Synthesis and biological evaluation of JL-A7 derivatives as potent ABCB1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.06.015

Cancer chemotherapy failure is often due to the overexpression of ATP-binding cassette (ABC) transporters (particularly ABCB1), resulting in a variety of structurally and pharmacologically unrelated drugs efflux. The multidrug resistance (MDR) phenomenon could be reversed by ABCB1 inhibitors. Now, JL-A7 as the lead compound based on a triazol-N-ethyl-tetrahydroisoquinoline scaffold, 18 compounds were designed and synthesized. Substitution in para positions yielded high activities toward ABCB1. Moreover, compound 5 could effectively block the drug efflux function of ABCB1 and increase the accumulation of anti-cancer drugs to achieve effective treatment concentration in MDR cells.

Full text of DOI:10.1016/j.bmc.2017.06.015

Accepted Manuscript
Synthesis and biological evaluation of JL-A7 derivatives as potent ABCB1 in-
hibitors
Miaobo Pan, Jian Cui, Lei Jiao, Hesham Ghaleb, Chen Liao, Jiaqi Zhou, Mutta
Kairuki, Haiyan Lin, Wenlong Huang, Hai Qian
PII:
S0968-0896(17)30736-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.015
BMC 13798
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2017
11 June 2017
12 June 2017
Please cite this article as: Pan, M., Cui, J., Jiao, L., Ghaleb, H., Liao, C., Zhou, J., Kairuki, M., Lin, H., Huang, W.,
Qian, H., Synthesis and biological evaluation of JL-A7 derivatives as potent ABCB1 inhibitors, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.015
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Synthesis and biological evaluation of JL-A7 derivatives as potent ABCB1 inhibitors
†
†
†
Miaobo Pana , Jian Cuia , Lei Jiaoa,b , Hesham Ghaleba, Chen Liaoa, Jiaqi Zhoua, Mutta Kairukia,
Haiyan Linc, Wenlong Huanga,d*, Hai Qiana, d*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Pharmacology, State-Province Key Laboratories of Biomedicine-Pharmaceutics,
Key Laboratory of Cardiovascular Research, Ministry of Education , College of Pharmacy,
Harbin Medical University, Harbin 150000, PR China
c Department of Biochemistry and Molecular Biology, Nanjing Medical University, 140
Hanzhong Road, Nanjing 210029, PR China
d Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Abstract
Cancer chemotherapy failure is often due to the overexpression of ATP-binding cassette (ABC)
transporters (particularly ABCB1), resulting in a variety of structurally and pharmacologically
unrelated drugs efflux. The multidrug resistance (MDR) phenomenon could be reversed by
ABCB1
inhibitors.
Now,
JL-A7
as
the
lead
compound
based
on
a
triazol-N-ethyl-tetrahydroisoquinoline scaffold, 18 compounds were designed and synthesized.
Substitution in para positions yielded high activities toward ABCB1. Moreover, compound 5
could effectively block the drug efflux function of ABCB1 and increase the accumulation of
anti-cancer drugs to achieve effective treatment concentration in MDR cells.
Key words: ABCB1 inhibitors, MDR, p-glycoprotein, cancer resistance.
*Co-corresponding authors: Wenlong Huang and Hai Qian, Centre of Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271051; Fax: +86-25-83271051.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian).
† Contributed equally to the first author.
1

1. Introduction
The Multidrug resistance (MDR) is cross-resistant to structurally and pharmacologically
distinct class of drugs, remaining one of impediment for tumor chemotherapy in clinic.1 In general,
MDR-mediated xenobiotic efflux is mainly related to the over-expression of membrane-associated
transport proteins named ATP-binding cassette (ABC) transport proteins.2-4 These transport
proteins, especially ABCB1(P-glycoprotein/P-gp), ABCC1 (multidrug resistance protein 1/MRP)
and ABCG2(breast cancer resistance protein /BCRP) are believed to expel a wide variety of
anti-tumor drugs out of tumor cells, leading to the emergence of MDR.5, 6 In recent years, the
importance of ABCB1 in cancer chemotherapy is widely recognized.5
During the past decades, numerous ABCB1 inhibitors have been discovered in a continuing
quest to reverse MDR in tumor.7-9 Unfortunately，none of above compounds has been approved
present in clinic application.10 The failure may be attributed to many reasons with example
implicated in cytotoxicity，influence on P450 enzymes, and adverse drug interaction.6, 10, 11
Actually，some third-generation ABCB1 inhibitors, like HM30181,12 zosuquidar (LY335979),13
and WK-X-34,8, 9 have shown potent activity and remarkable promise, even tariquidar (XR9576)
(Figure.1) have reached phase III clinical trials.6, 14 As many deficiencies of the existing ABCB1
inhibitors, there is a need to develop more effective and selective inhibitors of the protein.
2

Figure 1. Structures of third generation p-gp inhibitors: HM30181, WK-X-34, Tariquidar and
Zosuquidar.
We have reported the synthesis and biological evaluation of various substituted
triazol-N-ethyl-tetrahydroisoquinoline that proved to be even more potent inhibitors of ABCB1.15
Moreover, compound JL-A7 exhibited better potency than verapamil (VRP) on increasing
anticancer drug accumulation in K562/A02 cells. It could significantly reverse MDR in a
dose-dependent manner and also persist longer chemo-sensitizing effect than VRP. Further
mechanism studies revealed that compound JL-A7 could inhibit the efflux function of P-gp. In
drug design, the strategy of amide reversal is always adopted to improve the biological activity of
the compound. To find more promising candidates for MDR cancer treatment, with JL-A7 as the
leading compound, 18 new compounds were synthesized by the optimization of amide inversion
(Figure.2). And then more comprehensive activity evaluation was performed, consist of toxicity
assessment, MDR reversal activity in vitro, and effects on ABCB1.
Figure 2. (A) Previous design of JL-A serious compounds. (B) Structures of JL-A7 and Design of
the target compounds 1–18.
2. Chemistry
Synthesis of target compounds 1–18 are outlined in Scheme 1 Compounds a–c were
synthesized according to literature procedures with minor modification. Compounds 1d-18d were
3

prepared by amide condensation，starting from the disparate Aromatic amines and salicylic acid.
Compounds 1e-18e were prepared by 1d-18d and 3-Bromo-1-propyne, which refluxed in the
mixture of acetonitrile with potassium carbonate for 6 h. Subsequently, compound c and
compounds 1e–18e were treated with sodium ascorbate and copper sulfate in 75% methanol
stirring at room temperature for 24–48 h to provide compounds 1–18. The structures of target
compounds obtained were listed in Table 1.
Scheme 1. Synthesis of the target compounds.
Reagents and conditions: (i) NaN3, water, 80°C, 24 h; (ii) TEA/DCM, TsCl, r.t, 24 h; (iii)
TEA/acetonitrile, 60°C, 24h; (iv) aromatic amines, EDCI/HOBT, DCM r.t.; (v)
3-bromoprop-1-yne,
K2CO3,
acetone,
reflux,
4
h;
(vi)
2-(4-Azidophenethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (c), ascorbate sodium, CuSO4,
75% CH3OH, 24–48 h.
Table 1 Structures and ADM-resistance reversal activity of the target compounds 1–18 at 5 µM
concentration in K562/A02 cellsa
4

R
Compounds
IC50 of ADM(µmol/L)
12.16 0.60
7.65 1.06
RF
8.0
1
2
3-CH3
4-CH3
12.6
11.6
12.4
33.5
14.0
11.1
4.3
3
4-OCH3
3,4-OCH3
4-C(CH3)3
4-Cl
8.38 1.63
7.82 0.45
2.89 0.43
6.93 0.73
8.73 1.18
4
5
6
7
3-OCH3
3-Cl
8
22.82 1.95
21.91 3.19
25.52 1.50
31.92 3.10
8.29 1.22
4.52 1.49
6.25 0.91
21.07 1.66
27.69 0.20
26.92 1.58
17.31 0.66
9
2-CH3
4.4
10
11
12
13
14
15
16
17
18
JL-A7
H
3.8
3,4,5-OCH3
4-F
3.0
11.7
21.4
15.5
4.6
4-OCF3
4-CH2CH2CH2CH3
4-NO2
3-NO2
3.5
2-NO2
3.6
3-OCH3
-
5.6
28.2
3.42±1.12
ADM
tariquidar
VRP
-
-
-
96.91 12.24
1.97 0.23
1.0
49.1
5.5
17.55 1.32
a The IC50 value was determined after exposure to a series of ADM concentration with different target
compounds at 5μM using K562/A02 cells. Reversal fold (RF) refers to fold-change in drug sensitivity.
5

RF = (IC50 without modulator)/(IC50 with 5μM modulator).
3. Biological evaluation
3.1 Reversal of MDR in K562/A02 cells
The activity of the compounds in reversing multidrug resistance in tumors toward K562/A02
cells
(ABCB1-overexpression)
were
preliminarily
evaluated
by
the
3-(4,
5-dimethyl-2-thia-zolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method.16 We chose
Verapamil (VRP) and tariquidar as positive controls，and tested all of compounds’ intrinsic toxity
towards K562/A02 cells. As the results summarized in Table 1, control group with Adriamycin
(ADM) alone displayed little inhibitory effect (IC50 of 96.91
12.24 µM). However, the
combination groups, ADM with target compounds increased the inhibitory effect in different
degrees. It revealed that most of the compounds showed significant MDR reversal activity.
Among them, compound 5 showed the best potent as reversal fold (RF) was up to 31.4, superior to
JL-A7 (RF = 28.2), tariquidar (RF = 49.1), VRP (RF = 5.5).
3.1.1 Chemo-sensitizing effect
To further investigate compound 5 activity on reversing MDR, we evaluated its therapeutic
effect and safety by testing the half-maximal effective concentration (EC50) and therapeutic index
(TI).
The reversal activity of compound 5 was determined at various concentrations (5.0 µM, 2.5
µM, 1.0µM, 0.5 µM, 0.25 µM, 0.1µM, 0.05 µM) towards K562/A02 cells by MTT assay, selecting
VRP as the positive control. As demonstrated in Table 2, VRP showed slight modulating activity
at 5.0 µM (RF = 2.3). However, compound 5 showed apparent dose dependent activity and still
exhibited potent MDR reversal activity (RF = 3.9) when the concentration decreased to 0.5µM.
Additionally, the EC50 value of compound 5 was 256.2±6.2 nM, which was calculated by
GraphPad Prism 6.0 software from the dose response curve as showed in Figure 3. The results
suggested that compound 5 possessed the significantly potential to enhance the sensitivity of
ABCB1-overexpressing cells to anticancer drug in a dose-dependent manner, at Non-toxic
concentration.
Table 2 Percentage of IC50 of ADM and RF in different concentrations of compound 5 in
6

K562/A02cells.
Compounds
IC50 of ADM (µM)
RF
None
43.75 1.65
19.84 0.82
1.27 0.07
3.33 0.50
6.02 0.52
12.52 1.08
18.39 0.86
34.90 2.10
37.18 2.37
37.21 2.21
1
VRP, 5.0 µM
2.3
24.1
13.5
7.5
3.9
2.4
1.6
1.4
1.4
Compound 5, 5.0 µM
Compound 5, 2.5 µM
Compound 5, 1.0 µM
Compound 5, 0.5 µM
Compound 5, 0.25 µM
Compound 5, 0.1 µM
Compound 5, 0.05 µM
Compound 5, 0.025 µM
Figure 3. Dose response curve for the compound 5 in K562/A02 cells.
Various concentrations of target compound (0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, or 5.0 μM) and
ADM were added to 96-well plates. The cells were incubated for 48 h. The absorption values were
measured with a Microplate Reader at 570 nm. Data were analyzed with GraphPad Prism 6.0
software and are the mean SD for 3 independent tests.
3.1.2 Duration of reversal effects
It is known that a relatively long duration of action with reversibility for an ABCB1 inhibitor
is required for safe and effective therapy of ABCB1-mediated MDR. Therefore, the duration of
MDR reversal effect of compound 5 was evaluated according to the procedure previously
described with minor modification.15 VRP was chosen as the control. As demonstrated in Table 3,
the IC50s of ADM towards K562/A02 cells which pretreated with VRP and compound 5, were
7

10.73 µM, 1.26 µM respectively (no wash group). The MDR- reversing effects of VRP (RF = 1.6)
and compound 5 (RF = 11.83) decreased immediately after their removal from the medium.
However, the reversal effect of VRP was disappeared after its removal from the medium for 6 h.
In contrast, compound 5 showed reversal activity even after its removal from the medium for 24 h
and the IC50 of ADM was 10.32 µM (RF = 4.93). These data indicated that compound 5 displayed
potent MDR-reversing effect which could persist for longer time compared with the positive
control VRP.
Table 3 Duration of MDR reversal in K562/A02 cells after incubation and washout of verapamil,
tariquidar and compound 5
IC50/ADM [μM] (RF)
Treatment
schedule
Control
VRP
Tariquidar
Compound 5
51.34 5.1
(1.0)
10.73 2.1
(4.98)
1.60 0.2
(31.98)
1.26 0.2
(40.34)
No wash
wash, 0 h
wash, 6 h
wash, 12 h
wash, 24 h
31.28 2.77
(1.6)
3.02 0.2
(16.86)
4.33. 0.3
(11.83)
nd
nd
nd
nd
4.97 1.0
(10.37)
6.32 0.6
(8.16)
nd
nd
nd
8.28 0.5
(6.10)
10.32 1.2
(4.93)
14.39 0.7
(3.55)
17.21 3.4
(2.96)
Numbers in parentheses, reversal fold (RF), RF = (IC50 without modulator)/ (IC50 with modulator).
Each experiment was carried out two to three times, and the values were presented as the mean SD
nd: not determined.
3.2 Cytotoxicity assay
Since most potent molecular failure due to toxicity within the therapeutic concentration
range,6 the intrinsic cytotoxicity of 18 compounds against parental sensitive K562 cells and
K562/A02 cells (ADM-resistant sub-lines) were evaluated by MTT assay. Chemotherapy drug
ADM and ABCB1 inhibitors VRP and tariquidar were selected as controls.
As shown in Table 4, VRP had weak cytotoxic effects toward K562 and K562/A02 cells
with IC50 values 61.23 and 47.34M, respectively. Nevertheless, tariquidar displayed a high level of
toxicity toward K562 cells (IC50 of 31.56 µM) and K562/A02 cells (IC50 of 27.19 µM). In contrast,
8

all of the synthesized compounds showed no toxicity with IC50 values higher than 100 µM to
K562/A02 cells. Compound 3 and 8 were found to exhibit quite high toxicity (IC50 < 30µM) in
K562 cells.
Table 4 Cytotoxicity of the target compounds against K562 and K562/A02 cell lines
Cytotoxicity IC50 (µM)
Compounds
K562
K562/A02
>100
>100
>100
>100
>100
1
2
3
79.24±5.61
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
5
6
7
8
87.40±3.09
>100
>100
>100
>100
9
10
11
>100
>100
12
>100
>100
13
>100
>100
14
>100
>100
15
>100
>100
16
>100
>100
17
>100
>100
18
>100
>100
JL-A7
VRP
tariquidar
>100
>100
65.28 3.54
31.56 2.32
4.29 2.28
56.24 2.12
27.19 1.41
96.91 12.24
ADM
a The IC50s for the target compounds were determined by MTT method. Each experiment was carried
out 3 times.
3.3 Effects on ABCB1
Investigation of the inhibitory activity of the test compounds against ABCB1 was performed
with K562/A02 cell line. As ADM and Rhodamine 123 (Rh123) are fluorescent dye and substrate
of ABCB1, an increase of fluorescence by intracellular accumulation can be correlated to the
inhibition effects on ABCB1 of the compound.17
3.3.1 ADM accumulation
Firstly, in order to investigate the mechanism of compound 5 in modulating the anticancer drug
9

substrates accumulation level inside K562/A02 cells. The mean fluorescence intensity (MFI) of
retained intracellular DOX was estimated by BD FACSCalibur flow cytometer through the FL2
tunnel. The classical P-gp inhibitor VRP was chosen as the positive control. As the results shown
in Figure 4, K562/A02 cells treated with compound 5, the MFI of cells increased much more than
treated with VRP at the same concentration. The result indicated that compound 5 was more
potent than VRP in DOX accumulation in K562/A02 cells.17, 18
Figure 4. The effect of target compounds on the DOX accumulation in K562 or K562/A02 cells.
K562 or K562/A02 Cells were incubated with 20μM Rh123 without or with compound 5 or VRP
(5μM) for 150 min at 37℃ before washing with ice PBS for three times. The MFI of retained
intracellular DOX was measured by BD FACSCalibur flow cytometer through the FL2 tunnel. The
shift of the histogram to the right indicates an increase in intracellular accumulation of DOX due
to the inhibition of P-gp. (A: K562 and K562/A02 without modulator; B: K562/A without
modulator vs K562/A02 with 5μM compound 5; C: K562/A without modulator vs K562/A02 with
5μM VRP)
Furthermore, when incubated with modulators at various concentrations, the MFI of DOX
accumulated in K562/A02 cells showed dose-effect dependence. As shown in Figure 5, in blank
groups，the level of ADM in K562 cells was about 4.5-fold higher than that of K562/A02 cells in
the absence of ABCB1 inhibitors. And groups treated with ABCB1 modulators (such as VRP or
compound 5) showed increasing concentration than blank in K562/A02 cells. Once treated with
compound 5 (2.5 µM), the retained amount of ADM in the K562/A02 cells significantly increased.
When the K562/A02 cells were treated with compound 4 at various concentrations (0.5, 2.5, 5
µM), the accumulation of ADM was remarkably increased in a dose-dependent manner.
Additionally, the intracellular ADM level of compound 5 (5 µM) was 1.4 times higher than that of
VRP in K562/A02 cells at the same dose. The results suggested that ABCB1 can pump ADM out
of the cells which leads to a lower ADM level in K562/A02 cells，and compound 5 was more
10

potent than VRP in inhibiting the drug efflux function of ABCB1.
Figure 5. Effect of compound 5 and verapamil (VRP) on ADM intracellular accumulation in
K562/A02 cells.
*, P < 0.05, **, P < 0.01 compared with 5 the group of K562/A02 cells without modulators. Data were
analyzed with GraphPad Prism 6.0 software and are the mean SD for 3 independent tests.
3.3.2 Inhibition on Rh123 efflux
Furthermore, another ABCB1 substrate with fluorescent Rh12319 was chosen to further assess
effects on ABCB1 by compound 5. As shown in Figure 6., both compound 5 and VRP can inhibit
Rh123 efflux from K562/A02 cells, but the inhibitory effect of compound 5 was obviously better
than that of VRP over 90 min interval on the same condition. The rate constant (K) of efflux in
compound-treated K562/A02 cells was 0.0067 0.0095 s-1, which was lower than the value of both
the Vehicle control group (0.0231 0.0058 s-1) and the VRP-treated group (0.0092 0.0035 s-1).
These results above verified our presumption that compound 5 was an effective ABCB1
inhibitor by blocking the drug efflux function of ABCB1. Moreover, the potency of compound 5 is
much higher than the classical ABCB1 inhibitor VRP under the same conditions.
11

Figure 6. The effect of compound 5 and verapamil (VRP) on the efflux of rhodamine 123 (Rh123)
in K562/A02 cells. Data were analyzed with GraphPad Prism 6.0 software and are the mean SD
for 3 independent tests.
4. Conclusion and discussion
In summary, 18 compounds were designed on the base of amide reversal and evaluated in
vitro as ABCB1-mediated MDR reversal agents. The reversal of the amide bond keeps the activity
of the compound maintained or even enhanced. Among them, compound 5 is a potent MDR
reverse agent showing a dose-dependent manner well below the toxic concentration. Additionally,
compound 5 could effectively block the drug efflux function of ABCB1 to increase the
accumulation of anti-cancer drugs to achieve effective treatment concentration in MDR cells.
Therefore, compound 5 could be served as a promising candidate for the development of
ABCB1-mediated MDR-Reversal agents.
According to data in Table 1 above, the structure and activity relationship can be derived:
The Electronic effect of the substituents at the aniline moiety play a minor role. Substitution in
para positions yielded high activities. For example, compounds 5, 6, 13 and 14 with substitution in
para positions showed higher MDR reversal activity than others, applicable in JL-A7. However,
no distinct trend for the electron withdrawing substitutions was found, such as compound 15. In
particular, the benzene ring para-substituted compounds show great difference in reversal activity.
Among the para-substituted compound, compound 14 (RF=15.5) with n-butyl substituted has a
better activity than the methyl (compound 2, RF=12.6), methoxy (compound 3, RF=11.6) and
12

chlorine (compound 6, RF=14.0) substituted compounds but is weaker than the t-butyl substituted
compound (compound 5, RF=33.5). This difference may be related to the steric hindrance at the
active site, and may also be related to the binding force of the compound to the hydrophobic
pocket, which is worth discussing further.
To analyze and further characterize the interaction between target compound and P-gp, the
most promoting compound 5 was chosen to docking with a model of P-gp (PDB code: 3G60) As
shown in Figure 7, the interacting mode of compound 5 revealed the structure occupied a small
hydrophobic pocket of Tyr-949 and PHE-339
.
Figure 7. Docking interaction of compound 5 with P-gp (PDB ID: 3G60).
5. Experimental section
5.1. General chemistry
All reagents were reagent grade and all solvents were dried by standards methods before
using. Column chromatography was carried out on silica gel or alumina (200–300 mesh). Melting
points were measured using a Mel-TEMP II melting point apparatus, which was uncorrected. All
1
of the target compounds were analyzed by H NMR, 13C NMR (Bruker ACF-300Q, 300 MHz),
MS (1100 LC/MSD spectrometer; Hewlett–Packard); Thin-layer chromatography (TLC) was
performed on GF/UV 254 plates and the chromatograms were visualized under UV light at 254
and 365 nm. Compound e was prepared as previously described.
5.2. Synthesis of 2-azidoethanol (a)
To a 100 mL round bottom flask was added 2-bromoethanol (5.3 g, 42.4 mmol) and sodium
azide (5.5 g, 84.6 mmol) in water (50 mL). The mixture was stirred at 80℃ for 24 h, and then,
cooled to room temperature. The solution was extracted with ethyl acetate (4×30 mL) and the
13

organic layer was dried with sodium sulfate overnight, then, filtered. After the removal of the
solvent under vacuum, compound a was obtained as a crude pale yellow liquid (3.3g, yield:
90.5%), which was used in the next step directly. 1H NMR (300 MHz, DMSO-d6) δ ppm: 3.78 (t,
J = 5.1 Hz, 2H, OCH2CH2N3), 3.45 (t, J = 5.1 Hz, 2H, OCH2CH2N3), 2.02 (s, 1H, OH).
5.3. Synthesis of 2-azidoethyl 4-methylbenzenesulfonate (b)
To the solution of 2-azidoethanol (3.33 g, 37.9 mmol) and triethylamine (10 mL, 72.1 mmol) in
dry dichloromethane (25 mL), 4-toluenesulfonylchloride (6.2 g, 32.5 mmol) in dry
dichloromethane (25 mL) was added dropwise under constant stirring at 0℃ and then the mixture
was stirred at room temperature for 24 h. The reaction mixture was washed with 1 N HCl (3×40
mL) and 1N NaHCO3 (3×40 mL) and brine (3×40 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product was
purified by silica gel column chromatography using a mixture of ethyl acetate/ petroleum ether
(1:15, v/v) as eluent to afford the compound b (4.13 g). As colorless liquid, yield: 52.6%
5.4. Synthesis of 2-(2-azidoethyl)-6, 7-dimethoxy-1, 2, 3, 4- tetrahydroisoquinoline (c)
To a solution of 2-azidoethyl-4-methylbenzenesulfonate (4.1 g, 17 mmol) in dry acetonitrile (50
mL) and triethylamine (5.0 mL, 36 mmol), 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline
hydrochloride (3.9 g, 17 mmol) was added. The mixture was refluxed for 24 h, evaporated in
vacuo, and chromatographed on a silica gel column (EtOAc/PE 3/2 to EtOAc/MeOH 16/1, v/v) to
give compound c (3.1 g). As pale yellow solid, yield: 68.7%. 1H NMR (300 MHz, CDCl3) δ ppm:
6.61 (s, 1H, ArH), 6.53 (s, 1H, ArH), 3.85 (s, 6H, 2×OCH3), 3.64 (s, 2H, ArNCH2), 3.52 (t, J = 6.1
Hz, 2H, ArCH2), 2.80 (dd, 6H, J = 13.3, 6.1 Hz, 3×CH2).
5.5. General procedure for the preparation of 1d–17d
To a solution of Salicylic acid (2 mmol) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (2.2 mmol) in dichloromethane (30 mL)，then aromatic amine (2 mmol) was added，
then the solution was stirred at temperature for 3h. Water (80 ml) added, and extracted with
dichloromethane. Then, the organic layer was dried by anhydrous Na2SO4. After filtration, the
solvent was evaporated and the crude product was purified by silica gel column chromatography
using a mixture of ethyl acetate/methanol (8:1, v/v) as eluent to give the desire white solid product
compounds.
14

5.5. General procedure for the preparation of 1e–18e
To a solution of 1d-18d (1 mmol) and 3-bromo-prop-1-yne (1.1 mmol) in acetonitrile (15 mL),
potassium carbonate (3 mmol) was added, then the mixture was heated to reflux for 6 h. Then the
reaction mixture was cooled to room temperature followed by filtration and evaporated in vacuo to
get the desired product compound 1e–18e.
5.6. General procedure for the preparation of compounds 1–18
To the solution of 1e–18e (1 mmol) and c (1 mmol) in 75% methanol (20 mL), ascorbate sodium
(30 mg) and CuSO4 (10 mg) were added, respectively. The reaction solution was stirred at room
temperature for 24–48 h. The solution was evaporated in vacuo and extracted with
dichloromethane. Then, the organic layer was dried by anhydrous Na2SO4. After filtration, the
solvent was evaporated and the crude product was purified by silica gel column chromatography
using a mixture of ethyl acetate/methanol (15:1, v/v) as eluent to give the desire white solid
product compounds, Yeild: 38.5%-71.7%.
5.6.1
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(m-tolyl)benzamid (1)
Yield 75.9%; mp: 127-129℃; pale yellow powder; 1H NMR(300 MHz, DMSO-d6, ) δ ppm: 10.11
(s, 1H, CONH), 8.24 (s, 1H, NCH=C), 7.77 (d, J = 6.6Hz, 1H, ArH), 7.45 (m, 4H, ArH), 7.16 (m,
2H, ArH), 6.89 (d, J = 7.4Hz, 1H,ArH), 6.61 (s, 1H, ArH), 6.54 (s, 1H, ArH), 5.36 (s, 2H, OCH2),
4.58 (t, J = 5.8Hz, 2H, CHNCH2), 3.68 (s, 6H, 2×OCH3), 3.49 (s, 2H, ArCH2N), 2.86 (t, J = 6.0Hz,
2H, NCH2), 2.62 (s, 4H, 2×CH2), 2.28 (s, 3H, CH3); 13C NMR (DMSO, 75 MHz) δ ppm:21.1,
28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.2, 109.8,111.7, 113.6, 116.5, 119.9, 121.2, 123.9, 124.2,
124.8, 125.7, 126.1, 128.5, 130.4, 132.4,137.9, 138.8, 141.9, 146.8, 147.1, 155.5, 163.5; 528.4 ;
ESI-HRMS m/z calcd for C30H33N5O4 [M+Na]+ 550.2425, found 550.2420.
5.6.2
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(ptolyl)benzamide (2)
1
Yield 56.9%; mp: 167-168℃; pale yellow powder; H NMR(300 MHz, DMSO-d6) δ ppm: 10.09
(s, 1H, CONH), 8.23 (s, 1H, NCH=C), 7.76(d, J = 7.6Hz, 1H, ArH), 7.50 (m, 3H, ArH), 7.37 (d, J
15

= 8.8Hz, 1H, ArH), 7.10 (m, 3H, ArH), 6.62 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.35 (s, 2H, OCH2),
4.58 (t, J = 5.9Hz, 2H, CHNCH2), 3.68 (s, 6H, 2×OCH3), 3.50 (s, 2H, ArCH2N), 2.86 (t, J = 5.9Hz,
2H, NCH2), 2.63 (s, 4H, 2×CH2), 2.25 (s, 3H, CH3); 13C NMR (DMSO, 75 MHz)δ ppm: 20.4,
28.0, 47.0, 50.0, 54.8, 55.4, 56.7, 62.2, 109.7,111.7, 113.6, 119.3, 121.1, 124.1, 124.7, 125.7,
126.1, 129.1, 130.3, 132.3, 132.4, 136.4,142.0, 146.8, 147.1, 155.5, 163.4; ESI-HRMS m/z calcd
for C30H33N5O4 [M+Na]+ 550.2425, found 550.2422.
5.6.3
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(4-methoxyphenyl)benzamide (3)
Yield 46.0%; pale yellow powder; mp: 172-173℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.07
(s, 1H, CONH), 8.23 (s, 1H, NCH=C), 7.75( d, J = 6.8Hz, 1H, ArH), 7.50 (m, 3H, ArH), 7.37 (d, J
= 7.8Hz, 1H, ArH), 7.10 (dd, J = 6.8Hz ,6.8Hz, 1H, ArH), 6.88 (d, J = 7.9Hz, 2H, ArH),6.62 (s,
1H, ArH), 6.55 (s, 1H, ArH), 5.35 (s, 2H, OCH2), 4.58 (t, J = 5.9Hz, 2H, CHNCH2), 3.73(s, 3H,
OCH3), 3.67(s, 6H, 2×OCH3), 3.49 (s, 2H, ArCH2N), 2.89 (s, 2H, NCH2), 2.63 (s, 4H, 2×CH2);
13C NMR (DMSO, 75 MHz) δ ppm: 28.0, 47.1, 50.0, 54.8, 55.1, 55.4, 56.7, 62.3, 109.8,111.7,
113.6, 113.8, 120.8, 121.1, 124.2, 124.7, 125.7, 126.2, 130.3, 132.1, 132.2, 142.1,146.8, 147.1,
155.4, 163.2; ESI-HRMS m/z calcd for C30H33N5O5 [M+Na]+ 566.2374, found 566.2380.
5.6.4
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(3,4-dimethoxyphenyl)benzamide (4)
Yield 59.3%; pale yellow powder; mp:118-120℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.11 (s,
1H, CONH), 8.24 (s, 1H, NCH=C), 7.82(d, J = 7.2Hz, 1H, ArH), 7.50(m, 1H, ArH), 7.37 (m, 2H,
ArH), 7.23 (d, J = 8.3Hz ,1H, ArH), 7.11 (m, 1H, ArH),6.89(d, J = 8.5Hz, 1H, ArH), 6.62 (s, 1H,
ArH), 6.55 (s, 1H, ArH), 5.36 (s, 2H, OCH2), 4.58 (s, 2H, CHNCH2), 3.73(m, 12H, OCH3), 3.48
(s, 2H, ArCH2N), 2.86 (s, 2H, NCH2), 2.62 (s, 4H, 2×CH2);13C NMR (DMSO, 75 MHz) δ
ppm:27.9, 47.1, 50.0, 54.8, 55.4, 55.7, 56.7, 62.2, 104.5,109.8, 111.2, 111.7, 111.9, 113.5, 121.2,
123.7, 124.6, 125.7, 126.1, 130.4, 132.4, 132.5,141.9, 145.0, 146.8, 147.1, 148.6, 155.5, 163.0;
ESI-HRMS m/z calcd for C31H35N5O6 [M+Na]+ 596.2480, found 596.2480.
16

5.6.5
N-(4-(tert-butyl)phenyl)-2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)benzamide (5)
Yield 61.4%; pale yellow powder; mp: 150-152℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.10
(s, 1H, CONH), 8.24 (s, 1H, NCH=C), 7.78(dd, J = 7.6, 1.3Hz, 1H, ArH), 7.52 (m, 3H, ArH), 7.35
(m, 3H, ArH), 7.10 (dd, 1H, J= 7.4, 7.4Hz, ArH), 6.62 (s, 1H, ArH), 6.53 (s, 1H, ArH), 5.35 (s,
2H, OCH2), 4.59 (t, J =5.9Hz, 2H, CHNCH2), 3.68 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.48 (s,
2H, CH2N), 2.88 (t, J =5.5Hz, 2H, NCH2), 2.63 (s, 4H, 2 × CH2), 1.27 (s, 9H, 3×CH3);13C NMR
(DMSO, 75 MHz) δ ppm: 8.0, 31.1, 34.0, 47.1, 50.0, 54.8, 55.4, 56.7, 62.3,109.8, 111.8, 113.6,
119.2, 121.2, 124.0, 124.7, 125.3, 125.7, 126.2, 130.4, 132.4, 136.3,142.0, 145.8, 146.9, 147.2,
155.5, 163.4; ESI-HRMS m/z calcd for C33H39N5O4 [M+Na]+ 592.2894, found 592.2896.
5.6.6
N-(4-chlorophenyl)-2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)benzamide (6)
Yield 70.8%; pale yellow powder; mp: 165-166℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.32
(s, 1H, CONH), 8.22 (s, 1H, NCH=C), 7.73(m, 3H, ArH), 7.51 (ddd, J = 8.4, 8.4, 1.3Hz, 1H, ArH),
7.37 (m, 3H, ArH), 7.10 (dd, J= 7.4, 7.4 Hz, 1H, ArH), 6.62 (s, 1H, ArH), 6.56 (s, 1H, ArH), 5.34
(s, 2H, OCH2), 4.58(t, J = 5.9Hz, 2H, CHNCH2), 3.68 (s, 6H, 2×OCH3), 3.50 (s, 2H, ArCH2N),
2.85 (t, J = 5.8Hz, 2H, NCH2), 2.63 (s, 4H, 2×CH2); 13C NMR (DMSO, 75 MHz) δ ppm: 28.5.
47.6, 50.5, 55.3, 55.9, 57.2, 62.8, 110.3,112.2, 114.1, 121.4, 121.6, 124.7, 125.1, 126.2, 126.7,
127.5, 129.1, 130.7, 132.9, 138.4, 142.6, 147.4, 147.7, 155.9, 164.6; ESI-HRMS m/z calcd for
C29H30ClN5O4 [M+Na]+ 570.1879 , found 570.1880.
5.6.7
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(3-methoxyphenyl)benzamide (7)
Yield 64.4%; pale yellow powder; mp: 127-129℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.19
(s, 1H, CONH), 8.24 (s, 1H, NCH=C), 7.78(d, J = 6.5Hz, 1H, ArH), 7.52 (dd, J = 7.4, 7.4 Hz, 1H,
ArH), 7.37 (m, 2H, ArH), 7.21(m, 2H, ArH), 7.11 (dd, J = 7.4, 7.4 Hz, 1H, ArH), 6.65 (m, 1H,
ArH), 6.62 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.35 (s, 2H, OCH2), 4.58 (t, J = 5.5 Hz, 2H, CHNCH2),
17

3.75(s, 3H, 6H, 2×OCH3), 3.49 (s, 2H, ArCH2N), 2.86 (s, 2H, NCH2), 2.50 (s, 4H, 2×CH2); 13C
NMR (DMSO, 75 MHz) δ ppm: 28.5, 47.6, 50.5, 55.3, 55.5, 55.9, 57.1, 62.7, 105.7, 109.5, 110.3,
112.2, 114.1, 121.7, 124.4, 125.2, 126.2, 126.7, 130.0, 131.0, 133.0, 140.5, 147.4, 147.6, 156.0,
160.0, 164.2; ESI-HRMS m/z calcd for C30H33N5O5 [M+Na]+ 566.2374, found 566.2382.
5.6.8
N-(3-chlorophenyl)-2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)benzamide (8)
Yield 68.6%; pale yellow powder; mp: 127-130℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 10.33
(s, 1H, CONH), 8.20 (s, 1H, NCH=C), 7.86 (s, 1H, ArH), 7.72(d, J = 6.4 Hz , 1H, ArH), 7.52 (m,
2H, ArH), 7.34 (m, 2H, ArH),7.12 (m, 2H, ArH), 6.61 (s, 1H, ArH), 6.54 (s, 1H, ArH), 5.34 (s, 2H,
OCH2), 4.57 (s, 2H, CHNCH2), 3.67 (s, 6H, 2×OCH3), 3.48 (s, 2H, ArCH2N), 2.85 (s, 2H, NCH2),
2.62 (s, 4H, 2×CH2); 13C NMR (DMSO, 75 MHz) δ ppm: 27.9, 47.1, 49.9, 54.8, 55.4, 56.6, 62.2,
109.8, 111.7, 113.6, 117.8, 118.8, 121.1, 123.2, 124.0, 124.7, 125.7, 126.1, 130.2, 130.3, 132.5,
133.1, 140.3, 146.8, 147.1, 155.5, 164.2 ; ESI-HRMS m/z calcd for C29H30ClN5O4 [M+Na]+
570.1879, found 570.1882.
5.6.9
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(o-tolyl)benzamide (9)
Yield 56.9%; pale yellow powder; mp: 131-133℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 9.69 (s,
1H, CONH), 8.28 (s, 1H, NCH=C), 7.98(d, J = 7.6 Hz, 1H, ArH), 7.90 (d, J = 7.9 Hz, 1H, ArH),
7.55 (dd, J = 7.0 Hz, 7.0 Hz, 1H, ArH), 7.44 (d, J = 8.2 Hz, 1H, ArH), 7.10 (m, 4H, ArH), 6.62 (s,
1H, ArH), 6.56 (s, 1H, ArH), 5.41 (s, 2H, OCH2), 4.58 (t, J = 5.7 Hz, 2H, CHNCH2), 3.68 (s, 6H,
2×OCH3), 3.50 (s, 2H, ArCH2N), 2.86 (t, J = 5.6 Hz, 2H, NCH2), 2.62 (s, 4H, 2×CH2), 1.91 (s, 3H,
CH3); 13C NMR (DMSO, 75 MHz) δ ppm: 28.0, 47.0, 50.0, 54.8, 55.4, 56.6, 62.1, 109.8, 111.7,
113.5, 121.2, 122.7, 124.3, 125.4, 125.7, 126.0, 126.1, 129.2, 130.1, 131.1, 132.8, 136.4, 141.4,
146.8, 147.1, 155.7, 162.9; ESI-HRMS m/z calcd for C30H33N5O4 [M+Na]+ 550.2425, found
550.2426.
5.6.10
18

2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-phenylbenzamide (10)
1
Yield 60.2%; pale yellow powder; mp: 143-145℃；H NMR(300 MHz, DMSO-d6) δ ppm: 10.19(s,
1H, CONH), 8.56 (s, 1H, NCH=C), 7.77 (d, J = 7.2 Hz, 1H, ArH), 7.65 (d, J = 7.8 Hz, 2H, ArH),
7.51 (dd, J = 7.2 Hz, 7.5 Hz,1H, ArH), 7.34 (m, 4H, ArH), 7.08 (dd, J = 7.5 Hz, 7.2Hz, 2H, ArH),
6.62 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.35 (s, 2H, OCH2), 4.57(t, J = 5.7 Hz, 2H, CHNCH2), 3.68
(s, 6H, 2 ×OCH3), 3.49 (s, 2H, ArCH2N), 2.85 (s, 2H, NCH2), 2.63 (s, 4H, 2×CH2); 13C NMR
(DMSO, 75 MHz) δ ppm: 27.9, 47.0, 49.9, 54.8, 55.4, 56.6, 62.2, 109.8, 111.7, 113.5.0, 119.3,
121.1, 123.4, 124.1, 124.7, 125.7, 126.1, 128.7, 130.3, 132.3, 138.9, 142.0, 146.8, 147.1, 155.4,
163.6; ESI-HRMS m/z calcd for C29H31N5O4 [M+Na]+ 536.2268, found 536.2268.
5.6.11
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(3,4,5-trimethoxyphenyl)benzamide (11)
Yield 56.2%; yellow powder; mp: 66-68℃; 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.19 (s, 1H,
CONH), 8.24 (s, 1H, NCH=C), 7.85(d, J = 7.2 Hz, 1H, ArH), 7.54 (dd, J =7.2 Hz, 7.2 Hz, 1H,
ArH), 7.37 (d, J = 8.1 Hz,1H, ArH), 7.12 (m, 3H, ArH), 6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.37
(s, 2H, OCH2),4.58 (s, 2H, CHNCH2), 3.76 (m, 6H, 2×OCH3), 3.65(s, 6H, 2×OCH3), 3.49 (s, 3H,
OCH3), 3.36 (s, 2H, ArCH2N), 2.86 (s, 2H, NCH2), 2.62 (s, 4H, 2×CH2); 13C NMR (DMSO, 75
MHz) δ ppm: 27.9, 47.1, 49.9, 54.8, 55.4, 55.7, 56.6, 60.0, 62.2,97.2, 109.8, 111.7, 113.5, 121.3,
123.4, 124.7, 125.7, 126.1, 130.5, 132.7, 135.0, 141.9,152.7, 163.0; ESI-HRMS m/z calcd for
C32H37N5O7 [M+Na]+ 626.2585, found 626.2587.
5.6.12
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(4-fluorophenyl)benzamide (12)
1
Yield 46.0%, pale yellow powder mp: 143-145℃; H NMR (300 MHz, DMSO-d6) δ ppm: 10.13
(s, 1H, CONH), 8.01 (s, 1H, NCH=C), 7.70 (s, 4H, NCH=C), 7.28 (d, J = 6.8 Hz, 1H, ArH), 7.23
– 7.05 (m, 2H, ArH), 6.83 (d, J = 7.3 Hz, 1H, ArH), 6.74 – 6.45 (m, 3H, ArH), 4.52 (s, 2H,
CHNCH2), 4.40 (d, J = 2.1 Hz, 2H), 3.67 (s, 6H, 2×OCH3), 3.51 (s, 2H, ArCH2N), 2.87 (s, 2H,
19

NCH2), 2.64 (s, 4H, 2×CH2); 13C NMR (75 MHz, DMSO) δ ppm: 167.87, 148.74, 147.19, 146.92,
144.56, 135.37, 132.66, 128.89, 126.29, 125.80, 123.04, 122.46, 115.76, 115.16, 114.80, 111.84,
111.50, 109.96, 56.71, 55.46, 54.82, 50.10, 47.04, 38.17, 28.04; ESI-HRMS m/z calcd for
C29H30FN5O4 [M+Na]+ 554.2174, found 554.2179.
5.6.13
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(4-(trifluoromethoxy)phenyl)benzamide (13)
Yield 68.6%; pale yellow powder; mp: 127-129℃; 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.25
(s, 1H, CONH), 8.02 (s, 1H, NCH=C), 7.82 (d, J = 7.4 Hz, 2H, ArH), 7.78 – 7.65 (m, 2H, ArH),
7.33 (d, J = 7.8 Hz, 3H, ArH), 6.84 (d, J = 7.9 Hz, 1H, ArH), 6.67 (t, J = 6.9 Hz, 1H, ArH), 6.59
(d, J = 11.1 Hz, 2H, ArH), 4.53 (s, 2H, CHNCH2), 4.40 (s, 2H), 3.68 (s, 6H, 2×OCH3), 3.52 (s, 2H,
ArCH2N)), 2.88 (s, 2H, NCH2), 2.65 (s, 4H, 2×CH2).13C NMR (75 MHz, DMSO) δ ppm: 168.05,
148.74, 147.09, 146.80, 144.49, 138.30, 132.86, 129.02, 126.19, 125.70, 123.10, 121.86, 121.36,
115.48, 114.73, 111.59, 109.75 (s), 56.74 (s), 55.36, 54.81, 50.08, 46.97, 38.10, 28.04; ESI-HRMS
m/z calcd for C30H30F3N5O5 [M+Na]+ 620.2091, found 620.2097.
5.6.14
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(4-nitrophenyl)benzamide (14)
1
Yield 68.6%; pale yellow powder; mp: 123-125℃; H NMR(300 MHz, DMSO-d6) δ ppm: 10.10
(s, 1H, CONH), 8.24 (s, 1H, NCH=C), 7.78 dd, J = 7.6, 1.3Hz, 1H, ArH), 7.52 (m, 3H, ArH), 7.35
(m, 3H, ArH), 7.10 (dd, 1H, J = 7.4, 7.4Hz, ArH), 6.62 (s, 1H, ArH), 6.53 (s, 1H, ArH), 5.35 (s,
2H, OCH2), 4.59 (t, J =5.9Hz, 2H, CHNCH2), 3.68 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.48 (s,
2H, CH2N), 2.88 (t, J =5.5Hz, 2H, NCH2); 13C NMR (DMSO, 75 MHz) δ ppm: 14.0,21.1, 28.0,
47.1, 50.0, 54.8, 55.4, 56.7, 62.3,109.8, 111.8, 113.6, 119.2, 121.2, 124.0, 124.7, 125.3, 125.7,
126.2, 130.4, 132.4, 136.3,142.0, 145.8, 146.9, 147.2, 155.5, 163.4; ESI-HRMS m/z calcd for
C33H39N5O4 [M+Na]+ 592.2894, found 592.2895.
5.6.15
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(ptolyl)benzamide (15)
20

Yield 68.6%; pale yellow powder; mp:117-118℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 9.91 (s,
1H, CONH), 8.15 (s, 1H, NCH=C), 8.12 (d, J = 8.0 Hz, 1H, ArH), 7.75 (m, 4H, ArH), 7.25 (d, J =
8.1 Hz, 1H, ArH), 7.13 (dd, J = 7.2 Hz, 7.2 Hz, 1H, ArH), 6.98 (dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH),
6.62 (s, 1H, ArH), 6.56 (s, 1H, ArH), 5.21 (s, 2H, OCH2), 4.55 (t, J = 5.7 Hz, 2H, CHNCH2), 3.68
(s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.51 (s, 2H, ArCH2N), 2.87 (t, J = 5.8 Hz, 2H, NCH2), 2.63 (s,
4H, 2×CH2); 13C NMR (DMSO, 75 MHz) δ ppm: 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.3, 109.9,
111.8, 113.6, 120.8, 123.9, 124.1, 124.7, 125.6, 125.7, 126.2, 127.1, 129.2, 130.7, 132.8, 133.9,
142.5, 146.5, 146.9, 147.2, 149.8, 164.4; ESI-HRMS m/z calcd for C29H31N6O6 [M+Na]+
581.2119, found 581.2114.
5.6.16
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(3-nitrophenyl)benzamide (16)
Yield 53.8%; pale yellow powder; mp: 120-122℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 9.94 (s,
1H, CONH), 8.73 (s, 1H, NCH=C), 8.40 (m, 2H, ArH), 8.18 (s, 1H, ArH), 7.80 (dd, J = 8.0 Hz,
8.0 Hz, 1H, ArH), 7.69 (d, J =7.4 Hz, 1H, ArH), 7.31 (d, J = 7.9 Hz, 1H, ArH), 7.20 (dd, J = 8.0
Hz, 8.0 Hz, 1H, ArH),7.01 (dd, J = 7.3 Hz, 7.3 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 6.54 (s, 1H, ArH),
5.23 (s, 2H, OCH2), 4.55 (t, J = 5.9 Hz, 2H, CHNCH2), 3.68 (s, 3H, OCH3), 3.67 (s, 3H, OCH3),
3.49 (s, 2H, ArCH2N), 2.85 (t, J = 5.6Hz, 2H, NCH2), 2.61 (s, 4H, 2×CH2); 13C NMR (DMSO, 75
MHz) δ ppm: 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.6, 109.8,111.7, 113.9, 120.9, 122.4, 124.5,
125.2, 125.7, 126.0, 126.1, 126.3, 126.9, 130.2, 133.9, 136.0, 142.6, 146.9, 147.1, 147.7, 150.9,
163.1; ESI-HRMS m/z calcd for C29H31N6O6 [M+Na]+ 581.2119, found 581.2118.
5.6.17
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(2-nitrophenyl)benzamide (17)
Yield 71.7%; pale yellow powder; mp: 119-122℃; 1H NMR(300 MHz, DMSO-d6) δ ppm: 9.91 (s,
1H, CONH), 8.15 (s, 1H, NCH=C), 8.12 (d, J = 8.0 Hz, 1H, ArH), 7.75 (m, 4H, ArH), 7.25 (d, J =
8.1 Hz, 1H, ArH), 7.13 (dd, J = 7.2 Hz, 7.2 Hz, 1H, ArH), 6.98 (dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH),
6.62 (s, 1H, ArH), 6.56 (s, 1H, ArH), 5.21 (s, 2H, OCH2), 4.55 (t, J = 5.7 Hz, 2H, CHNCH2), 3.68
(s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.51 (s, 2H, ArCH2N), 2.87 (t, J = 5.8 Hz, 2H, NCH2), 2.63 (s,
4H, 2× CH2); 13C NMR (DMSO, 75 MHz) δ ppm: 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.3, 109.9,
21

111.8, 113.6, 120.8, 123.9, 124.1, 124.7, 125.6, 125.7, 126.2, 127.1, 129.2, 130.7, 132.8, 133.9,
142.5, 146.5, 146.9, 147.2, 149.8, 164.4; ESI-HRMS m/z calcd for C29H31N6O6 [M+Na]+
581.2119, found 581.2110.
5.6.18
2-((1-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methox
y)-N-(3-methoxyphenyl)benzamide (18)
1
Yield 38.5%; pale yellow powder; mp:115-118℃; H NMR (300 MHz, CDCl3) δ ppm: 8.62 (s,
1H, CONH), 8.47 (dd, J = 5.1 Hz, 4.3 Hz, 1H, ArH), 7.78 (s, 1H, NCH=C), 7.38 (m, 3H, ArH),
7.06 (m, 4H, ArH), 6.56 (s, 1H, ArH), 6.46 (s, 1H, ArH), 5 .30 (s, 2H, OCH2), 4.58 (t, J = 4.2 Hz,
2H, CHNCH2), 3.86 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.64 (s, 2H,
ArCH2N), 3.01 (s, 2H, NCH2), 2.74 (s, 4H, 2×CH2); 13C NMR (DMSO, 75 MHz) δ ppm: 27.9,
47.1, 50.0, 54.8, 55.3, 55.4, 56.6, 62.6, 109.9, 111.8, 112.4, 113.8, 117.5, 119.4, 121.0, 123.9,
124.5, 125.5, 125.7, 126.2, 127.5, 129.7, 135.9, 142.6, 146.9, 147.2, 150.0, 159.3, 164.5;
ESI-HRMS m/z calcd for C30H33N5O5 [M+Na]+ 566.2374, found 566.2372.
6. Biological assays
6.1. Cytotoxicity assay
The exponentially growing K562 and K562/A02 cells were grown in 96-well plates at 1×104
cells per well and incubated for 24 h. In the assay of cytotoxic evaluation, a graded dose of
compounds diluted with medium were added into the wells. In the assay of drug resistant
modulation, 5µM of the target compounds were added into the wells followed by various
concentrations of ADM. And the cells were incubated for another 48 h in an atmosphere of 95%
air with 5% CO2 at 37 °C. Then, MTT was added directly to the cells. After additional incubation
for 4 h at 37 °C, the absorbance at 570 nm was read on a microplate reader (Thermo Fisher
Scientific). The IC50 values of the compounds for cytotoxicity were calculated by GraphPad Prism
6.0 software from the dose–response curves.
6.2. MTT assay
K562/A02 cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine
22

serum at 37°C in a 5% CO2 humidified atmosphere. K562/A02 cells were seeded in 96-well plates
at 1×104 cells per well. After 24 h incubation, cells were treated with various concentrations of
ADM in absence or presence of target compounds for 48 h in an atmosphere of 95% air with 5%
CO2 at 37 °C. Then, MTT was added directly to the cells. After additional incubation for 4 h at
37°C, the absorbance at 570 nm was read on a microplate reader (Thermo Fisher Scientific). The
IC50 values of the compounds for cytotoxicity were calculated by GraphPad Prism 6.0 software
from the dose–response curves. Experiments were conducted in triplicates and repeated three
times independently.
6.3. Adriamycin intracellular accumulation
K562 and K562/A02 cells were seeded into 24-well plates at 1.5×105 /well. Different
concentrations of compound 5 and VRP were pre-incubated with cells for 60 min. Then 20µM
ADM was added into each well and incubated for 90 min, washed three times with PBS at 4°C for
intracellular mean fluorescence intensity (MFI) analysis. The mean fluorescence intensity of
retained was measured by fluorescence spectrophotometer. Data were expressed as means SD of
three independent experiments.
6.4. Rhodamine123 efflux assay
K562 or K562/A02 cells were seeded into 24-well plates at 1.5×105/well and incubated with
5µM Rh123 for 60 min before washing with ice PBS for three times. Then the cells were
incubated with or without various concentrations of compound 5 or VRP (0.5, 2.5, 5µM) for
another 90 min. Afterwards the cells were washed twice with ice-cold PBS. The mean
fluorescence intensity of retained intracellular Rh123 was estimated by BD FACSCalibur flow
cytometer.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China
(81370878 and 81673299), the ‘333’ Project of Jiangsu Province (BRA2015393) and the National
Science and Technology Major Project of the Ministry of Science and Technology of China
(2009ZX09102-033).
23

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25

•
•
High potency, EC = 256.2±6.2nM;
50
Low cytotoxity, with high therapeutic index
(TI >780);
•
•
Long activity duration, >24h;
Significant increase of ADM cellular
concentration ;
•
Inhibition of Rh123 efflux
Effect of compound 5 on intracellular ADM
26