Chalcogenopyrylium compounds as modulators of the ATP-binding cassette transporters P-glycoprotein (P-gp/ABCB1) and multidrug resistance protein 1 (MRP1/ ABCC1)

Article abstract of DOI:10.1021/jm3004398

Twenty-seven chalcogenopyrylium derivatives varying in the heteroatom of the pyrylium core and substituents at the 2-, 4-, and 6-positions were examined for their effect on human MRP1-mediated uptake of tritiated estradiol glucuronide into inside-out membrane vesicles, their affinity for and ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His ₁₀, and their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant cells. Differences in their effects on MRP1 and P-gp activity were noted, and a second set of thiopyrylium compounds with systematic substituent changes was examined to refine these differences further. Derivatives with tert-butyl substituents in the 2- and 6-positions had the lowest inhibitory activity toward both transporters. Derivatives with thioamide functionality in the 4-position were more active against MRP1 than derivatives with amide functionality. Conversely, derivatives with amide functionality in the 4-position were more active in P-gp than derivatives with thioamide functionality.

Full text of DOI:10.1021/jm3004398

Article
pubs.acs.org/jmc
Chalcogenopyrylium Compounds as Modulators of the ATP-Binding
Cassette Transporters P-Glycoprotein (P-gp/ABCB1) and Multidrug
Resistance Protein 1 (MRP1/ABCC1)
Sean P. Ebert,† Bryan Wetzel,† Robert L. Myette,‡ Gwenaelle Conseil,‡ Susan P. C. Cole,‡
̈
Geri A. Sawada,§ Tip W. Loo,∥ M. Claire Bartlett,∥ David M. Clarke,∥ and Michael R. Detty*,†
†Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, United States
‡Department of Pathology & Molecular Medicine, Division of Cancer Biology & Genetics, Queen’s University, Kingston,
Ontario K7L 3N6, Canada
§Drug Disposition, Eli Lilly and Company, Indianapolis, Indiana 46285, United States
∥Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
ABSTRACT: Twenty-seven chalcogenopyrylium derivatives vary-
ing in the heteroatom of the pyrylium core and substituents at the
2-, 4-, and 6-positions were examined for their effect on human
MRP1-mediated uptake of tritiated estradiol glucuronide into inside-
out membrane vesicles, their affinity for and ability to stimulate the
ATPase activity of purified human P-glycoprotein (P-gp)-His10, and
their ability to promote uptake of calcein AM and vinblastine in
multidrug-resistant cells. Differences in their effects on MRP1 and
P-gp activity were noted, and a second set of thiopyrylium com-
pounds with systematic substituent changes was examined to refine
these differences further. Derivatives with tert-butyl substituents
in the 2- and 6-positions had the lowest inhibitory activity toward
both transporters. Derivatives with thioamide functionality in the
4-position were more active against MRP1 than derivatives with amide functionality. Conversely, derivatives with amide
functionality in the 4-position were more active in P-gp than derivatives with thioamide functionality.
crystal structure (3.8 Å resolution) of cys-less mouse P-gp in
the absence of nucleotide shows an internal cavity of ∼6000 Å3
with the NBDs separated by ∼30 Å and portals to both the
cytoplasm and inner leaflet of the membrane.9 Discrete drug-
binding pockets were also observed in crystals of drug-P-gp
complexes.9 P-gp is able to transport a diverse array of
anticancer drugs including anthracyclines, Vinca alkaloids,
taxanes, epipodophyllotoxins, and agents such as mitomycin
C and trimetrexate.10−12 Many approaches to the development
of inhibitors/modulators of P-gp have been examined,7,8 yet
there are currently no approved reversal agents available in the
clinic.5,13,14
The ABCC subfamily of ABC proteins includes nine MRP-
related transporters.15−17 In addition to two NBDs and two
TMDs, MRP1 and MRP2, 3, 6, and 7 have a third TMD while
MRP4, 5, 8, and 9 lack the third TMD and, thus, are
structurally more similar to P-gp. While there are no high-
resolution structures of the intact MRP1 protein, low resolution
studies (∼22 Å) suggest it forms dimers with each monomer
containing a central pore of ∼80 × 100 Å2.18 MRP1 has 15%
amino acid sequence homology with P-gp and, like P-gp,
INTRODUCTION
■
The effective treatment of cancer with chemotherapeutic agents
is often limited by the emergence of multidrug resistance
(MDR) in the malignant cells. While drug resistance is a
consequence of a variety of mechanisms, efflux proteins that are
members of the ATP-binding cassette (ABC) superfamily of
membrane proteins are most commonly associated with the
emergence of MDR.1,2 Within this family of 48 proteins, three
transporters [P-glycoprotein or P-gp (encoded by ABCB1),
multidrug resistance protein 1 or MRP1 (encoded by ABCC1),
and breast cancer resistance protein or BCRP (encoded by
ABCG2)] are the most important in drug-resistant tumors.2−5
The onset of human P-gp expression and drug resistance can
be quite rapid, with elevated expression of the gene for P-gp
(ABCB1) being observed within an hour of treatment.6
The core structure of functional ABC transporters consists of
two nucleotide binding domains (NBDs) that bind and hydrolyze
ATP and two transmembrane domains (TMDs). Transporters
encoded as a single polypeptide, such as P-gp, contain two NBDs
and two TMDs in a single protein, while half transporters, such as
BCRP, contain only one TMD and one NBD and must dimerize
to carry out their function.6
P-gp was the first efflux protein identified and associated with
Received: February 8, 2012
Published: April 25, 2012
multidrug resistance in cancer chemotherapy.1,2,7,8 A recent
© 2012 American Chemical Society
4683
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Chart 1. Structures of Chalcogenopyrylium Compounds Evaluated As Modulators of MRP1 and P-gp
transports a variety of different classes of compounds19
including anticancer compounds such as the anthracyclines
and the Vinca alkaloids (in a GSH-dependent manner) as well
as methotrexate.16,17 In addition, and unlike P-gp, MRP1 is a
very efficient transporter of a vast array of conjugated organic
anions and mediates the physiological efflux of the pro-
inflammatory leukotriene C4 from mast cells.17 Relatively few
MRP1-specific inhibitors have been described, and none of
these have yet been tested in humans.16,17
cells/tumors27 and structurally related chalcogenorhodamine/
rosamine derivatives have targeted multidrug-resistant cells in
vitro.28,29 Chalcogenopyrylium compounds with high specificity
for one or multiple ABC transporters may be quite useful in vivo
as photosensitizers for multidrug-resistant tumors via irreversible
photodynamic damage to the efflux pumps.
Herein, we evaluate the 27 chalcogenopyrylium dyes shown
in Chart 1 as modulators/inhibitors of MRP1 and P-gp in order
to compare and contrast reactivity patterns with the two
structurally and functionally distinct transporters. Compounds
3 and 4 in Chart 1 have shown activity as inhibitors of P-gp
(IC50 ∼5 μM) by increasing calcein AM (CAM) uptake in
MDCKII-MDR1 cells,26 which suggested that the compounds
of Chart 1 would provide an initial comparison of the SAR for
the two transporters. On the basis of the initial screen of the
compounds in Chart 1 with MRP1 and P-gp, the compounds
28−32 shown in Chart 2 were synthesized and the individual
To understand the ABC drug transporters, we need to study
the similarities and differences between transporters with a
different number of TMDs. In particular, differences in ATP
hydrolysis at the two ATP-binding sites may be critical to the
design of specific inhibitors of individual ABC drug tranporters.
The two sites in P-gp are similar,19,20 while the two sites in
MRP1 are different.21 In some situations, it would be useful to
inhibit only one transporter to prevent side effects. For example,
if one wanted enhanced delivery to the brain then it would be
useful only to inhibit P-gp activity. In other cases, such as
overexpression of both P-gp and MRP1 in a cancer cell, it would
be useful to inhibit both pumps with a single compound.
Chalcogenorhodamine/rosamine derivatives23−25 and chalco-
genopyrylium derivatives26 are lipophilic cations that bind to
P-gp and can act as stimulators or inhibitors of the transporter’s
ATPase activity and can act as inhibitors of the transporter’s
ability to efflux small molecules from the cell. With respect to
inhibition of P-gp, submicromolar IC50’s for inhibition of
verapamil(VER)-induced stimulation of ATPase activity have
been observed with several chalcogenorhodamine25 and
chalcogenopyrylium compounds.26 The two classes of modu-
lators share some structural similarities and, in both cases, have
slow rates of active transport in MDCKII-MDR1 cells.25,26
Reactivity with other ABC transporters has not yet been
examined. With the chalcogenopyrylium scaffold, myriad
analogues are readily prepared with variation at the 2-, 4-, and
6-positions around the ring and at the heteroatom in the ring.
The chalcogenopyrylium compounds are model systems that
allow exploration of structure−activity space while readily
accessible synthetically. Furthermore, chalcogenopyrylium com-
pounds have been successfully utilized as photosensitizers
in vitro and in vivo in the photodynamic therapy of cancer
Chart 2. Structures of Chalcogenopyrylium Compounds
Evaluated As Modulators of MRP1 and P-gp
compounds, as well as compound 21 from Chart 1, were
evaluated as inhibitors of both MRP1 and P-gp in order to
examine the effect of small structural variations on reactivity
with the two transporters.
CHEMISTRY RESULTS AND DISCUSSION
Synthesis of Chalcogenopyrylium Analogues. The
chalcogenopyrylium compounds evaluated in this study can
■
4684
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of Class II Chalcogenopyrylium Derivatives 6−11
Scheme 2. Synthesis of Class II Chalcogenopyrylium Derivatives 12−14 and 18−20
be broadly grouped into four classes as shown in Chart 1. The
class I compounds all contain 4-dimethylaminophenyl sub-
stituents at the 2- and 4-positions of the chalcogenopyrylium
ring. Compounds 1−5 were selected from a library of
chalcogenopyrylium dyes that we have prepared as photo-
dynamic antimicrobial agents and were prepared by literature
methods.30,31 The class II compounds are characterized by
two identical substituents at the 2- and 6-positions (tert-butyl
groups for 6−16 and 18−20 and phenyl groups for 17).
Among the class II chalcogenopyrylium compounds, com-
pounds 15−17 are intermediates for the preparation of other
dyes and were prepared by literature methods.32,33 The class III
chalcogenopyrylium compounds are characterized by a 2-tert-
butyl substituent and a 6-(4-dimethylaminophenyl) substituent
on the thiopyrylium core. The class IV compounds 25−27
were selected from a library of AA1-related34 photosensitizers
for photodynamic therapy (PDT) and also were prepared by
literature methods.35
The class II compounds 6−11 were prepared as shown in
Scheme 1. The addition of excess PhMgBr to 2,6-di-tert-
butylchalcogenopyran-4-ones (33)36 followed by the addition of
aqueous HBr gave the 4-phenylchalcogenopyrylium bromides
6−8 in 40−56% isolated yield. Compounds 9−11 were prepared
by the addition of lithium 5-lithiothiophene-5-carboxylate (34)
to chalcogenopyranones 33. Dianion 34 was prepared from
2-carboxy-5-bromothiophene (35) using only 2.1 equiv of tert-
BuLi (1 equiv to deprotonate the acid and only 1 equiv for the
metal−halogen exchange) in the presence of 0.25 equiv of
TMEDA.37 Elimination of HBr from the tert-BuBr generated in
the metal−halogen exchange was not competitive kinetically in
these reactions.38 The addition of 34 to chalcogenopyranones 33
gave chalcogenopyrylium compounds 9−11 in 68−74% isolated
yield following workup with aqueous HPF6.
containing derivatives 12−14 were prepared by the addition of
chalcogenopyranones 33 to N,N-diethyl 2-lithiobenzamide
(36).25,39 Anion 36 was prepared by the deprotonation of
N,N-diethyl benzamide (37) with sec-BuLi in the presence of
1.0 equiv of TMEDA at −78 °C. Chalcogenopyrylium
compounds 12−14 were isolated in 23−47% isolated yield
following workup with aqueous HPF6. Yields were much
poorer with the inverse addition of anion 36 to chalcogenopyr-
anones 33. Thioamide 38 was prepared in 81% isolated yield
from thiophene-2-carboxaldehyde under Willgerodt−Kindler
conditions with elemental sulfur and piperidine.25 Deprotona-
tion of 38 with lithium diisopropylamide (LDA) occurred from
the sterically least hindered 5-position to give N-piperidyl
5-lithio-2-thiocarboxythiophene (39), which was added to
chalcogenpyranones 33 to give chalcogenopyrylium com-
pounds 18−20 in 66−74% isolated yield following workup
with aqueous HPF6. Unlike the tertiary amide group,39 which is
highly directing as in the preparation of 36, the thioamide
functionality does not direct lithiation in thiophenes. Only the
more acidic α-proton was removed, and none of the cor-
responding 2,3-disubstituted thiophenes were detected in the
product mixtures.
The class III chalcogenopyrylium compounds 21−24 were
prepared as shown in Scheme 3. The addition of PhMgBr, anion
36, or dianion 37 to 2-tert-butyl-6-(4-dimethylaminophenyl)-
thiopyran-4-one (40)30 gave thiopyrylium derivatives 22−24 in
45−87% isolated yields. Addition of the anion 39 from
thioamide 38 and LDA to thiopyranone 40 followed by workup
with aqueous HPF6 gave the PF6 salt of 21 (compound 41 in
Scheme 4), which was converted to the chloride salt 21 with an
ion-exchange resin.
As described below, thiopyrylium analogue 21 was an
effective modulator/inhibitor of MRP1 and P-gp. We prepared
a series of five additional compounds as shown in Scheme 4
that were related in structure to 21 by varying all combinations
Chalcogenopyrylium derivatives 12−14 and 18−20 were
prepared as shown in Scheme 2. The N,N-diethylbenzamide-
4685
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Scheme 3. Synthesis of Class III thiopyrylium derivatives 21−24
Scheme 4. Synthesis of the Amide and Thioamide Thiopyrylium Derivatives 21 and 28−32
of tert-butyl and 4-dimethylaminophenyl substituents at the
2- and 6-positions in the thiopyrylium core and varying amide
and thioamide functionality at the 4-position. The addition of
anion 39 to thiopyranones 33-S, 40, and 4240 followed by
workup with aqueous HPF6 gave thiopyrylium compounds 18,
41, and 43, which were converted to the chloride salts 29, 21,
and 28, respectively, with a chloride exchange resin.
Thiopyrylium thioamides 18, 41, and 43 were converted to
the thiopyrylium amide derivatives 30−32 with trifluoroacetic
anhydride.25 The amide compounds 30−32 were evaluated as
the hexafluorophosphate salts because the corresponding
chloride salts were hygroscopic and difficult to store. These
six compounds were evaluated as modulators of both MRP1
and P-gp to examine the impact of small structural
modifications on reactivity patterns in the two transporters.
n-Octanol/Water Partition Coefficients. Experimental
values of the n-octanol/water (PBS) partition coefficient (log P)
were measured using the “shake flask” method.41 Saturated
n-octanol solutions of chalcogenopyrylium compounds 21 and
28−32 were shaken with an equal volume of phosphate
buffered saline (PBS) at pH 7.4, and the concentrations in the
two layers were determined spectrophotometrically. Exper-
imental values of log P are compiled in Table 2 for these
compounds.
BIOLOGICAL RESULTS
■
Effect of Chalcogenopyrylium Compounds 1−27 on
MRP1-Mediated Vesicular Uptake of [3H]E217βG. To
determine whether the 27 chalcogenopyrylium dyes of Chart 1
could modulate MRP1 transport activity, their effect on MRP1-
mediated uptake of tritiated estradiol glucuronide ([3H]-
E217βG) into inside-out membrane vesicles was assessed.42
A stably transfected HEK-MRP1 cell line was used as a source
for membrane vesicles.43 Following immunoblot analysis to
confirm the expression of MRP1 (Figure 1), the vesicles were
initially tested for their [3H]E217βG uptake activity to assess
the integrity of the vesicles as reflected by their baseline
transport levels.
Having confirmed the transport competence of the MRP1-
enriched membrane vesicles, the 27 chalcogenopyrylium com-
pounds were screened at a single concentration of 30 μM, with
the exception of 1, 7, 9, 17, and 26, which were tested at 1 μM,
and chalcogenopyrylium compounds 2, 10, 11 and 20, which
were tested at 5 μM. Testing these latter compounds at a con-
centration <30 μM was necessary because of either insufficient
solubility and/or because they generated an unacceptably
high background which interfered with accurate scintillation
counting in the transport assay.
4686
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
20, 23, and 24) ranged from 30 to 50%. However, it should be
noted that 1, 7, 9, 17, and 26 could only be tested at a
maximum concentration of 1 μM, while chalcogenopyrylium
compounds 2, 10, 11, and 20 could only be tested at a
maximum concentration of 5 μM. Therefore, the efficacy of
these latter nine chalcogenopyrylium compounds (six of which
were from class II) may be underestimated.
Four of five class I chalcogenopyrylium compounds (2−5)
were effective inhibitors of MRP1-mediated [3H]E217βG
uptake, with mean inhibition values ranging from 70 to 96%
when tested at 30 μM (Table 1). The remaining compound 1
inhibited uptake of [3H]E217βG by only 15% at 1 μM.
In contrast to the class I compounds, the 15 class II
chalcogenopyrylium compounds 6−20 were relatively ineffec-
tive inhibitors of MRP1 transport activity. Only 18 and 19
inhibited [3H]E217βG uptake by more than 70% (Table 1) at
71 and 81%, respectively. Inhibition by 20 (the telluropyrylium
analogue of 18 and 19) was 50%. For the remaining 12 class II
compounds, inhibition ranged from 0 to 45%. However, for
reasons stated earlier, 6 of these 12 were tested at concentrations
<30 μM and, thus, their relative efficacy may be underestimated.
Figure 1. Expression of human MRP1 protein in membrane vesicles.
Immunoblots of membrane vesicles (MV) prepared from transfected
(1.0 and 2.0 μg of protein) and untransfected (1.0 μg of protein)
HEK293T cells are shown. mAb QCRL-1 was used to detect MRP1.
HEK refers to control membrane vesicles prepared from untransfected
cells.
As summarized in Table 1, the modulatory effects of the 27
chalcogenopyrylium compounds on MRP1-mediated uptake of
[3H]E217βG varied significantly. Ten of 27 (2−5, 18, 19, 21,
22, 25, and 27) inhibited MRP1-mediated uptake by 70−96%.
Nine of the remaining 17 (1, 7, 9−11, 15−17, and 26)
inhibited [3H]E217βG uptake by less than 30%. Inhibition by
the other eight chalcogenopyrylium compounds (6, 8, 12−14,
Table 1. Effect of Chalcogenopyrylium Compounds 1−27 on Human MRP1-Mediated [3H]E217βG Uptake, Stimulation of
Human P-gp-His10 ATPase Activity, and P-gp Inhibition in MDCKII-MDR1 Cells
% inhibition
(44, LSN 335984)
MRP1
human P-gp-His10
b
conc,
Vmax
,
a
c
compd
μM
% inhibition
15 (22, 8)
fold
KM, μM
5 μM
25 μM
VER
1
17.9 2.0
1.4 0.2
5.9 0.8
7.2 1.1
10.1 0.5
8.4 1.0
2.9 0.4
1.4 0.2
1.5 0.1
3.5 0.7
<1.4
24 2.6
13.2 0.3
37 12
1
5
2
70 (77, 61)
94 (94, 95)
96 (93, 99)
96 (94, 98)
33 (29, 36)
0 (0, 0)
d
d
d
d
3
30
30
30
30
1
13.2 0.7
25
23
60
67
4
31
56
95
89
1
8
6
9
5
6
0
0
0
1
2
7
8
30
1
34 (33,36)
8 (1, 15)
86 17
98
9
9
0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
5
15 (10, 21)
24 (15, 34)
34 (28, 41)
31 12
(111 9)
5
<1.4
(162 52)
30
30
30
30
30
1
7.9 1.8
5.9 1.1
6.9 0.8
52
17
30
8
4
8
45 (53, 38)
0 (0, 0)
0 (0, 0)
0 (0,0)
30
30
5
71 (80, 62)
81 (82, 81)
4.4 1.2
4.3 1.0
7.4 1.9
5.2 0.8
13.0 3.4
13.5 1.2
6.9 0.9
4.5 0.8
52 12
101 10
85 13
3.6 1.3
27
5
50
2
30
30
30
30
30
1
93 (94, 93)
96 (95, 98)
43 (47, 40)
50 (45, 56)
96 (97, 96)
8 (8, 9)
24.3 3.8
997 100
13.7 3.2
20.0 1.7
59 10
31
2
3
74
13
1
4
3
e
e
ND
ND
30
89 (91, 87)
4.0 0.3
24.3 4.9
a
% inhibition of [3H]E217βG uptake relative to vehicle control (1% DMSO). Results shown are the mean SD of three independent experiments.
b
Where only two experiments were performed, the results of both experiments are shown in parentheses. Vmax is the ratio of the maximum
stimulation in the presence of compound relative to that in the absence of compound (the basal activity). KM is the apparent Michaelis−Menten
constant or the concentration of compound required for half maximal stimulation of P-gp ATPase activity. Values in parentheses are IC50 values: the
c
d
e
concentration of compound required for 50% inhibition of VER-stimulated (400 μM) human P-gp-His10 ATPase activity. Values from ref 26. ND,
not determined because of spectral interference with the P-gp ATPase activity assay.
4687
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Among the class III compounds, 21 and 22 were strong
inhibitors of MRP1 transport activity (93% and 96% inhibition
of [3H]E217βG uptake, respectively) while 23 and 24 displayed
more modest inhibition (43% and 50%, respectively). Both
class IV compounds that could be used at 30 μM (25 and 27)
gave strong inhibition of [3H]E217βG uptake (96% and 89%
inhibition, respectively), while compound 26, which could be
used at only 1 μM, displayed 8% inhibition.
Effect of Chalcogenopyrylium Compounds on Human
P-gp-His10 ATPase Activity. The effect of chalcogenopyry-
lium compounds 1−14 and 18−27 (Chart 1) on P-gp ATPase
activity was examined using human P-gp-His10, which was
activated with sheep brain phosphatidylethanolamine.44−46
The apparent Michaelis−Menten constant (KM) for ATPase-
stimulating compounds was determined as well as the drug-
induced stimulation of maximal ATPase activity (Vmax) using
human P-gp-His10 (Table 1). P-gp ATPase activity was
determined as the release of inorganic phosphate from ATP.47
A typical ATPase activity vs concentration profile is shown in
Figure 2 for chalcogenopyrylium compound 23, which was the
give sufficient P-gp ATPase stimulation for an accurate determina-
tion of KM, the IC50s for 400 μM VER-induced stimulation were
111 and 162 μM, respectively. Among the class III compounds
21−24, Vmax values were stimulated 6.2−13.5-fold. Compounds
21, 23, and 24 had KM values between 13.7 and 20.0 μM. In
contrast, compound 22 highly stimulated Vmax (13.1-fold), but its
KM was quite large at 997 μM. Among the class IV compounds,
25 and 27 were assayed and found to be mildly P-gp ATPase
stimulating (Vmax stimulated by 4.5-fold and 4.0-fold, respectively),
with corresponding KM values of 59 and 24.3 μM.
CAM Uptake into MDCKII-MDR1 Transfected Cells.
Selected chalcogenopyrylium compounds were also evaluated
for their ability to facilitate CAM uptake into MDCKII-MDR1
transfected cells, which overexpress P-gp.50 CAM uptake was
determined at chalcogenopyrylium concentrations of 5 and
25 μM and P-gp inhibition was measured as a percentage of the
inhibition observed with 5 μM (R)-4-[(1a,6,10b)-1,1-dichloro-
1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-
yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol (44, LSN
335984, IC50 = 0.4 μM), which completely inhibits P-gp
(Chart 3).14 Compound 44 is structurally related to the P-gp-
Chart 3. Structures of LSN 335984 (44) and LSN 335979
(45)
specific inhibitor (R)-4-[(1a,6,10b)-1,1-difluoro-1,1a,6,10b-
tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-
quin-olinyloxy)methyl]-1-piperazineethanol (45, LSN 335979
or zosuquidar).14,51 The CAM fluorescence observed with the
chalcogenopyrylium compounds is reported as a percentage of
the CAM fluorescence observed with 5 μM 44 (100%
inhibition). The percentage inhibition at the two chalcogeno-
pyrylium concentrations is summarized in Table 1 for
compounds 3, 4, 6, 9, 12, 18, 21, and 23. Compounds 3 and
4 showed comparable activity in this assay with inhibition of 25
and 23%, respectively, at 5 μM and 60 and 67%, respectively, at
25 μM relative to inhibition by 44.26
Interactions of Amide- and Thioamide-Containing
Thiopyrylium Compounds 21 and 28−32 with MRP1
and P-gp. Thiopyrylium compounds 21 and 28−32 (Chart 2)
were evaluated at concentrations of 1 and 10 μM for their
ability to inhibit MRP1-mediated uptake of [3H]E217βG in
membrane vesicles (Table 2).42 At 1 μM, compounds 28
and 30 showed weak inhibition of MRP1-mediated transport
(25 and 18%, respectively) while compounds 29, 31, and 32
showed no detectable inhibition. At 10 μM, 21 gave 93%
inhibition and 28 and 30 were similarly effectively causing 81%
and 78% inhibition, respectively; 31 gave 59% inhibition and 29
gave 32% inhibition. Compound 32 was least effective in this
series, giving only 11% inhibition at 10 μM.
Figure 2. Effect of thiopyrylium compounds on P-gp ATPase activity.
His-tagged human P-glycoprotein was expressed in BHK cells, isolated
by Ni2+-chelate chromatography, and mixed with lipid. P-Glycoprotein
ATPase activity was then measured in the presence of various
concentrations of thiopyrylium derivatives 23 (filled circles, compound
with the largest Vmax), 30 (open circles, compound with lowest KM), or
VER (open squares). Data points represent the mean of triplicate
measurements and error bars represent one standard deviation.
most effective stimulator of the chalcogenopyrylium analogues
examined. Verapamil (VER) was included as a control com-
pound because of its well-known ability to stimulate P-gp
ATPase activity.48,49 For compounds 10 and 11, which only
weakly stimulated P-gp ATPase activity, the concentration
of compound required for 50% inhibition of VER-stimulated
(400 μM) ATPase activity (IC50) was determined (Table 1).
Within the class I compounds 1−5, the stimulation of P-gp
ATPase Vmax ranged from 1.4-fold for compound 1 to 10.1-fold
for compound 4 while KM ranged from 13.2 μM for com-
pounds 1 and 3 to 56 μM for 5. Among the class II compounds
6−14 and 18−20, stimulation of Vmax ranged from <1.4-fold for
compounds 10 and 11 to 7.9-fold stimulation for compound
12; KM values were higher in this series (52−101 μM), with the
exception of compounds 13 and 14, with KM values of 17 and
30 μM, respectively. For compounds 9 and 10, which did not
The effect of the thiopyrylium compounds 21 and 28−32
(Chart 2) on P-gp ATPase activity was also examined using
4688
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Table 2. Experimental Values of log P and the Effect of Thiopyrylium Amide and Thioamide Analogues on MRP1-Mediated
a
Transport, P-gp ATPase Activity, and P-gp-Mediated Calcein AM Uptake and Vinblastine Efflux
MRP1
P-gp
c
d
% inhibition
Vmax
,
KM,
IC50 CAM uptake,
IC50 for VIN efflux,
μM
b
compd
log P
1 μM
% inhibition 10 μM
fold
μM
μM
21
28
29
30
31
32
2.1
1.6
0.7
2.1
1.4
0.2
36
8
93 (94, 93)
81 (84, 79)
32 (28, 37)
78 (80, 76)
59 (60, 59)
11 (13, 10)
5.2 0.8
5.7 1.0
3.7 0.8
6.0 1.3
6.6 1.4
5.5 0.9
24.3 3.8
7.7 0.8
74 12
38.0 1.2
34.0 1.2
ND
11.5 1.0
6.6 1.0
ND
25 (24, 25)
0 (0, 0)
18 (29, 7)
0 (0, 0)
2.2 0.7
11.7 3.8
72 12
18
1
5.9 1.1
ND
24.0 1.3
ND
0 (0, 0)
ND
a
Details for methods are provided in Experimental Section. Values shown are means one standard deviation when three or more experiments were
performed. Where only two experiments were performed, means are shown with individual values of the two experiments in parentheses.
b
c
Experimental values of log P determined in n-octanol and PBS at pH 7.4 using the “shake flask” method. Vmax is the ratio of the maximum
d
stimulation in the presence of compound relative to that in the absence of compound (the basal activity). KM is the apparent Michaelis−Menten
constant and corresponds to the concentration of compound required for half-maximal stimulation of P-gp ATPase activity.
human P-gp-His10 activated with sheep brain phosphatidyletha-
nolamine.44−46 Values for KM and Vmax are compiled in Table 2.
All six compounds weakly stimulated P-gp ATPase activity
(3.7- to 6.6-fold) with a 35-fold range in KM values (2.2−74
μM). Thiopyrylium compounds 29 and 32 had the two highest
KM values (74 and 72 μM, respectively), compounds 21 and 31
had intermediate KM values (24.3 and 11.7 μM, respectively),
and compounds 28 and 30 had the lowest KM values (7.7 and
2.2 μM, respectively). The P-gp ATPase activity vs concen-
tration curve for 30 is shown in Figure 2.
Enhancement of CAM Uptake into MDCKII-MDR1
Cells. Thiopyrylium compounds 21, 28, 30, and 31 were
evaluated for their ability to facilitate uptake of CAM into
MDCKII-MDR1 cells.50 IC50 values for CAM uptake (Table 2)
were determined by measuring relative fluorescence values
obtained after a 20 min incubation with CAM at 37 °C. Amide
analogues 30 and 31 (IC50s of 24 and 18 μM, respectively) were
more effective modulators than the corresponding thioamide
analogues 21 and 28 (IC50s of 38 and 34 μM, respectively).
The 2,6-di-tert-butylthiopyrylium analogues 29 and 32 were not
evaluated in this assay due to their negligible inhibition of P-gp
at 5 and 25 μM in the two-concentration CAM uptake experi-
ments described above (≤7% inhibition at 25 μM).
Table 3. Effect of Chalcogenopyrylium Analogues 21 and
28−32 on Efflux Activity of MDCKII-MDR1 Cells
a
normalized ratio,
c
PAB
PBA
P
PPassive
BA/ABb
compd
nm s−1
nm s−1
PBA/AB
(∓ inh)
nm s−1
2
21
(+ inh)
28
0.3 0.1
1.6 1.3
1.1 0.1
0.4 0.1
1.8 0.2
320 29
2.4 0.1
1015
14
705
16
4
15
8
15
79
4
0.6
14
(+ inh)
29
0.8 0.1
300 10
2
167
11
(+ inh)
30
2.3 0.7 25.0 0.2
1.7 0.6 620 37
370
4.7
3.9
0.9
16
14
(+ inh)
31
4.9 2.4 23.0 0.2
5.6 2.0 22.0 9.0
3.7
5.3
(+ inh)
32
3.9 0.1
3.8 0.2 59.0 3.9
4.2 0.3 6.4 0.1
3.5 0.1
10
(+ inh)
1.5
a
Experiments were run with 5 μM dye and 4.3 mg mL−1 BSA. Values
of transport in the absorptive (PAB) and secretory (PBA) mode in the
absence or presence of inhibitor, the ratio of secretory to absorptive
transport (PBA/AB) in the absence or presence of inhibitor, the
normalized ratio [PBA/AB (no inhibitor)/PBA/AB (with inhibitor)].
Details for methods are provided in the Experimental Section. Values
b
shown are means
standard deviation. The normalized ratio
Inhibition of Vinblastine Efflux by Thiopyrylium
Compounds 21, 28, and 30 in MDCKII-MDR1 Cells.
Vinblastine (VIN) is a well-known chemotherapeutic agent,
and inhibition of VIN efflux by the chalcogenopyrylium com-
pounds may be of potential clinical value. [3H]-VIN was
introduced in an appropriate dilution series with 21, 28, or 31
and BSA to the basolateral chamber of a monolayer of
MDCKII-MDR1 transfected cells. The appearance of [3H]-
VIN in the apical chamber was monitored by scintillation
counting and gave IC50 values of 11.5, 6.6, and 5.9 μM for 21,
28, and 30, respectively. While these IC50 values are lower than
the corresponding IC50 values for CAM uptake, the trend
observed for CAM uptake is continued with the IC50 for amide
30 being 2-fold lower than the IC50 for the thioamide 21.
Transport of Thiopyrylium Compounds Across Mono-
layers of MDCKII-MDR1 Cells. The transport of the
thiopyrylium amide and thioamide derivatives 21 and 28−32
was examined in monolayers of MDCKII-MDR1 transfected
cells (Table 3).50 MDCKII-MDR1 monolayers display apical
and basolateral polarized membranes and are widely used as
a physiological model for studying P-gp mediated drug efflux.
In the monolayer, P-gp is asymmetrically distributed, being
represents the PBA/AB ratio in the absence of inhibitor divided by the
PBA/AB ratio in the presence of inhibitor. PPassive represents the mean of
PAB and PBA in the fully inhibited system.
c
solely present at the apical membrane. To evaluate the role of
P-gp in the transport of the compounds of this series, transport
was measured in the absorptive (apical to basolateral or AB)
and secretory (basolateral to apical or BA) transport direction
of the cell monolayer, and then an efflux ratio (PBA/AB) was
calculated.52 Addition of bovine serum albumin (BSA) to the
buffer was required because a marked fraction of thiopyrylium
mass added to the donor equilibrated with the cell monolayer
for some of the compounds, and this resulted in gross
underestimation of the permeability coefficient.53 The assay
was repeated in the presence of 44 (LSN 335984)14 to measure
transport when P-gp was fully inhibited.
Values of passive transport (PPassive) in the presence of
inhibitor, transport in the absorptive (PAB) and secretory (PBA)
mode in the absence or presence of inhibitor, the ratio of
secretory to absorptive transport (PBA/AB) in the absence or
presence of inhibitor, and the normalized ratio [PBA/AB (no
inhibitor)/PBA/AB (with inhibitor)] are compiled in Table 3.
4689
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
For 21 and 28−32, the large normalized efflux ratios for PBA/AB
(≥4) are assumed to be due to efficient P-gp-mediated efflux of
the compound and suggest that thiopyrylium compounds 21
and 28−32 are P-gp substrates. Thiopyrylium compound 21
has the highest normalized ratio for PBA/AB (705) and 31, the
lowest (4).
Table 4. Scoring Summary for Modulation/Inhibition of
MRP1 and P-gp by Chalcogenopyrylium Compounds
a
Transport rates in the absorptive direction (PAB) were slow
(≤5.6 nm s−1) for all six compounds as were rates of PPassive (14
nm s−1 for 29 and 30 and ≤5.3 nm s−1 for 21, 28, 31, and 32)
in fully inhibited systems. In the secretory direction (PBA), the
thiopyrylium compounds sorted into two groups. Compounds
28, 31, and 32 (1 thioamide, 2 amides) exhibited relatively slow
secretory transport rates (PBA 16−59 nm s−1), while compounds
21, 29, and 30 (2 thioamides, 1 amide) were transported at a
much faster rate (PBA 300−620 nm s−1).
DISCUSSION OF BIOLOGICAL RESULTS
■
The three major transporters [P-gp (encoded by ABCB1),10−12
MRP1 (encoded by ABCC1),16,17,22 and BCRP (encoded by
ABCG2)54,55] show considerable diversity in transportable
substrates as well as overlap of transportable substrates.
Understanding the differences in the biology and biochemistry
of the transporters can lead to mechanistic understanding of
why the different transporters prefer different substrates.56,57
Within the chalcogenopyrylium compounds of this study,
differences were noted in their inhibitory activity toward MRP1
and P-gp.
Comparative Interactions of Chalcogenopyrylium
Compounds with MRP1 and P-gp. As demonstrated by
the results in Tables 1 and 2, chalcogenopyrylium compounds
function as inhibitors of MRP1 in membrane vesicles, as
modulators (ATPase stimulating as well as inhibiting) of purified
human P-gp-His10 activated with sheep brain phosphatidyletha-
nolamine, and as P-gp inhibitors in human P-gp expressing
MDCKII-MDR1 cells. However, P-gp and MRP1 share only
15% amino acid sequence identity,19 and one might expect
differences in binding as well as differences in modulatory/
inhibitory activity within a series of related structures.
The chalcogenopyrylium compounds examined in this study
allow some qualitative correlations to be made among the
various chalcogenopyrylium classes described here. These results
are compiled as a qualitative comparison between MRP1 and
P-gp in Table 4. The class I chalcogenopyrylium compounds 2−5
act as strong inhibitors of MRP1 and weaker modulators of
P-gp as measured by KM (Tables 1 and 4) These compounds
all share 4-N,N-dimethylaminophenyl substituents at the 2- and
4-positions of the chalcogenopyrylium core. The 6-substituent
for the derivatives in Chart 1 is an alkyl group: methyl, n-butyl, or
tert-butyl. Related thiopyrylium compounds 46 and 47 (Chart 4)
with methyl and n-propyl substituents at the 6-position also
displayed inhibitory activity toward P-gp at 25 μM (74 and 87%
inhibition, respectively).26 Structures within this class appear to
interact more strongly with MRP1 than with P-gp.
The telluropyrylium compounds 25−27 of class IV (Chart 1)
share structural similarities with the class I compounds. These
molecules have three aromatic groups at the 2-, 4-, and
6-positions, with at least two of these bearing a 4-aminophenyl
and/or a 4-N-morpholinophenyl substituent. Whether the
aminoaryl substituents are located at the 2,4- or 2,6-positions
of the chalcogenopyrylium core, N···N distances will be
comparable as well as dihedral angles between the aromatic
substituents and the chalcogenopyrylium core. One might expect
these compounds to exhibit similar modulatory characteristics as
a
For MRP1: (+++) denotes >90% inhibition, (++) denotes 70−89%
inhibition, (+) denotes 50−69% inhibition, (−) denotes 30−49%
inhibition, and (−−) denotes <30% inhibition in the one-dose assays
of Tables 1 and 2. For PGP: (+++) denotes KM < 10 μM, (++)
denotes KM of 10−19 μM, (+) denotes KM of 20−49 μM, (−) denotes
KM of 50−74 μM, (−−) denotes KM of 75−199 μM, and (−−−)
denotes KM > 200 μM from the data of Tables 1 and 2.
those observed for the class I compounds and, indeed,
compounds 25 and 27 both strongly inhibited MRP1-mediated
transport at 30 μM (Tables 1 and 4). The interactions of these
two compounds with P-gp appear to be weaker than the
interactions with MRP1 (Tables 1 and 4). The thiopyrylium
compounds 48−50, which are closely related in structure to
compounds 25−27, inhibited VER-induced P-gp ATPase
activity with IC50 values of 30, 3.1, and 11 μM, respectively
(Chart 4).26
The class II compounds 6−20 (Chart 1) are characterized by
identical substituents at the 2- and 6-positions of the
chalcogenopyrylium core. With the exception of compound
17 with 2,6-diphenyl substituents, the remaining compounds in
class II have tert-butyl substituents at the 2- and 6-positions.
4690
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
Chart 4. Structures of Thiopyrylium Compounds with Modulatory/Inhibitory Activity toward P-gp
Among the class II compounds, 6−14 and 18−20 were
evaluated with both MRP1 and P-gp. Compounds 6−12 were
all weak inhibitors of MRP1. Compounds 13 and 14 with
N,N-diethyl benzamide functionality at the 4-position were
weak inhibitors of MRP1 and better modulators of P-gp
(Tables 1 and 4). Compounds 18−20 with thioamide func-
tionality at the 4-position were inhibitors of MRP1 and weak
modulators of P-gp (Tables 1 and 4).
Compounds 6, 9, and 12 exhibited weak but measurable
inhibition of MRP1-mediated uptake of [3H]E217βG into
inside-out membrane vesicles at 30 μM, while these same
compounds showed no inhibition of P-gp at 25 μM with
respect to CAM uptake into MDCKII-MDR1 transfected cells
(Table 1). Compound 18, which exhibited 71% inhibition of
[3H]E217βG transport by MRP1 at 30 μM, exhibited only
27% inhibition of P-gp at 25 μM in the CAM uptake assay
(Table 1). These results suggest that the 2,6-di-tert-butyl-
substituted derivatives among the class II compounds may have
differences in both binding to and inhibiting the activity of
MRP1 and P-gp.
In the class I and class IV compounds, two aminoaryl
substituents gave chalcogenopyrylium compounds that inhibited
both P-gp and MRP1, while in the class II compounds, two tert-
butyl substituents gave compounds with mixed effects. The class
III compounds 21−24 (Chart 1) have one tert-butyl substituent
at the 2-position and one 4-N,N-dimethylaminophenyl sub-
stituent at the 6-position of the chalcogenopyrylium core. The
functionality at the 4-position was diverse with thioamide (21)
and amide (23) hydrogen bond acceptors, carboxylic acid (22)
hydrogen bond donors, and an unsubstituted phenyl substituent
(24). Among these compounds, 21 and 22 are strong inhibitors
of MRP1-mediated [3H]E217βG uptake at 30 μM while
compounds 23 and 24 are less inhibitory toward MRP1 at
30 μM but still have measurable inhibition (Tables 1 and 4).
Interactions of 21 and 23 with P-gp followed similar trends.
Compound 21 exhibited 74% inhibition of P-gp at 25 μM in the
CAM uptake assay, while compound 23 exhibited only 13%
inhibition of P-gp at 25 μM in the same assay (Tables 1 and 4).
Role of the Chalcogen Atom in Modulatory/Inhibitory
Behavior of Chalcogenopyrylium Compounds toward
MRP1 and P-gp. Several S, Se, Te-triads (3−5, 6−8, 9−11,
12−14, and 18−20) are present in the compounds of Chart 1.
As shown in Table 1, all three chalcogen derivatives behaved
similarly with respect to inhibition of MRP1-mediated uptake
of [3H]E217βG into inside-out membrane vesicles. Thus, the
identity of the chalcogen atom in the chalcogenopyrylium does
not influence the inhibitory behavior of these materials.
With respect to P-gp ATPase activity, the chalcogen atom is
similarly inconsequential because KM values for the three
chalcogen analogues were either comparable or their differences
followed no predictable order (Table 1). In the 3−5 triad, the
thiopyrylium compound 3 has the lower KM for human P-gp-
His10 by a factor of 2.5 relative to selenopyrylium compound 4
and by a factor of 4.5 relative to telluropyrylium compound 5,
i.e., the rank order of the KM values for this triad is Te > Se > S.
The thiopyrylium compound 18 also has the lowest KM value in
the 18−20 triad by a factor of 2 relative to selenopyrylium
compound 19 and a factor of 1.6 relative to telluropyrylium com-
pound 20, giving a rank order of the KM values of Se > Te > S.
In other triads, the selenopyrylium analogue had the lowest
KM value. In the 12−14 triad, the KM for selenopyrylium
compound 13 was roughly half that for telluropyrylium
compound 14 and roughly one-third that for thiopyrylium
compound 12 and, thus, the rank order of the KM, values of this
triad is S > Te > Se. Within these triads, the variability of Vmax
within each triad is (overall low to high) 2-fold or less, again
suggesting minimal impact from the individual chalcogen atoms
(Table 1).
Effect of Thiopyrylium Compounds 21 and 28−32 on
MRP1 and P-gp Activities. This series of compounds 21 and
28−32 permitted the examination of reactivity with very
well-defined structural changes. All six compounds have a
thiopyrylium core and allow systematic examination of class II,
class III, and class IV substituent changes. As shown in Table 2
and summarized in Table 4, the most efficacious inhibitor of
MRP1-mediated [3H]E217βG uptake is the class III-related
compound 21 while the class II related compounds 29 and 32
(tert-Bu substituents at the 2- and 6-positions) give the least
inhibition. Class IV-related compounds 28 and 30 also are
effective inhibitors of MRP1, while thiopyrylium compound 31
is somewhat less effective.
Interestingly, the effectiveness of compounds 21 and 28−32
as inhibitors of MRP1-mediated [3H]E217βG uptake correlates
with lipophilicity as indicated by the experimental values of log
P found in Table 2. Compounds 21, 28, 30, and 31 are the
most effective inhibitors of MRP1 with values of log P in the
1.4−2.1 range, while compounds 29 and 32 with the lowest
values of log P (0.7 and 0.2, respectively) are the least effective
inhibitors.
The reactivity patterns with P-gp were quite similar. As
observed for MRP1 inhibition, the class II-related compounds
29 and 32 had the least effect on P-gp activity and the highest
values of KM (Tables 2 and 4), while class IV-related
thiopyrylium compound 30 interacts most strongly with P-gp
(lowest KM and the lowest IC50 value for VIN and CAM uptake
(Table 2)). The activity of compound 30 is followed by class
IV-related compounds 28 and 31 with respect to KM values,
IC50 value for VIN uptake for 28, and IC50 values for CAM
uptake (Tables 2 and 4).
4691
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
One difference in reactivity pattern was noted. Compound 21,
which most strongly affected MRP1 activity (Tables 1 and 4),
was less effective than 28, 30, and 31 with respect to inhibiting
P-gp based on values of KM, IC50 for VIN uptake, and IC50 for
CAM uptake (Tables 2 and 4). Interactions of 21 and 28−32
with P-gp showed no correlations with values of log P.
transporters differently. This is perhaps not surprising because
MRP1 and P-gp share very limited (approximately 15%)
homology and are functionally distinct in many ways.17
In the 4-position, a thioamide imparts greater inhibitory
efficacy than an amide in MRP1, while in P-gp, an amide
imparts greater inhibitory efficacy than a thioamide. With
respect to P-gp, the differences in inhibitory activity between
amide and thioamide groups in the 4-position are opposite to
the effects observed in an earlier study of rosamine/rhodamine
structures as modulators/inhibitors of P-gp, where a thioamide
was more inhibitory than the corresponding amide.25 These
data suggest that the pyrylium structures may bind to a different
site on P-gp than do the rosamine/rhodamine structures. Like
MRP1,58 P-gp has been shown to have multiple drug-binding
sites59 and binding could occur through an “induced-fit
mechanism” where the chalcogenopyrylium scaffold is more
flexible than the rhodamine scaffold.60,61
From the structure−activity relationships developed here,
comparison of the effectiveness of S-, Se-, and Te-containing
analogues of the amide/thioamide-containing structures 13, 14,
18−21, and 28−32 as photosensitizers for the treatment of
MRP1- and P-gp-expressing cells by PDT would be of interest.
Inactivation of the efflux pump by PDT might allow a dual
treatment modality involving both PDT and conventional
chemotherapy.
Thioamide vs Amide Functionality. In the series of
compounds 21 and 28−32, the amide or thioamide substituent
at the 4-position impacts inhibitory activity. In a recent study in
the laboratories of M. Wiese, thiourea functionality imparted
greater inhibitory activity toward MRP1 in a series of drug
derivatives relative to P-gp or BCRP.57 Among the class III-
related compounds, the thioamide 21 displays greater inhibition
than the corresponding amide 31 with respect to MRP1-
mediated [3H]E217βG uptake into inside-out membrane vesicles
as does the class II thioamide 29 with respect to amide 32
(Tables 2 and 4). The amide and thioamide functionalities
resulted in similar inhibition of MRP1 transport activity when
class IV-related compounds 28 and 30 at 10 μM are compared
(Table 2). With respect to effects on P-gp transport activity,
compounds with amide substituents were more efficacious
because KM and IC50 values for CAM uptake were lower for
amide-containing compounds 30 and 31 than for the thioamide-
containing compounds 21 and 28 (Table 2).
These observations were also supported by the impact of
amide-containing compounds 13, 14, and 23 and thioamide-
containing compounds 18−20. The amide-containing com-
pounds 13, 14, and 23 interacted weakly with MRP1 but were
moderate inhibitors of P-gp (Tables 1 and 4). In contrast, the
thioamide-containing compounds 18−20 were strong inhibitors
of MRP1 and were weak modulators of P-gp (Tables 1 and 4).
The amide functionality in 13, 14, 23, and 30−32 offers a
better hydrogen-bond acceptor and a stronger dipole than the
thioamide functionality in 18−21, 28, and 29 while conversely,
the thioamide groups are more hydrophobic and less polar.
For chalcogenopyrylium compounds 1−32, interactions with
MRP1 appear to be favored by the presence of a thioamide
group while interactions with P-gp are favored by the presence
of an amide group. These differences are not evident in
experiments, demonstrating 21 and 28−32 transport across
monolayers of MDCKII-MDR1 cells. All six compounds
show very slow passive transport (PPassive ≤ 14 nm s−1) and
absorptive transport (PAB ≤ 5.6 nm s−1) (Table 3). The slowest
secretory transport rates (PBA, Table 3) were exhibited by
thioamide 28 and amide 31 while thioamide 29 and amide 30
(PBA ≥ 300 nm s−1) exhibited fast secretory rates, which were
only slower than thioamide 21 (PBA = 620 nm s−1).
EXPERIMENTAL SECTION
■
General Methods. Chalcogenopyrylium compounds 1−5,24,25
15−17,26,30 and 25−2732 were prepared by literature methods.
Reactions were run under Ar. Tetrahydrofuran was distilled from
sodium benzophenone ketyl prior to use. Concentration in vacuo was
performed on a Buchi rotary evaporator. NMR spectra were recorded
̈
on an Inova 500 instrument (500 MHz for 1H, 125 MHz for 13C) with
residual solvent signal as internal standard. Infrared spectra were
recorded on a Perkin-Elmer FTIR instrument. UV−vis near-IR spectra
were recorded on a Perkin-Elmer Lambda 12 spectrophotometer or
on a Shimadzu UV-3600 spectrophotometer in quartz cuvettes with a
1 cm path length. Melting points were determined with a Buchi
̈
capillary melting point apparatus and are uncorrected. All compounds
tested have a purity of at least 95%, which was determined by an
analysis on a C18 reverse-phase HPLC column [Protein RP] using
75% CH3CN/H2O with a flow rate of 0.4 mL min−1 and monitoring
by a UV−visible detector operating at 254 or 442 nm. Nonhygroscopic
compounds have a purity of ≥95% as determined by elemental
analyses for C, H, and N, which were performed by Atlantic Microlab,
Inc., Norcross, GA. Experimental values of C, H, and N are within
0.4% of theoretical values.
2,6-Di-tert-butyl-4-phenylthiopyrylium Bromide (6). 2,6-Di-
tert-butyl-4H-thiopyran-4-one (33-S, 0.10 g, 0.46 mmol) was dissolved
in dry THF (4.5 mL). Phenylmagnesium bromide (5.5 M, 0.96 mL,
5.4 mmol) was added to the solution of 33-S. The reaction was heated
at reflux for 1 h and then cooled to 0 °C. The reaction was quenched
by the dropwise addition of 48% HBr (5 mL), and the resulting
mixture was stirred 1 h at 0 °C and was then poured into 25 mL of
water. The pyrylium compound was extracted with CH2Cl2 (3 ×
20 mL). The combined organic extracts were washed with water (2 ×
15 mL), washed with brine (2 × 15 mL), dried over Na2SO4, and
concentrated. The residue was recrystallized from CH3CN/diethyl
ether to give 0.064 g (40%) of 6 as a yellow powder, mp 206−209 °C.
1H NMR [400 MHz, CD2Cl2] δ 8.72 (s, 2 H), 8.055 (d × d, 2 H, J =
1.2, 8.0 Hz), 7.72 (m, 3 H), 1.74 (s, 18 H). 13C NMR [300 MHz,
CD3CN] δ 185.53, 162.12, 137.82, 133.85, 131.56, 130.78, 118.23,
43.01, 31.18. HRMS (ES) m/z 285.1680 (calcd for C19H25S+:
285.1671); λmax (EtOH) 357 nm (ε 2.56 × 104 M−1 cm−1). Anal.
Calcd for C19H25S·Br: C, 62.46; H, 6.90. Found: C, 62.11; H, 6.96.
2,6-Di-tert-butyl-4-phenylselenopyrylium Bromide (7). 2,6-
Di-tert-butyl-4H-selenopyran-4-one (33-Se, 0.10 g, 0.37 mmol) and
CONCLUSIONS
■
We have demonstrated that within the series of chalcogeno-
pyrylium compounds 1−32 studied herein, inhibitors of both
MRP1 and P-gp activities were identified. Pyrylium, thiopyry-
lium, selenopyrylium, and telluropyrylium derivatives were
examined and the identity of the chalcogen atom shown to have
minimal impact on this modulatory activity. The implications
here support the idea that effective selenopyrylium and/or
telluropyrylium photosensitizers for the PDT of multidrug-
resistant cells can be designed based on the SAR of pyrylium
and thiopyrylium analogues, which are somewhat easier to
synthesize. 4-Aminoaryl substituents on the chalcogenopyry-
lium ring appear to be important for inhibitory activity in both
proteins, while substituent changes at the 2-, 4-, and 6-positions
of the chalcogenopyrylium compounds impact the two
4692
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
5.5 M PhMgBr (0.80 mL, 4.4 mmol) were treated as described for the
preparation of 6. The crude product was recrystallized from CH3CN/
diethyl ether to yield 0.085 g (56%) of 7 as an orange powder, mp 198−
201 °C. 1H NMR [400 MHz, CD2Cl2] δ 8.53 (s, 2 H), 7.92 (d × d, 2 H,
J = 1.2, 8.4 Hz), 7.72 (m, 3 H), 1.743 (s, 18 H). 13C NMR [300 MHz,
CD3CN] δ198.15, 162.10, 139.73, 133.36, 131.59, 130.68, 118.26, 44.56,
31.67. HRMS (ES) m/z 333.1122 (calcd for C19H2580Se+: 333.1116);
λmax (EtOH) 373 nm (ε 2.2 × 104 M−1 cm−1). Anal. Calcd for
C19H25Se·Br: C, 55.35; H, 6.11. Found: C, 55.05; H, 6.03.
2,6-Di-tert-butyl-4-(2-(N,N-diethylaminocarbonyl)phenyl)-
thiopyrylium Hexafluorophosphate (12). s-BuLi (1.3 M in
cyclohexane/hexane, 0.77 mL, 1.0 mmol) was added dropwise to a
stirring solution of N,N-diethylbenzamide (0.177 g, 1.00 mmol) and
TMEDA (0.15 mL, 1.0 mmol) dissolved in 8.0 mL of dry THF
at −78 °C. A solution of thiopyranone 33-S (0.112 g, 0.5 mmol)
dissolved in 1.0 mL of dry THF was then added to the benzamide
solution via cannula. The reaction mixture was stirred at ambient
temperature for 1 h and was then quenched with glacial acetic acid
(0.25 mL). The reaction mixture was poured into 100 mL of aqueous
HPF6 (3.0% wt/wt) and stirred for 1 h. The crude 12 was extracted
with CH2Cl2 (3 × 25 mL). The combined organic extracts were dried
over Na2SO4 and concentrated. The residue was recrystallized from
CH3CN/ether to give 0.123 g (47%) of a purple−brown solid, mp
180−183 °C. 1H NMR [400 MHz, CD2Cl2] δ 8.63 (s, 2 H), 7.72 (m,
2 H), 7.65 (m, 1 H), 7.50 (m, 1 H), 3.39 (q, 2 H, J = 7.2 Hz), 3.05 (q,
2 H, J = 7.2 Hz), 1.66 (s, 18 H), 1.06 (t, 3 H, J = 7.2 Hz), 0.91 (t, 3 H,
J = 7.2 Hz). 13C NMR [300 MHz, CD3CN] δ 186.06, 161.88, 138.12,
135.76, 133.30, 132.55, 131.75, 130.63, 130.44, 128.40, 43.84, 43.04,
39.75, 31.06, 14.12, 13.05. HRMS (ES) m/z 384.2356 (calcd for
C24H34NOS+: 384.2361); λmax (EtOH) 329 nm (ε = 1.50 × 104 M−1
cm−1). Anal. Calcd for C24H34NOS·PF6: C, 54.43; H, 6.47; N, 2.64.
Found: C, 54.16; H, 6.62; N, 2.67.
2,6-Di-tert-butyl-4-(2-(N,N-diethylaminocarbonyl)phenyl)-
selenopyrylium Hexafluorophosphate (13). s-BuLi (1.3 M in
cyclohexane/hexane, 0.77 mL, 1.0 mmol), N,N-diethylbenzamide
(0.177 g, 1.00 mmol), TMEDA (0.15 mL, 1.0 mmol), and seleno-
pyranone 33-Se (0.136 g, 0.50 mmol) were treated as described for the
preparation of 12. The crude product was recrystallized from CH3CN
to give 0.058 g (23%) of 13 as a purple−brown solid, mp 155−157 °C.
1H NMR [400 MHz, CD2Cl2] δ 8.535 (s, 2 H), 7.71 (m, 2 H), 7.62
(d, 1 H, J = 7.2 Hz), 7.50 (d × d, 1 H, J = 2.0, 7.8 Hz), 3.37 (q, 2 H,
J = 7.2 Hz), 3.01 (q, 2 H, J = 7.2 Hz) 1.68 (s, 18 H), 1.02 (t, 3 H, J =
7.2 Hz), 0.88 (t, 3 H, J = 7.2 Hz). 13C NMR [300 MHz, CD3CN] δ
199.37, 169.42, 161.89, 137.85, 137.41, 133.35, 132.21, 131.33, 130.60,
128.39, 44.69, 43.64, 39.50, 31.57, 14.06, 12.99. HRMS (ES) m/z
432.1808 (calcd for C24H34NO80Se+: 432.1806); λmax (EtOH) 344 nm
(ε 1.40 × 104 M−1 cm−1). Anal. Calcd for C24H34NOSe·PF6: C, 50.00;
H, 5.94; N, 2.43. Found: C, 49.76; H, 5.96; N, 2.54.
2,6-Di-tert-butyl-4-(2-(N,N-diethylaminocarbonyl)phenyl)-
telluropyrylium Hexafluorophosphate (14). s-BuLi (1.3 M in
cyclohexane/hexane, 0.77 mL, 1.0 mmol), N,N-diethylbenzamide
(0.177 g, 1.00 mmol), TMEDA (0.15 mL, 1.0 mmol), and telluro-
pyranone 33-Te (0.160 g, 0.50 mmol) were treated as described for
the preparation of 12. The crude product was recrystallized from
CH3CN to give 0.077 g (25%) of a dark yellow−green solid, mp 174−
175 °C. 1H NMR [400 MHz, CD2Cl2] δ 8.52 (s, 2 H), 7.68 (m, 2 H),
7.57 (d, 1 H, J = 7.5 Hz), 7.47 (d, 1H, J = 7.5 Hz), 3.33 (q, 2 H, J = 7.2
Hz), 2.94 (q, 2 H, J = 7.2 Hz), 1.66 (s, 18 H), 0.945 (t, 3 H, J = 7.2
Hz), 0.82 (t, 3 H, J = 7.2 Hz). 13C NMR [300 MHz, CD3CN] δ
208.15, 169.61, 161. 49, 139.88, 137.19, 136.90, 131.38, 130.55,
130.42, 128.33, 46.75, 43.39, 39.17, 32.44, 14.03, 13.03. HRMS (ES)
m/z 482.1679 (calcd for C24H34NO130Te+: 482.1703); λmax (EtOH)
383 nm (ε 1.48 × 104 M−1 cm−1). Anal. Calcd for C24H34NOTe·PF6:
C, 46.11; H, 5.48; N, 2.24. Found: C, 45.98; H, 5.60; N, 2.32.
2,6-Di-tert-butyl-4-(5-(piperidine-1-carbonothioyl)thiophen-
2-yl)thiopyrylium Hexafluorophosphate (18). Lithium diisopro-
pylamide (LDA) was prepared by adding n-BuLi (2.0 M in
cyclohexane, 0.68 mL, 1.4 mmol) to a solution of diisopropylamine
(0.20 mL, 1.4 mmol) in 4.0 mL of dry THF at 0 °C. The solution was
stirred for 15 min and then cooled to −78 °C, and piperidin-1-yl-
thien-2-yl-methanethione (38, 0.284 g, 1.34 mmol), dissolved in
2.0 mL of dry tetrahydrofuran, was added via syringe. The reaction
mixture was stirred for 30 min, and an aliquot (1.55 mL, 0.31 mmol)
was added via syringe to thiopyranone 33-S (0.045 g, 0.20 mmol)
dissolved in 4.5 mL of dry THF. The reaction mixture was stirred for
30 min at ambient temperature. The mixture was then quenched with
glacial acetic acid (0.25 mL), poured into 100 mL of aqueous HPF6
(3.0% w/w), and stirred for 1 h. The crude 18 was extracted with
CH2Cl2 (3 × 25 mL). The combined organic extracts were dried over
2,6-Di-tert-butyl-4-phenyltelluropyrylium Bromide (8). 2,6-
Di-tert-butyl-4H-telluropyran-4-one (33-Te, 0.13 g, 0.40 mmol) and
PhMgBr (0.87 mL, 4.8 mmol) were treated as described for the
preparation of 6. The crude product was recrystallized from CH3CN/
diethyl ether to yield 0.083 g (45%) of product as a light-green
1
powder, mp 169−171 °C. H NMR [400 MHz, CD2Cl2] δ 8.29 (s,
2 H), 7.73 (d × d, 2 H, J = 1.2, 8.0 Hz), 7.595 (m, 3 H), 1.71 (s, 18 H).
13C NMR [300 MHz, CD3CN] δ 143.01, 135.08, 134.93, 131.41,
130.65, 129.40, 129.33, 45.76, 33.94, 32.22, 32.05, 28.19, 26.29. HRMS
(ES) m/z 383.1017 (calcd for C19H25130Te+: 383.1013); λmax (EtOH)
399 nm (ε 1.81 × 104 M−1 cm−1). Anal. Calcd for C19H25Te·Br: C,
49.51; H, 5.47. Found: C, 49.55 ; H, 5.45.
2,6-Di-tert-butyl-4-(5-carboxythien-2-yl)thiopyrylium Hexa-
fluorophosphate (9). t-BuLi (1.3 M in pentane, 2.59 mL, 3.36
mmol) was added dropwise to 5-bromothiophene-2-carboxylic acid
(35, 0.264 g, 1.60 mmol) and TMEDA (0.066 mL, 0.44 mmol)
dissolved in 2.0 mL of dry tetrahydrofuran at −50 °C under an argon
atmosphere. The reaction mixture was stirred at ambient temperature
for 45 min. This solution was then transferred via syringe to a solution
of thiopyranone 33-S (0.090 g, 0.40 mmol) dissolved in 9.0 mL of dry
THF. After 1 h, the reaction mixture was treated with glacial acetic
acid (0.25 mL) and poured into 100 mL of aqueous HPF6 (3.0% w/
w), and the resulting mixture was stirred for 1 h. The dye was
extracted with CH2Cl2 (3 × 25 mL). The combined organic extracts
were dried over Na2SO4 and concentrated. The residue was dissolved
in a minimal amount of CH3CN, which was then diluted with ether
(3 mL). The product was collected by filtration to give 0.136 g (71%)
of 9 as a yellow solid, mp 239−241 °C. 1H NMR [400 MHz, CD2Cl2]
δ 8.59 (s, 2 H), 8.16 (d, 1 H, J = 4.0 Hz), 8.04 (d, 1 H, J = 4.0 Hz),
1.68 (s, 18 H). 13C NMR [300 MHz, CD3CN] δ 185.60, 153.17,
145.54, 143.71, 135.76, 135.31, 128.76, 42.84, 30.91. HRMS (ES) m/z
+
335.1134 (calcd for C18H23O2S2 :335.1139); λmax (EtOH) 421 nm
(ε 2.80 × 104 M−1 cm−1). Anal. Calcd for C18H23O2S2·PF6: C, 45.00;
H, 4.83. Found: C, 44.91; H, 4.85.
2,6-Di-tert-butyl-4-(5-carboxythien-2-yl)selenopyrylium
Hexafluorophosphate (10). t-BuLi (1.3 M in pentane, 2.6 mL, 3.4
mmol), 5-bromothiophene-2-carboxylic acid (35, 0.264 g, 1.60 mmol),
TMEDA (0.066 mL, 0.44 mmol), and 33-Se (0.11 g, 0.40 mmol) were
treated as described for the preparation of 9. The crude product was
recrystallized from CH3CN/ether to give 0.144 g (68%) of 10 as a
1
dark-yellow solid, mp 217−223 °C. H NMR [400 MHz, CD2Cl2] δ
8.54 (s, 2 H), 8.22 (s, 1 H), 8.02 (s, 1 H), 1.698 (s, 18 H). 13C NMR
[300 MHz, CD3CN] δ 162.55, 153.21, 147.52, 138.37, 135.92, 135.59,
135.02, 128.82, 44.46, 31.48. HRMS (ES) m/z 383.0593 (calcd for
C18H23O2S80Se+: 383.0584); λmax (EtOH) 449 nm (ε 2.21 × 104 M−1
cm−1). Anal. Calcd for C18H23O2SSe·PF6: C, 41.00; H, 4.40. Found: C,
40.93, 4.39.
2,6-Di-tert-butyl-4-(5-carboxythien-2-yl)telluropyrylium
Hexafluorophosphate (11). t-BuLi (1.3 M in pentane, 2.6 mL, 3.4
mmol), 5-bromothiophene-2-carboxylic acid (35, 0.264 g, 1.60 mmol),
TMEDA (0.066 mL, 0.44 mmol), and 33-Te (0.13 g, 0.40 mmol) were
treated as described for the preparation of 9. The crude product was
recrystallized from CH3CN/ether to give 0.17 g (74%) of a brown
solid, mp 215−218 °C. 1H NMR [400 MHz, CD2Cl2] δ 8.61 (s, 2 H),
8.18 (s, H), 7.98 (s, 1 H), 3.46 (s, 1 H), 1.69 (s, 18 H). 13C NMR [300
MHz, DMSO] δ 167.67, 162.67, 147.32, 139.34, 135.03, 133.42,
132.50, 127.34, 33.85, 27.38. HRMS (ES) m/z 433.0459 (calcd for
C18H23O2S130Te+: 433.0481); λmax (EtOH) 471 nm (ε 2.64 × 104 M−1
cm−1). Anal. Calcd for C18H23O2STe·PF6: C, 37.53; H, 4.02. Found:
C, 37.29; H, 4.12.
4693
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
MgSO4 and concentrated. The resulting residue was purified via column
chromatography on SiO2 eluted with 10% diethyl ether/CH2Cl2,
followed by 5% methanol/CH2Cl2 to yield 0.075 g (66%) of a dark-red
(washed three times with 5 mL of methanol prior to use) was added,
and the resulting mixture was stirred for 1 h at ambient temperature.
The resin was removed by filtration, and the filter cake was washed
with 3 mL of methanol. The exchange was repeated twice more. The
final filtrates were concentrated, and the residue was purified via
recrystallization from CH3CN to give 0.089 g (71%) of 21 a dark-
1
solid, mp 107−110 °C. H NMR [400 MHz, CDCl3] δ 8.43 (s, 2 H),
8.16 (d, 1 H, J = 4.0 Hz), 7.20 (d, 1 H, J = 4.0 Hz), 4.27 (br s, 2 H), 3.81
(br s, 2 H), 1.78 (s, 6 H), 1.62 (s, 18 H). 13C NMR [300 MHz,
CD3CN] δ 184.17, 175.85, 172.51, 155.23, 153.20, 135.12, 128.40,
127.60, 126.00, 59.74, 42.42, 30.72, 24.31. HRMS (ES) m/z 418.1695
(calcd for C23H32NS3+: 418.1697); λmax (EtOH) 416 nm (ε 1.86 × 104
M−1 cm−1). Anal. Calcd for C22H32NS3·PF6: C, 49.01; H, 5.72; N, 2.48.
Found: C, 49.00; H, 5.75; N, 2.47.
1
purple solid, mp 138−140 °C. H NMR [400 MHz, CD2Cl2] δ 8.33
(s, 1 H), 7.96 (s, 1 H), 7.94 (d, 1 H, J = 3.2 Hz), 7.90 (AA′BB′, 2 H,
J = 8 Hz), 7.22 (d, 1 H, J = 3.2 Hz), 6.88 (AA′BB′, 2 H, J = 8 Hz), 4.29
(br s, 2 H), 3.84 (br s, 2 H), 3.22 (s, 6 H), 1.81 (br s, 6 H), 1.60 (s, 9
H). 13C NMR [300 MHz, CD2Cl2] δ 187.6, 175.5, 167.4, 155.5, 153.3,
150.5, 142.5, 135.2, 131.2, 128.1, 123.5, 122.4, 121.3, 113.3, 41.1, 40.7,
31.9, 31.2, 24.4, 23.0, 14.2. HRMS (ES) m/z 481.1787 (calcd for
2,6-Di-tert-butyl-4-(5-(piperidine-1-carbonothioyl)thiophen-
2-yl)selenopyrylium Hexafluorophosphate (19). n-BuLi (2.0 M
in cyclohexane, 0.68 mL, 1.4 mmol), diisopropylamine (0.20 mL,
1.4 mmol), piperidin-1-yl-thien-2-yl-methanethione (38, 0.284 g, 1.34
mmol), and selenopyranone 33-Se (0.054 g, 0.20 mmol) were treated
as described for the preparation of 18 to give 0.090 g (74%) of 19 as a
+
C27H33N2S3 : 481.1806); λmax (EtOH) 629 nm (ε 1.33 × 104 M−1
cm−1). Anal. Calcd for C27H33N2S3Cl: C, 62.70; H, 6.43; N, 5.42.
Found: C, 62.43; H, 6.13; N, 5.21.
2-tert-Butyl-6-(4-(dimethylamino)phenyl)-4-(5-carboxy-
thien-2-yl)-thiopyrylium Hexafluorophosphate (22). t-BuLi
(1.48 M in pentane, 2.84 mL, 4.22 mmol), 5-bromothiophene-2-
carboxylic acid (35, 0.331 g, 2.00 mmol), TMEDA (0.080 mL,
0.55 mmol), and thiopyranone 40 (0.13 g, 0.40 mmol) were treated as
described for the preparation of 9. The crude product was
recrystallized from CH3CN/ether to give 0.141 g (52%) of 22 as a
1
dark-red solid, mp 102−104 °C. H NMR [400 MHz, CDCl3] δ 8.40
(s, 2 H), 8.20 (d, 1 H, J = 4.0 Hz), 7.21 (d, 1 H, J = 4.0 Hz), 4.27 (br s,
2 H), 3.81 (br s, 2 H), 1.79 (s, 6 H), 1.64 (s, 18 H). 13C NMR [300
MHz, CD3CN] δ 196.32, 187.49, 155.00, 153.50, 143.84, 135.21,
128.80, 127.86, 54.75, 52.10, 44.17, 31.48, 27.50, 26.10, 24.54. HRMS
(ES) m/z 466.1142 (calcd for C23H32NS280Se+ 466.1141); λmax (EtOH)
431 nm (ε 1.57 × 104 M−1 cm−1). Anal. Calcd for C22H32NS2Se·PF6: C,
45.24; H, 5.28; N, 2.29. Found: C, 45.42; H, 5.56; N, 2.38.
1
dark-blue solid, mp 154−156 °C. H NMR [400 MHz, (CD3)2SO] δ
8.72 (s, 1 H), 8.56 (d, 1 H, J = 4.4 Hz), 8.28 (s, 1 H), 8.18 (d, 2 H, J =
9.2 Hz), 7.95 (d, H, J = 4.0 Hz), 6.94 (d, 2 H, J = 9.2 Hz), 3.18 (s, 6
H), 1.57 (s, 9 H). 13C NMR [300 MHz, CD3CN] δ 177.6, 172.7,
169.1, 162.15, 156.3, 150.2, 146.9, 140.8, 140.8, 135.8, 133.4, 131.3,
124.6, 123.5, 121.5, 113.9, 40.6, 30.9. HRMS (ES) m/z 398.1247
2,6-Di-tert-butyl-4-(5-(piperidine-1-carbonothioyl)thiophen-
2-yl)telluropyrylium Hexafluorophosphate (20). n-BuLi (2.0 M
in cyclohexane, 0.68 mL, 1.4 mmol), diisopropylamine (0.20 mL,
1.4 mmol), piperidin-1-yl-thien-2-yl-methanethione (38, 0.284 g, 1.34
mmol), and selenopyranone 33-Te (0.064 g, 0.20 mmol) were treated
as described for the preparation of 18 to give 0.098 g (74%) of a dark-
red solid, mp 92−94 °C. 1H NMR [400 MHz, CDCl3] δ 8.47 (s, 2 H),
8.13 (d, 1 H, J = 4.0 Hz), 7.16 (d, 1 H, J = 4.4 Hz), 4.27 (br s, 2 H),
3.82 (br s, 2 H), 1.78 (s, 6 H), 1.62 (s, 18 H). 13C NMR [300 MHz,
DMSO] δ 196.73, 187.74, 164.14, 155.32, 142.37, 134.49, 128.82,
127.86, 53.54, 41.78, 33.35, 31.30, 28.01, 26.66, 22.35. HRMS (ES)
m/z 516.1044 (calcd for C23H32NS2130Te+ 516.1038); λmax (EtOH)
486 nm (ε 1.716 × 104 M−1 cm−1). Anal. Calcd for C23H32NS2Te·PF6:
C, 41.91; H, 4.89; N, 2.12. Found: C, 42.00; H, 4.88; N, 2.21.
2-tert-Butyl-6-(4-(dimethylamino)phenyl)-4-(5-(piperidine-
1-carbonothioyl)thiophen-2-yl)thiopyrylium Chloride (21) and
Hexafluorophosphate (41). n-Butyllithium (1.6 M in hexanes, 0.947
mL, 1.39 mmol) was added dropwise to a solution of diisoproplyamine
(0.21 mL, 1.5 mmol) in THF (5 mL) at −78 °C. The resulting mixture
was stirred for 40 min before it was transferred via cannula into a
solution of piperidin-1-yl(thiophen-2-yl)methanethione (38, 0.151 g,
0.713 mmol) dissolved in THF (5 mL) at −78 °C. The reaction
mixture was stirred 30 min before it was transferred via cannula into a
solution of 2-tert-butyl-6-(4-(dimethylamino)phenyl)-4H-thiopyran-4-
one (40, 0.100 g, 0.348 mmol) in THF (10 mL) at −78 °C. The
resulting mixture was stirred for 15 min at −78 °C before warming to
40 °C and stirred for an additional 15 min. Acetic acid (1 mL) was
added dropwise, and the mixture was poured into a 10% v/v solution of
cold aqueous HPF6 (100 mL). The crude product was extracted with
CH2Cl2 (3 × 100 mL). The combined organic extracts were washed
with water (3 × 100 mL), dried over MgSO4, filtered, and
concentrated. The crude solid was purified via recrystallization from
acetonitrile to give 0.33 g (75%) of the hexafluorophosphate salt 41,
+
(calcd for C22H24NO2S2 : 398.1248); λmax (EtOH) 616 nm (ε = 2.83
× 104 M−1 cm−1). Anal. Calcd for C22H24NO2S2: C, 48.62; H, 4.45; N,
2.58. Found: C, 48.23; H, 4.41; N, 2.48.
2-tert-Butyl-4-(2-(N,N-diethylaminocarbonyl)phenyl)-6-(4-
(dimethylamino)phenyl)thiopyrylium Bromide (23). s-BuLi
(1.3 M in cyclohexane/hexane, 0.77 mL, 1.0 mmol), N,N-diethylben-
zamide (0.177 g, 1.00 mmol), TMEDA (0.15 mL, 1.0 mmol), and
thiopyranone 40 (0.144 g, 0.500 mmol) were treated as described for
the preparation of 12. The crude product was recrystallized from
CH3CN/ether to give 0.119 g (45%) of 23 as a purple solid, mp 74−
1
75 °C. H NMR [400 MHz, CD2Cl2] δ 8.48 (s, 1 H), 8.03 (s, 1 H),
8.00 (d, 1 H, J = 6.4 Hz), 7.70 (m, 3 H), 7.47 (m, 2 H), 6.92 (d, 2 H,
J = 8.8 Hz), 3.41 (q, 2 H, J = 7.2 Hz), 3.24 (s, 6 H), 3.00 (q, 2 H, J =
7.2 Hz), 1.60 (s, 9 H), 0.98 (t, 3 H, J = 7.2 Hz), 0.90 (t, 3 H, J = 7.2
Hz). 13C NMR [300 MHz, CD3CN] δ 177.56, 158.69, 136.38, 132.02,
131.24, 130.86, 130.46, 128.70, 128.24, 127.55, 113.95, 43.75, 40.60,
39.60, 30.97, 13.99, 12.84. HRMS (ES) m/z 447.2470 (calcd for
C28H35N2OS+: 447.2470); λmax (EtOH) 601 (ε 2.59 × 104 M−1 cm−1).
Anal. Calcd for C28H35N2OS·Br: C, 64.48; H, 6.12; N, 3.27. Found: C,
64.43; H, 6.09; N, 3.11.
2-tert-Butyl-6-(4-(dimethylamino)phenyl)-4-phenylthiopyry-
lium Bromide (24). 2-tert-Butyl-6-(4-(dimethylamino)phenyl)-4H-
thiopyran-4-one (40, 0.14 g, 0.50 mmol) and 5.5 M PhMgBr (1.1 mL,
6.0 mmol) were treated as described for the preparation of 6. The
crude product was recrystallized from CH3CN/ether to give 0.187 g
1
(87%) of 24 as a dark-purple powder, mp >250 °C. H NMR [400
MHz, CD2Cl2] δ 8.52 (s, H), 8.08 (s, 2 H), 7.94 (s, 2 H), 7.68 (s, 3
H), 6.92 (d, 2 H, J = 4.8 Hz), 3.23 (s, 6 H), 1.64 (s, 9H). 13C NMR
[300 MHz, CD3CN] δ 178.23, 169.57, 159.57, 156.04, 138.51, 133.14,
131.37, 130.53, 129.94, 127.47, 126.16, 121.63, 113.76, 41.78, 40.59,
31.05. HRMS (ES) m/z 348.1775 (calcd for C23H26NS+: 348.1786);
λmax (EtOH) 595 nm (ε 2.6 × 104 M−1 cm−1). Anal. Calcd for
C23H26NS·PF6: C, 55.98; H, 5.31; N, 2.84. Found: C, 56.03; H, 5.41;
N, 2.83.
2,6-Bis(4-(dimethylamino)phenyl)-4-(5-(piperidine-1-
carbonothioyl)thien-2-yl)thiopyrylium Chloride (28) and Hex-
afluorophosphate (43). n-Butyllithium (1.6 M in hexanes, 0.693 mL,
1.02 mmol) was added dropwise to a solution of diisoproplyamine
(0.17 mL, 1.2 mmol) in THF (5 mL) at −78 °C. The resulting
mixture of LDA was stirred for 40 min before it was transferred via
cannula into a solution of 38 (0.151 g, 0.713 mmol) dissolved in THF
1
mp 138−140 °C. H NMR [400 MHz, CD2Cl2] δ 8.33 (s, 1 H), 7.96
(s, 1 H), 7.94 (d, 1 H, J = 3.2 Hz), 7.90 (AA′BB′, 2 H, J = 8 Hz), 7.22
(d, 1 H, J = 3.2 Hz), 6.88 (AA′BB′, 2 H, J = 8 Hz), 4.29 (br s, 2 H), 3.84
(br s, 2 H), 3.22 (s, 6 H), 1.81 (br s, 6 H), 1.60 (s, 9 H). 13C NMR
[300 MHz, CD2Cl2] δ 187.6, 175.5, 167.4, 155.5, 153.3, 150.45, 142.5,
135.2, 131.2, 128.1, 123.5, 122.4, 121.3, 113.3, 41.1, 40.7, 31.9, 31.2,
+
24.4, 23.0, 14.2. HRMS (ES) m/z 481.1787 (calcd for C27H33N2S3 :
481.1806); λmax (EtOH) 630 nm (ε 1.35 × 104 M−1 cm−1). Anal. Calcd
for C27H33N2S3·PF6: C, 51.74; H, 5.31; N, 4.47. Found: C, 51.43; H,
5.13; N, 4.21.
Compound 41 (0.126 g, 0.20 mmol) was dissolved in 3 mL of
methanol and 0.50 g of Amberlite-400 (Cl) ion-exchange resin
4694
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
(5 mL) at −78 °C. The reaction mixture was stirred 30 min before it
was transferred via cannula into a solution of 2,6-bis(4-(dimethylamino)-
phenyl)-4H-thiopyran-4-one (42,40 0.100 g, 0.285 mmol) in THF
(10 mL) at −78 °C. The resulting mixture was stirred for 15 min
at −78 °C and was then warmed to 40 °C and stirred for an additional
15 min. Acetic acid (1 mL) was added dropwise, and the mixture was
poured into a 10% v/v solution of cold aqueous HPF6 (100 mL). The
crude product was extracted with CH2Cl2 (3 × 100 mL). The combined
organic extracts were washed with water (3 × 100 mL), dried over
MgSO4, filtered, and concentrated to give the hexafluorophosphate salt
43, which was recrystallized from CH3CN to give 0.16 g (80%) of 43,
mp 195−197 °C. 1H NMR [500 MHz, CD2Cl2] δ 8.50 (d, 1 H, J = 4.0
Hz), 8.28 (s, 2 H), 7.98 (d, AA′BB′, 4 H, J = 9.0 Hz), 7.22 (d, 1 H, J =
4.0 Hz), 6.89 (d, AA′BB′, 4 H, J = 9.0 Hz), 4.29 (br s, 2 H), 3.86 (br s,
2 H), 3.17 (s, 12 H), 1.80 (m, 6 H). 13C NMR [75.6 MHz, CD2Cl2] δ
187.5, 162.5, 154.7, 152.3, 149.3, 142.8, 131.4, 129.4, 127.8, 120.6, 119.2,
113.0, 52.4, 40.4, 27.4, 25.9, 24.4. HRMS (ES) m/z 544.1912 (calcd
for C31H34N3S3+: 544.1909); λmax (CH2Cl2) 716 nm (ε 3.9 × 104
M−1cm−1). Anal. Calcd for C31H34N3S3·PF6: C, 53.98; H, 4.97; N, 6.09.
Found: C, 54.01; H, 4.95; N, 6.12.
J = 9.0 Hz), 7.44 (d, 1 H, J = 4.0 Hz), 6.90 (d, AA′BB′, 2 H, J = 9.0
Hz), 3.68 (t, 4 H, J = 6.0 Hz), 3.22 (s, 12 H), 1.73 (m, 6 H), 1.60 (s, 9
H). 13C NMR [75.6 MHz, CD2Cl2] δ 176.0, 167.8, 161.4, 155.6, 150.2,
146.7, 142.4, 132.8, 130.8, 130.5, 123.3, 121.9, 121.0, 113.4, 49.9 (br),
41.1, 40.6, 31.1, 26.4, 24.7. HRMS (ESI) m/z 465.2029 (calculated
+
for C27H33O1N2S2 : 465.2029); λmax (CH2Cl2) 644 nm (ε 3.67 × 104
M−1cm−1).
2,6-Di-tert-butyl-4-(5-(piperidine-1-carbonothioyl)thien-2-
yl)thiopyrylium Hexafluorophosphate (32). Trifluoroacetic anhy-
dride (0.62 mL, 4.4 mmol) and thioamide 18 (0.25 g, 0.44 mmol)
were treated as described for the preparation of 31. The crude product
was recrystallized from acetonitrile to give 0.079 g (34%) of 32 as a
copper−brown solid, mp 110−113 °C. 1H NMR [500 MHz, CD2Cl2]
δ 8.54 (s, 2 H), 8.25 (d, 1 H, J = 4.0 Hz), 7.48 (d, 1 H, J = 4.0 Hz),
3.66 (br s, 4 H), 1.73 (m, 6 H), 1.66 (s, 18 H). 13C NMR [75.6 MHz,
CD2Cl2] δ 184.5, 161.0, 153.4, 149.2, 141.3, 134.9, 131.1, 127.2, 49.3
(br), 42.4, 31.2, 26.4, 24.6. HRMS m/z 402.1913 (calcd for
+
C23H32O1N1S2 : 402.1920); λmax (CH2Cl2) = 442 nm (ε 3.21 × 104
M−1cm−1).
Determination of Partition Coefficients. The n-octanol/water
partition coefficients were all measured at pH 7.4 in PBS using
UV−visible spectrophotometry. The measurements were done using a
“shake flask” direct measurement.41 Mixing for 3−5 min was followed
by 1 h of settling time. Equilibration and measurements were made at
23 °C using a Perkin-Elmer Lambda 12 spectrophotometer. High-
performance liquid chromatography grade n-octanol was obtained
from Sigma-Aldrich.
Cell Culture for MRP1 Studies. A stably transfected MRP1 over-
expressing HEK cell line was generated using a pcDNA3.1(−) expres-
sion vector containing the entire human MRP1 cDNA transcript.39
Following transfection using Lipofectamine 2000 (Gibco-Invitrogen;
Burlington, ON). HEK cells were selected in G418 (ONBIO;
Richmond Hill, ON) (1 mg mL−1), and clones of surviving cells
were isolated using colony rings. Clonal cell lines were then expanded
and screened for MRP1 by immunoblotting with mouse mAb QCRL-1.62
Homogeneity of MRP1 expressing cell populations was confirmed by
flow cytometry using mouse mAb QCRL-363 on an Epics Altra HSS
flow cytometer (Beckman Coulter). A homogeneous stable cell line
thus obtained was designated HEK-MRP1. All cultured cells were
incubated in Dulbecco's Modified Essential Medium with 7.5% fetal
bovine serum (FBS) at 37 °C in 5% CO2/95% air. For membrane
vesicle preparations, HEK-MRP1 cells were seeded onto 150 mm plates
at 18 × 106 cells per 20 mL and harvested at confluence.
Preparation of MRP1-Enriched Membrane Vesicles. Mem-
brane vesicles were prepared essentially as described by Loe et al.64
Cell pellets were thawed slowly on ice, resuspended in homoge-
nization buffer [250 mM sucrose/50 mM Tris pH 7.4/0.25 mM CaCl2
with protease inhibitors (Roche, Mississauga, ON)], and disrupted by
argon cavitation (350 psi, 4 °C, 5 min). After centrifugation at 1400,
the supernatant was transferred to high speed centrifuge tubes and
the remaining pellet was resuspended in homogenization buffer with
0.5 mM EDTA. Following a second centrifugation at 1400g, the
supernatants from the first and second centrifugations were combined
and overlaid onto a cushion comprised of 35% (w:v) sucrose/1 mM
EDTA/50 mM Tris, pH 7.4. After centrifugation at 100000g, the
interface layer containing membranes was removed, placed in 25 mM
sucrose/50 mM Tris, pH 7.4 buffer, and the membranes collected by
centrifugation again at 100000g. The membranes were then washed
with Tris-sucrose buffer (TSB) (250 mM sucrose, 50 mM Tris, pH 7.4)
and centrifuged at 55000g. The pellet was resuspended in TSB
and vesicles prepared by passaging through a 1 mL syringe with a
27-gauge needle. Vesicles were stored at −80 °C until needed. Vesicular
protein concentrations were determined using the Bradford method
with BSA as a standard.
The hexafluorophosphate salt of 43 was exchanged for chloride as
described for the preparation of 21. The final filtrates were con-
centrated, and the residue was purified via recrystallization from
CH3CN to give 0.062 g (31%) of 28 as a dark-purple solid, mp 190−
1
193 °C. H NMR [500 MHz, CD2Cl2] δ 8.50 (d, 1 H, J = 4.0 Hz),
8.28 (s, 2 H), 7.98 (d, AA′BB′, 4 H, J = 9.0 Hz), 7.22 (d, 1 H, J = 4.0
Hz), 6.89 (d, AA′BB′, 4 H, J = 9.0 Hz), 4.29 (br s, 2 H), 3.86 (br s,
2 H), 3.17 (s, 12 H), 1.80 (m, 6 H). 13C NMR [75.6 MHz, CD2Cl2] δ
187.5, 162.5, 154.7, 152.3, 149.3, 142.8, 131.4, 129.4, 127.8, 120.6,
119.2, 113.0, 52.4, 40.4, 27.4, 25.9, 24.4. HRMS (ES) m/z 544.1912
+
(calcd for C31H34N3S3 : 544.1909); λmax (CH2Cl2) 717 nm (ε 3.8 ×
104 M−1cm−1).
Preparation of 2,6-Di-tert-butyl-4-(5-(piperidine-1-
carbonothioyl)thiophen-2-yl)thiopyrylium Chloride (29). The
hexafluorophosphate salt of 18 was exchanged for chloride as
described for the preparation of 21. The crude solid was purified via
recrystallization from CH3CN to give 0.059 g (29%) of 29 as a dark-
1
red solid, mp 107−110 °C. H NMR [500 MHz, CD2Cl2] δ 9.02 (s,
2 H), 8.78 (d, 1 H, J = 4.0 Hz), 7.29 (d, 1 H, J = 4.0 Hz), 4.29 (br s,
2 H), 3.84 (br s, 2 H), 1.80 (m, 6 H), 1.68 (s, 18 H). 13C NMR [75.6
MHz, CD2Cl2] δ 186.8, 184.0, 156.0, 153.4, 141.1, 135.1, 128.3, 126.7,
52.0, 42.4, 31.3, 30.0, 27.4, 25.8, 24.3. HRMS (ES) m/z 418.1691
+
(calcd for C23H32N1S3 : 418.1691); λmax (EtOH) 416 nm (ε 1.86 ×
104 M−1cm−1).
2,6-Bis(4-(dimethylamino)phenyl)-4-(5-(piperidine-1-
carbonyl)thien-2-yl)thiopyrylium Hexafluorophosphate (30).
Thioamide 43 (0.250 g, 0.362 mmol) and trifluoroacetic anhydride
(0.50 mL, 3.6 mmol) were treated as described for the preparation of
31. The crude solid was purified via recrystallization from CH3CN to
1
give 0.127 g (52%) of 30 as a dark-blue solid, mp 142−145 °C. H
NMR [500 MHz, CD2Cl2] δ 8.14 (s, 2 H), 8.08 (br s, 1 H), 7.875 (d,
AA′BB′, 4 H, J = 9.0 Hz), 7.41 (d, 1 H, J = 4.0 Hz), 6.85 (d, AA′BB′,
4 H, J = 9.0 Hz), 3.684 (t, 4 H, J = 6.0 Hz), 3.16 (s, 12 H), 1.73 (m,
6 H). 13C NMR [75.6 MHz, CD2Cl2] δ 162.0, 161.4, 154.5, 149.0,
145.3, 142.7, 132.0, 130.7, 129.3, 120.4, 119.1, 112.8, 45.1 (br), 40.2,
+
26.4, 24.8. HRMS (ES) m/z 528.2130 (calcd for C31H34O1N3S2 :
528.2138); λmax (CH2Cl2) 717 nm (ε 4.68 × 104 M−1cm−1).
2-tert-Butyl-6-(4-(dimethylamino)phenyl)-4-(5-(piperidine-
1-carbonyl)thien-2-yl)thiopyrylium Hexafluorophosphate (31).
Trifluoroacetic anhydride (0.55 mL, 4.0 mmol) was added dropwise
to a stirred solution of hexafluorophosphate salt 41 (0.250 g, 0.339
mmol) dissolved in CH2Cl2 (25 mL). The resulting mixture was
heated at reflux for 14 h, and then 10% sodium carbonate solution
(5 mL) was added dropwise and the reaction was poured into distilled
water (200 mL). The crude product was extracted with CH2Cl2 (3 ×
100 mL). The combined organic extracts were washed with water (3 ×
100 mL), dried over Na2SO4, and concentrated. The crude solid
was purified via recrystallization from CH3CN to give 0.177 g (73%)
of 31 as a blue solid, mp 194−197 °C. 1H NMR [500 MHz, CD2Cl2] δ
8.47 (s, 1 H), 8.16 (br s, 1 H), 8.01 (s, 1 H), 7.98 (d, AA′BB′, 2 H,
Immunoblotting for MRP1. The presence of MRP1 in the
membrane vesicles was confirmed by immunoblot analysis as follows:
proteins were resolved by electrophoresis on a 7% polyacrylamide gel
and then electrotransferred to polyvinylidene fluoride membranes (Pall
Corporation; Ville St. Laurent, QC). After transfer, the membranes
were washed in TBS plus 0.1% Tween-20 (TBST) and then blocked in
4695
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
4% (w:v) skim milk powder in TBST for 1 h. Following blocking, the
membranes were incubated overnight at 4 °C with mAb QCRL-1
(diluted 1:10000) in blocking solution.65 After washing, blots were
incubated with horseradish peroxidase-conjugated secondary antibody
(Pierce Biotechnology; Rockford, IL) in blocking solution for 1−2 h at
room temperature and then washed in TBST before incubating with
chemiluminescence blotting substrate (PerkinElmer; Woodbridge,
ON) and exposing to film.
Cells were washed once with Dulbecco’s phosphate-buffered saline
containing 10 mM HEPES buffer at pH 7.4 (DPBSH) (Gibco) and
incubated with solutions of the chalcogenopyrylium analogue or
control compound in DPBSH at 37 °C in room atmosphere. IC50
values were calculated from 1:1 serial dilution series. After 30 min,
the test compound was replaced to include 0.5 μg mL−1 CAM and
incubated an additional 20 min. Calcein fluorescence was read on a
Cytofluor series 4000 Multi Well Plate Reader (PerSeptive
Biosystems) with λEX and λEM set at 485 and 530 nm, respectively.
Negative (0.25% DMSO in DPBSH) and positive [5 μM, 44 (LSN
335984)] controls were included in each plate. IC50 values were
calculated from the serial dilution curves using GraphPad PRISM
version 4.03 software. Briefly, compound concentration was plotted as
log μM concentration versus relative fluorescence units (rfu) and a
sigmoidal dose−response (variable slope) analysis with no weighting
or restrictions was applied. The CAM fluorescence observed with
5 μM 44 was taken as complete inhibition.
P-gp-Mediated Transport across MDCKII-MDR1 Monolayers.
MDCKII-MDR1 cells that were seeded at 50000 cells cm−2 onto 12-
well (1.13 cm2 surface area) Transwell polycarbonate filters (Costar)
were fed on days 3 and 5 and used on day 6. The upper and lower
chamber volumes were 0.5 and 1.0 mL, respectively. Cells were rinsed
10 min in DPBSH at 37 °C with mixing on a nutator (Clay Adams).
Cells were preincubated with 4.3 mg mL−1 BSA in DPBSH alone or
containing 5 μM 44 (LSN 335984). After 30 min, 5 μM test
compound (21, 28−32) in BSA/DPBSH with or without the inhibitor
44 was added to the donor chamber (0.5 mL upper or apical, 1.0 mL
lower or basolateral). Initial samples from the donor chamber were
taken at t = 0. For apical-to-basolateral (AB) flux, D0 was taken from
the mixing tube before addition to the cell monolayer. For basolateral-
to-apical (BA) flux, the sample was taken from the 12-well plate
10 min after transfer but before cell wells were added. Samples were
taken from both the donor and receiver chambers following a 1 h
incubation at 37 °C with constant mixing by nutation. Cell monolayers
were rinsed briefly twice with cold DPBS and extracted with 500 μL of
methanol for 3 min. Fifty μL samples (n = 3) were placed in a 96-deep
well assay plate, and protein was precipitated by adding 450 μL of
acetonitrile and shaken to mix. Plates were centrifuged 5 min at
5000 rpm. Compound concentrations were determined with an LC-
MS/MS assay. Chromatography was performed using a Betasil C18
2 mm × 20 mm 5 μm Javelin column (Thermo Scientific, Waltham,
MA) and 1 of 2 mobile phase systems. System 1 consisted of 5 mM
ammonium bicarbonate in water (mobile phase A) and 5 mM
ammonium bicarbonate in methanol (mobile phase B), with elution
accomplished by a methanol gradient at 1.5 mL min−1. System 2
consisted of 0.4% trifluoracetic acid (TFA), 1 mM ammonium
bicarbonate in water (mobile phase A), and 0.4% TFA/1 mM
ammonium bicarbonate in acetonitrile (mobile phase B), with elution
accomplished by an acetonitrile gradient at 1.5 mL min−1. Mass
spectrometric detection was performed with an API4000 mass
spectrometer (Applied Biosystems, Foster City, CA) equipped with
a turbo ion spray source, using selected reaction monitoring in positive
ion mode with precursor and product ion transitions specific to each
analyte.
MRP1-Mediated Uptake of [3H]E217βG by Membrane
Vesicles. ATP-dependent uptake of [6,7-3H]E217βG (45 Ci
mmol−1) (PerkinElmer,Woodbridge, ON) by MRP1-enriched mem-
brane vesicles was measured using a 96-well rapid filtration method
previously described.65 Reactions were carried out in duplicate in
96-well round-bottomed plates in a final reaction volume of 30 μL TSB.
Vesicle protein (2 μg) was incubated with reaction mix containing
[3H]E217βG (400 nM, 20 nCi) for 3 min at 37 °C. Stock solutions of
chalcogenopyrylium compounds were diluted as needed in TSB in the
dark and then added to both the reaction mix (containing either AMP
or ATP (4 mM), MgCl2 (10 mM), and E217βG/[3H]E217βG) and the
membrane vesicle preparations. The final concentration of DMSO
never exceeded 1%. Uptake of [3H]E217βG was stopped by rapid
dilution in ice-cold TSB followed by rapid filtration of the wells’
contents onto a Unifilter-96 GF/B filter plate using a 96-well Filtermate
Harvester apparatus (Packard BioScience, Meriden, CT). Radioactivity
was measured by liquid scintillation counting on a TopCount NXT
microplate scintillation and luminescence counter (Perkin-Elmer), and
uptake was expressed as pmol [3H]E217βG mg protein−1 min−1. To
determine ATP-dependent uptake, uptake in the presence of AMP was
subtracted from uptake in the presence of ATP.
Expression and Purification of P-gp, and Measurement of
P-gp ATPase Activity. The cDNA for human P-gp45 was modified to
contain a tag of 10 His residues at the COOH-terminus (P-gp-His10)
to facilitate purification by metal-chelate chromatography.46 Baby
hamster kidney (BHK) cells were cotransfected with P-gp-His10 and
pWL-neo (Stratagene, Cedar Creek, TX), and the transfected cells
selected with G418 as described previously.66 G418-resistant colonies
were selected and clones overexpressing P-gp were identified by
subjecting whole cell extracts to immunoblot analysis with a rabbit
polyclonal antibody to P-gp.67 BHK cells overexpressing P-gp-His10
were then expanded and used to obtain purified protein.
His-tagged P-gp was isolated by Ni-chelate chromatography as
described previously.46 A sample of the isolated His-tagged P-gp was
mixed with an equal volume of 10 mg mL−1 sheep brain
phosphatidylethanolamine (Type II−S, Sigma) that had been washed
and suspended in TBS (Tris-buffered saline: 10 mM Tris/HCl, pH 7.4
and 150 mM NaCl). The sample was sonicated and ATPase activity
measured in the absence of drug substrate or in the presence of various
concentrations of chalcogenopyrylium compounds. The samples were
incubated for 30 min at 37 °C, and the amount of inorganic phosphate
released was determined.47
To test for inhibition of drug-stimulated P-gp ATPase activity,
samples of P-gp-His10 in lipid were preincubated with various con-
centrations of the chalcogenopyrylium compounds for 15 min at
20 °C. The ATPase reactions were then started by addition of ATPase
reaction mix containing VER (final concentration of 0.4 mM) and
ATPase activity determined as described above. VER was used to test
for inhibition because it is one of the most commonly used and potent
stimulators of P-gp ATPase activity.45
Inhibition of VIN Efflux from MDCKII-MDR1 Cells by
Chalcogenopyrylium Compounds 21, 28, and 30. MDCKII-
MDR1 cells were seeded onto Costar Transwell polycarbonate
membranes and maintained as described above. On day 6, cells were
rinsed once for 10 min in DPBSH with nutation at 37 °C and then
were exposed to 0.0001, 0.01, 0.05, 0.1, 0.5, 1, or 10 μM 21, 28, or 30
in BSA/DPBSH. After 30 min at 37 °C, 1.0 mL [3H]-VIN (0.25 μCi
mL−1 from 0.1 mCi mL−1 ethanol stock) in appropriate 0.0001,
0.01, 0.05, 0.1, 0.5, 1, or 10 μM 21, 28, or 30 solution was introduced
to the basolateral chamber and 0.5 mL of fresh 0.0001, 0.01, 0.05, 0.1,
0.5, 1, or 10 μM chalcogenopyrylium solution was added to the apical
chamber. Initial donor samples were taken from the basolateral
chamber at t = 0. The apical solution was replaced every 10 min with
fresh buffer, and appearance of [3H]-VIN in the apical chamber was
measured by scintillation counting. An IC50 was calculated using XLfit
software.
Enhancement of CAM Uptake into MDCKII-MDR1 Cells by
Chalcogenopyrylium Compounds. MDCK cells transfected with
wild-type MDR1 (ABCB1) were obtained at passage number 12 from
Dr. Piet Borst at The Netherlands Cancer Institute. Cell growth was
maintained in Dulbecco’s Modified Eagle’s Medium (Gibco)
supplemented with 10% fetal bovine serum (FBS), 100 units mL−1
penicillin, and 100 μg mL−1 streptomycin in 75-cm2 flasks. Cultures
were passaged by trypsinization 1:10 twice a week and used at passage
numbers 16−42. Cells were seeded at 40000 cells per well in 96-well
flat bottom plates (Falcon) using a medium volume of 200 μL, which
was replaced on day 3 prior to their use on day 4.
4696
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
(10) Dantzig, A. H.; de Alwis, D. P.; Burgess, M. Considerations in
the design and development of transport inhibitors as adjuncts to drug
therapy. Adv. Drug Delivery Rev. 2003, 55, 133−150.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (716) 645-4228. Fax: (716) 645-6963. E-mail:
mdetty@buffalo.edu.
(11) Sandor, V.; Fojo, T.; Bates, S. E. Future perspectives for the
development of P-glycoprotein modulators. Drug Resist. Updates 1998,
1, 190−200.
Notes
(12) Mahon, F. X.; Deininger, M. W. N.; Schultheis, B.; Chabrol, J.;
Reiffers, J.; Goldman, J. M.; Melo, J. V. Selection and characterization
of BCR-ABL positive cell lines with differential sensitivity to the
tyrosine kinase inhibitor STI571: diverse mechanisms of resistance.
Blood 2000, 96, 1070−1079.
(13) Eckford, P. D. W.; Sharom, F. J. ABC efflux pump-based
resistance to chemotherapy drugs. Chem. Rev. 2009, 109, 2989−3011.
(14) Raub, T. J. P-Glycoprotein recognition of substrates and
circumvention through rational drug design. Mol. Pharmaceutics 2006,
3, 3−25.
(15) Cole, S. P.C.; Bhardwaj, G.; Gerlach, J. H.; Mackie, J. E.; Grant,
C. E.; Almquist, K. C.; Stewart, A. J.; Kurz, E. U.; Duncan, A. M.;
Deeley, R. G. Overexpression of a transporter gene in a multidrug-
resistant human lung cancer cell line. Science 1992, 258, 1650−1654.
(16) Slot, A. J.; Molinski, S. V.; Cole, S. P. C. Mammalian multidrug
resistance proteins (MRPs). Essays Biochem. 2011, 50, 197−207.
(17) Deeley, R. G.; Westlake, C.; Cole, S. P. C. Transmembrane
transport of endo- and xenobiotics by membrane ATP-binding cassette
multidrug resistance proteins. Physiol. Rev. 2006, 86, 849−899.
(18) Rosenberg, M. F.; Mao, Q. C.; Holzenburg, A.; Ford, R. C.;
Deeley, R. G.; Cole, S. P.C. The structure of the multidrug resistance
protein (MRP1/ABCC1). Crystallization and single particle analysis. J.
Biol. Chem. 2001, 276, 16076−16082.
(19) Loo, T. W.; Clarke, D. M. Covalent modification of P-
glycoprotein mutants containing a single cysteine in either nucleotide-
binding fold abolishes drug-stimulated ATPase activity. J. Biol. Chem.
1995, 270, 22957−22961.
(20) Urbatsch, I. L.; Sankaran, B.; Bhagat, S.; Senior, A. E. Both P-
glycoprotein nucleotide-binding sites are catalytically active. J. Biol.
Chem. 1995, 270, 26956−26961.
(21) Nagata, K.; Nishitani, M.; Matsuo, M.; Kioka, N.; Amachi, T.;
Ueda, K. Nonequivalent nucleotide trapping in the two nucleotide
binding folds of the human multidrug resistance protein MRP1. J. Biol.
Chem. 2000, 275, 17626−17630.
(22) de Jong, M. C.; Slootstra, J. W.; Scheffer, G. L.; Schroeijers, A.
B.; Puijk, W. C.; Dinkelberg, R.; Icool, M.; Broxterman, H. J.; Meloen,
R. H.; Scheper, R. J. Peptide transport by the multidrug resistance
protein MRP1. Cancer Res. 2001, 61, 2552−2557.
(23) Tombline, G.; Donnelly, D. J.; Holt, J. J.; You, Y.; Ye, M.;
Gannon, M. K.; Nygren, C. L.; Detty, M. R. Stimulation of P-
glycoprotein ATPase by analogues of tetramethylrosamine: coupling of
drug binding at the “R” site to the ATP hydrolysis transition state.
Biochemistry 2006, 45, 8034−8047.
(24) Tombline, G.; Holt, J. J.; Gannon, M. K.; Donnelly, D. J.;
Wetzel, B.; Sawada, G. A.; Raub, T. J.; Detty, M. R. ATP occlusion by
P-glycoprotein as a surrogate measure of drug coupling. Biochemistry
2008, 47, 3294−3307.
(25) Gannon, M. K.; Holt, J. J.; Bennett, S. M.; Wetzel, B. R.; Loo, T.
W.; Bartlett, M. C.; Clarke, D. M.; Sawada, G. A.; Higgins, J. W.;
Tombline, G.; Raub, T. J.; Detty, M. R. Rhodamine inhibitors of P-
glycoprotein: an amide thioamide “switch” for ATPase activity. J. Med.
Chem. 2009, 52, 3328−3341.
(26) Sawada, G. A.; Raub, T. J.; Higgins, J. W.; Brennan, N. K.;
Moore, T. M.; Tombline, G.; Detty, M. R. Chalcogenopyrylium dyes
as inhibitors/modulators of P-glycoprotein in multidrug-resistant cells.
Bioorg. Med. Chem. 2008, 16, 9745−9756.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Piet Borst at The Netherlands Cancer Institute
for supplying the MDCKII-MDR1 cells. This research was
supported in part by the NIH (GM-94367) to M.R.D. and
grants from the Canadian Cancer Society (19074) and the
Canadian Institutes for Health Research (no. 25043) to D.M.C.
and (no. 10519) to S.P.C.C. D.M.C. is the recipient of the
Canada Research Chair in Membrane Biology, and S.P.C.C. is
the recipient of the Canada Research Chair in Cancer Biology,
and Queen’s University Bracken Chair in Genetics and
Molecular Medicine.
ABBREVIATIONS USED
■
ABC, ATP-binding cassette; BCRP, breast cancer resistance
protein; BHK, baby hamster kidney; BSA, bovine serum
albumin; CAM, calcein AM; DMSO, dimethylsulfoxide;
DPBSH, Dulbecco’s HEPES-containing phosphate-buffered
saline; E217βG, estradiol glucuronide; FBS, fetal bovine
serum; HEK, human embryonic kidney; LDA, lithium
diisopropylamide; MDCK, Madin−Darby canine kidney;
MDR, multidrug resistance; MRP, multidrug resistance protein;
NBD, nucleotide binding domain; P-gp, P-glycoprotein; TBS,
Tris-buffered saline; THF, tetrahydrofuran; TMD, trans-
membrane domain; TMEDA, tetramethylethylenediamine;
TSB, Tris sucrose buffer; TBST, TBS plus 0.1% Tween-20;
VER, verapamil; VIN, vinblastine
REFERENCES
■
(1) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in
cancer: role of ATP-dependent transporters. Nature Rev. Cancer 2002,
2, 48−58.
(2) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;
Gottesman, M. M. Targeting multidrug resistance in cancer. Nature
Rev. Drug Discovery 2006, 5, 219−234.
(3) Dean, M.; Rzhetsky, A.; Allikmets, R. The human ATP-binding
cassette (ATP) transporter superfamily. Genome Res. 2001, 11, 1156−
1165.
́
(4) Huang, Y.; Sadee, W. Membrane transporters and channels in
chemoresistance and -sensitivity of tumor cells. Cancer Lett. 2006, 239,
168−182.
(5) Sharom, F. J. ABC multidrug transporters: structure, function and
role in chemoresistance. Pharmacogenomics 2008, 9, 105−127.
(6) Bhatia, A.; Schafer, H.-J.; Hrycyna, C. A. Oligomerization of the
̈
human ABC transporter ABCG2: evaluation of the native protein and
chimeric dimers. Biochemistry 2005, 44, 10893−10904.
(7) Gottesman, M. M.; Ling, V. The molecular basis of multidrug
resistance in cancer: the early years of P-glycoprotein research. FEBS
Lett. 2006, 580, 998−1009.
(8) Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra, M.;
Pastan, I.; Gottesman, M. M. Biochemical, cellular, and pharmaco-
logical aspects of the multidrug transporter. Annu. Rev. Pharmacol.
Toxicol. 1999, 39, 361−368.
(9) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.;
Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G.
Structure of P-glycoprotein reveals a molecular basis for poly-specific
drug binding. Science 2009, 323, 1718−1722.
(27) Detty, M. R.; Gibson, S. L.; Wagner, S. Current clinical and pre-
clinical photosensitizers for use in photodynamic therapy. J. Med.
Chem. 2004, 47, 3897−3915.
(28) Gibson, S. L.; Holt, J. J.; Ye, M.; Donnelly, D. J.; Ohulchanskyy,
T. Y.; You, Y.; Detty, M. R. Structure−activity studies of uptake and
phototoxicity with heavy-chalcogen analogues of tetramethylrosamine
4697
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
in vitro in chemosensitive and multidrug-resistant cells. Bioorg. Med.
Chem. 2005, 13, 6394−6403.
(46) Loo, T. W.; Clarke, D. M. Rapid purification of human P-
glycoprotein mutants expressed transiently in HEK-293 cells by nickel-
chelate chromatography and characterization of their drug-stimulated
ATPase activities. J. Biol. Chem. 1995, 270, 21449−21452.
(47) Chifflet, S.; Torriglia, A.; Chiesa, R.; Tolosa, S. A method for the
determination of inorganic-phosphate in the presence of labile organic
phosphate and high-concentrations of proteinapplication to LENS
ATPases. Anal. Biochem. 1988, 168, 1−4.
(48) Loo, T. W.; Clarke, D. M. Identification of residues in the drug-
binding domain of human P-glycoproteinanalysis of transmembrane
segment 11 by cysteine-scanning mutagenesis and inhibition by
dibromobimane. J. Biol. Chem. 1999, 274, 35388−35392.
(49) Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Overcoming of
vincristine resistance in P388 leukemia in vivo and in vitro through
enhanced cytotoxicity of vincristine and vinblastine by verapamil.
Cancer Res. 1981, 41, 1967−1972.
(29) Holt, J. J.; Gannon, M. K.; Tombline, G.; McCarty, T. A.; Page,
P. M.; Bright, F. V.; Detty, M. R. A cationic chalcogenoxanthylium
photosensitizer effective in vitro in chemosensitive and multidrug-
resistant cells. Bioorg. Med. Chem. 2006, 14, 8635−8643.
(30) McKnight, R. E.; Ye, M.; Ohulchanskyy, T. Y.; Sahabi, S.;
Wetzel, B. R.; Wagner, S. J.; Skripchenko, A.; Detty, M. R. Synthesis of
analogues of a flexible thiopyrylium photosensitizer for purging blood-
borne pathogens and binding mode and affinity studies of their
complexes with DNA. Bioorg. Med. Chem. 2007, 15, 4406−4418.
(31) Ohulchanskyy, T. Y.; Gannon, M. K., II; Ye, M.; Wagner, S. J.;
Skripchenko, A.; Prasad, P. N.; Detty, M. R. Switched-on” flexible
chalcogenopyrylium photosensitizers upon binding to DNA. Purging
of viral pathogens through singlet-oxygen induced DNA cleavage. J.
Phys. Chem. B 2007, 111, 9686−9692.
(32) Detty, M. R.; Merkel, P. B.; Hilf, R.; Gibson, S. L.; Powers, S. K.
Chalcogenapyrylium dyes as potential photochemotherapeutic agents.
II. Mitochondrial targeting and inhibition of cytochrome c oxidase in
isolated mitochondrial suspensions. J. Med. Chem. 1990, 33, 1108−
1116.
(33) Simard, T. P.; Yu, J. H.; Zebrowski-Young, J. M.; Haley, N. F.;
Detty, M. R. Soluble infrared-absorbing croconate dyes from 2,6-di-
tert-butyl-4-methylchalcogenopyrylium salts. J. Org. Chem. 2000, 65,
2236−2238.
(34) Sun, X.; Wong, J. R.; Song, K.; Hu, J.; Garlid, K. D.; Chen, L. B.
AA1, A newly synthesized monovalent lipophilic cation, expresses
potent in vivo antitumor activity. Cancer Res. 1994, 54, 1465−1471.
(35) Brennan., N. K.; Hall, J. P.; Davies, S. R.; Gollnick, S. O.;
Oseroff, A. R.; Gibson, S. L.; Hilf, R.; Detty, M. R. In vitro
photodynamic properties of chalcogenopyrylium analogues of the
thiopyrylium antitumor agent AA1. J. Med. Chem. 2002, 45, 5123−
5135.
(50) Evers, R.; Kool, M.; Smith, A. J.; van Deemter, L.; de Haas, M.;
Borst, P. Inhibitory effect of the reversal agents V-104, GF120918 and
Pluronic L61 on MDR1P-gp-, MRP1- and MRP2-mediated transport.
Br. J. Cancer 2000, 83, 366−374.
(51) Dantzig, A. H.; Shepard, R. L.; Law, K. L.; Tabas, L.; Pratt, S.;
Gillespie, J. S.; Binkley, S. N.; Kuhfeld, M. T.; Starling, J. J.; Wrighton,
S. A. Selectivity of the multidrug resistance modulator, LY335979, for
P-glycoprotein and effect on cytochrome P450 activities. J. Pharm. Exp.
Ther. 1999, 290, 854−890.
(52) Troutman, M. D.; Thakker, D. R. Rhodamine 123 requires
carrier-mediated influx for its activity as a P-glycoprotein substrate in
Caco-2 Cells. Pharm. Res. 2003, 20, 1192−1199.
(53) Sawada, G. A.; Barsuhn, C. L.; Lutzke, B. S.; Houghton, M. E.;
Padbury, G. E.; Ho, N. F. H.; Raub, T. J. Increased lipophilicity and
subsequent cell partitioning decrease passive transcellular diffusion of
novel, highly lipophilic antioxidants. J. Pharm. Exp. Ther. 1999, 288,
1317−1326.
(36) Leonard, K.; Nelen, M.; Raghu, M.; Detty, M. R.
Chalcogenopyranones from disodium chalcogenide additions to 1,4-
pentadiyn-3-ones. The role of enol ethers as intermediates. J.
Heterocycl. Chem. 1999, 36, 707−717.
(37) Gannon, M. K., II; Detty, M. R. Generation of 3- and 5-
lithiothiophene-2-carboxylates via metal−halogen exchange and their
addition reactions to chalcogenoxanthones. J. Org. Chem. 2007, 72,
2647−2650.
(38) Parnham, W. E.; Sayed, Y. A. Synthesis of benzoylbenzoic acids.
J. Org. Chem. 1974, 39, 2051−2053.
(39) Beak, P.; Brown, R. A. The tertiary amide as an effective director
of ortho lithiation. J. Org. Chem. 1982, 47, 34−46.
(40) Leonard, K. A.; Nelen, M. I.; Anderson, L. T.; Gibson, S. L.;
Hilf, R.; Detty, M. R. 2,4,6-Triarylchalcogenopyrylium dyes related in
structure to the antitumor agent AA1 as in vitro sensitizers for the
photodynamic therapy of cancer. J. Med. Chem. 1999, 42, 3942−3952.
(41) Sangster, J. In Octanol−Water Partition Coefficients: Fundamen-
tals and Physical Chemistry; Wiley Series in Solution Chemistry; Fogg,
P. G. T., Ed.; John Wiley and Sons: New York, 1997.
(42) Loe, D. W.; Almquist, K. C.; Deeley, R. G.; Cole, S. P. C.
Multidrug resistance protein (MRP)-mediated transport of leukotriene
C4 and chemotherapeutic agents in membrane vesicles. Demonstration
of glutathione-dependent vincristine transport. J. Biol. Chem. 1996,
271, 9675−9682.
(43) Ito, K.; Olsen, S. L.; Qiu, W.; Deeley, R. G.; Cole, S. P. C.
Mutation of a single conserved tryptophan in multidrug resistance
protein 1 (MRP1/ABCC1) results in loss of drug resistance and
selective loss of organic anion transport. J. Biol. Chem. 2001, 276,
15616−15624.
(54) Juvale, K.; Pape, V. F. S.; Wiese, M. Investigation of chalcones
and benzochalcones as inhibitors of breast cancer resistance protein.
Bioorg. Med. Chem. 2012, 20, 346−355.
(55) Pick, A.; Mueller, H.; Mayer, R.; Haenisch, B.; Pajeva, I. K.;
Weigt, M.; Boenisch, H.; Mueller, C. E.; Wiese, M. Structure−activity
relationships of flavonoids as inhibitors of breast cancer resistance
protein (BCRP). Bioorg. Med. Chem. 2011, 19, 2090−2102.
(56) Haimeur, A.; Conseil, G.; Deeley, R. G.; Cole, S. P. C. The
MRP-related and BCRP/ABCG2 multidrug resistance proteins:
biology, substrate specificity and regulation. Curr. Drug Metab. 2004,
5, 21−53.
(57) Haecker, H.-G.; Leyers, S.; Wiendlocha, J.; Guetschow, M.;
Wiese, M. Aromatic 2-(thio)ureidocarboxylic acids as a new family of
modulators of multidrug resistance-associated protein 1: synthesis,
biological evaluation, and structure−activity relationships. J. Med.
Chem. 2009, 52, 4586−4595.
(58) Maeno, K.; Nakajima, A.; Conseil, G.; Rothnie, A.; Deeley, R.
G.; Cole, S. P. C. Molecular basis for reduced estrone sulfate transport
and altered modulator sensitivity of TM6 and TM17 mutants of
MRP1 (ABCC1). Drug Metab. Dispos. 2009, 37, 1411−1412.
(59) Loo, T. W.; Bartlett, M. C.; Clarke, D. M. Methanethiosulfonate
derivatives of rhodamine and verapamil activate human P-glycoprotein
at different sites. J. Biol. Chem. 2003, 278, 50136−50141.
(60) Loo, T. W.; Clarke, D. M. Location of the rhodamine-binding
site in the human multidrug resistance P-glycoprotein. J. Biol. Chem.
2002, 277, 44332−44338.
(61) Loo, T. W.; Bartlett, M. C.; Clarke, D. M. Substrate-induced
conformational changes in the transmembrane segments of human P-
glycoprotein. Direct evidence for the substrate-induced fit mechanism
for drug binding. J. Biol. Chem. 2003, 278, 13603−13606.
(62) Hipfner, D. R.; Almquist, K. C.; Stride, B. D.; Deeley, R. G.;
Cole, S. P. C. Location of a protease-hypersensitive region in the
multidrug resistance protein (MRP) by mapping of the epitope of
MRP-specific monoclonal antibody QCRL-1. Cancer Res. 1996, 56,
3307−3314.
(44) Shapiro, A. B.; Ling, V. Stoichiometry of rhodamine 123
transport to ATP hydrolysis by P-glycoprotein. Eur. J. Biochem. 1998,
254, 189−193.
(45) Loo, T. W.; Clarke, D. M Functional consequences of proline
mutations in the predicted transmembrane domain of P-glycoprotein.
J. Biol. Chem. 1993, 268, 3143−3149.
4698
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699

Journal of Medicinal Chemistry
Article
(63) Hipfner, D. R.; Gauldie, S. D.; Deeley, R. G.; Cole, S. P. C.
Detection of the Mr 190,000 multidrug resistance protein, MRP, with
monoclonal antibodies. Cancer Res. 1994, 5, 5788−5792.
(64) Loe, D. W.; Almquist, K. C.; Deeley, R. G.; Cole, S. P. C.
Multidrug resistance protein (MRP)-mediated transport of leukotriene
C4 and chemotherapeutic agents in membrane vesicles. Demonstration
of glutathione-dependent vincristine transport. J. Biol. Chem. 1996,
271, 9675−9682.
́
(65) Letourneau, I. J.; Slot, A. J.; Deeley, R. G.; Cole, S. P. C.
Mutational analysis of a highly conserved proline residue in MRP1,
MRP2, and MRP3 reveals a partially conserved function. Drug Metab.
Dispos. 2007, 35, 1372−1379.
(66) Loo, T. W.; Bartlett, M. C.; Clarke, D. M. Rescue of ΔF508 and
other misprocessed CFTR mutants by a novel quinazoline compound.
Mol. Pharmaceutics 2005, 2, 407−413.
(67) Loo, T. W.; Clarke, D. M. P-glycoproteinassociations
between domains and molecular chaperones. J. Biol. Chem. 1995,
270, 21839−21844.
4699
dx.doi.org/10.1021/jm3004398 | J. Med. Chem. 2012, 55, 4683−4699