Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid

Article abstract of DOI:10.1021/jm401966m

P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC ₅₀ = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [¹²⁵I]-iodoarylazidoprazosin with IC₅₀ = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC₅₀ ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp.

Full text of DOI:10.1021/jm401966m

Article
pubs.acs.org/jmc
Design and Synthesis of Human ABCB1 (P-Glycoprotein) Inhibitors
by Peptide Coupling of Diverse Chemical Scaffolds on Carboxyl and
Amino Termini of (S)‑Valine-Derived Thiazole Amino Acid
Satyakam Singh,† Nagarajan Rajendra Prasad,‡,§ Eduardo E. Chufan,‡ Bhargav A. Patel,† Yi-Jun Wang,†
Zhe-Sheng Chen,† Suresh V. Ambudkar,*,‡ and Tanaji T. Talele*,†
†Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway,
Queens, New York 11439, United States
‡Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20892, United States
ABSTRACT: P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In
this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-
based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified
compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino
and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [125I]-
iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent
manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-
expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable
binding site for 28 within the drug-binding pocket of human P-gp.
hydrophobic drug-binding site that is capable of binding two
to three molecules simultaneously.5,6 Drug transport process by
P-gp is driven by ATP binding/hydrolysis to the drug-binding
competent state which results in a rotation of the NBDs
followed by adoption of a closed conformation and eventually
NBD dimer returns to the open conformation after ATP
hydrolysis.7 The closed conformation of NBD dimer is
responsible for the substrate translocation in the TMDs’
drug-binding sites, thus initiating the release of the substrate to
the extracellular side of the membrane.7
The ABC transporter-mediated MDR hinders the clinical
cure of cancer by chemotherapy. Therefore, many research
groups have been focusing on developing P-gp inhibitors to
enhance the therapeutic concentration of chemotherapeutic
drug inside cancer cells. Because of the unavailability of high-
resolution 3D-crystal structure of human P-gp, we used the
INTRODUCTION
■
P-glycoprotein (P-gp, ABCB1 or MDR1) is a cell membrane
ATP-binding cassette (ABC) transporter that is linked with the
development of multidrug resistance (MDR) in cancer cells.
Resistance to chemotherapy in cancer cells is a serious issue
which can arise due to numerous mechanisms, including the
overexpression of ABC drug transporters such as P-gp and
breast cancer resistance protein (BCRP, ABCG2, or MXR).
These transporters have been shown to contribute to decreased
intracellular concentrations of chemotherapeutic drugs.1,2 P-gp
utilizes energy derived from ATP hydrolysis to efflux an
extremely diverse set of hydrophobic compounds typified by
different chemical classes such as amphipathic, neutral, or
weakly basic agents including a number of recently developed
tyrosine kinase inhibitors.3 Human P-gp is composed of two
transmembrane domains (TMDs), each containing six helices
and two nucleotide-binding domains (NBDs). The TMDs
house the drug/substrate-binding sites and a translocation
conduit.4 Moreover, the TMD region contains a large
Received: December 20, 2013
Published: April 28, 2014
© 2014 American Chemical Society
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Figure 1. (A) Structures of the reported potent P-gp inhibitors containing chemical fragments such as methoxy substituted tetrahydroisoquinoline,
various methoxy substituted aryl rings, biphenyl, and benzophenone. (B) Structures of compounds 1 and 1a−1d.
recently corrected crystal structure of mouse P-gp as a template
to develop homology model of human P-gp,8 which was
eventually used for understanding the binding mode of
synthesized P-gp inhibitors. Numerous strategies such as
random and focused screening, systematic chemical modifica-
tions, and combinatorial chemistry have been applied to
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a
Scheme 1. Synthesis of Monomer Acid Derivative
a
Reagents and conditions: (i) HCTU, HOBt, DIEA, DMA, rt, 18 h.
develop the first three generations of P-gp inhibitors; however,
they suffer from toxicity and drug−drug interactions. Although
the first three generations of P-gp and/or ABCG2 modulators
(valspodar, biricodar, laniquidar, zosuquidar, elacridar, and
tariquidar) designed to reverse MDR failed in various stages of
clinical trials, ABC transporters undoubtedly play a crucial role
in the development of MDR, and researchers should not
underestimate their importance. The clinical failures were only
partly associated with ABC transporter modulation.9 They were
also due to inadequate bioavailability at tumor sites,9
nonselective inhibition of P-gp across all tissues including the
blood−brain barrier, simultaneous inhibition of the drug-
metabolizing enzyme CYP3A4,10 and interference with the
function of other ABC transporters that play a crucial role in
antitumor immune response.11 Another problem was inappro-
priate selection of the patient population.12
With more carefully designed clinical trials, fourth generation
P-gp modulators with low toxicity may be found to circumvent
modulator-associated problems. New approaches leading to the
development of fourth generation P-gp inhibitors exhibiting
high P-gp selectivity and potency are highly desirable. One such
approach is to append various chemical fragments that are
frequently seen in P-gp inhibitors, to a novel chemotype such as
thiazole amino acid. Using this approach, chemical fragments
from reported potent P-gp inhibitors, as shown in Figure 1A,
are inserted into chemically modified natural products.
The first available cocrystal structures of murine P-gp bound
to cyclic selenazole derivatives 1c (QZ59Se-SSS)13 and 1d
(QZ59Se-RRR)13 provided significant insight into various drug-
binding sites of P-gp transporter (Figure 1B). The cyclic trimer
compound 1a (QZ59S-SSS), which is bioisosteric to compound
1c, exhibited an IC50 value of 2.7 μM, whereas 1b, an isostere of
1d, showed an IC50 of 8.4 μM against mouse P-gp efflux
function (Figure 1B).14 Because of this distinguishable ability of
P-gp for stereoisomers of cyclic peptides, we maintained (S)
chirality for all the synthesized compounds. Moreover, several
natural products have been shown to contain thiazole ring.15
Our recent study encompasses the investigation of the
inhibitory effect of (S)-valine-based thiazole derivatives
including compound 1a on human P-gp efflux activity.16 The
results obtained from the exploration indicated the linear
thiazole chain (linear trimer, 1; IC50 = 1.5 μM) to be equally
effective as that of 1a (IC50 = 1.5 μM) in terms of inhibition of
efflux activity of human P-gp. Also, we noticed that deviation
from the trimer size (three consecutive thiazole units) of the
thiazole chain had a detrimental effect on the inhibitory activity.
Docking analysis of compound 1 within the drug-binding
pocket of homology-modeled human P-gp showed interactions
with certain key amino acid residues.16 The terminal thiazole
fragments of compound 1 were found to capture hydrogen
bonding and electrostatic interactions with the glutamine
(Q990 and Q725) and tyrosine (Y307 and Y953) residues
while being predominantly surrounded by hydrophobic amino
acid side chains in the drug-binding pocket of P-gp. On the
basis of these results, we decided to optimize compound 1 by
replacing the terminal thiazole units with chemical scaffolds
containing mono-, di-, and tri-methoxy aryl rings. For a few
analogues, we also borrowed chemical fragments from the
reported P-gp inhibitory structures that might increase the π−π
interaction surface area within the drug-binding pocket of P-gp.
Our choice of fragments was based on the chemical moieties
which are frequently observed in various reported preclinical
and clinical candidates such as tariquidar (XR9576),17
elacridar,18 LY402913,19 reserpine,20 XR9051,21 saracatinib,22
galloyl-based inhibitors,23 and benzophenone derivatives24 as
illustrated in Figure 1A. Additionally, Didziapetris et al.25
estimated the “rule of fours” which states that the compounds
with (N+O) ≥ 8, MW > 400, and acid pKa > 4 are likely to act
as P-gp substrates. Therefore, we synthesized a series of (S)-
valine derived thiazole analogues by extending the carboxyl and
the amino termini of monomer (mono thiazole unit) as well as
dimer (bis thiazole unit), taking into account the trimer size as
well as the “rule of fours”. This optimization strategy was
supported by the inhibitory activity of two representative
compounds (2 and 3) mentioned in our recent report.16
Certain critical aspects were considered for the selection of
fragments for extensions: (a) the coupled fragments must
impart the desired hydrophobicity (ClogP = 3−6),26−28 (b) the
selected fragments should present hydrogen bond acceptor
atoms such as oxygen and nitrogen, and (c) both amino and
carboxyl termini should be attached to various methoxy-
substituted moieties and extended aromatic fragments to
establish critical electrostatic interactions with hydrogen bond
donor residues and the π-stacking interactions at the drug-
binding pocket of the P-gp. Additionally in this regard, the
presence of the methoxy groups has been shown previously to
increase selective affinity toward P-gp.23 The synthesized
derivatives were assessed for their inhibitory efficacies on the
P-gp transport function by calcein-AM assay. Reversal of MDR
due to ABCB1 inhibition in SW620/Ad300 and HEK/ABCB1
cell lines was also investigated using paclitaxel in the presence
of compounds 28, 1a, and cyclosporine A. The effect of
compound 28 was tested with biochemical studies including
photolabeling with [125I]-iodoarylazidoprazosin (IAAP) and
measurement of ATPase activity of P-gp using crude
membranes of High Five insect cells expressing this transporter.
Furthermore, to obtain insights into binding interactions of
these analogues within the large drug-binding pocket, we
performed docking of compounds on all the possible binding
sites of the homology model of human P-gp.
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a
Scheme 2. Synthesis of Monomer Amine Derivatives
a
Reagents and conditions: (i) HCTU, HOBt, DIEA, DMA, rt, 18 h; (ii) DIEA, THF, rt, 12 h.
a
Scheme 3. Synthesis of Dimer Acid Derivatives
a
Reagents and conditions: (i) HCTU, HOBt, DIEA, DMA, rt, 18−24 h.
a
Scheme 4. Synthesis of Dimer Amine Derivatives
a
Reagents and conditions: (i) DIEA, THF, rt, 12 h.
respectively (Scheme 2). Alternatively, compound 9 was
prepared by reacting trimethoxybenzoyl chloride (9a) with a
monomer amine (6) using DIEA in THF. Our next objective
was to synthesize dimer derivatives that could mimic the trimer
structure using compounds 10 and 13. In Scheme 3, a dimer
acid (10) was coupled with di- and tri-methoxy anilines (11a
and 12a), respectively, to obtain compounds 11 and 12.
Consequently, compounds 14 and 15 were synthesized by
reaction of a dimer amine (13) with acid chlorides 14a and 15a
using DIEA in THF (Scheme 4). Further, target compounds
17−29 were synthesized starting from compound 9 by use of
the coupling reagents HCTU, HOBt, and DIEA in DMA
(Scheme 5). These target compounds were the size of a trimer
molecule, with both ends featuring chemical scaffolds borrowed
from potent P-gp modulators. At first, the ethyl ester present in
RESULTS AND DISCUSSION
■
Chemistry. A library of 21 thiazole-based compounds
targeted for P-gp efflux inhibition was synthesized as shown in
Schemes 1−5. The synthesis of compounds 2 and 3 along with
that of the required precursors (4, 6, 10, and 13) has been
described in our recent report.16 Target compounds 5, 7−9,
11−12, and 14−15 were synthesized by peptide coupling
reactions using thiazole acids (4 and 10) and thiazole amines (6
and 13) with various chemical scaffolds containing amine and
acid derivatives, respectively. Schemes 1 and 2 shows the
synthesis of monomer derivatives. Monomer acid 4 was reacted
with a piperidine derivative (5a) to obtain compound 5 in the
presence of the coupling agents HCTU, HOBt, and DIEA, as
shown in Scheme 1. Monomer amine 6 was coupled with acid
derivatives 7a and 8a to obtain compounds 7 and 8,
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a
Scheme 5. Synthesis of Monomer Zwitter-ion Derivatives
a
Reagents and conditions: (i) NaOH, THF/methanol/H2O (10:2:3), rt, 4 h; (ii) HCTU, HOBt, DIEA, DMA, rt, 18−24 h.
compound 9 was converted to acid 16 by aqueous basic
hydrolysis. Compound 16 was then coupled with various aryl
and arylalkyl amines to generate target compounds (17−29).
Structure−Activity Relationship. To optimize the
efficacy of a linear trimer derivative (1, IC50 = 1.5 μM), 21
compounds (5, 7−9, 11−12, 14−15, and 17−29) were
synthesized by appending the extremities of the monomer
and dimer thiazole units with the selected fragments from the
reported potent P-gp inhibitors. The target compounds were
evaluated for their ability to inhibit transport function of human
P-gp using BacMam-P-gp baculovirus-transduced HeLa cells
overexpressing the multidrug transporter. The assay utilized for
the evaluation involves measurement of fluorescence intensity
imparted by calcein, which accumulates in the cells due to
inhibition of P-gp efflux by the tested compounds (Table 1).
Initially, four compounds (5, 7, 8, and 9) comprising
substitutions on either side of the monomer unit were
synthesized and evaluated for P-gp transport inhibition. The
calcein-AM inhibition assay data (≤15% inhibition at 10 μM)
suggests that straight monomer module substitutions were
insufficient for any perceptible activity even with extended
appendages such as 5,6-dimethoxyindanonemethylpiperidine,
quinoline, and 3,4-dimethoxycinnamoyl, as seen in compounds
5, 7, and 8, respectively. This might be due to the lack of
appropriate orientation of these derivatives within the P-gp
drug-binding site, resulting in ineffective interactions. These
results imply that a compound similar to a trimer in terms of
length as well as shape is critical for effective P-gp efflux
inhibitory activity for this class of compounds. In view of this
substantiation, dimer derivatives 11−12 and 14−15 containing
methoxy-substituted aryl moieties were prepared and tested for
inhibitory potencies against P-gp transport function. Com-
pounds 11 (IC50 = 2.5 μM) and 12 (IC50 = 6.5 μM), both
dimer acid derivatives, were found to possess appreciable
inhibition, comparable to that of compounds 2 and 3. Likewise,
the dimer amine derivatives 14 (IC50 = 16 μM) and 15
(maximum 55% inhibition at 10 μM) were moderate inhibitors
of the P-gp mediated efflux process. These outcomes show a
significant improvement in P-gp efflux inhibition efficiency of
the compounds on advancing from dimer to trimer structural
size. Further, according to our strategy, we required
concomitant incorporation of chemical scaffolds on either
end of the mono-thiazole (monomer) unit. To achieve this, we
decided to maintain the trimethoxybenzoyl fragment at the
amino terminus because the presence of a trimethoxybenzoyl
group has been shown to increase the potency as well as
selectivity toward P-gp inhibition.23 To this end, 13 compounds
(17−29) were synthesized and examined in the calcein-AM
assay. Compounds 17 and 18 containing 4-methoxyphenylethyl
amine and 3,5-dimethoxyaniline fragments, respectively, were
poor to moderately active (24% and 37% inhibition at 10 μM,
respectively), whereas compound 19 comprising a 3,4,5-
trimethoxyaniline fragment showed improvement with 58%
inhibition at 10 μM. It appears that an increase in the number
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Table 1. (S)-Valine-Based Thiazole Derivatives As Inhibitors of P-gp Transport Function
a
BacMam-P-gp baculovirus-transduced HeLa cells were incubated with 0.5 μM calcein-AM for 10 min at 37 °C under dark in the presence and
absence of 10 μM (S)-valine-based thiazole derivatives. Percentage transport inhibition was derived by considering the level of inhibition obtained
with the standard inhibitor, tariquidar at 1 μM, and the values SD shown are the average of two independent experiments done in triplicate at 10
b
μM concentrations of inhibitors. IC50 values shown in brackets ( SD) were determined by at least two independent experiments done in triplicate.
c
d
Compounds reported in our recently published work.16 Ligand efficiency was calculated using formula, LE = (−RT ln IC50/number of non-
hydrogen atoms); IC50 is in molar, R = 1.987 kcal K−1 mol−1, and T = 300 K.
of methoxy groups on the phenyl ring of the compounds
enhances the binding affinity for P-gp. However, compound 20,
with a 3,4,5-trimethoxybenzyl amine fragment, lost the P-gp
inhibitory activity (4% inhibition at 10 μM). Compounds 21
and 22 with methylenedioxybenzyl amine and methylenedioxy
aniline showed 20% and 40% inhibition of P-gp, respectively.
Comparing compounds 19 with 20 and 21 with 22, the
insertion of a methylene spacer between the aryl and the amine
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Figure 2. Inhibition of calcein-AM transport. BacMam P-gp baculovirus-transduced HeLa cells were assayed for calcein-AM transport (A) in the
presence of selected derivatives at 10 μM and (B) in the presence of increasing concentrations of 11, 12, 14, and 28. (A) Representative histograms
show low, moderate, and high P-gp inhibitory activity of selected thiazole derivatives. Tariquidar (1 μM), a known inhibitor of P-gp, was used for
comparison. The values shown are the average of two independent experiments each done in triplicate. (B) Concentration-dependent inhibition of
calcein-AM efflux by selected derivatives was studied. The average values from two independent experiments each done in triplicate were plotted and
the IC50 values for compounds 11 (filled squares), 12 (filled diamonds), 14 (filled triangles), and 28 (filled circles) are 2.5, 6.5, 16, and 1 μM,
respectively.
Table 2. Reversal Effect of Compounds 28, 1a, and Cyclosporine A on the Cytotoxicity of Paclitaxel to SW620, SW620/Ad300,
HEK293, and HEK/ABCB1 Cell Lines
SW620
SW620/Ad300
HEK293
HEK/ABCB1
a
b
a
b
a
b
a
b
compd
paclitaxel
IC50 SEM (μM)
FR
IC50 SEM (μM)
FR
IC50 SEM (μM)
FR
IC50 SEM (μM)
FR
0.006 0.001
0.007 0.001
0.006 0.001
0.006 0.001
0.006 0.001
0.006 0.001
0.007 0.001
0.006 0.001
[1.0]
[1.2]
[1.0]
[1.0]
[1.0]
[1.0]
[1.2]
[1.0]
4.019 0.215
0.186 0.016
0.051 0.005
0.011 0.001
0.706 0.068
0.340 0.035
0.125 0.011
0.079 0.007
[669.8]
[31.0]
[8.5]
0.049 0.004
0.048 0.003
0.047 0.003
0.048 0.004
0.049 0.003
0.048 0.005
0.048 0.004
0.050 0.003
[1.0]
[1.0]
[1.0]
[1.0]
[1.0]
[1.0]
[1.0]
[1.0]
4.138 0.132
0.633 0.042
0.154 0.015
0.098 0.004
1.230 0.063
0.780 0.047
0.256 0.024
0.205 0.017
[84.4]
[12.9]
[3.1]
+ 28(1 μM)
+ 28 (3 μM)
+ 28 (10 μM)
+ 1a (0.3 μM)
+ 1a (1 μM)
+ 1a (3 μM)
+ CsA (3 μM)
[1.8]
[2.0]
[117.7]
[56.7]
[20.8]
[13.2]
[25.1]
[15.9]
[5.2]
[4.2]
a
IC50, concentration of indicated compound required for 50% inhibition of cell survival were calculated from the killing curves shown in Figure 3.
b
Mean values ( SEM) are from four independent experiments, each performed in triplicate. FR: fold-resistance was calculated by dividing the IC50
value for paclitaxel of SW620 (or HEK293) and SW620/Ad300 (or HEK/ABCB1) cells in the absence or presence of compounds 28, 1a, and
cyclosporine A by IC50 value for paclitaxel of SW620 (or HEK293) cells. CsA, cyclosporine A.
group proved detrimental for the P-gp inhibitory activity. This
finding suggests potential steric clashes within the drug-binding
pocket of P-gp for compounds 20 and 21 resulting from the
introduction of the methylene spacer group. The 6,7-
dimethoxytetrahydroisoquinoline group containing compound
23 was found to be devoid of P-gp inhibitory activity (16% at
10 μM). Furthermore, incorporation of a 2-aminoindane
substitution resulted in moderate activity of compound 24
(47% inhibition at 10 μM); however, incorporation of 2-
aminoethylpyridine (25) and 4-phenylbenzyl amine (26) were
found to have a detrimental effect on P-gp inhibitory activity
(5% and 23% inhibition at 10 μM, respectively), supporting our
previous observation of the unfavorable effect of an alkyl spacer
group. Weak inhibition of calcein-AM transport by compounds
22, 23, and 24 indicates a potential steric hindrance by the
bicyclic ring structure at the drug-binding pocket of P-gp.
Compound 27, containing a 4-aminobenzophenone substitu-
tion, lacks any significant inhibitory activity (18% at 10 μM),
while compound 28 with a 2-aminobenzophenone substitution
was found to have efficient P-gp inhibitory activity with IC50
value of 1 μM. Also, compound 29 showed appreciable
inhibition (54% inhibition at 10 μM) of P-gp transport activity.
Compound 27, with a benzoyl group at the para-position,
might be similar to compounds 22, 23, and 24 with respect to
steric hindrance. Moreover, effective inhibitory data for
compounds 28 and 29 indicates that it is essential for inhibitors
to have an angular shape to increase the contact surface with P-
gp. Compound 28, bearing the trimethoxybenzoyl group at the
amino terminus and 2-aminobenzophenone at the carboxyl
terminus, proved to be the most effective P-gp inhibitor (IC50 =
1.0 μM) among the synthesized compounds, as evident from
the dose−response curve shown in Figure 2B. Panel A of
Figure 2 shows a representative histogram with low, moderate,
and high inhibitory activity of selected thiazole derivatives,
whereas panel B shows the concentration-dependent inhibition
of calcein-AM efflux by compounds 11, 12, 14, and 28. We
attempted to correlate the pIC50 values of compounds 1, 2, 3,
11, 12, 14, and 28 with their respective ClogP values 5.81, 5.22,
4.93, 5.47, 5.87, 5.03, and 5.49 in order to appraise the function
of lipophilicity for P-gp inhibition. However, we were unable to
find any significant correlation within the narrow range of
ClogP values, which could be a result of a small sample size.
Among various parameters that govern P-gp inhibition, one
such parameter is ClogP, which should be in the range of 3−6
as mentioned earlier. Therefore, apart from ClogP other
parameters, such as a molecular framework including hydrogen
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Figure 3. Effect of compounds 28, 1a, and cyclosporine A (CsA) on ABCB1-mediated resistance to paclitaxel in ABCB1 overexpressing drug
selected (A) and transfected (B) cell lines. (A) Concentration-dependent curves of paclitaxel with or without compounds 28, 1a, and CsA at 3 μM in
parental SW620 and ABCB1 overexpressing SW620/Ad300 cells. The IC50 values of SW620/Ad300 cell line were compared with those of parental
SW620 cells (see Table 2). (B) Concentration-dependent curves of paclitaxel with or without compounds 28, 1a, and CsA at 3 μM in parental
HEK293/pcDNA3.1 and ABCB1 overexpressing HEK/ABCB1 cells. The IC50 values of HEK/ABCB1 cell line were compared with those of
parental HEK293 cells (see Table 2). Points with error bars represent the mean
SEM. The figure is a representative of four independent
experiments, each done in triplicate.
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bonding ability and interaction surface area, also play critical
role in inhibition of P-gp transport function. Ligand efficiency
calculations showed a marginal improvement for compound 28
(0.20) as compared to compound 1 (0.17) (Table 1). In
addition, the molecular weight decreased by 119 Da from
compound 1 to benzophenone analogue 28.
Effect of Compounds 28 and 1a on ABCB1-Mediated
Resistance to Paclitaxel in ABCB1 Overexpressing Drug-
Selected Cell Lines. Does compound 28 reverse resistance to
anticancer drugs in P-gp overexpressing cell lines? To address
this question, we performed MDR reversal experiments. To
select a nontoxic or relatively low drug concentration for
compounds 28 and 1a, cytotoxicity assays were performed on
parental and P-gp-overexpressing cell lines (data not shown).
On the basis of these results, concentrations of 10 μM for 28
and 3 μM for 1a demonstrated >85% cell survival.
To determine whether 28 and 1a could reverse P-gp-
mediated MDR, cell survival assays were performed in the
presence or absence of 28 and 1a, using the parental SW620
and drug-selected SW620/Ad300 cell lines. The drug-selected
SW620/Ad300 cell line showed 688.2-fold resistance to
paclitaxel, as compared to the parental SW620 cell line
(Table 2). Compound 1a, at 0.3, 1, and 3 μM, significantly
decreased the resistance of the SW620/Ad300 cell line to
paclitaxel from 669.8-fold to 117.7-, 56.7-, and 20.8-fold,
respectively, compared to SW620 cell line (Table 2).
Compound 28, at 1, 3, and 10 μM, further reduced the
resistance of SW620/Ad300 cell line to paclitaxel from 669.8-
fold to 31.0-, 8.5-, and 1.8-fold, respectively, compared to
SW620 cell line (Table 2). Moreover, at 10 μM, compound 28
can almost completely reverse the ABCB1-mediated drug
resistance in SW620/Ad300 cells. As expected, a known
modulator of P-gp, cyclosporine A (3 μM) significantly
decreased the resistance of SW620/Ad300 to 13.2-fold for
paclitaxel as compared to the control SW620 cell line (Table
2). Interestingly, at 3 μM, compound 28 showed more potent
reversal activity than cyclosporine A and 1a (Figure 3A). These
results suggest that compound 28 and 1a have the potential to
enhance the sensitivity of P-gp-overexpressing drug-selected
cell lines to anticancer drug substrates.
sensitivity was not significantly altered (Table 2). Interestingly,
at 3 μM, compound 28 demonstrated more potent reversal
activity than cyclosporine A and 1a (Figure 3B). These results
suggest that compounds 28 and 1a enhance the sensitivity of P-
gp-overexpressing transfected cell lines to paclitaxel.
Effect of Compound 28 on IAAP Photoaffinity
Labeling of P-gp. To assess the interaction of compound
28 at the drug-binding pocket of P-gp, its effect on biochemical
assays including photolabeling of P-gp with [125I]-IAAP and
ATP hydrolysis by P-gp was determined. Both compounds 28
and 1a inhibited the photoaffinity labeling of P-gp with [125I]-
IAAP in a concentration-dependent manner, reaching a
maximum of 90% inhibition at concentrations higher than 5
μM (see Figure 4A, data shown only for compound 28).16 The
concentration required for 50% inhibition of photolabeling by
compound 28 is 0.72 μM (Figure 4A). These results clearly
show that compound 28, similar to the other modulators, binds
Effect of Compounds 28 and 1a on P-gp-Mediated
Resistance to Paclitaxel in P-gp-Overexpressing Trans-
fected Cell Lines. There are multiple factors contributing to
the drug resistance mechanisms in drug-selected cell lines.
Therefore, we directly determined whether compounds 28 and
1a reverse P-gp-mediated MDR by performing cell survival
assays in the presence or absence of compounds 28 and 1a,
using the parental HEK293 cell line and ABCB1-transfected
HEK/ABCB1 cell line. The transfected HEK/ABCB1 cell line
showed 84.5-fold resistance to paclitaxel, as compared to the
parental HEK293 cell line (Table 2). Compound 1a, at 0.3, 1,
and 3 μM, significantly decreased the resistance of HEK/
ABCB1 cell line to paclitaxel from 84.4-fold to 25.1-, 15.9-, and
5.2-fold, respectively, compared to HEK293 cell line (Table 2).
Compound 28, at 1, 3, and 10 μM, further reduced the
resistance of the HEK/ABCB1 cell line to paclitaxel to 12.9-,
3.1-, and 2.0-fold, respectively, compared to HEK293, for which
resistance was not significantly altered by compound 28 (Table
2). At 10 μM concentration, compound 28 can almost
completely reverse ABCB1-mediated drug resistance in HEK/
ABCB1 cells. Being a positive control, cyclosporine A (3 μM)
significantly decreased the resistance of HEK/ABCB1 to 4.2-
fold for paclitaxel as compared to HEK293, for which drug
Figure 4. Effect of compound 28 on the photoaffinity labeling and
ATPase activity of P-gp. (A) Compound 28 inhibits the photoaffinity
labeling of P-gp with [125I]-IAAP. Crude membranes of High Five
insect cells expressing P-gp (65 μg protein/100 μL) in 50 mM MES-
Tris pH 6.8 were incubated with increasing concentrations of
compound 28 (0−20 μM), at 37 °C for 10 min. Samples were then
transferred to 4 °C bath, and 4−5 nM [125I]-IAAP was added under
subdued light. The samples were photo-cross-linked with [125I]-IAAP
as described previously.36 A representative autoradiogram from one of
two independent experiments is shown. In the graph, points represent
the average of two independent experiments. The data were fitted (R2
= 0.94) with a one-phase decay equation using GraphPad Prism 6.01.
(B) Compound 28 stimulates the basal ATPase activity of ABCB1.
Crude membranes of High Five insect cells expressing P-gp (10 μg
protein/100 μL) were incubated with increasing concentrations of
compound 28 (0−10 μM), in the presence and absence of sodium
orthovanadate (0.3 mM), in ATPase assay buffer as described
previously.37 The data obtained with compound 28 up to 2.5 μM
concentration are shown in (B). Points with error bars represent the
mean and SEM of three independent experiments. The data were
fitted (R2 = 0.92) with the Michaelis−Menten equation using
GraphPad Prism 6.01.
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at the drug-binding pocket located in the transmembrane
domains of human P-gp.
Effect of Compound 28 on the Basal ATPase Activity
of P-gp. The effect of compound 28 on the basal ATPase
activity of P-gp was investigated in crude membranes of High
Five cells expressing this transporter. Compound 28 stimulated
the ATP hydrolysis by P-gp up to 2-fold (see Figure 4B), at
concentrations ranging from 0.5 to 2.5 μM. At higher
concentrations, although basal ATPase activity was still
stimulated, the extent of the stimulation was slightly lower,
approximately 1.8-fold. The apparent affinity or concentration
required for 50% stimulation of ATP hydrolysis was calculated
with a concentration up to 2.5 μM, and the EC50 value was
0.027 μM. These data demonstrate that compound 28 is a
potent stimulator of the ATPase activity of P-gp.
Glide-XP (Schrodinger, LLC., New York, NY, 2013) docking
̈
experiments were performed to understand the molecular
interactions of these compounds within the drug-binding sites
of P-gp. Docking experiments were targeted to all possible
binding sites of P-gp, as proposed by Shi et al. (site-1, site-2,
site-3, and site-4) using “Extra Precision” (XP) glide mode.29
Analysis of the binding energy data indicated site-1 as the
preferred site of binding. For closer inspection of the possible
binding conformation of compound 28 within site-1, induced-
fit docking was performed to emulate the flexible receptor
binding model, as suggested by Loo et al.30 It may be noted
that the actual binding mode can be discerned through site-
directed mutagenesis and/or cocrystal structural studies of
human P-gp in the presence of compound 28.
Docking Interaction of Compound 28 with Homol-
ogy-Modeled Human P-gp. The binding interactions of
compound 28 were analyzed within site-1 of homology-
modeled human P-gp (Figure 5A,B). Compound 28 is
stabilized through specific interactions such as hydrogen
bonding and nonspecific interactions such as hydrophobic
interactions with residues in the drug-binding pocket of P-gp.
The hydrogen bond acceptor oxygen atom at the para-position
of the trimethoxyphenyl moiety showed hydrogen bonding
interaction with the side chain of Q990 (H3CO---HN-Q990).
The amide bond between the trimethoxyphenyl ring and the
thiazole ring forms a hydrogen bond with Q725 (NH---O
CNH2-Q725). The two phenyl rings of the benzophenone
group interacts with F336 through π−π stacking in which the
phenyl group attached to the thiazole ring via amide linkage
does so in a parallel displaced (offset) setting while the distal
benzoyl group forms an edge-to-face contact. Additionally, the
benzoyl group forms similar π−π stacking interactions with the
side chain phenyl ring of Y310 and F335. The amide bond
connecting the benzophenone and the thiazole group is in close
proximity to S979. Compound 28 was able to establish some
nonspecific interactions with the surrounding hydrophobic
residues. The trimethoxyphenyl, benzophenone, isopropyl, and
thiazole groups are mainly stabilized through hydrophobic
contacts within the large hydrophobic pocket formed by the
side chains of M69, F72, F303, I306, Y307, F314, L332, L339,
I340, F343, A729, F732, F759, Y953, F957, F978, F983, M986,
and A987.
Figure 5. Induced-fit docking model of compound 28 at site-1 of
human P-gp homology model. (A) A portion of the transmembrane
region of homology modeled human P-gp is shown in ribbon
presentation. Selected amino acids are depicted as sticks with the
atoms colored as carbon, purple−blue; hydrogen, white; nitrogen,
blue; oxygen, red; sulfur, yellow), whereas the inhibitor is shown as a
ball and stick model with the same color scheme as above except
carbon atoms are represented in orange. Hydrogen bonds are shown
as black dashes. The ribbon representation for portions of TM3 and
TM6 was undisplayed for better view. (B) A two-dimensional ligand−
receptor interaction diagram with important interactions observed in
the docked complex of compound 28 with the drug-binding site
residues of human P-gp is shown. The amino acids within 5 Å are
shown as colored bubbles, cyan indicates polar, and green indicates
hydrophobic residues. Hydrogen bonds are shown by purple dotted
arrows, while π-stacking aromatic interactions are shown by green
lines.
Analysis of the binding model of 28 at site-1 of P-gp attempts
to rationalize its effective P-gp inhibitory activity as well as
provides a potential possibility for further optimization with
respect to the residues around it and substitution pattern on N-
terminal benzoyl and C-terminal benzophenone moieties. For
example, the N-benzoyl group may be scanned for various
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Method A. General Procedures for Peptide Coupling of
Thiazole Amino Acid Units to Linear Oligomers.16 Di-
isopropylethylamine (DIEA) (1.5 equiv) was added to a well-stirred
suspension of carboxylic acid derivatives in anhydrous N,N-
dimethylacetamide (DMA). Upon cooling the reaction mixture to 0
°C, HCTU (1.5 equiv) and HOBt (1.5 equiv) were sequentially
added. The reaction mixture was allowed to stir at 0 °C for 10 min and
then treated with a precooled solution of the corresponding amines
(1.2 equiv) in DMA. The reaction mixture was stirred at rt, and after
completion as monitored by TLC, the reaction mass was concentrated
in vacuo. The solution was partitioned between ethyl acetate and
aqueous citric acid (10% w/v), and the separated aqueous phase was
extracted again with ethyl acetate. The organic extracts were combined
and washed sequentially with saturated aqueous sodium bicarbonate,
water, and brine, then dried over sodium sulfate, and the solvent was
removed under reduced pressure. The residue was purified by flash
chromatography on silica gel using n-hexane−ethyl acetate (1:1) as
eluent to provide desired peptide products.
combinations of hydroxyl and methoxy groups to capture
electrostatic interactions with proximal polar residues such as
N721, Q838, N839, N842, and Q990. Because the C-terminal
benzophenone group occupied a subsite formed by side chains
of aromatic residues, the benzoyl portion of the benzophenone
moiety may be similarly scanned for substitutions with small
electron donating/withdrawing groups to capture aromatic
interactions through inductive increase in π−π stacking.
CONCLUSIONS
■
We synthesized a series of (S)-valine derived thiazole analogues
that are comparable in size to a trimer length, composed of
various chemical scaffolds to inhibit human P-gp efflux activity.
This investigation resulted in identification of compound 28
bearing a trimethoxyphenyl ring and a benzophenone moiety at
the amino and carboxyl termini of the monothiazole zwitter-
ion, respectively. Intracellular accumulation of calcein (from
calcein-AM) in P-gp-transduced HeLa cells clearly demon-
strated that compound 28 inhibits the transport activity of P-gp.
Compound 28 was found to be superior to 1a and cyclosporine
A for reversal of resistance to paclitaxel in both SW620/Ad300
and HEK/ABCB1 cell lines. Moreover, compound 28 did not
exhibit any toxicity up to 10 μM concentration in the cell lines
studied in this report. Future in vivo reversal effects of 28 will
prove its worth as an effective MDR reversal agent for
combination with conventional chemotherapy. The inhibition
of IAAP-labeling and stimulation of basal ATP hydrolysis of P-
gp provide further evidence that the effect observed in intact
cells is mediated by the specific interaction of compound 28 at
the drug-binding pocket of P-gp. Consistent with cell- and
membrane-based assays, docking analysis revealed interactions
of compound 28 within site-1 in the drug-binding pocket of
homology-modeled human P-gp. Further efforts will be focused
on optimization of the substitution pattern on the terminal
phenyl ring of the benzophenone moiety to obtain not only
highly potent P-gp inhibitors but also to develop photoactivable
derivatives for identification of residues in site-1 that interact
with the inhibitor.
Method B. General Procedures for Peptide Coupling of
Thiazole Amines with Acyl Chlorides. To the cooled suspension of
thiazole amine derivatives (0 °C) in anhydrous THF were added DIEA
(1.5 equiv) and commercially available acyl chlorides (1.2−1.5 equiv).
The reaction mixture was then allowed to warm to rt and stirred for 12
h. The solvent was then removed by evaporation, and the resulting
reaction mass was diluted with ethyl acetate. The ethyl acetate layer
was then washed with aqueous citric acid (10% w/v), saturated sodium
bicarbonate, water, and brine. The organic fractions were dried over
anhydrous sodium sulfate and evaporated under vacuum. The crude
product was purified by flash chromatography on silica gel using n-
hexane−ethyl acetate (1:1) as eluent to obtain the required peptides.
(1-(4-(4-(5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)-
methyl)piperidine-1-carbonyl)thiazol-2-yl)-(S)-2-methylpro-
pylcarbamic Acid tert-Butyl Ester (5). Compounds 4 (0.30 g, 0.99
mmol) and 5a (0.65 g, 1.99 mmol) were reacted together by following
method A to obtain compound 5 as a white solid (0.638 g, 85%); mp
1
80−84 °C; Rf = 0.25 (EtOAc/n-hexane 1:1). H NMR (400 MHz;
CDCl3; TMS) δ 7.91 (s, 1H), 7.71 (d, 1H, J = 6.6 Hz), 7.09 (s, 1H),
7.06 (s, 1H), 4.61 (t, 1H, J = 7.1 Hz), 3.86 (s, 3H), 3.79 (s, 3H), 3.21−
3.28 (m, 3H), 2.66−2.76 (m, 4H), 2.45−2.47 (m, 1H), 1.71−1.78 (m,
4H), 1.40 (s, 9H), 1.24−1.31 (m, 3H), 0.88 (dd, 6H, J = 17.7 Hz, J =
5.5 Hz). m/z (ESI-MS) 594.33 (C30H41N3O6SNa requires 594.27, [M
+ Na]+). HPLC tR = 4.7 min, purity 100%.
(S)-2-{2-Methyl-1-[(quinoline-3-carbonyl)amino]propyl}-
thiazole-4-carboxylic Acid Ethyl Ester (7). Compounds 6 (0.30 g,
1.31 mmol) and 7a (0.453 g, 2.62 mmol) were reacted together
according to method A to obtain compound 7 as yellow foam (0.443
g, 88%); Rf = 0.40 (EtOAc/n-hexane 1:1). 1H NMR (400 MHz;
CDCl3; TMS) δ 9.38 (s, 1H), 8.68 (s, 1H), 8.16 (d, 1H, J = 2.8 Hz),
8.13 (s, 1H), 7.96 (d, 1H, J = 8.2 Hz), 7.83 (t, 1H, J = 7.5 Hz), 7.64 (t,
1H, J = 7.5 Hz), 7.55 (d, 1H, J = 8.48 Hz), 5.50 (t, 1H, J = 8.68 Hz)
4.44 (q, 2H, J = 5.8 Hz), 2.53−2.59 (m, 1H), 1.44 (t, 3H, J = 5.8 Hz),
1.13 (d, 6H, J = 6.5 Hz). m/z (ESI-MS) 384.33 (C20H22N3O3S
requires 384.13, [M + H]+). HPLC tR = 3.3 min, purity 99%.
(E)-2-{1-[3-(3,4-Dimethoxyphenyl)acryloylamino]-(S)-2-
methylpropyl}thiazole-4-carboxylic Acid Ethyl Ester (8). Com-
pounds 6 (0.30 g, 1.31 mmol) and 8a (0.328 g, 1.57 mmol) were
reacted together as per method A to obtain compound 8 as a light-
EXPERIMENTAL SECTION
■
General Synthesis. Chemicals were purchased from Aldrich
Chemical Co. (Milwaukee, WI), AK scientific (Union City, CA),
Oakwood Products (West Columbia, SC), Alfa Aesar (Ward Hill,
MA), and TCI America (Portland, OR) and were used as received. All
compounds were checked for homogeneity by TLC using silica gel as a
stationary phase. Melting points were determined on a Thomas−
Hoover capillary melting point apparatus and were uncorrected. NMR
spectra were recorded on a Bruker 400 Avance DPX spectrometer (1H
at 400 MHz) outfitted with a z-axis gradient probe. The chemical shifts
1
for H NMR were reported in parts per million (δ ppm) downfield
from tetramethylsilane (TMS) as an internal standard. The H NMR
1
data are reported as follows: chemical shift, multiplicity s (singlet), d
(doublet), t (triplet), dd (doublet of doublets), m (multiplet), and bs
(broad singlet). Flash chromatography was performed using silica gel
(0.060−0.200 mm) obtained from Dynamic adsorbents. The purity of
all target compounds was assessed using an Agilent 1260 Infinity
HPLC system. The column used was a C18 reverse phase column
(Phenomenex-Kinetex, 150 mm × 4.6 mm, 5 μ, 100 Å, serial no.
660057-3) eluting with an isocratic mobile phase (acetonitrile/water
60:40) at a flow rate of 1.0 mL/min, and samples were monitored at
UV = 254 nm. All tested compounds were confirmed to be ≥95% pure
based on the area of the major peak when compared to the total
combined area.
1
yellow foam (0.462 g, 84%); Rf = 0.50 (EtOAc/n-hexane 1:1). H
NMR (400 MHz; CDCl3; TMS) δ 8.30 (s, 1H), 7.90 (1H, s), 7.53 (d,
1H, J = 15.68 Hz), 7.15−7.16 (m, 1H), 7.13 (d, 1H, J = 8.3 Hz), 6.95
(d, 1H, J = 8.2 Hz), 6.68 (d, 1H, J = 15.64 Hz), 5.23 (d, 1H, J = 6.7
Hz), 4.37 (q, 2H, J = 7.12 Hz), 3.85 (s, 3H), 3.84 (s, 3H), 2.44−2.42
(m, 1H), 1.38 (t, 3H, J = 7.0 Hz), 1.03 (d, 3H, J = 6.7 Hz), 0.98 (d,
3H, J = 6.7 Hz). m/z (ESI-MS) 441.25 (C21H26N2O5SNa requires
441.16, [M + Na]+). HPLC tR = 3.4 min, purity 99%.
(S)-2-[2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid Ethyl Ester (9). Compounds 6 (2 g,
8.77 mmol) and 9a (2.42 g, 10.52 mmol) were reacted together by
following method B to obtain compound 9 as a white solid (3.03 g,
Synthesis. The synthesis and characterization data for compounds
2, 3, 4, 6, 10, and 13 was described in our recent report.16
82%); mp 144−150 °C; Rf = 0.60 (EtOAc/n-hexane 1:1). H NMR
1
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(400 MHz; CDCl3; TMS) δ 9.03 (d, 1H, J = 8.12 Hz), 8.45 (s, 1H),
7.23 (s, 2H), 5.07 (t, 1H, J = 8.16 Hz), 4.30 (q, 2H, J = 6.4 Hz), 3.83
(s, 6H), 3.70 (s, 3H), 2.47−2.55 (m, 1H), 1.31 (t, 3H, J = 5.8 Hz),
1.05 (d, 3H, J = 6.1 Hz), 0.90 (d, 3H, J = 6.24 Hz). m/z (ESI-MS)
445.33 (C20H26N2O6SNa requires 445.15, [M + Na]+). HPLC tR = 3.7
min, purity 99%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid [1-(4-Methoxyphenyl)ethyl]amide
(17). Compounds 16 (0.1 g, 0.25 mmol) and 17a (0.046 g, 0.30
mmol) were reacted together following method A to obtain
compound 17 as a light-yellow solid (0.098 g, 74%); mp 50−54 °C;
Rf = 0.45 (EtOAc/n-hexane 1:1). 1H NMR (400 MHz; CDCl3; TMS)
δ 8.01 (s, 1H), 7.41 (d, 1H, J = 7.7 Hz), 7.32 (d, 1H, J = 8.3 Hz),
7.01−7.07 (m, 2H), 6.86−6.89 (m, 2H), 6.67−6.79 (m, 2H), 5.38−
5.26 (m, 2H), 3.90 (s, 6H), 3.87 (s, 3H), 3.80 (s, 3H), 2.47−2.45 (m,
1H), 1.59 (d, 3H, J = 6.4 Hz), 1.05 (d, 6H, J = 7.2 Hz). m/z (ESI-MS)
550.17 (C27H33N3O6SNa requires 550.21, [M + Na]+). HPLC tR = 3.9
min, purity 97%.
[1-(4-{1-[4-(2,4-Dimethoxyphenylcarbamoyl)thiazol-2-yl]-
(S)-2-methylpropylcarbamoyl}thiazol-2-yl)-(S)-2-
methylpropyl]carbamic Acid tert-Butyl Ester (11). Compounds
10 (0.10 g, 0.21 mmol) and 11a (0.065 g, 0.42 mmol) were reacted
together as per method A to obtain compound 11 as a white solid
1
(0.104 g, 81%); mp 70−72 °C; Rf = 0.55 (EtOAc/n-hexane 1:1). H
NMR (400 MHz; CDCl3; TMS) δ 9.10 (s, 1H), 8.13 (s, 1H), 8.08 (s,
1H), 7.90 (d, 1H, J = 9.12 Hz), 7.61 (s, 1H), 7.27 (s, 1H), 7.09 (d, 1H,
J = 8.76 Hz), 6.89 (d, 1H, J = 8.6 Hz), 5.41−5.36 (m, 1H), 5.13 (bs,
1H), 3.96 (s, 3H), 3.91 (s, 3H), 2.60−2.55 (m, 1H), 2.39 (bs, 1H),
1.48 (s, 9H), 1.10 (d, 6H, J = 6.2 Hz), 1.04 (d, 3H, J = 6.68 Hz), 0.98
(t, 3H, J = 7.12 Hz). m/z (HRMS) 640.2236 (C29H39N5O6S2Na
requires: 640.2239, [M + Na]+). HPLC tR = 7.6 min, purity 95%.
[ 2 - M e t h y l - 1 - ( 4 - { 2 - m e t h y l - 1 - [ 4 - ( 3 , 4 , 5 -
t r i m e t h o x y p h e n y l c a r b a m o y l ) t h i a z o l - 2 - y l ] - ( S ) -
propylcarbamoyl}thiazol-2-yl)-(S)-propyl]carbamic Acid tert-
Butyl Ester (12). Compounds 10 (0.10 g, 0.21 mmol) and 12a
(0.077 g, 0.42 mmol) were reacted together following method A to
obtain compound 12 as oil (0.11 g, 71%); Rf = 0.45 (EtOAc/n-hexane
1:1). 1H NMR (400 MHz; CDCl3; TMS) δ 8.09 (s, 1H), 8.06 (s, 1H),
7.84 (d, 1H, J = 9.4 Hz), 7.65 (s, 1H), 7.29 (s, 1H), 6.62 (s, 1H),
6.61(s, 1H), 5.36 (m, 1H), 4.66 (m, 1H), 3.88 (s, 6H), 3.78 (s, 3H),
2.54−2.49 (m, 1H), 2.30 (bs, 1H), 1.48 (s, 9H), 1.05−0.90 (m, 12H).
m/z (HRMS) 670.2348 (C30H41N5O7S2Na requires: 670.2345, [M +
Na]+). HPLC tR = 8.0 min, purity 95%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (3,5-Dimethoxyphenyl)amide (18).
Compounds 16 (0.1 g, 0.25 mmol) and 18a (0.046 g, 0.30 mmol)
were reacted together using method A to obtain compound 18 as a
light-yellow solid (0.099 g, 74%); mp 124−130 °C; Rf = 0.55 (EtOAc/
1
n-hexane 1:1). H NMR (400 MHz; CDCl3; TMS) δ 9.05 (s, 1H),
8.12 (s, 1H), 7.06 (s, 2H), 6.93 (s, 2H), 6.66 (t, 1H, J = 8.7 Hz), 6.28
(s, 1H), 5.41 (dd, 1H, J = 9.33 Hz, 6.6 Hz), 3.89−3.92 (m, 9H), 3.81
(s, 6H), 2.49−2.58 (m, 1H), 1.06−1.10 (m, 6H). m/z (HRMS)
552.1770 (C26H31N3O7SNa requires: 552.1780, [M + Na]+). HPLC tR
= 4.3 min, purity 100%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (3,4,5-Trimethoxyphenyl)amide
(19). Compounds 16 (0.1 g, 0.25 mmol) and 19a (0.055 g, 0.30
mmol) were reacted together following method A to obtain
compound 19 as off-white solid (0.106 g, 75%); mp 58−62 °C; Rf
1
= 0.40 (EtOAc/n-hexane 1:1). H NMR (400 MHz; CDCl3; TMS) δ
9.03 (s, 1H), 8.15 (s, 1H), 7.06 (s, 2H), 7.02 (s, 2H), 6.61 (d, 1H, J =
8.68 Hz), 5.45 (dd, 1H, J = 9.3 Hz, 6.6 Hz), 3.94 (s, 6H), 3.91 (s, 9H),
3.86 (s, 3H), 2.52−2.60 (m, 1H), 1.09−1.12 (m, 6H). m/z (HRMS)
582.1900 (C27H33N3O8SNa requires: 582.1886, [M + Na]+). HPLC tR
= 3.3 min, purity 99%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid 3,4,5-Trimethoxybenzylamide (20).
Compounds 16 (0.1 g, 0.25 mmol) and 20a (0.059 g, 0.30 mmol)
were reacted together by following method A to obtain compound 20
as a light-yellow solid (0.104 g, 72%); mp 76−80 °C; Rf = 0.30
(EtOAc/n-hexane 1:1). 1H NMR (400 MHz; CDCl3; TMS) δ 8.07 (s,
1H), 7.57 (t, 1H, J = 5.8 Hz), 7.01 (s, 2H), 6.68 (d, 1H, J = 8.9 Hz),
6.58 (s, 2H), 5.37 (dd, 1H, J = 8.9 Hz, 6.5 Hz), 4.56 (t, 2H, J = 5.0
Hz), 3.88 (s, 9H), 3.84 (s, 6H), 3.83 (s, 3H), 2.48−2.43 (m, 1H), 1.05
(d, 3H, J = 6.8 Hz), 1.01 (d, 3H, J = 6.8 Hz). m/z (ESI-MS) 596.25
(C28H35N3O8SNa requires 596.21, [M + Na]+). HPLC tR = 2.7 min,
purity 100%.
2-[1-({2-[1-(3,4-Dimethoxybenzoylamino)-(S)-2-
methylpropyl]thiazole-4-carbonyl}amino)-(S)-2-
methylpropyl]thiazole-4-carboxylic Acid Ethyl Ester (14).
Compounds 13 (0.1 g, 0.24 mmol) and 14a (0.058 g, 0.29 mmol)
were reacted together according to method B to obtain compound 14
1
as a white foam (0.096 g, 69%); Rf = 0.40 (EtOAc/n-hexane 1:1). H
NMR (400 MHz; CDCl3; TMS) δ 8.12 (d, 1H, J = 8.5 Hz), 8.05 (d,
1H, J = 3.2 Hz), 7.96 (t, 1H, J = 6.2 Hz), 7.49 (d, 1H, J = 5.8 Hz), 7.40
(dd, 1H, J = 8.3 Hz, 2.0 Hz), 6.92 (dd, 1H, J = 8.3 Hz, 4.4 Hz), 6.80
(m, 1H), 5.40 (m, 1H), 5.31 (m, 1H), 3.95−3.93 (m, 8H), 2.64−2.58
(m, 1H), 2.54−2.47 (m, 1H), 1.25 (bs, 3H), 1.98−0.97 (m, 12H). m/z
(HRMS) 597.1826 (C27H34N4O6S2Na requires: 597.1817, [M +
Na]+). HPLC tR = 3.5 min, purity 95%.
2 - [ ( S ) - 2 - M e t h y l - 1 - ( { 2 - [ - (S ) - 2 - m e t h y l - 1 - ( 3 , 4 , 5 -
trimethoxybenzoylamino)propyl]thiazole-4-carbonyl}amino)-
propyl]thiazole-4-carboxylic Acid Ethyl Ester (15). Compounds
13 (0.1 g, 0.24 mmol) and 15a (0.063 g, 0.29 mmol) were reacted
together following method B to obtain compound 15 as a yellow solid
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (Benzo[1,3]dioxol-5-ylmethyl)-
amide (21). Compounds 16 (0.1 g, 0.25 mmol) and 21a (0.045 g,
0.30 mmol) were reacted together following method A to obtain
compound 21 as a dark-brown solid (0.105 g, 79%); mp 54−58 °C; Rf
1
(0.106 g, 72%); mp 58−62 °C; Rf = 0.40 (EtOAc/n-hexane 1:1). H
NMR (400 MHz; CDCl3; TMS) δ 8.12 (d, 1H, J = 7.3 Hz), 8.07 (d,
1H, J = 2.16 Hz), 8.02−7.94 (m, 1H,), 7.11 (s, 1H), 7.08 (s, 1H), 6.89
(dd, 1H, J = 12.7 Hz, 9.1 Hz), 5.42−5.31 (m, 2H), 3.96−3.89 (m,
11H), 2.61−2.47 (m, 2H), 1.27 (t, 3H, J = 5.8 Hz), 1.12−0.97 (m,
12H). m/z (ESI-MS) 627.28 (C28H36N4O7S2Na requires 627.19, [M +
Na]+). HPLC tR = 3.6 min, purity 99%.
1
= 0.35 (EtOAc/n-hexane 3:2). H NMR (400 MHz; CDCl3; TMS) δ
8.07 (s, 1H), 7.49 (t, 1H, J = 5.8 Hz), 7.00 (s, 2H), 6.85 (s, 1H), 6.81
(s, 1H), 6.76 (d, 1H, J = 8.1 Hz), 6.62 (d, 1H, J = 8.8 Hz), 5.95 (s,
2H), 5.35 (dd, 1H, J = 9.1 Hz, 6.4 Hz), 4.55 (t, 2H, J = 8.1 Hz), 3.88
(s, 9H), 2.41−2.49 (m, 1H), 1.04 (d, 3H, J = 6.8 Hz), 1.00 (d, 3H, J =
6.8 Hz). m/z (ESI-MS) 550.25 (C26H29N3O7SNa requires 550.17, [M
+ Na]+). HPLC tR = 3.1 min, purity 100%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid Benzo[1,3]dioxol-5-ylamide (22).
Compounds 16 (0.1 g, 0.25 mmol) and 22a (0.041 g, 0.30 mmol)
were reacted together following method A to obtain compound 22 as a
light-yellow solid (0.087 g, 67%); mp 60−64 °C; Rf = 0.50 (EtOAc/n-
hexane 3:2). 1H NMR (400 MHz; CDCl3; TMS) δ 9.00 (s, 1H), 8.12
(s, 1H), 7.42 (s, 2H), 7.02 (s, 1H), 6.97 (d, 1H, J = 6.72 Hz), 6.80 (d,
1H, J = 8.3 Hz), 6.62 (d, 1H, J = 8.7 Hz), 5.98 (s, 2H), 5.40 (dd, 1H, J
= 8.8 Hz, 6.4 Hz), 3.92 (s, 6H), 3.90 (s, 3H), 2.48−2..57 (m, 1H), 1.08
(t, 6H, J = 6.9 Hz). m/z (HRMS) 536.1478 (C25H27N3O7SNa
requires: 536.1467, [M + Na]+). HPLC tR = 3.5 min, purity 99%.
N-{1-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinoline-2-
carbonyl)thiazol-2-yl]-(S)-2-methylpropyl}-3,4,5-trimethoxy-
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (16). Compound 9 (2.2 g, 5.20 mmol)
was added to the solvent mixture [THF:methanol:water (10:2:3)], and
cooled to 0 °C. Sodium hydroxide (10 equiv) was added, and the
mixture was stirred at rt for 12 h. The reaction mixture was then
concentrated in vacuo and partitioned between ethyl acetate (30 mL)
and water (20 mL). The aqueous phase containing compound was
collected and acidified to pH 4 with 10% potassium hydrogen sulfate
and then extracted with ethyl acetate (3 × 20 mL). Organic fractions
were dried over sodium sulfate and concentrated under reduced
pressure to yield the intermediate compound 16 as a white solid (1.88
g, 92%); Rf = 0.20 (MeOH/CH2Cl2 5:95). 1H NMR (400 MHz;
DMSO-d6) δ 12.34 (s, 1H), 8.10 (s, 1H), 7.27 (s, 1H), 7.08 (s, 2H),
5.36 (t, 1H, J = 7.1 Hz), 3.93 (s, 6H), 3.88 (s, 3H), 2.47−2.55 (m,
1H), 1.08 (d, 3H, J = 8.0 Hz), 0.99 (d, 3H, J = 6.5 Hz).
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Journal of Medicinal Chemistry
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benzamide (23). Compounds 16 (0.1 g, 0.25 mmol) and 23a (0.058
g, 0.30 mmol) were reacted together as per method A to obtain
compound 23 as a light-yellow solid (0.116 g, 81%); mp 62−64 °C; Rf
(29). Compounds 16 (0.1 g, 0.25 mmol) and 29a (0.065 g, 0.30
mmol) were reacted together following method A to obtain
compound 29 as a light-yellow solid (0.095 g, 64%); mp 90−94 °C;
Rf = 0.40 (EtOAc/n-hexane 3:2). 1H NMR (400 MHz; CDCl3; TMS)
δ 12.29 (s, 1H), 8.81 (d, 1H, J = 8.2 Hz), 8.20 (s, 1H), 7.63−7.68 (m,
3H), 7.57 (d, 1H, J = 7.1 Hz), 7.50 (d, 1H, J = 8.8 Hz), 7.29 (s, 1H),
7.20 (s, 2H), 7.15 (t, 2H, J = 8.2 Hz), 5.51 (dt, 1H, J = 6.2 Hz), 3.91
(s, 3H), 3.85 (s, 6H), 2.55−2.47 (m, 1H), 1.11 (d, 3H, J = 6.8 Hz),
1.06 (d, 3H, J = 6.7 Hz). m/z (HRMS) 614.1736 (C31H30FN3O6SNa
requires: 614.1737, [M + Na]+). HPLC tR = 9.5 min, purity 100%.
Biological Procedures. Chemicals. Dulbecco’s Modified Eagle’s
Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin,
and trypsin 0.25% were products of Hyclone, Thermo Scientific
(Logan, UT). Phosphate buffered saline (PBS) 20× concentrate (PH
7.5) was purchased from AMRESCO (Solon, OH). Paclitaxel,
cyclosporine A, paraformaldehyde, 3-(4,5-dimethylthiazol-yl)-2,5-
diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO),
and other chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO). [125I]-Iodoarylazidoprazosin (2200 Ci/mmol) was
obtained from PerkinElmer Life Sciences (Wellesley, MA). OPSYS
microplate reader was purchased from Dynex Technologies (Chantilly,
VA).
Cell Lines and Cell Culture. HeLa cells were cultured in DMEM
media supplemented with 10% FBS, 1% glutamine, and 1% penicillin.
The human colon cancer cell line SW620 and its doxorubicin-selected
P-gp-overexpressing subline SW620/Ad30031 were cultured at 37 °C,
5% CO2, with DMEM containing 10% FBS and 1% penicillin/
streptomycin. The human embryo kidney parental cell line HEK293
and HEK/ABCB1 transfected with human ABCB1 cDNA were
maintained in the DMEM with G418 (2 mg/mL) and cultured in a
manner similar to that of the above cell lines. All cells were grown as
adherent monolayers in drug-free culture media for more than 2 weeks
before the assay.
Transduction of HeLa Cells with BacMam-P-gp Baculovirus.
HeLa cells were transduced with BacMam WT-P-gp virus, which was
added at a titer of 10−15 viral particles per cell.32 After an hour,
DMEM medium was added and the cells were incubated further. Then
10 mM butyric acid was added after 3−4 h, and the cells were grown
overnight at 37 °C. The cells were trypsinized, washed, counted, and
analyzed by flow cytometry for inhibition of transport function of WT
P-gp by the derivatives.
Calcein-AM Efflux Assay. The ability of the synthesized derivatives
to inhibit the transport function of P-gp was checked using flow
cytometry as described previously.33,34 Briefly, the transfected cells
were trypsinized and incubated with various concentrations of the
derivatives followed by calcein-AM (0.5 μM).32 The cells were
analyzed after washing with cold PBS. Fluorescence produced by
calcein was measured on a FACSort flow cytometer equipped with a
488 nm argon laser and 530 nm bandpass filter. The results are
reported as an average of two independent experiments, each done in
triplicates. The IC50 of these derivatives was calculated using GraphPad
Prism 5.0.
1
= 0.30 (EtOAc/n-hexane 3:2). H NMR (400 MHz; CDCl3; TMS) δ
7.83 (s, 1H), 7.06 (s, 2H), 6.90 (d, 1H, J = 8.64), 6.65 (s, 1H), 6.59 (s,
1H), 5.44−5.48 (m, 1H), 4.80−4.88 (m, 2H), 3.89 (s, 9H), 3.86 (s,
6H), 2.83−2.88 (m, 2H), 2.46−2.52 (m, 1H), 1.8 (s, 2H), 1.03−1.07
(m, 6H). m/z (ESI-MS) 592.17 (C29H35N3O7SNa requires 592.22, [M
+ Na]+). HPLC tR = 2.8 min, purity 100%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid Indan-2-ylamide (24). Compounds 16
(0.1 g, 0.25 mmol) and 24a (0.040 g, 0.30 mmol) were reacted
together following method A to obtain compound 24 as a light-yellow
solid (0.117 g, 91%); mp 74−78 °C; Rf = 0.50 (EtOAc/n-hexane 3:2).
1H NMR (400 MHz; CDCl3; TMS) δ 8.04 (s, 1H), 7.40 (d, 1H, J =
7.84), 7.17−7.24 (m, 4H), 6.99 (s, 2H), 6.55 (d, 1H, J = 8.64 Hz),
5.32 (dd, 1H, J = 8.9 Hz, 6.6 Hz), 4.89−4.95 (m, 1H), 3.89 (s, 6H),
3.88 (s, 3H), 3.46 (m, 2H), 2.98 (m, 2H), 2.49 (m, 1H), 1.04 (d, 3H, J
= 6.76 Hz), 1.04 (d, 3H, J = 6.68 Hz). m/z (HRMS) 532.1893
(C27H31N3O5SNa requires: 532.1882, [M + Na]+). HPLC tR = 4.1
min, purity 99%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (2-Pyridin-2-yl-ethyl)amide (25).
Compounds 16 (0.1 g, 0.25 mmol) and 25a (0.037 g, 0.30 mmol)
were reacted together by using method A to obtain compound 25 as a
1
light-yellow oil (0.119 g, 94%); Rf = 0.20 (EtOAc/n-hexane 3:2). H
NMR (400 MHz; CDCl3; TMS) δ 8.40 (s, 1H), 8.14 (s, 1H), 7.92 (d,
1H, 10.3 Hz), 7.59 (td, 1H, J = 7.7 Hz, 6.4 Hz), 7.38 (d, 1H, J = 8.1
Hz), 7.14−7.18 (m, 3H), 7.08−7.11 (m, 1H), 5.35−5.39 (m, 1H)
3.87−3.89 (m, 9H), 3.77−3.82 (m, 2H), 3.06 (t, 2H, J = 6.4 Hz),
2.43−2.51 (m, 1H), 1.07 (d, 3H, J = 6.8 Hz), 1.02 (d, 3H, J = 6.7 Hz).
m/z (ESI-MS) 499.42 (C25H31N4O5S requires 499.19, [M + H]+).
HPLC tR = 2.2 min, purity 99%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (Biphenyl-4-ylmethyl)amide (26).
Compounds 16 (0.1 g, 0.25 mmol) and 26a (0.055 g, 0.30 mmol)
were reacted together following method A to obtain compound 26 as a
white solid (0.120 g, 85%); mp 70−74 °C; Rf = 0.50 (EtOAc/n-hexane
1
3:2). H NMR (400 MHz; CDCl3; TMS) δ 8.11 (s, 1H), 7.57−7.60
(m, 5H), 7.42−7.47 (m, 4H), 7.38 (d, 1H, J = 7.4 Hz), 7.02 (s, 2H),
6.66 (d, 1H, J = 7.6 Hz), 5.34−5.38 (m, 1H), 4.67−4.69 (m, 2H), 3.88
(s, 9H), 2.41−2.49 (m, 1H), 1.01−1.05 (m, 6H). m/z (ESI-MS)
560.17 (C31H34N3O5S requires 560.21, [M + H]+). HPLC tR = 6.3
min, purity 100%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (4-Benzoylphenyl)amide (27). Com-
pounds 16 (0.1 g, 0.25 mmol) and 27a (0.060 g, 0.30 mmol) were
reacted together following method A to obtain compound 27 as a
white solid (0.118 g, 81%); mp 72−76 °C; Rf = 0.50 (EtOAc/n-hexane
3:2). 1H NMR (400 MHz; DMSO-d6) δ 10.49 (s, 1H), 9.00 (d, 1H, J
= 8.4 Hz), 8.46 (s, 1H), 8.06 (d, 2H, J = 8.7 Hz), 7.81 (d, 2H, J = 8.6
Hz), 7.75 (d, 2H, J = 7.1 Hz), 7.65−7.70 (m, 1H), 7.57 (t, 2H, J = 7.6
Hz), 7.24 (s, 2H), 5.24 (t, 1H, J = 8.2 Hz), 3.85 (s, 6H), 3.71 (s, 3H),
2.38−2.46 (m, 1H), 1.08 (d, 3H, J = 6.6 Hz), 0.98 (d, 3H, J = 6.7 Hz).
m/z (ESI-MS) 574.17 (C31H32N3O6S requires 574.19, [M + H]+).
HPLC tR = 6.8 min, purity 96%.
Cytotoxicity Determination by MTT Assay. We used a modified
MTT colorimetric assay to detect the sensitivity of cells to anticancer
drugs in vitro.35 Briefly, cells were seeded in 180 μL of medium in 96-
well plates in triplicate at 5000−6000 cells/well and incubated at 37
°C, 5% CO2, for 24 h to allow the cells to attach to the wells. Cells in
96-well plates were preincubated with or without the reversal agents
(20 μL/well) for 2 h, and then different concentrations of
chemotherapeutic drugs (20 μL/well) were added into designated
wells. After 72 h of incubation at 37 °C, 20 μL of MTT solution (4
mg/mL) was added to each well. The plates were further incubated at
37 °C for 4 h, allowing viable cells to change the yellow-colored MTT
into dark-blue formazan crystals. Subsequently, the MTT/medium was
removed from each well without disturbing the cells, and 100 μL of
DMSO was added into each well. Plates were placed on a shaking table
to thoroughly mix the formazan into the solvent. Finally, the
absorbance was determined at 570 nm by Opsys microplate reader
(Dynex Technologies, Chantilly, VA). The MTT assays were
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid (2-Benzoylphenyl)amide (28). Com-
pounds 16 (0.1 g, 0.25 mmol) and 28a (0.060 g, 0.30 mmol) were
reacted together using method A to obtain compound 28 as a light-
yellow solid (0.097 g, 67%); mp 78−82 °C; Rf = 0.45 (EtOAc/n-
1
hexane 3:2). H NMR (400 MHz; DMSO-d6) δ 11.73 (s, 1H), 9.02
(d, 1H, J = 7.9 Hz), 8.52 (d, 1H, J = 8.5 Hz), 8.39 (s, 1H), 7.65−7.73
(m, 4H), 7.55 (t, 3H, J = 7.7 Hz), 7.24−7.29 (m, 3H), 5.17 (t, 1H, J =
8.3 Hz), 3.83 (s, 6H), 3.71 (s, 3H), 2.38−2.46 (m, 1H), 1.09 (d, 3H, J
= 6.7 Hz), 1.02 (d, 3H, J = 6.7 Hz). m/z (HRMS) 596.1851
(C31H31N3O6SNa requires: 596.1831, [M + Na]+). HPLC tR = 9.4
min, purity 96%.
2-[(S)-2-Methyl-1-(3,4,5-trimethoxybenzoylamino)propyl]-
thiazole-4-carboxylic Acid [2-(4-Fluorobenzoyl)phenyl]amide
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Journal of Medicinal Chemistry
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performed four times independently, and each independent experi-
ment was done in triplicate.
for compound 28 was used as input for grid generation and ligands to
be docked, respectively. In this, the centroid of the ligand in the
refined protein structure was selected to generate grid. The top scoring
conformation of 28 at site-1 of human P-gp was used for graphical
analysis. Computational work was carried out on a Dell Precision 490n
dual processor with the Linux OS (Ubuntu 12.04 LTS).
Photoaffinity Labeling of ABCB1 with [125I]-Iodoarylazidoprazo-
sin. ABCB1-transfected High Five insect cell membranes expressing
ABCB1 (P-gp) (65 μg protein/100 μL) were incubated with varying
concentrations of compound 28 (0−20 μM) for 10 min at 37 °C in 50
mM MES-Tris pH 6.8. [125I]-IAAP (2200 Ci/mmol; 4−6 nM) was
added and membranes incubated for an additional 5 min with minimal
exposure to light. The samples were then illuminated with a 365 nm
UV lamp for 10 min and photoaffinity labeling of P-gp with [125I]-
IAAP was determined as previously described.36 Results from two
independent experiments are reported (Figure 4A).
AUTHOR INFORMATION
Corresponding Authors
*For S.V.A.: phone, +1 301 402 4178; fax, +1 301 435 8188; E-
■
mail, ambudkar@mail.nih.gov.
*For T.T.T.: phone, +1 718 990 5405; fax, +1 718 990 1877; E-
mail, talelet@stjohns.edu.
ATP Hydrolysis by P-gp. The vanadate-sensitive ATPase activity of
P-gp in crude membranes of High Five insect cells, in the presence of
concentrations of compound 28 ranging from 0 to 10 μM, was
measured as previously described.37 Three independent experiments in
duplicates were carried out and results are reported as mean SEM
(Figure 4B).
Molecular Modeling. Ligand Preparation. The structures of the
linear thiazole derivatives were built using the fragment dictionary of
Maestro v9.5 and energy minimized by Macromodel program v10.1
Present Address
§For N.R.P.: Department of Biochemistry and Biotechnology,
Annamalai University, Annamalainagar 608 002, Tamilnadu,
India.
Author Contributions
The manuscript was written through contributions of all the
authors. All authors have given approval to the final version of
the manuscript.
(Schrodinger, LLC, New York, NY, 2013). LigPrep v2.7 tool was used
̈
to generate low energy 3D conformers of the minimized structures.
Default settings were used, but the “Generate Tautomers” option was
not selected. The resultant ligand structures were eventually docked at
all four grids (site-1 to site-4) generated on homology-modeled human
P-gp.
Notes
The authors declare no competing financial interest.
Homology Modeling. The refined crystal structure of mouse P-gp
in complex with compounds 1c (PDB ID: 4M2T)8 and 1d (PDB ID:
4M2S)8 served as templates to generate ligand-bound homology
models of human P-gp. The alignment of human P-gp and mouse P-gp
sequences resulted in 87% sequence identity and 93% similarity. The
protocol for homology modeling using the default parameters of Prime
ACKNOWLEDGMENTS
■
This research was supported by the Department of
Pharmaceutical Sciences of St. John’s University and St.
John’s University Seed Grant no. 579-1110 to T.T.T. Drs.
N.R.P., E.E.C., and S.V.A. were supported by the Intramural
Research Program of the NIH, National Cancer Institute,
Center for Cancer Research. Financial support from Indian
Council of Medical Research, New Delhi, in the form of
International Fellowship for Young Indian Biomedical
Scientists to Dr. N.R.P. is gratefully acknowledged (Indo/
FRC/452(Y-19)/2012-13IHD). We acknowledge Drs. Susan E.
Bates and Robert W. Robey (NCI, NIH, Bethesda, MD) for
providing SW620 and SW620/Ad300 cell lines. We thank Dr.
Stephen Aller (The University of Alabama at Birmingham,
Birmingham, AL) for generously providing human apo-state P-
gp homology model. We thank George Leiman for editorial
assistance. S.S. and T.T.T. are thankful to Dr. Sanjai Kumar and
Dibyendu Dana (Queens CollegeCUNY, Queens, NY) for
assisting in ESI-MS and HRMS analyses.
v3.3 implemented in Maestro v9.5 (Schrodinger, LLC, New York, NY,
̈
2013) is essentially the same as reported earlier.29 Validation of the
generated homology models was performed using Ramachandran plot
analysis, which suggested more than 91% residues in the core allowed
region, 5−8% residues in the allowed regions, and <0.7% residues in
the sterically disallowed regions. The backbone root-mean-square
deviation (RMSD) was calculated for the homology models from the
corresponding experimental structures. The RMSD was found to be
less than 0.33 Å for all of the generated models, which is not surprising
considering that mouse P-gp and human P-gp share high (93%)
sequence similarity. The homology modeled human P-gp generated
from the mouse model in complex with 1c and 1d were used as
templates for generating grids for site-2 and site-1, respectively. The
human P-gp homology model based on mouse P-gp in apoprotein
state was generously provided by Dr. S. Aller and was used to generate
grids for site-3 and site-4.
Protein Preparation and Grid Generation. All homology models
were refined by default parameters in Protein Preparation Wizard
ABBREVIATIONS USED
■
ABC, ATP-binding cassette; calcein-AM, calcein acetoxyme-
thylester; HCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxy-
benzotriazole; IAAP, iodoarylazidoprazosin; IPTG, isopropyl β-
D-1-thiogalactopyranoside; MDR, multidrug resistance; MES, 2-
(N-morpholino)ethanesulfonic acid; P-gp, P-glycoprotein; TM,
transmembrane
implemented in Maestro v9.5 and Impact program v6.0 (Schrodinger,
̈
LLC, New York, NY, 2013), in which the protonation states of the
ionizable residues were adjusted to the dominant ionic forms at
physiological pH. On the basis of refined human P-gp homology
model, different receptor grids were generated by selecting 1c (site-2)
and 1d (site-1) bound ligands, all amino acid residues that are known
to contribute to verapamil binding (site-3), and two residues (Phe728
and Val982) common in the previous three sites (site-4).
Docking Protocol. To determine the probable binding site for
compound 28, the LigPrep derived ligand structure was docked at each
of the generated grids (site-1 to site-4) of P-gp using the “Extra
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Journal of Medicinal Chemistry
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