Discovery of 5-Cyano-6-phenylpyrimidin Derivatives Containing an Acylurea Moiety as Orally Bioavailable Reversal Agents against P-Glycoprotein-Mediated Mutidrug Resistance

Article abstract of DOI:10.1021/acs.jmedchem.8b00335

P-glycoprotein (ABCB1)-mediated multidrug resistance (MDR) has become a major obstacle in successful cancer chemotherapy, which attracted much effort to develop clinically useful compounds to reverse MDR. Here, we designed and synthesized a novel series o

Full text of DOI:10.1021/acs.jmedchem.8b00335

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Article
Discovery of 5-cyano-6-phenylpyrimidin derivatives containing
an acylurea moiety as orally bioavailable reversal agents
against P-glycoprotein-mediated mutidrug resistance
Bo Wang, Li-Ying Ma, Jing-Quan Wang, Zi-Ning Lei, Pranav Gupta, Yuan-Di Zhao, Zhong-
Hua Li, Ying Liu, Xin-Hui Zhang, Ya-Nan Li, Bing Zhao, Zhe-sheng Chen, and Hong-Min Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00335 • Publication Date (Web): 05 Jul 2018
Downloaded from http://pubs.acs.org on July 5, 2018
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Discovery of 5ꢀcyanoꢀ6ꢀphenylpyrimidin derivatives containing
an acylurea moiety as orally bioavailable reversal agents
against Pꢀglycoproteinꢀmediated mutidrug resistance
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1#
1#
2#
2
Bo Wang , Li-Ying Ma , Jing-Quan Wang , Zi-Ning Lei , Pranav
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Gupta , Yuan-Di Zhao , Zhong-Hua Li , Ying Liu , Xin-Hui Zhang ,
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1*
2*
1*
Ya-Nan Li , Bing Zhao , Zhe-Sheng Chen and Hong-Min Liu
1
Collaborative Innovation Center of New Drug Research and Safety Evaluation,
Henan Province; Key Laboratory of Advanced Drug Preparation Technologies,
Ministry of Education; Key Laboratory of Henan Province for Drug Quality and
Evaluation; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou
450001, PR China
2
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,
St. John’s University, Queens, NY 11439, USA
ABSTRACT：Pꢀglycoprotein (ABCB1)ꢀmediated multidrug resistance (MDR) has
become a major obstacle in successful cancer chemotherapy, which attracted much
effort to develop clinically useful compounds to reverse MDR. Here, we designed and
synthesized a novel series of derivatives with a 5ꢀcyanoꢀ6ꢀphenylpyrimidin scaffold
and evaluated their potential reversal activities against MDR. Among these
compounds, 55, containing an acylurea appendage, showed most potent activity in
reversing paclitaxel resistance in SW620/AD300 cells. Further studies demonstrated
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5 could increase accumulation of PTX, interrupt ABCB1ꢀmediated Rh123
accumulation and efflux, stimulate ABCB1 ATPase activity and especially have no
effect on CYP3A4 activity, which avoid drug interaction caused toxicity. More
importantly, 55 significantly enhanced the efficacy of PTX against the SW620/AD300
cell xenograft without obvious side effect for orally intake. Given all that, the
pyrimidineꢀacylurea based ABCB1 inhibitor may be a promising lead in developing
new efficacious ABCB1ꢀdependent MDR modulator.
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INTRODUCTION
Multidrug resistance (MDR) has become a major obstacle in successful cancer
1
,2
chemotherapy.
Although it’s reported that MDR is involved with multiple
mechanisms, the overexpression of some members of the ATPꢀbinding cassette (ABC)
protein family is thought to be a major contributor to the development of MDR in
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ꢀ5
cancer cells. Pꢀglycoprotein (ABCB1), a main member of ABC transport proteins,
is encoded by MDR1 gene and pumps specific chemotherapeutic agents out of the
cancer cells by the energy of adenosine triphosphate (ATP) hydrolysis, thereby
decreasing the intracellular drug accumulation and resulting in drug
6,7
resistance. ABCB1 was found to be widely overexpressed in human solid tumors
and hematologic malignancies, such as acute lymphocytic leukemia, colon, liver,
8
pancreas and kidney cancers. It was shown that a broad variety of important
chemotherapeutic drugs with different structures such as paclitaxel, camptothecin
analogues, anthracyclines, vinca alkaloids, and tyrosine kinase inhibitors (e.g.
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ꢀ13
imatinib, nilotinib, everolimus) are substrates of ABCB1.
Down regulation or
inhibition of ABCB1 can effectively enhance the anticancer efficacy of conventional
14
chemotherapeutic agents.
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Because of its overexpression in cancer cells, ABCB1 has become a therapeutic
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target for getting around MDR. Several reviews concerning the discovery of reversal
16ꢀ19
agents targeting ABCB1 protein have been published.
In clinical, combining
ABCB1 modulators with chemotherapeutic agents has been acknowledged as a
promising therapeutic strategy to get around ABCB1ꢀmediated MDR. Therefore, over
the past few decades, considerable efforts have been made in developing ABCB1
20ꢀ32
inhibitors.
According to their specificity, affinity, and toxicity, ABCB1
modulators are classified to three generations. Firstꢀgeneration MDR reversal agents
are represented by drugs in clinical use for other indications (e.g. quinidine,
cyclosporine A, and verapamil) with limited selectivity and higher drug
33
concentrations requirement. The secondꢀgeneration agents are most analogues based
on first generation drugs such as valspodar, dexverapamil and elacridar. Despite
undesirable toxic characteristics inherited limited their pharmacological utilization,
34ꢀ36
secondꢀgeneration agents possessed higher activity and selectivity.
In particular,
the thirdꢀgeneration ABCB1 inhibitors such as tariquidar possessed high affinity to
ABCB1 at a nanomolar concentration to acquire more specific and effective inhibition
against ABCB1 function. However, although several ABCB1 inhibitors have entered
the clinical trial stage, there still have been no satisfactory results so far because of
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some limitations such as insignificant clinical effests, concerns about safely, or
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pharmacokinetic interaction.
Therefore, developing more selective and effective
novel ABCB1 inhibitors to reverse MDR has become an urgent requirement.
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In an effort to design and develop more selective and effective novel ABCB1
inhibitors to combat drug resistance, we previously conducted an activity test on our
inꢀhouse structurally diverse molecular library (ca.500 compounds) and identified the
potent pyrimidine skeleton and subsequent extensive medicinal chemistry efforts
leading to the discovery of potent triazole contained pyrimidineꢀbased MDR mediator
PRAꢀ1 (Fig. 1), which could effectively reverse PTX resistance in SW620/AD300
39ꢀ41
cells by increasing intracellular accumulation of PTX.
Furthermore, lipophilicity
has been identified as an important factor correlated to potent inhibitory activity
against ABCB1, since the inhibitors majorly bind to the big hydrophobic pocket
located within transmembrane portion of ABCB1 and lipophilicity helps them enter
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the cavity. Besides of hydrophobicity, several features of ABCB1 inhibitors have
been indicated as contributors of passive diffusion into cell membrane and/or
inhibitory function, including halogen group, aromatic ring center, hydrogen bond
43,44
acceptor, and positive ionizable groups.
Our strategy of inhibitor design involved
maximizing active site interactions, particularly by promoting a network of strong
hydrogen bonding interactions with the large hydrophobic pocket for ABCB1.
Therefore, in this work, we first evaluated the reversal activity of series I compounds
with hydrogen bond donors and receptors properties (acetamide group) which have
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been identified form PRAꢀ1. In addition, series II derivatives without 1,2,3ꢀtriazole
group were synthesized based on series I to explore the effect of molecular size and
lipophilicity on reversal activity. And then, after preliminarily reversal activity
evaluations, compound 55, which showed the most potency, and other two compound
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2 and 60 with relative potent reversal activity were investigated for further
mechanism of reversing ABCB1ꢀmediated MDR.
Figure 1. Structure of PRAꢀ1 and newly designed compounds containing an acylurea moiety.
RESULTS AND DISCUSSION
Chemistry. The general synthesis route of the target pyrimidineꢀthiourea hybrids is
depicted in Scheme 1. Benzaldehydes 1aꢀf, ethyl cyanoacetate 2, and thiourea 3 were
prolonged heated in ethanol containing potassium carbonate to obtain
6
ꢀarylꢀ5ꢀcyanoꢀ2ꢀthiouracils 4aꢀf . Then, compounds 4aꢀd reacted with the propargyl
bromide in dioxane to obtain the target derivatives 5aꢀd. Compound 12 and 13 were
readily synthesized from 11 and the corresponding arylamines following literature
procedures. Compound 12 was allowed to react with sodium azide to yield 14.
Compounds 6aꢀd and 15aꢀd was prepared via click reaction of compound 5aꢀd with
appropriately substituted benzyl azides and compound 14, respectively. Compounds
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3aꢀh was prepared via reaction of compound 4aꢀf with appropriately 12, 13,
respectively. Then, these highly activated intermediates (6aꢀd, 15aꢀd, and 43aꢀf) were
reacted with appropriately substituted arylamines to obtain compounds 7ꢀ10, 16ꢀ42,
and 44ꢀ72.
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Scheme 1. Synthesis of the target pyrimidineꢀbased derivatives.
2 3
Reagents and conditions: a: absolute ethanol, absolute K CO , reflux, 10 h; b: (i)propargyl
4 2
bromide, dioxane, reflux; (ii)phosphorous oxychloride, reflux, 1 h; c: CuSO ·5H O, sodium
ascorbate, THFꢀH
2
O (1:1), rt. d: appropriate aniline, absolute ethanol, reflux, 6h; e: (i)oxalyl
chloride,1,2ꢀdichloroethane, 90°C; (ii)2ꢀaminopyridine or aniline, 0°C; f: NaN , acetone–H O
3
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Biological Testing. Effect of target compounds to reverse PTX resistance in
SW620/AD300 cells. In order to rule out the possibility that the potential toxic of
compounds themselves may cause false positive results, we first evaluated the
cytotoxicity of all tested compounds against sensitive SW620 cells and
PTXꢀresistance SW620/AD300 cells, which overexpressed ABCB1. The data showed
that almost all target compounds had no toxicity against both cell lines, and the
survival rates of compounds 7ꢀ10, 16ꢀ42, and 44ꢀ72 at the concentration of 2.0 ꢁM
were all above 90%, which were suitable for testing their reversal acitivity in
PTXꢀreisitance cell line SW620/AD300 (Table S1, S2 and S3 in Supporting
Information). Therefore, we chose 2ꢁM as the suitable incubation concentration for
further study.
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The reversalꢀfold (RF) was usually used to represented the potency of MDR
reversers, via the ratio of PTX IC50 values on SW620/AD300 cells between in the
absence and in the presence of MDR modulators, and 0.1% DMSO was used as
solvent control. The nontoxic concentration of 2 ꢁM was used to explore the effects
of target compounds on reversal of PTXꢀresistance in SW620/AD300 cells based on
survival rate results. Data are depicted in Table 1ꢀ4. PTX singleꢀtreatment
demonstrated little toxicity on SW620/AD300 cells (IC50=4.186±0.513 ꢁM).
However, in the present of compounds or verapamil (VRP), PTX showed a improved
toxic effects on SW620/AD300 cells to different extents, suggesting that majority of
the test compounds could reverse PTXꢀresistance.
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First, the reversal activity of 7ꢀ10 and 16ꢀ19 in PTX resistance was evaluated
preliminary in SW620/AD300 cells by MTT (Table 1). Unexpectedly, the extension
of the side chain by introducing the Nꢀ(pyridinꢀ2ꢀylcarbamoyl)acetamide group
(16ꢀ19) resulted in decreased activity, compared with the corresponding
PRAꢀ1ꢀanalogs 7ꢀ10. Further structureꢀactivity relationship (SAR) studies found that
the substitution pattern and electronic effect on the aromatic ring were important for
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the reverse activity. Compounds 16ꢀ18 with electronꢀdonating groups at R have more
potent reversal effect (RF 6.23ꢀ17.37) than compound 19 (RF 1.41) with
electronꢀwithdrawing groups. A similar trend was also observed (31, 36 vs 42). In
terms of the substitution pattern at R
ꢀdonating group (20ꢀ25 and 27ꢀ31), displayed increased reversal activity compared
with 26. In addition, the compounds 27ꢀ31 with electronꢀdonating groups at R also
2
, whether they had an electronꢀwithdrawing or
2
showed increased activity compared with 20ꢀ25 with electronꢀwithdrawing groups. As
mentioned in the introduction, lipophilicity has been identified as an important factor
correlated to potent inhibitory activity against ABCB1. To explore the reasons for the
decreased reversal activity of the series I compounds, the lipophilicity of compounds
7
ꢀ10 and 16ꢀ19 has been tested and the results are shown in Figure S1, compounds
6ꢀ19 containing acetamide group showed the lower lipophilicity than the responding
1
compounds 7ꢀ10 with higher reversal activity (Table 1). In addition, docking analysis
found that the weaker activity of 18 may be resulted from more groups that solvate in
water (such as the triazole group) and larger molecular size through docking
analysis.(Figure S2) Along with the lower binding energy score for 18 (XP Glide
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gscore: ꢀ6.833 kcal/mol) than 9 (XP Glide gscore: ꢀ8.453 kcal/mol) at topꢀscoring
poses, the weak binding affinity and reversal effect toward ABCB1 may be resulted
from lower lipophilicity and more groups that solvate in water, and larger molecular
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size. However, two carbonyl oxygen atom of acetamide group of compound 18 were
engaged in hydrogen bond with ABCB1 which inspired us to further optimization.
Table 1. PTXꢀresistance reversal activity of 7ꢀ10 and 16ꢀ19 at 2 ꢁM in
a
SW620/AD300 cells
IC50/PTX
ꢁM)
IC50/PTX
(ꢁM)
Compd.
R
1
RF
Compd.
R
1
RF
(
7
8
9
pꢀCH(CH
3
)
2
0.091±0.003 46.00
0.193±0.021 21.69
0.152±0.011 27.54
1.235±0.052 3.39
0.211±0.012 19.84
4.186±0.513 1.00
16
17
18
19
pꢀCH(CH
3
)
2
0.610±0.052 6.86
0.241±0.019 17.37
0.672±0.073 6.23
2.978±0.474 1.41
p ꢀCH
p ꢀCH
3
3
m,p,m-triOCH
3
m,p,m-triOCH
3
10
pꢀBr
pꢀBr
VRP
control
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
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Table 2. PTXꢀresistance reversal activity of 20ꢀ42 at 2 ꢁM in SW620/AD300
a
Cells
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IC50/PTX
Compd.
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(ꢁM)
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22
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
3
)
3
)
3
)
3
)
)
3
3
)
3
)
3
)
3
)
)
3
3
)
2
2
2
2
2
2
2
2
2
2
2
oꢀCl
pꢀF
0.882±0.054
1.980±0.297
2.523±0.402
1.545±0.189
1.402±0.147
1.529±0.184
2.532±0.403
0.664±0.078
0.893±0.049
0.411±0.038
0.307±0.051
4.75
2.11
1.66
2.71
2.99
2.74
1.65
6.30
4.69
10.18
13.64
pꢀCl
mꢀCl
mꢀNO
oꢀF
23
24
25
26
27
28
29
30
2
H
pꢀOCH
3
oꢀCH
3
oꢀOCH
oꢀOH
3
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3
3
3
3
1
2
3
4
pꢀCH(CH
3
)
2
pꢀCH(CH
pꢀCH(CH
3
)
)
2
2
0.441±0.035
0.318±0.027
3.092±0.490
2.445±0.388
3.055±0.485
3.535±0.548
3.560±0.545
0.391±0.048
0.312±0.050
2.222±0.347
1.743±0.241
2.462±0.391
0.211±0.012
4.186±0.513
9.49
13.16
1.35
1.71
1.37
1.18
1.18
10.71
13.42
1.88
2.40
1.70
19.84
1.00
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
3
3
3
3
3
mꢀCF
pꢀCl
3
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
35
36
37
38
mꢀCl
pꢀCH
pꢀCH
pꢀCH
pꢀCH
pꢀCH
pꢀBr
pꢀCH(CH
)
3
3
3
3
3
3
2
pꢀOCH
oꢀOCH
mꢀCl
3
3
39
40
41
42
mꢀCF
mꢀCF
3
3
pꢀBr
3 2
pꢀCH(CH )
VRP
control
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
On the basis of above results, to improve lipophilicity and reduce molecular size,
compounds 44ꢀ69 without 1,2,3ꢀtriazole group were synthesized and their reversal
effect are shown in Table 3. Unexpectedly, compounds 44 and 45 with
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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
electronꢀdonating at R
2
showed less potent than corresponding compounds 17 and 27.
2
In contrast, the compound 47 with electronꢀwithdrawing at R displayed the
significantly increased reversal activity, about 10ꢀfold more potent than 44ꢀ46. An
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
opposite trend was observed at R . Compounds 49, 50, and 64ꢀ68 with
1
electronꢀwithdrawing groups at R
1
showed decreased reversal effect. These findings
and R may be important contributors in
indicate that the electronic effect at R
1
2
determining activity. Based on above findings, further modifications were mainly
focused on the position of substituent and the hetero atoms substitution. Compared
with compounds (51, 55, and 56) at the 3ꢀsubstitution at R , compounds (57, 53, and
2
54) at the 2,4ꢀsubstitution performed a relatively weak reversal effect. In particular,
compound 55 with chlorine substitution in meta position at the phenyl ring in R
2
displayed the most potent reversal activity compared to the ortho and paraꢀsubstituted
compounds (53 and 54), about 40ꢀfold more potent than Verapamil. Surprisingly,
compound 52 with bromine substitution in para position at the phenyl ring in R2
displayed about 10ꢀfold more potent than compound 54 with chlorine substitution,
2
which indicated that halogen substitutions at R may play an important role. During
the SAR study, we also found the hetero atoms substitution at X was important for the
reversal activity: the nitrogen atom substitution derivatives 58, 56, and 55 were more
potent than the corresponding carbon hetero substitution derivatives 59ꢀ61. In order to
further investigate the relationship of strong activity and lipophilicity of series II, the
hdrophilicity of representative compounds for series II has also been tested.
Compounds 51, 54, 55, 48 without 1,2,3ꢀtriazole group exhibited higher lipophilicity,
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as well as much more potent reversal activity, than the responding compounds 33ꢀ35
and 40 (Figure S3). These results further indicated that the high reversal activity may
be closely related to lipophilicity. Moreover, amide bond length is critical for MDR
reversal activity, and compound 55 with acetamide group (n = 2) displayed the best
reversal activity than 71 (n = 1) and 72 (n = 0), respectively, as well as 54 vs 70,
which may be related to the stronger hydrogen bonds interaction with ABCB1 (Table
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
4). SAR was summarized and indicated in Figure 2.
Table 3. PTXꢀresistance reversal activity of 44ꢀ69 at 2 ꢁM in SW620/AD300
a
cells
IC50/PTX
Compd.
R
1
R
2
X
RF
(ꢁM)
44
45
46
47
48
49
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
pꢀCH(CH
)
3
3
)
3
)
3
)
2
2
2
2
p –CH
3
N
N
N
N
N
N
2.547±0.406
1.926±0.285
1.598±0.204
0.113±0.011
0.691±0.061
1.466±0.166
1.64
2.17
2.62
37.04
6.06
2.86
pꢀOCH
3
mꢀCH
3
mꢀCF
mꢀCF
mꢀCF
3
p ꢀCH
pꢀCl
3
3
3
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2
3
4
5
6
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
5
5
5
5
0
1
2
3
m,p-diF
mꢀCF
mꢀCF
pꢀBr
oꢀCl
3
3
N
N
N
N
N
N
N
N
N
C
C
C
C
C
N
N
N
1.345±0.129
0.072±0.002
0.022±0.001
0.110±0.008
0.332±0.021
0.005±0.000
0.051±0.003
1.177±0.071
0.483±0.052
1.144±0.058
0.016±0.000
0.199±0.003
0.165±0.014
0.167±0.021
0.294±0.031
0.958±0.019
0.312±0.029
3.11
58.14
190.27
38.05
12.61
837.20
82.08
3.56
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,mꢀtriOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,p,m-triOCH
m,pꢀdiF
3
3
3
3
3
3
3
3
3
3
3
3
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
54
55
56
57
pꢀCl
mꢀCl
mꢀNO
2
pꢀCF
3
58
59
60
61
62
63
64
65
66
oꢀOCH
oꢀOCH
mꢀCl
3
3
8.67
3.66
261.63
21.04
25.37
25.07
14.24
4.37
mꢀNO
2
pꢀF
pꢀCH(CH
oꢀCl
3 2
)
m,pꢀdiF
mꢀCl
m,pꢀdiF
oꢀOCH
3
13.42
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9
m,pꢀdiF
pꢀCl
H
pꢀCl
pꢀCl
N
N
N
1.195±0.077
1.808±0.257
2.506±0.399
0.211±0.012
4.186±0.513
3.50
2.32
1.67
19.84
1.00
p–CH
3
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
VRP
control
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
Table 4. PTXꢀresistance reversal activity of 70ꢀ72 at 2 ꢁM in SW620/AD300
a
cells
IC50/PTX
Compd.
R
1
R
2
X
n
RF
(ꢁM)
5
7
5
7
7
4
0
5
1
2
m,p,mꢀtriOCH
m,p,mꢀtriOCH
m,p,mꢀtriOCH
m,p,mꢀtriOCH
m,p,mꢀtriOCH
3
3
3
3
3
pꢀCl
pꢀCl
N
H
N
N
H
2
0
2
1
0
0.332±0.021
3.375±0.264
0.005±0.000
0.759±0.032
3.986±0.126
0.211±0.012
4.186±0.513
12.61
1.24
mꢀCl
mꢀCl
mꢀCl
837.20
5.51
1.05
VRP
19.84
1.00
control
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
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2
3
4
5
6
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
Figure 2. SAR for MDR reversal activity of seriesⅡagainst ABCB1ꢀmediated MDR.
Molecular Docking Analysis. The topꢀscoring docked pose of compound 55 (IFD
Glide gscore: ꢀ15.084 kcal/mol) into the drug binding pocket of human homology
ABCB1 is presented in Figure 3. The Glide gscore is an index that indicates the
4
6
approximate ligand binding free energy. The core structure of 55 was mainly
stabilized in a large hydrophobic cavity formed on the basis of a number of aromatic
and hydrophobic and residues, including Ile299, Ala302, Phe303, Ile306, Tyr307,
Tyr310, Phe335, Phe336, Leu339, Ile340, Phe343, Leu724, Phe728, Ala729, Phe732,
Phe770, Phe983, Met 986, and Ala987. From the in silico modeling analysis, twoπꢀπ
stacking interactions were found between the trimethoxyphenyl ring of 55 and the
phenyl ring of hydroxyphenyl ring of Tyr310 and the phenyl ring of Phe983. In
addition, several hydrogen bonds were predicted between compound 55 and the
surrounding residues in the drug binding pocket of human homology ABCB1. Gln725
was involved in two hydrogen bonding interactions by one of the carbamoyl hydrogen
atom interacting with the carbonyl oxygen atom of acetamide group of 55
(ꢀCO…H
2
NꢀGln725, 2.05 Å), and the other carbamoyl hydrogen atom interacting
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2
with the nitrogen atom in the pyridine ring of 55 (ꢀN…H NꢀGln725, 2.72 Å).
Similarly, the hydrogen atom in the carbamoyl group of Gln990 was engaged in a
hydrogen bond with the N atom in the pyrimidine ring of 55 (ꢀN…H NꢀGln990, 2.28
3
2
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
Å). The carbamoyl oxygen in Gln990 also interacted, by hydrogen bonding, with the
two secondary amides in the pyridinꢀ2ꢀylcarbamoyl group (ꢀNH…OCꢀGln990, 1.96 Å)
and the acetamide group (ꢀNH…OCꢀGln990, 2.02 Å) of 55, respectively. Moreover,
hydrogen bonds were formed between the carbamoyl oxygen in 55 and the phenolic
group of Tyr307 (ꢀCO…HOꢀTyr307, 1.82 Å), as well as between the 4ꢀmethoxy
group in the trimethoxyphenyl ring of 55 and the phenolic group of Tyr310
3
(CH O…HOꢀTyr310, 1.65 Å).
Docking analysis predicted relatively high binding affinity of compound 55 to
human ABCB1, which supports the observations from cellꢀbased reversal activity test.
The high binding affinity of compound 55 to ABCB1 central hydrophobic
drugꢀbinding cavity may be closely related to its pharmacophoric features such as
43,44
hydrophobicity, Hꢀbond acceptor, aromatic ring center, and halogen group.
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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
Figure 3. IFD predicted binding mode of compound 55 to human ABCB1 homology model. (A)
The ribbon diagram of 3D structural inꢀward facing conformation of a homology model of human
ABCB1 ground on the crystal structure counterparts of mouse ABCB1. The location of 55 (orange)
molecule, as illustrated by a ball and stick model is shown within the ABCB1 internal cavity. (B)
The docked conformation of 55 (ball and stick model) is shown within the ABCB1 drugꢀbinding
cavity, with the atoms colored as carbon–orange, hydrogen–white, oxygen–red, nitrogen–blue,
sulfurꢀyellow, chlorideꢀgreen. Important amino acid residues are depicted (sticks model) with the
same color scheme as above for all atoms but carbon atoms in grey. Dotted yellow lines
represent hydrogenꢀbonding interactions, whereas dotted blue lines represent π ꢀπ stacking
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interactions. The values of the relevant distances are indicated in Å. (C) The schemic diagram of
ligand−receptor interaction between 55 and human ABCB1. The amino acids within 4 Å are
depicted as colored bubbles, polar residues are in blue color, and hydrophobic residues are in
green color. Grey circles indicate solvent exposure. Purple arrows represent Hꢀbonds, and green
lines represent πꢀπ stacking aromatic interactions.
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
Effect of compound 52, 55 and 60 at lower concentration on reversing PTX resistance.
In order to investigate the doseꢀdependent relationship, we chose compounds 52, 55
and 60 with relatively more potent reversal activity than others at 2 ꢁM to evaluate
their effects at lower concentrations. The results (Table 5) showed that effects of
compound 55, 52 and verapamil followed a concentrationꢀdependent pattern where
higher concentration showed more potent reversal activity. The effects of compound
60 at 0.5 and 1 ꢁM had no significant difference, but when at 2ꢁM, compound 60
showed more effective activity. These findings suggested that compound 52, 55 and
60 could reverse the PTX resistance in doseꢀdependent manner.
a
Table 5. PTXꢀresistance reversal activity of 52, 55, 60 in SW620/AD300 cells
Compd.
PTX
IC50/PTX (ꢁM)
4.186±0.513
0.058±0.002
0.031±0.001
RF
1.00
+
55 (0.5ꢁM)
72.17
135.03
+
55 (1ꢁM)
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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
+
55 (2ꢁM)
0.005±0.000
0.552±0.003
0.130±0.002
0.022±0.001
0.113±0.018
0.159±0.022
0.016±0.000
0.211±0.012
837.20
8.019
32.20
190.27
37.04
26.33
261.63
19.84
+
52 (0.5ꢁM)
+52 (1ꢁM)
0
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0
+
52(2ꢁM)
+60 (0.5ꢁM)
+60(1ꢁM)
+60 (2ꢁM)
+
Verapamil (2ꢁM)
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
Effect of compound 55 on ABCB1 expression and subcellular localization.
Downꢀregulating the expression of ABCB1 or inhibiting its function may also cause
the reversal of ABCB1ꢀmediated MDR. We first evaluated if compound 55 could
effectively regulated on the expression level of ABCB1 expression. The
SW620/AD300 cells possessed much higher level of ABCB1 than SW620 cells
(
Figure 4A) and no significant changes on the level of ABCB1 under treatment of
compound 55 (0, 1, 2, or 4 ꢁM) for 48 h (Figure 4B). Further immunofluorescence
assay showed similar results (Figure 4C). What’s more, compound 55 did not alter the
subcellular localization compared to the control. Compounds 52 and 60 performed
similar results (Figure S4). These findings indicated that the reversal activity of
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compound 55 was not due to a downꢀregulation of ABCB1 expression but its
functional inhibition.
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Figure 4. Effect of compound 55 on ABCB1 expression and subcellular localization. (A) The
protein level of ABCB1 in SW620 and SW620/AD300 cell lines, and the GAPDH was used as a
loading control. (B) The protein level of ABCB1 in SW620/AD300 cells treated with compound
55 for 48 h was examined, and the GAPDH was used as a loading control. (C) Effect of compound
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5 on the subcellular localization of ABCB1 (green) after treatment for 48 h. Hoechst 33258 (blue)
was stained for the cell nuclei.
Inhibitory Effect of ABCB1-Mediated Rhodamine 123 accumulation and Efflux.
Because of the unchanged expression of ABCB1 after treatment of compound 55, we
hypothesized that it may be involved in the function of ABCB1. ABCB1ꢀdependent
fluorescent rhodamine 123 (Rh123) efflux assay was widely used to evaluate the
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47
reversal activity of ABCB1ꢀmediated MDR modulators. As shown in Figure 5A, the
intracellular Rh123 was notably lower in ABCB1ꢀoverexpressed SW620/AD300 than
parental cells, and the VRP was used as positive control. Compound 55 could
significantly increase the accumulation of Rh 123 in a doseꢀdependent pattern in
ABCB1ꢀoverexpressed SW620/AD300 cells but not in ABCB1ꢀlowexpressed SW620
cells, which may be related to the specific interaction with ABCB1.
Furthermore, Rh123 efflux assay showed (Figure 5B) that after removing Rh123
incubation, the intracellular Rh123 in SW620/AD300 decreased much faster than
parental cells over time, suggesting a ABCB1ꢀmediated positive process of drug
efflux. In ABCB1ꢀlowexpressed SW620 cells, compound 55 did not significantly
effect its intracellular Rh123 efflux. While in ABCB1ꢀoverexpressed SW620/AD300
cell line, compound 55 could inhibit Rh123 efflux in a doseꢀdependent manner, which
is also more potent than VRP. Compounds 52 and 60 performed similar results
(Figure S5). These data may suggest that compound 55 sensitized SW620/AD300 to
PTX by increasing its intracellular accumulation and inhibiting its efflux, which may
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due to the inhibition of ABCB1 activity.
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Figure 5. Effect of compound 55 and VRP on accumulation (A) and efflux (B) of Rh 123 in
SW620 and SW620/AD300 cells. Each data is presented as mean ± SD of three independent
experiments and a representative experiment is shown.
Effect of compound 55 on PTX Accumulation. For further investigation of the reversal
mechanism, we first evaluated the exact amount of PTX in SW620 or
ABCB1ꢀoverexpressed SW620/AD300 cell lines after treatment of compound 55 and
VRP by HPLC. The results were depicted in Figure 6A. PTX accumulation in SW620
cells was higher than that of SW620/AD300 cells in the absence of inhibitors, which
was due to the ABCB1 mediated drug efflux. Importantly, the intracellular PTX was
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maintained at a significantly higher amount compared to the control in
SW620/AD300 cells under treatment with compound 55 or VRP. A similar trend was
3
also observed by measuring the accumulation of tritiumꢀlabeled PTX ([ H]ꢀPTX) in
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SW620/AD300 cells (Figure 6B). Moveover, [ H]ꢀPTX efflux assay was also tested.
As is shown in Figure 6C, after 120 min of efflux, 29% and 69% normalized loss of
3
[
H]ꢀpaclitaxel occurred in SW620 and SW620/Ad300 cells, respectively. Also, at the
3
end of 120 min efflux, 36% and 69% of normalized [ H]ꢀpaclitaxel was removed in
SW620/Ad300 cells with or without coꢀincubation with compound 55. Moreover,
effects of compound 55 followed a concentrationꢀdependent pattern where 2 and 4
3
ꢁM
compound 55 exhibited significant increase of [ H]ꢀpaclitaxel in
ABCB1ꢀoverexpressing cells (Figure 6C, right). Efflux pattern of parental cells were
not significantly altered by compound 55 (Figure 6C, left). These findings further
suggested that 55 was a potent ABCB1 inhibitor by increasing PTX intracellular
accumulation and inhibiting its efflux.
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Figure 6. Effects of compound 55 and VRP on accumulation and maintenance of PTX. (A) The
exact amount of PTX in SW620 or SW620/AD300 cell lines was measured by HPLC. (B) Effect
3
of compound 55 and VRP on accumulation of [ H]ꢀpaclitaxel in SW620 and SW620/AD300 cell
lines. (C) After 0, 30, 60 or 120 min, the same numbers of SW620 (left) and SW620/Ad300 (right)
were measured the radioactivity with the scintillation fluid. Data was obtained from three
independent assays and shown as mean ± SD.
Effect of compound 55 on the ABCB1 ATP hydrolysis. The activity of ATPase could
be reflected by ATP consumption because the ABCB1 transporter takes advantage of
energy from ATP hydrolysis to pump its substrates across the cell membrane against
concentration gradient. Therefore, we measured ATP hydrolysis mediated by ABCB1
in the presence of compound 55 at gradient concentrations from 0ꢀ40 ꢁM. The result
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shows that compound 55 could stimulated the ATPase activity of ABCB1 with a
maximal stimulation of 4.56ꢀfold and the concentration of compound 55 to obtain
50% stimulation is 0.97 ꢁM (Figure 7).
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Figure 7. Effect of compound 55 on the ABCB1 ATP hydrolysis. The ATPase assay was carried
out using PREDEASY ATPase Kit.
Effect of compound 55 on Cytochrome P3A4 (CYP3A4). The overlaped substrate
specificity between ABCB1 and CYP3A4 often generates unexpected
pharmacokinetic parameters when combining ABCB1 inhibitors with anticancer
agents. In order to identify whether compound 55 was a safe ABCB1 modulator at
effective dose, we evaluated the inhibition activity of compound 55 against CYP3A4.
The widely used specific CYP3A4 inhibitor Ketoconazole could notably inhibit the
activity of CYP3A4 in a doseꢀdependent pattern with IC 0.065±0.007 ꢁM. While,
5
0
compound 55 possessed a poor inhibitory effect against CYP3A4 with IC50 17.243±
.237 ꢁM, even poorer than Verapamil (IC50 8.193±0.913 ꢁM). And compounds 52
1
and 60 performed weaker activity against CYP3A4 (Figure S6) than 55. These data
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indicated that this series of compounds has the potential as leading compounds to
further develop safe ABCB1 inhibitors.
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Figure 8. Doseꢀresponse curves after different treatment: the canonical competitive CYP3A4
inhibitor Ketoconazole, positive reversal modulator of ABCB1ꢀmediated resistance Verapamil,
compound 55.
Cellular thermal shift assay (CETSA). As ABCC1 is the second most prevalent ABC
transporter in MDR, most ABCB1 inhibitors have been evaluated their inhibitory
48
activity against ABCC1 and usually exhibited potent inhibitory activity.
Furthermore, compounds containing pyrimidine scaffold have been proved as potent
4
9ꢀ52
dual inhibitors of ABCB1 and ABCC1.
Therefore, the binding affinity of 55 with
53
ABCB1 and ABCC1 was tested by CETSA, respectively. In brief, the lysate of
SW620/AD300 was incubated with compound 55, and then heated at different
temperatures. The target protein would maintain stable after heating once it was
bounded by compound compared to the DMSO control group. As shown in Figure 9,
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compound 55 efficiently could stabilize ABCB1 but not ABCC1 at higher
temperatures. This data suggested that the reversal activity of compound 55 may be
associated with its specific target to ABCB1.
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Figure 9. Compound 55 engaged toABCB1 (upper) and ABCC1 (lower) in SW620/AD300.
Vincristine-resistance reversal activity of compound 55 on ABCB1-overexpressed and
ABCC1-overexpressed cell lines. Considering the fact that drug selected cell line may
possess other drug resistance factors, we established the ABCB1 and ABCC1 selected
overexpressing cell system HEK293/ABCB1 and HEK293/ABCC1 to investigate the
predominant role of ABCB1 in reversal activity of compound 55.Correspondingly, we
also used other two drug selected cancer cell lines KBꢀC2 and KBꢀCV60
overꢀexpressed ABCB1 and ABCC1, respectively. What’s more, other than PTX, we
used another clinical antitumor drug vincristine, which is the common substrate for
ABCB1 and ABCC1. The data in Table 6 showed that compound 55 could reverse
vincristine resistance against both ABCB1ꢀoverexpressed cell lines, KBꢀC2 and
HEK293/ABCB1. While in ABCC1ꢀoverexpressed cell lines, KBꢀCV60 and
HEK293/ABCC1, compound 55 did not exhibit obviously reversal activity. These
findings indicated that ABCB1 may play an important and selective role in resistance
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reversal activity of compound 55.
Table 6. Vincristineꢀresistance reversal activity of compound 55 on
a
ABCB1ꢀoverexpressed and ABCC1ꢀoverexpressed cell lines.
1
1
1
1
1
1
1
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1
1
2
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2
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IC50 (ꢁM)
Treatment
KBꢀC2
RF
HEK293/ABCB1
RF
Vincristine
1.138±0.012 1.00
0.295±0.032 3.85
1.095±0.071
1.00
+
55 1 µM
0.112±0.030
0.051±0.016
9.75
+
Verapamil 3µM 0.064±0.018 17.92
21.52
IC50 (ꢁM)
Treatment
Vincristine
KBꢀCV60
RF
HEK/ABCC1
0.113±0.023
RF
0.249±0.050 1.00
0.221±0.025 1.13
1.00
+
55 1µM
0.099±0.019
0.029±0.009
1.14
3.90
+
MK571 25 µM 0.056±0.014 4.45
a
IC50 values were obtained from three independent repeats and represented as mean ± SD.
Xenograft Study. Based on the potent reversal activity of ABCB1ꢀmediated MDR, the
reversal activity in vivo was also examined after single or combined treatment of
compound 55 and PTX on xenograft model bearing ABCB1ꢀoverexpressed
SW620/AD300 cells by subcutaneous implantation. After the treatment of single or
combined treatment of compound 55 and PTX, the tumor weight and the tumor
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