Designed P-glycoprotein inhibitors with triazol-tetrahydroisoquinoline-core increase doxorubicin-induced mortality in multidrug resistant K562/A02 cells

Article abstract of DOI:10.1016/j.bmc.2019.06.013

Multidrug resistance (MDR) refers to the cross-resistance of cancer cells to one drug, accompanied by other drugs with different mechanisms and structures, which is one of the main obstacles of clinical chemotherapy. Overexpression of P-glycoprotein (P-gp) was an extensively studied cause of MDR. Therefore, inhibiting P-gp have become an important strategy to reverse MDR. In this study, two series of triazole-tetrahydroisoquinoline-core P-gp inhibitors were designed and synthesized. Among them, compound I-5 had a remarkable reversal activity of MDR activity and the preliminary mechanism study was also carried out. All the results proved that compound I-5 was considered as a promising P-gp-mediated MDR reversal candidate.

Full text of DOI:10.1016/j.bmc.2019.06.013

Accepted Manuscript
Designed P-glycoprotein inhibitors with triazol-tetrahydroisoquinoline-core in-
crease doxorubicin-induced mortality in multidrug resistant K562/A02 cells
Mutta Kairuki, Qianqian Qiu, Miaobo Pan, Qifei Li, Jiaqi Zhou, Hesham
Ghaleb, Wenlong Huang, Hai Qian, Cheng Jiang
PII:
S0968-0896(19)30775-8
https://doi.org/10.1016/j.bmc.2019.06.013
BMC 14950
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
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9 May 2019
4 June 2019
6 June 2019
Please cite this article as: Kairuki, M., Qiu, Q., Pan, M., Li, Q., Zhou, J., Ghaleb, H., Huang, W., Qian, H., Jiang,
C., Designed P-glycoprotein inhibitors with triazol-tetrahydroisoquinoline-core increase doxorubicin-induced
mortality in multidrug resistant K562/A02 cells, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.06.013
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Designed
P-glycoprotein
inhibitors
with
triazol-
tetrahydroisoquinoline-core increase doxorubicin-induced mortality
in multidrug resistant K562/A02 cells
Mutta Kairuki 1,, Qianqian Qiu 2,#, Miaobo Pan1, Qifei Li1, Jiaqi Zhou1, Hesham
Ghaleb1, Wenlong Huang1,3, Hai Qian1,3,* and Cheng Jiang4,*
1
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
2
School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland
Bioresources and Environmental Protection, Yancheng Teachers’ University,
Yancheng, PR China
3
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
4
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing
210009, PR China
 Mutta Kairuki and Qianqian Qiu contributed equally as first authors.
*Co-corresponding authors: Hai Qian, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. Phone, +86-25-83271051; Fax, +86-25-
83271051; E-mail, qianhai24@163.com.
Cheng Jiang, Department of Medicinal chemistry, China Pharmaceutical University, Nanjing 210009, PR China
Phone, +86-25-83271051; Fax, +86-25-83271051; E-mail, jc@cpu.edu.cn

Abstract
Multidrug resistance (MDR) refers to the cross-resistance of cancer cells to one drug,
accompanied by other drugs with different mechanisms and structures, which is one
of the main obstacles of clinical chemotherapy. Overexpression of P-glycoprotein (P-
gp) was an extensively studied cause of MDR. Therefore, inhibiting P-gp have
become an important strategy to reverse MDR. In this study, two series of triazole-
tetrahydroisoquinoline-core P-gp inhibitors were designed and synthesized. Among
them, compound I-5 had a remarkable reversal activity of MDR activity and the
preliminary mechanism study was also carried out. All the results proved that
compound I-5 was considered as a promising P-gp-mediated MDR reversal candidate.
Keywords: Multidrug resistance; P-glycoprotein; reversal activity; K562/A02 cells
1. Introduction
Malignant cancer is one of the diseases that seriously threaten human survival.[1]
Chemotherapy is one of the main treatments for cancer. With the development of
chemotherapeutics, the mortality rate of cancer patients was declined significantly,
but the resistance of cancer cells to chemotherapeutics hinders the cure of clinical
malignant tumors, and greatly increases the recurrence and mortality of cancer[2, 3].
Multidrug resistance is the most serious one. Multidrug resistance (MDR) refers to the
cross-resistance of cancer cells to one drug, accompanied by other drugs with
different mechanisms and structures. The mechanisms of MDR have been extensively
studied [4-7]. Among these mechanisms, overexpression of ATP-binding cassette
subfamily (ABC) transmembrane transporter was the most significant one, which
enhances the translocation of cytotoxic drugs and results in the reduction of
intracellular drug concertation lower than the effective concentration[8, 9]. P-
glycoprotein (P-gp) is encoded by MDR1 gene, consisting of 1280 amino acids. It is
one of the most thoroughly studied ABC transporter families[10]. As a key protein
regulating multidrug resistance in many kinds of cancer cells, overexpression of P-gp
results in the concentration of antineoplastic drugs in tumor cells lower than the

effective concentration, making loss of cytotoxicity or apoptotic activity. Therefore,
inhibiting the function of P-gp and reducing the efflux of anti-cancer drugs have
become an important strategy to reverse MDR [11, 12].
Verapamil (VRP), a calcium antagonist, was firstly discovered to have the power of
reversing MDR in cancer cells by Tsuruo et al. in 1981[13]. In the past forty years,
many P-gp inhibitors with different structures and mechanisms have been found,
some of which have entered the stage of clinical research [14-17]. However, an
ABCB1 inhibitor has not been approved for the market due to a lack of significant
clinical efficacy, pharmacokinetic interaction, or concerns about its safety [14]. As a
result, developing novel, targeted, and potent ABCB1 inhibitors to reverse drug
resistance is in a great need.
It has been reported that the tetrahydroisoquinolinethylphenylamine-based P-gp
inhibitors showed potent MDR reversal activity, including WK-X-34, Tariquidar (Tar)
and HM30181[18-21]. In addition, triazole, as a crucial nitrogen-heterocycle, also
attracted our attention due to its wide application as ABCB1 inhibitors to reverse
MDR, as well as its easy accessibility. 1,2,3-triazole has the similar bond length and
plane structure with amide bond, which can play better π-π interaction with receptor
proteins [22]. Heterocycles such as indole, quinoline, thiophene and furan ring play an
important role in anti-cancer drugs. From the above, we designed series I compounds
as shown in Fig.1A. In order to reduce molecular weight and improve the druggability,
the designed structures were in further simplification to get series II compounds. As
the docking results shown in Fig.1B, one of the typical designed structure of series I
(I-1) and series II (II-1) both occupied hydrophobic pocket of P-gp protein (PDB
ID:6FN1) in a similar interaction mode. I-1 has Pi-Pi stacking action with PHE982
and PHE302, and forms hydrogen-bond interaction with GlN945. Similarly, II-1 has
PI-PI stacking interaction with PHE982 and hydrogen-bond with TYR952. Hence, it
is believed that the two series of compounds may have similar activities.
Consequently, 24 novel tetrahydroisoquinoline P-gp inhibitors were synthesized and
founded that some of them exhibited a powerful improvement of reversal activity.

Figure 1. (A) Design of the target compounds; (B) Structure docking results with P-gp
(PDB ID:6FN1).
2. Results and discussion
2.1 chemistry
As depicted in scheme 1, the two series compounds I-1-12 and II-1-12 with triazol-
tetrahydroisoquinoline-core were synthesized. Compound a was prepared from the
starting material 1-fluoro-2-nitrobenzene refluxed in the mixture of DMF with prop-2-
yn-1-amine and potassium carbonate for 8h.[23] Treatment of compound a with
sodium hyposulfite in 50% ethanol afforded compound b which was then reacted with
disparate aromatic formic acid in dry dichloromethane with 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDCI),
1-
Hydroxybenzotriazole (HOBT) as coupling agents to get the compound c1-12 [24].

Compounds g and j were synthesized according to literature procedures with minor
modification [25, 26]. Subsequently, compound g or j and compounds c1-12 were
treated with ascorbate sodium and copper sulfate in 75% methanol stirring at room
temperature for 48 h to provide compounds I-1-12 and II-1-12. Most of the target
compounds could be precipitated from the reaction solution directly, for higher purity
they should be purified by column chromatography. The structures of target
compounds obtained were listed in Table 1.
H
N
H
N
H
N
F
i
ii
iii
NH
NH2
NO2
NO2
O
Het
a
b
c1-12
O
O
NH2
O
O
O
iv
v
NH.HCl
vii
N
O
d
e
NO2
O
O
O
O
vi
N
N
NH2
N3
f
g
N
N
N
H
N
N
viii
O
NH
O
I-1-12
O
Het
OH
O
OH
h
ix
x
Br
N3
S
N3
O
O
i
N
O
O
NH
N
N
O
O
viii
xi
O
O
NH
.HCl
N
N
NH
O
N3
Het
d
j
II-1-12
Scheme 1. Synthesis of the target compounds.
Reagents and conditions: (i) 2-Propynylamine, K2CO3, DMF, 80°C, 8h；(ii) Na2S2O4,
NaHCO3, MeOH: H2O: THF=1:1:1, 0°C~ r.t., 12 h; (iii) EDCI, HOBT r.t., 24 h; (iv)
(CH2O)n, EtOH, r.t., 3h; con. HCl, reflux, 4h; (v) 1-(2-bromoethyl)-4-nitrobenzene,
K2CO3, CH3CN, reflux, 17h; (vi) H2/Pd-C, DCM/EtOH, r.t., 24h; (vii) NaNO2, 50%
AcOH, 0°C~5°C, 30 min; NaN3, 0°C~5°C, 50 min; (viii) c1-12, sodium ascorbate,

CuSO4, 75% CH3OH, 24h~48h; (ix) NaN3, H2O, 80 °C, 24 h; (x)TEA/DCM, TsCl, r.t.,
24 h; (xi) i, TEA, acetonitrile, 60 °C.
Table 1. Structures of Target Compounds
N
N
N
N
H
N
NH
NH
N
N
N
O
N
NH
O
Het
O
O
I-1-12
II-1-12
Het
Het
Het
Compound
I-1 / II-1
I-2 / II-2
I-3 / II-3
I-4 / II-4
I-5 / II-5
I-6 / II-6
Compound
I-7 / II-7
S
N
Cl
I-8 / II-8
N
S
I-9 / II-9
N
H
S
H3C
N
CH3
Cl
I-10 / II-10
I-11 / II-11
I-12 / II-12
S
N
S
N
O
2.2 Cytotoxicity evaluation
Due to toxicity at the therapeutic concentration range, the majority of potent P-gp
inhibitors are failed for further studies, the intrinsic cytotoxicity of target compounds
against parental human erythroleukemia sensitive cell line K562 and its doxorubicin
(DOX)-selected derivative P-gp overexpressing K562/A02 cells was evaluated by
MTT assay. As shown in Table 2, the anticarcinogen DOX exhibited weak inhibitory
effect on the survival of K562/A02 cells (IC50 of 55.47 ± 1.67 μM), which was about
108.8-fold greater resistance than K562 cells (IC50 = 0.51 ± 0.09 μM). Except the
potent inhibitor Tariquidar (18.42 ± 1.12 μM for K562 cells and 30.10 ± 2.16 μM for

K562/A02 cells), most of the 24 target compounds had low intrinsic cytotoxicity
(IC50s > 40 μM) against both K562 and K562/A02 cells. It can be seen from data
points of IC50 that the survival rates of all compounds at 5.0 μM were all over 95%,
appropriate to determine its reversal effect on K562/A02 cells. Therefore, the reversal
activity of I-1-12 and II-1-12 in DOX-resistance were determined preliminarily at
non-toxic concentration of 5.0 μM by MTT.
Table 2. Cytotoxicity of the target compounds on K562 and K562/A02 cellsa.
IC50(μM)
IC50(μM)
K562/A02
Cpd.
Cpd.
K562
K562/A02
K562
I-1
I-2
>100
>100
60.71 ± 2.3
65.93 ± 4.3
59.55 ± 1.9
>100
II-1
II-2
II-3
II-4
II-5
II-6
II-7
II-8
II-9
II-10
II-11
II-12
Tar
>100
>100
>100
>100
I-3
>100
>100
>100
1-4
>100
>100
>100
I-5
>100
63.23 ± 2.3
52.08 ± 1.5
>100
57.88 ± 0.56
>100
>100
I-6
45.09 ± 1.2
>100
>100
I-7
>100
>100
I-8
42.7 ± 1.33
>100
46.99 ± 0.92
>100
>100
>100
I-9
>100
>100
I-10
I-11
I-12
VRP
DOX
>100
>100
>100
>100
>100
58.13±2.4
58.39±1.9
59.24 ± 3.12
55.47 ± 1.67
>100
43.71 ± 1.34
>100
25.76 ± 0.92
68.27 ± 3.54
0.51 ± 0.09
>100
18.42 ± 1.12
30.10 ± 2.16
a
Cytotoxicity of target compounds toward cells were determined by MTT assay and
data are presented as mean ± SD for three independent tests.
2.3 P-gp modulating activity of the target compounds on reversing DOX
resistance in K562/A02 cells and structure-activity relationships
Based on the results above, all the target compounds were investigated for further
reversal activity at 5.0 μM with verapamil (VRP) and Tariquidar (Tar) as positive
controls. As the results summarized in Table 3, the combination of most target
compounds enhanced the toxic effect of DOX in K562/A02 cells. P-gp modulating
activity of compounds was evaluated by a parameter known as reversal fold (RF).

Most of the target compounds showed much more active MDR reversal activity than
VRP at the same concentration. Especially, compound I-5 exerted the strongest
reversal activity with RF of 42, which similar to Tar (RF = 42.3).
According to Table 3, the activities of series I compounds with the triazole-N-
phenylethyl tetrahydroisoquinoline moiety were much better than that of simplified
series II compounds. It was speculated that the simplification of the benzene ring
structure reduced the hydrophobicity of the compounds and thus affected the reversal
activity. For the docking results in Fig.1B, the most difference between the two
compounds is that the benzene ring of tetrahydroisoquinoline in I-1 can have Pi-Pi
stacking with PHE302, while II-1 loses its interaction with PHE302 because of
shortening the length of the whole molecule. However, tertiary amine N atom of II-1
could capture hydrogen ion, forming positive charge center, to interact with residue
GLU874. Based on the determined results in reversal activity, it is concluded that the
molecular structure contributes more to the P-gp active conformation through
PHE302, which directly affects the final reversal activity. Besides, in series I
compounds, several thiophene ring derivatives exhibited prominent activity,
presumably because the introduction of sulfur atoms in heterocycles can change the
charge distribution of compounds and increase the hydrophobicity of compounds. In
addition, the introduction of chlorine atoms can further increase lipophilicity, enhance
its affinity with binding sites of P-gp hydrophobic cavity, and improve the metabolic
instability of thiophene rings. Therefore, the compound I-5 showed the strongest
reversal activity.
Table 3. DOX-resistance reversal activity of target compounds at 5.0 M
concentration in K562/A02 cells a.
Compound
(5.0 μM)
I-1
IC50/DOX
(μM)
Compound
(5.0 μM)
II-1
IC50/DOX
(μM)
RF
RF
2.62 ± 0.3
2.43 ± 0.35
5.10 ± 0.21
21.17
22.82
10.87
27.30 ± 0.23
24.37 ± 0.89
17.90 ± 0.33
2.03
2.27
3.09
I-2
II-2
I-3
II-3

I-4
I-5
2.54 ± 0.19
1.32 ± 0.07
4.69 ± 0.32
51.06 ± 2.3
19.88 ± 1.2
8.44 ± 1.11
14.52 ± 0.92
4.17 ± 0.89
4.93 ± 0.23
7.084 ± 0.67
1.33 ± 0.22
21.83
42.02
11.83
1.09
II-4
II-5
19.37 ± 0.78
14.37 ± 1.27
14.29 ± 1.82
14.81 ± 2.38
43.26 ± 5.43
13.32 ± 2.09
29.57 ± 2.33
4.11 ± 0.45
43.46 ± 5.65
55.47 ± 1.67
2.86
3.38
3.88
3.74
1.28
4.16
1.88
13.5
1.28
1.00
I-6
II-6
I-7
II-7
I-8
2.79
II-8
I-9
6.57
II-9
I-10
I-11
I-12
VRP
Tar
3.82
II-10
II-11
II-12
DOX
13.30
11.25
7.83
42.34
a The IC50 value was determined after exposure to a series of DOX concentration with
different target compounds at 1.0 M using K562/A02 cells; Reversal fold (RF, fold-
change in drug sensitivity) = (IC50 without inhibitor / (IC50 with inhibitor). Data were
analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three
independent tests.
2.4 Chemo-sensitizing effect of target compound
Based on the reversal activity, compound I-5 was considered as the most promising
candidate for further dose-response reversal effect at different concentrations (5.0 μM,
2.0 μM, 1.0 μM, 0.5 μM, 0.25 μM, 0.1 μM, 0.075 μM, 0.05 μM, 0.025 μM, 0.01μM
and 0.005 μM) towards K562/A02 cells. As shown in Figure 2, there was still weak
reversal activity of I-5 even at 10 nM and the EC50 value of I-5 was only (46.83 ± 4.6)
nM calculated by the doseresponse curve in GraphPad Prism 7.0 software, indicating
I-5 was so powerful in inversing DOX resistance.

Figure 2. Impact of I-5 on inversing DOX resistance in K562/A02 cells. (A) IC50
values of DOX in K562/A02 cells dealt with DOX with/without different
concentrations of I-5; (B) EC50 value for I-5 in depressing DOX-resistance of
K562/A02 cells. The percentage of DOX IC50 was charted with log concentration of
I-5, which is equal to (DOX IC50 in each modulator concentration/DOX IC50 without
modulator) × 100%. Thus, EC50 can be noted for the modulator concentration, which
can diminish the DOX IC50 by 50%.
2.5 Duration of reversal effects
The relatively long duration of reversal action is a required condition for P-gp
inhibitors. To further ensure the combination of I-5 and anti-cancer drugs could exert
great activity, the duration of reversal effect of I-5 was determined. As the results
shown Table 4, the duration of reversal effects for both compound 3 and positive
tariquidar at a concentration of 5 μM were over 24 h resulting in a significantly longer
duration of action than VRP with the same concentration.
Table 4. Duration of MDR reversal effect in K562/A02 cells after incubation and
washout of I-5.
IC of DOX(μM) (RF)a
Treatment
50
schedule
Control
VRP (5 μM)
Tar (5 μM)
I-5 (5 μM)
55.47 ± 1.67
(1.00)
7.08 ± 0.67
(7.83)
1.33 ± 0.22
(42.34)
1.32 ± 0.07
(42.02)
No wash
Wash, 0h
26.24 ± 2.68
(2.11)
2.89 ± 0.34
(19.19)
3.26 ± 0.91
(17.05)
NDb

5.13 ± 0.54
(10.18)
7.84 ± 0.68
(7.08)
Wash, 6h
Wash, 12h
Wash, 24h
ND
ND
ND
>50
ND
ND
13.45 ± 1.22
(4.12)
16.84 ± 1.68
(3.29)
23.14 ± 2.14
(2.40)
33.47 ± 2.63
(1.27)
a
Reversal fold (RF) = (IC50 without inhibitor)/(IC50 with inhibitor).The values were
presented as the mean ± SD for three independent tests. b ND: not determined.
2.6 Effect of target compound on P-gp function
2.6.1 DOX accumulation
Furthermore, to validate our assumption that whether the reversal effect of compound
I-5 is associated with a concomitant increase in DOX accumulation, we utilized
fluorescence microscope to give a further evaluation.[27] As shown in Figure 3A,
sensitive K562 cells retained most of the red fluorescence after incubation. While it
was almost dark in K562/A02 cells, for the overexpressed P-gp had pumped most of
the DOX out of the cells. It was encouraging to find that incubation with compound I-
5 could induce more DOX accumulation than VRP at the same condition.
When incubated with modulators at various concentrations, the fluorescence intensity
of DOX accumulated in K562/A02 cells showed dose-effect dependence. The results
demonstrated in Figure 3B indicated that with the absence of modulators, DOX
accumulation in K562 cells was more than 4-fold (p<0.01) than that in K562/A02
cells. In comparison, 0.1μM compound I-5 exhibited higher efficiency in enhancing
accumulation of DOX in K562/A02 than VRP at 5μM. Meanwhile, 5 μM Tar and I-5
showed similar potency in increasing DOX accumulation in K562/A02, almost
restored the DOX level to the parental K562’s level. Results indicated that compound
I-5 was a potent P-gp inhibitor.

Figure 3. Effect of target compounds on intracellular DOX accumulation in K562 or
K562/A02 cells. (A) with inverted fluorescence microscope: A) K562 cells + 20 μM
DOX; B) K562/A02 cells + 20 μM DOX; C) K562/A02 cells + 5 μM I-5 + 20 μM
DOX; D) K562/A02 cells + 5 μM VRP + 20 μM DOX. (B) with fluorescence
spectrophotometer. The results are presented as the mean ± SD for three independent
experiments; (**) p < 0.01, (*) p < 0.05 relative to the negative control (K562/A02).
2.6.2 Inhibitory effect on Rh123 efflux
Further exploration of the effect of I-5 on P-gp efflux function was assayed by Rh123
efflux. As shown in Fig. 4, the efflux of Rh123 in the control group was increased
during 90 min, while 2.0 μM I-5-treated K562/A02 cells , the efflux of Rh123 was
significantly inhibited, the remaining mean fluorescence intensity (MFI) at 90 min
was over 50% compared with control and 5.0μM-treated VRP group, suggesting that
I-5 can reverse MDR by inhibiting P-gp-mediated drug efflux significantly..

Figure 4. Effect of 2.0 μM I-5 and 5.0 μM VRP on efflux of Rh123 from K562/A02.
Each data point is presented as mean ± SD for three independent experiments.
2.7 Effect of I-5 on P-gp expression
The presence of P-gp protein was proved by a band with a molecular weight of ≈170
kDa in K562/A02 cell lysates by western blotting, while it was not present in parental
K562 cells, suggesting the absence of P-gp protein (Figure 5). Inhibiting the function
or lowering expression of P-gp will contribute to the reversal of P-gp-mediated MDR.
Therefore, whether the reversal ability of I-5 was also due to a decrease in protein
expression should be confirmed. As demonstrated in Fig.5, there was no significant
alteration in P-gp expression in K562/A02 cells treated with different compounds or
concentrations, indicating that MDR reversal by I-5 was not caused by a decreased P-
gp expression but instead most likely due to direct inhibition of P-gp efflux.

Figure 5. Western blot analysis showing the expression of ABCB1 after exposure to
I-5. (A) Effect of I-5 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 β-Tubulin.
Representative result is shown here, and similar results were obtained in two other
independent trials.
3. Conclusion
In summary, in this paper, two series of triazole-tetrahydroisoquinoline-core P-gp
inhibitors were designed and synthesized, series II was simplified based on series I.
This investigation contributed to the identification of I-5 as a potent P-gp inhibitor in
reversing P-gp-mediated DOX-resistance in K562/A02 cells and possess outstanding
reversal potency with EC50 in namomolar range (EC50 = 46.84 ± 4.6 nM). The longer
duration of reversal activity was demonstrated in I-5, more than 24h.
Besides, I-5 can significantly increase DOX accumulation in K562/A02 cells and
suppress the efflux of Rh123 simultaneously. MDR reversal by I-5 was not caused by
a decreased P-gp expression but instead most likely due to direct inhibition of P-gp

efflux. As a result, I-5 could be served as a promising candidate for the development
of P-gp inhibitors in cancer chemotherapy.
4. Experimental Section
4.1 Reagents and apparatus
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 (200-300 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
RY-1 melting-point apparatus and were uncorrected. NMR spectra were recorded in
1
DMSO-d6 on a Bruker ACF-300Q instrument (300 MHz for H, 75 MHz for 13C;
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 = multiplet, dd = doublets of doublet, br = broad.
Infrared spectras were recorded on a Perkin-Elmer FTIR instrument. ESI-MS datas
were recorded with Waters ACQUITY UPLC Systems with Mass (Waters, Milford,
MA). The intermediates a, b, c1-12, d, e, f, g, h, i, and j were synthesized by previous
reports[28,29].
4.2 General procedure for preparation of compounds I-1-12 and II-1-12.
To the solution of c1-12 (1mmol) and intermediates g or i (1mmol) in 75% methanol
(40mL), ascorbate sodium (30mg) and CuSO4 (10mg) were added. The crude product
was purified by chromatography on silica gel utilized a mixture of chloroform and
acetone (80:1, v/v) as eluent to furnish the desired target compounds I-1-12 and II-1-
12.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)thiophene-2-carboxamide (I-1)
1
Yield: 39.6%; pale-yellow solid; m.p.107 - 109℃; ESI-MS m/z: 595.5 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.73 (s, 1H), 8.59 (s, 1H), 8.00 (s, 1H), 7.82 (s,
1H), 7.74 (d, J = 7.8 Hz, 2H), 7.47 (d, J = 7.4 Hz, 2H), 7.21 (s, 1H), 7.19 – 7.03 (m,

2H), 6.86 (d, J = 7.8 Hz, 1H), 6.64 (d, J = 7.4 Hz, 3H), 5.70 (t, J = 4.4 Hz, 1H), 4.46
13
(d, J = 4.4 Hz, 2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.90 (s, 2H), 2.70 (s, 6H); C NMR
(75 MHz, DMSO-d6) δ ppm:160.53 ,147.12, 146.82, 143.36, 141.22, 134.68, 131.29,
130.01, 129.19, 127.93, 127.57, 127.26, 120.99, 119.79, 116.23, 111.76, 111.49,
109.95, 58.99, 55.46, 55.00, 50.43, 38.94, 38.59, 32.26, 28.21; Anal. Calcd for
C33H34N6O3S: C, 66.65; H, 5.76; N, 14.13. Found: C, 66.55; H, 5.64; N, 14.18.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)thiophene-3-carboxamide (I-2)
1
Yield: 45.3%; pale-yellow solid; m.p.104 - 106℃; ESI-MS m/z: 595.5 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.55 (s, 1H), 8.58 (s, 1H), 8.32 (s, 1H), 7.80 –
7.68 (m, 2H), 7.63 (d, J = 4.5 Hz, 2H), 7.47 (d, J = 6.3 Hz, 2H), 7.13 (m, 2H), 6.86 (d,
J = 6.6 Hz, 1H), 6.65 (d, J = 6.0 Hz, 3H), 5.58 (t, J = 4.4 Hz, 1H), 4.45 (d, J = 4.4 Hz,
2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.90 (s, 2H), 2.70 (s, 6H); 13C NMR (75 MHz, CDCl3)
δ ppm: 162.12, 147.51, 147.08, 142.14, 141.26, 135.13, 129.89, 129.41, 127.64,
126.44, 126.04, 124.38, 120.43, 120.10, 118.78, 113.85, 111.30, 109.40, 59.76, 56.18,
55.54, 51.04, 40.10, 33.50, 28.67; Anal. Calcd for C33H34N6O3S: C, 66.65; H, 5.76; N,
14.13. Found: C, 66.65; H, 5.64; N, 14.28.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-methylthiophene-3-carboxamide
(I-3)
1
Yield: 39.8%; pale-yellow solid; m.p.89 - 91℃; ESI-MS m/z: 609.3 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.21 (s, 1H), 8.62 (s, 1H), 7.75 (d, J = 8.0 Hz,
2H), 7.65 (d, J = 5.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 6.8 Hz, 1H), 7.08
(d, J = 7.4 Hz, 1H), 7.02 (d, J = 5.0 Hz, 1H), 6.86 (d, J = 7.4 Hz, 1H), 6.65 (d, J = 8.0
Hz, 3H), 5.58 (t, J = 5.4 Hz, 1H), 4.45 (d, J = 5.4 Hz, 2H), 3.69 (s, 6H), 3.55 (s, 2H),
13
2.89 (d, J = 6.8 Hz, 2H), 2.70 (s, 6H), 2.48 (s, 3H); C NMR (75 MHz, CDCl3) δ
ppm: 161.70, 147.12, 142.86, 142.60, 142.34, 141.86, 141.22, 135.25, 132.35, 129.92,
127.62, 127.34, 126.56, 125.70, 120.73, 120.53, 120.29, 119.05, 114.06, 111.32,
109.41, 76.89, 59.76, 55.81, 51.04, 40.26, 33.52, 28.65, 16.04; Anal. Calcd for
C34H36N6O3S: C, 67.08; H, 5.96; N, 13.81. Found: C, 66.95; H, 5.94; N, 13.98.

N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-5-methylthiophene-2-carboxamide
(I-4)
Yield: 45.3%; white solid; m.p.101 - 103℃; ESI-MS m/z: 609.3 ([M+H]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 9.62 (s, 1H), 8.59 (s, 1H), 7.79 (s, 1H), 7.74 (d, J = 8.2
Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 7.11 (m, 2H), 6.91 (s, 1H), 6.85 (d, J = 8.2 Hz, 1H),
6.64 (d, J = 7.8 Hz, 3H), 5.65 (t, J = 5.1 Hz, 1H), 4.45 (d, J = 5.1 Hz, 2H), 3.70 (s,
6H), 3.56 (s, 2H), 2.90 (s, 2H), 2.71 (s, 6H), 2.49 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ ppm: 161.19, 147.14, 146.41, 141.20, 135.21, 129.90, 127.61, 126.90, 125.90,
120.46, 120.14, 118.88, 114.00, 111.32, 109.42, 59.75, 56.29, 55.54, 51.03, 40.21,
33.49, 28.64, 15.74; Anal. Calcd for C34H36N6O3S: C, 67.08; H, 5.96; N, 13.81.
Found: C, 67.15; H, 6.04; N, 13.95.
5-chloro-N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)thiophene-2-
carboxamide (I-5)
1
Yield: 43.1%; pale-yellow solid; m.p.104 - 106℃; ESI-MS m/z: 629.3 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.79 (s, 1H), 8.58 (s, 1H), 7.88 (s, 1H), 7.74 (d, J
= 7.7 Hz, 2H), 7.46 (d, J = 7.0 Hz, 2H), 7.26 (s, 1H), 7.10 (t, J = 8.6 Hz, 2H), 6.85 (d,
J = 8.0 Hz, 1H), 6.64 (d, J = 7.7 Hz, 3H), 5.76 (t, J = 5.0 Hz, 1H), 4.45 (d, J = 5.0 Hz,
2H), 3.69 (s, 6H), 3.54 (s, 2H), 2.90 (s, 2H), 2.70 (s, 6H); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 159.57, 146.77, 143.45, 141.24, 137.24, 134.90, 133.22, 130.01,
129.10, 128.75, 127.88, 126.55, 125.87, 122.58, 120.99, 119.78, 116.15, 111.76,
111.43, 109.94, 59.02, 55.45, 55.04, 50.46, 36.68, 32.27, 28.26; Anal. Calcd for
C33H33ClN6O3S: C, 63.00; H, 5.29; N, 5.63. Found: C, 62.85; H, 5.34; N, 5.28.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)picolinamide (I-6)
1
Yield: 33.6%; pale-yellow solid; m.p.144 - 146℃; ESI-MS m/z: 590.4 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 10.09 (s, 1H), 8.73 (d, J = 4.1 Hz, 1H), 8.60 (s,
1H), 8.15 (d, J = 7.8 Hz, 1H), 8.05 (t, J = 7.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.70 –
7.61 (m, 1H), 7.46 (d, J = 8.3 Hz, 3H), 7.07 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.3 Hz,

1H), 6.71 (t, J = 7.3 Hz, 1H), 6.64 (d, J = 7.8 Hz, 2H), 5.71 (t, J = 5.4 Hz, 1H), 4.44
(d, J = 5.4 Hz, 2H, -NHCH2-), 3.70 (s, 6H, -OCH3), 3.55 (s, 2H, -NCH2Ar), 2.88 (d, J
13
= 7.1 Hz, 2H), 2.70 (s, 6H); C NMR (75 MHz, DMSO-d6) δ ppm: 162.78, 149.96,
148.36, 147.14, 146.83, 142.07, 141.20, 137.90, 134.72, 129.97, 126.70, 126.58,
126.47, 125.90, 125.45, 125.21, 124.29, 122.23, 121.01, 119.74, 117.00, 112.32,
111.78, 109.99, 58.98, 55.46, 55.01, 50.43, 32.27, 28.23; Anal. Calcd for C34H35N7O3:
C, 69.25; H, 5.98; N, 16.63. Found: C, 69.35; H, 5.90; N, 16.78.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)isonicotinamide (I-7)
Yield: 29.8%; brown solid; m.p.178 - 180℃; ESI-MS m/z: 590.3 ([M+H]+); 1H NMR
(300 MHz, , DMSO-d6) δ ppm: 9.92 (s, 1H), 8.58 (s, 1H), 7.74 (s, 3H), 7.46 (s, 3H),
7.30 (s, 1H), 7.16 (s, 2H), 7.11 (s, 2H), 6.84 (s, 1H), 6.65 (s, 4H), 5.79 (t, J = 5.4 Hz,
1H), 4.45(d, J = 5.4 Hz, 2H), 3.70 (s, 6H), 2.90 (d, J = 7.1 Hz, 2H), 2.73 (s, 6H); 13C
NMR (75 MHz, DMSO-d6) δ ppm: 164.19, 150.07, 147.14, 146.85, 145.49, 143.28,
141.59, 127.38, 126.23, 125.78, 122.90, 121.78, 115.86, 111.74, 111.27, 109.84,
56.76, 55.43, 54.87, 50.04, 46.97, 40.82 – 40.53, 40.14, 39.59, 39.18, 39.18, 28.02;
Anal. Calcd for C34H35N7O3: C, 69.25; H, 5.98; N, 16.63. Found: C, 69.45; H, 5.86; N,
16.68.
2-chloro-N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)nicotinamide (I-8)
Yield: 34.5%; white solid; m.p.129 - 131℃; ESI-MS m/z: 624.3 ([M+H]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 9.88 (s, 1H), 8.63 (s, 1H), 8.52 (s, 1H), 8.17 (d, J = 7.5
Hz, 1H), 7.75 (d, J = 6.3 Hz, 2H), 7.57 (s, 1H), 7.47 (d, J = 6.4 Hz, 2H), 7.34 (d, J =
7.5 Hz, 1H), 7.12 (s, 1H), 6.88 (d, J = 6.4 Hz, 1H), 6.69 (s, 1H), 6.65 (m, 2H), 5.47 (t,
J = 5.4 Hz, 1H), 4.47 (d, J = 5.4 Hz, 2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.90 (s, 2H), 2.71
13
(s, 6H); C NMR (75 MHz, CDCl3) δ ppm: 159.23, 146.32, 143.37, 142.81, 142.62,
141.83, 137.19, 136.62, 134.83, 130.37, 126.77, 125.20, 123.41, 121.91, 121.69,
121.43, 118.70, 118.06, 115.72, 115.26, 114.07, 108.80, 106.59, 104.69, 54.97, 51.49,
50.77, 46.28, 35.32, 28.73, 23.89; Anal. Calcd for C34H34ClN7O3: C, 65.43; H, 5.49;
N, 15.71. Found: C, 65.35; H, 5.64; N, 15.88.

N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-1H-indole-2-carboxamide (I-9)
Yield: 38.1%; orange solid; m.p.126 - 128℃; ESI-MS m/z: 626.9 ([M-1]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 11.70 (s, 1H), 9.74 (s, 1H), 8.61 (s, 1H), 7.74 (s, 2H),
7.68 (s, 1H), 7.47 (d, J = 7.4 Hz, 3H), 7.37 (s, 1H), 7.28 – 7.16 (m, 2H), 7.16 – 6.99
(m, 2H), 6.90 (s, 1H), 6.65 (d, J = 7.4 Hz, 3H), 5.80 – 5.58 (t, J = 5.4 Hz, 1H), 4.47 (d,
13
J = 5.4 Hz, 2H), 3.70 (s, 6H), 3.57 (s, 2H), 2.90 (s, 2H), 2.72 (s, 6H); C NMR (75
MHz, CDCl3) δ ppm: 153.14, 142.15, 141.32, 139.05, 137.45, 136.60, 131.72, 130.59,
129.60, 124.76, 123.26, 121.09, 120.81, 118.83, 118.18, 116.29, 115.31, 114.91,
106.49, 106.10, 104.48, 98.73, 54.41, 51.04, 50.29, 45.76, 35.69, 28.25, 23.40; Anal.
Calcd for C37H37N7O3: C, 70.79; H, 5.94; N, 15.62. Found: C, 70.75; H, 5.84; N,
15.48.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)quinoline-3-carboxamide (I-10)
1
Yield: 37.2%; pale-yellow solid; m.p.109 - 111℃; ESI-MS m/z: 640.4([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 10.02 (s, 1H), 9.43 (s, 1H), 9.01 (s, 1H), 8.61 (s,
1H), 8.18 – 8.09 (m, 2H), 7.93 – 7.85 (m, 1H), 7.74 (d, J = 8.2 Hz, 3H), 7.46 (d, J =
8.2 Hz, 2H), 7.25 (d, J = 7.4 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 7.9 Hz,
1H), 6.69 (d, J = 7.0 Hz, 1H), 6.64 (d, J = 7.9 Hz, 2H), 5.91 (t, J = 5.2 Hz, 1H), 4.49
(d, J = 5.2 Hz, 2H), 3.69 (s, 6H), 3.54 (s, 2H), 2.94 – 2.84 (m, 2H), 2.70 (s, 6H); 13C
NMR (75 MHz, DMSO-d6) δ ppm: 164.52, 153.30, 149.38, 148.40, 147.01, 143.30,
141.24, 138.59, 136.16, 134.71, 131.21, 130.00, 129.10, 128.93, 127.50, 127.37,
126.57, 125.88, 123.24, 121.00, 119.77, 116.14, 111.75, 111.42, 109.95, 59.02, 55.50,
55.26, 50.44, 38.52, 32.28, 28.26; Anal. Calcd for C38H37N7O3: C, 71.34; H, 5.83; N,
15.33. Found: C, 71.25; H, 5.66; N, 15.25.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-
1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)quinoline-2-carboxamide (I-11)
Yield 39.5%; yellow solid; m.p.136 - 138℃; ESI-MS m/z: 640.4 ([M+H]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 10.28 (s, 1H), 8.63 (m, 2H), 8.25 (d, J = 8.4 Hz, 1H),
8.15 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 7.2 Hz, 1H), 7.75 (s, 3H), 7.55 (d, J = 7.2 Hz,

1H), 7.47 (d, J = 7.6 Hz, 2H), 7.11 (s, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.76 (s, 1H), 6.65
(d, J = 7.2 Hz, 2H), 5.79 (t, J = 5.2 Hz, 1H), 4.46 (d, J = 5.2 Hz), 3.70 (s, 6H), 3.56 (s,
2H), 2.91 (s, 2H), 2.71 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ ppm: 162.90,
150.14, 147.05, 145.85, 142.07, 141.21, 137.96, 134.75 , 130.47, 129.99, 129.27,
128.87, 128.11, 126.58, 126.50, 125.90, 125.22, 124.55, 121.04, 119.78, 118.73,
117.23, 112.64, 111.83, 110.02, 58.99, 55.49, 55.03, 50.44, 38.98, 32.27, 28.25; Anal.
Calcd for C38H37N7O3: C, 71.34; H, 5.83; N, 15.33. Found: C, 71.40; H, 5.96; N,
15.35.
N-(2-(((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)furan-2-
carboxamide (I-12)
1
Yield: 29.6%; pale-yellow solid; m.p.94 - 96℃; ESI-MS m/z: 579.3 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.57 (s, 1H), 8.57 (s, 1H), 7.90 (s, 1H), 7.74 (d, J
= 8.3 Hz, 2H), 7.46 (d, J = 8.3Hz, 2H), 7.29 (s, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.09 (t,
J = 7.4 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.73 – 6.59 (m, 4H), 5.66 (t, J = 5.4 Hz, 1H),
4.45 (d, J = 5.4 Hz, 2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.89 (d, J = 7.4 Hz, 2H), 2.70 (s,
6H); 13C NMR (75 MHz, DMSO-d6) δ ppm: 162.44, 147.77, 146.88, 145.28, 143.16,
138.59, 136.82 130.00, 127.19, 126.50, 125.98, 125.71, 122.96, 121.00, 119.78,
116.86, 116.30, 114.34, 111.89, 110.02, 59.00, 55.47, 55.03, 50.45, 37.04, 32.29,
28.24; Anal. Calcd for C33H39N6O4: C, 68.50; H, 5.92; N, 14.52. Found: C, 68.55; H,
5.84; N, 14.48.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)thiophene-2-carboxamide (II-1)
1
Yield: 35.4%; pale-yellow solid; m.p. 94 - 96℃; ESI-MS m/z: 519.4 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.68 (s, 1H), 7.94 (d, J = 7.5 Hz, 2H), 7.82 (s,
1H), 7.17 (m, 2H, ArH), 7.04 (d, J = 7.5 Hz,, 1H), 6.78 (s, 1H), 6.63 (s, 3H), 5.55 (t, J
= 5.7 Hz, 1H), 4.52 (t, J = 6.0 Hz, 2H), 4.36 (d, J = 5.7 Hz,, 2H), 3.69 (s, 6H), 3.52 (s,
13
2H), 2.87 (t, J = 6.0 Hz, 2H), 2.64 (s, 4H); C NMR (75 MHz, DMSO-d6) δ ppm:
160.38, 147.22, 147.06, 145.38, 143.33, 139.72, 131.23, 129.13, 127.92, 127.64,
127.10, 126.29, 125.83, 122.99, 116.05, 111.88, 111.42, 109.97, 56.72, 55.50, 54.85,
50.07, 47.02, 40.10, 38.98, 38.67, 28.01; Anal. Calcd for C27H30N6O3S: C, 62.53; H,

5.83; N, 16.20. Found: C, 62.58; H, 5.80; N, 16.13.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)thiophene-3-carboxamide (II-2)
1
Yield: 33.5%; pale-yellow solid; m.p. 95 - 97℃; ESI-MS m/z: 519.4 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.52 (s, 1H), 8.29 (s, 1H), 7.93 (s, 1H), 7.62 (s,
2H), 7.13 (d, J = 6.4 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.63
(s, 2H), 6.57 (s, 1H), 5.52 (t, J = 5.7 Hz, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.35 (d, J = 5.7
13
Hz, 2H), 3.75 – 3.62 (s, 6H), 3.51 (s, 2H), 2.86 (t, J = 6.0 Hz, 2H), 2.64 (s, 4H); C
NMR (75 MHz, DMSO-d6) δ ppm: 161.51, 147.42, 146.90, 145.41, 143.22,
139.23,129.52, 127.28, 126.94, 126.55, 125.82, 123.41, 123.00, 116.07, 111.85,
111.40, 109.97, 56.72, 55.50, 54.85, 50.06, 47.01, 42.50, 40.33, 38.96, 38.67, 28.00;
Anal. Calcd for C27H30N6O3S: C, 62.53; H, 5.83; N, 16.20. Found: C, 62.48; H, 5.85;
N, 16.25.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)-3-methylthiophene-2-carboxamide (II-3)
1
Yield: 47.5%; pale-yellow solid; m.p. 84 - 86℃; ESI-MS m/z: 555.6 ([M+ Na]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.18 (s, 1H), 7.95 (s, 1H), 7.64 (d, J = 4.5 Hz,
1H), 7.20 (d, J = 6.8 Hz, 1H), 7.00 (d, J = 4.5 Hz, 2H), 6.77 (d, J = 6.8 Hz, 1H), 6.63
(s, 2H), 6.58 (s, 1H), 5.43 (t , J = 4.5 Hz, 1H), 4.53 (t, J = 4.8 Hz, 2H), 4.34 (d, J = 4.5
Hz, 2H), 3.69 (s, 6H), 3.52 (s, 2H), 2.87 (t, J = 4.8 Hz, 2H), 2.64 (s, 4H), 2.45 (s,
3H);13C NMR (75 MHz, CDCl3) δ ppm: 161.61, 145.98, 132.29, 127.26, 125.90,
125.58, 122.53, 119.06, 114.32, 111.33, 109.33, 57.11, 55.91, 55.48, 50.82, 47.99,
40.38, 28.47, 15.99; Anal. Calcd for C28H32N6O3S: C, 63.14; H, 6.06; N, 15.78.
Found: C, 63.05; H, 6.09; N, 15.83.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)-5-methylthiophene-2-carboxamide (II-4)
Yield: 35.9%; brown solid; m.p. 92 - 94℃; ESI-MS m/z: 555.6 ([M+ Na]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 9.57 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H), 7.12 (d, J = 6.3
Hz, 1H), 7.07 – 6.98 (m, 1H), 6.90 (s, 1H), 6.75 (d, J = 6.3 Hz, 1H), 6.63 (s, 2H), 6.58
(s, 1H), 5.51 (t, J = 4.5 Hz, 1H), 4.52 (t, J = 4.8 Hz, 2H), 4.35 (d, J = 4.5 Hz, 2H),

13
3.69 (s, 6H), 3.52 (s, 2H), 2.87 (t, J = 4.8 Hz, 2H), 2.64 (s, 4H), 2.46 (s, 3H); C
NMR (75 MHz, DMSO-d6) δ ppm: 160.32, 147.15, 146.86, 145.35, 143.24, 129.40,
127.30, 126.98, 126.41, 126.23, 126.55, 125.72, 125.43, 123.17, 123.09, 123.01,
116.07, 111.78, 111.40, 109.88, 56.72, 55.45, 54.83, 50.05, 46.97, 38.64, 27.99, 15.23;
Anal. Calcd for C28H32N6O3S: C, 63.14; H, 6.06; N, 15.78. Found: C, 63.22; H, 6.01;
N, 15.76.
5-chloro-N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-
1,2,3-triazol-4-yl)methyl)amino)phenyl)thiophene-2-carboxamide (II-5)
1
Yield: 32.3%; pale-yellow solid; m.p. 93 - 95℃; ESI-MS m/z: 553.4([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.74 (s, 1H), 7.90 (s, 1H), 7.83 (s, 1H), 7.23 (d, J
= 7.8 Hz,, 1H), 7.05 (d, J = 6.6 Hz, 2H), 6.74 (d, J = 7.8 Hz, 1H), 6.62 (s, 2H), 6.56 (s,
1H), 5.52 (t, J = 4.5 Hz, 1H), 4.49 (t, J = 4.8 Hz, 2H), 4.34 (d, J = 4.5 Hz, 2H), 3.67 (s,
6H), 3.50 (s, 2H), 2.84 (t, J = 4.8 Hz, 2H), 2.63 (s, 4H); 13C NMR (75 MHz, DMSO-
d6) δ ppm: 159.28, 147.18, 146.88, 145.38, 143.40, 138.24, 136.82, 133.16,129.03,
128.02, 127.38, 126.03, 125.43, 122.72, 122.14, 119.78, 115.94, 111.57, 109.91,
56.72, 55.47, 54.85, 50.03, 46.98, 38.57, 28.00; Anal. Calcd for C27H29ClN6O3S: C,
58.63; H, 5.29; N, 15.20. Found: C, 58.76; H, 5.21; N, 15.16.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)picolinamide (II-6)
1
Yield: 23.5%; brown solid; m.p. 133 - 135℃ ; ESI-MS m/z: 536.5 ([M+Na]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 10.09 (s, 1H), 8.72 (d, J = 4.5 Hz, 1H), 8.13 (d,
J = 7.7 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.96 (s, 1H), 7.69 – 7.63 (m, 1H), 7.45 (d, J
= 7.7 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.69 (t, J = 7.6 Hz,
1H), 6.62 (s, 1H), 6.57 (s, 1H), 5.65 (t, J = 5.5 Hz, 1H), 4.52 (t, J = 5.8 Hz, 2H), 4.34
(d, J = 5.5 Hz, 2H), 3.68 (s, 6H), 3.51 (s, 2H), 2.86 (t, J = 5.8 Hz, 2H), 2.62 (s, 4H);
13C NMR (75 MHz, DMSO-d6) δ ppm: 162.59, 149.91, 149.11, 148.37, 146.84,
145.44, 141.97, 137.92, 126.70, 126.23, 125.75, 125.21, 124.26 , 123.10, 122.19,
116.84, 112.31, 111.74, 109.85, 56.76 , 55.43, 54.84, 50.08, 46.99, 36.68,28.00; Anal.
Calcd for C28H31N7O3: C, 65.48; H, 6.08; N, 19.09. Found: C, 65.41; H, 6.11; N,
19.06.

N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)isonicotinamide (II-7)
Yield: 28.9%; pale-yellow solid; m.p. 98 - 100℃; ESI-MS m/z: 536.5 ([M+Na]+); 1H
NMR (300 MHz, DMSO-d6) δ ppm: 9.90 (s, 1H), 8.77 (d, J = 3.6 Hz, 2H), 7.93 (s,
1H), 7.91 (s, 1H), 7.89 (s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.77
(d, J = 8.0 Hz, 1H), 6.66 – 6.60 (m, 2H), 6.57 (s, 1H), 5.69 (t, J = 5.7 Hz, 1H), 4.52 (t,
J = 6.0 Hz, 2H), 4.36 (d, J = 5.7 Hz, 2H), 3.69 (s, 6H), 3.51 (s, 2H), 2.86 (t, J = 6.0
Hz, 2H), 2.64 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ ppm: 164.19, 150.06, 147.16,
146.86, 145.49, 143.27, 141.59, 127.35, 126.24, 125.79, 123.02, 122.77, 121.79,
115.87, 111.76, 111.28, 109.86, 56.76, 55.44, 54.87, 50.04, 46.98, 28.03; Anal. Calcd
for C28H31N7O3: C, 65.48; H, 6.08; N, 19.09. Found: C, 65.51; H, 6.13; N, 19.01.
2-chloro-N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-
1,2,3-triazol-4-yl)methyl)amino)phenyl)nicotinamide (II-8)
Yield: 33.5%; pale-yellow solid; m.p. 99 - 101℃; ESI-MS m/z: 570.5 ([M+Na]+); 1H
NMR (300 MHz, DMSO-d6) δ ppm: 9.88 (s, 1H), 8.53 (s, 1H), 8.15 (s, 1H), 7.98 (d, J
= 6.0 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.33 (d, J = 6.0 Hz, 1H), 7.06 (s, 1H), 6.79 (d,
J = 7.2 Hz, 1H), 6.73 – 6.61 (m, 2H), 6.58 (s, 1H), 5.42 (t, J = 4.5 Hz, 2H, 1H), 4.54 (t,
J = 6.0 Hz, 2H), 4.38 (d, J = 4.5 Hz, 2H), 3.69 (s, 6H), 3.53 (s, 2H), 2.88 (t, J = 6.0
13
Hz, 2H), 2.65 (s, 4H); C NMR (75 MHz, CDCl3) δ ppm: 150.99, 147.23, 145.60,
141.86, 139.57, 131.60, 127.93, 125.86, 123.58, 122.80, 122.52, 118.70, 113.63,
111.32, 109.34, 57.10, 55.91, 55.47, 50.81, 47.96, 40.09, 28.48; Anal. Calcd for
C28H30ClN7O3: C, 61.37; H, 5.52; N, 17.89. Found: C, 61.32; H, 5.50; N, 17.95.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)-1H-indole-2-carboxamide (II-9)
1
Yield: 38.9%; pale-green solid; m.p. 115 - 117℃; ESI-MS m/z: 552.5 ([M+H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 11.69 (s, 1H), 9.70 (s, 1H), 7.96 (s, 1H), 7.66 (d,
J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.22 (t, J = 7.3 Hz, 2H), 7.07 (t,
J = 7.5 Hz, 2H), 6.82 (d, J = 8.0 Hz, 1H), 6.68 (t, J = 7.5 Hz, 1H), 6.59 (s, 1H), 6.56
(s, 1H), 5.57 (t, J = 5.2 Hz, 1H), 4.52 (t, J = 5.4 Hz, 2H), 4.37 (d, J = 5.2 Hz, 2H),

3.68 (s, 6H), 3.51 (s, 2H), 2.87 (t, J = 5.4 Hz, 2H), 2.62 (s, 4H); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 160.27, 147.16, 146.87, 145.29 , 143.16, 136.62, 131.47, 127.05,
126.23, 125.77, 123.55, 123.15, 121.57, 119.83, 116.24, 112.34, 111.66, 109.86,
103.81, 56.74, 55.44, 54.82, 50.08, 47.01, 38.83, 28.24, 27.99; Anal. Calcd for
C31H33N7O3: C, 67.50; H, 6.03; N, 17.77. Found: C, 67.59; H, 6.01; N, 17.72.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)quinoline-3-carboxamide (II-10)
Yield: 37.5%; pale-yellow solid; m.p. 105 - 107℃; ESI-MS m/z: 564.5 ([M+H]+); 1H
NMR (300 MHz, DMSO-d6) δ ppm: 9.99 (s, 1H), 9.41 (s, 1H), 8.99 (s, 1H), 8.13 (s,
2H), 7.94 (s, 2H), 7.73 (s, 1H), 7.23 (s, 1H), 7.06 (s, 1H), 6.78 (s, 1H), 6.75 – 6.48 (m,
3H), 5.76(t, J = 5.4 Hz, 1H), 4.51(t, J = 5.4 Hz, 2H), 4.38(d, J = 5.2 Hz, 2H), 3.67 (s,
6H), 3.50 (s, 2H), 2.86 (t, J = 5.4 Hz, 2H), 2.62 (s, 4H); 13C NMR (75 MHz, DMSO-
d6) δ ppm: 165.45, 149.36, 148.55, 147.03, 145.53, 143.30, 136.10, 131.18, 130.67,
130.46, 129.09, 128.76, 127.29, 126.82, 126.48, 126.03, 123.08, 115.93, 111.80,
111.31, 109.91, 56.73, 55.46, 54.88, 50.01, 46.99, 38.77, 38.65, 28.00; Anal. Calcd
for C32H33N7O3: C, 68.19; H, 5.90; N, 17.39. Found: C, 68.26; H, 5.93; N, 17.31.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methyl)amino)phenyl)quinoline-3-carboxamide (II-11)
Yield: 45.3%; yellow solid; m.p. 94 - 95℃; ESI-MS m/z: 586.7 ([M+Na]+); 1H NMR
(300 MHz, DMSO-d6) δ ppm: 10.22 (s, 1H), 8.61 (d, J = 7.8 Hz, 1H), 8.28 – 8.16 (m,
2H), 8.11 (d, J = 7.3 Hz, 1H), 7.98 (s, 1H), 7.91 (d, J = 6.4 Hz, 1H), 7.76 (d, J = 6.4
Hz, 1H), 7.50 (d, J = 6.7 Hz, 1H), 7.06 (d, J = 5.7 Hz, 1H), 6.83 (d, J = 7.3 Hz, 1H),
6.73 (d, J = 6.4 Hz, 1H), 6.59 (s, 1H), 6.54 (s, 1H), 5.71 (t, J = 5.4 Hz, 1H), 4.52 (t, J
= 5.4 Hz, 2H), 4.38 (d, J = 5.2 Hz, 2H), 3.67 (s, 6H), 3.50 (s, 2H), 2.86 (t, J = 5.4 Hz,
13
2H), 2.61 (s, 4H); C NMR (75 MHz, DMSO-d6) δ ppm: 162.87, 150.13, 147.18,
146.89, 145.87, 145.52, 142.18, 137.93, 130.54, 129.30, 128.87, 128.13, 126.37,
125.78, 125.35, 124.22, 123.05, 120.39, 118.73, 116.90, 112.42, 111.81, 109.92,
56.74, 55.47, 54.83, 50.07, 47.05, 36.68, 27.99; Anal. Calcd for C32H33N7O3: C, 68.19;
H, 5.90; N, 17.39. Found: C, 68.10; H, 5.88; N, 17.44.
N-(2-(((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-

triazol-4-yl)methyl)amino)phenyl)furan-2-carboxamide (II-12)
1
Yield: 39.6%; pale-yellow solid; m.p. 96 - 98℃; ESI-MS m/z: 503.3 ([M+ H]+); H
NMR (300 MHz, DMSO-d6) δ ppm: 9.55 (s, 1H), 7.92 (d, J = 11.1 Hz, 2H), 7.26 (s,
1H), 7.15 (d, J = 7.3 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.63
(m, 4H), 5.55 (t, J = 5.4 Hz, 1H), 4.52 (t, J = 5.4 Hz, 2H), 4.34 (d, J = 4.8 Hz, 2H),
3.69 (s, 6H), 3.52 (s, 2H), 2.87 (t, J = 5.4 Hz, 2H), 2.64 (s, 4H); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 156.80, 147.74, 147.16, 146.86, 145.32, 143.14, 127.06, 126.25,
125.78, 122.94, 118.99, 116.11, 114.31, 111.86, 111.48, 109.88, 56.74, 55.45, 54.84,
50.08, 46.99, 38.65, 27.99; Anal. Calcd for C27H30N6O4: C, 64.53; H, 6.02; N, 16.72.
Found: C, 64.46; H, 5.98; N, 16.80.
4.2 Molecular docking studies
P-gp protein (PDB ID: 6FN1) was setout and optimized with relevant module by
Schrodinger software. Receptor Grid Generation Module was utilized to produce
docking area. Structure of ligands were drawn by ChemOffice2017 software and
prepared using LigPrep module. Ultimately, molecular docking between ligands and
receptor was conducted with extra precision.
4.3 Cytotoxicity and MDR reversal assay
The cell viability was tested by MTT assay with a minor modification.[30-32] K562
and K562/A02 cells were harvested during logarithmic growth phase, and were
seeded in 96-well micro-titer plates at 1×104 cells per well. In the MTT assay for
anticancer MDR reversal experiments, cells were exposed to the presence of DOX
with or without P-gp inhibitors for 48 h. 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% CO2, the absorbance at 490 nm was read on a microplate reader
(Thermo, USA). The IC50 values of the compounds for cytotoxicity were calculated
by GraphPad Prism 6.0 software (GraphPad software, San Diego, CA, USA) from the
dose–response curves.
4.4 Duration of the MDR reversal
The experiment was carried out as the reported procedures with minor
modification.[33] In brief, K562/A02 cells were seeded in 96-well micro-titer plates

at 1 × 104 per well during logarithmic growth phase, cells were incubated for 24 h
with or without 5.0 μM of VRP, Tar, I-5 or PBS before being washed 0 or 3 times
with culture medium. Then, the cells were incubated for 0, 6, 12, or 24 h before the
addition of varying concentrations of DOX or vehicle. The incubation was continued
for 48 h prior to the MTT assay.
4.5 DOX intracellular accumulation
The reported procedures with minor modification was employed for the detection of
accumulation of DOX [25]. K562 and K562/A02 cells were seeded into 24-well
plates 1.5×104 per well. Different concentrations of VRP, Tar and I-5 (0.5 μM, 2.5
μM, 5.0 μM) were pre-incubated with cells for 60 min. Then 20 μM DOX was added
into each well and incubated for 90 min, washed with ice-cold PBS for three times at
4℃. The fluorescence intensity of cells can be observed through fluorescence
microscope. Afterwards the cells were disintegrated by Triton×100 liquid. The mean
fluorescence intensity (MFI) of accumulation intracellular DOX was measured by
fluorescence spectrophotometer. Data were expressed as means ± SD of three
independent experiments.
4.6 Rh123 efflux assay
K562 and K562/A02 cells were seeded into 24-well plates 1.5×104 per well and
incubated with 5μM Rh123 for 60 min before washing with ice-cold PBS for three
times. Then the cells were incubated with or without 2.0 μM I-5 or 5.0 μM VRP for
another 90 min. Afterwards the cells were washed thrice in ice-cold PBS. Afterwards
the cells were disintegrated by Triton×100 liquid [35]. The MFI of retained
intracellular Rh123 was estimated by BD FACSCalibur flow cytometer through the
FL1 tunnel. Data were expressed as means ± SD of three independent experiments.
4.7 Western blotting
After 48h incubation with VRP (5.0μM), Tar (5.0μM), I-5 (0.5μM, 5.0μM),
K562/A02 cells were harvested and washed trice with ice-cold PBS and lysed with
RIPA lysis buffer containing 10% PMSF. Total protein was extracted by centrifuging
at 12000r for 15 min at 4℃. Total protein content was determined by BCA Protein
Assay kit. The Protein samples were separated by 8% SDS-PAGE gel electrophoresis

and the proteins were transferred to PVDF membranes. Then the membranes were
blocked with TBST(10mM Tris-HCl, pH 7.5, 150mM NaCl and 0.1% Tween 20)
containing 5% nonfat dried milk for 1h and probed with the specific P-gp antibody, β-
tublin antibody overnight at 4℃ . After being washed with TBST 3 times, the
membranes were incubated with the secondary antibodies for 2h at room temperature.
After washing for another three times, proteins were visualized using the enhanced
chemiluminescence detection system and Quantity One software were used to
analyses the protein’s band intensity [35, 36].
Acknowledgements
This study was supported by National Natural Science Foundation of China (No.
81872733, 81872734 and 81803353).
Conflict of interest
The authors declare no competing financial interest.
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