Design, Synthesis, and Pharmacological Characterization of N-(4-(2 (6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)yl)ethyl)phenyl)quinazolin-4-amine Derivatives: Novel Inhibitors Reversing P-Glycoprotein-Mediated Multidrug Resistance

Article abstract of DOI:10.1021/acs.jmedchem.6b01787

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a principal obstacle for successful cancer chemotherapy. A novel P-gp inhibitor with a quinazoline scaffold, 12k, was considered to be the most promising for in-depth study. 12k possessed high potency (EC₅₀ = 57.9 ± 3.5 nM), low cytotoxicity, and long duration of activity in reversing doxorubicin (DOX) resistance in K562/A02 cells. 12k also boosted the potency of other MDR-related cytotoxic agents with different structures, increased accumulation of DOX, blocked P-gp-mediated Rh123 efflux, and suppressed P-gp ATPase activity in K562/A02 MDR cells. However, 12k did not have any effects on CYP3A4 activity or P-gp expression. In particular, 12k had a good half-life and oral bioavailability and displayed no influence on DOX metabolism to obviate the side effects closely related to increased plasma concentrations of cytotoxic agents in vivo.

Full text of DOI:10.1021/acs.jmedchem.6b01787

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Article
Design, Synthesis, and Pharmacological Characterization of N-
(4-(2 (6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)yl)ethyl)phenyl)
quinazolin-4-amine Derivatives: Novel Inhibitors
Reversing P-glycoprotein-Mediated Multidrug Resistance
Qianqian Qiu, Baomin Liu, Jian Cui, Zheng Li, Xin Deng, Hao Qiang, Jieming
Li, Chen Liao, Bo Zhang, Wei Shi, Miaobo Pan, Wenlong Huang, and Hai Qian
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01787 • Publication Date (Web): 29 Mar 2017
Downloaded from http://pubs.acs.org on March 29, 2017
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Design, Synthesis, and Pharmacological Characterization of N-(4-(2
(
6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)yl)ethyl)phenyl)
quinazolin-4-amine Derivatives: Novel Inhibitors Reversing
P-glycoprotein-Mediated Multidrug Resistance
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a,#
a,#
a
a
a
a
a
Qianqian Qiu , Baomin Liu , Jian Cui , Zheng Li , Xin Deng , Hao Qiang , Jieming Li , Chen
a
a
a
a
a,b,*
a,b,*
Liao , Bo Zhang , Wei Shi , Miaobo Pan , Wenlong Huang
and Hai Qian
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing, PR China
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing, PR China
ABSTRACT
Pꢀglycoprotein (Pꢀgp)ꢀmediated multidrug resistance (MDR) is a principal obstacle for successful
cancer chemotherapy. A novel Pꢀgp inhibitor with a quinazoline scaffold, 12k, was considered to
be the most promising for inꢀdepth study. 12k possessed high potency (EC50 = 57.9 ± 3.5 nM),
low cytotoxicity and long duration of activity in reversing doxorubicin (DOX) resistance in
K562/A02 cells. 12k also boosted the potency of other MDRꢀrelated cytotoxic agents with
different structures, increased accumulation of DOX, blocked Pꢀgpꢀmediated Rh123 efflux, and
suppressed Pꢀgp ATPase activity in K562/A02 MDR cells. However, 12k did not have any effects
on CYP3A4 activity or Pꢀgp expression. In particular, 12k had a good halfꢀlife and oral
bioavailability, and displayed no influence on DOX metabolism to obviate the sideꢀeffects closely
related to increased plasma concentrations of cytotoxic agents in vivo.
KEYWORDS: Pꢀglycoprotein; multidrug resistance; quinazoline; reversal activity
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INTRODUCTION
The development of multidrug resistance (MDR) to anticancer drugs is a leading cause of
1ꢀ3
failure for cancer treatment. Mechanisms involved in MDR have been characterized extensively
and shown to be quite complicated. Overexpression of Pꢀglycoprotein (Pꢀgp) is the most common
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mechanism of MDR.
Pꢀgp, also known as adenosine triphosphate (ATP) bindingꢀcassette
subfamily B member 1 (ABCB1), is encoded by MDR1 gene and extrudes various cytotoxic
agents out of cells by the energy of adenosine triphosphate (ATP) hydrolysis, resulting in low and
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, 7
ineffective intracellular drug concentrations and MDR to cancer cells.
Coꢀadministration of Pꢀgp modulators with anticancer drugs in the clinic has long been
recognized as a promising strategy to circumvent Pꢀgpꢀmediated MDR, and considerable efforts
8
have been made to develop three generations of Pꢀgp inhibitors over the past few decades. In
particular, thirdꢀgeneration Pꢀgp inhibitors comprise compounds such as WKꢀXꢀ34 (1), HM30181
(2) and tariquidar (XR9576, 3) with high affinity to Pꢀgp at a nanomolar concentration to acquire
9
ꢀ11
inhibition
of
Pꢀgp
function
specifically
and
effectively
(Figure
1A).
6
,7ꢀdimethoxyꢀ2ꢀphenethylꢀ1,2,3,4ꢀtetrahydroꢀisoquinoline is the most characteristic structure in
these excellent inhibitors, and has been considered to be the foremost active domain to have an
12, 13
efficient role in Pꢀgp inhibition.
However, a Pꢀgp inhibitor has not been approved for the
market due to a lack of significant clinical efficacy, pharmacokinetic interaction or concerns about
1
4
15, 16
its safety. Therefore, there is an urgent need to develop new agents to overcome MDR.
The quinazoline core, as a crucial aromatic heterocycle, is commonly present in various
tyrosine kinase inhibitors (TKIs), including gefitinib (4), lapatinib (5) and erlotinib (6), which
have been identified as substrates or modulators of Pꢀgp and other ABC transporters (Figure
17ꢀ19
1B).
In addition, some preꢀclinical studies and clinical trials are underway to evaluate the
coꢀadministration of anticancer drugs with these TKIs to conquer drug resistance and improve
therapeutic outcome in cancer patients. Quinazoline is thought to interact with the active region of
Pꢀgp, and 4ꢀanilinoꢀquinazolines have been reported to be inhibitors of breast cancer resistance
20
protein. Extensive efforts have been devoted to develop further bioactive molecules on the basis
of quinazoline fragment. Structural modifications of promising Pꢀgp inhibitors by introduction of
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quinazoline scaffolds is a convenient and rewarding method to exploit new Pꢀgp reversal agents
with strong bioactivity, outstanding affinity to Pꢀgp targets and high safety profile.
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Figure 1. A) Selected Pꢀgp inhibitors; B) TKIs with Pꢀgp inhibitory activity
In the present study, we designed and synthesized a series of 2ꢀarylquinazolines containing the
group 6,7ꢀdimethoxyꢀ2ꢀphenethylꢀ1,2,3,4ꢀtetrahydroꢀisoquinoline (12a–s) via substitution (Figure
2
). Then, we evaluated their effects on cell viability and MDR reversal activity in cells sensitive or
resistant to DOX. After preliminary biological evaluations, compound 12k, which showed the
most potency, was investigated further with regard to its potency and mechanism of reversing
Pꢀgpꢀmediated drug resistance.
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Figure 2. Design of novel target compounds
RESULTS AND DISCUSSION
Chemistry
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The intermediate 7a–s was readily prepared by the reaction of commercial substituted
ꢀaminobenzoic acid and aromatic acyl chloride. The synthesized 7a–s was heated in
NH OH/EtOH at 80°C for 12 h to afford quinazolinꢀ4(3H)ꢀones 8a–s, which were then reacted
2
4
with 10 eq. SOCl
conditions, commercial 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline hydrochloride and
ꢀ(2ꢀbromoethyl)ꢀ4ꢀnitrobenzene were reacted for 17 h to produce compound 10. Then, reduction
2
at 50°C for 6 h to produce the desired intermediate 9a–s. Under reflux
1
of compound 10 under hydrogen atmosphere with Pd/C as catalyst delivered compound 11.
Further nucleophilic reaction of compounds 9a–s and 11 gave target compounds 12a–s. Structures
of all target compounds are presented in Table 1. All structures of the new compounds were
1
13
confirmed by H NMR, C NMR, HRMS and IR spectra. Moreover, the purity of target
compounds 12a–s was ascertained by ultra high performance liquid chromatography (UHPLC).
Results demonstrated that all compounds had a purity of ≥ 95% (Experimental Section).
a
Scheme 1. Synthesis of target compounds 12a-s
a
Reagents and conditions: (i) R
, 50°C, 6 h; (iv) 1ꢀ(2ꢀbromoethyl)ꢀ4ꢀnitrobenzene, K
Pd/C, EtOH/CH Cl , r.t., 24 h; (vi) CH SO H, EtOH, reflux.
2
COCl, pyridine, r.t., 6 h; (ii) NH
4
OH/EtOH, 80°C, 12 h; (iii) 10
eq. SOCl
2
2
CO
3
, CH CN, reflux, 17 h; (v) H ,
3
2
2
2
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3
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Table 1 Structures of target compounds
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1
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compound
R
R
compound
12k
R
R
12a
H
H
12b
H
12l
H
12c
H
H
12m
12n
H
H
12d
12e
H
H
12o
12p
H
H
12f
12g
H
H
H
12q
12r
12s
H
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2h
2i
7ꢀCl
1
6,7ꢀdiOCH
3
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12j
H
Biological evaluation
Effects of target compounds on cell viability
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Before evaluation of MDR reversal potency, the effects of these compounds at 1.0 ꢁM on the
viability of sensitive K562 cells and its DOXꢀresistant K562/A02 cells with overexpressed Pꢀgp
were determined preliminarily by the MTT assay. Anticancer drug DOX and Pꢀgp inhibitor
verapamil (VRP) were served as controls. As present in Table 2, 19 target compounds showed
hardly any toxicity toward these two cell lines. Survival of K562 cells using 5.0 ꢁM DOX was
only 8.8%, whereas toxicity of 5.0 ꢁM DOX towards K562/A02 cells was inferior to 4 %.
a
Table 2 Survival rate of target compounds at 1.0 ꢁM toward K562 and K562/A02 cell (%)
Compound
K562
K562/A02 Compound
K562
K562/A02
92.1 ± 1.5
> 99.0
1
1
2a
2b
> 99.0
12m
12n
12o
12p
12q
12r
12s
98.7 ± 1.5
> 99.0
> 99.0
>
>
>
99.0
99.0
99.0
> 99.0
12c
12d
12e
94.7 ± 2.1
> 99.0
> 99.0
> 99.0
85.2 ± 0.8
> 99.0
95.1 ± 1.6 > 99.0
> 99.0
> 99.0
1
1
1
1
2f
> 99.0
> 99.0
96.5 ± 2.1
95.3 ± 1.3
97.3 ± 3.2
> 99.0
2g
2h
2i
> 99.0
91.4 ± 3.0 89.2 ± 4.2
92.9 ± 1.8 92.1 ± 2.2
1
97.5 ± 3.0
> 99.0
93.4 ± 0.5 94.2 ± 1.3 5.0 ꢁM VRP
88.3 ± 5.1
1
2j
> 99.0
91.3 ± 1.0 5.0 ꢁM DOX 8.8 ± 1.1
96.4 ± 3.2
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2k
96.5 ± 2.2 > 99.0
Control
> 99.0
> 99.0
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> 99.0
97.9 ± 0.7
a
Survival rates of target compounds toward cells were determined by MTT assay and data are
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presented as mean ± SD for three independent tests. 0.1% DMSO was used as solvent control.
Reversal of DOX resistance
Based on survival results, a concentration of 1.0 µM was used to investigate the effects of 19
target compounds on reversal of resistance to DOX in K562/A02 cells. Data on the reversal
activity of all compounds are depicted in Table 3. DOX alone demonstrated little toxic effect on
K562/A02 cells (halfꢀmaximal inhibitory concentration IC50 = 89.8 ± 4.52 µM). However,
coꢀadministration of DOX and target compounds or VRP led to an increase of toxic effects on
K562/A02 cells to different extents, suggesting that all test compounds could reverse resistance to
DOX.
First, 12a with unsubstituted phenyl increased reversal activity considerably from 4.2ꢀ
reversal fold (RF) of the positive control VRP to 7.7ꢀRF. The para positionꢀsubstituted
compounds 12d, i, j, o, p and q, irrespective of whether they had an electronꢀwithdrawing or
ꢀ
donating group, displayed similar or lower reversal activity compared with 12a. In particular, 12j,
o and q were inferior to VRP. Interestingly, the ortho and meta positionꢀsubstituted 12b, c, h and
n exhibited increased activity at different extents. Furthermore, the effects of multiple substitution
with methoxy groups at the phenyl moiety were investigated. Results suggested that disubstitution
with methoxy groups contributed greatly to the increase in reversal activity (12e, and g), whereas
a positive effect was not observed from trisubstitution with methoxy groups (12f), perhaps due to
steric hindrance. To explore this reversal activity further, we synthesized compounds containing
2
1
R replaced with nitrogen heterocycles and R substituted with electronꢀwithdrawing or ꢀdonating
groups, most of which exhibited no or minor benefit to reversal activity (12l, m, r and s), except
12k. Surprisingly, 1.0 ꢁM 12k presented more remarkable MDR reversal activity than 5.0 ꢁM
VRP, with much higher RF than 1.
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Some factors related to the activity of a substance can be derived from these results. The
electronic effect (electronꢀwithdrawing and electronꢀdonating) of the substituents at the phenyl
moiety as well as steric requirements plays a minor part. Substitution at meta and ortho positions
yielded high activities. However, a distinct advantage for disubstitution with methoxy substituents
was found, whereas trisubstitution was a disadvantage. Simultaneously, a quinazoline moiety with
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nitrogen heterocycles made a distinct difference in reversal activity. The 4ꢀpyridine moiety (12k)
had dramatic capacity against Pꢀgp, and had the most potential. After reversal evaluation of all
target compounds, a clear SAR picture was depicted in Figure 3.
Table 3 DOXꢀresistance reversal activity of target compounds at 1.0 µM concentration in
a
K562/A02 cells
.
Compound IC /DOX (µM) RF
Compound
12l
IC /DOX (µM) RF
50
5
0
12a
11.66 ± 1.04
8.24 ± 0.78
6.15 ± 0.69
11.37 ± 1.31
5.22 ± 0.41
14.25 ± 1.30
5.79 ± 0.41
10.09 ± 1.02
11.97 ± 1.22
7.7
15.22 ± 2.21
34.54 ± 3.72
6.70 ± 0.31
29.93 ± 3.22
14.97 ± 1.38
27.21 ± 2.74
10.95 ± 1.70
8.89 ± 0.65
4.83 ± 0.71
5.9
12b
10.9
14.6
7.9
12m
12n
2.6
12c
13.4
3.0
12d
12o
12e
17.2
6.3
12p
6.0
12f
12q
3.3
12g
15.5
8.9
12r
8.2
12h
12s
10.1
18.6
1
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1
2i
7.5
1
2j
2k
47.26 ± 4.21
1.9
5.0 ꢁM VRP 21.38 ± 3.71
4.2
1.0
b
2.91 ± 0.12
30.9
Control
89.80 ± 4.52
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a
The IC50 value was determined after exposure to a series of DOX concentration with different
target compounds at 1.0 µM using K562/A02 cells; Reversal fold (RF, foldꢀchange in drug
b
sensitivity) = (IC50 without inhibitor / (IC50 with inhibitor). 0.1% DMSO was added as solvent
control. Data were analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for
three independent tests.
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Figure 3. SARs summary of derivatives with quinazoline scaffolds.
Dose–response relationship and selective index (SI) of 12k
Looking forward the quantitative efficacy of the most promising compound 12k to reverse
DOXꢀresistance, EC50 value was determined in K562/A02 cells. EC50 refers to the concentration
of inhibitor required to reduce the IC50 of DOX by half compared with a control without a
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modulator. Compound 12k had an EC50 of 57.9 ± 3.5 nM for reversing DOX resistance in
K562/A02 cells (Figure 4A).
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Figure 4. (A) EC50 value of 12k in lowering DOXꢀresistance in K562/A02 cells. The percent of
IC50 of DOX was plotted against log concentration of 12k. The percent of IC50 of DOX = [(IC50 of
DOX at each modulator concentration/ IC50 of DOX without modulator) × 100%. EC50 is defined
as the concentration of modulator that can reduce the % of IC50 of DOX by half; (B) IC50 values of
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12k and DOX in inhibition of K562, K562/A02 and HUVEC cell lines determined by MTT
method. The inhibition was plotted against log concentration of 12k and DOX. Each date point is
presented as mean ± SD for three independent tests.
To confirm that the increased cytotoxicity with compound 12k shown in Table 3 and Figure 4A
was due to modulation and not due to the cytotoxicity of 12k itself, the IC50 values were
determined for K562 cells and K562/A02 cells. DOX showed a high toxic effect on K562 cells
(IC50
= 0.62 ± 0.07 ꢁM) and low toxic effect on K562/A02 cells (IC50 = 89.80 ± 4.52 ꢁM)
(
Figure 4B), and K562/A02 cells displayed ≈144.8ꢀfold greater resistance than K562 cells. IC50
values for compound 12k to K562 cells and K562/A02 cells were 4.00 ꢁM and 12.78 ꢁM,
respectively, which were 75.6ꢀ and 241.6ꢀfold of the EC50 for compound 12k in reversing DOX
resistance in K562/A02 cells, respectively. Results of the cytotoxicity assay suggested that the
enhanced cytotoxicity seen in Table 3 and Figure 4A was a result of the modulatory effect of
compound 12k. The toxicity of 12k against human umbilical vein endothelial cells (HUVECs)
was determined to evaluate its safety: this compound showed no toxic effects towards HUVECs
(IC50 = 73.58 ± 2.32 ꢁM). The SI (ratio of IC50 towards HUVECs to EC50 for reversing DOX
resistance in K562/A02 cells) for 12k was very high, up to 1270.8, suggesting that 12k may be a
safe MDR modulator for normal human cells when coꢀadministrated with anticancer agents.
Effect of compound 12k on reversing P-gp-mediated resistance to other
anticancer drugs
Resistance to other chemotherapeutic drugs with diverse structures and mechanisms of action
is a common phenomenon in MDR. Therefore, whether 12k can also reverse Pꢀgpꢀmediated
resistance to other Pꢀgp substrates including paclitaxel (PTX), vinblastine (VLB) and
daunorubicin (DNR) was evaluated. A nonꢀMDR anticancer drug, cyclophosphamide (CTX), was
also selected to verify the reversal potency of compound 12k on Pꢀgp inhibition. Concentrations
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of anticancer drugs were set to 0, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 ꢁM (Figure 5). When
coꢀadministered with 1.0 ꢁM 12k in K562/A02 cells, enhanced toxicity of PTX, VLB and DNR
was observed. However, reversal activity was not demonstrated when CTX was coꢀadministered
with 1.0 ꢁM 12k in K562/A02 cells. The results indicated that inhibition of the Pꢀgp function of
compound 12k was responsible for the reversal of resistance.
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Figure 5. Effect of compound 12k on reversing other anticancer drugs resistance in K562/A02
cells: A. DNR; B. PTX; C. VLB; D. CTX. DNR, PTX, and VLB are Pꢀgp substrates, whereas
CTX is not. Each data point is presented as mean ± SD for three independent tests.
Duration of drug effect
To evaluate the duration of action of compound 12k as a modulator, K562/A02 cells were
incubated with 1.0 ꢁM compound 12k for 24 h followed by removal of the modulator by washing
and incubation for various times in the absence of the reversal agent before DOX addition. VRP
(5.0 ꢁM) and phosphateꢀbuffered saline were selected as comparators in this experiment, which
9
, 22
was carried out according to a previous report.
5.0 ꢁM VRP and 1.0 ꢁM 12k displayed strong
MDR reversal activity in resistant K562/A02 cells (noꢀwash group) (Figure 5). The
MDRꢀreversing potency of VRP decreased immediately after its removal from the medium. After
its removal for 6 h, a MDRꢀreversing effect was not observed, indicating that the reversal potency
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of VRP lasted ≤6 h. In contrast, the MDR reversal effect of compound 12k was remarkable
immediately after its removal. Moreover, 12k showed effective reversal activity 24 h after its
removal from the medium. These results also demonstrated that the MDRꢀreversing potency of
1
2k persisted even after 24ꢀh washout.
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Figure 6. Duration of reversal effect of 12k and VRP toward DOX in K562/A02 cells after
incubation and subsequent washout. Cell viability was determined by MTT assay. Data represents
means ± SD of triplicate determinations.
Effect of 12k on DOX accumulation
Due to a defect in accumulation of cytotoxic moieties caused by Pꢀgp, the reversal ability of 12k
correlated to a concomitant increase in DOX accumulation was explored. The autoꢀfluorescence
property of DOX allows an accumulation assay by spectrofluorometry to be carried out. DOX
accumulation in K562 cells was about 5.46ꢀfold (P < 0.001) higher than that of K562/A02 cells in
the absence of a modulator (Figure 7), which was due to Pꢀgpꢀmediated DOX efflux. Even at
modulator concentrations down to 0.1 ꢁM, 12k and 1 enhanced 2.5ꢀh accumulation of DOX into
K562/A02 cells significantly (P < 0.05), whereas VRP enhanced DOX accumulation only if
present at 5.0 ꢁM. These data suggested that 12k exhibited greater potency in increasing DOX
accumulation into K562/A02 cells than VRP, and was also superior to the positive control 1.
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Figure 7. Effect of compound 12k on intracellular DOX accumulation in K562/A02 cells, with
0
.1% DMSO as negative control. The results are presented as the mean ± SD for three
independent experiments; (***) P < 0.001, (**) P < 0.01, (*) P < 0.05 relative to the negative
control (K562/A02).
Inhibitory effect of 12k on P-gp-mediated Rh123 efflux
It is well known that the potency of MDR modulators on Pꢀgp efflux employs Pꢀgpꢀdependent
2
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efflux of the fluorescent dye Rh123 via flow cytometry to evaluate. As demonstrated in Figure 8,
1.0 ꢁM 12k could inhibit Rh123 efflux from K562/A02 cells significantly compared with the
control (P < 0.05). Moreover, at different time points of 5, 10, 25, 30, 60 and 90 min, intracellular
Rh123ꢀassociated mean fluorescence intensity (MFI) in 1.0 ꢁM 12kꢀtreated cells was superior to
that in 5.0 ꢁM VRPꢀ and 1.0 ꢁM 1ꢀtreated cells, suggesting that the inhibitory activity of 12k was
more powerful than that of positive controls. These consequences demonstrated 12k can reverse
MDR by inhibiting Pꢀgpꢀmediated drug efflux.
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Figure 8. Effect of 12k on efflux of Rh123 from K562/A02, with 0.1% DMSO as negative control.
Each data point is presented as mean ± SD for three independent experiments.
Effect of 12k on P-gp-ATPase activity
In further evaluation, the effect of 12k on PꢀgpꢀATPase activity was also determined. VRP is not
only an inhibitor but a substrate of Pꢀgp, which is proved to be also a stimulator of PꢀgpꢀATPase.
PꢀgpꢀATPase activity was strongly increased over the basal level by 2.6ꢀfold by 50.0 ꢁM VRP
(
Figure 8). However, 12k and 1 exerted inhibition of PꢀgpꢀATPase even at 0.5 ꢁM, which was
obviously lower than the basal level (Figure 9), suggesting that similar to 1, compound 12k could
inhibit the activity of Pꢀgp ATPase.
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Figure 9. Effect of 12k on PꢀgpꢀATPase activity. Sodium vanadateꢀinhibitable ATPase (Pꢀgp
ATPase) was studied as described in Experimental Section. Pꢀgp ATPase was measured in the
absence (basal activity) or presence of Pꢀgp modulators (VRP, 12k and 1). Results are presented
as the mean ± SD for three independent experiments.
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Effect of 12k on P-gp expression
The presence of Pꢀgp protein was proved by a band with a molecular weight of ≈170 kDa in
K562/A02 cell lysates by western blotting. However, it was not present in parental K562 cells,
suggesting the absence of Pꢀgp protein (Figure 10). Inhibiting the function or lowering expression
of Pꢀgp will contribute to the reversal of Pꢀgpꢀmediated MDR. Therefore, whether the reversal
ability of 12k was due to a decrease in protein expression should be confirmed. Firstly, the cells
were incubated with 12k (0.1, 0.5, or 2.0 ꢁM). Subsequently, the Pꢀgp expression in the cell
lysates were determined respectively. There was no evident alteration in Pꢀgp expression in
K562/A02 cells (Figure 10), indicating that MDR reversal by 12k was not caused by a decreased
protein expression but instead most likely due to direct inhibition of Pꢀgp efflux.
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Figure 10. Western blot analysis indicating the expression of ABCB1 after exposure to 0.1, 0.5, or
2.0 ꢁM 12k. (A) Effect of 12k at 0.1, 0.5, or 2.0 ꢁM on expression level of ABCB1 in K562/A02
cells for 72 h. (B) Band intensity was analyzed by Quantity One software and protein expression
was presented as the ratio of target protein's band intensity to that of βꢀTubulin. Representative
result is shown here and similar results were obtained in two other independent trials.
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Effect of 12k on cytochrome P3A4 (CYP3A4)
There is an impressive overlap between Pꢀgp and CYP3A4 with regard to the characteristics of
substrate and tissue distribution. This overlap suggests the presence of several Pꢀgp inhibitors as
substrates for CYP3A4 with unexpected pharmacokinetic interactions with chemotherapeutics. To
investigate further the characteristics of 12k as a safe Pꢀgp inhibitor, its in vitro inhibitory effect
was determined on rat liver CYP3A4 (Figure 11). A specific inhibitor of CYP3A4, ketoconazole
inhibited CYP3A4 activity in a concentrationꢀdependent manner, whereas 12k has no effect on
CYP3A4 activity even at 40 µM, which was much higher than the EC50 in vitro. These results
demonstrated that 12k may be a relatively safe Pꢀgp modulator even if coꢀadministered with
chemotherapeutics metabolized by CYP3A4.
Figure 11. The effect of 12k and ketoconazole on CYP3A4. Ketoconazole, a specific inhibitor
of CYP3A4, was the positive control. 6βꢀOH testosterone (6βꢀOHT) is the specific product of
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testosterone metabolized by CYP3A4. The data represent the mean ± SD for three independent
experiments.
Pharmacokinetics of 12k in vivo
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To measure the pharmacokinetics of 12k in vivo, three SD rats in each group were selected to be
given 12k via oral or intravenous routes. The standard curve for 12k was y = 0.0045x + 0.0086
2
with R = 0.999. Main pharmacokinetic parameters were calculated using Phoenix WinNonlin
v6.3 (Certara, Princton, NJ, USA) and are presented in Table 4. The peak concentration of 12k in
plasma was 569.6 ± 83.5 ng/mL (1100 ± 161.5 nM), much higher than the EC50 value in
K562/A02 cells. The halfꢀlife (T1/2) for 12k in the intravenous group was 5.9 ± 0.3 h, which was
an appropriate duration and much longer than that of the free DOX group (T1/2 = 1.6
± 0.2 h). In
addition, 12k had good bioavailability (≤95.3%).
Table 4 Pharmacokinetic parameters of 12k (i.v. dose of 5 mg/kg, p.o. of 10 mg/kg) in the plasma
a
of male Sprague–Dawley rats.
Parameter
I.V. (5mg/kg)
1655 ± 151
1754 ± 173
5.9 ± 0.3
P.O. (10mg/kg)
3131 ± 94
−1
AUC(0–24) (ng·mL ·h)
−1
AUC(0–∞) (ng·mL ·h)
3344 ± 119
6.2 ± 0.7
T1/2 (h)
–
1
Cmax (ng·mL )
569.6 ± 83.5
47.8 ± 2.0
761.2 ± 63.0
49.9 ± 1.8
ꢀ
1
ꢀ1
CL (ml.min kg )
F (%)
95.3
a
Note: Data are shown as the mean ± SD (n = 3). AUC, area under the curve from zero to 24h or
infinity; T , elimination halfꢀlife; Cmax, maximum plasma concentration; CL, plasma clearance; F
1
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(bioavailability) = (AUCpo/AUCiv)*(doseiv/dosepo) *100%
Pharmacokinetics of DOX in rats treated with 12k
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In a further study of the effect of 12k on DOX metabolism, the rat group coꢀadministered VRP
and DOX showed an increased plasma concentration of DOX compared with the freeꢀDOX group,
suggesting an elevated risk of anthracyclineꢀinduced cardiotoxicity. The rat group administered
DOX combined with 12k or 1 had an almost identical metabolism as the group administered free
DOX (Figure 12). The MDR inhibitor 12k showed potent MDR reversal activity in vitro, but also
reached the necessary plasma concentration without drug interactions. Other data also suggested
that pharmacokinetic indices (T , plasma clearance, mean residence time; data not shown) were
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not significantly different from that of the freeꢀDOX group in vivo. These results suggested that
2k application could obviate the sideꢀeffects closely related to increased plasma concentrations
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of cytotoxic agents in vivo.
Figure 12. Plasma concentration–time profiles for DOX for the four groups of male Sprague–
Dawley rats: group A (free DOX, i.v. dose of 10 mg/kg body mass); group B (DOX + VRP, i.v.
dose of 10 mg/kg DOX and 5 mg/kg VRP); group C (DOX + 1, i.v. dose of 10 mg/kg DOX and 5
mg/kg 1); and group D (DOX + 12k, i.v. dose of 10 mg/kg DOX and 5 mg/kg 12k). GraphPad
Prism 5.0 software was used for data analysis and the data points are presented as the mean ± SD
for three independent tests.
CONCLUSIONS
In this study, a series of novel Pꢀgpꢀmediated MDR modulators with quinazoline scaffolds were
designed and synthesized via a convenient procedure. All the structures of the novel compounds
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were characterized by NMR, HRMS, and IR spectra. Biological evaluation in vitro demonstrated
that a few compounds exhibited potent MDR reversal activity. In particular, 12k exhibited
outstanding activity against the Pꢀgpꢀoverexpressing daughter line K562/A02 with a low EC50
(57.9 ± 3.5 nM).
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This compound showed high survival towards normal cells (IC50 = 73.58 ± 2.32 ꢁM), suggesting
that the SI of 12k was very high (1270.8). 12k also exhibited reversal effects on the other
chemotherapeutic drugs that may have different structures and mechanisms of action, such as Pꢀgp
substrates (PTX, VLB, DNR) but had no effect on the nonꢀPꢀgp substrate CTX. The duration of
action of 12k lasted even after 24ꢀh washout.
12k exhibited remarkable potency not in increasing accumulation of DOX into K562/A02 cells,
but in inhibiting Pꢀgpꢀmediated drug efflux of Rh123. Also, 12k (different from VRP) contributed
to inhibition of PꢀgpꢀATPase, which accounted (at least in part) for the mechanism action of MDR
reversal. Westernꢀblotting suggested that MDR reversal by 12k was not due to a decrease in
protein expression. More importantly, 12k did not affect CYP3A4 activity even at high
concentrations, and avoided pharmacokinetic interactions in vitro. A followꢀup inꢀdepth study in
vivo demonstrated 12k to have superior pharmacokinetic profiles and not to be susceptible to
DOX metabolism. In conclusion, 12k holds potential for reversing Pꢀgp mediated MDR.
EXPERIMENTAL SECTION
General chemistry
All starting materials, reagents and solvents were obtained from commercial sources and used
without further purification unless otherwise indicated. Purifications by column chromatography
were carried out over silica gel (200ꢀ300 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 taken on a RYꢀ1 meltingꢀpoint apparatus and were uncorrected. NMR spectra
1
13
6
were recorded in DMSOꢀd on a Bruker ACFꢀ300Q instrument (300 MHz for H, 75 MHz for C;
Bruker Instruments, Inc., Billerica, MA, USA), chemical shifts are expressed as values (ppm)
relative to tetramethylsilane as 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 =
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multiplet, dd = doublets of doublet, br = broad. Infrared spectras were recorded on a
PerkinꢀElmer FTIR instrument. ESIꢀMS datas were recorded with Waters ACQUITY UPLC
Systems with Mass (Waters, Milford, MA). High Resolution Mass measurement was performed
on Agilent QꢀTOF 6520 mass spectrometer with electron spray ionization (ESI) as the ion source.
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The intermediate 7a-s , 8a-s , 9a-s , 10 and 11 were prepared the following reported
procedures (supporting information).
Purities of Target Compounds
The purity of all target compounds was established by UHPLC of Shimadzu LCꢀ30A
(
2 3
SHIMADZU, Japan). The LC was operated as follows: solvent A, 10mM aqueous NaH PO ;
solvent B, methanol; column, Shimꢀpack XRꢀODS II (2.2 ꢁm) 100 * 2.0 mm (SHIMADZU,
Japan); column temperature, 40 °C; flow rate, 0.4 mL/min; gradient program, 0ꢀ5 min: %B =
30ꢀ95 gradient, 5ꢀ6.5 min: %B = 95, 6.5ꢀ7.5 min: %B = 95ꢀ30 gradient, 7.5ꢀ10 min: %B = 30. UV
signals were recorded at 254 nm. The content results of compounds 12a-s were intelligently
calculated with the LabSolution. The purities of all target compounds were >95% for biological
evaluation.
General Procedure for the Synthesis of 12a-s
The mixtures of compounds 9a-s (1 mmol), 11 (1 mmol) and methanesulfonic acid (0.1 mmol) in
ethanol (10 ml) were heated to reflux for 4 h. Then the reaction solutions were cooled to room
temperature and filtered to give a yellow solid. The resulting solid was added into the 1 N NaOH
solution (15 ml) followed by strong stirring for 1 h, and then organic compounds were extracted
with DCM (20 ml × 2). Organic layers were washed with water (20 ml), brine (20 ml) respectively
2 4
and dried over Na SO . The solvents were filtered and evaporated under reduced pressure to give
the crudes. The final compounds 12a-s was recrystallized from ethanol.
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-phenylquinazolin-4-a
mine (12a).
1
Yellow solid; Yield 52.5%; m.p. 88ꢀ90°C ; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.82 (s, 1 H, NH),
8
.57 (d, J = 8.4 Hz, 1 H, ArH), 8.46ꢀ8.43 (m, 2 H, ArH), 7.91ꢀ7.85 (m, 4 H, ArH), 7.51ꢀ7.49 (m, 3
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H, ArH), 7.37ꢀ7.34 (m, 3 H, ArH), 6.66, 6.64 (2s, 2 H, ArH), 3.69 (s, 6 H, 2 × OCH ), 3.57 (s, 2 H,
3
1
3
ArCH
2
N), 2.89ꢀ2.72 (m, 8 H, 4 × CH
37.15, 135.80, 133.11, 130.21, 128.62, 128.35, 128.06, 127.84, 126.65, 125.92, 125.85, 122.94,
22.07, 113.97, 111.73, 109.9, 59.54, 55.42, 55.09, 50.55, 32.47, 28.31; HRMS (ESI) m/z calcd
2 6
); C NMR (75 MHz, DMSOꢀd ) δ: 157.82, 150.41, 138.32,
1
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
ꢀ1
for [C33H32N4O2+H] 517.2598, found 517.2588；IR (KBr，cm ) ν：2932.06, 1560.51, 1515.26,
1416.87, 1358.35, 1255.17, 1227.06, 1227.96, 760.47, 709.58. Purity 96.106 %; t 4.128 min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(2-methoxyphenyl)
quinazolin-4-amine (12b).
1
Yellow solid; Yield 43.7%; m.p. 107ꢀ109°C ; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.73 (s, 1 H, NH),
8
7
7
3
1
1
.57 (d, J = 8.1 Hz, 1 H, ArH), 7.94ꢀ7.79 (m, 4 H, ArH), 7.64ꢀ7.59 (m, 2 H, ArH), 7.42 (dd, J =
.5, 1.7 Hz, 1 H, ArH), 7.24 (d, J = 8.4 Hz, 2 H, AH), 7.14 (d, J = 8.4 Hz, 1 H, ArH), 7.03 (dd, J =
.5, 7.5 Hz, 1 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 3.80 (s, 3 H, OCH
3
), 3.73 (s, 6 H, 2 × OCH
3
),
13
.53 (s, 2 H, ArCH
2
N), 2.80ꢀ2.49 (m, 8 H, 4 × CH
2
); C NMR (75 MHz, DMSOꢀd
6
) δ: 157.06,
46.83, 137.3, 135.3,, 132.8, 130.8, 130.1, 128.4, 127.9, 126.6, 125.8, 122.7, 121.6, 119.9, 113.4,
11.9, 111.7, 109.9, 59.59, 55.49, 55.42, 55.03, 50.55, 32.43, 28.27; HRMS (ESI) m/z calcd for
+
ꢀ1
[
C34H34N4O3+H] 547.2704, found 547.2699；IR (KBr, cm ) ν: 2832.44, 1245.63, 1601.30,
516.81, 1463.32, 754.71. Purity 98.180 %; t 4.013 min.
1
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(3-methoxyphenyl)
quinazolin-4-amine (12c).
1
Yellow solid; Yield 60.6%; m.p.76ꢀ77°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.83 (s, 1 H, NH),
8.56 (d, J = 7.8 Hz, 1 H, ArH), 8.05ꢀ8.01 (m, 2 H, ArH), 7.89ꢀ7.84 (m, 4 H, ArH), 7.62ꢀ7.59 (m, 1
H, ArH), 7.42ꢀ7.31 (m, 3 H, ArH), 7.06ꢀ7.04 (m, 1 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 3.82 (s, 3
1
3
H, OCH
3
), 3.68 (s, 6 H, 2 × OCH
) δ: 159.3, 158.7, 157.7, 150.4, 147.1, 146.8, 139.8, 137.1, 135.8, 133.0,
29.3, 128.5, 128.0, 126.6, 125.9, 125.8, 122.9, 122.2, 120.2, 116.2, 114.0, 112.6, 111.7, 110.0,
3
), 3.55 (s, 2 H, ArCH
2
N), 2.85ꢀ2.49 (m, 8 H, 4 × CH
2
);
C
NMR (75 MHz, DMSOꢀd
6
1
5
+
9.5, 55.4, 55.0, 54.9, 50.5, 32.4, 28.2; HRMS (ESI) m/z calcd for [C34H34N4O3+H] 547.2704,
ꢀ1
found 547.2691; IR (KBr, cm ) ν：3356.93, 2837.76, 1596.32, 1560.54, 1515.47, 1463.21,
254.07, 1127.30, 762.07, 737.73. Purity 98.480 %; t 4.051 min.
1
r
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N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(4-methoxyphenyl)
quinazolin-4-amine (12d).
1
Yellow solid; Yield 63.2%; m.p. 125ꢀ127°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.77 (s, 1 H, NH),
8
.54 (d, J = 8.4 Hz, 1 H, ArH), 8.41 (d, J = 8.7 Hz, 2 H, ArH), 7.89 (d, J = 8.7 Hz, 2 H, ArH), 7.82
d, J = 3.8 Hz, 2 H, ArH), 7.57ꢀ7.52 (m, 1 H, ArH), 7.32 (d, J = 8.4 Hz, 2 H, ArH), 7.04 (d, J = 8.9
Hz, 2 H, ArH), 6.63, 6.61 (2s, 2 H, ArH), 3.82 (s, 3 H, OCH ), 3.68 (s, 6 H, 2 × OCH ), 3.53 (s, 2
); C NMR (75 MHz, DMSOꢀd ) δ: 161.10, 158.89,
57.67, 150.54, 147.10, 146.86, 137.20, 129.49, 128.57, 127.85, 126.65, 125.91, 125.30, 122.92,
21.99, 113.68, 111.73, 109.96, 59.52, 55.44, 55.40, 55.19, 55.08, 50.51, 32.48, 28.30; HRMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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7
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9
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7
8
9
0
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2
3
4
5
6
7
8
9
0
(
3
3
1
3
H, ArCH
2
N), 2.85ꢀ2.49 (m, 8 H, 4 × CH
2
6
1
1
+
ꢀ1
(
ESI) m/z calcd for [C34H34N4O3+H] 547.2704, found 547.2703；IR (KBr，cm ) ν：3309.73,
2825.96, 1606.34, 1572.40, 1515.62, 1452.92, 1228.80, 1170.10, 697.67. Purity 98.419 %; t
r
4
.089 min.
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(3,4-dimethoxyphenyl)
quinazolin-4-amine (12e).
1
Yellow solid; Yield 56.2%; m.p.158ꢀ160°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.79 (s, 1 H, NH),
8
.54 (d, J = 8.4 Hz, 1 H, ArH), 8.06ꢀ8.03 (m, 2 H, ArH), 7.79 (d, J = 8.4 Hz, 2 H, ArH), 7.82 (d, J
= 3.9 Hz, 2 H, ArH), 7.57ꢀ7.53 (m, 1 H, ArH), 7.34 (d, J = 8.4 Hz, 2 H, ArH), 7.07 (d, J = 8.7 Hz,
2 H, ArH), 6.66, 6.64 (2s, 2 H, ArH), 4.04, 4.01 (2s, 6 H, 2 × OCH ), 3.85ꢀ3.83 (2s, 6 H, 2 ×
3
1
3
OCH ), 3.56 (s, 2 H, ArCH N), 2.85ꢀ2.49 (m, 8 H, 4 × CH ); C NMR (75 MHz, DMSOꢀd ) δ:
3
2
2
6
158.76, 157.58, 150.74, 148.35, 147.11, 146.87, 137.17, 135.79, 132.98, 130.93, 128.46, 127.86,
126.63, 125.90, 125.31, 122.93, 122.26, 120.92, 113.70, 111.75, 110.97, 109.96, 59.63, 55.50,
+
5
5.42, 55.11, 50.55, 32.46, 28.28; HRMS (ESI) m/z calcd for [C35H36N4O4+H] 577.2809,
ꢀ1
found 577.2806; IR (KBr, cm ) ν: 3380.53, 2837.76, 1593.37, 1570.14, 1516.67, 1463.43,
1
235.73, 1177.90, 764.32. Purity 96.705 %; t 3.957 min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(3,4,5-trimethoxyphen
yl)quinazolin-4-amine (12f).
1
Pale yellow solid; Yield 48.4%; m.p. 174ꢀ176°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.86 (s, 1 H,
NH), 8.54 (d, J = 8.4 Hz, 1 H, ArH), 7.89ꢀ7.81 (m, 6 H, ArH), 7.61ꢀ7.56 (m, 1 H, ArH), 7.33 (d, J
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= 8.7 Hz, 2 H, ArH), 6.66, 6.64 (2s, 2 H, ArH), 3.87 (s, 6 H, 2 × OCH ), 3.73, 3.70 (2s, 9 H, 3 ×
3
1
3
OCH
3
), 3.55 (s, 2 H, ArCH
2 2 6
N), 2.87ꢀ2.68 (m, 8 H, 4 × CH ); C NMR (75 MHz, DMSOꢀd ) δ:
1
1
5
58.37, 157.59, 152.67, 147.11, 146.86, 139.42, 137.06, 135.98, 133.61, 128.38, 127.99, 126.62,
25.89, 125.63, 122.94, 122.52, 113.78, 111.75, 109.97, 104.97, 60.04, 59.68, 55.57, 55.44, 55.08,
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46
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48
49
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55
56
57
58
59
60
+
0.55, 32.46, 28.27; HRMS (ESI) m/z calcd for [C36H38N4O5+Na] 629.2734, found 629.2739;
ꢀ
1
IR (KBr, cm ) ν：3374.06, 2833.53, 1591.22, 1572.10, 1515,52, 1461.03, 1222.36, 1124.83,
7
41.71, 766.49. Purity 98.106 %; t 3.875min.
r
2
-(benzo[d][1,3]dioxol-5-yl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phe
nyl)quinazolin-4-amine (12g)
Pale yellow solid; Yield 57.1%; m.p.136ꢀ139°C; H NMR (300 MHz, DMSOꢀd
NH), 8.57 (d, J = 8.2 Hz, 1 H, ArH), 8.04 (dd, J = 8.2, 1.6 Hz, 1 H, ArH), 7.89ꢀ7.86 (m, 3 H, ArH),
1
6
) δ: 9.86 (s, 1 H,
7
1
3
1
1
.81ꢀ7.93 (m, 2 H, ArH), 7.59ꢀ7.53 (m, 1 H, ArH), 7.35 (d, J = 8.4 Hz, 2 H, ArH), 7.03 (d, J = 8.2,
.6 Hz, 1 H, ArH), 6.71, 6.68 (2s, 2 H, ArH), 6.11 (s, 2 H, OCH ), 3.82 (s, 2 H, ArCH N), 3.71,
); C NMR (75 MHz, DMSOꢀd ) δ: 158.48,
2
2
13
.70 (2s, 6 H, 2 × OCH
3
), 2.95ꢀ2.83 (m, 8 H, 4 × CH
2
6
57.67, 150.41, 149.15, 147.48, 147.08, 137.36, 134.74, 133.08, 132.69, 128.93, 128.60, 127.87,
25.50, 125.16, 123.02, 122.56, 122,20, 113.79, 111.65, 109.85, 108.08, 107.60, 101.39, 58.46,
+
56.39, 55.48, 55.43, 54.06, 50.08, 31.49, 27.19; HRMS (ESI) m/z calcd for [C34H32N4O4+Na]
ꢀ1
583.2316, found 583.2318; IR (KBr, cm ) ν: 3327.43, 2902.65, 1593.37, 1558.01, 1517.18,
1443.09，1425.41, 1124.86, 741.80, 774.22. Purity 99.882%; t 3.986 min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(m-tolyl)quinazolin-4-
amine (12h).
1
Pale yellow solid; Yield 53.8%; m.p.138ꢀ140°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.95 (s, 1 H,
NH), 8.66 (d, J = 8.4 Hz, 1 H, ArH), 8.30ꢀ8.23 (m, 2 H, ArH), 8.01 (d, J = 8.4 Hz, 2 H, ArH), 7.86
d, J = 3.6 Hz, 2 H, ArH), 7.62ꢀ7.57 (m, 1 H, ArH), 7.42ꢀ7.37 (m, 3 H, ArH), 7.31 (d, J = 8.4 Hz,
H, ArH), 6.78, 6.75 (2s, 2 H, ArH), 4.16 (s, 2 H, ArCH N), 3.73, 3.72 (2s, 6 H, 2 × OCH ),
(
1
3
1
1
2
3
1
3
.12ꢀ2.97 (m, 8 H, 4 × CH ), 2.42 (s, 3 H, CH ); C NMR (75 MHz, DMSOꢀd ) δ: 159.06,
2
3
6
57.77, 150.45, 148.01, 147.45, 138.32, 137.91, 137.33, 133.08, 130.84, 128.59, 128.49, 128.26,
27.99, 125.75, 125.11, 124.04, 123.19, 122.21, 114.01, 111.59, 109.75, 56.91, 55.54, 55.49,
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+
52.32, 49.30, 29.96, 25.58, 21.21; HRMS (ESI) m/z calcd for [C34H34N4O2+H] 531.2755,
ꢀ1
found 531.2741; IR (KBr, cm ) ν: 3287.91, 2934.68, 1618.62, 1559.93, 1520.64, 1463.98,
449.78, 1123.00, 729.48, 766.31. Purity 97.801%; t 4.082 min.
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N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(p-tolyl)quinazolin-4-a
0
1
2
3
4
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6
7
8
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0
1
2
3
4
5
6
7
8
9
0
1
2
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2
3
4
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8
9
0
mine (12i).
1
Pale yellow solid; Yield 61.1%; m.p.97ꢀ100°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.80 (s, 1 H,
NH), 8.57 (d, J = 8.1 Hz, 1 H, ArH), 8.35 (d, J = 8.1 Hz, 2 H, ArH), 7.90 (d, J = 8.3 Hz, 2 H, ArH),
.84 (d, J = 3.4 Hz, 2 H, ArH), 7.60ꢀ7.54 (m, 1 H, ArH), 7.34ꢀ7.29 (m, 4 H, ArH), 6.63 (d, J = 6.8
Hz, 2 H, ArH), 3.78, 3.75 (2s, 6 H, 2 × OCH ), 3.54 (s, 2 H, ArCH N), 2.84ꢀ2.70 (m, 8 H, 4 ×
); C NMR (75 MHz, DMSOꢀd ) δ: 159.11, 157.75, 150.48, 147.12,
46.88, 139.84, 137.19, 135.69, 132.98, 128.94, 128.57, 127.97, 127.88, 126.67, 125.93, 125.54,
122.95, 122.00, 113.93, 111.76, 109.98, 59.48, 55.42, 55.09, 50.49, 32.48, 28.30, 20.95; HRMS
7
3
2
13
CH
2
), 1.91 (s, 3 H, CH
3
6
1
+
ꢀ1
(ESI) m/z calcd for [C34H34N4O2+H] 531.2755, found 531.2749; IR (KBr, cm ) ν: 3380.53,
2920.35, 1571.61, 1557.21, 1514.96, 1451.24, 1417.11, 1175.87, 739.87, 763.08. Purity 96.471%;
t
r
4.055 min.
2
-(4-(tert-butyl)phenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)q
uinazolin-4-amine (12j).
1
Pale yellow solid; Yield 47.1%，m.p.120ꢀ122°C. H NMR (300 MHz, DMSOꢀd ) δ: 9.79 (s, 1 H,
6
NH), 8.57 (d, J = 8.4 Hz, 1 H, ArH), 8.37 (d, J = 8.4 Hz, 2 H, ArH), 7.91 (d, J = 8.4 Hz, 2 H, ArH),
7
.84 (d, J = 3.9 Hz, 2 H, ArH), 7.58ꢀ7.51 (m, 3 H, ArH), 7.35 (d, J = 8.4 Hz, 2 H, ArH), 6.65 (d, J
5.2 Hz, 2 H, ArH), 3.69 (s, 6 H, 2 × OCH ), 3.57(s, 2 H, ArCH N), 2.87ꢀ2.49 (m, 8 H, 4 × CH ),
); C NMR (75 MHz, DMSOꢀd ) δ: 159.11, 157.73, 152.88, 150.51, 147.13,
=
3
2
2
1
3
1
1
1
.33 (s, 9 H, 3CH
3
6
46.89, 137.23, 135.73, 135.63, 132.97, 128.57, 128.01, 127.74, 126.65, 125.91, 125.57, 125.11,
22.94, 121.95, 113.94, 111.76, 109.99, 59.53, 55.42, 55.08, 50.54, 34.47, 32.50, 30.99, 28.30;
+
ꢀ1
HRMS (ESI) m/z calcd for [C37H40N4O2+H] 573.3224, found 573.3223; IR (KBr, cm ) ν:
3
386.43, 2949.85, 1572.74, 1558.01, 1515.61, 1448.99, 1354.70, 1325.23, 1130.76, 763.96,
05.78. Purity 97.291%; t 4.039 min.
7
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N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(pyridin-4-yl)quinazoli
n-4-amine (12k).
1
Pale yellow solid; Yield 61.1%; m.p. 180ꢀ182°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.94 (s, 1 H,
NH), 8.74 (d, J = 5.5 Hz, 2 H, ArH), 8.64 (d, J = 8.0 Hz, 1 H, ArH), 8.26 (d, J = 5.5 Hz, 2 H, ArH),
.91 (d, J = 8.4 Hz, 4 H, ArH), 7.91ꢀ7.85 (m, 1 H, ArH), 7.36 (d, J = 8.3 Hz, 2 H, ArH), 6.65 ((d,
J = 7.0 Hz, 2 H, ArH), 3.70, 3.69 (2s, 6 H, 2 × OCH ), 3.58 (s, 2 H, ArCH N), 2.90ꢀ2.49 (m, 8 H,
) δ: 158.07, 157.25, 150.15, 150.08, 147.11, 146.87,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
3
2
1
3
4
1
1
2 6
× CH ); C NMR (75 MHz, DMSOꢀd
45.54, 136.82, 136.10, 133.34, 128.65, 128.28, 126.66, 125.91, 123.07, 122.28, 121.72, 114.41,
11.77, 109.98, 59.47, 55.46, 55.43, 55.09, 50.51, 32.49, 28.30; HRMS (ESI) m/z calcd for
+
ꢀ1
[
C32H32N5O2+H] 518.2551, found 518.2547; IR (KBr, cm ) ν: 3238.94, 1618.77, 1604.08,
553.35, 1516.65, 1452.10, 1132.92, 771.17, 705.14, 599.42. Purity 99.507%; t 3.916 min.
1
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(pyridin-3-yl)quinazoli
n-4-amine (12l).
1
Pale yellow solid; Yield 44.8%; m.p. 110ꢀ112°C. H NMR (300 MHz, DMSOꢀd ) δ: 9.92 (s, 1 H,
6
NH), 9.54 (s, 1 H, ArH), 8.68 (d, J = 5.5 Hz, 2 H, ArH), 8.58 (d, J = 8.2 Hz, 1 H, ArH), 7.88ꢀ7.84
(m, 4 H, ArH), 7.65ꢀ7.61 (m, 1 H, ArH), 7.55ꢀ7.50 (m, 1 H, ArH), 7.35 (d, J = 8.2 Hz, 2 H, ArH),
6
.64 (d, J = 6.2 Hz, 2 H, ArH), 3.69 (s, 6 H, 2 × OCH
3
), 3.55 (s, 2 H, ArCH
) δ: 157.89, 157.44, 150.76, 150.53, 150.16, 149.17, 136.90,
35.98, 135.01, 133.65, 133.21, 130.35, 129.79, 128.94, 128.58, 128.03, 126.51, 126.15, 123.48,
23.07, 122.43, 122.31, 114.13, 111.73, 109.95, 59.41, 56.65, 56.06, 55.44, 50.02, 50.45, 38.43,
2
N), 2.88ꢀ2.50 (m, 8 H,
13
ArH); C NMR (75 MHz, DMSOꢀd
6
1
1
2
+
ꢀ1
8.21; HRMS (ESI) m/z calcd for [C32H31N5O2+H] 518.2551, found 518.2545; IR (KBr, cm )
ν: 3333.33, 1601.53, 1570.88, 1559.77, 1516.46, 1450.97, 1125.62, 765.85, 724.87, 704.41. Purity
7.221%; t 3.899 min.
9
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(quinolin-2-yl)quinazo
lin-4-amine(12m).
1
Pale yellow solid; Yield 43.7%; m.p. 149ꢀ151°C; H NMR (300 MHz, DMSOꢀd ) δ: 9.94 (s, 1 H,
6
NH), 8.67 (d, J = 8.4 Hz, 1 H, ArH), 8.58ꢀ8.49 (m, 2 H, ArH), 8.19 (t, J = 8.7 Hz, 3 H, ArH),
8.07ꢀ7.82 (m, 4 H, ArH), 7.72ꢀ7.65 (m, 2 H, ArH), 7.37 (d, J = 8.4 Hz, 2 H, ArH), 6.67 (d, J = 5.2
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6
1
3
Hz, 2 H, ArH), 3.69ꢀ3.65 (m, 8 H, 2 × OCH , ArCH N), 2.89ꢀ2.77 (m, 8 H, 4 × CH ); C NMR
3
2
2
(
75 MHz, DMSOꢀd
6
) δ: 158.38, 158.14, 154.84, 149.17, 148.29, 147.65, 147.35, 138.11, 136.81,
33.60, 132.33, 130.01, 129.67, 128.73, 128.08, 127.89, 127.61, 127.48, 126.89, 123.52, 123.38,
22.34, 120.72, 120.03, 114.27, 111.52, 109.66, 55.95, 55.57, 55.52, 51.26, 48.85, 29.05, 24.53;
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
ꢀ1
HRMS (ESI) m/z calcd for [C36H33N5O2+H] 568.2707, found 568.2703; IR (KBr, cm ) ν:
3
345.13, 2932.15, 1625.09, 1554.08, 1519.24, 1430.03, 1242.20, 1121.92, 776.29, 680.98. Purity
5.851%; t 3.934 min.
9
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(3-nitrophenyl)quinaz
olin-4-amine (12n).
1
Pale yellow solid; Yield 59.5%，m.p.140ꢀ142°C. H NMR (300 MHz, DMSOꢀd
6
) δ: 10.01 (s, 1 H,
NH), 9.16 (d, J = 1.4 Hz, 1 H, ArH), 8.76 (dd, J = 7.8, 1.0 Hz, 1 H, ArH), 8.61 (d, J = 8.1 Hz, 1 H,
ArH), 8.31ꢀ8.27 (m, 1 H, ArH), 7.91ꢀ7.83 (m, 4 H, ArH), 7.75 (t, J = 8.0 Hz, 1 H, ArH), 7.65ꢀ7.60
(
m, 1 H, ArH), 7.36 (d, J = 8.4 Hz, 2 H, ArH), 6.69 (d, J = 5.2p Hz, 2 H, ArH), 3.82 (s, 2 H,
13
2 3 2 6
ArCH N), 3.70 (s, 6 H, 2 × OCH ), 2.96ꢀ2.83 (m, 8 H, 4 × CH ); C NMR (75 MHz, DMSOꢀd )
δ: 157.94, 156.80, 150.09, 148.01, 147.45, 147.09, 139.96, 137.10, 135.05, 133.62, 133.28, 129.92,
1
28.55, 128.11, 126.36, 125.14, 124.53, 123.13, 122.45, 122.24, 114.18, 111.67, 109.86, 58.50,
+
55.44, 54.04, 50.06, 31.54, 27.24; HRMS (ESI) m/z calcd for [C33H31N5O4+H] 562.2449,
ꢀ1
found 562.2447; IR (KBr, cm ) ν: 3321.53, 1618.38, 1561.53, 1529.34, 1514.99, 1465.83,
1345.84, 1118.46, 734.62, 714.80, 527.67. Purity 97.189%; t 3.972 min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(4-nitrophenyl)quinaz
olin-4-amine (12o).
1
Pale yellow solid; Yield 55.4%; m.p.181ꢀ183°C. H NMR (300 MHz, DMSOꢀd
6
) δ: 9.95 (s, 1 H,
NH), 8.65ꢀ8.59 (m, 3 H, ArH), 8.33 (d, J = 7.8 Hz, 2 H, ArH), 7.92ꢀ7.85 (m, 4 H, ArH), 7.70ꢀ7.64
(
3
m, 1 H, ArH), 7.37 (d, J = 8.4 Hz, 2 H, ArH), 6.66, 6.64 (2s, 2 H, ArH), 3.69 (s, 6 H, 2 × OCH ),
13
3
1
1
3
.58 (s, 2 H, ArCH
2
N), 2.88ꢀ2.73 (m, 8 H, 4 × CH
2
); C NMR (75 MHz, DMSOꢀd
6
) δ: 157.91,
57.10, 150.09, 148.32, 147.12, 146.88, 144.32, 136.82, 136.02, 133.30, 128.78, 128.63, 128.25,
26.62, 125.90, 123.48, 123.02, 122.23, 114.11, 111.74, 109.97, 59.48, 55.45, 55.40, 55.08, 50.52,
+
2.53, 28.30; HRMS (ESI) m/z calcd for [C33H31N5O4+H] 562.2449, found 562.2442; IR (KBr,
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ꢀ
1
cm ) ν: 3427.73, 1598.89, 1565.44, 1517.05, 1408.10, 1340.47, 1129.12, 847.85, 760.95, 716.46.
Purity 97.685%; t 3.991 min.
r
2
-(4-chlorophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)quina
zolin-4-amine (12p).
10
11
12
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55
56
57
58
59
60
1
Pale yellow solid; Yield 41.2%，m.p. 113ꢀ115°C; H NMR (300 MHz, DMSOꢀd ) δ: 9.86 (s, 1 H,
6
NH), 8.57 (d, J = 8.4 Hz, 1 H, ArH), 8.44ꢀ8.40 (m, 2 H, ArH), 7.86ꢀ7.83 (m, 4 H, ArH), 7.64ꢀ7.54
(
m, 3 H, ArH), 7.34 (d, J = 8.4 Hz, 2 H, ArH), 6.64 (d, J = 6.3 Hz, 2 H, ArH), 3.69 (s, 6 H, 2 ×
1
3
3 2 2 6
OCH ), 3.56 (s, 2 H, ArCH N), 2.88ꢀ2.71 (m, 8 H, 4 × CH ); C NMR (75 MHz, DMSOꢀd ) δ:
1
1
1
58.02, 157.84, 150.27, 147.11, 146.86, 137.88, 137.23, 137.02, 135.80, 135.01, 133.08, 130.38,
29.81, 129.52, 128.95, 128.57, 128.34, 128.01, 126.58, 125.88, 123.03, 122.14, 121.10, 113.99,
11.69, 59.43, 56.04, 55.43, 55.38, 55.05, 50.44, 32.45, 28.26; HRMS (ESI) m/z calcd for
+
ꢀ1
[
C33H31ClN4O2+H] 551.2208, found 551.2213; IR (KBr, cm ) ν: 3356.93, 2914.45, 1599.26,
1
571.54, 1557.29, 1516.07, 1255.77, 1166.55, 762.99. Purity 97.037%; t 3.945 min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-(4-fluorophenyl)quina
zolin-4-amine (12q).
1
Pale yellow solid; Yield 61.1%，m.p.125ꢀ127°C. H NMR (300 MHz, DMSOꢀd
6
) δ: 9.91 (s, 1 H,
NH), 8.57 (d, J = 8.3 Hz, 1 H, ArH), 8.49ꢀ8.44 (m, 2 H, ArH), 7.87ꢀ7.82 (m, 4 H, ArH), 7.62ꢀ7.57
(m, 1 H, ArH), 7.44ꢀ7.28 (m, 4 H, ArH), 6.66, 6.64 (2s, 2 H, ArH), 3.69 (s, 6 H, 2 × OCH ), 3.59
) δ: 165.23, 161.95,
3
13
2 2 6
(s, 2 H, ArCH N), 2.89ꢀ2.50 (m, 8 H, 4 × CH ); C NMR (75 MHz, DMSOꢀd
158.09, 157.81, 150.34, 147.13, 146.86, 138.22, 137.10, 135.68, 134.84, 133.39, 132.97, 130.03,
128.92, 128.53, 127.93, 126.48, 125.67, 123.05, 122.14, 121.07, 115.23, 114.94, 113.90, 111.66,
109.89, 105.40, 59.69, 59.36, 56.50, 55.95, 55.40, 55.34, 54.98, 50.35, 32.39, 28.20; HRMS (ESI)
+
ꢀ1
m/z calcd for [C33H31FN4O2+H] 535.2504, found 535.2496; IR (KBr, cm ) ν: 3268.44,
825.96, 1572.93, 1560.94, 1516.70, 1454.88, 1254.06, 1144.73, 748.55, 760.48. Purity 97.018%;
tr 3.938 min.
2
7
-chloro-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2-phenylquinaz
olin-4-amine (12r).
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Pale yellow solid; Yield 61.1%; m.p.105ꢀ107°C. H NMR (300 MHz, DMSOꢀd ) δ: 9.93 (s, 1 H,
6
NH), 8.60 (d, J = 9.0 Hz, 1 H, ArH), 8.46ꢀ8.43 (m, 2 H, ArH), 7.89ꢀ7.86 (m, 3 H, ArH), 7.62 (dd,
J = 8.8, 1.9 Hz, 1 H, ArH), 7.51ꢀ7.49 (m, 4 H, ArH), 7.34 (d, J = 8.3 Hz, 2 H, ArH), 6.63 (d, J =
7
.7 Hz, 2 H, ArH), 3.70 (s, 6 H, 2 × OCH
3
), 3.54 (s, 2 H, ArCH
2 2
N), 2.87ꢀ2.70 (m, 8 H, 4 × CH );
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
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8
9
0
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2
3
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0
1
2
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8
9
0
13
C NMR (75 MHz, DMSOꢀd
6
) δ: 160.22, 157.67, 151.48, 147.10, 146.86, 137.97, 137.67,
1
36.83, 136.00, 130.47, 128.59, 128.33, 128.01, 126.69, 126.62, 125.97, 125.89, 125.22, 122.14,
12.66, 111.72, 109.94, 59.44, 55.43, 55.40, 55.07, 50.46, 32.46, 28.29; HRMS (ESI) m/z calcd
1
+
ꢀ1
for [C33H31ClN4O2+H] 55.2208, found 551.2192; IR (KBr, cm ) ν: 2932.15, 1612.18, 1556.36,
1
514.65, 1450.47, 1226.96, 1127.38, 768.53, 706.75. Purity 98.670%; t 3.850min.
r
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-6,7-dimethoxy-2-phenyl
quinazolin-4-amine (12s)
1
Pale yellow solid; Yield 52.3%; m.p. 120ꢀ122°C; H NMR (300 MHz, DMSOꢀd ) δ: 9.50 (s, 1 H,
6
NH), 8.44 (dd, J = 8.0, 2.0 Hz, 2 H, ArH), 7.88ꢀ7.83 (m, 3 H, ArH), 7.51ꢀ7.44 (m, 3 H, ArH), 7.33
(d, J = 8.4 Hz, 2 H, ArH), 7.27 (s, 1 H, ArH), 6.62 (d, J = 7.9 Hz, 2 H, ArH), 3.97, 3.95 (2s, 6 H, 2
1
3
× OCH
3
), 3.70, 3.69 (2s, 6 H, 2 × OCH
) δ: 157.50, 156.47, 154.29, 148.73, 147.56, 147.10, 146.86, 138.73,
37.47, 135.30, 129.66, 128.53, 128.23, 127.50, 126.66, 125.92, 121.95, 111.71, 109.94, 107.64,
07.50, 102.09, 59.51, 56.18, 55.70, 55.43, 55.39, 55.08, 54.84, 50.47, 32.48, 28.30; HRMS (ESI)
3 2 2
), 3.54 (s, 2 H, ArCH N), 2.87ꢀ2.69 (m, 8 H, 4 × CH );
C NMR (75 MHz, DMSOꢀd
6
1
1
+
ꢀ1
m/z calcd for [C35H36N4O4+H] 577.2809, found 577.2811; IR (KBr, cm ) ν: 3374.63, 2825.96,
1
602.21, 1560.96, 1514.62, 1460.77, 1225.42, 1163.17, 780.11, 702.90. Purity 98.227%; t 3.786
r
min.
Material and Methods
Materials and animals
3
ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT), doxorubicin (DOX),
daunorubicin (DNR), vinblastine (VLB), paclitaxel (PTX), cyclophosphamide (CTX), verapamil
VRP), rhodamine 123 (Rh123) and dimethyl sulfoxide (DMSO) were purchased from
(
SigmaꢀAldrich (St. Louis, MO, USA). WKꢀXꢀ34 was synthesized by our laboratory before.
Monoclonal antibody against ABCB1 (ab170904) was obtained from Abcam plc, USA.
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α/βꢀTubulin (#2148) and antiꢀrabbit IgG (#7074) were provided by Cell Signaling Technology,
TM
Inc, USA. PgpꢀGlo assay systems were purchased from Promega Corporation, USA. RPMI
1640s were from Life Technologies, Inc. All common chemicals were in analytical or higher
grade. Sprague−Dawley (SD) rats (male, 200−250 g) were purchased from the Comparative
Medical Center of Yangzhou University (Jiangsu, China). All animal experimental protocols
adhered to the Guide for the Care and Use of Laboratory Animals published by the National
Institutes of Health (NIH Publication 85ꢀ23, revised 1986), and our experiments have been
approved by the institutional committee of China Pharmaceutical University.
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Cell lines and cell culture
Human leukemia sensitive cell line K562 and its doxorubicinꢀselected Pꢀgp overexpressing
daughter cell line K562/A02 were kindly provided by Professor BaoꢀAn Chen (Department of
Hematology, The Affiliated ZhongꢀDa Hospital, Southeast University (Nanjing, China). Human
umbilical vein endothelial cell line (HUVEC) was purchased from KeyGEN BioTECH (Nanjing,
China). The cell lines were grown in RPMIꢀ1640 medium supplemented with 10%
2
heatꢀinactivated fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO . To
confirm drug resistance characteristics, 1 mg/ml ADM was added to K562/A02 cultures and
maintained in drugꢀfree medium for 2 weeks before usage. The cells in exponential growth can be
used for experiments.
Methods
Cytotoxicity assay
28ꢀ31
Cell viability was determined by MTT method with a minor modification.
K562 and
K562/A02 cells during logarithmic growth phase were seeded in 96ꢀwell microꢀtiter plates at
4
1
×10 cells per well. Pꢀgp inhibitors were prepared as 5 mM DMSO stocks. In the MTT assay for
MDR reversal experiments, cells were incubated in the presence of anticancer agents (DOX, DNR,
VLB, PTX or CTX) with or without Pꢀgp inhibitors for 48h. MTT dye (10 µl of 2.5 mg/ml in PBS)
was added to each well 4 hours priors to experiment termination in a 37 °C incubator containing 5%
2
CO . The plates were then centrifuged at 1500 RPM for 15 min and the supernatant was discarded
without disturbing the formazan crystals and cells in the wells, while the MTT formazan crystals
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