Discovery of Potent Inhibitors against P-Glycoprotein-Mediated Multidrug Resistance Aided by Late-Stage Functionalization of a 2-(4-(Pyridin-2-yl)phenoxy)pyridine Analogue

Article abstract of DOI:10.1021/acs.jmedchem.0c00337

SIS3 is a specific inhibitor of Smad3 that inhibits the TGFβ1-induced phosphorylation of Smad3. In this article, a variety of SIS3 derivatives were designed and synthesized to discover potential inhibitors against P-glycoprotein-mediated multidrug resistance aided by late-stage functionalization of a 2-(4-(pyridin-2-yl)phenoxy)pyridine analogue. A novel class of potent P-gp reversal agents were investigated, and a lead compound 37 was identified as a potent P-gp reversal agent with strong bioactivity and outstanding affinity for P-gp.

Full text of DOI:10.1021/acs.jmedchem.0c00337

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Article
Discovery of potent inhibitors against P-glycoprotein-
mediated multidrug resistance aided by late-stage
functionalization of 2-(4-(pyridin-2-yl)phenoxy)pyridine analogue
Yao Ma, Dawei Yin, Jingjia Ye, Xiduan Wei, Yameng Pei, Xueyuan Li, Guangxu Si, Xuan-Yu
Chen, Zhe-sheng Chen, Yi Dong, Feng Zou, Wei Shi, Qianqian Qiu, Hai Qian, and Gang Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00337 • Publication Date (Web): 24 Apr 2020
Downloaded from pubs.acs.org on April 24, 2020
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Discovery of potent inhibitors against P-glycoprotein-mediated
multidrug resistance aided by late-stage functionalization of 2-(4-
(pyridin-2-yl)phenoxy)pyridine analogue
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,#
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Yao Ma , Dawei Yin , Jingjia Ye , Xiduan Wei , Yameng Pei , Xueyuan Li ,
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Guangxu Si , Xuan-Yu Chen , Zhe-Sheng Chen , Yi Dong , Feng Zou , Wei Shi ,
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3,
2,
Qianqian Qiu , Hai Qian *, Gang Liu *
1. Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, 2A Nanwei Rd, Xicheng Dist, Beijing 100050, P.R. China
2
. School of Pharmaceutical Sciences, Tsinghua University, Haidian Dist，Beijing
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00084, P.R. China
3. Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
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. College of Pharmacy and Health Science, St. John’s University, Queens, New York,
NY 11439, USA
. College of Integrated Chinese and Western Medicine, Hebei Medical University,
Shijiazhuang, Hebei 050017, China
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Abstract
SIS3 is a specific inhibitor of Smad3 that inhibits the TGFβ1-induced
phosphorylation of Smad3. In this article, a variety of SIS3 derivatives were designed
and synthesized to discover potential inhibitors against P-glycoprotein-mediated
multidrug resistance to cancer cells aided by late-stage functionalization of 2-(4-
(pyridin-2-yl)phenoxy)pyridine analogue. A novel class of potent P-gp reversal agents
were investigated and a lead compound 37 was identified as a potent P-gp reversal agent
with strong bioactivity and outstanding affinity for ABCB1.
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Introduction
The resistance of cancer cells to a broad spectrum of structurally and mechanistically
distinct anticancer drugs is known as multidrug resistance (MDR),1,2 which is a major
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obstacle in cancer chemotherapy. Many mechanisms of MDR have been established,
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while one of the well-characterized mechanisms is the overexpression of ATP binding
cassette (ABC) transporters.7-9 Among ABC transporter family members, P-
glycoprotein (P-gp, also called multidrug resistance 1, MDR1, and ABCB1) is the first
member to be identified as related to MDR.10,11 It can recognize exogenous anticancer
drugs and efflux them from the cancer cells, resulting in decreased intracellular drug
accumulation. Many chemotherapeutics in clinical practice are susceptible to P-gp-
mediated efflux, such as paclitaxel, doxorubicin, topotecan, dasatinib and gefitinib,
among many others.12
Since it was associated with drug resistance, P-gp has been an ideal target for reversing
MDR by co-administration of its modulators with anticancer drugs. Three generations
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of P-gp inhibitors have been developed over the past few decades (Figure 1). However,
they all failed in clinical investigations for various reasons, such as high intrinsic
toxicity, poor target specificity, drug-drug interactions, or insignificant clinical
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efficacy. Therefore, exploring selective and effective novel P-gp inhibitors with low
cytotoxicity to circumvent MDR is urgently needed.15,16
Figure 1. Examples of three generations of P-gp inhibitors
SIS3 is a specific inhibitor of Smad3 that inhibits TGFβ1-induced phosphorylation of
Smad3.17 Since the SIS3 molecule contains
tetrahydroisoquinoline fragment, which is a typical structure of some P-gp inhibitors
Figure 2), we speculated that SIS3 may also possess P-gp inhibitory activity as well.
a
6,7-dimethoxy-1,2,3,4-
(
Therefore, the reversal activity of SIS3 in P-gp overexpressing DOX-resistance
SW620/AD300 cells was tested (Table 1). As expected, SIS3 showed a reversal activity
that was 1.5-fold times of positive compound Verapamil (VPR). During our studies,
Ambudkar and co-workers also reported that SIS3 reverses ABCB1- and ABCG2-
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mediated MDR in cancer cell lines, which coincides with our result. Therefore, it is
reasonable for further development of novel P-gp inhibitors with enhanced potency and
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selectivity through efficient modifications of SIS3 derivatives.
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Figure 2. Structure of SIS3 and two reported P-gp inhibitors
The key factor in medicinal chemistry is to balance the relationship of the molecular
structure of drug candidates with its physicochemical properties, target specificity and
binding affinity, metabolic stability and cytotoxicity. Commonly, a subtle molecular
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change can determine the direction and success of a drug development program. The
development of transitional metal-catalyzed C-H activation obviously promotes drug
molecule synthesis, and late-stage functionalization (LSF), which utilizes the C-H
bonds of drug leads as sites of diversification for generating new analogs, is especially
useful.20-22 In this study, we designed and synthesized a variety of SIS3 analogues; then,
the aforementioned LSF method was applied to rapidly generate new derivatives,
leading to the discovery of lead compound 37, a novel and potent P-gp inhibitor, with
strong bioactivity and outstanding affinity for P-gp.
Results and Discussion
Molecular Design and Activity in Reversal of Doxorubicin (DOX) Resistance
According to the chemical structure of SIS3, we initially explored several SIS3
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derivatives targeting its double bond including saturated compound 1 and compounds
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-4 with substituents at the α or β position of the double bond (Figure 3), aiming to
avoid its potential ability of Michael addition reaction to the off-target proteins. Next,
a variety of bioisosteres of the amide bond were designed to investigate the necessity
of the tetrahydroisoquinoline fragment in SIS3 (Figure 3, 5-9). Some heterocycles were
also integrated onto the desired compounds to explore the possibility of novel P-gp
modulators (Figure 3, 10-14).
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The reversal activities of these compounds in DOX resistance were tested in P-gp
overexpressing SW620/AD300 cells at nontoxic concentration of 1.0 μM. Meanwhile,
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.5 μM DOX was employed for the assay, a concentration at which it showed
insignificant cytotoxicity on SW620/AD300 cells (Table 1, control group). Co-
administration of tested compounds or verapamil (VRP) with DOX led to a
significantly increase of cytotoxicity on SW620/AD300 cells, suggesting that the tested
compounds reversed the resistance of DOX to SW620/AD300 cells. Compounds that
showed more than 50% inhibition were selected to test their reversal-folds (RF), which
was calculated from dividing the DOX IC50 values without P-gp inhibitors by DOX
IC50 value with P-gp inhibitors.
As mentioned above, DOX alone (0.5 μM) showed insignificant cytotoxicity on
SW620/AD300 cells, with only 5.3% growth inhibition (Table 1, control group). In the
presence of SIS3, DOX showed improved toxic efficacy with 40.0% growth inhibition
(Table 1). Unexpectedly, compounds 1 and 2 (Table 1) showed significantly decreased
activity (17.7% and 12.2%). Methylated compound 3 maintained a similar potency
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(44%, Table 1), while trifluoromethylated compound 4 showed a decreased activity
(32.5%, Table 1). Except for compound 10, which still had 44.7% inhibition, all the
other synthesized bioisosteres of the SIS3 amide bond were useless (5-9, 10.0-37.9%,
and 11-14, 14.0-20.7%, Table 1).
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Figure 3. Molecular design based on the double bond and amide bond
Table 1. Activities of Compounds 1-14 With Regard to Reversing DOX Resistance at
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Compd
% Inhibition
Compd
% Inhibition
10.9 ± 1.4
Control
VRP
5.3 ± 3.0
26.1 ± 2.6
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SIS3
40.0 ± 2.3
17.7 ± 1.5
12.2 ± 4.6
44.0 ± 4.2
32.5 ± 1.0
37.9 ± 2.2
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aAll compounds were tested for their reversal activities of DOX resistance at 1.0 μM in
SW620/AD300 cells. Cells were incubated with 0.5 µM DOX and 1.0 μM tested
compound together. Percentage inhibition was determined using SRB assay after 48 h
incubation and normalized to the group containing no DOX. Percentage inhibition was
presented as the mean ± SD here.
In the subsequent structural design, a series of derivatives based on the aza-indole
fragment were synthesized to increase the skeleton diversity (Table 2) via various
approaches, including ring fusing strategy (15-19, Table 2), ring splitting strategy (20,
Table 2), N-walking strategy (21, Table 2), and pharmacophore integration of the 2-(4-
(
pyridin-2-yl)phenoxy)pyridine group (22, Table 2) that is the skeleton of some calcium
channel blockers. 23-25
Many structurally different drugs are substrates of P-gp, implying the binding pocket
of P-gp is very flexible. Compounds 23-25 were then designed to prolong the molecular
length and/or to increase the molecular hydrophobicity through the introduction of
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aromatic rings or other motifs that often produces efficient inhibitors of P-gp. Natural
flavonoids and coumarins motifs were also integrated into compounds 26 and 27.
Compounds 28 and 29 were designed for the same reasons as 22 (Table 2).
Molecules derived from the ring fusing strategy showed a comparable activity to SIS3
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(16-19, 40.1-45.5%, Table 2), apart from compound 15, with only 22.9% inhibition,
suggesting that six-membered cycles are acceptable in this type of skeleton.
Compounds obtained from the ring splitting and N-walking strategy or introduction of
a 2-(4-(pyridin-2-yl)phenoxy)pyridine group showed lower inhibitory percentage (20-
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2, 26.7-37.5%, Table 2) because of the large changes in the molecular skeleton.
Compounds with prolonged molecular length and increased hydrophobicity showed
potent reversal activity (23-29, 41.8%-64.0%, Table 2). Counting for the unified RF
values, the positive control of VRP was 11.20-reversal fold (Table 2). RF values of 23,
24, or 25 was 40.72, 30.35, and 60.71, respectively (Table 2). RFs of 26 and 27,
containing flavonoids or coumarins motif, were 24.55 and 16.95, respectively, that the
corresponding modifications were not necessary (Table 2). Compound 28 did not gain
corresponding RF value due to its weak reversal activity with less than 50% inhibition
at tested concentration. Interestingly, 29 with the 2-(4-(pyridin-2-yl)phenoxy)pyridine
group exhibited optimal reversal potency (RF 60.71) at current stage.
Table 2. Activities of Compounds 15-29 with regard to Reversing DOX Resistance at
1.0 μM in SW620/AD300 Cells
Compd
R
1
% Inhibition
IC50 / DOX (µM)
RF
(0.5 μM DOX+
1.0 μM Compd)
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Control
VRP
---
---
---
5.3 ± 3.0
26.1 ± 2.6
40.0 ± 2.3
33.39 ± 7.08
2.98 ± 0.49
ND
1.00
11.20
ND
SIS3
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22.9 ± 1.9
45.4 ± 1.8
ND
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0.82 ± 0.29
1.10 ± 0.23
40.72
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ND
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a
The Percentage inhibition was determined after exposure to 0.5 µM DOX with
different compounds at 1.0 μM in SW620/AD300 cells, and normalized to the group
containing no DOX. The IC50 value was determined after exposure to a series of DOX
concentrations with different compounds at 1.0 μM in SW620/AD300 cells. Reversal
fold (RF, fold-change in drug sensitivity) = (IC50 without inhibitor) / (IC50 with
inhibitor). Data were analyzed with GraphPad Prism 7.0 software and presented as
mean ± SD for three independent tests. ND: not determined.
LSF is a useful approach for medicinal chemistry to rapidly explore structure activity
27
relationships (SARs). As indicated in table 2, compound 28 (with 41.8% inhibition)
showed a comparable activity with SIS3 in the primary screening. Aside from its
suitability for derivatization via LSF, we think that its chemical skeleton is promising
for potent P-gp inhibitors, because some known P-gp inhibitors contains one or more
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pyridine groups. We firstly explored the influence of different sites on the pyridine
group and the double bond, leading to compounds 30-34 (Table 3). Compounds 35-45
were then prepared by using the LSF method that was previously developed by our
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
group (Table 3). Apart from compound 33, 36 and 38 with 36.1-49.8% inhibition, the
other compounds showed better activity with more than 50% inhibition and their RF
values were further determined. The compound 31 with 3-pyridyl group showed the
highest RF value of (RF 85.62 in this article. For the derivatives with substituents at the
pyridyl-linked phenyl group, 3-substituted analogues, e.g. 37, 44-45, with RF values of
59.63, 54.74, and 68.14, respectively, turned out to be more potent.
Table 3. Activities of Compounds 30-34 (R
1
) and 35-45 (R ) for Reversal of DOX
2
Resistance at 1.0 μM in SW620/AD300 Cells
Compd
R
1
or R
2
% Inhibition (0.5 IC50 / DOX (µM)
μM DOX + 1.0
RF
μM Compd)
Control
VRP
---
---
5.3 ± 3.0
33.39 ± 7.08
2.98 ± 0.49
1.00
26.1 ± 2.6
11.20
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SIS3
---
40.0 ± 2.3
ND
ND
6
0.2 ± 1.6
0.99 ± 0.30
33.73
3
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
62.6 ± 1.5
56.5 ± 1.2
0.39 ± 0.10
0.75 ± 0.11
85.62
44.52
3
2
3
3
3
4
36.1 ± 2.5
59.0 ± 0.8
ND
ND
0.76 ± 0.13
43.93
3
3
5
6
2-Me
3-F
55.1 ± 6.2
49.8 ± 0.9
60.3 ± 1.1
49.5 ± 2.5
56.4 ± 0.7
56.3 ± 2.3
53.2 ± 1.6
59.2 ± 1.6
60.1 ± 1.3
58.3 ± 1.6
58.2 ± 0.4
0.74 ± 0.13
ND
45.12
ND
37
3-Cl
0.56 ± 0.08
ND
59.63
ND
3
3
4
4
8
9
0
1
2,3,5,6-tetra-D
3-OH
0.82 ± 0.14
0.78 ± 0.15
1.18 ± 0.28
0.76 ± 0.15
0.70 ± 0.14
0.61 ± 0.10
0.49 ± 0.09
40.72
42.81
28.30
43.93
47.70
54.74
68.14
3,5-di-OH
2,6-di-I
2-I
42
4
4
4
3
4
5
2-Br
3-CN
3-Me
a
The Percentage inhibition was determined after exposure to 0.5 µM DOX with
different compounds at 1.0 μM in SW620/AD300 cells, and normalized to the group
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containing no DOX. The IC50 value was determined after exposure to a series of DOX
concentrations with different compounds at 1.0 μM in SW620/AD300 cells. Reversal
fold (RF, fold-change in drug sensitivity) = (IC50 without inhibitor) / (IC50 with
inhibitor). Data were analyzed with GraphPad Prism 7.0 software and presented as
mean ± SD for three independent tests. ND: not determined.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemistry. To synthesize compound 1-4 (Scheme 1), intermediates A-C were
previously prepared as the methods of literature.15,28 Compound 1 was synthesized by
direct reduction of SIS3. For 2-4, the corresponding carboxylic acids (2c, 3b, 4d) were
prepared, then the desired compounds were obtained by condensation reactions with
6
,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. E/Z isomers of compounds 2 and 4 were
1
gained as detected by H-NMR. The synthetic routes of 5-8 are depicted in Scheme 2.
Scheme 1. Synthesis of compounds 1-4
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reagents and conditions: a) 5% Pd/C, H
2
, MeOH, r.t., 3h; b) NaH, anhydrousTHF, r.t.,
2
h; c) LiOH, EtOH/H
2
O, r.t., 3h; d) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline,
EDCI, HOBT , TEA, DMF, r.t., 12h; e) Pd(OAc)
2
, PPh
3
, K
2
CO , DMF, 110°C, 2h; f) n-
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BuLi, anhydrous THF, -78°C, 2h.
Scheme 2. Synthesis of compounds 5-8
Reagents and conditions: a) TEA, THF, r.t. 3h; b) 1. A (Scheme 1) for 5 or 1-methyl-2-
phenyl-1H-indole-3-carbaldehyde for 6, n-BuLi, THF, -40°C-r.t., 12h; 2. P , 12h; c)
EDCI, HOBT, TEA, DMF, r.t., 12h; d) Bergess reagent, THF, MW, 150°C, 20min.
2
O
5
2
9
Compound 9 was attained through Heck reacton of intermediate 9a and C (Scheme
3
). Compounds 10-12 were obtained by reaciton of acyl chloride with corresponding
amine. 30,31 For the synthesis of compounds 13 and 14, the brominated intermediate 3a1
was prepared, followed by cyclization with 3,4-dimethoxybenzohydrazide or 3,4-
dimethoxybenzylamine to gain the desired products.
To synthesize 15-22 (Scheme 4), their corresponding carboxylic acids were prepared
according to the reported methods 22,32-35 and then reacted with 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline to provide the aimed products.
Compounds 23-36 were obtained by reaction of corresponding carboxyl acid with 4-
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(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)aniline (Scheme 5). The
intermediates of 24 and 25 were synthesized by a one-pot reaction of indole, ethyl
glyoxylate and morpholine. To synthesze 26 and 27, the starting materials 26a and 27a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
6
were gained based on reported methods (Scheme5). The corresponding carboxyl acid
intemediates of compounds 28, 30-33, 35 and 36 were obtained through the same
synthetic route with conpound 22 (Scheme 4). For compound 29, the iodinatd
intermediate 29a was obtained based on our reported method (Scheme 5).22 The
synthetic method of 34 was depicted in Scheme 5 too.
Scheme 3. Synthesis of compounds 9-14
Reagents and conditions: a) Pd(OAc)
2
, PPh
3
, TEA, DMF, 110°C, 10h; b)
, reflux, 1h; d)
,4-dimethoxybenzohydrazide or (3,4-dimethoxyphenyl)methanamine, Toluene, reflux,
h.
correspongding amine or azide, Toluene, reflux, 10h; c) NBS, BPO, CCl
4
3
4
Scheme 4. Synthesis of compounds 15-22
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: a) 1. POCl
acid, piperidine, pyridine, reflux, 12h; c) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline,
EDCI, HOBT, TEA, DMF, r.t., 12h; d) I /H IO , H SO , AcOH/H O, 70°C; e) methyl
acrylate, Pd(OAc) , PPh , K CO , DMF, 110°C, 2h; f) LiOH, EtOH/H O, r.t., 2h; g)
NIS, KOH, MeCN, r.t., 4h.
3
, DMF, 0°C, 0.5h; 2. H
2
O, r.t., 0.5h; b) malonic
2
5
5
2
4
2
2
3
2
3
2
Scheme 5. Synthesis of compounds 24-37, 29 and 34
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: a) HFIP/Toluene, r.t., 0.5h; b) LiOH, EtOH/H O, r.t., 2h, c)
2
4
-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)aniline, EDCI, HOBT,
DMF, r.t., 12h; d) Pd(OAc)
2
, PPh
3
, K
2
CO
3
, DMF, 110°C, 2h; e) Pd(OAc) , NIS, TFA,
2
t
1
20 °C, 12h; f) NaO Bu, DMSO, 0°C-r.t., 5h.
Late-Stage Functionalization (LSF). The synthesis of 37-45 is depicted in Scheme 6.
Initially, we intended to directly modify 2-(4-(pyridin-2-yl)phenoxy)pyridine group of
22
2
8 by our LSF method via metal-catalyzed C-H activation reactions. The reaction
were successfully performed, however, the reaction mixture was difficult to be
separated, which is a major challenge for LSF owing to only a small functional group’s
introduction (data not shown). We then had to make a second choice to performed LSF
on intermediate 22a (Scheme 6), followed by hydrolysis and condensation reactions to
gain the desired products. It is noteworthy that our Pd-catalyzed C-H activation method
is a regioselective reaction, for instance, the modification occurred at the C-3 position
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of 22a in AcOH, while at the C-2 position 22a in TFA that enable us to efficiently
synthesize intermediates 37a-45a, and final products of 37-45 (Scheme 6).
Scheme 6. Synthesis of compounds 37-45 by Late-Stage Functionalization of 22a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Yield %d
Compd
Conditions a + Pd(OAc)
2
10 mol%
R
3-Cl
3
3
3
4
7
8
9
0
NCS, AcOH, 80 C
45
d-TFA, 120 C(twice), d
4
-AcOH, 120 C(twice)
2,3,5,6-tetraD
3-OH
75(D>95%)
PhI(OAc)
PhI(OAc)
2
2
(1.0 equiv), Ac
2
O / AcOH, 120 C
O / AcOH, 120 C
56
67
35
40
44
51
40
(2.0 equiv), Ac
2
3.5-diOH
2,6-diI
2-I
41
42
43
44
45
NIS, TFA, 120 C
NIS, TFA, 120 C
NBS, TFA, 120 C
2-Br
K
4
6
Fe(CN) , CuBr
2
, DMF, 130 C
3-CN
MeB(OH) , [CpRhCl
2
2 2
]
, Ag CO
2
3
, DCE, 130 Ce
3-Me
Reagents and conditions: b) NaOH, EtOH/H
2
O, 50°C, 1h; c) 4-(2-(6,7-dimethoxy-3,4-
d
dihydroisoquinolin-2(1H)-yl)ethyl)aniline, EDCI, HOBT, DMF, r.t., 12h. yields of
e
3
7a-45a. Non Pd(OAc)
2
.
Intrinsic Toxicity and Effective Concentration (EC50) of Target Compounds. This
new class of P-gp inhibitors was further investigated by selecting certain compounds
for additional experiments in which their intrinsic cytotoxicity at 10.0 µM were
determined (listed in Table 4). Compounds 25, 29 and 45, which caused cell growth
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inhibition rates greater than 15%, were excluded accordingly (Table 4). To
quantitatively determine the efficacy of reversal of DOX resistance, we then determined
the EC50 values of the superior compounds 31, 37 and 44 in reversing DOX resistance
in SW620/AD300 cells. As shown in Figure 4, these three compounds were very potent
in reversing DOX resistance with EC50 values in the nanomolar range (103.4 ± 32.0
nM, 37.2 ± 5.4 nM and 51.0 ± 5.4 nM, respectively), among which compound 37 had
the lowest EC50 value of 37.2 ± 5.4 nM. Based on the SARs described above, and in
terms of in vitro cytotoxicity and potency, compound 37 was finally selected for in-
depth study.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Cytotoxicity of Tested Compounds in SW620/AD300 cells.
Compd
Cell growth inhibition rate (%) at 10 µM
2
2
5
9
42.0 ± 2.84
17.0 ± 4.05
13.9 ± 5.98
9.7 ± 5.96
31
37
44
45
13.9 ± 1.85
17.1 ± 3.78
a
SRB method was used for determination of the IC50 values for the target
compounds. Data were analyzed with GraphPad Prism 7.0 software and presented as
the mean ± SD of three independent tests.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. EC50 values of compounds 31, 37 and 44 for reversal of DOX resistance in
SW620/AD300 cells. The percentage of the IC50 of DOX was calculated as follows:
[
(IC50 of DOX at each modulator concentration/IC50 of DOX without modulator) ×
100)]. EC50 refers to the concentration of modulator that can reduce the percentage of
IC50 of DOX by half. Each data point is presented as the mean ± SD of three
independent tests.
Molecular Docking Analysis. A docking study of 37 was performed using human
14
homology ABCB1 on the basis of mouse ABCB1 protein (PDB code 4M1M). The
results indicated that 37 was stabilized in a hydrophobic pocket formed by Leu 65, Pro
6
6, Met 69, Phe 303, Phe 335, Phe 336, Leu724, Phe 728, Phe 983 and Met 949 of
ABCB1 (Figure 5A). Three main interactions were predicted between 37 and the
ABCB1 including that a π-π interaction was indicated between the C-pyridine group of
3
7 with the phenyl ring of Phe 303 of ABCB1; another π-π interaction was observed
between the O-pyridine group of 37 with the phenyl ring of Phe 983 of ABCB1; and a
hydrogen bond occurred between the amide group of 37 with the hydroxyl group of Tyr
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953 (Figure 5B). The docking analysis indicates relatively high affinity of 37 for
ABCB1, which is consistent with the results of the cell-based reversal fold test.
A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
Figure 5. Docking analysis of 37 with the human ABCB1 homology model on the basis
of mouse ABCB1 protein (PDB code 4M1M). (A) Compound 37 (ball and stick model)
docked with the human ABCB1 homology model (ribbon). The blue grids represent
hydrophobic regions in the binding pocket. (B). Predicted interactions between 37 and
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5
5
5
5
6
ABCB1. Carbon atoms of 37 are labeled green; oxygen and nitrogen atoms of 37 are
labeled red and blue, respectively. ABCB1 is shown as a ribbon. Only relevant residues
discussed in the text are shown as sticks and labeled. The proposed hydrogen bonds and
π-π stacking involved in the binding results are represented as yellow and green dashed
lines.
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Effect of 37 on ABCB1 Expression. Downregulation of the expression ofABCB1 may
also cause the reversal of ABCB1-mediated MDR. As illustrated in Figure 6,
SW620/AD300 cells exhibited a much higher ABCB1 level than SW620 cells (Figure
6A), and no significant changes in the ABCB1 level were observed after treatment with
3
7 (0, 1.0, 2.0, or 5.0 μM) for 48 h (Figure 6B). These findings revealed that the reversal
activity of 37 was not due to downregulated expression of ABCB1.
Figure 6. Effect of 37 on ABCB1 expression. (A) Western blot analysis showing the
expression of ABCB1 in the SW620 and SW620/AD300 cell lines. (B) Western blot
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analysis showing the expression of ABCB1 in SW620/AD300 cell line after exposure
to 1.0, 2.0 and 5.0 μmol/Lof 37. Band intensity was analyzed by Image Lab, and protein
expression is presented as the ratio of the target protein band intensity to that of β-actin
(n=4).
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Inhibitory Effect of 37 on ABCB1-Mediated Rhodamine 123 Accumulation and
Efflux. To assess the potency of 37 on ABCB1 efflux function, an ABCB1-dependent
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fluorescent rhodamine 123 (Rh123) efflux assay was performed. When exposed to 1
μg/mL Rh123, ABCB1-overexpressing SW620/AD300 cells accumulated much less
Rh123 than SW620 cells (Figure 7A). Addition of 37 led to an obvious increase in
Rh123 content in SW620/AD300 cells but not in SW620 cells. The inhibitory effect of
37 on ABCB1 function was further confirmed by monitoring the efflux of Rh123. As
shown in Figure 7B, when Rh123 was pre-incubated with cells, it was then expelled
from SW620/AD300 cells much faster than from SW620 cells. In ABCB1-low-
expressing SW620 cells, compound 37 did not significantly affect intracellular Rh123
efflux, while in the ABCB1-overexpressing SW620/AD300 cell line, compound 37
inhibited Rh123 efflux in a dose-dependent manner. These results demonstrated that 37
could restore Rh123 accumulation and delay its efflux, which further indicated that the
effects of ABCB1 might be abrogated by 37.
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Figure 7. Effect of 37 on accumulation (A) and efflux (B) of Rh123 in SW620 and
SW620/AD300 cells. Data are presented as the mean ± SD of three independent
experiments.
Reversal Activity of 37 on ABCG2-Overexpressing and ABCC1- Overexpressing
Cell Lines. To determine whether 37 could block the drug efflux function of both
ABCG2 and ABCC1, we next examined the chemosensitization effect of 37 in the
ABCG2 overexpressing H460/MX20 and ABCC1 overexpressing KB-CV60 cancer
cell lines. The extent of reversal of multidrug-resistant cells for a particular drug by 37
was calculated as the reversal fold (RF) value and summarized in Tables 5-6. MK571
and Ko143 were used as positive controls for the reversal of drug resistance mediated
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by ABCG2 and ABCC1, respectively. We found that at nontoxic concentrations and
without affecting the proliferation of cells, 37 did not exhibit reversal activity in either
cell line. Such findings implied that compound 37 selectively reverses ABCB1-
mediated drug resistance.
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Table 5. Reversal Activity of 37 for Mitoxantrone Resistance in H460/MX20 Cells
(ABCG2-Overexpressing Cell Line)
Compd
IC50(μM)
RF
-
Mitoxantrone
2.246 ± 0.781
0.179 ± 0.015
0.966 ± 0.211
2.258 ± 0.307
Mitoxantrone + KO143 10 μM
Mitoxantrone + 37 5 μM
Mitoxantrone + 37 2.5 μM
12.57
2.32
0.99
Table 6. Reversal Activity of 37 for Vincristine Resistance in KB-CV60 Cells (ABCC1-
Overexpressing Cell Line)
Compd
IC50(μM)
RF
-
Vincristine
1.018 ± 0.290
0.291 ± 0.027
0.711 ± 0.125
0.911 ± 0.020
Vincristine + MK571 25 μM
Vincristine + 37 5 μM
Vincristine + 37 2.5 μM
3.50
1.43
1.12
Compound 37 did not affect ABCB1 ATP Hydrolysis. ABCB1-mediated hydrolysis
of ATP was measured in the presence of 37 at concentrations ranging from 0 to 5.0 μM.
VRP, which is an inhibitor as well as a substrate of P-gp, is a stimulator of ABCB1-
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mediated ATP hydrolysis. As shown in Figure 8, VRP strongly increased the ABCB1-
ATPase activity relative to the basal level. However, 37 did not affect ABCB1-mediated
ATP hydrolysis.
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Figure 8. Effect of 37 on ABCB1-ATPase activity. Sodium vanadate inhibitable
ATPase (ABCB1 ATPase) was studied as described in the Experimental Section.
ABCB1 ATPase was measured in the absence (basal activity) or presence of ABCB1
modulators (VRP and 37). The results are presented as the mean ± SD of three
independent experiments: (****) P < 0.0001 compared to the basal activity.
Cellular Thermal Shift Assay (CETSA). The binding affinity of 37 with ABCB1 was
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assessed by CETSA. SW620/AD300 cell lysate was incubated with 37 and then
heated to different temperatures. Unbound proteins were denatured and precipitated at
elevated temperatures, while compound-bound proteins remained in solution. As shown
in Figure 9, 37 efficiently stabilized ABCB1 at higher temperatures, suggesting that 37
directly interacts with ABCB1.
Figure 9. Compound 37 directly interacts with ABCB1. The binding affinity of 37 with
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ABCB1 was assessed by a CETSA. SW620/AD300 cell lysate was incubated with 37
(20 µM) and then heated to different temperatures. ABCB1 expression was assessed by
Western blots.
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Xenograft Study. To determine whether 37 has the reversal activity of ABCB1-
mediated MDR in vivo, the antitumor activities of DOX combined with or without 37
were tested on a DOX-resistant xenograft model bearing ABCB1-overexpressing
K562/A02 cells by subcutaneous implantation. The images of animals and tumors after
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4 days of treatment are displayed in Figure 10A. Based on the data collected every
two days, the tumor sizes of mice treated with 37 or DOX alone did not differ from
those of mice in the control group, whereas the tumor sizes in the combination groups
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were significantly reduced (Figure 10B). The average tumor size was 610 mm in the
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DOX alone group, 508 mm in the combination VRP group, and 437 mm in animals
treated with DOX and 37. Regarding the tumor weight at Day 14, mice treated with
coadministration of 37 and DOX showed a significant difference compared to the DOX
alone group (Figure 10C). These results indicate the ability of 37 to inhibit tumor
growth in K562/A02 xenografts.
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Figure 10. Antitumor efficacy of 37 in the K562/A02 xenograft mice model. (A)
Photographs of mice and tumors in each group. (B) The tumor volumes of K562/A02
xenograft mice in each group were measured every two days. (C) The tumor weights
of K562/A02 xenograft mice in each group after the 14-day treatment. *P <0.05, **P
<0.01, ***P <0.001 vs. the DOX alone group.
 Conclusion
In this study, a series of 2-(4-(pyridin-2-yl)phenoxy)pyridine derivatives was designed
and used to identify novel P-gp-mediated MDR modulators. In particular, compound
3
7 exhibited outstanding reversal activity against MDR in the P-gp overexpressing
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SW620/AD300 cell line with a low EC50 (37.2 ± 5.4 nM). This compound exhibited
remarkable potency in increasing the accumulation of Rh123 in SW620/AD300 cells
as well in inhibiting P-gp-mediated drug efflux of Rh123. Compound 37 (different from
VRP) has no effect on P-gp-ATPase. Western blotting results indicated that MDR
reversal by 37 was not due to a decrease in protein expression but due its functional
inhibition. Further in vivo studies demonstrated that the growth of ABCB1-
overexpressing K562/A02 xenografts was significantly inhibited by DOX combined
with 37 compared to single treatment of DOX. These results indicated that 37 may serve
as a leading compound to target ABCB1-dependent MDR.
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
EXPERIMENTAL SECTION
Chemistry.
General Information on Synthesis. DCM, THF, DMF, MeOH, and other commercial
reagents were purchased from domestic corporations and used without further
purification. Silica gel for column chromatography was purchased from Qingdao
Haiyang Chemical and Special Silica Gel Co, Ltd. The automatic liquid
chromatography−mass spectrometry (LC-MS) analysis was performed on a Waters SQ
Advantage mass spectrometer equipped with an ultraperformance LC (UPLC) system
and an eluent splitter (5% eluent was split into the MS system). High-resolution LC-
MS was conducted using Agilent LC/MSD TOF with an Agilent ZORBAX SB-C18
(rapid resolution, 3.5 μm, 2.1 mm × 30 mm) column at a flow rate of 0.40 mL/min. The
solvent was MeOH/water (75:25 (v/v)) containing 5 mmol/L ammonium formate. The
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