Rhodamine inhibitors of P-glycoprotein: An amide/thioamide "switch" for ATPase activity

Article abstract of DOI:10.1021/jm900253g

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compou

Full text of DOI:10.1021/jm900253g

3328
J. Med. Chem. 2009, 52, 3328–3341
Rhodamine Inhibitors of P-Glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity
Michael K. Gannon, II,† Jason J. Holt,† Stephanie M. Bennett,† Bryan R. Wetzel,† Tip W. Loo,‡ M. Claire Bartlett,‡
David M. Clarke,‡ Geri A. Sawada,§ J. William Higgins,§ Gregory Tombline,| Thomas J. Raub,* and Michael R. Detty*,†
Department of Chemistry, UniVersity at Buffalo, The State UniVersity of New York, Buffalo, New York 14260-3000, Department of Medicine
and Department of Biochemistry, UniVersity of Toronto, Toronto, Ontario M5S 1A8, Canada, Drug Disposition, Eli Lilly and Company,
Indianapolis, Indiana 46285, Department of Microbiology and Immunology, UniVersity of Rochester Medical Center, 601 Elmwood AVenue,
P.O. Box 607, Rochester, New York 14642
ReceiVed February 28, 2009
We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom
of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl
group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated
MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine
in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells.
Thioamide 31-S gave KM of 0.087 µM in human P-gp. Small changes in structure among this set of compounds
affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for
P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary
thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary
thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive,
and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide
31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ∼2 µM in
MDCKII-MDR1 cells.
Introduction
of rates, yet mechanistic studies suggest that transport of these
structural classes involves a common transition state in the
transporter.13 Related chemical structures within a single class
can have markedly divergent rates of transport. Among the
rhodamines, tetramethylrosamine (TMR, Chart 1) is the best
transport substrate for P-gp both in viable MDR cells and in
reconstituted P-gp.14-16 The transport of TMR is 5- to 10-fold
faster than the reported transport of other rhodamine deriva-
tives.16
The treatment of cancer with chemotherapeutic agents is often
thwarted by the emergence of multidrug resistance (MDRa) in
the transformed cells. P-glycoprotein (P-gp, also known as
MDR1 or ABCB1)1-4 is a member of the ATP-binding cassette
(ABC) superfamily and was the first efflux protein identified
and associated with multidrug resistance in cancer chemo-
therapy. The onset of P-gp expression and drug resistance can
be quite rapid, with mdr gene expression being observed within
an hour of treatment.5 Since the discovery of verapamil (VER)
as an inhibitor of P-gp,6 many approaches to the development
of inhibitors/modulators of P-gp have been examined.7,8 Al-
though a large number of compounds, possessing diverse
chemical structures and biological activities, are able to reverse
MDR, there are currently no approved reversal agents available
in the clinic.9,10
An obvious starting point to circumvent MDR in P-gp
expressing cells is to design chemotherapeutic agents that are
not recognized/transported by the pump.9 This approach led to
two new agents (irinotecan and imatinib) that were thought to
be nonsubstrates for P-gp. However, both are now recognized
to be P-gp substrates.17,18 In a corollary to this approach, one
might ask what structural features in a given class of compounds
are responsible for recognition by the pump and what structural
features are required for transportseither fast or slow? If critical
structural features could be identified, could one then design
either more effective drugs in known classes or, perhaps, identify
additional classes of inhibitors/modulators for the pump?
P-gp is able to transport a diverse array of anticancer drugs
including anthracyclines, vinca alkaloids, taxanes, epipodophyl-
lotoxins, and agents such as mitomycin C, dactinomycim, and
trimetrexate.9-12 This varied set of chemical structures as well
as other substrates for P-gp are transported with a wide range
Identifying the pharmacophore that enables drug transport is
not a trivial issue, especially because it is recognized that both
competitive and noncompetitive interactions exist within the
drug pocket19-21 and that P-gp also has allosteric sites that
appear to reside outside the common pocket.22,23 Two nonover-
lapping binding sites were first identified on P-gp and were
called the “H” and “R” sites for binding by Hoechst 33342 and
rhodamine 123, respectively.24,25 The H-site is well studied and
includes many of the known inhibitors of P-gp.26,27 The Clarke
laboratory has reported that methanethiosulfonate derivatives
of rhodamine and VER cross-link human P-gp at different
sites.28,29 In contrast, Pajeva and Wiese in their pharmacophore
model for rhodamines27 suggest that VER and rhodamines have
structural features that can adopt comparable spatial orientations.
* To whom correspondence should be addressed. For T.J.R.: phone, (317)
651-2330; fax, (317) 433-9287; E-mail, raubtj@lilly.com. For M.R.D.:
phone, (716) 645-6800, ext. 2200; fax, (716) 645-6963; E-mail, mdetty@
buffalo.edu.
†
Department of Chemistry, University at Buffalo, The State University
of New York, Buffalo.
‡
Department of Medicine and Department of Biochemistry, University
of Toronto.
§
Drug Disposition, Eli Lilly and Company.
Department of Microbiology and Immunology, University of Rochester
|
Medical Center.
a Abbreviations list: ABC, ATP-binding cassette; BSA, bovine serum
albumin; CDI, carbonyl diimidazole; DABCO, 1,4-diaza[2.2.2]bicyclooctane;
DMSO, dimethylsulfoxide; DPBSH, Dulbecco’s Hepes-containing phosphate-
buffered saline; MDR, multidrug resistance; NBD, nucleotide binding
domain; P-gp, P-glycoprotein; TMEDA, tetramethylethylenediamine.
10.1021/jm900253g CCC: $40.75
2009 American Chemical Society
Published on Web 04/29/2009

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3329
Chart 1. Structures of Tetramethylrosamine Analogues
Seelig discusses the importance of tight-binding between a
substrate and its transporter and the importance of both
π-electron interactions with a cation and hydrogen-bonding
interactions between a substrate and its transporter.30-33 How-
ever, there is no clear algorithm for predicting either substrates
or inhibitors.27,34
Chart 2. Chalcogenoxanthone Precursors to
Tetramethylrosamine and Rhodamine Analogues
Rhodamine dyes have been used to assay P-gp-mediated
transport. Efflux of rhodamine 123 from cells was used to define
P-gp transport substrates/antagonists in a cross-correlation of
drug resistance patterns in the NCI 60 set of cells with the NCI
Drug Screen Database of compounds.35,36 It seemed plausible
to design rhodamine-related structures that would behave as
modulators/inhibitors of P-gp because small changes in rhodamine
structure had large impacts on rates of transport by P-gp. The
Tombline/Detty lab has recently described the synthesis of a
small library of rhodamine-related structures and the impact of
these molecules on ATPase activity in mouse cys-less P-gp
(MDR3 CL).37,38 The initial library of rhodamines appeared to
validate the presence of the “R” binding site in P-gp first
described by Shapiro and Ling24,25 and also suggested that the
pocket may be pliable because roughly a 1000-fold range of
ATPase activities was observed. On the basis of structures in
this initial library, we characterized a smaller set of TMR
analogues that initially displayed a relatively negligible stimula-
tion of ATPase, suggesting that they were not good transport
substrates.39 However, their ability to promote ATP occlusion,
inhibit VER-dependent ATPase activity competitively, and be
transported in cells revealed a high affinity for P-gp at a
legitimate transport-competent site albeit at a slower rate.
Building on these initial data, we examined a series of 46
rhodamine derivatives to identify structural features that promote
a high affinity for P-gp with minimal stimulation of ATPase
activity with the expectation that these derivatives would be
inhibitors of P-gp-mediated efflux. The rhodamine derivatives
were evaluated for their ability to stimulate ATPase activity
not only in MDR3 CL but also in wild-type human P-gp, their
ability to be transported in multidrug-resistant cells, and their
ability to inhibit/modulate P-gp in multidrug-resistant cells. This
library of rhodamine derivatives contains structural diversity at
the heteroatom atom of the xanthylium core (S, Se, Te) and the
3,6-diamino substituents (-NH2, -NMe2, julolidyl) in addition
to systematic variation of the 9-substituent (aryl, alkyl, het-
eroaryl) to introduce hydrogen-bond donors and acceptors with
a variety of spatial orientations. Structural features were
identified that gave high affinity for P-gp with limited stimula-
tion of ATPase activity in isolated protein and inhibited/
modulated P-gp in multidrug-resistant cells to increase uptake
of calcein AM (CAM) and vinblastine (VIN).
Results and Discussion
Synthesis of Rhodamine Analogues. The rhodamine ana-
logues investigated in this study fall into two categories. The
first category shown in Chart 1 includes analogues of TMR,
which lacks the carboxylic acid oxidation state that characterizes
most rhodamine derivatives. The derivatives 1-E with S, Se,
and Te replacing O in the xanthylium core help delineate the
role of the chalcogen atom, while derivatives 1-E, 2-E, 3-S,
and 4-S were prepared to vary the character of the amino group.
Derivatives 5-E through 12-E examine the role of hydrogen-
bond acceptors on 9-aryl substituents, while derivative 13-S has
a hydrogen-bond acceptor on a more flexible 9-alkyl substituent.
Chalcogenorhodamines 1-E, 2-E, and 5-E through 13-S38-40
were synthesized via the addition of the appropriate Grignard
reagent (prepared from the corresponding bromoalkane or
arylbromide) to chalcogenoxanthones 14-E41 or 15-E42 (Chart
2). The initial addition products were dehydrated in aqueous
HPF6 or HBr to give the indicated salts. The chloride salt of
7-S was obtained using an ion-exchange resin to exchange
-
chloride for the PF6
.
The TMR analogues 3-S and 4-S were synthesized as shown
in Scheme 1. 3-Aminothioxanthone 16-S43 was converted to

3330 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
Scheme 1. Synthesis of Tetramethylrosamine Analogues 3-S and 4-S
Chart 3. Structures of Rhodamine Analogues with Carboxylic Acid Oxidation States in the 9-Substituents
3-dimethylaminothioxanthone 17-S with excess iodomethane,
which resulted in the formation of some quaternary salt. Heat-
ing the reaction mixture with 1,4-diaza[2.2.2]bicyclooctane
(DABCO) demethylated any quaternary product that was formed
and thioxanthone 17-S was isolated in 72% yield overall. The
nitro group of 17-S was reduced to the primary amino group
with SnCl2 in ethanol to give thioxanthone 18-S in 81% yield.
The primary amino group was protected with trityl chloride and
triethylamine to give thioxanthone 19-S in 75% yield. The
addition of excess phenylmagnesium bromide to 19-S followed
by treatment with aqueous HPF6 gave thioxanthylium dye 3-S
in 68% isolated yield. The reduction of 16-S with SnCl2 in
ethanol gave diaminothioxanthone 20-S43 in 98% isolated yield.
Both amino groups were protected with trityl chloride and
triethylamine to give thioxanthone 21-S in 59% isolated yield.
The addition of excess phenylmagnesium bromide to 21-S
followed by treatment with aqueous HPF6 gave thioxanthylium
dye 4-S in 46% isolated yield.
mide) are found in this series. Carboxylic acid groups in different
spatial orientations on a 9-thienyl substituent are found in 22-E
and 23-E. The carboxylic acid group of 22-S is replaced with
an ethyl ester and a primary carboxamide group in 24-S and
25-S, respectively, and with tertiary N,N-diethylamide substit-
uents in 26-E. Compound 27-S is the thioxanthylium analogue
of rhodamine II with a 9-2-carboxyphenyl substituent,40 while
28-S and 29-S are the methyl ester and N,N-diethylamide
analogues, respectively. Compounds 30-E, 31-E, and 32-E
incorporate tertiary thioamide functionality.
The addition of lithium 3-lithiothiophene-2-carboxylate (33)44
to xanthones 14-E followed by treatment with aqueous HPF6
gave rhodamines 22-S and 22-Se (Scheme 2). The addition of
lithium 5-lithiothiophene-2-carboxylate (34) followed by treat-
ment with aqueous HPF6 gave rhodamines 23-E.44 The addition
of lithium 2-lithiobenzoate (35) to thioxanthone 15-S followed
by treatment with aqueous HPF6 gave thiorhodamine 27-S in
75% isolated yield (Scheme 2).40 Carboxylic acid derivative
23-S was converted to ethyl ester 24-S in 85% isolated yield
with carbonyl diimidazole (CDI) and ethanol and to carboxa-
mide derivative 25-S in 83% isolated yield with CDI and
ammonia.45 Similarly, carboxylic acid derivative 27-S was
converted to methyl ester 28-S with CDI and methanol.
Compounds in the second category are shown in Chart 3 and
introduce the carboxylic acid oxidation state into the 9-aryl or
heteroaryl substituent. Groups that can be either a hydrogen-
bond donor or acceptor on the 9-substituent (carboxylic acid or
primary carboxamide) or groups that can only function as a
hydrogen-bond acceptor (ester, tertiary amide, or tertiary thioa-

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3331
Scheme 2. Synthesis of 9-Thienyl Carboxylic Acid Derivatives 22-E, 23-E, 24-S, 25-S, 27-S, and 28-S
Scheme 3. Synthesis of Tertiary Amide Containing Derivatives 26-E and 29-S
Scheme 4. Synthesis of Thioamide Derivatives 30-E-32-E
The addition of N,N-diethyl 5-lithiothiophene-2-carboxamide
(36) to chalcogenoxanthones 14-E gave chalcogenorhodamines
26-S and 26-Se (Scheme 3).38 Similarly, the addition of N,N-
diethyl 2-lithiobenzamide (37) to 14-S gave chalcogenor-
hodamine 29-S in 43% isolated yield (Scheme 3).
Thioamide derivatives 30-E-32-E were prepared as shown
in Scheme 4.Thiophene-2-carboxaldehyde was oxidized under
Willgerodt-Kindler conditions with elemental sulfur and pip-
eridine to give thioamide 3846 in 81% isolated yield and with
elemental sulfur and morpholine to give thioamide 3947 in 99%
isolated yield. Both thioamides were deprotonated with lithium
diisopropylamide to give 5-lithiothiophenes 40 and 41. The
addition of a solution of 40 to solutions of chalcogenoxanthones
14-E gave 30-S, 30-Se, and 30-Te in 75, 79, and 94% isolated
yields, respectively, following a workup with aqueous HPF6.
Similarly, the addition of a solution of 40 to chalcogenoxan-
thones 15-E followed by an acidic workup gave 31-S, 31-Se,
and 31-Te in 62, 31, and 54% isolated yields, respectively.
Chalcogenorhodamines 32-S and 32-Se were prepared in 57 and
70% isolated yields, respectively, following the addition of a

3332 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
Table 1. Stimulation of Human P-gp-His10 ATPase Activity by the
Rosamine and Rhodamine Analogues of Charts 1 and 3
several compounds, which were weakly stimulating for P-gp
ATPase activity, we also determined an IC50, the concentration
of compound required for 50% inhibition of VER-stimulated
(400 µM) ATPase activity.
human P-gp-His10
a
compd
Vmax
,
fold
KM,b µM
IC50,c µM
Rosamine Analogues of Chart 1. Compounds 2-Se, 6-Se,
and 8-Se with the julolidyl fragments as one amino group gave
very little stimulation of P-gp (Vmax <1.5-fold stimulation), which
precluded accurate determination of KM. Values of IC50 for 50%
inhibition of VER-stimulated (400 µM) ATPase activity for
2-Se, 6-Se, and 8-Se were 2.3, 6.8, and 23.6 µM (Table 1),
respectively, indicating that these compounds could inhibit VER-
induced stimulation even if they were not stimulating ATPase
activity. For the remaining compounds investigated, Vmax ranged
from 2.2-fold stimulation for 2-S to 9-fold stimulation for 5-S,
which is roughly 50% of the 17.9-fold stimulation observed for
VER. Values of KM with human P-gp ranged from 1.3 µM for
2-S to 32 µM for 4-S (Table 1).
In general for the rosamine analogues of Chart 1, the identity
of the heteroatom (S, Se, Te) in the chalcogenoxanthylium core
had little impact on either Vmax or KM. Replacing one dimethy-
lamino group with an amino group had no impact on either
Vmax or KM (1-S vs 3-S). Replacing both dimethylamino
substituents with amino substituents in 4-S had little impact on
Vmax but gave a 4-fold higher value of KM (Supporting
Information Table S1). A hydrogen-bond acceptor in the
3-position of the 9-aryl substituent gave lower values of KM
(7-E and 10-E) relative to compounds with a hydrogen-bond
acceptor in the 4-position (5-E). The flexible 9-(3-phenoxy)pro-
pyl substituent of 13-S also gave a KM of 3 µM. The
incorporation of two additional rings in the julolidyl fragment
of 2-E, 6-Se, and 8-Se gave reduced values of Vmax. For
compounds 2-E, values of KM and IC50 were lower (1.3-4.4
µM) than values of KM observed for compounds 1-E (8.3-9.1
µM), again suggesting higher affinity for P-gp from the two
additional rings.
Rhodamine Analogues of Chart 3. Values of Vmax, KM, and
IC50 (Table 1) were determined for selected rhodamine ana-
logues in Chart 3 with human P-gp-His10 as described above.
In this set of compounds, the carboxylic acid groups of 22-E,
23-E, and 27-S and the primary carboxamide of 25-S can act
as either a hydrogen-bond donor or acceptor while the esters
24-S and 28-S and tertiary amides 26-E and 29-S can only act
as hydrogen-bond acceptors. Dye 26-Se with a tertiary amide
was more stimulating (Vmax of 10.5-fold stimulation) with respect
to ATPase activity and had higher affinity (KM of 0.68 µM)
than carboxylic acid-containing derivatives 22-E and 23-E (Vmax
e 4.3-fold; KM > 9 µM). Within the carboxylic acid-containing
derivatives 22-E and 23-E, KM decreased as the heteroatom
became larger. Tertiary amide-containing derivatives 26-E and
29-S also showed high affinity and high ATPase stimulation
with mouse MDR3 CL with Vmax >10-fold above basal levels
and with values of KM in the 3-6 µM range (Supporting
Information Table S1).
VER
1-S
1-Se
1-Te
2-S
2-Se
2-Te
3-S
4-S
5-S
5-Se
5-Te
6-Se
7-S
7-Se
7-Te
8-Se
10-S
10-Se
10-Te
11-S
11-Se
12-S
12-Se
13-S
22-S
22-Se
23-S
23-Se
23-Te
26-Se
30-S
30-Se
30-Te
31-S
17.9 ( 2.0
7.7 ( 1.0
7.2 ( 1.1
8.4 ( 1.9
2.2 ( 0.3
<1.5
3.0 ( 0.7
7.5 ( 1.5
6.4 ( 0.6
9.0 ( 0.5
6.8 ( 1.0
4.5 ( 0.3
<1.5
24 ( 2.6
8.5 ( 1.2
9.1 ( 1.1
8.3 ( 0.8
1.3 ( 0.5
NDd
2.3 ( 0.4
4.4 ( 0.8
9.3 ( 1.0
32 ( 3
8.3 ( 1.2
10.1 ( 0.7
9.0 ( 0.8
NDd
3.4 ( 0.6
3.5 ( 0.2
2.0 ( 0.1
NDd
3.9 ( 0.1
3.3 ( 0.6
3.2 ( 1.0
4.9 ( 0.8
8.3 ( 0.5
10.0 ( 1.3
10.4 ( 1.0
3.2 ( 0.4
111 ( 4
56 ( 11
140 ( 11
89 ( 7
9.6 ( 1.1
0.68 ( 0.14
0.36 ( 0.08
0.35 ( 0.02
0.41 ( 0.03
0.087 ( 0.012
6.8 ( 0.2
7.7 ( 0.5
7.6 ( 0.6
5.5 ( 0.3
<1.5
23.6 ( 3.3
5.9 ( 1.3
2.5 ( 0.2
5.7 ( 0.7
8.2 ( 0.4
7.5 ( 0.9
4.8 ( 0.3
3.3 ( 0.6
6.2 ( 0.8
4.3 ( 0.3
2.1 ( 0.3
2.7 ( 0.2
2.8 ( 0.6
1.8 ( 0.1
10.5 ( 1.6
9.0 ( 0.5
5.0 ( 0.6
7.0 ( 0.4
3.1 ( 0.3
7.9 ( 1.1
9.0 ( 0.2
5.3 ( 1.2
0.67 ( 0.14
a Vmax is the ratio of the maximum stimulation in the presence of
compound relative to that in the absence of compound (the basal activity).
b KM is the apparent Michaelis-Menten constant or the concentration of
compound required for half maximal stimulation of ATPase activity. c IC50
is the concentration of compound required for 50% inhibition of verapamil-
stimulated (400 µM in human P-gp-His10) ATPase activity. d ND, not
determined because of low stimulation of ATPase activity.
solution of 41 to chalcogenoxanthones 15-E with an acidic
workup. Unlike the tertiary amide group,48 which is highly
directing, the thioamide functionality does not direct lithiation
in thiophenes. Only the more acidic R-proton was removed and
none of the corresponding 2,3-disubstituted thiophenes were
detected in the product mixtures.
P-gp ATPase Activity of TMR Analogues of Chart 1. We
examined the ATPase activity of the TMR analogues of Chart
1 using MDR3 CL P-gp,49 which is 87% identical to human
MDR1 P-gp in sequence, and using human P-gp-His10 activated
with sheep brain phosphatidylethanolamine.29,50,51 Many of the
thio- and selenoxanthylium analogues of Chart 1 have shown
activity as photosensitizers.52,53 Consequently, all interactions
of these derivatives with P-gp were conducted in the dark.
Results from the mouse MDR3 Cl P-gp are found in the
Supporting Information (Table S1).
ATPase Activity with Human P-gp. We determined the
apparent Michaelis-Menten constant (KM) for the stimulating
rosamine/rhodamine compound of Charts 1 and 3 and the drug-
induced stimulation of maximal ATPase activity (Vmax) using
human P-gp-His10 (Table 1). VER was included as a control
compound (generally considered relatively robust and one of
the most stimulating drugs known for P-gp).54,55 ATPase activity
was determined from the release of inorganic phosphate.55 For
Thioamide-Containing Rhodamine Derivatives of Chart
3. The tertiary amide-containing derivatives 26-E and 29-S were
highly stimulating toward ATPase activity with high affinity
for P-gp. Are both attributes due to the strength of the amide
carbonyl as a hydrogen-bond acceptor or due to the increased
hydrophobicity from the two alkyl substituents on the amide
nitrogen? To probe this question, the affinity of tertiary
thioamides 30-E and 31-S (Chart 3) for P-gp was determined
as well as the impact of the tertiary thioamides on ATPase
activity. Values of Vmax, KM, and IC50 are compiled in Table 1.
All four compounds displayed submicromolar values of KM

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3333
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.57 With the exception of carboxylic
acid-containing derivative 23-Se, bovine serum albumin (BSA)
addition to the buffer was required because a marked fraction
of mass added to the donor equilibrated with the cell monolayer
for some of the compounds and this resulted in gross underes-
timation of the permeability coefficient.58 The assay was
repeated in the presence of 2.5 µM (R)-4-[(1a,6,10b)-1,1-
dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclo-
hepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol
(LSN335984, 42), which is structurally related to the P-gp-
specific inhibitor (R)-4-[(1a,6,10b)-1,1-difluoro-1,1a,6,10b-tet-
rahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quino-
linyloxy)methyl]-1-piperazineethanol (LY335979 or zosu-
quidar),59 to measure transport when P-gp was fully inhibited.
Compound 42 completely inhibits P-gp with an IC50 of 0.4 µM.
The ratio PBA/AB reported in Table 2 is normalized by dividing
PBA/AB in the absence of inhibitor by the secretory/basal ratio
observed in the fully inhibited system. Large efflux ratios are
assumed to be due to efficient P-gp-mediated efflux of the
compound. Values of passive transport (Ppassive) in the presence
Figure 1. Stimulation of human P-gp ATPase activity by compounds
30-S and 31-S. Histidine-tagged P-glycoprotein was expressed in BHK
cells, isolated by nickel-chelate chromatography and mixed with lipid.
P-glycoprotein ATPase activity was then measured in the presence of
various concentrations of rhodamine derivatives 30-S or 31-S. Error
bars represent one standard deviation from the mean.
of inhibitor, transport in the absorptive (PAB) and secretory (PBA
)
mode in the absence of inhibitor, the normalized ratio [PBA/AB
(no inhibitor)/PBA/AB (with inhibitor)], and the % cell-associated
dye in the AB direction in the absence or presence of inhibitor
are compiled in Table 2. Of the 14 compounds examined in
the MDCKII-MDR1 cell monolayer system, all were shown
to be P-gp substrates, as indicated by net efflux defined by
normalized PBA/AB ratios of >2, with TMR having the highest
ratio (217) and carboxylic acid-containing derivative 23-Se, the
lowest (2.6).
Rates of transport in the absorptive direction were comparable
for compounds with no hydrogen-bond acceptor in the 9-aryl
substituent (TMR, 1-S, 2-S, 2-Se) and fell in the PAB range of
0.8-16.0 nm s-1. Compounds with weak hydrogen-bond
acceptors (thioamide-containing derivatives 30-S, 30-Se, 30-
Te, and 31-S) had even slower transport in AB direction with
values of PAB in the range of 0.8-1.5 nm s-1. The addition of
a stronger hydrogen-bond acceptor on the 9-aryl or heteroaryl
group gave an order of magnitude or greater increase in
absorptive transport (PAB of 116-169 nm s-1 for methoxy-
containing derivatives 9-S, 10-S, 11-S, and 11-Se and PAB of
101 nm s-1 for tertiary amide-containing derivative 26-Se).
Substituents that function as both a hydrogen-bond donor and
acceptor gave intermediate values of absorptive transport (PAB
of 45 nm s-1 for 23-Se with a carboxylic acid group and PAB
of 20 nm s-1 for 25-S with a primary amide group). Rates of
transport in the secretory direction were maximal for TMR and
Figure 2. Inhibition of human P-gp VER-stimulated ATPase activity.
Histidine-tagged P-glycoprotein was expressed in BHK cells, isolated
by nickel-chelate chromatography and mixed with lipid. P-glycoprotein
ATPase activity was then measured in the presence of 0.4 mM VER
with various concentrations of rhodamine derivatives 30-S, 30-Se, 30-
Te, or 31-S. Data represent the average for triplicate measurements,
and error bars represent the standard deviation.
toward human P-gp with 31-S having the highest affinity for
human P-gp (KM of 0.087 µM). The substitution of the julolidyl
fragment for a dimethylamino group gave reduced stimulation
of ATPase activity (Figure 1 comparing 30-S and 31-S).
Rhodamines 30-E displayed values of Vmax between 5- and
9-fold stimulation for derivatives 30-E and 3.1-fold stimulation
for 31-S, as one might expect from the trends observed in the
other julolidyl-containing compounds of this study. This com-
parison also held with respect to values of IC50 for VER-induced
stimulation of ATPase activity in human P-gp as shown in
Figure 2. Compounds 30-E gave values of IC50 of 5.3-9.0 µM
toward human P-gp, while 31-S displayed an IC50 of 0.67 µM
(Table 1).
Transport Across Monolayers of MDCKII-MDR1
Cells. The transport of selected rosamine and rhodamine dyes
was examined in monolayers of MDCKII-MDR1 transfected
cells, which overexpress P-gp (Table 2).56 MDCKII-MDR1
monolayers display apical and basolateral polarized membranes
and are considered to be a near-physiological model for studying
P-gp drug transport. In the monolayer, P-gp is asymmetrically
distributed being 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
the structurally related 1-S (PBA of 1010 and 900 nm s-1
,
respectively), were minimal for primary amide derivative 25-S
and the thioamide-containing 30-E and 31-S (PBA of 22-80
nm s-1), and were intermediate for the remaining 10 derivatives
(PBA of 200-693 nm s-1). The normalized PBA/AB ratios were
smallest for those derivatives with a hydrogen-bond acceptor
in the 9-substituent (normalized PBA/AB ratios of 2.6-11) and
much larger for derivatives with either a 9-phenyl group
(normalized PBA/AB ratios of 17-217) or a tertiary thioamide
group on the 9-substituent (normalized PBA/AB ratios of 14-29).
Passive transport, Ppassive, in the presence of 42 was markedly
lower for derivatives 30-E and 31-S with tertiary thioamide
groups (1.2-2.8 nm s-1). For compounds 2-S and 2-Se, Ppassive
was 7 and 12.3 nm s-1, respectively (Table 3). For the remaining
compounds, Ppassive fell in the range of 11-56 nm s-1
.

3334 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
Table 2. Transport and Cell Association Studies of Rosamine and Rhodamine Analogues with MDCK-MDR1 Cellsa
c
compd
PAB nm s-1
PBA nm s-1
PBA/AB normalized ratio, PBA/AB (( inh)b Ppassive nm s-1 % cell associatedd ratio (( inh)e
log P
TMR
(+ inh)
1-S
(+ inh)
2-S
(+ inh)
2-Se
(+ inh)
9-S
(+ inh)
10-S
(+ inh)
11-S
(+ inh)
11-Se
(+ inh)
23-Sef
(+ inh)
25-S
(+ inh)
26-Se
(+ inh)
30-S
(+ inh)
30-Se
(+ inh)
30-Te
(+ inh)
31-S
3.0 ( 0.1
14 ( 1
6.0 ( 0.1
30 ( 1
1010 ( 10
21 ( 2
900 ( 120
39 ( 2
360 ( 50
11 ( 3
250 ( 40
12 ( 1
338
1.6
149
1.3
450
3.4
15
0.9
4.6
0.5
4.1
0.5
5.5
0.6
2.6
0.5
4.4
1.7
3.7
0.7
4.2
0.5
54
217
114
130
17
17 ( 5
63 ( 2
14.5 ( 0.9
4.3
5.1
2.1
2.0
1.4
4.3
3.3
1.5
0.9
0.6
2.2
1.8
2.4
1.1
34 ( 7
14 ( 1
1.2
3.2
71 ( 2
0.8 ( 0.1
3.3 ( 0.1
16.0 ( 0.1
13.0 ( 0.8
116 ( 3.4
39.0 ( 0.2
169 ( 7
59 ( 4
118 ( 1
48 ( 2.6
131 ( 7
31 ( 2
45 ( 19
16 ( 4
20 ( 1
12 ( 0
101 ( 1.6
63 ( 0.7
1.5 ( 0.1
1.8 ( 0.5
1.1 ( 0.2
1.3 ( 0.1
0.9 ( 0.1
1.2 ( 0.1
0.8 ( 0.1
0.8 ( 0.1
7 ( 6
30 ( 2
64 ( 3
12.3 ( 0.8
64 ( 4
32 ( 1
2.4
530 ( 33
18.0 ( 0.3
693 ( 155
30.0 ( 0.4
653 ( 39
26 ( 0.1
340 ( 100
15 ( 0.3
200 ( 24
28 ( 0.1
73 ( 8
10
29 ( 14
66 ( 18
45 ( 21
56 ( 18
37 ( 3
53 ( 3.1
23 ( 11
57 ( 9.7
22 ( 6
4.8 ( 0.8
11 ( 2.3
6.7 ( 2.9
46 ( 24
24 ( 0.73
2.8 ( 1
29 ( 1.5
2.1 ( 0.8
21 ( 3.3
1.6 ( 0.4
23 ( 1.5
1.2 ( 0.4
30 ( 1.5
47 ( 26
13 ( 3
-0.009
0.14
0.28
0.18
8.2
10
16 ( 12
37 ( 24
5.6 ( 0.2
12 ( 9
5.3
2.6
5.1
9.2
25
9 ( 0.1
424 ( 9
29 ( 0.03
80 ( 19
3.9 ( 0.1
41 ( 3
11 ( 5.5
16 ( 1
-0.39
1.7
2.1
37
2.3
24
1.7
61
2.1
16
8.6 ( 0.3
12 ( 3
2.9 ( 0.1
22 ( 8
2.0 ( 0.1
50 ( 1
0.1.6 ( 0.1
14
29
23 ( 3
1.3
2.7
(+ inh)
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)], the % cell associated rhodamine analogue in the absence or presence of inhibitor, the ratio of cell associated rhodamine in the
presence or absence of inhibitor, and values of log P (the n-octanol/water partition coefficient). Details for methods are provided in Experimental Section.
Error limits represent ( standard deviation. b The normalized ratio represents the PBA/AB ratio in the absence of inhibitor divided by the PBA/AB ratio in the
c
presence of inhibitor. Ppassive represents the mean of PAB and PBA in the fully inhibited system. d % Cell associated is the fraction of mass extracted from
the cell monolayer by methanol wash after 1 h flux in the AB direction. e For % cell associated dye. f No added BSA.
Table 3. IC50 for the Uptake of CAM in Rhodamine-Treated
MDCKII-MDR1 Cells
compd
CAM uptake IC50, µMa
VER
1-S
1-Se
2-S
2-Se
30-S
30-Se
30-Te
31-S
31-Se
31-Te
32-S
32-Se
42
14 ( 3
11 ( 3
11 ( 3
4.7 ( 2.0
4.9 ( 0.6
19 ( 2
9 ( 1
13 ( 1
2.1 ( 1.2
5.3 ( 1.2
14 ( 1
Figure 3. Uptake of CAM into MDCKII-MDR1 cells as a function
of concentration of rhodamine dyes 30-S, 30-Se, 30-Te, and 31-S.
Values of IC50 were determined by a sigmoidal dose-response (variable
slope) analysis and are compiled in Table 3. Data represent the average
and standard error mean for duplicate measurements.
20 ( 1
14 ( 1
0.93 ( 0.08
a Details for methods are provided in Experimental Section. Error limits
represent ( standard deviation.
cell-associated dye in the inhibited (30%) and uninhibited (23%)
systems [ratio (( inhibitor) of 1.3].
The % cell-associated dye for derivatives with a 9-phenyl
substituent (TMR, 1-S, 2-S, 2-Se) or a 9-3-methoxyaryl sub-
stituent (10-S, 11-S, 11-Se) was high in the inhibited system
(54-71%) but was significantly lower when the pump was
active (7.5-32%), giving ratios between the inhibited and
uninhibited systems from ∼2 for 2-S and 2-Se to 7.2 for 11-S.
Compound 9-S with a 9-4-methoxyphenyl substituent was an
anomaly with high cell association in both the uninhibited (47%)
and inhibited (66%) systems. Amide-containing derivative 25-
S, diethylamide-containing derivative 26-Se, and the thioamide-
containing derivatives 30-E and 31-S showed less cell-associated
dye in both inhibited (6.7-30%) and uninhibited (12-23%)
systems. Compound 31-S had essentially identical values of %
Enhancement of CAM Uptake into MDCKII-MDR1
Cells. Rosamine analogues 1-S, 1-Se, 2-S, and 2-Se and
rhodamine analogues 30-E, 31-E, and 32-E were next evaluated
for their ability to facilitate uptake of CAM into MDCKII-MDR1
cells.56 Values of IC50 for CAM uptake (Table 3) were
determined by measuring relative fluorescence values obtained
after 20-min incubation with CAM at 37 °C. Typical curves
are shown in Figure 3 for CAM uptake in the presence of various
concentrations of 30-E and 31-S. Substitution of the julolidyl
fragment reduced IC50 from 11 µM for 1-S and 1-Se to an IC50
of 4-5 µM for 2-S and 2-Se. Similarly, values of IC50 were
lowered from 19 and 9 µM for 30-S and 30-Se, respectively, to

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3335
by Shapiro and Ling revealed the presence of at least two
binding sites on P-gp described as “H” and “R” sites for Hoechst
33342 and rhodamine 123, respectively.24,25 However, it is not
at all clear that these sites in P-gp are “rigid.” The Clarke
laboratory described an “induced fit” model for binding of small
molecules to P-gp in which the shape of the drug-binding site
changes upon binding of compounds with different shapes.62
The binding pocket also may have many different ways of
binding to a single molecule. QacR, a member of the TetR
family of repressors, binds to many of the same molecules as
QacA, the MDR pump that QacR regulates.63 Structural studies
with QacR suggested that five glutamate residues were critical
for charge neutralization in the binding of cationic molecules
to the protein.64 However, mutations of QacR showed that
binding of cations to the protein was still observed when several
of the glutamate residues had been replaced.65 In the case of
one cationic compound, the positive charge was neutralized by
side-chain oxygen atoms and by aromatic residues. Conse-
quently, the drug-binding pocket of P-gp may not have well-
defined binding sites for molecules of diverse structure but may
be a highly flexible domain in which conformational changes
can have pronounced impact on binding. A model of human
P-gp generated by comparison to the crystal structures of two
bacterial transporters featured a large cavity within the trans-
membrane domains into which drug substrates such as rhodamine
B, VER, and VIN could be successfully docked.66 The docked
positions of rhodamine and other drug substrates were found
to interact with residues that alter substrate specificity upon
mutation.
Figure 4. Analyses for inhibition of VIN transport by wild-type, human
Pgp using 31-S in membrane vesicles from PEAK-MDR1 cells. The
line represents the nonlinear least-squares regression fits of the data.
Data represent the average and standard error mean for duplicate
measurements.
2.1 and 5.3 µM for 31-S and 31-Se, respectively, by introduction
of the julolidyl fragment. Replacing a methylene group of the
piperidine-containing thioamides 31-E with the O atom of
morpholino thioamides 32-S and 32-Se gave higher values of
IC50 (20 and 14 µM, respectively).
Inhibition of VIN Efflux by 31-S in MDCKII-MDR1
Cells. Because VIN is an actual chemotherapeutic drug used
clinically, inhibition of VIN transport by the rhodamines is likely
to indicate translational value. [3H]-Vinblastine in an appropriate
dilution series with 31-S and BSA was introduced 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 an
IC50 of 2.4 ( 0.4 µM (Figure S1, Supporting Information),
which is essentially identical to the IC50 observed for CAM
uptake.
The rhodamines of this study were selected to have similar
shapes and similar noncovalent interactions with P-gp in the
xanthylium core in the hope that one might examine the impact
of subtle structural changes in the substrate to a single binding
region of P-gp. The rosamines of Chart 1 and rhodamines of
Chart 3 share certain structural similarities: (1) All derivatives
have two N-atoms as hydrogen-bond acceptors at the 3- and
6-positions. In most of the derivatives, the N-atoms are part of
dimethylamino substituents. Compounds 3-S and 4-S have NH2
substituents, while compounds 2-E, 6-Se, 8-Se, 31-E, and 32-E
incorporate one N-atom as part of a julolidyl fragment with the
other as a dimethylamino substituent. (2) All derivatives have
a chalcogen atom (O, S, Se, or Te) in the xanthylium core, which
can also function as an H-bond acceptor. (3) All derivatives
bear a positive charge, which is delocalized over the xanthyllium
π-framework. Seelig has discussed the importance of π-electron
interactions with a cation and hydrogen-bonding interactions
between a substrate and its transporter for tight binding and
substrate recognition to occur.30-32 The rosamines and rhodamines
of this study are all delocalized aromatic cations and they each
contain two of the so-called Seelig type II hydrogen bond
acceptor units, with two hydrogen bond-accepting atoms 4.6 (
0.6 Å apartsthe chalcogen atom-nitrogen atom arrays, whose
separation is on the order of 5.0-5.15 Å, as determined by X-ray
crystallographic studies.38,40
Inhibition of VIN Transport in a Membrane Vesicle
Model. Compound 31-S was evaluated for its ability to inhibit
VIN transport by human P-gp in an inside-out membrane vesicle
model, prepared from human embryonic kidney (PEAKSTABLE
)
cells overexpressing human Pgp.60,61 The accumulation of [3H]-
VIN into P-gp membrane vesicles in the presence of 31-S (0.01
to 10 µM) was monitored, using 5 µM 42 as a standard for
complete P-gp inhibition. Previously, the inhibition of [3H]-
VIN accumulation in P-gp membrane vesicles by 42 gave an
IC50 value of 6.5 ( 1.1 nM and, thus, complete inhibition of
P-gp was assumed at 5 µM.60 Mixtures of [3H]-VIN and 31-S
were added to the vesicles and, following a reaction time of
2.5 min, the vesicles were removed by filtration and the residual
radiation on the filter was measured by scintillation counting
to give an IC50 of 0.13 ( 0.03 µM as shown in Figure 4. The
IC50 measured in this model is similar to the 0.67 µM value of
IC50 observed with isolated human P-gp-His10 (Table 1).
Most of the significant structural variation within these
compounds comes at the 9-position where a variety of aliphatic,
aromatic, and heteroaromatic substituents have been incorpo-
rated. Among these, compounds 11-E, 12-E, and 22-E through
32-E add a Seelig type I unit to the 9-aryl or heteroaryl
substituent. The Seelig type I units separate the hydrogen bond-
accepting atoms by 2.5 ( 0.5 Å.30-32 This latter group of
compounds varies in the relative spatial orientation of the type
I unit as well as in the strength of the hydrogen-bond acceptor.
Discussion
Interactions of the Rosamines/Rhodamines with P-gp.
Within a library of closely related molecules that all bind to
the same protein, the assumption is often made that binding
occurs at a common site. P-glycoprotein and other ABC
transporters appear to have multiple drug-binding sites that allow
a diverse array of structures to bind to the protein. Early studies

3336 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
We can also examine the affinity of the rhodamine molecules
for P-gp as well as the impact of the small molecule on ATPase
activity (Vmax). For derivatives that stimulate ATPase activity,
affinity can be approximated by KM. In systems that do not
stimulate ATPase activity, affinity can be measured with respect
to IC50 for the inhibition of VER-induced ATPase activity.
Affinity for P-gp measured by binding in the transmembrane
domains and impact upon ATPase activity driven by the
nucleotide binding domains (NBDs) can be separated. The
Clarke laboratory has shown that a truncation mutant of P-gp
lacking both NBDs could still interact with rhodamine-type
compounds.67
can be utilized as a higher throughput and equally sensitive
primary assay and was deemed more efficient and practical for
these initial studies using fluorescence as a method of detection.
While most of the rhodamines of Table 3 were comparable to
VER with respect to IC50 for CAM uptake in the MDCKII-
MDR1 cells, compound 31-S was more potent with an IC50 more
similar to those of quinidine, cyclosporin A, zosuquidar, and
42 (Table 3). Compound 31-S also inhibited the efflux of VIN,
a clinically used chemotherapeutic drug, with a similar IC50 of
2.4 µM in MDCKII-MDR1 cells. In this assay as well as in
the vesicle assay, the more passively permeable VIN gave a
more robust signal relative to digoxin.
Certain structural features appear critical for affinity and
ATPase activity. Comparison of KM and Vmax for compounds
1-E and 2-E show significant increases in affinity (p < 0.05)
upon replacement of one dimethylamino substituent with the
julolidyl fragment and significant decreases in ATPase activity
in human P-gp-His10. Similarly, comparison of KM and Vmax for
compounds 30-E and 31-E show significant increases in affinity
(p < 0.05) upon introduction of the julolidyl fragment and
significant decreases in ATPase activity.
Simple hydrogen-bond acceptors in the 9-substituents of the
rosamine analogues of Chart 1 had little impact on either affinity
(KM) or ATPase stimulation (Vmax) in human P-gp. The
introduction of Seelig type I hydrogen-bond acceptors on the
9-substituents had a much greater impact. The addition of a
carboxylic acid group, which is also a hydrogen-bond donor as
well as a Seelig type I acceptor, gave a decrease in ATPase
stimulation relative to the 9-phenyl substituent and affinity was
either reduced or unchanged. The tertiary amide substituents in
compounds 26-E and 29-S impart high affinity (KM on the order
of 3-6 µM) and high ATPase activity (>10-fold stimulation
above basal) in both human and mouse P-gp (Tables 1 and S1).
Replacing the carbonyl of the amide with the thiocarbonyl group
of the tertiary thioamides 30-E gave slightly increased affinity
and reduced ATPase activity. However, incorporation of the
julolidyl fragment and the thioamide functionality in 31-S gave
the highest affinity observed in this study (KM of 0.087 µM)
with low ATPase activity (Vmax of 3-fold stimulation above basal
in human P-gp-His10) and inhibition of VER-induced stimulation
with an IC50 of 0.67 µM.
The compound TMR has been recognized as one of the best
transport substrates for P-gp14-16 with, in our hands, KM of 77
µM and ATPase stimulation 3.7-fold above basal (Supporting
Information Table S1) and with a rate of efflux, PBA, of 1010
nm s-1 in MDCKII-MDR1 cells (Table 2). One might conclude
from this work that a series of closely related rhodamine and
rosamine structures can be tuned for high affinity and high
ATPase activity (for 26-Se, KM of 0.68 µM and Vmax of 10.5-
fold stimulation in human Pgp-His10) or for high affinity and
high inhibitory activity (for 31-S, KM of 0.087 µM and IC50 of
0.67 µM for inhibition of VER-induced stimulation of ATPase
activity in human P-gp-His10). The highly stimulating 26-Se has
a secretory rate of transport of 424 nm s-1 for PBA, while the
highly inhibitory 31-S has a slower secretory rate of 50 nm
s-1
.
Earlier studies demonstrated that TMR is transported by P-gp
5- to 10-times more rapidly than other rhodamine derivatives.16
TMR was transported the fastest among the 15 compounds
examined in Table 3. For rosamine analogues 1-S, 2-S, 2-Se,
9-S, 10-S, 11-S, and 11- Se PBA was 250-900 nm s-1, which
is on the same order of magnitude as PBA for TMR. The
rhodamine analogues 23-Se, 25-S, 26-Se, 30-E, and 31-S
displayed more varied transport, but values of PBA reflected
affinity and ATPase stimulation. TMR was transported 5-fold
faster than 5-carboxy-2-thienyl derivative 23-Se, which dis-
played low affinity for P-gp and low stimulation of ATPase
activity. TMR was transported 13- to 46-times more rapidly
than thioamides 30-E and 31-S, which displayed high affinity
and low stimulation of ATPase activity. In contrast, the high
affinity, highly stimulating tertiary amide derivative 26-Se was
transported with a rate more comparable to that of TMR (424
nm s-1). Again, the amide/thioamide “switch” changed the
fundamental interaction of the small molecule with P-gp.
Cell Studies. The rosamine and rhodamine dyes of this study
enter MDCKII-MDR1 cells as indicated by their active
transport by P-gp (normalized PBA/AB > 2, Table 2) and by their
facilitation of CAM uptake (Table 3). Thus, all of the com-
pounds of Table 2 bind to transport sites including those that
have Vmax values at or near basal levels. With respect to
inhibition of P-gp in whole cells, the same structural features
that gave higher affinity and reduced ATPase activity in the
isolated protein also gave lower values of IC50 for CAM uptake
in the MDCK-MDR1 cells. The incorporation of the julolidyl
fragment in 2-S and 2-Se lowered IC50 for CAM uptake to 4.7
and 4.9 µM, respectively, relative to 1-S and 1-Se with two
dimethylamino substituents at the 3- and 6-positions (IC50’s of
11 µM for both). Addition of the thioamide functionality in 31-S
lowered IC50 to 2.1 µM. Interestingly, the addition of another
hydrogen bond-accepting atom in the morpholino thioamides
32-E gave increased values of IC50 for CAM uptake (20 µM
for 32-S vs 2.1 µM for 31-S and 14 µM for 32-Se vs 5.3 µM
for 31-Se, Table 3).
One would anticipate that the performance of 31-S as an
inhibitor would be similar in isolated protein or in whole cells.
Compound 31-S effectively interacts with P-gp in a native
membrane environment and facilitates the uptake of CAM and
inhibits the efflux of VIN. However, the values of IC50 of ∼2
µM are significantly higher than values IC50 for inhibition of
VER-induced stimulation of P-gp (0.67 µM) in human P-gp-
His10 activated with sheep brain phosphatidylethanolamine and
IC50 for inhibition of [3H] VIN uptake (0.13 µM) in an inside-
out P-gp membrane vesicle model.
There are several reasons why effective concentrations in
whole cells might be different than those in isolated protein. It
is known that lipid composition and fluidity greatly influence
drug interactions with P-gp,20,31,69 and differences between
experimental conditions or Pgp environment (i.e., membrane
composition) may account for differences in behavior between
ATPase and [3H]-VIN experiments with isolated protein and
CAM and [3H]-VIN experiments in whole cells.
Digoxin has been used as a default substrate for P-gp because
of the clinical implications of recognizing a drug-drug trans-
porter (P-gp) interaction where digoxin with a very narrow
margin of safety is unusually affected.68 However, CAM uptake

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3337
An alternative explanation is that 31-S has limited access to
the pump in the whole cell experiments. This may be because
Ppassive into the favored membrane microenvironment is rate
limiting, or other efflux transporters assist in escorting 31-S from
the cell,70 or because 31-S has high binding affinity, specific or
nonspecific, to the membrane and the unbound fraction at the
pump is very low. Compounds 30-E and 31-S show particularly
slow Ppassive rates (1.2-2.8 nm s-1) that correlate with relatively
slower efflux rates in the uninhibited system (PBA e 80 nm
s-1). This is consistent with passive replenishing of the driving
membrane concentration lagging behind active removal. Com-
plete inhibition of efflux by inhibitor 42 implies that P-gp is
solely responsible; however, involvement of one or more
additional unknown efflux transporters cannot be excluded
unequivocally if 42 also happens to inhibit these putative
transporters. One important comparison is between 31-S with
tertiary thioamide functionality, where Ppassive is 1.2 nm s-1 and
PAB is 50 nm s-1 in the uninhibited system, and 26-Se with
tertiary amide functionality, where Ppassive is 46 nm s-1 and PAB
is 101 nm s-1 in the uninhibited system. The former is inhibitory
toward P-gp while the latter is highly stimulating. The 40-fold
difference in Ppassive suggests that membrane concentrations of
the former will be replenished much more slowly than the latter.
If the available membrane concentrations are indeed low, then
more will be required to be added to the whole cell system to
raise these membrane concentrations effectively to levels that
result in pump inhibition. For the membrane vesicle systems,
this limitation is unlikely to exist because the favored membrane
microenvironment should be more readily accessible due to
inside-out nature of the membrane vesicle.
increase in affinity for the thioamides. The amide/thioamide
“switch” also slows the rate of transport of the rhodamine
derivatives in both absorptive and secretory directions in the
cell. The substitution of a julolidyl fragment for a dimethylamino
substituent in the xanthylium core also gives higher affinity for
P-gp in both isolated protein and in whole cells.
Compound 31-S represents an interesting lead compound for
the development of more potent inhibitors of P-gp. In particular,
understanding the factors that limit the availability of 31-S to
the pump in the native membrane environment is critical for
the development of more potent rhodamine inhibitors of P-gp.
Experimental Section
General Methods. Dyes 1-E, 2-E, and 5-E through 13-E were
prepared from xanthones 14-E41 and 15-E42 as described in refs
38-40. Dyes 22-E and 23-E were prepared according to ref 44.
Compound 27-S was prepared according to ref 40. Tetrahydrofuran
was distilled from sodium benzophenone ketyl prior to use.
Reactions were run under Ar. Concentration in vacuo was per-
formed on a Bu¨chi rotary evaporator. NMR spectra were recorded
on an Inova 500 instrument (500 MHz for 1H NMR, 125 MHz for
13C NMR) 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 Bu¨chi capillary melting point apparatus and are
uncorrected. New compounds have a purity of g95% 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.
3,6-Bis(N,N-dimethylamino)-9-(2-thienyl-5-carboxamido)-
thioxanthylium Hexafluorophosphate (25-S). Dye 27-S40 (0.055
g, 0.10 mmol) and CDI (50 mg, 0.31 mmol) in 4.5 mL of CH2Cl2
were stirred for 4 h. Gaseous NH3, generated by heating an NH4OH
solution under a stream of N2, was bubbled through the reaction
mixture for 1.0 h. Water (3 mL) was added to the reaction mixture,
and the products were extracted with CH2Cl2 (2 × 10 mL). The
combined organic extracts were washed with 20 mL of saturated
NaHCO3 solution (2 ×) and concentrated in vacuo. The crude dye
was recrystallized from CH3CN/Et2O (2×) to give 0.045 g (83%)
The role of the carboxylic acid oxidation state in the
9-position of the rosamine and rhodamine derivatives is
important. For the “stimulating” drugs TMR, 1-S, 9-S, 10-S,
11-S, and 11-Se and the “inhibitory” compounds 2-S and 2-Se,
which lack the carboxylic acid oxidation state, the % cell
associated dye (% cell) in the uninhibited system is 14-47%
and in the inhibited system is 53-71% with ratios between
1.5 and 5.1 for the uninhibited to inhibited systems (Table 2).
The addition of a substituent with a carboxylic acid oxidation
state in compounds 23-S, 25-S, 26-Se, 30-E, and 31-S gives a
lower cell association in both the uninhibited (5.6-23%) and
inhibited (4.8-30%) systems, with ratios between 0.6 and 2.4
for the uninhibited to inhibited systems. These differences are
not a simple function of lipophilicity because values of log P,
the n-octanol/water partition coefficient, fall in the range of
-0.09-2.4 for TMR, 1-S, 9-S, 10-S, 11-S, and 11-Se and
-0.39-2.7 for compounds 23-S, 25-S, 26-Se, 30-E, and 31-S
(Table 2). All compounds should have access to aqueous and
lipophilic environments. For 31-S in particular, the % cell is
essentially unchanged in the presence or absence of inhibitor
with a ratio of 1.3 for the uninhibited (23% cell associated) to
inhibited (30% cell associated) systems. Again, these results
suggest that the thioamides, and 31-S in particular, have limited
access to the pump.
1
of 25-S as a purple solid, mp 239-242 °C. H NMR (CD3CN) δ
7.75 (d, 1H, J ) 3.6 Hz), 7.58 (d, 2H, J ) 9.6 Hz), 7.23 (d, 1H,
J ) 3.6 Hz), 7.19 (d, 2H, J ) 2.8 Hz), 7.03 (d × d, 2H, J ) 2.8,
9.6 Hz), 3.23 (s, 12H). 13C NMR (CD2Cl2) δ 163.5, 154.5, 152.0,
144.6, 143.3, 140.7, 136.4, 132.4, 129.5, 120.2, 116.7, 106.6, 41.0.
λmax (H2O) 595 nm (ε 6.1 × 104 M-1 cm-1). HRMS-ES m/z
408.1202 (calcd for C22H22ON3S2: 408.1199).
3,6-Bis(N,N-dimethylamino)-9-(2-methylcarboxyphenyl)thiox-
anthylium Hexafluorophosphate (28-S). Dye 27-S40 (0.055 g, 0.10
mmol) and CDI (50 mg, 0.31 mmol) in 4.5 mL of CH2Cl2 were
stirred for 4 h. Methanol (30 µL, 24 mg, 0.75 mmol) was added,
and the resulting mixture was stirred for 24 h. Water (3 mL) was
added, and products were extracted with CH2Cl2 (2 × 10 mL). The
organic extracts were washed with saturated NaHCO3 (2 × 20 mL),
dried, and concentrated in vacuo. The residue was recrystallized
from CH3CN/Et2O to give 0.041 g (80%) of 28-S, mp 263-264
1
°C. H NMR (CD2Cl2) δ 8.32 (d × d, 1H, J ) 1.6, 7.8 Hz), 7.82
(t × d, 1H, 1.6, 7.8 Hz), 7.76 (t × d, 1H, 1.6, 7.8 Hz), 7.31 (d ×
d, 1H, J ) 1.6, 7.8 Hz), 7.22 (d, 2H, J ) 9.6 Hz), 7.10 (d, 2H,
J ) 2.4 Hz), 6.88 (d × d, 2H, J ) 2.4, 9.6 Hz), 3.59 (s, 3H), 3.26
(s, 12H). λmax (CH3OH) 571 nm (ε 74000 M-1 cm-1). HRMS (ESI)
m/z 417.1635 (calcd for C25H25N2O2S2+: 417.1637).
Conclusions
We have demonstrated that simple structural changes in a
series of rosamine/rhodamine structures can give molecules with
high affinity for P-gp and that are highly stimulating for ATPase
activity or molecules with high affinity and high inhibitory
activity toward P-gp. Tertiary amide and thioamide groups on
the 9-substituent, in particular, dictate ATPase stimulation for
the former and inhibition for the latter. The amide/thioamide
“switch” markedly decreases ATPase stimulation with an
3,6-Bis(N,N-dimethylamino)-9-(2-N,N-diethylcarboxamidophe-
nyl)thioxanthylium Hexafluorophosphate (29-S). tert-Butyllith-
ium (2.2 mL of a 1.5 M solution, 3.3 mmol) was added dropwise
to N,N-diethylbenzamide (0.53 g, 3.0 mmol) and tetramethyleth-
ylenediamine (TMEDA, 0.5 mL, 3.0 mmol) in THF (5 mL) and
cooled to -78 °C to generate a solution of N,N-diethyl 2-lithioben-

3338 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
zamide (37). Xanthone 14-S (0.10 g, 0.34 mmol) in 1 mL of THF
was added immediately. The reaction mixture was stirred at ambient
temperature for 14 h and then was cooled to 0 °C. Acetic acid (3
mL) was added, followed by 10% aqueous HPF6 (10 mL) until a
deep-purple color was observed. Ice water (50 mL) was added, the
purple precipitate was collected by filtration, and the resulting solid
was recrystallized from CH3CN/ether to yield 85 mg (42%) of 29-S
1H, J ) 2.5 Hz), 7.07 (d, 1H, J ) 3.5 Hz), 6.98 (d × d, 1H, J )
2.5, 9.5 Hz), 4.28 (br s, 2H), 4.04 (br s, 2H), 3.56 (t, 4H, J ) 5.5
Hz), 3.25 (s, 6H), 2.86 (t, 2H, J ) 6.0 Hz), 2.78 (t, 2H, J ) 6.0
Hz), 2.17 (quintet, 2H, J ) 6.0 Hz), 2.01 (quintet, 2H, J ) 6.0
Hz), 1.82 (s, 6H). 13C NMR (DMSO-d6) δ 186.1, 152.0, 148.3,
147.1, 147.0, 140.0, 137.9, 137.6, 133.6, 130.9, 129.8, 125.4, 125.0,
118.2, 117.1, 114.9, 113.4, 105.6, 50.5, 49.4, 26.7, 23.0, 19.2, 18.7.
λmax (H2O) 607 nm (ε ) 3.4 × 104 M-1 cm-1). HRMS (ESI) m/z
544.1905 (calcd for C31H34N3S3+: 544.1909).
1
as a green crystalline solid, mp >300 °C. H NMR (CD3CN) δ
7.74 (m, 2H), 7.61 (m, 1H), 7.54 (m, 1H), 7.47 (m, 1H), 7.38 (d,
2H, J ) 9.9 Hz), 7.24 (d, 2H, J ) 2.8), 7.02 (d × d, 2H, J ) 9.8,
2.8 Hz), 3.27 (s, 12H), 3.13 (q, 2H, J ) 7.0 Hz), 3.01(q, 2H, J )
7.0 Hz) 1.08 (t, 3H, J ) 6.7 Hz), 0.507 (t, 3H, J ) 6.7 Hz). 13C
NMR (CD2Cl2) δ 167.9, 158.9, 154.0, 144.4, 137.2, 133.8, 130.4,
130.0, 129.4, 129.4, 126.7, 119.7, 115.2, 105.7, 43.6, 40.9, 38.4,
14.1, 11.8, 2.0. λmax (EtOH) 578 nm (ε ) 6.2 × 104 M-1 cm-1).
HRMS (ESI) m/z 458.2261 (calcd for C28H32N3S+: 458.2266).
3,6-Bis(dimethylamino)-9-(N-piperidyl-2-thienyl-5-thiocar-
boxamido)thioxanthylium Hexafluorophosphate (30-S). n-Bu-
tyllithium (2.5 M in hexane, 0.30 mL, 0.78 mmol) was added to a
stirred solution of diisopropylamine (0.11 mL, 0.78 mmol) in 2.0
mL of anhydrous THF at 0 °C. After 15 min, the reaction mixture
was cooled to -78 °C and 38 (0.15 g, 0.71 mmol) in 1 mL of
anhydrous THF was added. The resulting mixture was stirred for
0.5 h at -78 °C. An aliquot (1.0 mL, 0.22 mmol) of the 1-(5-
lithio-2-thienyl)piperidin-1-ylmethanethione (40) solution was added
to a solution of xanthone 14-S (50 mg, 0.17 mmol) in 1 mL of dry
THF, and the resulting mixture was warmed at 35 °C for 0.5 h and
then cooled to ambient temperature. Acetic acid (0.25 mL) was
added, and the resulting mixture was then poured into a 10% w/w
solution of aqueous HPF6. After 1 h of stirring, the precipitate was
collected by filtration, washed with water and ether, and recrystal-
lized from CH3CN/Et2O to give 0.080 g (75%) of 30-S as a green
12-(Dimethylamino)-2,3,6,7-tetrahydro-9-(N-piperidyl-2-thie-
nyl-5-thiocarboxamido)-1H,5H-selenoxantheno[2,3,4-ij]quinolizin-
14-ium Hexafluorophosphate (31-Se). From xanthone 15-Se (50.0
mg, 0.126 mmol) and 1.9 M 40 (1.0 mL, 0.19 mmol), 31-Se was
obtained as described above for the preparation of 30-S. Yield: 70
1
mg (31%), mp 175-176 °C. H NMR (CD2Cl2) δ 7.64 (d, 1H, J
) 9.5 Hz), 7.40 (s, 1H), 7.24 (s, 1H), 7.20 (d, 1H, J ) 2.5 Hz),
7.03 (d, 1H, J ) 3.0 Hz), 6.90 (d, 1H, J ) 9.5 Hz), 4.27 (br s, 2H),
4.07 (br s, 2H), 3.54 (m, 4H), 3.23 (s, 6H), 2.75 (br s, 4H), 2.19
(br s, 2H), 2.00 (br s, 2H), 1.82 (br s, 6H). 13C NMR (CD2Cl2) δ
188.6, 152.6, 150.8, 149.6, 148.6, 143.4, 142.4, 140.8, 137.2, 135.2,
130.1, 125.9, 125.3, 121.0, 120.0, 117.3, 114.9, 108.8, 52.0, 51.1,
40.6, 28.1, 26.2, 24.5, 20.7, 20.3. λmax (H2O) 621 nm (ε ) 2.9 ×
104 M-1 cm-1). HRMS (ESI) m/z 592.1349 (calcd for C31H34-
N3S280Se+: 592.1354).
12-(Dimethylamino)-2,3,6,7-tetrahydro-9-(N-piperidyl-2-thie-
nyl-5-thiocarboxamido)-1H,5H-telluroxantheno[2,3,4-ij]quino-
lizin-14-ium Hexafluorophosphate (31-Te). From xanthone 15-
Te (50.0 mg, 0.112 mmol) and 0.19 M 40 (0.80 mL, 0.15 mmol),
31-Te was obtained as described above for the preparation of 30-
1
S. Yield: 50 mg (54%), mp 172-174 °C. H NMR (CD2Cl2) δ
7.45 (d, 1H, J ) 9.8 Hz), 7.51 (s, 1H), 7.47 (d, 1H, J ) 2.5 Hz),
7.18 (d, 1H, J ) 4.0 Hz), 7.00 (d, 1H, J ) 3.5 Hz), 6.84 (d × d,
1H, J ) 2.5, 9.8 Hz), 4.27 (br s, 2H), 4.07 (br s, 2H), 3.50 (m,
4H), 3.20 (s, 6H), 2.73 (t, 2H, J ) 6.0 Hz), 2.61 (t, 2H, J ) 6.0
Hz), 2.21 (quintet, 2H, J ) 6.0 Hz), 2.00 (m, 2H), 1.82 (s, 6H).
13C NMR (DMSO-d6) δ 186.8, 151.5, 151.1, 148.1, 146.8, 143.0,
138.3, 136.5, 136.3, 134.1, 129.7, 125.3, 125.0, 121.9, 121.3, 120.6,
115.7, 114.9, 51.0, 50.3, 39.9, 29.6, 27.0, 23.5, 19.9. λmax (H2O)
635 nm (ε ) 1.9 × 104 M-1 cm-1). HRMS (ESI) m/z 642.1231
(calcd for C31H34N3S2130Te+: 642.1256).
1
solid, mp 264-266 °C. H NMR (CD3CN) δ 7.68 (d, 2H, J )
10.0 Hz), 7.45 (d, 1H, J ) 4.0 Hz), 7.20 (d, 2H, J ) 2.0 Hz), 7.12
(d, 1H, J ) 3.5 Hz), 7.07 (d × d, 2H, J ) 2.5, 9.5 Hz), 4.28 (br s,
2H), 4.01 (br s, 2H), 3.24 (s, 12H), 1.79 (m, 6H). 13C NMR
(CD3CN) δ 188.7, 154.6, 152.2, 149.5, 144.7, 138.8, 136.6, 131.1,
126.1, 120.4, 116.7, 106.7, 41.1, 24.8. λmax (H2O) 595 nm (ε ) 4.5
× 104 M-1 cm-1). HRMS (ESI) m/z 492.1586 (calcd for
C27H30N3S3+: 492.1596).
3,6-Bis(dimethylamino)-9-(N-piperidyl-2-thienyl-5-thiocar-
boxamido)selenoxanthylium Hexafluorophosphate (30-Se). From
xanthone 14-Se (50.0 mg, 0.145 mmol) and 0.22 M 40 (1.00 mL,
0.22 mmol), 30-Se was obtained as described for the preparation
of 30-S. Yield: 80 mg (79%), mp 215-217 °C. 1H NMR (CD2Cl2)
δ 7.76 (d, 2H, J ) 9.5 Hz), 7.26 (d, 2H, J ) 2.5 Hz), 7.21 (d, 1H,
J ) 3.5 Hz), 7.07 (d, 1H, J ) 3.5 Hz), 6.95 (d × d, 2 H, J ) 2.5,
9.8 Hz), 4.29 (br s, 2H), 4.02 (br s, 2H), 3.28 (s, 12H), 1.81 (br s,
6H). 13C NMR (DMSO-d6) δ 186.1, 152.2, 150.5, 146.9, 143.7,
139.0, 136.4, 129.6, 124.8, 118.7, 115.2, 109.1, 39.8, 23.0. λmax
(H2O) 606 nm (ε ) 4.8 × 104 M-1 cm-1). HRMS (ESI) m/z
540.1034 (calcd for C27H30N3S280Se+: 540.1041).
12-(Dimethylamino)-2,3,6,7-tetrahydro-9-(N-morpholino-2-
thienyl-5-thiocarboxamido)-1H,5H-thioxantheno[2,3,4-ij]quino-
lizin-14-ium Hexafluorophosphate (32-S). n-Butyllithium (2.4 M
in hexane, 300 µL, 0.72 mmol) was added to a stirred solution of
diisopropylamine (109 µL, 0.774 mmol) in 2.0 mL of anhydrous
THF at 0 °C. The solution was stirred for 15 min before cooling to
-78 °C. Thioamide 39 (150 mg, 0.703 mmol) in 1 mL of anhydrous
THF was added. The reaction mixture was stirred for 0.5 h to give
a 0.22 M solution of 1-N-morpholino-1-(5-lithio-2-thienyl)methaneth-
ione 41. An aliquot (1.0 mL, 0.22 mmol) of this solution was added
to xanthone 15-S (50.0 mg, 0.143 mmol) in 1 mL of THF. The
resulting mixture was heated at 35 °C for 0.5 h and cooled to room
temperature.Workup and purification as described for the prepara-
tion of 30-S gave 32-S. Yield: 60 mg (57%), mp 194-196 °C. 1H
NMR (CD2Cl2) δ 7.62 (d, 1H, J ) 9.5 Hz), 7.38 (s, 1H), 7.24 (d,
1H, J ) 3.5 Hz), 7.09 (m, 2 H), 6.98 (d × d, 1H, J ) 2.5, 9.5 Hz),
4.26 (br s, 2H), 3.86 (br s, 2H), 3.56 (t, 4H, J ) 5.5 Hz), 3.25 (s,
6H), 2.86 (t, 2H, J ) 6.5 Hz), 2.78 (t, 2H, J ) 6.5 Hz), 2.17 (quintet,
2H, J ) 6.5 Hz), 2.01 (quintet, 2H, J ) 6.5 Hz). 13C NMR (CD2Cl2)
δ 189.9, 153.2, 149.8, 149.3, 148.2, 142.4, 140.1, 139.9, 135.3,
132.9, 130.5, 126.6, 126.1, 120.3, 119.2, 115.4, 114.5, 106.0, 100.4,
66.9, 52.0, 51.0, 40.7, 28.2, 24.4, 20.7, 20.2. λmax (H2O) 612 nm
(ε ) 2.6 × 104 M-1 cm-1). HRMS (ESI) m/z 546.1704 (calcd for
C30H32N3OS3+: 546.1702).
3,6-Bis(dimethylamino)-9-(N-piperidyl-2-thienyl-5-thiocar-
boxamido)telluroxanthylium Hexafluorophosphate (30-Te).
From xanthone 15-Te (50.0 mg, 0.127 mmol) and 0.19 M 40 (1.00
mL, 0.19 mmol), 30-Te was obtained as described for the
1
preparation of 30-S. Yield: 0.090 g (94%), mp 156-158 °C. H
NMR (CD2Cl2) δ 7.85 (d, 2H, J ) 10.0 Hz), 7.51 (d, 2H, J ) 2.5
Hz), 7.19 (d, 1H, J ) 2.5 Hz), 7.02 (d, 1H, J ) 3.5 Hz), 6.86 (d
× d, 2H, J ) 2.5, 10.0 Hz), 4.28 (br s, 2H), 4.04 (br s, 2H), 3.22
(s, 12H), 1.81 (br s, 6H). 13C NMR (DMSO-d6) δ 186.7, 153.5,
151.6, 147.0, 142.5, 139.2, 138.4, 129.6, 125.2, 121.3, 116.3, 115.5,
23.5. λmax (H2O) 621 nm (ε ) 4.0 × 104 M-1 cm-1). HRMS (ESI)
m/z 590.0918 (calcd for C27H30N3S2130Te+: 590.0938).
12-(Dimethylamino)-2,3,6,7-tetrahydro-9-(N-piperidyl-2-thie-
nyl-5-thiocarboxamido)-1H,5H-thioxantheno[2,3,4-ij]quinolizin-
14-ium Hexafluorophosphate (31-S). From xanthone 15-S (50.0
mg, 0.143 mmol) and 0.19 M 40 (1.1 mL, 0.21 mmol), 31-S was
obtained as described above for the preparation of 30-S. Yield:
0.060 g (62%), mp 169-171 °C. H NMR (CD2Cl2) δ 7.65 (d,
1H, J ) 9.5 Hz), 7.40 (s, 1H), 7.22 (d, 1H, J ) 3.5 Hz), 7.09 (d,
12-(Dimethylamino)-2,3,6,7-tetrahydro-9-(N-morpholino-2-
thienyl-5-thiocarboxamido)-1H,5H-selenoxantheno[2,3,4-ij]quin-
olizin-14-ium Hexafluorophosphate (32-Se). From xanthone 15-
Se (50.0 mg, 0.126 mmol) and a 0.20 M solution 41 (0.10 mL,
0.20 mmol), 32-Se was obtained as described above for the
preparation of 32-S. Yield: 70 mg (70%), mp 171-172 °C. 1H NMR
(CD2Cl2) δ 7.62 (d, 1H, J ) 9.5 Hz), 7.40 (s, 1H), 7.24 (d, 1H, J
1

Rhodamine Inhibitors of P-gp
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3339
) 2.5 Hz), 7.22 (d, 1H, J ) 4.0 Hz), 7.06 (d, 1H, J ) 4.0 Hz),
6.91 (d × d, 1H, J ) 2.5, 9.8 Hz), 4.25 (br s, 2H), 3.86 (br s, 2H),
3.55-3.52 (m, 4H), 3.23 (s, 6H), 2.77-2.75 (m, 4H), 2.19 (quintet,
2H, J ) 6.5 Hz), 2.00 (quintet, 2H, J ) 6.5 Hz). 13C NMR (CD2Cl2)
δ 190.1, 152.8, 150.8, 149.7, 148.0, 143.5, 142.5, 141.7, 137.2,
135.3, 130.3, 126.1, 126.0, 121.0, 120.0, 117.4, 115.1, 108.9, 67.0,
52.1, 51.2, 40.7, 28.1, 26.3, 20.7, 20.4. λmax (H2O) 621 nm
(ε ) 2.9 × 104 M-1 cm-1). HRMS (ESI) m/z 594.1158 (calcd for
C30H32N3OS280Se+: 594.1147).
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.
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 rhodamine analogue in DPBSH
at 37 °C in room atmosphere. EC50 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 [2.5 µM 42] controls were included in each
plate. EC50 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.
Pgp-Transport Studies Across MDCK-MDR1 Monolay-
ers. MDCK-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 for 10 min in DPBSH at 37 °C with mixing on
a nutator (Clay Adams). Cells were preincubated with 4.3 mg mL-1
bovine serum albumin (BSA) in DPBSH alone or containing 2.5
µM 42. After 30 min, 5 µM test compound in BSA/DPBSH with
or without inhibitor was added to the donor chamber (0.5 mL upper
or apical, 1.0 mL lower or basolateral). Initial donor samples 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, this 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 two times using cold
DPBS and extracted with 500 µL of methanol for 3 min. All
samples were 200 µL and transferred directly to a black-wall, flat
bottom 96-well plate (Costar). The plate was centrifuged for 5 min
at 1500 rpm to reduce bubbles and then analyzed using a
Spectromax M2 fluorescent plate reader (Molecular Devices) with
λEX and λEM set at 584 and 612 nm, respectively.
Inhibition of Vinblastine Efflux in MDCKII-MDR1 Cells
by 31-S. MDCKII-MDR1 cells were seeded onto Costar Transwell
polycarbonate membranes and maintained as previously described.
On day 6, cells were rinsed 1 × 10 min in DPBSH with nutation
at 37 °C and then conditioned in 0.0001, 0.01, 0.05, 0.1, 0.5, 1, or
10 µM 31-S 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 EtOH stock) in
appropriate 31-S solution was introduced to the basolateral chamber
and 0.5 mL fresh 31-S 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.
Inhibition of Vinblastine Efflux in an Inside-Out P-gp
Membrane Vesicle Model by 31-S. The inhibition assay was
conducted using 0.1 µM [3H]-VIN (0.25 µCi mL-1) as a P-gp
substrate. The accumulation of [3H]-VIN into P-gp membrane
vesicles was inhibited by 17-S (0.01-10 µM), using 5 µM 42 as a
standard for complete P-gp inhibition. Previously, the inhibition
of [3H]-VIN accumulation in P-gp membrane vesicles by 42 was
shown to exhibit an IC50 value of (6.5 ( 1.1) nM, and thus a
complete inhibition of P-gp was assumed at 5 µM. The reaction
mixtures contained 4 mM ATP, ATP with different concentrations
of 17-S, ATP with 5 µM 42, or 4 mM AMP-PNP. All reaction
mixtures contained 0.1 µM [3H]-VIN, the regenerating system
including 100 µg mL-1 creatine kinase, 0.010 M creatine phosphate,
and 0.010 M MgCl2. The reaction was started by addition of 30
µL of reaction mixtures to vesicles (20 µg/20 µL) or 20 µL sucrose
buffer. The reactions were incubated for 2.5 min at 37 °C with
1-N-Piperidinyl-1-(2-thienyl)methanethione (38). Piperidine
(5.30 mL, 53.5 mmol), thiophene-2-carboxyaldehyde (1.40 mL, 17.8
mmol), and elemental sulfur (1.40 g, 44.6 mmol) in 12.0 mL of
dry DMF were stirred at 110 °C for 0.5 h, cooled to ambient
temperature, and poured into 60 mL of CH2Cl2. The resulting
solution was washed with water (2 × 15 mL), brine (2 × 15 mL),
dried over Na2SO4, and concentrated in vacuo. Chromatography
(1:9 EtOAc/hexanes, then 1:1 EtOAc/hexanes) on SiO2 followed
by recrystallization from EtOAc/hexanes gave 3.0 g (81%) of 38
as yellow crystals, mp 83-84 °C (lit.46 mp 85-86 °C). H NMR
1
(CDCl3) δ 7.35 (d × d, 1H, J ) 1.5, 5.3 Hz), 7.03 (d × d, 1H,
J ) 1.5, 3.5 Hz), 6.93 (d × d, 1H, J ) 3.5, 4.8 Hz), 4.26 (br s,
2H), 3.82 (br s, 2H), 1.75 (br m, 6H). LRMS (ESI) m/z 212.1 (calcd
for C10H13NS2 + H+: 212.0).
1-N-Morpholino-1-(2-thienyl)methanethione (39). From thio-
phene-2-carboxyaldehyde (1.90 mL, 20.0 mmol), elemental sulfur
(1.60 g, 50.0 mmol), and morpholine (5.2 mL, 60 mmol), 3947 was
obtained as described for the preparation of 38. Yield: 4.5 g (99%),
1
mp 64-66 °C. H NMR (CDCl3) δ 7.35 (d × d, 1H, J ) 1.5, 5.3
Hz), 7.03 (d × d, 1H, J ) 1.5, 3.5 Hz), 6.93 (d × d, 1H, J ) 3.5,
4.8 Hz), 4.26 (br s, 2H), 3.82 (br s, 2H), 1.75 (br m, 6H). LRMS
(ESI) m/z 212.1 (calcd for C10H13NS2 + H+: 212.0).
Expression of P-gp, Purification, and Measurement of
ATPase Activity. The cDNA for human P-glycoprotein (P-gp)50
was modified to contain a 10-histidine tag at the COOH-terminal
end (P-gp-His10) to facilitate purification by metal-chelate chro-
matography.51 Baby hamster kidney (BHK) cells were cotransfected
with P-gp-His10 and pWL-neo (Stratagene, Cedar Creek, TX) and
the transfected cells treated with the cytotoxic agent G418 as
described previously.71 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.72 BHK cells overexpressing P-gp-His10 were then
expanded and then used to purify the protein.
Histidine-tagged P-gp was isolated by nickel-chelate chroma-
tography as described previously.51 A sample of the isolated
histidine-tagged P-gp was mixed with an equal volume of 10 mg/
mL 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 rhodamine
compounds. The samples were incubated for 30 min at 37 °C, and
the amount of inorganic phosphate released was determined.55
To test for inhibition of P-gp drug-stimulated ATPase activity,
samples of P-gp-His10 in lipid were preincubated with various
concentrations of the rhodamine compounds for 15 min at 20 °C.
The ATPase reactions were then started by addition of ATPase
reaction mix containing verapamil (final concentration of 0.4 mM)
and ATPase activity determined as described above. Verapamil was
used as a substrate to test for inhibition because it is one of the
most potent activators of P-gp ATPase activity.54
Enhancement of Calcein-AM Uptake into MDCK-MDR1
Cells by Rhodamine Analogues. 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
penicillin, and 100 µg/mL streptomycin in 75 cm2 flasks. Cultures
were passaged by trypsinization 1:10 twice a week and used at
passage number 16-42. Cells were seeded at 40000 cells/well in

3340 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Gannon et al.
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P-glycoprotein-mediated exclusion of rhodamine dyes from multidrug-
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P-glycoprotein in proteoliposomes. Effect of substrates and modulators,
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gentle agitation. The reactions were stopped by the addition of ice-
cold sucrose buffer to wells and filtered onto a membrane filter
(GF/B glass fiber plate soaked overnight in 10% FBS/0.25 M
sucrose buffer, v/v) with a 96-channel cell harvester (PerkinElmer,
Boston, MA). Radioactivity retained on the filter was determined
by liquid scintillation counting.
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Acknowledgment. This research was supported in part by
NIH Grant T32 CA09363 (Postdoctoral Training Grant) to G.T.,
and grants from the Canadian Cancer Society (19074) and the
Canadian Institutes for Health Research (25043) to DMC. DMC
is the recipient of the Canada Research Chair in Membrane
Biology. G.T. gratefully acknowledges Alan Senior, Robert
Bambara, and Barbara Iglewski for encouragement and the use
of their laboratory space and supplies. We thank Dr. Piet Borst
at The Netherlands Cancer Institute for supplying the MDCKII-
MDR1 cells, William Perry at Eli Lilly and Company for the
human embryonic kidney (PEAKSTABLE) cells, and Anastasiya
Sokolova at the University at Buffalo for technical assistance
in the preparation of intermediates.
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Exp. Physiol. 1998, 83, 221–232.
Supporting Information Available: Experimental procedures
for the preparation of 3-S, 4-S, 5-Te, 6-Se, 7-Se, 7-Te, 8-Se, 9-Te,
12-S, 17-S, 18-S, 19-S, 20-S, and 21-S and for the preparation of
vesicles from human embryonic kidney (PEAKSTABLE) cells;
calculations for vesicle inhibition studies, table for mouse MDR3
CL P-gp data, and figure showing the inhibition of [3H]-VIN efflux
from MDCK-MDR1 with 31-S. This material is available free of
charge via the Internet at http://pubs.acs.org.
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by P-glycoprotein with distinct drug specificities. Eur. J. Biochem.
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