Novel 5-oxo-hexahydroquinoline derivatives: Design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

Article abstract of DOI:10.2147/DDDT.S119995

Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C₄ and various carboxamide substituents at C₃ were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells’ sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C₄ position and compounds with 4-chlorophenyl carboxamide at C₃ demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 μM, and it also significantly reduced the IC₅₀ of DXR by 70.1% and 88.7% at 10 and 25 μM, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal.

Full text of DOI:10.2147/DDDT.S119995

Drug Design, Development and erapy
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O r i g i n a l r e s e a r c h
novel 5-oxo-hexahydroquinoline derivatives:
design, synthesis, in vitro P-glycoprotein-mediated
multidrug resistance reversal profile and
molecular dynamics simulation study
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
14 February 2017
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Omolbanin shahraki1,2
najmeh edraki1
Mehdi Khoshneviszadeh1,2
Farshid Zargari1
sara ranjbar1,2
Abstract: Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important
mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-
hexahydroquinoline derivatives containing different nitrophenyl moieties at C4 and various
carboxamide substituents at C3 were designed, synthesized and evaluated for their ability to
inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma
cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds
with highest MDR reversal activities was further evaluated by measuring the alterations of
MES-SA/Dx5 cells’ sensitivity to doxorubicin (DXR) using MTT assay. The results of both
biological assays indicated that compounds bearing 2-nitrophenyl at C4 position and compounds
with 4-chlorophenyl carboxamide at C3 demonstrated the highest activities in resistant cells,
while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active
derivatives, 5c, significantly increased intracellular Rh123 at 100 µM, and it also significantly
reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 µM, respectively, in MES-SA/Dx5
cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also
investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular
dynamics simulation study was also performed to investigate the possible binding site of 5c in
complex with human P-gp, which showed that this compound formed 11 average H-bonds with
Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found
between the results of the computational and experimental studies. The findings of this study
show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for
the discovery of new agents for P-gp-mediated MDR reversal.
luciano saso3
Omidreza Firuzi1
ramin Miri1
1Medicinal and natural Products
chemistry research center,
2Department of Medicinal chemistry,
school of Pharmacy, shiraz University
of Medical sciences, shiraz, iran;
3Department of Physiology and
Pharmacology “Vittorio ersparmer”,
sapienza University of rome,
rome, italy
Keywords: cancer, P-glycoprotein, multidrug resistance, 1,4-dihydropyridine, molecular
dynamics simulation
Introduction
Despite recent progress in the elucidation of cancer biology and development of novel
strategies in cancer diagnosis and treatment, this disease is still among the leading
causes of death around the world.1 The resistance of malignant cells to structurally
and mechanistically unrelated classes of anticancer agents is recognized as MDR.2
Different mechanisms are involved in drug resistance; a very important one is overex-
pression of P-gp (ABCB1, MDR1), which has been one of the first members of ABC
transporters to be studied.3 P-gp extrudes a wide variety of endogenous molecules
and xenobiotics and plays an important physiological role in detoxifying cells from
correspondence: ramin Miri;
Omidreza Firuzi
Medicinal and natural Products
chemistry research center,
shiraz University of Medical sciences,
PO Box 71345-3388, shiraz, iran
Tel +98 713 230 7869
Fax +98 713 230 2225
email ramin.miri.15@gmail.com;
foomid@yahoo.com
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and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you
hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
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http://dx.doi.org/10.2147/DDDT.S119995

shahraki et al
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exogenous toxic agents.4 However, overexpression of P-gp Results and discussion
in cancer cells leads to reduced accumulation of chemothera-
peutic drugs and results in resistance against these agents.5 In
Fragment-based design
Several efforts have been made to clarify the basic features
that are responsible for the MDR reversal effect of DHPs.
In our previous studies, we described the design, synthe-
sis and in vitro data of a series of new inhibitors of P-gp
with 1,4-DHP structure carrying 2- or 3-pyridyl methyl
carboxylate at C3 with different alkyl carboxylate moieties
at C5 position as well as nitrophenyl or heteroaromatic rings
at C4 of central DHP core.26,27 Herein, we report a fragment-
based design for a new set of 5-oxo-hexahydroquinoline
derivatives based on a previously established SAR for
reported DHPs (Scheme 1).
this context, inhibition of P-gp by small-molecule inhibitors
seems to be a promising approach for overcoming MDR in
cancer cells.6
Three different generations of P-gp inhibitors have been
discovered. The first-generation inhibitors such as verapamil,
cyclosporine A and quinidine7 were developed for other
applications and then tested for P-gp inhibition. The second-
(such as valspodar8) and third-generation inhibitors (such as
elacridar,9 tariquidar,10 laniquidar11 and zosuquidar12) were
specifically designed for MDR reversal and did not display
other pharmacological effects. However, clinical results of
these agents were unsatisfactory due to insufficient therapeu-
tic benefit and unacceptable systemic toxicity.
–
–
–
The 5-oxo-hexahydroquinoline scaffold was chosen
based on the observation that DHP pharmacophore
exhibits MDR reversal effect.15
Initial studies on calcium channel blockers demonstrated
that verapamil and diltiazem were able to reverse MDR by
increasing intracellular levels of chemotherapeutic agents.13
Afterward, Safa et al confirmed P-gp-inhibitory effect of
calcium channel blocker azidopine belonging to the family
of 1,4-DHP derivatives.14 Other authors have also reported
the MDR reversal effect of a set of synthesized DHPs.15
SAR studies of DHPs show that replacement of car-
boxylate esters at C3 and C5 positions with aryl carboxamide
groups dramatically diminishes their cardiovascular effects.
We have previously studied the synthesis and biological eval-
uation of symmetric and asymmetric 1,4-DHPs containing
different carboxamide substitutions at mentioned positions
and observed that these compounds had poor calcium-
channel-blocking activity compared to nifedipine.16–19
The compounds bearing DHP core have been shown to
display a broad range of biological and pharmacological
effects.16 Besides their calcium-channel-blocking activity,20–22
they have shown therapeutic effects against Alzheimer’s
disease and atherosclerosis23 and have also shown MDR
reversal effect.20,24,25
The presence of nitroaryl substituents at C4 position
has previously proved to enhance MDR reversal effect
compared to heteroaryl rings.
Substitution of different moieties bearing nitrogen at C3
such as carboxamide group resulted in increasing MDR
reversal effect of designed compounds. Accordingly,
different pyridyl methyl carboxylates at C3 position26,27
were bioisosterically replaced with alkyl or aryl
carboxamides.
Many studies have revealed that natural products with
fused rings such as quercetin are MDR reversal agents.28
Based on these findings, bioisosteric replacement and
hybridization of 4H-chromen-4-one ring of quercetin
with 4,6,7,8-tetrahydroquinolin-5(1H)-one in DHP-based
designed compounds would be expected to improve MDR
reversal activity.29,30
Thus, in this study, a library of 26 compounds with 5-oxo-
hexahydroquinolone skeleton bearing different nitrophenyl
moieties at C4 and different aryl or alkyl carboxamides at
C3 was designed.
Three decades after the discovery of P-gp, lots of efforts chemistry
have been made on the development of novel P-gp inhibi- The applied route for synthesis of designed compounds is
tors, but no small-molecule inhibitor has been identified for outlined in Scheme 2, and the structures are presented in
clinical use. In an attempt to respond to this crucial need for Table 1. By the reaction of the commercially available differ-
novel therapeutic agents and as part of our research program ent primary and secondary amines (1) with 2,2,6-trimethyl-
toward the discovery of useful MDR reversal agents, herein 4H-1,3-dioxin-4-one (2), corresponding 3-oxobutanamides
we report synthesis, biological evaluation and MD simula- (1a–6a) were obtained (Table 2). The final products were
tions study of a set of 26 novel 5-oxo-hexahydroquinoline synthesized by the reaction of obtained intermediates (1a–6a)
derivatives. The experimental results showed that these with different aryl aldehydes and 1,3-cyclohexadione in the
compounds can indeed inhibit P-gp, and these findings were presence of excess amounts of ammonium acetate (Table 1).
in good agreement with the computational findings.
All compounds were completely purified and characterized
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a study of 5-oxo-hexahydroquinoline derivatives
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Scheme 1 Fragment-based design of P-gp inhibitors using DhP backbone.
Abbreviations: P-gp, P-glycoprotein; DhP, dihydropyridine; MDr, multidrug resistance.
1
using H NMR, 13C NMR and mass and IR spectroscopy continuous exposure to DXR. Alterations in the amount of
(Supplementary materials).
the fluorescent Rh123 retained inside the MES-SA-Dx5 cells
can be logically related to the inhibition of the activity of
P-gp efflux pump in the cells. Figure 1 shows a representative
histogram of the effect of different doses of compound 2f
Biological evaluation
rh123 accumulation assay
The P-gp-mediated Rh123 efflux was determined by flow on the intracellular accumulation of Rh123 in resistant cells.
cytometry on the DXR-resistant uterine sarcoma MES-SA/ A dose-dependent response can be clearly observed at 5, 25
Dx5 cell line and its parental nonresistant MES-SA cell line.31 and 100 µM on Rh123 accumulation as the histogram shifts
Verapamil was used as a positive control. MES-SA/Dx5 is to the right. All the active compounds except 2c showed a
a resistant cell line, which overexpresses P-gp as a result of dose-dependent effect. The results are expressed as Rh123
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Scheme 2 synthesis of the 5-oxo-hexahydroquinoline derivatives 1c–4f. Reagents and conditions: (i) xylene, reflux, 2–4 h; and (ii) CH3cOOnh4, ethanol, reflux, 24 h.
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shahraki et al
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Table 1 chemical structures of synthesized 5-oxo-hexahyroquinoline derivatives
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a study of 5-oxo-hexahydroquinoline derivatives
Table 2 chemical structure of synthesized intermediates (1a–6a)
DHP ring showed the highest levels of Rh123 accumulation
and P-gp inhibition at 100 µM. Compounds 2c and 5c also
exhibited a considerable MDR reversal activity at 25 and
100 µM, respectively.
Compounds Product
MW (g/mol) Yield (%)
1a
177.20
82
+
1
2
2
Considering the data presented in Figure 2, a brief SAR
2a
211.65
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can be deduced as shown in Figure 3.
1
2
2
In different series of synthesized compounds, those
bearing 2-nitrophenyl substitution at C4 such as 1c, 2c, 3c,
5c and 6c were the most potent derivatives. This reveals that
the presence of 2-nitrophenyl moiety is crucial in enhancing
the potency.
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Compounds containing N-(4-chlorophenyl) carboxamide
at C3 (2c, 2d and 2f) exhibited good MDR reversal effect.
For example, compound 2f showed 6.0-fold Rh123 accu-
mulation relative to the negative control.
2
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5a
6a
202.21
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78
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None of the compounds bearing 4-carboxyphenyl moi-
ety at C3 (4c, 4d and 4e) were effective MDR reversal
agents. 4c and 4d exhibited only 1.32-fold Rh123
accumulation.
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As shown in Figure 4, the effects of synthesized com-
pounds were specific against resistant MES-SA/Dx5 cell
line, because Rh123 accumulation was not observed in the
parental MES-SA cells.
accumulation fold increase, representing the ratio of Rh123
fluorescence in the presence and absence of synthesized com-
pounds (Figure 2). Compound 2f bearing phenyl substitute at
C4 and N-(4-chlorophenyl) carboxamide at C3 position of the
Determination of chemosensitization in resistant
cells induced by synthesized compounds
Effects of synthesized compounds on the alteration of
MES-SA/Dx5’s sensitivity to DXR were evaluated by MTT
assay, in which cell viability is measured spectrophotometri-
cally by the amount of reduced formazan. For this purpose,
the most potent compounds were selected based on the results
of Rh123 accumulation assay. To select a nontoxic concentra-
tion (cell survival .85%), cytotoxicity assay was performed
on P-gp-expressing MES-SA/Dx5 cell line. On the basis
of the obtained results, maximum concentration was set to
25 µM (data not shown). The effects of coadministration of
synthesized compounds and DXR were then studied in these
cells. The percent reduction of DXR’s IC50 was calculated
(Figure 5). DXR showed a relative low activity in the assay
of cell survival (IC50: 2.7 µM). The compounds 1c and 3c
at 25 µM decreased the DXR’s IC50 by 47.7% and 69.8%,
respectively, and did not show any considerable chemosensi-
tization effect. Compound 2f, which was the most potent com-
pound in the Rh123 accumulation assay, showed a moderate
activity in this assay. The most potent compound 2c bearing
2-nitrophenyl at C4 and 4-chlorophenycarboxamide at C3
showed 92.3% and 99.2% decrease in the IC50 of DXR at 10
and 25 µM, respectively. The other 2-nitrophenyl-containing
compound 5c with 4-methoxyphenyl substitute at C3 also
256
25 µM
Control
5 µM
100 µM
0
100
101
102
103
104
Rh 123
Figure 1 Flow cytometric histogram of 2f demonstrating Rh123 fluorescence in
Mes-sa/Dx5 cell line. cells were pretreated with 5, 25 and 100 µM of synthesized
compounds for 20 min and then exposed to 5 µM rh123 for another 20 min at 37°c.
The cells were then washed three times with ice-cold PBs. Fluorescence intensity
caused by intracellular Rh123 accumulation was measured by an FACSCalibur flow
cytometer at 488 and 530 nm excitation and emission wavelengths, respectively.
Abbreviations: rh123, rhodamine 123; PBs, phosphate-buffered saline.
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Figure 2 The effect of synthesized compounds on the intracellular accumulation of rh123 in Mes-sa/Dx5 cell line. cells were pretreated with 5, 25 or 100 µM of synthesized
compounds for 20 min and then exposed to 5 µM rh123 for another 20 min at 37°c. The cells were then washed three times with ice-cold PBs. rh123 retained in cells
was measured by flow cytometry at 488 and 530 nm excitation and emission wavelengths, respectively. Rh123 accumulation fold increases were calculated by dividing
the mean fluorescence intensity obtained for each compound by that of control cells. The effects of synthesized compounds with 2-nitrophenyl (A), 3-nitrophenyl (B),
4-nitrophenyl (C) and phenyl (D) at c4 position and verapamil as positive control are shown. Data are presented as mean ± seM of three to four independent measurements.
*The difference between Rh123 accumulation folds in the absence and presence of test compound is significant (P,0.05).
Abbreviations: rh123, rhodamine 123; PBs, phosphate-buffered saline; seM, standard error of the mean.
resulted in 70.1% and 88.7% decrease in the IC50 of DXR observed between the results of Rh123 accumulation assay
at 10 and 25 µM, respectively. The other tested compounds and DXR uptake in resistant cells.
were able to induce a statistically significant decrease in the
IC50 of DXR at 10 and 25 µM, and a good correlation was
cytotoxicity against heK293 cells
In order to determine the effect of synthesized compounds
on HEK293 (human embryonic kidney) cells used as a nor-
mal cell line, the MTT assay was performed. The cells were
treated with the test compounds at concentrations of 5, 10
and 25 µM for 48 h. The obtained results (Figure 6) revealed
that all compounds exhibited no cytotoxicity against normal
cells (viability .80%) except compound 2c which showed
54.9% viability at 25 µM.
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Different studies have been focused on the elucidation of
the mechanism of action of synthesized DHPs on P-gp and
identification of possible binding sites of DHPs. To this
purpose, domain mapping using photoaffinity labeling along
with immunoprecipitation of different DHP derivatives has
confirmed that the binding site is located near the N-terminal
Figure 3 structure–activity relationship study and the effect of substituted moieties
on MDr reversal activity of target compounds obtained from rh123 accumulation
assay against Mes-sa/Dx5 cells. Upside and downside arrows demonstrate that the
substitutions had a positive or a negative influence on the MDR reversal potential,
respectively.
Abbreviations: MDr, multidrug resistance; rh123, rhodamine 123.
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a study of 5-oxo-hexahydroquinoline derivatives
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Figure 4 The effect of synthesized compounds on the intracellular accumulation of rh123 in Mes-sa cell line. cells were pretreated with 5, 25 and 100 µM of synthesized
compounds or 5 and 25 µM of verapamil for 20 min and then exposed to 5 µM of rh123 for another 20 min at 37°c. The cells were then washed three times with ice-
cold PBS. Mean fluorescence intensity caused by Rh123 was evaluated by flow cytometry at 488 and 530 nm excitation and emission wavelengths, respectively. The Rh123
accumulation fold increases were calculated by dividing the mean fluorescence intensity obtained for each compound by that of control cells. The effects of synthesized
compounds with 2-nitrophenyl (A), 3-nitrophenyl (B), 4-nitrophenyl (C) and phenyl (D) at c4 position and verapamil as positive control are shown. Data represent mean ±
SEM of three independent measurements. No significant difference was observed between Rh123 accumulation in the absence and presence of synthesized compounds for
any of the derivatives (P.0.05).
Abbreviations: rh123, rhodamine 123; PBs, phosphate-buffered saline; seM, standard error of the mean.
nucleotide-binding domain of P-gp.32 Ferry et al proposed that
ꢀꢁꢂ
DHP-binding site that is located in the cytoplasmic region of
P-gp comprises residues 491–525.33 Other investigators have
ꢀ
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used a dexniguldipine-HCl ([3H] B9209-005) to probe the
DHPs’ binding site showing that residues 468–527 are the
ligand-interacting region of the protein. These findings show
that DHP derivatives probably bind to the nucleotide-binding
domain of P-gp, inhibiting the action of this efflux pump.34
In order to explore the structural requirements of 5-oxo-
hexahydroquinoline derivatives which determine their inter-
action with P-gp, we chose the most favorite pose of one of
the most potent and nontoxic compounds 5c from docking
study and placed it in the proposed active site of homology-
modeled P-gp. The crystal structure of murine P-gp (PDB
ID: 3G5U, chain A, resolution: 3.8 Å) served as a template
to create homology models of human P-gp (UNIPORT ID:
P08183). The alignment of human P-gp and mouse P-gp
sequences shows 87% identity. The QMEAN Z-score of
the obtained models ranged from -2.35 (best) to -4.14.
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Figure 5 The effect of synthesized compounds on the reduction of doxorubicin’s
cytotoxicity in Mes-sa/Dx5 cell line. cells were seeded in 96-well microplates and
cultured overnight. They were then treated with 5, 10 and 25 µM of test compounds.
after 90 min, doxorubicin was added. The cells were further incubated for 48 h,
and then the MTT assay was performed. in the absence and presence of synthesized
compounds, ic50 values and percent reduction of doxorubicin’s ic50 values were
calculated. Meanvalues(±seM)aretheaverageofthreeindependentexperimentseach
done in triplicate. *The difference between the percent reduction of doxorubicin’s
ic50 values in the absence and presence of test compounds is significant (P,0.05).
Abbreviation: seM, standard error of the mean.
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ꢀꢁꢂ
backbone atoms, least squares alignment and the Gromos
algorithm35 (cutoff: 0.15 nm) by g_cluster module as
implemented in Gromacs 4.6.5. As shown in Figure 7B,
cyclohexanone moiety of the ligand participates in hydro-
phobic interactions with the side chains of Arg905, Phe904
and Val472.36 There is another hydrophobic interaction that
occurs between N-(4-methoxyphenyl) part of ligand and
side chain of Val478 as well as phenyl moiety of Tyr490.
In order to investigate hydrogen-bonding profile of ligand
and receptor, we used g_h bond module implemented in
Gromacs 4.6.5 with H-bond forming a distance of 3.5 Å
and an angle of 120°. Table 3 includes all information
about hydrogen-bonding pattern obtained during 10 ns
MD simulation. The analysis revealed that compound 5c
forms eleven average H-bonds sharing nine H-bonds with
Ser909, Thr911, Arg547, Arg543 and Ser474 as accep-
ꢀꢂꢂ
ꢀ
ꢀ
ꢁꢂ
ꢂ
ꢀ
ꢀꢆ—0
ꢃꢇꢁꢆ—0
ꢁꢆ—0
ꢀꢂꢆ—0
ꢃꢁꢆ—0
Figure 6 The effect of synthesized compounds on heK293 cells. heK293 cells
were seeded in 96-well culture plates and cultured overnight. They were then
treated with the synthesized compounds at 5, 10 and 25 µM. The cells were further
incubated for 48 h, and then MTT assay was performed. *The difference between
the test compound and control untreated cells is significant (P,0.05).
All models obtained from GA341 showed the highest tors. Oxygen atom of N-(4-methoxyphenyl) carboxamide
possible value of 1. The derived backbone conformation was pendant at C3 formed the most stable H-bond with Arg543
inspected by Ramachandran plot which showed that 83.8% with occupancy of 81% (some interactions are not shown
of the residues lie in most favored, 12.1% in additionally for clarity). This is in good agreement with our SAR
allowed, 3.3% in generously allowed and 0.8% in outlier studies which proves that the N-(4-methoxyphenyl) moiety
regions. These results indicate that the phi and psi backbone increases the MDR reversal effect of this ligand. Further
dihedral angles in the obtained model are precise.
analysis of ligand interaction with active site residues
We incorporated our results into the model for the P-gp revealed a π–cation interaction between nitrogen atom
ligand–receptor interaction that is shown in Figure 7. This of Arg543 and 2-nitrophenyl ring. In summary, we here
configuration is the midpoint structure of most populated propose 3D models of complexes of P-gp with a promising
cluster obtained by clustering analysis of MD trajectories 5-oxo-hexahydroquinoline reversal agent that is consistent
(data not shown). Clustering was performed using Cα with our SAR studies.
$
%
3KHꢆꢇꢀ
$UJꢆꢇꢄ
6HUꢆꢇꢆ
7KUꢆꢈꢈ
7\Uꢀꢆꢇ
*OXꢀꢁꢃ
$UJꢄꢀꢅ
9DOꢀꢁꢂ
Figure 7 interaction of compound 5c with P-gp protein. P-gp binding pocket (A) and the direct contact of 5c with the active site of P-gp are depicted (B). ligand is shown as
orange sticks and the side chains as cyan. hydrogen bonds, hydrophobic interactions and π–cation interaction are shown as dashed blue, gray and orange lines, respectively.
solvent and lipids are not shown for clarity.
Abbreviation: P-gp, P-glycoprotein.
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a study of 5-oxo-hexahydroquinoline derivatives
Table 3 analysis of hydrogen bond formation between inhibitor
and P-gp
primary or secondary amines (5 mmol). The reaction mixture
was refluxed for 2–4 h. After completion of the reaction as
monitored by thin-layer chromatography, the mixture was
cooled and transferred to a separating funnel and washed with
petroleum ether (30–50 mL). The solid product was collected
and dried (Table 2). Compounds 3a and 4a were reported
previously.37 N,N-Diethyl acetoacetamide and N,N-dimethyl
acetoacetamide were commercially available. The NMR data
are reported in the Supplementary materials.
±
ꢂꢃ
2
ꢂꢁ
ꢀ
1
ꢂꢄ
2
ꢂꢂ
2
ꢂꢆ
2
2
1
ꢅ
+
reagents
1
+
ꢁꢂ
ꢁꢁ
All reagents were purchased from Sigma-Aldrich (St Louis,
MO, USA) if not specifically stated, and were ultrapure grade.
Penicillin/streptomycin was purchased from Invitrogen.
FBS, PBS, RPMI 1640 and trypsin were purchased from
Biosera. DXR was obtained from EBEWE Pharma. Rh123
was purchased from Sigma-Aldrich.
Donor residue Acceptor residue* Distance
Occupancy***
(atom)
(atom)
D–A (Å)** (%)
5c (h11)
5c (h11)
ser909
ser909
ser909
ser909
Thr911
arg547
arg543
arg543
ser474
glu476
glu476
5c (O26)
5c (n9)
5c (O23)
5c (n21)
5c (O26)
5c (O22)
5c (O24)
5c (O24)
5c (n12)
2.25
2.29
3.71
3.60
3.01
3.51
2.99
5.17
1.83
3.45
2.95
55.1
56.1
32.1
26.5
10.9
11.3
10.8
17.0
80.9
12.9
18.2
cell culture and treatment
Human uterine sarcoma cell line MES-SA and its multidrug-
resistant counterpart MES-SA/Dx538 obtained by continuous
in vitro exposure to DXR were used in this study. Both
cell lines were obtained from Sigma-Aldrich. Cells were
grown in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS and 1% penicillin/streptomycin at
37°C and in incubators having humidified air containing 5%
CO2. Resistant cells were cultured in the presence of 100 nM
DXR, which was removed from the media 24 h before each
experiment. HEK293 cell line (obtained from Pasteur Insti-
tute of Iran) was also used in this study. This cell line was
maintained in Dulbecco’s Modified Eagle’s Medium F12
supplemented with 10% FBS, 1% l-glutamine and 1 mL
penicillin/streptomycin.
Notes: *atom names are based on amber nomenclature. **Distance between
donor and acceptor atoms. ***Percentage of time the hydrogen bond is intact.
Abbreviations: P-gp, P-glycoprotein; D–a, Donor–acceptor distance.
Experimental section
chemistry
All melting points were taken on a hot stage apparatus
(Electrothermal, Essex, UK) and were reported uncorrected.
IR spectra were recorded with a Perkin-Elmer spectrometer
(KBr disk) (PerkinElmer, Waltham, MA, USA). NMR
spectra were recorded on a Bruker Avance 300 spectrometer
A 0.05% trypsin solution was used to detach the cells
from culture flasks. Test compounds and verapamil were
prepared in DMSO at 40 mM stock solutions, and DXR
was prepared in complete medium at 10 mM. Drugs and test
compounds were then diluted with complete culture medium
to obtain the final test concentrations. Maximum concentra-
tion of DMSO in wells did not exceed 0.25%.
1
(300 MHz for H NMR, 75 MHz for 13C NMR). Mass
spectra were obtained with an Agilent spectrometer (9575c
inert MSD; Agilent Technologies, Santa Clara, CA, USA).
Chromatographic separations were performed on a silica
gel column by gravity chromatography (Kieselgel 40,
0.063–0.200 mm; Merck) or flash chromatography (Kieselgel
40, 0.040–0.063 mm; Merck). Yields are given after purifi-
cation, unless otherwise stated. All compounds were named
following IUPAC guidelines as defined by ChemBioDraw
Ultra 12.0 software.
Flow cytometric determination of rh123
accumulation
Accumulation of intracellular Rh123, a P-gp substrate,
inside resistant and nonresistant cells was measured by flow
3-Oxo-n-substituted phenylbutanamide
Ten milliliters of xylene was added to a mixture of 2,2,6- cytometry as an index of P-gp inhibition. A suspension of
trimethyl-4H-1,3-dioxin-4-one (6 mmol) and different MES-SA/Dx5 or MES-SA cells was prepared at a density
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of 5×105 cells/mL in serum-free RPMI 1640. Half of the for 4 h. The medium was then removed, and 200 µL DMSO
cell suspensions was placed in 1.5 mL tubes. Different was added into each well to dissolve formazan crystals. After
concentrations of target compounds taken from prepared another hour of incubation, the mixture was gently shaken
stock solutions in DMSO were added in a volume of 0.4 mL. for 30 min. The absorbance was measured using a microplate
After 20 min of incubation at 37°C, the P-gp substrate Rh123, reader at the wavelength of 570 nm. Each concentration
was added in a volume of 100 µL at a final concentration was tested in duplicate, and the experiments were repeated
of 5 µM. Incubation was continued for another 20 min at three times.
37°C. Later, the cells were centrifuged and washed three
times with ice-cold PBS and were resuspended in PBS for
MD simulation study of 5c in complex
measurements. The control cells were treated in the same with human P-gp
way without being treated with any inhibitor. A total of In order to better understand the mode of interaction of
2×104 cells were counted. The fluorescence uptake of Rh123 synthesized compounds with P-gp receptor, MD simula-
within a number of cells was determined by an FACSCalibur tion was applied to the complex of ligand and receptor.
flow cytometer (Becton Dickinson, Franklin Lakes, NJ, We precisely constructed the homology model of P-gp
USA). The results are expressed as Rh123 accumulation based on the murine P-gp (PDB entry: 3G5U)39 using
fold increase, which was defined as geometric mean value of Clustal Omega program from its website http://www.ebi.
Rh123 fluorescence in the cells treated with the synthesized ac.uk/Tools/msa/clustalo/. One thousand 3D models of
compounds or verapamil relative to the geometric mean value P-gp were generated, using MODELLER 9v2 program,40
of the control cells.
and they were evaluated using GA341 method. The
models with lowest DOPE score values were selected for
further computational studies. The loops were remodeled
using the quantum mechanical method in the MOD-
Determination of alteration of resistant
cells’ sensitivity to DXr by MTT assay
The assessment of DXR’s cytotoxicity was performed on ELLER software. QMEAN Z-score from the website
exponentially growing resistant MES-SA/Dx5 cells. Cells http://swissmodel.expasy.org/qmean/cgi/index.cgi41 was
were seeded in 96-well plates at a density of 5×104 cells/mL employed for checking the stereochemical quality of the
in a final volume of 100 µL for 24 h. They were then treated selected models.
with 1, 3 or 10 µM DXR in the presence or absence of
Afterward, the model was inserted in the POPC phospho-
synthesized compounds at different concentrations (5, 10 lipids bilayer and subjected to 80 ns MD simulation to inves-
or 25 µM) for 90 min. After incubation for 48 h at 37°C, tigate the stability as well as structural and conformational
supernatants were carefully discarded, and 80 µL of MTT changes during simulation. In this work, one of the most
reagent (0.5 mg/mL) was added to each well, allowing viable potent inhibitors among all the synthesized ligands was cho-
cells to reduce the MTT solution to formazan crystals. After sen and placed in the active site of the P-gp. Ligand–receptor
4 h, the supernatants were discarded, and 200 µL DMSO was complex was then inserted in POPC lipid bilayer, and 20 ns
added to dissolve the formazan crystals. The absorbance of MD simulation was performed. Initial configuration of the
the solution was then read by a microplate reader at a wave- ligand was acquired via docking procedure. At first, ligand
length of 570 nm with background correction at 675 nm. The was sketched using MarvinSketch, and then the structure
final results are expressed as percent reduction of DXR’s IC50, was optimized by density functional theory, B3LYP/3-21
which is calculated based on DXR’s IC50 in the absence and level using ORCA software. Optimized structure of the
presence of synthesized compounds.
ligand was then docked to P-gp model, which was obtained
previously. The molecular docking was conducted using
AutoDock Vina.42,43 The grid box was set at 18×18×18 with
a spacing value of 1 Å. The best conformation with the least
assessment of cytotoxicity against
heK293 cells
HEK293 cells were seeded in 96-well culture plates at a binding energy and better interacting residues was selected.
density of 2,000 cells/well and allowed to attach. After 24 h Full general amber force field44 topology/coordinate files
incubation at 37°C in 5% CO2, the culture medium was were created using the programs Parmchk and Tleap of the
removed and replaced with fresh medium containing the AmberTools package45 to describe van der Waals and bonded
target compounds at different concentrations and further parameters for the ligand. Partial atomic charges were then
incubated at 37 CC for 48 h. Afterward, 80 µL of MTT assigned based on the RESP ESP charge Derive Server.46
solution (0.5 mg/mL) was added to all wells and incubated The AMBER format files of the ligands were converted to
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a study of 5-oxo-hexahydroquinoline derivatives
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Abbreviations
3D, three-dimensional; ABC, ATP-binding cassette; DHPs,
dihydropyridines; DMSO, dimethyl sulfoxide; DXR,
doxorubicin; FBS, fetal bovine serum; IR, infrared; MD,
molecular dynamics; MDR, multidrug resistance; NMR,
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Acknowledgment
The authors thank the support of the Vice-Chancellor for
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Disclosure
The authors report no conflicts of interest in this work.
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