ch lor id e (12-S). A solution of ∆-4H-2-(4-
anilino)-6-(4-N-morpholinophenyl)thiopyran-4-one (16-S, 0.10
g, 0.27 mmol) in THF (5 mL) was added dropwise to a 1.0 M
solution of phenylmagnesium bromide in THF (1.4 mL, 1.4
mmol). The resulting mixture was heated at reflux for 0.5 h
and was treated as described for AA1. The crude product was
recrystallized from CH3CN and a small amount of ether to give
0.10 g (64%) of the hexafluorophosphate salt as a dark blue
solid, mp 170-173 °C: 1H NMR [400 MHz, CD2Cl2] δ 8.28 (s,
1 H), 8.27 (s, 1 H), 7.87 (m, 4 H), 7.82 (AA′BB′, 2 H, J ) 8.8
Hz), 7.69 (m, 3 H), 7.06 (AA′BB′, 2 H, J ) 9.2 Hz), 6.89
(AA′BB′, 2 H, J ) 8.8 Hz), 4.83 (s, 2 H), 3.86 (t, 4 H, J ) 4.8
Hz), 3.48 (t, 4 H, J ) 4.8 Hz); λmax (CH2Cl2) 616 nm, ꢀ ) 4.40
(( 0.06) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.020 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.014 g (85%) of 4-Se as a dark blue solid, mp >260
°C: 1H NMR [400 MHz, CD2Cl2] δ 8.24 (s, 2 H), 7.93 (AA′BB′,
2 H, J ) 9.2 Hz), 7.69 (AA′BB′, 4 H, J ) 8.8 Hz), 7.06 (AA′BB′,
2 H, J ) 9.2 Hz), 6.86 (AA′BB′, 4 H, J ) 8.8 Hz), 4.70 (s, 4 H),
3.87 (t, 4 H, J ) 5.1 Hz), 3.46 (t, 4 H, J ) 5.1 Hz); λmax (H2O)
553 nm, ꢀ ) (3.15 ( 0.01) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.050 g) was dissolved in 20
mL of CH3CN was treated with Amberlite IRA-400 Chloride
ion-exchange resin as described for AA1. The product was
recrystallized from CH3CN and a small amount of ether to give
0.040 g (98%) of 12-S as a dark blue solid, mp 176-180 °C:
1H NMR [500 MHz, CD2Cl2] δ 8.28 (s, 1 H), 8.27 (s, 1 H), 7.87
(m, 4 H), 7.82 (AA′BB′, 2 H, J ) 8.8 Hz), 7.69 (m, 3 H), 7.06
(AA′BB′, 2 H, J ) 9.2 Hz), 6.89 (AA′BB′, 2 H, J ) 8.8 Hz),
4.83 (s, 2 H), 3.86 (t, 4 H, J ) 4.8 Hz), 3.48 (t, 4 H, J ) 4.8
Hz); λmax (H2O) 597 nm, ꢀ ) 2.12 (( 0.09) × 104 M-1 cm-1
.
2,6-Bis(4-a n ilin o)-4-(4-N-m or p h olin op h en yl)t ellu r o-
p yr yliu m Ch lor id e (4-Te). A solution of ∆-4H-2,6-bis(4-
anilino)telluropyran-4-one (5-Te, 0.050 g, 0.13 mmol) in THF
(5 mL) was added dropwise to the Grignard reagent prepared
from N-(4-bromophenyl)morpholine (0.36 g, 1.5 mmol) and Mg
turnings, (0.08 g, 3.4 mmol) in THF (2 mL). The resulting
mixture was heated at reflux for 0.5 h and was treated as
described for AA1. The crude product was recrystallized from
CH3CN and a small amount of ether to give 0.017 g (17%) of
the product as a dark blue solid, mp > 260 °C: 1H NMR [500
MHz, CD2Cl2] δ 8.27 (s, 2 H), 7.93 (AA′BB′, 2 H, J ) 9.2 Hz),
7.61 (AA′BB′, 4 H, J ) 8.9 Hz), 7.07 (AA′BB′, 2 H, J ) 9.2
Hz), 6.84 (AA′BB′, 4 H, J ) 8.9 Hz), 4.64 (s, 4 H), 3.87 (t, 4 H,
J ) 5.0 Hz), 3.42 (t, 4 H, J ) 5.0 Hz); λmax (CH2Cl2) 618 nm, ꢀ
) 5.38 (( 0.02) × 104 M-1 cm-1. Anal. C, H, N.
Anal. C, H, N.
2-(4-An ilin o)-6-(4-N-m or p h olin op h en yl)-4-p h en ylsele-
n op yr yliu m Ch lor id e (12-Se). A solution of ∆-4H-2-(4-
anilino)-6-(4-N-morpholinophenyl)selenopyran-4-one (16-Se,
0.10 g, 0.24 mmol) in THF (5 mL) was added dropwise to a
1.0-M solution of phenylmagnesium bromide in THF (1.4 mL,
1.4 mmol). The resulting mixture was heated at reflux for 0.5
h and was treated as described for AA1. The crude product
was recrystallized from CH3CN and a small amount of ether
to give 0.11 g (71%) of the hexafluorophosphate salt as a dark
blue solid, mp 170-173 °C: 1H NMR [400 MHz, CD2Cl2] δ 8.19
(s, 1 H), 8.18 (s, 1 H), 7.84 (m, 4 H), 7.76 (AA′BB′, 2 H, J ) 8.8
Hz), 7.66 (m, 3 H), 7.04 (AA′BB′, 2 H, J ) 9.2 Hz), 6.88
(AA′BB′, 2 H, J ) 8.8 Hz), 4.93 (s, 2 H), 3.86 (t, 4 H, J ) 4.8
Hz), 3.47 (t, 4 H, J ) 4.8 Hz); λmax (CH2Cl2) 645 nm, ꢀ ) 4.9 ((
0.2) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.030 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.020 g (74%) of 4-Te as a dark blue solid, mp >260
°C: 1H NMR [400 MHz, CD2Cl2] δ 8.24 (s, 2 H), 7.90 (AA′BB′,
2 H, J ) 9.2 Hz), 7.60 (AA′BB′, 4 H, J ) 8.9 Hz), 7.06 (AA′BB′,
2 H, J ) 9.2 Hz), 6.84 (AA′BB′, 4 H, J ) 8.9 Hz), 4.74 (s, 4 H),
3.87 (t, 4 H, J ) 5.0 Hz), 3.42 (t, 4 H, J ) 5.0 Hz); λmax (H2O)
582 nm, ꢀ ) 2.28 (( 0.04) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.050 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.020 g (75%) of the product as a dark blue solid, mp
176-180 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.28 (s, 1 H), 8.27
(s, 1 H), 7.87 (m, 4 H), 7.82 (AA′BB′, 2 H, J ) 8.8 Hz), 7.69
(m, 3 H), 7.06 (AA′BB′, 2 H, J ) 9.2 Hz), 6.89 (AA′BB′, 2 H, J
) 8.8 Hz), 4.83 (s, 2 H), 3.86 (t, 4 H, J ) 4.8 Hz), 3.48 (t, 4 H,
J ) 4.8 Hz); λmax (H2O) 597 nm, ꢀ ) 2.12 (( 0.09) × 104 M-1
cm-1. Anal. C, H, N.
2-(4-An ilin o)-6-(4-N,N-d im eth yla n ilin o)-4-p h en ylsele-
n op yr yliu m Ch lor id e (11-Se). A solution of ∆-4H-2-(4-
anilino)-6-(4-N,N-dimethylanilino)selenopyran-4-one (15-Se,
0.074 g, 0.20 mmol) in THF (5 mL) was added dropwise to a
1.0-M solution of phenylmagnesium bromide in THF (1.0 mL,
1.0 mmol). The resulting mixture was heated at reflux for 0.5
h and was treated as described for AA1. The crude product
was recrystallized from CH3CN and a small amount of ether
to give 0.027 g (15%) of the product as a dark blue solid, mp
180-183 °C: 1H NMR [400 MHz, CD2Cl2] δ 8.24 (s, 1 H), 8.18
(s, 1 H), 7.96 (m, 2 H), 7.84 (AA′BB′, 2 H, J ) 9.2 Hz), 7.76
(AA′BB′, 2 H, J ) 8.8 Hz), 7.60 (m, 3 H), 7.04 (AA′BB′, 2 H, J
) 9.2 Hz), 6.88 (AA′BB′, 2 H, J ) 8.8 Hz), 4.93 (br s, 2 H),
3.18 (s, 6 H); λmax (CH2Cl2) 670 nm, ꢀ ) 5.1 (( 0.2) × 104 M-1
cm-1. Anal. C, H, N.
2-(4-An ilin o)-6-(4-N -m or p h olin op h e n yl)-4-p h e n yl-
tellu r op yr yliu m Ch lor id e (12-Te). A solution of ∆-4H-2-(4-
anilino)-6-(4-N-morpholinophenyl)telluropyran-4-one (16-Te,
0.10 g, 0.22 mmol) in THF (5 mL) was added dropwise to a
1.0-M solution of phenylmagnesium bromide in THF (1.1 mL,
1.1 mmol). The resulting mixture was heated at reflux for 0.5
h and was treated as described for AA1. The crude product
was recrystallized from CH3CN and a small amount of ether
to give 0.090 g (63%) of the hexafluorophosphate salt as a dark
blue solid, mp 190-193 °C: 1H NMR [400 MHz, CD2Cl2] δ 8.17
(s, 1 H), 8.16 (s, 1 H), 7.81 (m, 2 H), 7.74 (AA′BB′, 2 H, J ) 8.8
Hz), 7.65 (m, 5 H), 7.02 (AA′BB′, 2 H, J ) 8.8 Hz), 6.86
(AA′BB′, 2 H, J ) 8.4 Hz), 4.97 (s, 2 H), 3.86 (t, 4 H, J ) 4.8
Hz), 3.45 (t, 4 H, J ) 4.8 Hz); λmax (CH2Cl2) 674 nm, ꢀ ) 5.02
(( 0.07) × 102 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.030 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.020 g (75%) of 11-Se as a dark blue solid, mp 187-
The hexafluorophosphate salt (0.050 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400

5132 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.020 g (75%) of 12-Te as a dark blue solid, mp 176-
180 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.15 (s, 1 H), 8.14 (s, 1
H), 7.81 (m, 2 H), 7.74 (AA′BB′, 2 H, J ) 8.8 Hz), 7.65 (m, 5
H), 7.02 (AA′BB′, 2 H, J ) 8.8 Hz), 6.86 (AA′BB′, 2 H, J ) 8.4
Hz), 4.97 (s, 2 H), 3.86 (t, 4 H, J ) 4.8 Hz), 3.45 (t, 4 H, J )
H), 8.26 (s, 1 H), 7.93 (AA′BB′, 2 H, J ) 9.2 Hz), 7.70 (AA′BB′,
2 H, J ) 8.9 Hz), 7.61 (AA′BB′, 2 H, J ) 8.5 Hz), 7.07 (AA′BB′,
2 H, J ) 8.9 Hz), 7.02 (AA′BB′, 2 H, J ) 9.2 Hz), 6.84 (AA′BB′,
2 H, J ) 8.5 Hz), 4.67 (s, 2 H), 3.86 (m, 8 H), 3.42 (m, 8 H);
λmax (CH2Cl2) 638 nm, ꢀ ) 3.50 (0.09) × 104 M-1 cm-1. Anal.
C, H, N.
The hexafluorophosphate salt (0.053 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN ether to give 0.037 g (82%) of
13-Te as a dark blue solid, mp >260 °C:1H NMR [500 MHz,
CD2Cl2] δ 8.27 (s, 1 H), 8.26 (s, 1 H), 7.93 (AA′BB′, 2 H, J )
9.2 Hz), 7.70 (AA′BB′, 2 H, J ) 8.9 Hz), 7.61 (AA′BB′, 2 H, J
) 8.5 Hz), 7.07 (AA′BB′, 2 H, J ) 8.9 Hz), 7.02 (AA′BB′, 2 H,
J ) 9.2 Hz), 6.84 (AA′BB′, 2 H, J ) 8.5 Hz), 4.84 (s, 2 H), 3.86
(m, 8 H), 3.42 (m, 8 H); λmax (H2O) 584 nm, ꢀ ) 2.38 (( 0.03)
× 104 M-1 cm-1. Anal. C, H, N.
4.8 Hz); λmax (H2O) 653 nm, ꢀ ) 256 ( 9.5 × 102 M-1 cm-1
.
Anal. C, H, N.
2-(4-An ilin o)-4,6-bis(4-N-m or p h olin op h en yl)th iop yr y-
liu m Ch lor id e (13-S). A solution of ∆-4H-2-(4-anilino)-6-(4-
N-morpholinophenyl)thiopyran-4-one (16-S, 0.05 g, 0.14 mmol)
in THF (5 mL) was added dropwise to the Grignard reagent
prepared from N-(4-bromophenyl)morpholine (0.17 g, 0.69
mmol) and Mg turnings, (0.04 g, 1.7 mmol) in THF (2 mL).
The resulting mixture was heated at reflux for 0.5 h and was
treated as described for AA1. The crude product was recrystal-
lized from CH3CN and ether to give 0.10 g (64%) of the
hexafluorophosphate salt as a dark blue solid, mp >260 °C:
1H NMR [500 MHz, CD2Cl2] δ 8.31 (s, 1 H), 8.30 (s, 1 H), 7.96
(AA′BB′, 2 H, J ) 8.5 Hz), 7.85 (AA′BB′, 2 H, J ) 8.2 Hz),
7.76 (AA′BB′, 2 H, J ) 8.2 Hz), 7.06 (m, 4 H), 6.88 (AA′BB′, 2
H, J ) 8.5 Hz), 4.66 (s, 2 H), 3.86 (m, 8 H), 3.45 (m, 8 H); λmax
(CH2Cl2) 578 nm, ꢀ ) 6.9 (( 0.4) × 104 M-1 cm-1. Anal. C, H,
N.
2-(4-An ilin o)-4-(4-N,N-dim eth ylan ilin o)-6-(4-N-m or ph oli-
n op h en yl)th iop yr yliu m Ch lor id e (14-S). A solution of
∆-4H-2-(4-anilino)-6-(4-N-morpholinophenyl)thiopyran-4-one
(16-S, 0.10 g, 0.27 mmol) in THF (5 mL) was added dropwise
to the Grignard reagent prepared from 4-bromo-N,N-dimeth-
ylaniline (0.28 g, 1.4 mmol) and Mg turnings, (0.07 g, 2.8
mmol) in THF (2 mL). The resulting mixture was heated at
reflux for 0.5 h and was treated as described for AA1. The
crude product was recrystallized from CH3CN and ether to give
0.12 g (72%) of the hexafluorophosphate salt as a dark blue
solid, mp >260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.30 (s, 1
H), 8.27 (s, 1 H), 7.98 (AA′BB′, 2 H, J ) 9.2 Hz), 7.82 (AA′BB′,
2 H, J ) 8.9 Hz), 7.73 (AA′BB′, 2 H, J ) 9.2 Hz), 7.05 (AA′BB′,
2 H, J ) 9.2 Hz), 7.02 (AA′BB′, 2 H, J ) 9.2 Hz), 6.86 (AA′BB′,
2 H, J ) 8.9 Hz), 4.55 (s, 2 H), 3.86 (t, 4 H, J ) 4.9 Hz), 3.40
(t, 4 H, J ) 4.9 Hz), 3.20 (s, 6 H); λmax (CH2Cl2) 589 nm, ꢀ )
7.7 (( 0.2) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.038 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.028 g (88%) of 13-S as a dark blue solid, mp >260
°C:1H NMR [500 MHz, CD2Cl2] δ 8.29 (s, 1 H), 8.28 (s, 1 H),
7.96 (AA′BB′, 2 H, J ) 8.5 Hz), 7.85 (AA′BB′, 2 H, J ) 8.2
Hz), 7.76 (AA′BB′, 2 H, J ) 8.2 Hz), 7.06 (m, 4 H), 6.88
(AA′BB′, 2 H, J ) 8.5 Hz), 5.04 (s, 2 H), 3.86 (m, 8 H), 3.45
(m, 8 H); λmax (H2O) 546 nm, ꢀ ) 1.89 (( 0.05) × 104 M-1 cm-1
.
The hexafluorophosphate salt (0.050 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and ether to give 0.039 g (95%)
of 14-S as a dark blue solid, mp >260 °C: 1H NMR [500 MHz,
CD2Cl2] δ 8.30 (s, 1 H), 8.29 (s, 1 H), 7.98 (AA′BB′, 2 H, J )
9.2 Hz), 7.83 (AA′BB′, 2 H, J ) 9.2 Hz), 7.73 (AA′BB′, 2 H, J
) 8.9 Hz), 7.05 (AA′BB′, 2 H, J ) 9.2 Hz), 7.02 (AA′BB′, 2 H,
J ) 9.2 Hz), 6.86 (m, 4 H), 4.64 (s, 2 H), 3.86 (t, 4 H, J ) 4.8
Hz), 3.40 (t, 4 H, J ) 4.8 Hz), 3.20 (s, 6 H); λmax (H2O) 553 nm,
ꢀ ) 1.55 (( 0.09) × 104 M-1 cm-1. Anal. C, H, N.
Anal. C, H, N.
2-(4-An ilin o)-4,6-b is(4-N-m or p h olin op h en yl)selen o-
p yr yliu m Ch lor id e (13-Se). A solution of ∆-4H-2-(4-anilino)-
6-(4-N-morpholinophenyl)selenopyran-4-one (16-Se, 0.10 g,
0.24 mmol) in THF (5 mL) was added dropwise to the Grignard
reagent prepared from N-(4-bromophenyl)morpholine (0.29 g,
1.2 mmol) and Mg turnings, (0.060 g, 2.4 mmol) in THF (2
mL). The resulting mixture was heated at reflux for 0.5 h and
was treated as described for AA1. The crude product was
recrystallized from CH3CN and ether to give 0.13 g (77%) of
the hexafluorophosphate salt as a dark blue solid, mp >260
°C: 1H NMR [500 MHz, CD2Cl2] δ 8.27 (s, 1 H), 8.26 (s, 1 H),
7.96 (AA′BB′, 2 H, J ) 8.6 Hz), 7.79 (AA′BB′, 2 H, J ) 8.2
Hz), 7.71 (AA′BB′, 2 H, J ) 8.6 Hz), 7.07 (m, 4 H), 6.86
(AA′BB′, 2 H, J ) 8.2 Hz), 4.67 (s, 2 H), 3.86 (m, 8 H), 3.43
2-(4-An ilin o)-4-(4-N,N-dim eth ylan ilin o)-6-(4-N-m or ph oli-
n op h en yl)selen op yr yliu m Ch lor id e (14-Se). A solution of
∆-4H-2-(4-anilino)-6-(4-N-morpholinophenyl)selenopyran-4-
one (16-Se, 0.10 g, 0.24 mmol) in THF (5 mL) was added
dropwise to the Grignard reagent prepared from 4-bromo-N,N-
dimethylaniline (0.28 g, 1.4 mmol) and Mg turnings (0.07 g,
2.8 mmol) in THF (2 mL). The resulting mixture was heated
at reflux for 0.5 h and was treated as described for AA1. The
crude product was recrystallized from CH3CN and ether to give
0.12 g (72%) of the hexafluorophosphate salt as a dark blue
solid, mp 179-183 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.27 (s,
1 H), 8.26 (s, 1 H), 7.99 (AA′BB′, 2 H, J ) 9.2 Hz), 7.77 (AA′BB′,
2 H, J ) 9.1 Hz), 7.68 (AA′BB′, 2 H, J ) 8.5 Hz), 7.04 (AA′BB′,
2 H, J ) 9.2 Hz), 6.95 (AA′BB′, 2 H, J ) 9.1 Hz), 6.87 (AA′BB′,
2 H, J ) 8.5 Hz), 4.59 (s, 2 H), 3.86 (t, 4 H, J ) 4.6 Hz), 3.40
(t, 4 H, J ) 4.6 Hz), 3.19 (s, 6 H); λmax (CH2Cl2) 616 nm, ꢀ )
5.3 (( 0.2) × 104 M-1 cm-1. Anal. C, H, N.
(m, 8 H); λmax (CH2Cl2) 608 nm, ꢀ ) 5.1 (( 0.1) × 104 M-1 cm-1
.
Anal. C, H, N.
The hexafluorophosphate salt (0.049 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.039 g (95%) of 13-Se as a dark blue solid, mp >260
°C:1H NMR [500 MHz, CD2Cl2] δ 8.27 (s, 1 H), 8.26 (s, 1 H),
7.96 (AA′BB′, 2 H, J ) 8.6 Hz), 7.79 (AA′BB′, 2 H, J ) 8.2
Hz), 7.71 (AA′BB′, 2 H, J ) 8.6 Hz), 7.07 (m, 4 H), 6.86
(AA′BB′, 2 H, J ) 8.2 Hz), 4.97 (s, 2 H), 3.86 (m, 8 H), 3.43
(m, 8 H); λmax (H2O) 566 nm, ꢀ ) 2.28 (( 0.07) × 104 M-1 cm-1
.
Anal. C, H, N.
The hexafluorophosphate salt (0.047 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.037 g (95%) of 14-Se as a dark blue solid, mp 187-
190 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.27 (s, 1 H), 8.26 (s, 1
H), 7.97 (AA′BB′, 2 H, J ) 9.5 Hz), 7.77 (AA′BB′, 2 H, J ) 8.9
Hz), 7.68 (AA′BB′, 2 H, J ) 8.9 Hz), 7.04 (AA′BB′, 2 H, J )
8.9 Hz), 6.90 (AA′BB′, 2 H, J ) 9.5 Hz), 6.86 (AA′BB′, 2 H, J
) 8.5 Hz), 4.63 (s, 2 H), 3.86 (t, 4 H, J ) 4.6 Hz), 3.40 (t, 4 H,
J ) 4.6 Hz), 3.19 (s, 6 H); λmax (H2O) 557 nm, ꢀ ) 3.32 ((0.06)
× 104 M-1 cm-1. Anal. C, H, N.
2-(4-An ilin o)-4,6-b is(4-N-m or p h olin op h en yl)t ellu r o-
p yr yliu m Ch lor id e (13-Te). A solution of ∆-4H-2-(4-anilino)-
6-(4-N-morpholinophenyl)telluropyran-4-one (16-Te, 0.10 g,
0.22 mmol) in THF (5 mL) was added dropwise to the Grignard
reagent prepared from N-(4-bromophenyl)morpholine (0.26 g,
1.1 mmol) and Mg turnings, (0.060 g, 2.4 mmol) in THF (2
mL). The resulting mixture was heated at reflux for 0.5 h and
was treated as described for AA1. The crude product was
recrystallized from CH3CN and a small amount of ether to give
0.10 g (61%) of the hexafluorophosphate salt as a dark blue
solid, mp >260 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.27 (s, 1

Chalcogenopyrylium Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5133
2-(4-An ilin o)-4-(4-N,N-dim eth ylan ilin o)-6-(4-N-m or ph oli-
n op h en yl)tellu r op yr yliu m Ch lor id e (14-Te). A solution of
∆-4H-2-(4-anilino)-6-(4-N-morpholinophenyl)selenopyran-4-
one (16-Te, 0.10 g, 0.22 mmol) in THF (5 mL) was added
dropwise to the Grignard reagent prepared from 4-bromo-N,N-
dimethylaniline (0.28 g, 1.4 mmol) and Mg turnings, (0.07 g,
2.8 mmol) in THF (2 mL). The resulting mixture was heated
at reflux for 0.5 h and was treated as described for AA1. The
product was recrystallized from CH3CN and ether to give 0.13
g (85%) of the hexafluorophosphate salt as a blue solid, mp
180-184 °C: 1H NMR [500 MHz, CD2Cl2] δ 8.30 (s, 1 H), 8.28
(s, 1 H), 7.98 (AA′BB′, 2 H, J ) 9.2 Hz), 7.68 (AA′BB′, 2 H, J
) 8.9 Hz), 7.59 (AA′BB′, 2 H, J ) 8.9 Hz), 7.02 (AA′BB′, 2 H,
J ) 8.5 Hz), 6.95 (AA′BB′, 2 H, J ) 9.2 Hz), 6.84 (AA′BB′, 2
H, J ) 8.5 Hz), 4.58 (s, 2 H), 3.86 (t, 4 H, J ) 4.8 Hz), 3.39 (t,
4 H, J ) 4.8 Hz), 3.16 (s, 6 H); λmax (CH2Cl2) 646 nm, ꢀ ) 7.3
(( 0.1) × 104 M-1 cm-1. Anal. C, H, N.
°C: 1H NMR [500 MHz, CD2Cl2] δ 8.30 (s, 2 H), 8.02 (AA′BB′,
2 H, J ) 9.2 Hz), 7.84 (AA′BB′, 4 H, J ) 9.2 Hz), 7.07 (m, 6
H), 3.86 (t, 12 H, J ) 4.8 Hz), 3.45 (m, 12 H); λmax (H2O) ) 582
nm, ꢀ ) 1.78 (( 0.02) × 104 M-1 cm-1. Anal. C, H, N.
Electr och em ica l P r oced u r es. A BAS 100 potentiostat/
galvanostat and programmer were used for the electrochemical
measurements. The working electrode for cyclic voltammetry
was a platinum disk electrode (diameter, 1 mm) obtained from
Princeton Applied Research. The auxiliary and reference
electrodes were silver wires. The reference for cyclic voltam-
metry 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
and freshly distilled prior to use. Tetrabutylammonium fluo-
roborate (Aldrich Chemical Co.) was recrystallized from EtOAc-
ether and then dried overnight at 80 °C before it was used as
supporting electrolyte at 0.2 M. Nitrogen was used for sample
deaeration.
The hexafluorophosphate salt (0.045 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and ether to give 0.037 g (95%)
of 14-Te as a dark blue solid, mp 187-190 °C: 1H NMR [500
MHz, CD2Cl2] δ 8.28 (s, 1 H), 8.27 (s, 1 H), 7.96 (AA′BB′, 2 H,
J ) 9.2 Hz), 7.67 (AA′BB′, 2 H, J ) 9.2 Hz), 7.58 (AA′BB′, 2
H, J ) 8.5 Hz), 7.01 (AA′BB′, 2 H, J ) 9.2 Hz), 6.90 (AA′BB′,
2 H, J ) 9.2 Hz), 6.83 (AA′BB′, 2 H, J ) 8.5 Hz), 4.70 (s, 2 H),
3.85 (t, 4 H, J ) 4.8 Hz), 3.39 (t, 4 H, J ) 4.8 Hz), 3.15 (s, 6
H); λmax (H2O) 583 nm, ꢀ ) 3.56 (( 0.08) × 104 M-1 cm-1. Anal.
C, H, N.
2-(4-An ilin o)-4-(4-N-m or p h olin op h en yl)-6-p h en ylsele-
n op yr yliu m Ch lor id e (21-Se). A solution of ∆-4H-2-(4-
anilino)-6-phenylselenopyran-4-one8c (0.050 g, 0.15 mmol) in
THF (5 mL) was added dropwise to the Grignard reagent
prepared from N-(4-bromophenyl)morpholine (0.37 g, 1.5
mmol) and Mg turnings, (0.07 g, 2.8 mmol) in THF (2 mL).
The resulting mixture was heated at reflux for 0.5 h and was
treated as described for AA1. The crude product was recrystal-
lized from CH3CN and ether to give 0.080 g (90%) of the
hexafluorophosphate salt as a dark blue solid, mp >260 °C:
1H NMR [500 MHz, CD2Cl2] δ 8.44 (s, 1 H), 8.38 (s, 1 H), 8.04
(AA′BB′, 2 H, J ) 9.2 Hz), 7.73 (m, 7 H), 7.08 (AA′BB′, 2 H, J
) 9.2 Hz), 6.89 (AA′BB′, 2 H, J ) 8.9 Hz), 6.87 (AA′BB′, 2 H,
J ) 8.5 Hz), 4.81 (s, 2 H), 3.87 (t, 4 H, J ) 5.0 Hz), 3.51 (t, 4
H, J ) 5.0 Hz); λmax (CH2Cl2) 611 nm, ꢀ ) 5.5 (( 0.2) × 104
M-1 cm-1. Anal. C, H, N.
Deter m in a tion of P a r tition Coefficien ts. The octanol/
water partition coefficients were all measured at pH 6 (phos-
phate-buffered) using UV-visible spectrophotometry. The
measurements were done using a “shake flask” direct mea-
surement.20 Mixing for 3-5 min was followed by 1 h of settling
time. Equilibration and measurements were made at 23 °C
using a Perkin-Elmer Lambda 12 spectrophotometer. HPLC
grade 1-octanol was obtained from Sigma-Aldrich.
Cells a n d Cu ltu r e Con d ition s. Colo-26, a murine colon
carcinoma cell line, was maintained in a growth medium of
RPMI 1640 supplemented with 10% fetal calf serum (FCS) and
antibiotics (all components purchased from GIBCO Labora-
tories, Grand Island, NY) at 37 °C, 5% CO2. R3230AC, a rat
mammary adenocarcinoma cell line, was maintained in a
growth medium of Minimum Essential Media (R-MEM) supple-
mented with 10% fetal bovine serum (FBS, Atlanta Biologicals,
Atlanta, GA), 50 units/mL of penicillin G, 50 mg/mL of
streptomycin, and 1.0 mg/mL of Fungizone.
In Vitr o P h ototoxicity Mea su r em en ts. Colo-26 cells
were plated at 5 × 103 cells/well of a 96-well tissue culture
plate the evening before the assay. The day of the assay, the
cells were washed twice with phosphate-buffered saline (PBS),
and 100 µL of Hank’s basic salt solution (HBSS) with 1% FCS
containing various concentrations of dyes AA1, 1-4, 11-14,
21-Se, or 27-Se was added to each well. The dye and cells were
incubated for 2 h at 37 °C followed by a wash with PBS and
the addition of 100 µL of PBS supplemented with 1% FCS.
The plates were irradiated with filtered 360-to-800-nm light
for a total light dose of 15 J cm-2. Following irradiation, 100
µL of growth media was added and the plates were incubated
for 24 h at 37 °C, 5% CO2. Control cells (no dye + light; dye +
no light) were treated using the same conditions. Cell survival
was monitored using the MTT assay as described in Mos-
mann.24
Cytoch r om e c Oxid a se Mea su r em en ts in Cu ltu r ed
R3230AC Tu m or Cells. Cells cultured from the rodent
mammary adenocarcinomas (R3230AC) were used for these
studies. The R3230AC tumors were maintained by transplan-
tation in the abdominal region of 100-120 g Fischer female
rats, using the sterile trochar technique.25 R3230AC cells were
cultured from tumor homogenates using the method described
earlier.26 Cells were maintained in passage culture on 100-
mm diameter polystyrene dishes (Becton Dickinson, Franklin
Lakes, NJ ) in 10 mL of R-MEM supplemented with 10% FBS,
50 units/mL penicillin G, 50 ug/mL streptomycin, and 1.0ug/
mL Fungizone. Only cells from passages 1-10 were used for
experiments. A stock of cells, passages 1-4, were maintained
at -86 °C to initiate the experimental cultures. Cultures were
maintained at 37 °C in a 5% CO2 humidified atmosphere
(Forma Scientific, Marietta, OH). Passage was accomplished
by removing the culture medium, then adding a 1 mL solution
containing 0.25% trypsin, incubating at 37 °C for 2 to 5 min
until cells were detached from the surface followed by seeding
new culture dishes with an appropriate number of cells in 10
mL of R-MEM. Cell counts were performed using a particle
counter (Model ZM, Coulter Electronics, Hialeah, FL).
The hexafluorophosphate salt (0.050 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and ether to give 0.031 g (76%)
of 21-Se as a dark blue solid, mp >260 °C: 1H NMR [500 MHz,
CD2Cl2] δ 8.39 (s, 1 H), 8.32 (s, 1 H), 8.00 (AA′BB′, 2 H, J )
9.5 Hz), 7.76 (m, 7 H), 7.07 (AA′BB′, 2 H, J ) 9.2 Hz), 6.89
(AA′BB′, 2 H, J ) 8.9 Hz), 4.81 (s, 2 H), 3.86 (t, 4 H, J ) 4.6
Hz), 3.49 (t, 4 H, J ) 4.6 Hz); λmax (H2O) 580 nm, ꢀ ) 2.41
((0.05) × 104 M-1 cm-1. Anal. C, H, N.
2,4,6-Tr is(4-N-m or ph olin oph en yl)selen opyr yliu m Ch lo-
r id e (27-Se). A solution of ∆-4H-2,6-Bis(4-N-morpholinophe-
nyl)selenopyran-4-one (26-Se, 0.10 g, 0.21 mmol) in THF (5
mL) was added dropwise to the Grignard reagent prepared
from N-(4-bromophenyl)morpholine (0.25 g, 1.0 mmol) and Mg
turnings, (0.05 g, 2.1 mmol) in THF (2 mL). The resulting
mixture was heated at reflux for 2 h and was treated as
described for AA1. The crude product was recrystallized from
CH3CN and ether to give 0.080 g (90%) of the hexafluorophos-
phate salt as a dark blue solid, mp >260 °C: 1H NMR [500
MHz, CD2Cl2] δ 8.27 (s, 2 H), 7.97 (AA′BB′, 2 H, J ) 9.2 Hz),
7.81 (AA′BB′, 4 H, J ) 9.2 Hz), 7.06 (m, 6 H), 3.86 (t, 12 H, J
) 4.8 Hz), 3.45 (m, 12 H); λmax (CH2Cl2) ) 622 nm, ꢀ ) 6.71 ((
0.09) × 104 M-1 cm-1. Anal. C, H, N.
The hexafluorophosphate salt (0.049 g) was dissolved in 20
mL of CH3CN and was treated with Amberlite IRA-400
Chloride ion-exchange resin as described for AA1. The product
was recrystallized from CH3CN and a small amount of ether
to give 0.040 g (95%) of 21-Se as a dark blue solid, mp >260

5134 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brennan et al.
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To determine whether 2-Se photosensitized mitochondrial
cytochrome c oxidase activity, R3230AC cells were plated on
12 well culture plates using an initial cell seeding of 1.0 × 105
cells/well in 1.0 mL of R-MEM with 10% FBS. Cells were
incubated for 24 to 48 h at 37 °C in a 5% CO2 humidified
atmosphere until they reached 4 to 7 × 105 cells/well, a number
where the cells were still in log phase growth. The R-MEM
was removed and fresh R-MEM with 10% FBS, with dye at 1
× 10-8, 1 × 10-7, and 1 × 10-6 final concentration, was added.
Control cells, with no added dye, were maintained under the
same conditions. Cells were incubated for 24 h in the dark,
the medium was removed, the cells were washed once with
R-MEM minus FBS and phenol (no dye in the wash), and 1.0
mL of fresh R-MEM minus phenol but supplemented with 10%
FBS was added. Monolayers on culture plates with lids
removed were then exposed to 360-to-750-nm light from a
tungsten-halogen source (0.5 mW/cm2) at ambient tempera-
ture. At the end of the irradiation period (10, 20, or 30 min),
the medium was removed and replaced with 1.0 mL of fresh
R-MEM containing phenol and 10% FBS and incubated for 24
h. The media was removed, 0.1 mL of trypsin was added to
each well, and cells were incubated at 37 °C for 3-5 min until
all the cells detached from the surface. Cell suspensions were
transferred to 1.0-mL microcentrifuge tubes and centrifuged
at 8000g for 3 min. The supernatant was aspirated, and cell
pellets were immediately frozen and stored at -86 °C. Cyto-
chrome c oxidase activity was determined on cells that had
been thawed and sonicated for 10 s using a Bronson sonicator
(Model 185, Brinkmann Ind.) at a setting of 2. A sonicated
cell suspension, representing 1 to 1.5 × 106 cells, was used for
measurements of cytochrome c oxidase according to the method
described earlier.21 Data are expressed as percent of control
cytochrome c oxidase activity, moles of cytochrome c oxidized/
min/cell, which was determined from cells not exposed to dye
or light. Cytochrome c oxidase activity was also determined
in cells exposed to dye alone or light alone, drug, and light
controls, respectively.
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For dyes 1-S, 3-Se, 4-Se, 13-Te, and 14-Se, cells were
incubated for 24 h in the dark as above, with 1.0 × 10-6
M
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ated for 1 h with 360-to-750-nm light from a filtered tungsten-
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substrate and replaced with media (MEM + 10% FBS). Both
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Ack n ow led gm en t. The authors thank the National
Institutes of Health (Grant CA69155 to M.R.D., Grant
CA55791 to A. R. O., and Grant CA36856 to R.H.) and
the shared resources of the Roswell Park Cancer Center
support grant (P30 CA16056) for partial support of this
work.
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Su p p or tin g In for m a tion Ava ila ble: Figure S1 showing
LD50 as a function of E°; Figure S2 showing LD50 as a function
of log P; Figure S3 showing LD50/EC50 as a function of E°;
experimental procedures for the preparation of compounds 5,
6, N-(4-iodophenyl)morpholine, 15, 16, 18-20, and 23-26; and
plots of dark and phototoxicity for dyes 2-Se, 3-Se, 4-Se, 13,
14-Se, and 27-Se. This information is available free of charge
via the Internet at http://pubs.acs.org.
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