Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance

Article abstract of DOI:10.1016/j.ejmech.2019.06.066

Ko143, a potent ABCG2 inhibitor that reverses multidrug resistance in cancer, cannot be used clinically due to its unsuitable metabolic stability. We identified benzoyl indoles as reversal agents that reversed ABCG2-mediated multidrug resistance (MDR), wi

Full text of DOI:10.1016/j.ejmech.2019.06.066

Accepted Manuscript
Benzoyl indoles with metabolic stability as reversal compounds for ABCG2-Mediated
drug resistance
Chao-Yun Cai, Hong Zhai, Zi-Ning Lei, Cai-Ping Tan, Bao-Li Chen, Zhao-Yi Du, Jing-
Quan Wang, Yun-Kai Zhang, Yi-Jun Wang, Pranav Gupta, Bo Wang, Zhe-Sheng
Chen
PII:
S0223-5234(19)30593-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.066
EJMECH 11469
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 April 2019
Revised Date: 2 June 2019
Accepted Date: 22 June 2019
Please cite this article as: C.-Y. Cai, H. Zhai, Z.-N. Lei, C.-P. Tan, B.-L. Chen, Z.-Y. Du, J.-Q. Wang,
Y.-K. Zhang, Y.-J. Wang, P. Gupta, B. Wang, Z.-S. Chen, Benzoyl indoles with metabolic stability as
reversal compounds for ABCG2-Mediated drug resistance, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.066.
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ACCEPTED MANUSCRIPT
Benzoyl Indoles with Metabolic Stability as Reversal
Compounds for ABCG2-Mediated Drug Resistance
Chao-Yun Cai a, b, 1, Hong Zhai a, 1, Zi-Ning Lei b, Cai-Ping Tana, Bao-Li Chen a, Zhao-Yi Du a, Jing-Quan
Wang b, Yun-Kai Zhang b, Yi-Jun Wang b, Pranav Gupta b, Bo Wang*, a, Zhe-Sheng Chen *, b
a MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen
University, 135 Xingang West Road, Guangzhou, 510275, P. R. China
b Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University,
8000 Utopia Parkway, Queens, New York 11439, United States
1 The first two authors contribute equally to this work.
Benzoyl indoles as ABCG2 reversal compounds have greater synthetic tractability and
metabolic stability than the potent ABCG2 inhibitor, Ko143.
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Benzoyl Indoles with Metabolic Stability as Reversal Agents for
ABCG2-Mediated Multidrug Resistance
Chao-Yun Cai a, b, 1, Hong Zhai a, 1, Zi-Ning Lei b, Cai-Ping Tana, Bao-Li Chen a, Zhao-Yi Du a, Jing-Quan
Wang b, Yun-Kai Zhang b, Yi-Jun Wang b, Pranav Gupta b, Bo Wang*, a, Zhe-Sheng Chen *, b
a MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen
University, 135 Xingang West Road, Guangzhou, 510275, P. R. China
b Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University,
8000 Utopia Parkway, Queens, New York 11439, United States
1 The first two authors contribute equally to this work.
Abstract
Ko143, a potent ABCG2 inhibitor that reverses multidrug resistance, cannot be used
clinically due to its unsuitable metabolic stability. We identified benzoyl indoles as
reversal agents that reversed ABCG2-mediated multidrug resistance (MDR), with
synthetic tractability and enhanced metabolic stability compared to Ko143. Bisbenzoyl
indole 2 and monobenzoyl indole 8 significantly increased the accumulation of
mitoxantrone (MX) in ABCG2-overexpressing NCI-H460/MX20 cells, and sensitized
NCI-H460/MX20 cells to mitoxantrone. Mechanistic studies were conducted by [3H]-MX
accumulation assay, Western blot analysis, immunofluorescence analysis and ABCG2
ATPase assay. The results revealed that the reversal efficacies of compounds 2 and 8 were
not due to an alteration in the expression level or localization of ABCG2 in
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ABCG2-overexpressing cell lines. Instead, compounds 2 and 8 significantly stimulated
the ATP hydrolysis of ABCG2 transporter, suggesting that these compounds could be
competitive substrates of ABCG2 transporter. Overall, the results of our study indicated
that compounds 2 and 8 significantly reversed ABCG2-mediated MDR by blocking the
efflux of anticancer drugs.
Keywords
ABCG2 transporter; Multidrug resistance; Benzoyl indoles; Metabolic stability; Reversal
agents
Introduction
Multidrug resistance (MDR) is characterized by the resistance of cancer cells to drugs
that are structurally and mechanistically distinct. 1-3 There are several different factors
that contribute to MDR, including enhanced drug efflux, decreased cellular uptake, drug
detoxification, altered drug target, enhanced DNA repair, apoptosis inhibition, and altered
cell cycle check points. 4-6 ATP-binding cassette transporters (ABC transporters) are a
superfamily of proteins that transport various substrates by utilizing the energy of ATP
hydrolysis. 7 ABCG2, as a G-subfamily member of ABC transporter, is a
‘half-transporter’, with one nucleotide binding domains (NBD) and one transmembrane
domain (TMD) fused to a single polypeptide chain. 8 The functional form of the ABCG2
transporter is a homodimer. 9 The ABCG2 transporter is localized in the blood-brain
barrier (BBB), blood-testis barrier (BTB), blood-placental barriers (BPB), blood-retinal
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barriers (BRB), and the luminal surface of liver canaliculi and renal proximal convoluted
tubules. 10 The ABCG2 transporter, due to its localization on the surfaces of excretory
organs can alter the absorption, distribution, metabolism, and elimination (ADME) of
certain drugs. 11, 12 The substrates of the ABCG2 transporter are primarily hydrophobic
and are either negatively or positively charged molecules, e.g., mitoxantrone (MX),
methotrexate, topotecan, flavopiridol and SN-38. 13 It has been shown that the
overexpression of ABCG2 transporter in certain cancers significantly decreases or
abrogates the efficacy of various chemotherapeutic drugs. 14, 15
Ko143 (Figure 1) was found to be the most potent inhibitor of the ABCG2
transporter. 16 A major problem associated with Ko143 was the low total yield (5%) as its
synthesis required a multi-step synthesis method. 17, 18 Furthermore, Ko143 was rapidly
metabolized to a compound that lacked efficacy as an inhibitor of the ABCG2 transporter.
19 Thus, in order to develop efficacious ABCG2 inhibitors, it is essential to find
compounds that can be easily synthesized that have a suitable pharmacokinetic profile.
Jackson et al. determined the cryo-EM structure of ABCG2 protein (PDB: 6ETI)
bound to a Ko143 analog, MZ29, and two Fabs. 9 The result indicated that the –NH group
of the indole ring in the Ko143 analog formed a hydrogen bond with Asn436 and the
methoxy group formed a hydrogen bond with Thr435. Thus, the indole and methoxy
group may be important in the interaction of the Ko143 analogs and ABCG2. In this
study, we simplified the structure of Ko143 to a methoxy benzoyl indole (Figure 1),
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which has a molecular size similar to Ko143 but with higher conformational flexibility. In
addition, a benzoyl naphthalene compound (Figure 1) was synthesized to elucidate the
importance of the indole group in reversing ABCG2-mediated drug resistance.
Figure 1. Simplifying Ko143 to benzoyl indole
Since Ko143 is rapidly metabolized to an inactive compound in the plasma, the
metabolic stability of the indole derivatives would be a major concern. Previously, it has
been shown that the benzoyl indole, BPR0L075 (Figure 2), has a half-life of 2.8 h in
plasma following the oral administration of 20 mg/kg in rats. 20 Therefore, we
hypothesized that benzoyl indoles could be developed as a new series of ABCG2 reversal
compounds with an improved pharmacokinetic profile.
OMe
O
OMe
OMe
MeO
N
H
BPR0L075
Figure 2. The structure of BPR0L075
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Results and discussion
Chemistry. The general method used to synthesize the indole derivatives in this
study is shown in Scheme 1. The synthesis involves a straightforward reaction sequence.
Initially, the acylation reaction was performed with the use of methoxy indoles and
methoxy benzoyl chloride in the presence of POCl3 and AlCl3. Secondly, the
hydroxy-substituted compounds were afforded by the deprotection of the methoxy groups.
The synthesis of 3-acylindoles has recently gained considerable attention as 3-substituted
indoles are not only significant therapeutic compounds but are also used as synthetic
intermediates for alkaloid synthesis. 21, 22 Several methods for the synthesis of
3-acylindoles have been reported, including Friedel–Crafts or Vilsmeier–Haack
acylations, the acylation of indole Grignard reagents or 3-indolylzinc chloride, and the
use of iminium ions, dialkoxycarbenium, or N-(2-haloacyl)-pyridinium salts. 23-31
The synthetic route of the indole derivatives is shown in Scheme 1. The indoles
reacted with acyl chloride in the presence of AlCl3 and POCl3 to obtain compounds 1-12
(Scheme 1). Interestingly, when we synthesized benzoyl indole 1, we obtained 4-OMe
bisbenzoyl indole 2 in the same reaction. Similar results occurred in the synthesis of 3,
where 2,4-OMe bisbenzoyl indole 4 was obtained. The corresponding hydroxy
compounds 13-23 were synthesized by demethylation of compounds 1-12 with boron
tribromide. In addition, the benzoyl naphthalene compound 24 (Table 1) was synthesized
as a control to elucidate the importance of the indole group in reversing
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ABCG2-mediated multidrug resistance. Compound 24 was synthesized by the acylation
of methoxy naphthalene in the presence of AlCl3 and POCl3. The structures of all the
synthesized compounds were confirmed by HRMS (high resolution mass spectrometry),
IR (infrared radiation), 1H, 13C NMR and HMBC
correlation) data.
(heteronuclear multiple bond
Scheme 1. Synthesis of indole derivatives
The determination of the ABCG2 reversal activity of the indole derivatives at
selected (non-toxic) concentrations
The structure of indole derivatives and the selective non-toxic concentration is
shown in Table 1 and Table 2. The selective concentration is the concentration that did
not decrease the viability of the parental NCI-H460 and drug-resistant NCI-H460/MX20
cell lines by more than 20%. NCI-H460/MX20 cell line overexpressing ABCG2 was
selected by MX. 32 The reversal efficacies of the indole derivatives were determined
(Figure 3) at the selected concentrations by determining their effect on the intracellular
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levels of mitoxantrone (2.5 µM), a substrate of the ABCG2 transporter. 5 As shown in
Figure 3, 2.5 µM of MX alone produced a 24±3% inhibition of the proliferation of
NCI-H460/MX20 cells and the efficacy of MX increased when combined with the
different indole derivatives. For example, MX (2.5 µM) inhibited the proliferation of
NCI-H460/MX20 cells by more than 87% when combined with the monobenzoyl indoles
7, 8, 13, 15 and 17, or the bisbenzoyl indoles 2 and 4 at the selected concentrations.
However, the benzoyl naphthalene 24 (25 µM) did not significantly increase the efficacy
of 2.5 µM of MX. The reversal efficacy of 2,4-OMe benzoyl naphthalene 24 was lower
than that of 2,4-OMe benzoyl indoles 3, 9 and 12. Most of the methoxy indoles have
greater reversal efficacies than the corresponding hydroxy indoles except methoxy
indoles 1, 3 and 6. Interestingly, 5-OMe indole compounds 8 (4-OMe phenyl), 9
(2,4-OMe phenyl), and 10 (3,4,5-OMe phenyl) showed greater reversal efficacies than
those corresponding 4-OMe indole compounds 1, 3 and 5, respectively. However, the
4-OH indole compounds 13 and 15 showed better reversal efficacies than those 5-OH
indole compounds 19 and 20, respectively. Among the 5-OMe indole compounds 6~11,
compounds 7 and 8 with 3-OMe and 4-OMe benzoyl group, respectively, had better
reversal efficacies than the other indoles. The above results indicated that the reversal
efficacies of the indole compounds are influenced by the position and hydrophilicity of
the substituents at the indole ring and the benzoyl group. However, the benzoyl
naphthalene 24 (25 µM) did not significantly increase the efficacy of 2.5 µM of MX. The
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reversal efficacy of 2,4-OMe benzoyl naphthalene 24 was lower than that of 2,4-OMe
benzoyl indoles 3, 9 and 12, suggesting that the indole group was critical for the reversal
of ABCG2-mediated multidrug resistance.
Table 1. The structure and the selected concentration of monobenzoyl indoles and benzoyl
naphthalene
MeO
R2
O
OMe
O
R1
OMe
N
H
MeO
Scaffold A
24
*Selected
*Selected
Compd.
R1
R2
concentration
Compd.
R1
R2
concentration
(µM)
10
25
1
(µM)
25
25
4-OMe
4-OMe
4-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
7-OMe
4-OMe
2,4-OMe
3,4,5-OMe
2-OMe
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
5-OH
5-OH
5-OH
7-OH
/
4-OH
2,4-OH
3,4,5-OH
2-OH
1
3
13
15
16
17
18
19
20
21
22
23
24
25
5
1
25
6
10
10
25
1
25
3-OMe
3-OH
7
25
4-OMe
4-OH
8
25
2,4-OMe
3,4,5-OMe
2, 6-OMe
2, 4-OMe
2,4-OH
3,4,5-OH
2,6-OH
2,4-OH
/
9
25
10
11
12
1
10
10
10
10
*The concentrations that did not decrease the viability of NCI-H460 and NCI-H460/MX20 cells by
more than 20%.
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Table 2. The structure and the selected concentration of bisbenzoyl indoles
*Selected
Compd.
R1
R2
R3
concentration (µM)
4-OMe
2, 4-OMe
4-OH
4-OMe
2, 4-OMe
4-OH
OMe
OMe
OH
25
25
25
2
4
14
*The concentrations that did not decrease the viability of NCI-H460 and NCI-H460/MX20 cells by
more than 20%.
Figure 3. The reversal efficacy of the indole derivatives in combination with 2.5 µM of MX in
NCI-H460/MX20 cells. The inhibition of proliferation indicates the inhibition of the proliferation of
NCI-H460/MX20 cells after incubation with MX in the absence or presence of the indole
derivatives, compared to the proliferation of cells in the control group. The results were obtained
from three independent experiments performed in triplicate. *, P < 0.05, **, P < 0.01, ****, P <
0.0001 versus no indole derivatives group, two-way ANOVA.
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The determination of the ABCG2 reversal efficacy of the indole derivatives 2, 4, 7, 8,
13, 15 and 17
The reversal efficacies of bisbenzoyl indoles 2 and 4, and monobenzol indoles 7, 8, 13,
15 and 17 at different concentrations on the proliferation of NCI-H460/MX20 cells, in
combination with 2.5 µM of MX, were determined to ascertain which compound had the
greatest efficacy (Figure 4). The results suggested that reversal efficacy increased as the
concentration of the indole derivatives increased, which indicated that the reversal
efficacies of these indole compounds are dose-dependent. Monobenzoyl indole 8 had the
greatest anti-proliferative efficacy among the seven compounds at the same concentration,
while bisbenzoyl 2 has greater efficacy than bisbenzoyl 4 (Figure 4). Thus, compounds 2
and 8 exhibited the highest reversal efficacy among the bisbenzoyl and monobenzoyl
indoles, respectively. Subsequently, we further investigated the reversal efficacies and
inhibition mechanism of compounds 2 and 8 as reversal compounds for
ABCG2-mediated drug-resistance.
Figure 4. The reversal efficacy of the indole derivatives in combination with 2.5 µM of MX. The
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proliferation of NCI-H460/MX20 cells was inhibited by 25 ± 4 % with 2.5 µM of MX alone. The
inhibition of proliferation indicates the inhibition of the proliferation of NCI-H460/MX20 cells after
incubation with MX in the absence or presence of the indole derivatives, compared to the
proliferation of cells in the control group. The results were obtained from three independent
experiments performed in triplicate.
The cytotoxic curves of compounds 2 and 8 on H460, H460/MX20, S1, S1-M1-80,
HEK-293/pcDNA3.1, wild-type HEK-293/ABCG2-482-R2, mutant
HEK-293/ABCG2-482-G2 and HEK-293/ABCG2-482-T7 cell lines are shown in Figure
5, indicating that compounds 2 and 8 at the concentration of 25 µM and 5 µM exhibited
lower than 20% inhibition on all the cell lines we investigated. S1-M1-80 is an
ABCG2-mediated multi-drug resistant cell line selected by MX. 33 The previous studies
revealed that mutations at amino-acid 482 in the ABCG2 gene influence the substrate
specificity of the ABCG2 transporter. 34 It was also reported that the efficacy of
Novobiocin, a ABCG2 inhibitor, would be altered due to the mutations at amino-acid 482.
Novobiocin exhibited potent inhibition on wild-type ABCG2, however, almost no
inhibition on mutant ABCG2. 35 Thus, we used the transfected wild-type
HEK293/ABCG2-482-R2, mutant HEK293/ABCG2-482-G2 and
HEK293/ABCG2-482-T7 cells to investigate whether the reversal effects of the benzoyl
indoles would be different in these transfected HEK293 cell lines. As shown in Figure
6A, compounds 2 and 8 significantly increased the cytotoxicity of MX in
ABCG2-overexpressing NCI-H460/MX20 cell line. Compounds 2 and 8 did not
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significantly sensitize NCI-H460/MX20 cells to cisplatin, which is not a substrate of the
ABCG2 transporter (Figure 6B). These results suggested that the sensitizing effects of
compounds 2 and 8 is limited to ABCG2 substrates for ABCG2-mediated multidrug
resistance cancer cell lines. Furthermore, the reversal efficacy of compounds 2 and 8
were also determined in parental S1 and S1-M1-80 cells, where ABCG2 overexpression
was also induced using mitoxantrone. 36 Human embryonic kidney (HEK293) cells were
transfected with the pcDNA3.1 vector containing a full-length ABCG2, with either
coding arginine (R), glycine (G), or threonine (T) at amino acid position 482, to generate
wild-type HEK293/ABCG2-482-R2, mutant (HEK293/ABCG2-482-G2 and
HEK293/ABCG2-482-T7), respectively. Compounds 2 and 8 significantly increased the
cytotoxicity of 1) MX and doxorubicin in S1-M1-80 cells (Figure 6C, 6D) and 2) MX in
HEK293/ABCG2-482-R2, HEK293/ABCG2-482-G2 and HEK293/ABCG2-482-T7 cells
expressing theABCG2 transporter (Figure 6E). 37 Thus, it can be concluded that
compounds 2 and 8 might sensitize ABCG2-mediated multidrug resistance cell lines to
anticancer drugs by interacting with ABCG2. Even though the potencies of compounds 2
and 8 are not as strong as Ko143, the two compounds are also effective to reverse
ABCG2-mediated multi-drug resistance. The representative cytotoxic curves of
mitoxantrone, cisplatin and doxorubicin in combination with compounds 2, 8 and Ko143
on different cell lines were also shown in Figure 7 to indicate the effects of compounds 2,
8 and Ko143 on the cytotoxicity of the anti-cancer drugs.
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Figure 5. Cytotoxic curves of compounds 2 and 8 on H460, H460/MX20, S1, S1-M1-80,
HEK-293/pcDNA3.1, HEK-293/ABCG2-482-R2, HEK-293/ABCG2-482-G2 and
HEK-293/ABCG2-482-T7 cell lines.
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Figure 6. The effect of compounds 2 and 8 on the IC50 values of (A) mitoxantrone and (B) cisplatin
in parental NCI-H460 and the drug-resistant cell line, NCI-H460/MX20. The effect of compounds 2
and 8 on the IC50 values of (C) mitoxantrone and (D) doxorubicin in parental S1 and drug-resistant
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cell line, S1-M1-80. (E) The effect of compounds 2 and 8 on the IC50 values of mitoxantrone in
parental HEK293/pcDNA3 and transfected ABCG2-overexpressing HEK293/ABCG2-482-R2,
HEK293/ABCG2-482-G2 and HEK293/ABCG2-482-T7 cells. The error bars represent the SD
value. Ko143 (5 µM) was used as a positive control. ****, P < 0.0001 versus no indole derivatives
group, two-way ANOVA.
Figure 7. Cytotoxic curves of mitoxantrone, cisplatin and doxorubicin in combination with
compounds 2, 8 and Ko143 on H460, H460/MX20, S1, S1-M1-80, HEK-293/pcDNA3.1,
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HEK-293/ABCG2-482-R2, HEK-293/ABCG2-482-G2 and HEK-293/ABCG2-482-T7 cell lines.
The effect of the indole derivatives on the intracellular accumulation of MX in
ABCG2-overexpressing cells
The reversal efficacies of compounds 2 and 8 were tested at low concentrations which
were not toxic to both parental NCI-H460 and drug-resistant NCI-H460/MX20 cells.
Thus, the significant increase in the cytotoxicity of MX in combination of compounds 2
or 8 cannot be considered as additivity of the cytotoxicity. The effect of the compounds
on intracellular [3H]-MX accumulation was determined by comparing the accumulation
of [3H]-MX in parental and ABCG2-mediated MDR cells with or without the reversal
compounds. The intracellular level of [3H]-MX in NCI-H460/MX20 cells in the absence
of the reversal compounds was approximately 3.1-fold lower than NCI-H460 cells (which
do not overexpress ABCG2) (Figure 8). The incubation of NCI-H460/MX20 cells with
compounds 2 (25 µM) and 8 (5.0 µM) significantly increased the accumulation of
[3H]-MX by 3.0-fold and 2.7-fold, respectively (Figure 8). Ko143, at 5.0 µM, produced a
3-fold increase in the accumulation of [3H]-MX and there was no statistical difference in
the accumulation of [3H]-MX between Ko143 (5.0 µM) and compounds 2 (10 or 25 µM)
or compound 8 (5.0 µM). The results revealed that compounds 2 and 8 effectively
blocked the efflux of MX by NCI-H460/MX20 cells.
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Figure 8. The effects of 2 and 8 on the intracellular accumulation of [3H]-MX in NCI-H460 and
NCI-H460/MX20 cells. The error bars represent the SD value. Ko143 (5.0 µM) was used as
positive control for ABCG2-overexpressing cells. ****, P < 0.0001 versus no indole derivatives
group, two-way ANOVA.
The expression level and the intracellular localization of ABCG2 in cells
overexpressing ABCG2
The factors inhibiting the efflux function of ABCG2 transporter could include the
down-regulation of expression or the localization alteration of ABCG2 in cells. To
determine whether compounds 2 and 8 can cause the down-regulation of ABCG2 protein,
the ABCG2 expression levels were determined for cells incubated with 2 (25 µM) or 8
(5.0 µM) for 0, 24, 48, and 72 h. The results showed no significant alteration in the
expression level of ABCG2 in the drug-resistant NCI-H460/MX20 cells following
incubation with compounds 2 or 8 (Figure 9A, Figure 9B).
Alterations in the localization of the ABCG2 transporter in the membrane has been
shown to reverse ABCG2-mediated MDR. 38 Therefore, we used an immunofluorescence
assay to determine if the localization of ABCG2 transporter in NCI-H460/MX20 cells
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was altered following incubation with the indole derivatives. As shown in Figure 9C,
compounds 2 and 8 did not induce the internalization of cell surface ABCG2 transporter.
These results suggest that the reversal mechanism of action for compounds 2 and 8 was
not induced by the alteration the expression or localization of ABCG2 transporter.
Figure 9. (A) The effect of compound 2, at 25 µM, on the expression levels of ABCG2 protein in
NCI-H460/MX20 cells after 24, 48, and 72 h of incubation. Equal amounts of total cell lysate were
used for each sample. (B) The effect of compound 8, at 5 µM, on the expression level of ABCG2 in
NCI-H460/MX20 cells after 24, 48, and 72 h of incubation. (C)The effect of 2 (25 µM) and 8 (5 µM)
on the subcellular localization of ABCG2 in ABCG2-overexpressing NCI-H460/MX20 cells.
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The effect of the indole compounds on ABCG2 ATPase activity
Since there is no alteration of expression or localization of ABCG2, the inhibition of
the efflux function of ABCG2 transporter could be due to the inhibition of the activity of
ABCG2 transporter. To determine whether compounds 2 and 8 inhibit or stimulate the
activity of ABCG2 ATPase, we performed an ATPase assay using membranes of High
Five insect cells overexpressing ABCG2, in the presence of different concentrations of
compounds 2 or 8. Both compounds 2 or 8 significantly stimulated the ATPase activity of
ABCG2 in a concentration-dependent manner, with a maximal stimulation of 3.01-fold
and 2.03-fold greater, than the basal activity (Figure 10), respectively. The concentration
of 2 or 8 required to obtain 50% of maximal stimulation was 0.21 and 0.75 µM,
respectively. These results suggested that 2 and 8 were the substrates of ABCG2
transporter. Therefore, compounds 2 and 8 could acts as a potential competitive substrate,
thus blocking the efflux function of ABCG2 and increasing the accumulation of
anticancer drugs.
Besides the mechanisms we discussed above, inhibition of the phosphorylation of
ABCG2 could be another mechanism for reversing ABCG2-mediated multi-drug
resistance. As depicted in the previous study, BCRP/ABCG2 can be phosphorylated by
Pim-1L and thus the drug resistance would increase. 39 We suppose that inhibiting the
phosphorylation of ABCG2 could decrease the efflux function of ABCG2, thus increase
the accumulation of the anti-cancer drugs in drug-resistant cancer cells. Additional
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experiments are needed to determine whether the inhibition of phosphorylation of
ABCG2 was induced by the benzoyl indoles in our future study.
Figure 10. The effect of compounds 2 and 8 (0–40 µM) on orthovanadate (Vi)-sensitive ABCG2
ATPase activity. The concentrations of compounds 2 and 8 were plotted at linear in panel (A) and
(B), respectively.
Docking analysis of indole derivatives with ABCG2
The best-scored docked position of the indole derivatives within the large drug-binding
cavity of human ABCG2 is shown in Figure 11. The indole group of 2 forms a hydrogen
bond with Thr435 in the B chain of the ABCG2 protein (-NH•••O=C- Thr435). In
addition, the phenyl ring of the indole group forms a π-π interaction with Phe439 in the B
chain of ABCG2, while the other phenyl ring of 2 forms a π-π interaction with Phe439 in
the A chain of ABCG2. Compound 2 was stabilized into a hydrophilic pocket formed by
residues Val401, Leu405, Phe432, Val546, Phe547, Met549 of ABCG2. The amine group
in compound 8 forms a hydrogen bonding with Asn436 (-NH•••O=C- Asn436). Moreover,
the indole group of 8 was stabilized into a hydrophilic pocket formed by nearby residues
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Phe431, Phe432, Val546, Met549 and Leu555 of the ABCG2 protein. Ko143 formed a
hydrogen bond with Asn436 in the B chain of the ABCG2 transporter. Based on the
results of the induced-fit docking, compounds 2, 8 and Ko143 had a strong interaction
with the human ABCG2 transporter protein, with docking scores of -11.591 and -10.052
and 10.236 kcal/mol, respectively. Interestingly, compound 2 had a higher docking score
than 8, but 8 had greater reversal efficacy than 2 in the cytotoxicity and intramolecular
accumulation assays. Compound 2 may have a lower reversal efficacy due to its large
size, which could impede its entry into the binding site of the human ABCG2 protein. 40
As depicted in Figure 11G, even though compounds 2, 8 and Ko143 bind with the same
domain of ABCG2 protein, the binding modes of the three compounds are not
superimposable, which could contribute to the difference in the reversal efficacies of
compounds 2, 8 and Ko143.
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Figure 11. Predicted binding positions of compounds 2 and 8 with the human ABCG2 transporter
protein. (A) Docked position of compound 2 within the binding site of the human ABCG2
transporter protein. Compound 2 is shown as a ball and stick model, with the atoms colored as
follows: carbon = cyan, hydrogen = white, nitrogen = blue and oxygen = red. The important
residues are depicted as sticks with the same color scheme as above except that carbons are
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indicated by the grey color. Ring centroids were represented as dark-green dots. Dotted yellow
lines indicate hydrogen bonds. (B) A two-dimensional ligand−receptor interaction diagram shows
the important interactions of compound 2 with the binding site residues of human ABCG2. The
amino acids are shown as colored bubbles, cyan indicates polar residues and green indicates
hydrophobic residues. Hydrogen bonds are indicated by the purple dotted arrow, and π-π stacking
aromatic interactions are indicated by the green lines. (C) Docked position of compound 8 within
the binding site of the human ABCG2 transporter protein. (D) A two-dimensional ligand−receptor
interaction diagram shows the important interactions of compound 8 with the binding site residues
of the human ABCG2 transporter protein. (E) Docked position of Ko143 within the binding site of
the human ABCG2 protein. (F) A two-dimensional diagram shows the important contacts of Ko143
with the binding site residues of the human ABCG2 transporter protein. (G) The comparison of the
binding mode of 2, 8 and Ko143 with ABCG2 protein. Compounds are shown as a ball and stick
model, with the atoms colored as follows: hydrogen = white, nitrogen = blue, oxygen = red. The
carbon atoms are labeled in different colors: 2 (grey), 8 (cyan), Ko143 (orange).
Metabolic Stability of the indole derivatives
Approximately 60 % of the drugs are metabolized by CYP 450 in the human liver. 41
Human liver microsomes, derived from human liver, contain a wide variety of membrane
bound drug metabolizing enzymes. Thus, in vitro metabolic stability testing with human
liver microsomes is useful to predict the in vivo hepatic CYP-mediated metabolism of the
compounds in the human body. The in vitro metabolic stability testing with human liver
microsome has been commonly applied for investigating the metabolism of the drugs
prior to in vivo study. 42-44 The metabolic stabilities of compounds 2 and 8 were
investigated with human liver microsome, and the results indicated that compounds 2 and
8 are more metabolically stable than Ko143 (Table 3). The amount of compounds 2 and 8
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remaining after incubation with human liver microsome for 1 h was 56 and 78%,
respectively, compared to 23% for Ko143. Since the low metabolic stability of Ko143 is
the major problem for clinical application, we would like to discover metabolically stable
reversal agents for ABCG2-mediated multidrug resistance. Inspiringly, the results
revealed that compounds 2 and 8 are more metabolically stable than Ko143. Thus, indole
compounds 2 and 8 as reversal agents could be valuable and suitable for clinical
application in the future.
Table 3. In vitro metabolic stability of compounds 2, 8 and Ko143
compound
Metabolic stability a /%
2
8
56
78
23
±
±
±
4.6
1.2
1.4
Ko143
a Metabolic stability was calculated as the % compound remaining after 1 h of incubation with
human liver microsomes.
Conclusions
In this study, we synthesized 23 benzoyl indoles and determined their efficacy and
mechanism of action for reversing ABCG2-mediated MDR. Based on the
structure-activity relationship data, The reversal efficacies of 2,4-OMe benzoyl indoles 3,
9 and 12 were greater than that of 2,4-OMe benzoyl naphthalene 24, revealing the indole
group was more important in enhancing the reversal efficacy compared to naphthalene
group. Docking studies showed that the indole moiety of compounds 2 and 8 formed
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hydrogen bonds with the ABCG2 transporter protein, indicating the importance of the
indole group. Furthermore, in most cases, compounds that had methoxy substituents had
greater reversal efficacy than those with the hydroxy groups. The increase in reversal
efficacy may be due to the stronger interaction between the methoxy group and the
hydrophobic pocket residues in the human ABCG2 transporter protein. The methoxy
benzoyl indole compounds 2 and 8, had the greatest reversal efficacy among bisbenzoyl
and monobenzoyl indole compounds, respectively. Inspiringly, compounds 2 and 8 have
better metabolic stability than the potent ABCG2 inhibitor, Ko143, which revealing the
value of clinical application of these benzoyl indoles. The reversal efficacy of compound
8 was significantly greater than that of compound 2. The lower reversal efficacy of
compound 2 may result from its larger molecule size, thus hindering compound 2 from
entering the drug-binding site of the ABCG2 transporter protein. Compounds 2 and 8
could act as competitive substrates of ABCG2 transporter to block the efflux of
anticancer drugs, thus sensitizing ABCG2-mediated multidrug resistant cancer cells to
the anticancer drugs. Overall, the benzoyl indoles 2 and 8 had significant efficacy as
ABCG2-mediated drug resistance reversal agents, with synthetic tractability and
enhanced metabolic stability. These results give insight into the rational design of
reversal agents targeting ABCG2 transporter.
Experimental Section
General Chemistry. Chemicals were obtained from Materials and Instrumentation.
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Chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI), TCI America
(Portland, OR) and Alfa Aesar (Ward Hill, MA), and used without further purification
unless otherwise indicated. Purifications by column chromatography were carried out
over silica gel (300−400 mesh) and monitored by thin layer chromatography (TLC)
performed on GF/UV 254 plates and were visualized using UV light at 254 and 365 nm.
Melting points were determined using a SGW X-4 digital melting point apparatus, and
the temperatures were not corrected. IR spectra were determined on an EQUINOX 55
Fourier transformation infrared spectrometer. 1H and 13C NMR spectra were recorded on
Varian INOVA 500, Bruker AVANCE AV400, or Mercury-Plus 300 NMR spectrometer in
CDCl3, DMSO-d6, or Acetone-d6 and TMS as an internal standard. Chemical shifts were
expressed as values (ppm) relative to tetramethylsilane as an internal standard, and
coupling constants (J values) were given in hertz (Hz). Abbreviations are used as follows:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet,
doublet of doublet of doublets (ddd), br = broad. High resolution (HR) mass spectra were
measured on Thermo MAT95XP mass spectrometer.
General procedure for the synthesis of compounds 1-12.
A mixture of methoxy indole (2 mmol) and methoxy benzoylchloride (2 mmol) was
stirred in the presence of POCl3 (5 ml) and AlCl3 (0.5 g) in dichloroethane (10 ml) at 60
for 2.5 h. The reaction was quenched by 30 ml crushed ice and then extracted with
dichloromethane (3 × 30 ml). The combined organic layer was washed by saturated
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sodium carbonate until PH=7, washed with saturated aqueous sodium chloride, dried over
with anhydrous Na2SO4, and evaporated to give the crude product. The obtained residue
was purified by column chromatography on silica gel using petroleum ether-acetone as
the eluent to produce compounds 1-12.
(4-Methoxy-1H-indol-3-yl)(4-methoxyphenyl)methanone (1). Yield: 36 %, white solid,
m.p. 250-251
DMSO): (ppm) 11.81 (br, 1H), 7.74 (d,
(dd, =8.0, 7.6 Hz, 1H), 7.11 (dd, = 8.0, 0.8 Hz, 1H), 7.02 (d,
= 7.6, 0.8 Hz, 1H), 3.85 (s, 3H), 3.59 (s, 3H); 13C NMR (101 MHz, DMSO):
℃
; IR (KBr): 3182, 3003, 1620, 1420, 1093 cm-1; 1H NMR (400 MHz,
= 8.8 Hz, 2H), 7.64 (d, = 2.8 Hz, 1H), 7.15
= 8.8 Hz, 2H), 6.61 (dd,
(ppm)
δ
J
J
J
J
J
J
δ
189.28, 162.57, 153.89, 138.48, 133.27, 131.82, 130.96, 124.00, 116.72, 116.20, 113.61,
105.64, 102.16, 55.86, 55.38; HRMS for C17 H15 N O3 ([M-H]-) calculated: 280.0968,
found: 280.0986.
(4-methoxy-1H-indole-3,5-diyl)bis((4-methoxyphenyl)methanone) (2). Yield: 19 %,
yellow solid, m.p. 194-196
(400 MHz, DMSO): (ppm) 12.00 (br, 1H), 7.77 (dd,
2H), 7.13 (dd, = 8.8, 2.0 Hz, 2H), 7.05 (dd, = 8.8, 2.0 Hz, 2H), 6.78 (d,
1H), 3.89 (s, 3H), 3.86 (s, 3H); 13C NMR (101 MHz, DMSO):
(ppm) 193.86, 189.17,
℃
; IR (KBr): 3380, 2935, 1600, 1361, 1075 cm-1; 1H NMR
= 8.8, 2.4 Hz, 4H), 7.60-7.56 (m,
= 8.8 Hz,
δ
J
J
J
J
δ
162.86, 162.59, 158.10, 137.09, 132.84, 131.95, 131.86, 131.58, 131.17, 116.92, 116.89,
115.10, 114.21, 113.82, 101.80, 55.96, 55.94, 55.92; HRMS for C25 H21 N O5 ([M-H]-)
calculated: 414.1336, found: 414.1393.
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(4-Methoxy-1H-indol-3-yl)(2,4-dimethoxyphenyl)methanone (3). Yield: 40 %, white
solid, m.p. 224-226
MHz, DMSO): (ppm) 11.75 (s, 1H), 7.53 (d,
7.10 (t, = 8.0 Hz, 1H), 7.04 (dd, = 8.4, 0.8 Hz, 1H), 6.61 (d,
= 7.6, 0.8 Hz, 1H), 6.54 (dd, = 8.8, 2.0 Hz, 1H), 3.82 (s, 3H), 3.59 (s, 3H), 3.58 (s,
3H); 13C NMR (101 MHz, DMSO):
(ppm) 188.29, 162.37, 159.32, 154.21, 138.94,
℃
; IR (KBr): 3213, 2937, 1622, 1420, 1088 cm-1; 1H NMR (400
= 3.2 Hz, 1H), 7.25 (d, = 8.4 Hz, 1H),
= 2.4 Hz, 1H), 6.58 (dd,
δ
J
J
J
J
J
J
J
δ
133.35, 131.32, 125.42, 123.90, 119.73, 115.52, 105.65, 104.78, 102.51, 98.88, 55.89,
55.83, 55.53; HRMS for C18 H17 N O4 ([M-H]-) calculated: 310.1074, found: 310.1089.
(4-methoxy-1H-indole-3,5-diyl)bis((2,4-dimethoxyphenyl)methanone) (4). Yield: 8 %,
white solid, m.p. 181-184
(400 MHz, Acetone): (ppm) 11.56 (s, 1H), 7.68 – 7.73 (m, 1H), 7.47 (d,
7.43 (d, = 8.4 Hz, 1H), 7.31 (d, = 8.4 Hz, 1H), 6.72 (d, = 2.4 Hz, 1H), 6.70 – 6.65
(m, 2H), 6.64 (d, = 2.0 Hz, 1H), 6.61 (dd, = 8.4, 2.4 Hz, 1H), 3.92 (s, 3H), 3.90 (s,
3H), 3.77 (s, 3H), 3.71 (s, 3H), 3.63 (s, 3H); 13C NMR (101 MHz, Acetone):
(ppm)
℃
; IR (KBr): 3321, 2937, 1620, 1416, 1072 cm-1; 1H NMR
= 8.4 Hz, 1H),
δ
J
J
J
J
J
J
δ
194.97, 187.95, 162.96, 162.52, 159.83, 158.90, 158.41, 137.39, 132.00, 131.58, 131.50,
130.13, 125.17, 122.52, 120.82, 115.85, 115.73, 104.84, 104.36, 101.27, 98.55, 98.14,
55.07, 54.98, 54.94; HRMS for C27 H25 N O7 ([M-H]-) calculated: 474.1547, found:
474.1560.
(4-Methoxy-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)methanone (5). Yield: 58 %, white
solid, m.p. 239-243
℃
; IR (KBr): 3114, 2964, 1578, 1422, 1125 cm-1; 1H NMR (400
28