Thiorhodamines containing amide and thioamide functionality as inhibitors of the ATP-binding cassette drug transporter P-glycoprotein (ABCB1)

Article abstract of DOI:10.1016/j.bmc.2012.05.075

Twelve thiorhodamine derivatives have been examined for their ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His ₁₀, to promote uptake of calcein AM and vinblastine into multidrug-resistant, P-gp-overexpressing

Full text of DOI:10.1016/j.bmc.2012.05.075

Bioorganic & Medicinal Chemistry 20 (2012) 4290–4302
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiorhodamines containing amide and thioamide functionality as inhibitors
of the ATP-binding cassette drug transporter P-glycoprotein (ABCB1)
Alexandra Orchard a, Gregory A. Schamerhorn a, Brandon D. Calitree a, Geri A. Sawada b, Tip W. Loo c,d
,
M. Claire Bartlett c,d, David M. Clarke c,d, Michael R. Detty a,
⇑
a Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260-3000, United States
b Drug Disposition, Eli Lilly and Company, Indianapolis, IN 46285, United States
c Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
d Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve thiorhodamine derivatives have been examined for their ability to stimulate the ATPase activity
of purified human P-glycoprotein (P-gp)-His10, to promote uptake of calcein AM and vinblastine into mul-
tidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their rates of transport in monolayers
of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells. The thiorhodamine derivatives have
structural diversity from amide and thioamide functionality (N,N-diethyl and N-piperidyl) at the 5-posi-
tion of a 2-thienyl substituent on the thiorhodamine core and from diversity at the 3-amino substituent
with N,N-dimethylamino, fused azadecalin (julolidyl), and fused N-methylcyclohexylamine (half-julol-
idyl) substituents. The julolidyl and half-julolidyl derivatives were more effective inhibitors of P-gp than
the dimethylamino analogues. Amide-containing derivatives were transported much more rapidly than
thioamide-containing derivatives.
Received 2 April 2012
Revised 15 May 2012
Accepted 24 May 2012
Available online 7 June 2012
Keywords:
Multidrug resistance, P-glycoprotein
thiorhodamine
transport inhibition
ATPase stimulation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
P-gp can also be assayed for their ability to stimulate P-gp ATPase
activity. Our work with rhodamine/rosamine-related structures
Multidrug resistance (MDR) often emerges in the treatment of
cancer following exposure of the patient to chemotherapeutic
agents. While MDR appears from a variety of mechanisms, efflux
proteins that are members of the ATP-binding cassette (ABC)
super-family are most commonly associated with the emergence
of MDR.1–3 P-glycoprotein (P-gp, also known as MDR1, encoded
by ABCB1)1–5 is one member of the ABC superfamily and was the
first efflux protein identified and associated with MDR in cancer
chemotherapy. Many approaches to the development of inhibi-
tors/modulators of P-gp have been examined. While a large num-
ber 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.3,6,7
Rhodamine and rosamine dyes with small structural changes
have been used to assay P-gp-mediated transport giving large
changes (>100-fold) in rates of transport.8–10 Efflux of rhodamine
123 from cells was used to define P-gp transport substrates/antag-
onists in a cross-correlation of drug resistance patterns in the NCI
60 set of cells with the NCI Drug Screen Database of com-
pounds.11,12 Interaction of rhodamine and rosamine dyes with
has demonstrated that small structural changes give >1000-fold
range of P-gp affinities (as measured by KM) with some rhoda-
mine/rosamine molecules being highly stimulating for ATPase
activity. Some of the rhodamine/rosamine-related compounds
were found to be potent inhibitors of P-gp drug transport.13,14
In more recent work, it was demonstrated that essentially sin-
gle-atom changes—that is, interchanging amide and thioamide
functionality—in a small series of rosamine/rhodamine structures
gave either molecules with high affinity for P-gp and high stimula-
tion of ATPase activity or molecules with high affinity but low
stimulation of ATPase activity.15 Specifically, tertiary amide groups
on the 9-aryl or heteroaryl substituent gave high P-gp ATPase
stimulation while tertiary thioamide groups on the 9-aryl or het-
eroaryl substituent gave low stimulation of ATPase activity. The
single-atom change of amide to thioamide also slows the rate of
P-gp-mediated transport of the rhodamine derivatives in both
absorptive and secretory directions in the cell. In addition, the
substitution of a fused azadecalin for a dimethylamino substituent
in the xanthylium core also gave higher affinity for P-gp in both
isolated protein and in whole cells.
The chalcogenorhodamines are also photosensitizers for treat-
ment of multidrug-resistant cancer cells in vitro with photody-
namic therapy (PDT) via the generation of singlet oxygen from a
⇑
Corresponding author. Tel.: +1 716 645 4228; fax: +1 716 645 6963.
E-mail address: mdetty@buffalo.edu (M.R. Detty).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.075

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4291
biopolymer-bound chalcogenorhodamine.16–18 Understanding the
parameters that impact ATPase activity and binding affinity in rho-
damine-like modulators of P-gp and the factors that influence the
drug availability to the pump in the native membrane environment
are critical for the development of more potent rhodamine-like
inhibitors of P-gp that may have a dual application as photosensi-
tizers for treatment of multidrug-resistant cells by PDT.
To examine further the roles of the amide/thioamide ‘switch’
and the lipophilicity/hydrophobicity of one amino substituent on
P-gp ATPase stimulation, inhibition of P-gp-mediated efflux in
whole cells, and transport of the chalcogenorhodamines in whole
cells, the series of amide- and thioamide-containing thiorhodam-
ines 1–12 (Chart 1) were prepared. The thiorhodamine core was
selected 1) to avoid any ambiguities associated with the chalcogen
atom and 2) because a thiorhodamine was the best inhibitor in ear-
lier work.15 The thiorhodamines were evaluated for their ability to
stimulate ATPase activity in isolated, wild-type human P-gp, their
ability to be transported in multidrug-resistant, P-gp over-express-
ing cells, and their ability to inhibit P-gp drug transport in multi-
drug-resistant cells.
substituted with 1-chloro-3-methyl-2-butene in the presence of
K2CO3, and the aniline product 16 was obtained in 78–87% isolated
yields. Cyclization of 16 with concentrated sulfuric acid gave 17 in
isolated yields of 70–80%. Subsequent Vilsmeier–Haack reaction
with POCl3 and dimethylformamide (DMF) converted 17 to alde-
hyde 18 in yields of >96%. Oxidation of 18 to a carboxylic acid oxi-
dation state was problematic as was observed for the oxidation of
9-formyljulolidine.20 modified Willgerodt–Kindler21 reaction
A
oxidized 18 to the thioamide 19 in 75–83% isolated yields using
elemental sulfur and piperidine in refluxing DMF.22 Amide 20
was formed from 19 using trifluoroacetic anhydride (TFAA) in iso-
lated yields of >90%.19
Directed ortho-lithiation of 20 in THF at ꢀ78 °C with sec-butyl-
lithium and N,N,N0,N0-tetramethylethylenediamine (TMEDA) was
followed immediately by the addition of 3-dimethylaminophenyl
disulfide (21)19,23 at ꢀ78 °C to minimize the amount of self-con-
densed side product formation that has been seen in similar reac-
tions. The isolated yield of diaryl sulfide 22 was 35–45%.
Subsequent cyclization of 22 with POCl3 in acetonitrile19 gave the
desired thioxanthone 15 in 90–91% isolated yield, putting the over-
all yield for the synthesis of the half-julolidyl thioxanthone 15 at
20%.
2. Results and discussion
The reaction is regioselective for ortho-lithiation at the desired
position. None of the isomeric diaryl sulfide 23 (Chart 3) was ob-
served in the product mixture suggesting either that only ortho-
lithiated intermediate 24 was formed in the reaction or that
ortho-lithiated intermediate 25, if formed, then hindered sterically
approach of the electrophile and formation of 23 was prevented.
We have been unable to ortho-lithiate the tetramethyljulolidyl
derivative 2620 (Chart 3) under a variety of conditions suggesting
that kinetic deprotonation next to the gem-dimethyl group is ex-
tremely slow and that anion 24 is likely the only lithiated species
formed.
With thioxanthones 13–15 readily available, the syntheses of
dyes 1–12 followed the pathways outlined in Scheme 2.
Willgerodt–Kindler oxidation of thiophene-2-carboxaldehyde with
elemental sulfur and diethylamine gave thioamide 27 in 49% iso-
lated yield.24 Deprotonation of 27 with sterically bulky lithium
diisopropylamide (LDA) gave the 2-thienyl anion 28, which was
2.1. Synthesis of thiorhodamine analogues 1–12
Thiorhodamines 1–12 were prepared from thioxanthones 13–
15 (Chart 2), which were prepared by reported procedures.19,20
Compound 14 replaces a dimethylamino substituent with an
azadecalin substituent. The fused aniline equivalent of this substi-
tution is known as julolidine (Chart 2) and compounds derived
from 14 are referred to as ‘julolidyl’ rhodamines in the remainder
of the manuscript. Thioxanthone 15 incorporates a trimethyltetra-
hydroquinoline group (Chart 2) and compounds derived from 15
are referred to as ‘half-julolidyl’ rhodamines in the remainder of
the manuscript. The thioxanthone 15 has the same number of car-
bon atoms as 14, but the gem-dimethyl group adds the potential
for steric interactions absent in 14.
Thioxanthone 15 is a previously unknown structure and was
prepared as outlined in Scheme 1. N-Methylaniline was first
NEt2
N
NEt2
X
X
X
S
S
S
PF6
PF6
PF6
Me2N
S
NMe2
Me2N
S
NMe2
N
S
NMe2
1
5
, X = O
6, X = S
, X = O
3
, X = O
2, X = S
4, X = S
N
S
NEt2
S
N
S
X
X
X
PF6
PF6
PF6
Me Me
Me Me
N
S
NMe2
N
S
NMe2
N
S
NMe2
Me
Me
7
11
, X = O
12, X = S
, X = O
9
, X = O
8, X = S
10, X = S
Chart 1. Thiorhodamine analogues 1–12 examined in this study.

4292
A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
O
O
O
Me Me
Me Me
Me2N
S
NMe2
N
S
NMe2
N
S
NMe2
Me
13
14
15
N
N
Me
1,2,3,5,6,7-hexahydro-
pyrido[3,2,1-ij]quinolone
"julolidine"
1,4,4-trimethyl-1,2,3,4-
tetrahydroquinoline
"half-julolidine"
Chart 2. Thioxanthone precursors to thiorhodamines 1–12.
Me
Me
Me
O
Me
Me Me
MeN
Me
MeN
NH
Cl
POCl3
DMF
H2SO4
Me
Me
K2CO3, CH3CN
N
Me
16
17
18
S8, DMF
O
S
Me Me
piperidine
1) sec-BuLi
TMEDA,
THF
N
S
Me Me
O
Me Me
TFAA
CH2Cl2
POCl3,
Et3N
N
N
N
Me
22
2)
N
N
15
Me
DMF
Me
(Me2N
S)2
20
19
NMe2
21
Scheme 1. Synthesis of thioxanthone 15.
O
Me Me
Li
N
Li
Me2N
S
Me Me
O
Me Me
O
N
Me Me
O
N
N
N
Me
Me
N
Me
N
N
Me
Me
23
24
25
26
Chart 3. Possible directed lithiation intermediates, products and substrates.
S
S
NEt2
LDA, THF
-78 oC
NEt2
CHO
13 14
, or
15
1)
,
S8, Et2NH
DMF
S
S
2
1
6
10
(15%)
(61%), (56%), or
TFAA,
CH2Cl2
S
2) 10% HPF6
Li
28
27
(49%)
S8,
NH
5
9
(63%), (54%), or (51%)
DMF
S
S
N
LDA, THF
-78 o
13 14
15
1)
,
, or
N
S
4
3
8
12
(52%)
(69%), (60%), or
TFAA,
CH2Cl2
11
C
2) 10% HPF6
S
30
Li
29
4%)
(9
7
(70%), (54%), or
(50%)
Scheme 2. Syntheses of thiorhodamines 1–12 from thioxanthones 13–15.
then added to solutions of the thioxanthones 13–15. Workup with
aqueous HPF6 gave the diethyl thioamide-containing dyes 2, 6, and
10 in 61%, 56%, and 15% isolated yields, respectively.
The synthesis of 8 and 12 followed an earlier procedure that
was developed for the preparation of thioamide-containing thio-
rhodamine dye 4.15 Willgerodt–Kindler oxidation of thiophene-
2-carboxaldehyde with elemental sulfur and piperidine gave
thioamide 29 in 94% isolated yield (Scheme 2).27 Deprotonation
of 29 with LDA gave anion 30, which was added to thioxanthone
13 to give thioamide-containing dye 4 in 69% isolated yield follow-
ing workup with aqueous HPF6.15 Similarly, the addition of 30 to
Unlike the tertiary amide group,25 which is highly directing, the
thioamide functionality does not direct lithiation in thiophenes.
Only the more acidic a-proton was removed and none of the cor-
responding 2,3-disubstituted thiophenes were detected in the
product mixtures.26

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4293
thioxanthones 14 and 15 followed by workup with aqueous HPF6
gave piperidyl thioamide-containing dyes 8 and 12 in 60% and
52% isolated yields, respectively.
The amide-containing thiorhodamines 1, 3, 5, 7, 9 and 11 were
prepared from their thioamide counterpart via hydrolysis of the
thioamide. Trifluoroacetic anhydride was added to CH2Cl2 solu-
tions of thioamides 2, 4, 6, 8, 10 and 12 to give the corresponding
amides in isolated yields of 51–70%.19
2.2. Measurement of n-octanol/water partition coefficients
Experimental values of the n-octanol/water partition coefficient
(logP) were measured using the ‘shake flask’ method.28 A saturated
n-octanol solution of dye 1–12 was shaken with an equal volume
of phosphate buffered saline (PBS) at pH 7.4 and the concentrations
in the two layers were determined spectrophotometrically. Values
of logP are compiled in Table 1 and covered a range from logP = 1.2
for dyes 1 and 2 to logP ꢁ2.6–2.7 for dyes 8, 10 and 12. Based on
these values of logP, thiorhodamine compounds 1–12 would have
access to both aqueous and hydrophobic environments in the stud-
ies with whole cells described below.
Figure 1. Stimulation of human P-gp ATPase activity by compounds 1 and 7.
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 thiorhodamine
derivative 1 or 7. Error bars represent one standard deviation from the mean.
2.3. P-gp ATPase activity with isolated human P-gp-His10
Several trends emerged from the data shown in Table 1. With
respect to Vmax, values were significantly larger (p <0.05, Student
t-test) for dimethylamino analogues 1–4 (6.6–12.1-fold stimula-
tion) relative to their corresponding julolidyl analogues 5–8 (3.5–
5.6-fold stimulation) and half-julolidyl analogues 9–12 (64.7-fold
stimulation). The latter two groups did not show significant differ-
ences. There were no statistically significant differences in values
of Vmax for any specific amide/thioamide pair. With respect to
KM, values were significantly higher (p <0.03 for amides, p <0.01
for thioamides) for the dimethylamino analogues 1–4 [(5.5–
6.4) ꢂ 10ꢀ6 M] relative to either their corresponding julolidyl ana-
logues 5–8 [(1.9–3.6) ꢂ 10ꢀ6 M] or the corresponding half-julolidyl
analogues 9 and 10 (2.2 ꢂ 10ꢀ6 M and 3.1 ꢂ 10ꢀ6 M], respectively).
For 11 and 12 where values of KM could not be determined, values
of IC50 for 50% inhibition of VER-stimulated (4 ꢂ 10ꢀ4 M) P-gp ATP-
ase activity were 1.1 ꢂ 10ꢀ6 M and 1.7 ꢂ 10ꢀ6 M, respectively. It
The ATPase activity of thiorhodamine compounds 1–12 (Chart
1) was examined using isolated human P-gp-His10, which was
activated with sheep brain phosphatidylethanolamine.29–31 The
apparent Michaelis–Menten constant (KM) for ATPase-stimulating
compounds was determined as well as the drug-induced stimula-
tion of maximal ATPase activity (Vmax) using isolated human
P-gp-His10 (Table 1). Verapamil (VER) was included as a control
compound (generally considered relatively robust and one of the
most stimulating drugs known for P-gp).32,33 ATPase activity was
determined from the release of inorganic phosphate.33 Representa-
tive curves are shown in Figure 1 for compound 1, which was the
most stimulating rhodamine for ATPase activity, and for compound
7, which had the lowest value of KM. For compounds 11 and 12,
which were too weakly stimulating for P-gp ATPase activity to per-
mit determination of KM, the concentration of compound required
for 50% inhibition of VER-stimulated (4 ꢂ 10ꢀ4 M) P-gp ATPase
activity (IC50) was determined (Fig. 2 and Table 1).
should be noted that compound
8 showed a lower KM
(8.7 ꢂ 10ꢀ8 M) in a previous study15 because those assays were
Table 1
Stimulation of ATPase activity, inhibition of verapamil-induced ATPase activity, IC50’s for calcein AM (CAM) uptake and tritiated vinblastine (VIN) efflux by thiorhodamine amide
and thioamide analogues, and n-octanol/water partition coefficients (logP) for thiorhodamines 1–12 and VER.
Compd
Isolated
Human
P-gp-His10
MDCKII-MDR1
Cells
logP
a
b
c
Vmax
,
fold stimulation
KM,
10ꢀ6
M
IC50
,
10ꢀ6
M
IC50 CAM uptake, 10ꢀ6 Md
IC50 VIN efflux, 10ꢀ6 Md
VER
1
2
3
4
5
6
7
8
17.9 2.0
12.1 1.6
12.1 1.9
8.6 2.3
6.6 0.7
5.6 0.6
4.6 1.6
4.1 0.6
3.5 0.9
4.7 0.3
4.3 0.5
<2
24 2.6
5.7 1.0
5.8 0.6
6.4 0.4
5.5 1.3
3.6 0.4
2.3 0.4
1.9 0.4
2.5 0.4
2.2 1.0
3.1 0.7
NDe
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
>100
1.2
1.2
1.5
(1.7)g
1.9
1.7
1.4
(2.7)g
1.4
2.7
1.7
2.6
14
1
>100
NDf (19 2)g
10.0 1.6
9.6 1.2
56
1
12.0 1.1 (2.1 1.2)g
7.1 1.2
—
9
8.0 1.2
8.7 1.1
5.6 1.1
8.9 1.1
10
11
12
8.8 1.1
13.0 1.1
9.3 1.1
1.1 0.2
1.7 0.3
<2
NDe
a
b
c
d
e
f
Vmax is the ratio of maximum stimulation in the presence of compound to that without compound (basal activity).
KM is the concentration of compound required to achieve 50% maximal stimulation.
IC50 is the concentration of compound required to inhibit 50% of verapamil-stimulated ATPase activity.
Details for methods are provided in Section 4. Error limits represent standard deviation.
ND, not determined because stimulation of ATPase activity too low.
ND, not determined because of limited solubility in BSA-buffer.
Values in parentheses from Ref. 15.
g

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A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
Figure 3. % Inhibition of CAM uptake by 5 ꢂ 10ꢀ6 and 2.5 ꢂ 10ꢀ5 M thiorhodamines
1–12 relative to MDCKII-MDR1 cells fully-inhibited by LSN 335984. Values shown
are the average of duplicate runs and error bars show the reproducibility of the two
runs. White bars show values for amides 1, 3, 5, 7, 9 and 11 at 2.5 ꢂ 10ꢀ5 M while
gray bars show values for thioamide 2, 4, 6, 8, 10 and 12 at 2.5 ꢂ 10ꢀ5 M. The black
bars are for the adjacent thiorhodamine at 5 ꢂ 10ꢀ6 M.
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
M VER with various concentrations of
thiorhodamines 11 and 12. Data represent the average for triplicate measurements
several trends emerged. In pair-wise comparisons, the amides
were weaker inhibitors than the corresponding thioamides. With
a given amine functionalization (dimethylamino, julolidyl, half
julolidyl), the piperidyl thioamides and diethyl thioamides dis-
played comparable inhibition. Within the different amine function-
alization, the dimethylamino analogues 1–4 were weaker
inhibitors (3–19% inhibition) than their corresponding julolidyl
analogues 5–8 (41–74% inhibition), which in turn were weaker
inhibitors than the corresponding half-julolidyl analogues 9–12
(64–105% inhibition).
measured in the presence of 4 ꢂ 10ꢀ4
and error bars represent the standard deviation.
performed in the presence of 10-fold lower lipid (0.5 mg/ml) and
detergent (2.0 ꢂ 10ꢀ4 M dodecyl-b-
D-maltoside) concentrations.
2.4. Enhancement of CAM uptake into MDCKII-MDR1
transfected cells
Values of IC50 for the enhancement of CAM uptake into MDCKII-
MDR1 transfected cells by thiorhodamines 1–12 (Table 1) were
determined by measuring relative fluorescence values obtained
after 20-min incubation with CAM at 37 °C. Typical curves are
shown in Figure 4 for CAM uptake in the presence of various con-
centrations of thiorhodamines 1–12. Among the dimethylamino
analogues 1–4, the amides 1 and 3 were weaker inhibitors (IC50’s
for CAM uptake >1 ꢂ 10ꢀ4 M) than thioamide analogues 2 and 4
(IC50’s of 1.4 ꢂ 10ꢀ5 M and 1.9 ꢂ 10ꢀ5 M,15 respectively). Among
the julolidyl analogues 5–8, diethyl amide 5, diethyl thioamide 6,
and piperidyl thioamide 8 all had comparable values of IC50 for
CAM uptake [9.6–12.0) ꢂ 10ꢀ6 M], which were lower than the
IC50 for piperidyl amide 11 (5.6 ꢂ 10ꢀ5 M). All of the half-julolidyl
amides and thioamides 9–12 had comparable values of IC50 for
CAM uptake [(7.1–13.0) ꢂ 10ꢀ6 M], which were comparable to
those observed for 5, 6 and 8. With the exception of IC50 for CAM
uptake for half-julolidyl amide 11 relative to julolidyl amide 7
where there was a >4-fold difference in IC50, there were no signif-
icant differences in the other three amide/thioamide pairs for the
Thiorhodamine compounds 1–12 were also evaluated for their
ability to facilitate the uptake of calcein AM (CAM) into MDCKII-
MDR1 transfected cells, which over express P-gp.34 The CAM up-
take in these cells was determined at concentrations of 5 ꢂ 10ꢀ6
M and 2.5 ꢂ 10ꢀ5 M in the thiorhodamine and the inhibition of
P-gp was compared as a percentage of the inhibition observed
with 5 ꢂ 10ꢀ6 M (R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetra-
hydrodibenzo[a,e]-cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyl-
oxy)methyl]-1-piperazineethanol (31, LSN 335984, IC50 = 0.4 ꢂ
10ꢀ6 M, Chart 4), which completely inhibits P-gp. LSN 335984 is
structurally related to the P-gp-specific inhibitor (R)-4-
[(1a,6,10b)-1,1-difluro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclo-propa
[c]cyclohepten-6-yl]-[(5-quin-olinyloxy)methyl]-1-piperazineeth-
anol (32, LSN 335979 or zosuquidar, Chart 4).7,35 Values of the per-
centage inhibition at the two concentrations are shown in Figure
3.
As shown in Figure 3, all of the compounds 1–12 were weak
inhibitors at 5 ꢂ 10ꢀ6 M (0–41%). At 2.5 ꢂ 10ꢀ5 M in this assay,
julolidyl and half-julolidyl derivatives 5–12 with respect to IC50
.
2.5. Inhibition of vinblastine efflux by thiorhodamines 9–12 in
MDCKII-MDR1 cells
Cl
H
Cl
H
F
F
H
H
Vinblastine (VIN) is a clinical chemotherapeutic drug and
inhibition of its transport by the thiorhodamine compounds is
indicative of potential translational value. Inhibition of P-gp-
mediated VIN efflux by the half-julolidyl series 9–12 was exam-
ined in MDCKII-MDR1 transfected cells. [3H]-Vinblastine, in an
appropriate dilution series with 9–12 and BSA, was introduced to
the basolateral chamber of a monolayer of MDCKII-MDR1 transfec-
ted cells. The appearance of [3H]-VIN in the apical chamber was
monitored by scintillation counting and gave comparable values
of IC50 of (5.6–8.9) ꢂ 10ꢀ6 M for 9–12 (Table 1) for inhibition of
VIN efflux.
N
N
N
N
O
N
O
N
OH
(LSN 335984)
OH
(LSN 335979)
32
31
Chart 4. Structures of LSN 335984 (31) and LSN 335979 (32).

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4295
Figure 4. Uptake of CAM into MDCKII-MDR1 cells as a function of concentration of thiorhodamines 1–4 (Panel a), 5–8 (Panel b) and 9–12 (Panel c). Values of IC50 (Table 1)
were determined by a sigmoidal dose-response (variable slope) analysis. Data represent the average and standard error mean for triplicate measurements.
2.6. Transport across monolayers of MDCKII-MDR1 cells
fected cells (Table 2).34 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 underestimation of the
The transport of thiorhodamine amide and thioamide deriva-
tives 1–12 was measured in monolayers of MDCKII-MDR1 trans-
Table 2
Transport and cell association studies of thiorhodamine amide and thioamide analogues 1–12 with MDCK-MDR1 cellsa
b
Compd
PAB, 10ꢀ9 m sꢀ1
PBA, 10ꢀ9 m sꢀ1
PBA(ꢀinh)/PBA(+inh)
PPassive
,
10ꢀ9 m sꢀ1
% Cell associatedc
Ratio (+/ꢀinh)d
1
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
280 35
22
ꢁ7
2.1 0.4
12 3.5
5.7
(+inh)
2
(+inh)
3
(+inh)
4
(+inh)
5
(+inh)
6
(+inh)
7
(+inh)
8
(+inh)
9
(+inh)
10
(+inh)
11
(+inh)
12
(+inh)
13
2
210 17
5.1 0.1
41
38
73
12
24
27
69
14
69
31
170
ꢁ3
ꢁ4
ꢁ1
ꢁ9
ꢁ2
ꢁ4
ꢁ1
ꢁ14
ꢁ1
ꢁ4
<1
11
3
5.1
3.9
4.8
4.8
1.8
6.1
2.1
3.3
1.2
5.2
1.8
56 11
2.8 1.0
290
9
7.7 0.1
110 35
1.5 0.1
210 22
17.0 0.4
65.0 1.5
2.7 0.1
220 29
8.1 0.1
11
12
58
3
1
2
5.8 0.2
28
25.0 0.2
45
6.9 0.1
3
6
42
31
64
10
33
52
64
7
3
9
2
6
6
7
83
3
1.2 0.1
370 17
27.0 0.3
69.0 2.5
1.0 0.1
230 24
7.5 0.1
34 22
8.6 0.1
45.0 0.1
34.0 2.7
62.0 0.2
0.2 0.1
a
Experiments were run with 5 ꢂ 10ꢀ6 M dye and 4.3 mg mLꢀ1 BSA. Values of transport in the absorptive (PAB) and secretory (PBA) mode in the absence or presence of
inhibitor, the ratio [PBA (no inhibitor)/PBA (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. Details for methods are provided in Section 4. Error limits represent standard deviation.
b
PPassive represents the mean of PAB and PBA in the fully inhibited system.
% Cell associated is the fraction of mass extracted from the cell monolayer by methanol wash after 1-h flux in the AB direction.
For % cell associated dye.
c
d

4296
A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
permeability coefficient.36 The assay was repeated in the presence
of 31 (LSN 335984),35 to measure transport when P-gp was fully
inhibited.
transport in the secretory direction (PBA). One can ask whether
these observations are general for amide- and thioamide-substi-
tuted groups at the 9-position of chalcogenorhodamine analogues
or whether the observations are specific for the particular com-
pounds examined.
MDCKII-MDR1 monolayers display apical and basolateral polar-
ized membranes and are considered to be a near-physiological
model for studying P-gp drug transport. In the monolayer, P-gp is
solely present at the apical membrane. For thiorhodamines 1–12,
transport was measured in the absorptive (apical to basolateral
or AB) and secretory (basolateral to apical or BA) transport direc-
tion of the cell monolayer. Values of transport in the absorptive
(PAB) and secretory (PBA) mode in the absence of inhibitor, passive
transport (PPassive) in the presence of inhibitor, and the % cell-asso-
ciated dye in the AB direction in the absence or presence of inhib-
itor are compiled in Table 2.
One might assume that molecules with closely related struc-
tures would bind to a common site in P-gp, but this assumption
is challenged by several observations. The ‘H’ and ‘R’ sites for Hoe-
chst 33342 and rhodamine 123, respectively, on P-gp have been
described by Shapiro and Ling indicating that there are at least
two binding sites on the protein.29,38 Furthermore, the binding
sites may be fluid. 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 to accommodate the
shape of the small molecule.39 Modeling studies of P-gp have also
indicated a variety of different conformations for binding of small
molecules to P-gp.40–42 It is possible that the structurally related
amides and thioamides of this study could have different binding
to P-gp. Recent studies have shown that thiourea functionality im-
parted greater inhibitory activity toward MRP1 in a series of drug
derivatives relative to P-gp or BCRP, which is consistent with dif-
ferent types of binding.43
The thiorhodamines 1–12 of this study examined a very small
structure–activity space: compounds 1–7 and 9–12 were designed
as related structures to 8 to compare amide/thioamide-substituted
rhodamines with the tertiary amides/thioamides bearing diethyl-
amino or piperidyl groups and to examine the systematic changes
of one amino substituent at the 3-position of the thiorhodamine
core. Because the structure–activity space is limited, one might
not expect great differences among the compounds, but hopefully
trends are identified. In isolated protein, the series 1–12 gave a range
of values for Vmax (maximum stimulation of ATPase activity) from
<2-fold stimulation for 11 and 12 to 12.1-fold stimulation for 1
and 2 and for KM from 1.9 ꢂ 10ꢀ6 M for 7 to 6.4 ꢂ 10ꢀ6 M for 3. All
twelvecompoundsentered MDCKII-MDR1 cells as indicated by their
active transport by P-gp [PBA(ꢀinh)/PBA(+inh) >12, Table 2] and by
their facilitation of CAM uptake (Table 1). Thus, all of the compounds
1–12 bind to transport sites including the half-julolidyl amide 11
and thioamide 12 that have Vmax values at or near basal levels.
Compounds 11 and 12, while showing no apparent ATPase
stimulation in isolated P-gp, inhibited VER-induced ATPase activ-
ity with IC50’s of (1.1 0.2) ꢂ 10ꢀ6 and (1.7 0.3) ꢂ 10ꢀ6 M,
respectively. Compounds 11 and 12 act as inhibitors in the isolated
protein.
In the series of compounds 1–12, certain structural features
appear critical for high affinity and impact on ATPase activity.
Thiorhodamine compounds 5–12 bearing the more hydrophobic
julolidyl and half-julolidyl fragments displayed significantly higher
affinity for P-gp as indicated by lower values of KM (p <0.03,
Student t-test) relative to the corresponding thiorhodamine com-
pounds 1–4 bearing dimethylamino fragments (Table 1). Further-
more, the increased affinity for compounds 5–12 bearing the
julolidyl and half-julolidyl fragments gave decreased P-gp ATPase
stimulation that was statistically significant (p <0.05, Student
t-test) relative to the corresponding thiorhodamine compounds
1–4 bearing dimethylamino fragments. The presence of a thioam-
ide or an amide group in thiorhodamines 1–12 gave no predictable
trend in either Vmax or KM in isolated protein.
In whole cell experiments, values of IC50 for CAM uptake were
fivefold higher for amides 1, 3, and 7 relative to their correspond-
ing thioamides 2, 4, and 8 (Table 1). For the remaining six com-
pounds (5, 6, and 9–12), IC50’s for CAM uptake were similar for
both amides and thioamides and were similar to IC50 values for
2, 4 and 8. Values of IC50 for VIN efflux were also quite similar
for both compounds in the amide/thioamide pairs among com-
pounds 9–12 (Table 1).
Normally, the role of P-gp in the transport of various molecules
is determined by comparison of transport in the absorptive (apical
to basolateral or AB) and secretory (basolateral to apical or BA)
direction of the cell monolayer, and then an efflux ratio (PBA/AB
)
is calculated.37 In the case of thiorhodamine compounds 1–12,
transport in the absorptive direction was too slow
(PAB 6 1 ꢂ 10ꢀ9 m sꢀ1) for measurement and an accurate efflux ra-
tio (PBA/AB) could not be calculated. Consequently, a ratio of trans-
port in the secretory direction in the absence and presence of
inhibitor [PBA(ꢀinh)/PBA(+inh)] is included in Table 2 instead.
Large PBA/AB ratios are assumed to be due to efficient P-gp-med-
iated efflux of the compound and we assume that the large values
of PBA(ꢀinh)/PBA(+inh) observed for 1–12 (14–170, Table 2) are also
consistent with these compounds being actively transported by P-
gp. In every amide/thioamide pair, PBA for the amide is greater than
the thioamide. For the amides, PBA is in the range (2.1–
3.7) ꢂ 10ꢀ7 m sꢀ1 and, for the thioamides, PBA is in the range
(0.34–2.1) ꢂ 10ꢀ7 m sꢀ1. In the fully inhibited system, PPassive is
slow for all of the compounds (from <1 ꢂ 10ꢀ9 m sꢀ1 to
1.4 ꢂ 10ꢀ8 m sꢀ1) and values of PPassive are slower for the thioamide
in every amide/thioamide pair.
The % cell-associated dye for thiorhodamines 1–12 was higher
in the inhibited system (12–64%) relative to the uninhibited sys-
tem where the pump was active (2–52%). These differences were
twofold or greater for all compounds except for diethyl thioamide
10 for which the inhibited and uninhibited systems were quite
similar (64% and 52%, respectively). In every amide/thioamide pair,
the % cell-associated dye was higher for the thioamide relative to
the amide in both the inhibited and uninhibited system. The ratio
for % cell-associated dye between inhibited and uninhibited pump
was comparable for both amides and thioamides for the dimethyl-
amino analogues 1–4 (3.9–5.7). For the julolidyl analogues 5–8
and the half-julolidyl analogues 9–12, the ratio for % cell-associ-
ated dye between inhibited and uninhibited pump was much
greater for the amide analogues 5, 7, 9 and 11 (3.3–6.1) than the
thioamide analogues 6, 8, 10 and 12 (1.2–2.1).
2.7. Discussion of biological results
In earlier work, we saw differences among a group of amide-
and thioamide-containing chalcogenorhodamine compounds with
respect to rates of transport across monolayers of MDCKII-MDR1
transfected cells and with respect to ATPase stimulation/inhibition
in isolated protein.15 The amide and thioamide functionality were
located on aryl or heteroaryl substituents at the 9-position of the
chalcogenorhodamine core. The compound with the highest affin-
ity for P-gp in isolated protein and with the lowest values of IC50
for CAM uptake was thiorhodamine 8 with the piperidyl thioamide
at the 5-position of a 2-thienyl group. Compound 8 was trans-
ported extremely slowly in both secretory and absorptive
directions in monolayers of MDCKII-MDR1 transfected cells while
amide analogues related in structure to 8 gave much faster

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4297
One would anticipate that the performance of thiorhodamines
1–12 as inhibitors would be similar in isolated protein or in whole
cells. These compounds effectively interact with P-gp in a native
membrane environment and facilitate the uptake of CAM or inhibit
the P-gp-mediated efflux of VIN. If one compares the magnitude of
KM and the IC50 for inhibition of VER-induced ATPase activity in
isolated protein with the magnitude of the values of IC50 for CAM
uptake (via inhibition of P-gp efflux of CAM) and P-gp-mediated
VIN efflux in whole cells, values for the whole-cell data are 3- to
30-fold higher (i.e., are less effective in cells than in isolated pro-
tein) than values in isolated protein for the julolidyl and half-julol-
idyl derivatives 5–12.
An active pump should limit the % cell-associated dye relative
to the % cell-associated dye in the presence of inhibitor. The amide
systems 5, 7, 9 and 11 have ratios for % cell-associated dye between
inhibited and uninhibited pump that are between 3.3 and 6.1.
Clearly, the active pump [absence of added 31 (LSN 335984)] limits
the amount of cell-associated dye. For the thioamide analogues 6,
8, 10 and 12, ratios for % cell-associated dye between inhibited
and uninhibited pump were much closer to a value of 1 (1.2–2.1,
Table 2), which suggests that the pump is inhibited even in the ab-
sence of added 31.
3. Conclusions
There are numerous reasons why effective concentrations in the
cell may be different than effective concentrations in isolated pro-
tein. Differences between the local environment of P-gp as well as
lipid composition and fluidity can impact drug interactions with P-
gp.44,45 These differences are not a simple function of lipophilicity
among the thiorhodamines 1–12 since values of logP, the n-octa-
nol/water partition coefficient, fall in the range of 1.2–2.7 (Table
1). All compounds should have access to aqueous and lipophilic
environments.
Alternatively, these data suggest that the thiorhodamines may
have limited availability to the pump in the native environment
especially when one considers that rates of transport in the absorp-
tive direction (PAB) were all 61 nm sꢀ1 and that values of PPassive
were all 614 nm sꢀ1 (Table 2). This is consistent with both active
and passive replenishing of the membrane concentration in the
native environment lagging behind active removal mediated by
the pump [PBA in the uninhibited system in the range (0.34–
3.7) ꢂ 10ꢀ7 m sꢀ1]. If the available membrane concentrations are
indeed low, then more drug will be required in the whole cell
system to raise membrane concentrations effectively to levels that
result in pump inhibition.
The rate of secretory transport (PBA) of thiorhodamines 1–12
across monolayers of MDCKII-MDR1 transfected cells was sensitive
to both the amine fragment (dimethylamino, julolidyl, half-julol-
idyl) and the amide/thioamide functionality. In every example,
transport of the thioamide was slower than transport of the amide
both in the uninhibited and fully inhibited systems (Table 2). In the
fully inhibited system, PBA(+inh) was 65 ꢂ 10ꢀ9 m sꢀ1for all of the
thioamide derivatives while PBA(+inh) for the amide derivatives
were in the range (7.5–27) ꢂ 10ꢀ9 m sꢀ1. In the uninhibited sys-
tem, transport of the amide/thioamide pair was most similar for
thiorhodamines 1 and 2 with the dimethylamino fragment where
the ratio of PBA (amide)/PBA (thioamide) was 1.3. For the remaining
amide/thioamide pairs, the ratio of PBA (amide)/PBA (thioamide)
was in the range 2.6–6.8 and PBA (amide)/PBA (thioamide) ratios
were larger for the half-julolidyl derivatives (5.5 for 9/10 and 6.8
for 11/12) relative to the julolidyl derivatives (3.2 for 5/6 and 2.7
for 7/8).
An initial lead comparing amide and thioamide functionality in
two thiorhodamine derivatives suggested that thioamides might
be better inhibitors of P-gp than amide derivatives.15 We have
examined a small segment of structure–activity space surrounding
this observation through a series of thiorhodamine derivatives 1–
12 varying in amide versus thioamide functionality on a 2-thienyl
substituent at the 9-position of the thiorhodamine core and vary-
ing in an amine fragment (dimethylamino, julolidyl, half-julolidyl)
at the 3-position of the thiorhodamine core. While overall differ-
ences are small within this series, certain trends emerge. The net
effect of all the compounds 1–12 is to function as an inhibitor of
P-gp in the native environment of whole cells as measured by
IC50 values for the uptake of CAM. Compounds 9–12 also have
the net effect of functioning as inhibitors of P-gp as measured for
IC50 values for inhibition of P-gp-mediated efflux of tritiated VIN.
Derivatives with julolidyl and half-julolidyl fragments have lower
values of IC50 for CAM uptake in whole cells than derivatives with
a dimethylamino fragment suggesting that these derivatives have
higher affinity for P-gp. Values of KM for the julolidyl thiorhodam-
ines 5–8 and half-julolidyl thiorhodamines 9–12 are lower than
values of KM for the dimethylamino-derivatives 1–4, which is con-
sistent with a higher affinity
The thioamide derivatives are transported quite slowly in both
absorptive and secretory directions in monolayers of MDCKII-
MDR1 transfected cells suggesting that the thioamide derivatives
have limited availability to the pump in the native membrane envi-
ronment in this model system. Understanding the factors that
determine this availability in the amide/thioamide pairs may help
in the development of more potent inhibitors of P-gp that may
function also as photosensitizers for PDT of multidrug-resistant
cells. The slow transport of the thioamide analogues from P-gp-
expressing cells suggests that heavy-atom analogues of 6, 8, 10,
and 12 should be efficient photosensitizers for the treatment of
multidrug-resistant cells with PDT. The corresponding selenorhod-
amines should be available via similar synthetic chemistry.
4. Experimental
All of the thiorhodamines in the series 1–12 were substrates for
P-gp as indicated by the large values of PBA(ꢀinh)/PBA(+inh) (>12,
Table 2). An interesting correlation can be found with the % cell-
associated dye in the absence of the added inhibitor 31 (LSN
335984). The % cell-associated dye was higher for the thioamide
derivatives relative to the amide derivatives in the uninhibited sys-
tem (Table 2). An active pump should limit the % cell-associated
dye and the results are consistent with increased inhibition with
the thioamides. More hydrophobic compounds would be expected
to have a higher local concentration in the membrane (% cell-asso-
ciated dye) and have a greater potential both to inhibit and to in-
voke recognition by P-gp. This is reflected in the higher values of
% cell-associated dye for the more hydrophobic thioamides 6, 8,
10, and 12 relative to 2 and 4, suggesting more inhibition in the
former set of compounds relative to the latter.
4.1. General methods
Thiorhodamines 415 and 815 and thioxanthones 1319 and 1420
were prepared by literature methods. Reactions were run under ar-
gon. Tetrahydrofuran (THF) was distilled from sodium benzophe-
none ketyl prior to use. Reactions were concentrated in vacuo on
a Büchi 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 Büchi
capillary melting point apparatus and are uncorrected. All

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A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
compounds tested have a purity of at least 95%, which was deter-
mined 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 and mon-
itoring by a UV–visible detector operating at 254 or 442 nm.
from CH2Cl2/Et2O, to give 171 mg (70.3%) of thiorhodamine 3 as
a purple solid, mp 242–244 °C. 1H NMR (500 MHz, CD2Cl2) d 7.72
(d, 2H, J = 9.5 Hz), 7.43 (d, 1H, J = 4.0 Hz), 7.18 (d, 1H, J = 4.0 Hz),
7.10 (d, 2H, J = 2.5 Hz), 7.02 (d ꢂ d, 2H, J = 9.5, 2.5 Hz), 3.74 (t,
4H, J = 5.0 Hz), 3.30 (s, 12H), 1.79–1.72 (m, 2H), 1.71–1.66 (m,
4H); 13C NMR (75.5 MHz, CD2Cl2) d 162.0, 153.9, 152.0, 144.4,
141.9, 137.4, 136.3, 130.7, 128.5, 120.1, 116.0, 105.8, 47.1 (br),
41.0, 26.5 (br), 24.9; IR (film on NaCl) 2933, 2850, 1593 cmꢀ1; kmax
4.1.1. Preparation of 3,6-bis(dimethylamino)-9-(5-
(diethylcarbamothioyl)thiophen-2-yl)thioxanthylium
hexafluorophosphate (2)
n-Butyllithium (0.890 M in hexanes, 5.86 mL, 5.23 mmol,
in CH2Cl2 (loge) 308 (4.68), 338 (sh), 565 (sh), 609 nm (4.98);
3.9 equiv) was added to
a
solution of N,N-diisopropylamine
HRMS (ESI) m/z 476.1833 (calcd for C26H30N3S3+: 476.1825).
(0.889 mL, 6.30 mmol, 4.7 equiv) in THF (10 mL) at ꢀ78 °C. The
resulting mixture was stirred for 0.5 h before it was added to a
solution of N,N-diethylthiophene-2-carbothioamide (1.07 g,
5.36 mmol, 4 equiv) in THF (40 mL) at ꢀ78 °C. The resulting solu-
tion was stirred for 2 min before it was transferred via cannula into
a solution of 3,6-bis-(dimethylamino)-9-thioxanthen-9-one19 (13,
400 mg, 1.34 mmol) in THF (20 mL) at ambient temperature with
stirring. The reaction mixture was heated to 40 °C for 0.5 h, and
then cooled to ambient temperature. Glacial acetic acid (2 mL)
was added and the mixture was poured into a 10% v/v aqueous
HPF6 solution at 0 °C. The resulting precipitate was collected via
vacuum filtration after 3 h of stirring and washed with water
(30 mL) and diethyl ether (50 mL). The product was purified via
column chromatography (SiO2, 1:9 Et2O/CH2Cl2, Rf = 0.40), fol-
lowed by recrystallization from CH2Cl2/Et2O to give 514 mg
(61.3%) of thioamide 2 as a purple solid, mp 248–250 °C. 1H NMR
4.1.4. Preparation of 12-(dimethylamino)-2,3,6,7-tetrahydro-9-
(5-(diethylcarbamothioyl)-thiophen-2-yl)-1H,5H-thioxantheno
[2,3,4-ij]quinolizin-14-ium hexafluorophosphate (6)
n-Butyllithium (0.920 M in hexanes, 4.24 mL, 3.90 mmol), N,N-
diisopropylamine (0.663 mL, 4.70 mmol) in THF (10 mL) and N,N-
diethylthiophene-2-carbothioamide (797 mg, 4.00 mmol) in THF
(80 mL) at ꢀ78 °C were treated as described in the preparation of
thiorhodamine 2.
A
solution of thioxanthone 1420 (350 mg,
0.999 mmol) in THF (30 mL) was treated with the thienyl anion
as described for the preparation of thioamide 2. The crude product
was purified via column chromatography (SiO2 gel, 1:9 Et2O/
CH2Cl2, Rf = 0.50), followed by recrystallization from CH2Cl2/Et2O
to give 0.381 g (56.2%) of thiorhodamine 6 as a purple solid, mp
153–155 °C: 1H NMR (500 MHz, CD2Cl2) d 7.65 (d, 1H, J = 10 Hz),
7.39 (s, 1H), 7.24 (d, 1H, J = 4.0 Hz), 7.09 (d, 1H, J = 2.5 Hz), 7.07
(d, 1H, J = 4.0 Hz), 6.99 (dd, 1H, J = 2.5, 10 Hz), 4.12 (br s, 2H),
3.89 (br s, 2H), 3.56 (t, 4H, J = 6.0 Hz), 3.25 (s, 6H), 2.86 (t, 2H,
J = 6.0 Hz), 2.78 (t, 2H, J = 6.0 Hz), 2.18 (quintet, 2H, J = 6.0 Hz),
2.01 (quintet, 2H, J = 6.0 Hz), 1.41 (t, 6H, J = 7.0 Hz); 13C NMR
(75.5 MHz, CD2Cl2) d 188.6, 153.1, 149.7, 149.4, 149.1, 142.3,
140.0, 139.0, 135.3, 132.9, 130.3, 126.5, 120.3, 119.1, 115.2,
114.4, 105.9, 51.9, 50.9, 48.9 (br), 48.3 (br), 40.7, 28.2, 24.3, 20.6,
20.1, 14.3 (br), 11.1 (br); IR (film on NaCl) 2932, 1593, 1456,
(500 MHz, CD2Cl2)
d 7.78 (d, 2H, J = 10 Hz), 7.25 (d, 1H,
J = 3.5 Hz), 7.11 (d, 1H, J = 3.5 Hz), 7.09 (d, 2H, J = 2.5 Hz), 7.05
(dd, 2H, J = 2.5, 10 Hz), 4.12 (br s, 2H), 3.87 (br s, 2H), 3.30 (s,
12H), 1.41 (t, 6H, J = 2.0 Hz); 13C NMR (75.5 MHz, CD2Cl2) d
188.5, 153.9, 151.9, 149.6, 144.4, 137.9, 136.4, 130.5, 124.7,
120.1, 116.0, 105.8, 45.2 (br), 44.5 (br), 37.2, 10.6 (br), 7.4 (br);
IR (film on NaCl) 2933, 1594, 1498, 1446, 1391, 1364, 1343,
1252 cmꢀ1; kmax in CH2Cl2 (log
e) 308 (4.76), 339 (sh), 568 (sh),
610 nm (5.02); HRMS (ESI) m/z 480.1613 (calcd for C26H30N3S3
:
1387, 1364, 1315, 1180 cmꢀ1; kmax in CH2Cl2 (log
e) 310 (4.74),
+
480.1596).
339 (sh), 578 (sh), 621 nm (5.04); HRMS (ESI) m/z 532.1917 (calcd
for C30H34N3S3+: 532.1909).
4.1.2. Preparation of 3,6-bis(dimethylamino)-9-(5-
(diethylcarbamoyl)thiophen-2-yl)thioxan-thylium
hexafluorophosphate (1)15
4.1.5. Preparation of 12-(dimethylamino)-2,3,6,7-tetrahydro-9-
(5-(diethylcarbamoyl)thiophen-2-yl)-1H,5H-thioxantheno
[2,3,4-ij]quinolizin-14-ium hexafluorophosphate (5)
Trifluoroacetic anhydride (0.449 mL, 3.20 mmol, 10 equiv) was
added dropwise to a solution of thioamide 2 (200 mg, 0.320 mmol)
in CH2Cl2 (30 mL). The mixture was heated at reflux for 2 h, and
then cooled to ambient temperature. A solution of 10% Na2CO3
(20 mL) was added, and the resulting mixture was extracted with
CH2Cl2 (3 ꢂ 15 mL). The combined organic extracts were concen-
trated. The crude product was purified via column chromatography
(SiO2, 1:9 Et2O/CH2Cl2, Rf = 0.20), followed by recrystallization
from CH2Cl2/Et2O, to give 123 mg (63.0%) of thiorhodamine 1 as
a purple solid, mp 264–266 °C (lit.15 mp: 264–266 °C): 1H NMR
Trifluoroacetic anhydride (0.307 mL, 2.21 mmol, 7.5 equiv) and
thiorhodamine
6 (200 mg, 0.295 mmol, 1 equiv) in CH2Cl2
(30 mL) were treated as described for the preparation of 1. The
crude product was purified via column chromatography (SiO2,
1:9 Et2O/CH2Cl2, Rf = 0.25), followed by recrystallization from
CH2Cl2/Et2O, to give 105 mg (54.0%) of thiorhodamine 5 as a purple
solid, mp 159–161 °C. 1H NMR (500 MHz, CD2Cl2) d 7.58 (d, 1H,
J = 9.5 Hz), 7.48 (d, 1H, J = 3.5 Hz), 7.32 (s, 1H), 7.14 (d, 1H,
J = 3.5 Hz), 7.10 (d, 1H, J = 2.5 Hz), 6.96 (dd, 1H, J = 2.5, 9.5 Hz),
3.62 (br s, 4H), 3.56 (t, 4H, J = 6.0 Hz), 3.25 (s, 6H), 2.86 (t, 2H,
J = 6.0 Hz), 2.76 (t, 2H, J = 6.0 Hz), 2.18 (quintet, 2H, J = 6.0 Hz),
2.00 (quintet, 2H, J = 6.0 Hz), 1.38–1.23 (m, 6H); IR (film on NaCl)
(500 MHz, CD2Cl2)
d 7.72 (d, 2H, J = 10 Hz), 7.48 (d, 1H,
J = 3.5 Hz), 7.18 (d, 1H, J = 3.5 Hz), 7.10 (d, 2H, J = 2.5 Hz), 7.01
(dd, 2H, J = 2.5, 10 Hz), 3.61 (br s, 4H), 3.30 (s, 12H), 1.31 (br s,
6H); 13C NMR (75.5 MHz, CD2Cl2) d 162.4, 153.8, 151.8, 144.2,
142.7, 137.7, 136.2, 130.9, 128.0, 120.0, 115.9, 105.8, 43.3 (br),
2934, 1593 cmꢀ1; kmax in CH2Cl2 (log
(sh), 621 nm (5.01); HRMS (ESI) m/z 516.2144 (calcd for
30H34N3OS2+: 516.2138).
e) 310 (4.65), 341 (sh), 577
40.9, 13.5 (br); kmax in CH2Cl2 (log
e) 313 (4.85), 343 (sh), 574
C
(sh), 615 nm (5.13)
4.1.6. Preparation of 12-(dimethylamino)-2,3,6,7-tetrahydro-9-
(N-piperidyl-2-thienyl-5-carboxamido)-1H,5H-thioxantheno
[2,3,4-ij]quinolizin-14-ium hexafluorophosphate (7)
Trifluoroacetic anhydride (0.754 mL, 5.43 mmol) and thiorhod-
amine 815 (250 mg, 0.362 mmol) in CH2Cl2 (30 mL) were treated
as described for the preparation of 1. The crude product was
purified via column chromatography (SiO2, 1:9 Et2O/CH2Cl2,
Rf = 0.25), followed by recrystallization from CH2Cl2/Et2O, to give
132 mg (54.2% yield) of thiorhodamine 7 as a purple solid, mp
4.1.3. Preparation of 3,6-bis(dimethylamino)-9-(5-(piperidine-
1-carbonyl)thiophen-2-yl)thio-xanthylium hexafluorophos
phate (3)
Trifluoroacetic anhydride (0.272 mL, 1.96 mmol, 5 equiv) and
thiorhodamine 415 (250 mg, 0.392 mmol, 1 equiv) in CH2Cl2
(30 mL) were treated as described for the preparation of 1 from
2. The crude product was purified via column chromatography
(SiO2, 1:9 Et2O/CH2Cl2, Rf = 0.25), followed by recrystallization

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4299
183–185 °C. 1H NMR (500 MHz, CD2Cl2) d 7.59 (d, 1H, J = 10 Hz),
7.43 (d, 1H, J = 3.5 Hz), 7.34 (s, 1H), 7.13 (d, 1H, J = 3.5 Hz), 7.10
(d, 1H, J = 2.5 Hz), 6.96 (dd, 1H, J = 2.5, 10 Hz), 3.74 (t, 4H,
J = 5.0 Hz), 3.56 (t, 4H, J = 6.0 Hz), 3.24 (s, 6H), 2.86 (t, 2H,
J = 6.0 Hz), 2.76 (t, 2H, J = 6.0 Hz), 2.18 (quintet, 2H, J = 6.0 Hz),
2.01 (quintet, 2H, J = 6.0 Hz), 1.79–1.72 (m, 2H), 1.72–1.66 (m,
4H); 13C NMR (75.5 MHz, CD2Cl2) d 162.1, 153.0, 149.7, 149.5,
142.2, 141.5, 140.0, 138.3, 135.2, 132.9, 130.4, 128.5, 126.5,
120.4, 119.2, 115.2, 114.4, 105.9, 51.9, 50.9, 47.7 (br), 40.6, 28.1,
26.5 (br), 24.8, 24.3, 20.6, 20.1; IR (film on NaCl) 2937, 2854,
stirred 0.5 h at ꢀ78 °C and was then was added slowly to a solution
of piperidin-1-yl(thiophen-2-yl)methanethione (1.05 g, 4.97 mmol)
in THF (20 mL) at ꢀ78 °C. The resulting mixture was stirred 5 min
before it was transferred via cannula into a solution of thioxanthone
15 (500 mg, 1.42 mmol) in THF (10 mL) at ambient temperature. The
resulting mixture was stirred for 0.5 h at 35 °C. After cooling to
ambient temperature, glacial acetic acid (2 mL) was added, and
the reaction mixture was poured into a 10% v/v solution of stirring,
cold aqueous HPF6. The resulting precipitate was collected via filtra-
tion after 1 h of stirring and washed with water (10 mL) and diethyl
ether (10 mL). The product was purified via column chromatogra-
phy (SiO2, 4% MeOH/CH2Cl2) followed by recrystallization from
MeOH/ether to give 511 mg (52.1%) of 12 as dark blue crystals, mp
168–170 °C. 1H NMR (500 MHz, CD3CN) d 7.72 (d, 1H, J = 9.5 Hz),
7.50 (s, 1H), 7.25 (d, 1H, J = 3.6 Hz), 7.18 (d, 1H, J = 4.0 Hz), 7.13 (d,
2H, J = 2.8 Hz), 7.07 (d ꢂ d, 1H, J = 2.8, 9.5 Hz), 4.31 (br s, 2H), 3.99
(br s, 2H), 3.60 (t, 2H, J = 6.0 Hz), 3.22 (s, 9H), 1.78 (br s, 6H), 1.75
(t, 2H, 6.0 Hz), 1.13 (s, 6H); 13C NMR (75.6 MHz, CD2Cl2) d 188.9,
153.5, 151.2, 150.8, 149.1, 143.7, 143.4, 138.1, 136.0, 130.4, 125.2,
120.5, 119.7, 115.6, 105.8, 105.0, 52.4 (br), 49.0, 40.9, 40.4, 34.5,
1593 cmꢀ1; kmax in CH2Cl2 (log
e) 310 (4.65), 341 (sh), 578 (sh),
+
622 nm (5.00); HRMS (ESI) m/z 528.2153 (calcd for C31H34N3OS2
:
528.2138).
4.1.7. Preparation of 6-(5-(diethylcarbamothioyl)thiophen-2-
yl)-9-(dimethylamino)-1,4,4-trimethyl-2,3,4,6-tetrahydro-1H-
thiochromeno[3,2-g]quinolin-6-ylium hexafluorophosphate
(10)
n-Butyllithium (0.891 M, 2.79 mL, 2.48 mmol), diisopropyl-
amine (0.370 mL, 2.62 mmol), and piperidin-1-yl(thiophen-2-
yl)methanethione (495 mg, 2.48 mmol) in THF (20 mL) were treated
as described for the preparation of thioamide 2. A solution of
9-(dimethylamino)-1,4,4-trimethyl-3,4-dihydro-1H-thiochromeno
[3,2-g]quinolin-6(2H)-one (15, 250 mg, 0.709 mmol) in THF (10 mL)
was treated with the thienyl anion as described for the preparation
of 2. The crude product was purified via column chromatography
(SiO2, 4% MeOH/CH2Cl2) followed by recrystallization from MeOH/
ether to give 28.4 mg (15%) of 10 as dark blue crystals, mp 130–
132 °C. 1H NMR (500 MHz, CD2Cl2) d 7.79 (d, 1H, J = 9.5 Hz), 7.59
(s, 1H), 7.26 (d, 1H, J = 3.5 Hz), 7.10 (d, 1H, J = 3.5 Hz), 7.06 (d, 1H,
J = 2.0 Hz), 7.02 (s, 1H), 7.01 (d ꢂ d, 1H, J = 3.0 Hz, 10 Hz), 4.12 (br
s, 2H), 3.87 (br s, 2H), 3.65 (t, 2H, J = 6.0 Hz), 3.27 (s, 9H), 1.82 (t,
2H, 6 Hz), 1.20 (s, 6H); 13C NMR (75.6 MHz, CD2Cl2) d 188.8, 153.5,
151.2, 150.8, 149.3, 143.7, 143.4, 137.9, 136.0, 135.9, 130.3, 130.2,
124.7, 120.5, 119.7, 115.6, 105.7, 105.0, 48.9, 48.1 (br), 40.8, 40.4,
32.4, 28.6, 27.2 (br), 24.5; IR (film, CCl4)
1447, 1389, 1330, 1252 cmꢀ1; kmax in CH2Cl2 (log
(sh), 579 (sh), 620 nm (4.99); HRMS (ESI) m/z 546.2071 (calcd for
31H36N3S3+: 546.2066).
mmax: 2936, 1594, 1479,
e
) 312 (4.78), 345
C
4.1.10. Preparation of 9-(dimethylamino)-1,4,4-trimethyl-6-(5-
(piperidine-1-carbonyl)thiophen-2-yl)-3,4-dihydro-2H-
thiochromeno[3,2-g]quinolin-1-ium hexafluorophosphate (11)
Trifluoroacetic anhydride (0.080 mL, 0.58 mmol) and thiorhod-
amine 12 (200 mg, 0.289 mmol) in anhydrous CH2Cl2 (10 mL) were
treated as described for the preparation of amide 1. The product
was purified via column chromatography (SiO2, 10% Et2O/CH2Cl2)
to give 98.0 mg (50.1%) of 11 as dark blue crystals, mp 142–
147 °C. 1H NMR (500 MHz, CD2Cl2) d 7.72 (d, 1H, J = 9.5 Hz), 7.53
(s, 1H), 7.44 (d, 1H, J = 3.5 Hz), 7.17 (d, 1H, J = 3.5 Hz), 7.06 (d,
1H, J = 3.0 Hz), 7.02 (s, 1H), 6.99 (dd, 1H, J = 2.5, 9.5 Hz), 3.74 (t,
4H, J = 6.0 Hz), 3.65 (t, 2H, J = 6.5 Hz), 3.28 (s, 3H), 3.27 (s, 6H),
1.81 (t, 2H, J = 6.5 Hz), 1.74–1.77 (m, 2H), 1.66–1.72 (m 4H), 1.18
(s, 6H); 13C NMR (75.6 MHz, CD2Cl2) d 162.2, 153.5, 151.2, 150.7,
143.7, 143.5, 141.7, 137.9, 136.0, 135.9, 130.6, 130.2, 128.4,
120.5, 119.8, 115.6, 105.8, 105.2, 48.9, 47.1 (br) 40.9, 40.4, 34.5,
34.5, 32.3, 28.6, 14.7 (br); IR (film, CCl4)
1446, 1389, 1330, 1251 cmꢀ1; kmax in CH2Cl2 (log
(sh), 576 (sh), 619 nm (5.04). HRMS (ESI) m/z 534.2060 (calcd for
30H36N3S3+: 534.2066).
m
max: 2937, 1594, 1480,
e) 312 (4.83), 346
C
4.1.8. Preparation of 6-(5-(diethylcarbamoyl)thiophen-2-yl)-9-
(dimethylamino)-1,4,4-trimethyl-2,3,4,6-tetrahydro-1H-
32.3, 28.5, 26.5 (br), 24.9; IR (film, CCl4)
m
max: 2936, 1594, 1478,
thiochromeno[3,2-g]quinolin-6-ylium hexafluorophosphate (9)
Trifluoroacetic anhydride (0.082 mL, 0.59 mmol) and thiorhod-
amine 10 (200.0 mg, 0.294 mmol) in anhydrous CH2Cl2 (10 mL)
were treated as described for the preparation of 1. The crude product
was purified via column chromatography (SiO2, 10% Et2O/CH2Cl2) to
give 101 mg (51%) of 9 as dark blue crystals, mp 155–160 °C. 1H NMR
(500 MHz, CD2Cl2) d 7.69 (d, 1H, J = 9.5 Hz), 7.54 (s, 1H), 7.50 (d, 1H,
J = 3.5 Hz), 7.18 (d, 1H, J = 4.0 Hz), 7.07 (d, 1H, J = 2.0 Hz), 7.03 (s, 1H),
6.98 (dd, 1H, J = 9.5 Hz, 2.0 Hz), 3.65 (t, 2H, J = 6.5 Hz), 3.61 (br s, 4H,),
3.29 (s, 3H), 3.27 (s, 6H), 1.81 (t, 2H, J = 6.5 Hz), 1.32 (t, 6H, J = 6.5 Hz),
1.18 (s, 6H); 13C NMR (75.6 MHz, CD2Cl2) d 162.6, 153.5, 151.3, 150.9,
143.7, 143.4, 142.8, 138.1, 136.1, 135.9, 130.7, 130.3, 128.1, 120.5,
119.8, 115.6, 105.7, 105.1, 49.0, 42.8 (br), 40.9, 40.4, 34.5, 32.3,
1448, 1389, 1330, 1253 cmꢀ1; kmax in CH2Cl2 (log
e
) 313 (4.81),
344 (sh), 576 (sh), 619 nm (5.08); HRMS (ESI) m/z 530.2288 (calcd
for C31H36O1N3S2+: 530.2294).
4.1.11. Preparation of N-methyl-N-(3-methylbut-2-enyl)aniline
(16)46
N-Methylaniline (7.13 g, 66.6 mmol) and potassium carbonate
(9.20 g, 66.6 mmol) were stirred for 10 min at ambient temperature
in CH3CN (100 mL). 1-Chloro-3-methyl-2-butene (4.64 g,
88.7 mmol) was added slowly over 10 min. The resulting mixture
was heated at 40 °C for 20 h. After cooling to ambient temperature,
water (100 mL) was added and products were extracted with CH2Cl2
(5 ꢂ 50 mL). The organic fractions were dried over anhydrous
MgSO4 and concentrated. The resulting red-brown oil was purified
via column chromatography (SiO2, 30:70 CH2Cl2/hexanes) to give
10.1 g (86.7%) of 16. 1H NMR (500 MHz, CDCl3) d 7.25–7.20 (m,
2H), 6.74 (d, 2H, J = 8.0 Hz), 6.71 (t, 1H, J = 7.0 Hz), 5.22 (t, 1H,
J = 5.0 Hz), 3.89 (d, 2H, J = 6.0 Hz), 2.89 (s, 3H), 1.72 (d, 6H,
J = 1.0 Hz). HRMS (EI) m/z 175.1359 (calcd for C12H17N1+: 175.1356).
28.6, 13.9 (br); IR (film, CCl4)
1330, 1254 cmꢀ1; kmax in CH2Cl2 (log
(sh), 618 nm (5.08); HRMS (ESI) m/z 518.2299 (calcd for
30H36O1N3S2+: 518.2294).
m
max: 2935, 1593, 1477, 1448,1390,
e) 313 (4.81), 346 (sh), 575
C
4.1.9. Preparation of 9-(dimethylamino)-1,4,4-trimethyl-6-(5-
(piperidine-1-carbono-thioyl)thiophen-2-yl)-2,3,4,6-
tetrahydro-1H-thiochromeno[3,2-g]quinolin-6-ylium hexa-
fluorophosphate (12)
4.1.12. Preparation of 1,4,4-trimethyl-1,2,3,4-
tetrahydroquinoline (17)47
A solution of n-butyllithium (0.891 M in hexanes, 5.58 mL,
4.97 mmol) and N,N-diisopropylamine (0.740 mL, 5.25 mmol) was
Concentrated sulfuric acid (1.5 mL) was slowly added to 16
(0.500 g, 2.85 mmol) at 0 °C. After 1 h at 0 °C, ice water (150 mL)

4300
A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
+
was added. The product was extracted with CH2Cl2 (5 ꢂ 50 mL).
The combined organic extracts were dried over anhydrous MgSO4
and concentrated. The resulting brown oil was purified via chro-
matography (SiO2, 1:9 Et2O/CH2Cl2) to give 380 mg (76.0%) of 17.
1H NMR (500 MHz, CDCl3) d 7.20 (dd, 1H, J = 2.0, 7.2 Hz), 7.09 (td,
1H, J = 1.5, 8.4 Hz), 6.67 (td, 1H, J = 1.5, 7.2 Hz), 6.60 (dd, 1H,
J = 1.5, 8.0 Hz), 3.23 (t, 2H, J = 6.0 Hz), 2.90 (s, 3H), 1.79 (t, 2H,
J = 6.0 Hz), 1.28 (s, 6H). HRMS (EI) m/z 175.1357 (calcd for
786, 764 cmꢀ1; HRMS (EI) m/z 287.2119 (calcd for C18H27O1N2
287.2118).
:
4.1.16. Preparation of (7-(3-(dimethylamino)phenylthio)-1,4,4-
trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)(piperidin-1-
yl)methanone (22)
sec-Butyllithium (1.0 M in cyclohexane, 1.05 mL, 1.05 mmol)
was added dropwise to a stirred solution of N,N,N0,N0-tetramethyl-
ethylenediamine (150 mg, 1.14 mmol) and 21 (250 mg,
0.873 mmol) in 90 mL of THF at ꢀ78 °C. A solution of bis-3-(N,N-
dimethylamino)phenyldisulfide (1.38 g, 4.54 mmol) in tetrahydro-
furan at ꢀ78 °C was immediately added, and the resulting mixture
was stirred at ꢀ78 °C for 1 h and was then stirred at room temper-
ature for 1 h. Aqueous saturated ammonium chloride (15 mL) was
added to the resulting mixture and the products were extracted
with CH2Cl2 (3 ꢂ 50 mL). The organic extracts were dried over
anhydrous MgSO4 and concentrated. The resulting yellow oil was
purified via column chromatography (SiO2, 1:9 Et2O/CH2Cl2) to
give 55.5 mg (37.4%) of 22 as a yellow-brown oil. 1H NMR
(500 MHz, CDCl3) d 7.12 (t, 1H, J = 8.0 Hz), 7.02 (s, 1H), 6.80 (t,
1H, J = 2.0), 6.71 (d, 1H, J = 7.6 Hz), 6.59 (d ꢂ d, 1H, J = 2.8,
8.4 Hz), 6.50 (s, 1H), 3.67 (br s, 2H), 3.21 (t, 4H, J = 6.0 Hz), 2.91
(s, 6H), 2.73 (s, 3H), 1.72 (t, 2H, J = 6.0 Hz), 1.60 (br s, 4H), 1.46
(br s, 2H), 1.24 (s, 6H); 13C NMR (75.5 MHz, CDCl3) d 169.4,
151.0, 145.8, 136.3, 130.9, 130.5, 129.4, 126.2, 124.5, 119.6,
C
12H17N1+: 175.1356).
4.1.13. Preparation of 1,4,4-trimethyl-1,2,3,4-
tetrahydroquinoline-6-carbaldehyde (18)48
Phosphorus oxychloride (481 mg, 3.14 mmol) was added slowly
to N,N-dimethylformamide (DMF) (2.5 mL) at 0 °C. Compound 17
(500 mg, 2.85 mmol) in dry DMF (2.5 mL) was added and the
resulting mixture was heated at reflux for 1 h. After cooling to
ambient temperature, cold NaOH (100 mL, 1 M) was added, and
the product was extracted diethyl ether (5 ꢂ 50 mL). The combined
organic fractions were washed with water (4 ꢂ 50 mL) and brine
(5 ꢂ 50 mL), were dried over anhydrous MgSO4 and concentrated.
The product was purified via chromatography (SiO2, 1:9 Et2O/
CH2Cl2) to give 560 mg (96.6%) of 18 as a yellow solid, mp 48–
50 °C. 1H NMR (500 MHz, CDCl3) d 9.69 (s, 1H), 7.71 (s, 1H), 7.57
(d, 1H, J = 8.5 Hz), 6.58 (d, 1H, J = 8.5 Hz), 3.42 (t, 2H, J = 6.0 Hz),
3.03 (s, 3H), 1.76 (t, 2H, J = 6.0 Hz), 1.30 (s, 6H); 13C NMR
(75.5 MHz, CDCl3) d 190.9, 150.6, 131.9, 131.3, 127.2, 125.5,
110.2, 48.1, 39.6, 36.5, 32.4, 30.3.
115.5, 113.7, 111.2, 48.2, 47.5, 42.9, 40.5, 39.0, 36.8, 31.9, 30.6,
+
26.0, 24.7; HRMS (EI) m/z 438.2575 (calcd for C26H36O1N3S1
:
438.2574).
4.1.14. Preparation of piperidin-1-yl(1,4,4-trimethyl-1,2,3,4-
tetrahydroquinolin-6-yl)methanethione (19)
4.1.17. Preparation of 9-(dimethylamino)-1,4,4-trimethyl-3,4-
A mixture of 18 (3.19 g, 15.7 mmol), elemental sulfur (1.26 g,
39.2 mmol), piperidine (314 mg, 47.1 mmol) and DMF (5 mL) were
heated at reflux for 0.5 h. Cold water (100 mL) was added to the
resulting mixture and the orange solid that formed was collected
by filtration, dissolved in CH2Cl2, and the resulting solution was
dried over anhydrous MgSO4 and concentrated. The product was
purified via column chromatography (SiO2, CH2Cl2) yielding
3.85 g (81.0%) of 19 as a yellow crystalline solid, mp 150–152 °C.
1H NMR (500 MHz, CDCl3) d 7.18 (m, 1H), 7.16 (d, 1H, J = 2.0 Hz),
6.49 (d, 1H, J = 9.0 Hz), 4.32 (s, 2H), 3.67 (s, 2H), 3.28 (t, 2H,
J = 6.0 Hz), 2.93 (s, 3H), 1.81 (s, 2H), 1.76 (t, 4H, J = 6.0 Hz), 1.60
(s, 2H), 1.26 (s, 6H); 13C NMR (75.5 MHz, CDCl3) d 201.5, 146.1,
130.45, 130.0, 126.6, 124.7, 109.7, 53.6, 51.6, 47.5, 39.1, 36.8,
dihydro-1H-thiochromeno[3,2-g]quinolin-6(2H)-one (15)
Phosphorus oxychloride (496 mg, 3.24 mmol) was added drop-
wise to a mixture of triethylamine (328 mg, 3.24 mmol) and 22
(118 mg, 0.270 mmol) in acetonitrile (25 mL). The resulting red
mixture was stirred at ambient temperature for 5 min before stir-
ring for 2 h at 80 °C. After cooling to ambient temperature, the
resulting mixture was added to stirring, cold aqueous NaOH
(200 mL, 1 M). The products were extracted with dichloromethane
(5 ꢂ 50 mL), and the combined organic extracts were dried over
MgSO4 and concentrated. The product was then purified via col-
umn chromatography (SiO2, 1:9 Et2/CH2Cl2) to give 80.0 mg
(90.3%) of desired product, mp 204–206 °C. 1H NMR (500 MHz,
CDCl3) d 8.44 (d, 1H, J = 9.0 Hz), 8.39 (s, 1H), 6.79 (dd, 1H, J = 2.5,
9.0 Hz), 6.57 (s, 1H), 6.45 (s, 1H), 3.41 (t, 2H, J = 6.0 Hz), 3.07 (s,
6H), 3.02 (s, 3H), 1.78 (t, 2H, J = 6.5 Hz), 1.35 (s, 6H); 13C NMR
(75.5 MHz, CDCl3) d 179.8, 178.5, 165.5, 152.2, 148.3, 139.5,
137.5, 131.4, 126.9, 119.7, 119.1, 111.7, 105.7, 104.3, 48.0, 40.6,
32.0, 30.6, 27.0, 25.7, 24.4; IR (film, CCl4)
1603, 1516, 1473, 1440, 1410, 1330, 1310, 1264, 1238, 1209,
1115, 1017, 786 cmꢀ1
HRMS (EI) m/z 303.1890 (calcd for
18H27N2S1+: 303.1889).
mmax: 2934, 2853, 2363,
;
C
39.6, 36.7, 32.6, 30.4; IR (film, CCl4)
mmax: 2956, 1594, 1514,
4.1.15. Preparation of piperidin-1-yl(1,4,4-trimethyl-1,2,3,4-
tetrahydroquinolin-6-yl)methanone (20)
1320 cmꢀ1; HRMS (EI) m/z 353.1687 (calcd for C21H25O1N2S1
:
+
353.1682).
Trifluoroacetic anhydride (4.06 g, 19.3 mmol) was slowly added
to a solution of 19 (5.84 g, 19.3 mmol) in dry CH2Cl2 (5 mL). The
resulting mixture was stirred 10 min at room temperature. A solu-
tion of 10% aqueous Na2CO3 (100 mL) was added with stirring. The
products were extracted with CH2Cl2 (7 ꢂ 50 mL), and the com-
bined organic extracts were washed with brine (5 ꢂ 50 mL), dried
over MgSO4 and concentrated. The product was purified via col-
umn chromatography (SiO2, 1:9 Et2O/CH2Cl2) to give 5.00 g
(90.4%) of 20 as red-orange crystals, mp 66–68 °C; 1H NMR
(500 MHz, CDCl3) d 7.30 (d, 1H, J = 2.0 Hz), 7.18 (dd, 1H, J = 2.0,
2.5 Hz), 6.52 (d, 1H, J = 8.5 Hz), 3.56 (s, 4H), 3.29 (t, 2H,
J = 6.0 Hz), 2.93 (s, 3H), 1.77 (t, 2H, J = 6.0 Hz), 1.68–1.64 (m, 2H),
1.59 (br s, 4H), 1.27 (s, 6H); 13C NMR (75.5 MHz, CDCl3) d 172.01,
146.94, 130.97, 127.3, 126.3, 123.3, 110.2, 48.0, 47.0, 39.6, 37.3,
4.1.18. Preparation of N,N-diethylthiophene-2-carbothioamide
(27)
Thiophene-2-carbaldehyde (1.00 g, 8.92 mmol), elemental sul-
fur (713 mg, 22.3 mmol), and diethylamine (2.76 mL, 26.8 mmol)
and DMF (5 mL) were combined in a three-neck flask fitted with
reflux condenser. The resulting mixture was allowed to stir 3 h at
110 °C. Ice cold water (100 mL) was added with stirring, and the
mixture was extracted with diethyl ether (8 ꢂ 50 mL) and washed
with brine (5 ꢂ 50 mL). The organic fractions were combined, dried
over anhydrous magnesium sulfate, and concentrated under re-
duced pressure. The product was purified via chromatography (sil-
ica, 10% ethyl acetate/hexanes) to give 0.87 g (49%) of the desired
product as an orange oil. 1H NMR (500 MHz, CDCl3) d 7.35 (dd,
1H, J = 1.0, 5.5 Hz), 7.08 (dd, 1H, J = 1.0, 3.5 Hz), 6.96 (dd, 1H,
J = 3.5, 5.5 Hz), 4.10 (br s, 2H), 3.70 (br s, 2H), 1.34 (br s, 6H); 13C
32.5, 31.1, 26.7, 25.3. IR (film, CCl4)
mmax: 2932, 2852, 2360, 2341,
1607, 1518, 1468, 1428, 1406, 1331, 1265, 1210, 1115, 1008,

A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
4301
NMR (75.5 MHz, CDCl3) d 190.9, 145.0, 127.9, 126.3, 14.9, 48.0 (br),
14.1 (br), 11.1 (br); HRMS: m/z 199.0477 (calcd for C9H13NS2:
compound was replaced to include 0.5
l
g mLꢀ1 CAM and incu-
bated an additional 20 min. Calcein fluorescence was read on a
Cytofluor series 4000 Multi Well Plate Reader (PerSeptive Biosys-
tems) with kEX and kEM set at 485 nm and 530 nm, respectively.
Negative (0.25% DMSO in DPBSH), and positive [5 ꢂ 10ꢀ6 M LSN
335984] controls were included in each plate. IC50 values were cal-
culated from the serial dilution curves using GraphPad PRISM ver-
sion 4.03 software. Briefly, compound concentration was plotted as
199.0484); IR (film)
1418, 1380, 1354, 1305, 1276, 1245, 1210, 1175, 1131, 1095,
1074 cmꢀ1
mmax: 3070, 2969, 2931, 2869, 1520, 1489,
.
4.2. Determination of partition coefficients
The octanol/water partition coefficients were all measured at
pH 7.4 (PBS) using UV–visible spectrophotometry. The measure-
ments were done using a shake flask direct measurement.28 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 1-octanol was obtained from Sigma–Aldrich.
log lM concentration versus relative fluorescence units (rfu) and a
sigmoidal dose-response (variable slope) analysis with no weight-
ing or restrictions was applied.
4.5. P-gp-transport studies across MDCKII-MDR1 monolayers
MDCKII-MDR1 cells that were seeded at 50,000 cells cmꢀ2 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 mL and 1.0 mL, respectively.
Cells were rinsed 10 min in DPBSH at 37 °C with mixing on a nuta-
tor (Clay Adams). Cells were pre-incubated with 4.3 mg mLꢀ1 BSA
in DPBSH alone or containing 5 ꢂ 10ꢀ6 M LSN 335984. After
4.3. Expression of P-gp, purification and measurement of
ATPase activity
To facilitate purification by metal-chelate chromatography,31
the cDNA for human P-glycoprotein (P-gp)30 was modified to con-
tain a 10-histidine tag at the COOH-terminal end (P-gp-His10). His-
tidine-tagged P-gp was then stably expressed in Baby hamster
kidney (BHK) cells by co-transfecting with P-gp-His10 and pWL-
neo (Stratagene, Cedar Creek, TX). The transfected cells were then
treated with the cytotoxic agent G418 as described previously.49
Clones over-expressing P-gp were identified by subjecting whole
cell extracts of G418-resistant colonies to immunoblot analysis
with a rabbit polyclonal antibody to P-gp.50 Histidine-tagged
P-gp was then isolated by nickel-chelate chromatography as
described previously.31 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) and ATPase activity
was determined in the absence or presence of various concentra-
tions of thiorhodamine compounds. The samples were incubated
for 30 min at 37 °C and the reactions were stopped by addition of
SDS. The amount of inorganic phosphate released was then
determined.33
To test for inhibition of P-gp VER-stimulated ATPase activity,
samples of P-gp-His10 in lipid were pre-incubated with various
concentrations of thiorhodamine compounds for 15 min at 20 °C.
Verapamil was used as a substrate to test for inhibition because
it is one of the most potent activators of P-gp ATPase activity.32
The reactions were started by addition of ATPase reaction mix con-
taining VER (final concentration of 4 ꢂ 10ꢀ4 M) and ATPase activity
determined as described above.
30 min, 5 lM test compound (1–12) 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 basolat-
eral-to-apical (BA) flux this sample was taken from the 12-well
plate 10 min after transfer, but before cell wells were added. Sam-
ples were taken from both the donor and receiver chambers fol-
lowing
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 5 ꢂ 10ꢀ4 L methanol for 3 min. Samples
(5 ꢂ 10ꢀ5 L) were combined into n = 3 cassettes in a 96-deep well
assay plate and protein was precipitated by adding 4.5 ꢂ 10–4
L
acetonitrile, 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 2ꢂ20 mm 5 micron Javelin column; (Thermo Scientific, Wal-
tham, Massachusetts) and 1 of 2 mobile phase systems. System 1
consisted of 5 ꢂ 10ꢀ3 M ammonium bicarbonate in water (Mobile
Phase A), and, 5 ꢂ 10ꢀ3 M ammonium bicarbonate in methanol
(Mobile Phase B), with elution accomplished by a methanol gradi-
ent at 1.5 mL/min. System 2 consisted of 0.4% trifluoroacetic acid
(TFA), 1 ꢂ 10ꢀ3 M ammonium bicarbonate in water (Mobile Phase
A), and, 0.4% TFA/1 ꢂ 10ꢀ3 M ammonium bicarbonate in acetoni-
trile (Mobile Phase B), with elution accomplished by an acetonitrile
gradient at 1.5 mL/min. Mass spectrometric detection was per-
formed with an API4000 mass spectrometer (Applied Biosystems,
Foster City, California) equipped with a turbo ion spray source,
using selected reaction monitoring in positive ion mode with pre-
cursor and product ion transitions specific to each analyte.
4.4. Enhancement of calcein-AM uptake into MDCKII-MDR1
cells by thiorhodamine analogues
MDCK cells transfected with wild-type MDR1 (ABCB1) were ob-
tained at passage number 12 from Dr. Piet Borst at The Netherlands
Cancer Institute. Cell growth was maintained in Dulbecco’s Modi-
fied Eagle’s Medium (Gibco) supplemented with 10% fetal bovine
4.6. Inhibition of vinblastine efflux in MDCKII-MDR1 cells by
thiorhodamine compounds 9–12
serum (FBS), 100 units/ml penicillin and 100 lg/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 40,000 cells/well in 96-well flat bottom plates (Falcon)
using a medium volume of 2 ꢂ 10ꢀ4 L, which was replaced on
day 3 prior to their use on day 4.
Cells were washed once with Dulbecco’s phosphate-buffered
saline containing 10ꢀ2 M Hepes buffer at pH 7.4 (DPBSH) (Gibco)
and incubated with solutions of the thiorhodamine analogue or
control compound in DPBSH containing 4.3 mg mLꢀ1 bovine serum
albumin (BSA) at 37 °C in room atmosphere. IC50 values were
calculated from 1:1 serial dilution series. After 30 min, the test
MDCKII-MDR1 cells were seeded onto Costar Transwell polycar-
bonate membranes and maintained as described above. On day 6,
cells were rinsed 1 ꢂ 10 min in DPBSH with nutation at 37 °C,
and then conditioned in 1 ꢂ 10ꢀ10, 1 ꢂ 10ꢀ8, 5 ꢂ 10ꢀ8, 1 ꢂ 10ꢀ7
,
5 ꢂ 10ꢀ7, 1 ꢂ 10ꢀ6 or 1 ꢂ 10ꢀ5 M 9–12 in BSA/DPBSH. After
30 min at 37 °C, 1 ꢂ 10ꢀ3
L l
[3H]-VIN (0.25 Ci mLꢀ1 from
0.1 mCi mLꢀ1 EtOH stock) in appropriate solution of 9–12 was
introduced to the basolateral chamber and 5 ꢂ 10ꢀ4 M fresh thio-
rhodamine compound was added to the apical chamber. Initial do-
nor samples were taken from the basolateral chamber at t = 0. The
apical solution was replaced every 10 min with fresh buffer and

4302
A. Orchard et al. / Bioorg. Med. Chem. 20 (2012) 4290–4302
appearance of [3H]-VIN in the apical chamber was measured by
scintillation counting. An IC50 was calculated using XLfit software.
15. 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.
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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 (25043) to D.M.C. D.M.C. is the recipient of the
Canada Research Chair in Membrane Biology. We also acknowl-
edge Ms. Michelle Linder, Ms. Kellie Davies, and Ms. Jackie Hill
for their technical assistance in the synthesis of starting materials.
We also acknowledge Mr. Codi Vinci for his assistance with analyt-
ical HPLC.
18. Holt, J. J.; Gannon, M. K.; Tombline, G.; McCarty, T. A.; Page, P. M.; Bright, F. V.;
Detty, M. R. Biorg. Med. Chem. 2006, 14, 8635.
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Supplementary data
Supplementary data (1H and 13C NMR spectra and HPLC data for
compounds 2, 3, 5–7, and 9–12 and 1H and 13C NMR spectra for
thioxanthone 15) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.075.
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