Design, synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.065

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC₅₀ >100 μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors.

Full text of DOI:10.1016/j.bmc.2016.03.065

Accepted Manuscript
Design, synthesis and biological evaluation of LBM-A5 derivatives as potent
P-glycoprotein-mediated multidrug resistance inhibitors
Yuxiang Wu, Miaobo Pan, Yuxuan Dai, Baomin Liu, Jian Cui, Wei Shi,
Qianqian Qiu, Wenlong Huang, Hai Qian
PII:
S0968-0896(16)30222-X
http://dx.doi.org/10.1016/j.bmc.2016.03.065
BMC 12914
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2016
30 March 2016
31 March 2016
Please cite this article as: Wu, Y., Pan, M., Dai, Y., Liu, B., Cui, J., Shi, W., Qiu, Q., Huang, W., Qian, H., Design,
synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance
inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.065
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and biological evaluation of LBM-A5 derivatives as potent
P-glycoprotein-mediated multidrug resistance inhibitors
Yuxiang Wua,#, Miaobo Pana,#, Yuxuan Daia, Baomin Liua,b, Jian Cuia, Wei Shia, Qianqian Qiua,
Wenlong Huang a,* , Hai Qian a,*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Nan Jing Research and Development Center, CTTQ Pharmaceutical Research Institute, Building 9,
NO. 699-8, Xuanwu Dadao, Xuanwu District, Nanjing, Jiangsu, PR China
Abstract
A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with
triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were
designed and synthesized via click chemistry. Most of the synthesized compounds showed higher
reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a
comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity towards
K562 cells (IC50 > 100μM). Compared with VRP, compound 4 exhibited more potency in increasing
drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR
in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with
reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the
intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123
(Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising
candidate for developing P-gp-mediated MDR inhibitors.
Key words: click chemistry, multidrug resistance inhibitors, P-glycoprotein, reversal activity
1. Introduction
The resistance of tumour cells against cytotoxic agents is a significant obstruction for successful
tumor chemotherapy. As this resistance is structurally unrelated to cytostatics, it was named multidrug
resistance (MDR), which is characterized by a decreased sensitivity to cytotoxic agents. In MDR
tumour cells, various members of the ATP-binding cassette (ABC) family transporters, including
P-glycoprotein (P-gp, ABCB1), the breast cancer-resistance protein (BCRP, ABCG2) and the
multidrug resistance-associated protein 1 (MRP1,ABCC1), can simultaneously be overexpressed.1
Among them, P-gp is the most extensive drug transporter , which has the ability to expel a wide variety
of chemotherapeutic agents out of tumor cells resulting in MDR.2,3 Obviously, it should be a potential
strategy to develop P-gp inhibitors to suppress P-gp-mediated MDR.4 In the past three decades,
considerable efforts have been made to overcome MDR of tumor cells and have developed three
generations of P-gp inhibitors such as Verapamil, Valspodar (PSC-833) and Tariquidar (XR9576).5,6
Nevertheless, all of these P-gp inhibitors failed later on due to observed toxicity, low selectivity or
pharmacokinetic interactions. Up to now, no P-gp inhibitors have been approved for clinical
application.7 Therefore, it is still exigent to develop new non-toxic and potent P-gp inhibitors with high
# Contributed equally to the first author.
*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:
+86-25-83271302; Fax: +86-25-83271480.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian)

selectivity for cancer treatment.
For designing novel multidrug resistance inhibitors, an approach we adopted was to reserve the
chemical fragment, such as N-ethyl-tetrahydroisoquinoline and nitrogen heterocyclic ring, which are
frequently appeared in reported potent P-gp inhibitors .8-10 In the present study, LBM-A5 effectively
reversed MDR (EC50 = 483.6 ± 81.7 nmol·L−1) by inhibiting the function of P-gp, with relatively ideal
pharmacokinetics and in a safe manner.11,12 Thus, LBM-A5 could be served as a lead compound. The
third generation P-gp inhibitors with a phenethyl-tetrahydroisoquinoline structure, such as Tariquidar
and WK-X-34, contain multiple hydrophobic aromatic rings leading to the larger molecular weights,
which go against their druggability. Therefore, the novel multidrug resistance inhibitors were designed
to decrease the molecular weight through structural modification that the aromatic amide structures
were replaced by the different aromatic rings (Fig.1). 1,2,3-triazole ring, a hydrophobic aromatic group,
associating with biological targets through hydrogen bonding and dipole interactions was introduced to
the designed compounds by click chemistry.10 Click chemistry, commonly copper-(I)-catalyzed
1,2,3-triazole formation from azides and terminal acetylenes, has been widely applied in drug
discovery.13 Based on the principles above, we synthesized and biologically evaluated compounds 1–25
as P-gp-mediated MDR inhibitors.
Figure 1. (A) Structures of the reported potent P-gp inhibitors containing chemical fragments such
as N-ethyltetrahydroisoquinoline and nitrogen heterocyclic ring. (B) Design of the target
compounds 1–25.

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) 2-azidoethyl 4-methylbenzenesulfonate(b), TEA/acetonitrile, 60°C, 24
h; (iv) 3-bromoprop-1-yne, K2CO3, acetone, reflux, 4 h; (v) 2-(4-Azidophenethyl)-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (e), ascorbate sodium, CuSO4, 75% CH3OH, 24–48 h; (vi) 2-(2-
azidoethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (c), ascorbate sodium, CuSO4, 75% CH3OH,
24–48 h.
2. Chemsitry
The synthetic routes of target compounds 1–25 are outlined in Scheme 1. Compounds a–c were
synthesized according to literature procedures with minor modification.14–16 Compounds 1d-17d were
prepared starting from the disparate aromatic phenols and 3-bromo-prop-1-yne, which refluxed in the
mixture of acetone and potassium carbonate for 4 h. Subsequently, compound c or e and compounds
1d–17d were treated with ascorbate sodium and copper sulfate in 75% methanol stirring at room
temperature for 24–48 h to provide compounds 1–25. Interestingly, some of the target compounds
could precipitate from the reaction solution directly and were isolated with high purity. While others
which could not precipitate directly were purified by column chromatography. The structures of target
compounds obtained were listed in Table 1.

Table 1
Structures and ADM-resistance reversal activity of the target compounds 1–25 at 5 μM concentration
in K562/A02 cellsa
R'
Compounds
IC50 of ADM(μmol/L)
RF
7.4
1
5.11±0.32
2
4.48±0.17
8.5
3
4
7.02±0.23
1.21±0.18
5.4
31.4
5
6
1.81±0.13
3.79±0.08
21.0
10.0
7
8
6.53±0.11
6.45±0.31
10.96±0.42
4.17±0.07
4.15±0.12
4.11±0.06
5.8
5.9
3.5
9.1
9.2
9.3
9
10
11
12
13
14
10.58±0.55
12.35±0.61
3.6
3.1

15
6.68±0.33
5.7
16
17
18
19
20
6.17±0.56
11.22±0.79
6.23±0.65
4.95±0.29
1.86±0.15
6.2
3.4
6.1
7.7
20.4
21
4.17±0.38
9.1
22
23
8.35±0.48
9.35±0.56
4.6
4.1
24
25
3.98±0.41
9.6
2.7
14.08±1.06
Controlb
WK-X-34
LBM-A5
VRP
38.02±1.65
1.25±0.12
1.65±0.22
8.50±0.45
1
30.4
23.0
4.5
a The IC50s were 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.
RF = (IC50 without modulator)/(IC50 with 5μM modulator).
b 0.1% DMSO was added as solvent control for testing the P-gp modulating activity.
3. Results and discussion
3.1. Biological evaluation
3.1.1. Cytotoxicity assay
To identify ideal P-gp inhibitors reversing MDR at non-toxic concentrations, the intrinsic
cytotoxicity of the target compounds against parental sensitive K562 cells and their ADM-resistant
sublines K562/A02 cells which overexpress P-gp (induced by ADM) was evaluated by MTT assay.

Anticancer drug adriamycin (ADM) and P-gp inhibitors VRP, WK-X-34 and the lead compound
LBM-A5 were selected as controls. As shown in Table 2, VRP had weak cytotoxic effects toward
K562 and K562/A02 cells with IC50 values 65.28 and 56.24μM, respectively. Nevertheless, WK-X-34
displayed a high level of toxicity toward K562 cells (IC50 of 19.56±1.32μM) but a weak toxicity toward
K562/A02 cells (IC50 of 50.10 ±2.51μM). In contrast, LBM-A5 and all of the synthesized compounds
showed no toxicity with IC50 values higher than 100μM to K562/A02 cells. Compound 6 bearing a
quinoline ring and compound 12 with 4-tert-butylbenzene were found to exhibit high toxicity (IC50 <
30μM) in K562 cells. Compound 1 and 3 which both contain a nitro group in their formula showed
weak cytotoxic effects (IC50 ≈ 50μM) toward K562 cells. With a trifluoromethoxy group at the
para-position, compound 8 showed moderate cytotoxic effect (IC50 of 37.40±3.09μM). The other
synthesized compounds showed no toxicity to K562 cells with IC50 values higher than 100μM. In
addition, The cytotoxicity assays indicated that most of our compounds possess little cytotoxic effects
in tested cell lines and are suitable candidates for the development of P-gp inhibitors.
Table 2
Cytotoxicity of the target compounds against K562 and K562/A02 cell linesa
Cytotoxicity IC50 (μM)
Compounds
K562
48.77±1.21
>100
K562/A02
>100
1
2
>100
3
49.22±1.61
>100
>100
4
>100
5
6
>100
>100
28.02±1.97
>100
>100
7
>100
8
37.40±3.09
>100
>100
9
>100
10
>100
>100
11
>100
>100
12
21.10±1.07
>100
>100
13
>100
14
>100
>100
15
>100
>100
16
>100
>100
17
>100
>100
18
>100
>100
19
>100
>100
20
>100
>100
21
>100
>100
22
>100
>100
23
>100
>100
24
>100
>100
25
>100
>100
VRP
WK-X-34
65.28±3.54
19.56±1.32
56.24±2.12
50.10±2.51

LBM-A5
ADM
>100
>100
6.23±0.28
38.02±1.65
aThe IC50s for the target compounds were determined by MTT method. Each experiment was carried
out three times.
3.1.2. Effects of the target compounds on reversing ADM resistance in K562/A02 cells
The effects of the target compounds on reversing adriamycin (ADM) resistance towards
K562/A02 cells (P-gp-overexpression) were preliminarily investigated by the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. 11, 17 The
well-known classical P-gp inhibitor VRP, WK-X-34 and LBM-A5 were chosen as positive controls.
All compounds 1–25 and the positive controls were assayed at 5μM, a low concentration which could
result in less than 10% cytotoxicity towards K562/A02 cells after 48 h incubation. As the results
summarized in Table 1, anticancer drug ADM alone displayed little inhibitory effect on the survival of
K562/A02 cells (IC50 of 38.02±1.65μM). However, the combination treatment of ADM with target
compounds or the positive controls increased the inhibitory effect in different degrees. It revealed that
most of the target compounds exerted MDR reversal activities. Among them, compound 4 with a
significantly decreased IC50 of ADM (1.21± 0.18 μM) showed the strongest reversal activity and its
reversal fold (RF) was 31.4, which was close to WK-X-34 (RF=30.4) and a little higher than LBM-A5
(RF=23.0). In addition, some of the target compounds exhibited more active MDR reversal activity
than the positive control VRP, when co-administered with ADM at the same condition.
3.1.3. Chemo-sensitizing effect of target compound
To further investigate the reversal potency and dose–response effects, we have determined the
reversal activity of compound 4 at various other concentrations (2.5μM, 1.25μM, 0.625μM, 0.31μM,
0.156μM, 0.078μM) towards K562/A02 cells (P-gp-overexpression) by MTT assay, selecting VRP as
positive controls.18,19 As demonstrated in Table 3, VRP showed slight modulating activity at 2.5μM
(RF = 1.9). However, compound 4 showed apparent dose dependent activity and still exhibited potent
MDR reversal activity (RF = 7.2) when the concentration decreased to 0.31μmol/L. Additionally, the
EC50 value of compound 4 was 192.6±34.3nM, which was calculated by GraphPad Prism 5.0 software
from the dose–response curves. The results suggested that compound 4 had the significantly potential
to enhance the sensitivity of P-gp-overexpressing cells to anticancer drug substrates in a
dose-dependent manner.
Table 3
Sensitization of compound 4 on reversing MDR towards K562/A02 cells at different concentrationsa
Compounds
IC50 of ADM (μM)
RF
None
38.02±1.65
19.84±0.82
1.87±0.31
2.25±0.32
3.36±0.28
5.25±0.25
16.98±0.82
23.45±1.04
1
VRP, 2.5μM
1.9
Compound 4, 2.5μM
Compound 4, 1.25μM
Compound 4, 0.625μM
Compound 4, 0.31μM
Compound 4, 0.156μM
Compound 4, 0.078μM
20.3
16.9
11.3
7.2
2.2
1.6
a Reversal fold (RF), RF = (IC50 without modulator)/(IC50 with modulator). Each experiment was
carried out three times, and the values were presented as the mean±standard error of mean.

3.1.4. Effect of compound 4 on ADM accumulation
It is known that ADM is a fluorescent substrate of P-gp and can be used to monitor drug
accumulation in cells. In order to investigate the mechanism of compound 4 in modulating the
anticancer drug substrates accumulation level inside P-gp-overexpressing K562/A02 cells, the
intracellular accumulation of ADM was assessed by monitoring its fluorescence intensity through
flow cytometry assay. The classical P-gp inhibitor VRP was chosen as a positive control. As
shown in Figure 2, the level of ADM in K562 cells was about 4.5-fold higher than that of
K562/A02 cells in the absence of P-gp inhibitors. As probably P-gp can pump ADM out of the
cells, it leads to a lower ADM level in K562/A02 cells. Once treated with compound 4 (5μM), 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 (0.5μM, 2.5μM, 5μM), the accumulation
of ADM was remarkably increased in a dose-dependent manner. Additionally, the intracellular
ADM level of compound 4 (5μM) was 1280.5±35.62, which was 1.5 times higher than that of VRP
in K562/A02 cells at the same dose. The results suggested that compound 4 was more potent than
VRP in inhibiting the drug efflux function of P-gp.
Figure 2. The effect of compound 4 on intracellular ADM accumulation in K562/A02 cells. 0.1%
DMSO was used as vehicle control and VRP was chosen as a positive control. Different concentrations
of compound 4 and VRP were pre-incubated with cells for 60 min. Then 20μM ADM was added into
every well and incubated for 90 min, washed three times with PBS at 4°C. Data were expressed as
means±SD of two independent experiments. *P < 0.05, ** P < 0.01 relative to the negative control
(K562/A02).
3.1.5. Inhibitory effect of compound 4 on Rh123 efflux function
In order to further supplement the above assumption, the inhibitory effect of compound 4 on the
P-gp efflux function was assayed by detecting the retained intracellular Rh123, another fluorescence
substrate of P-gp, according to the method described in literatures with minor modification.20,21 The
results from the Rh123 efflux assay showed that 5μM of compound 4 or VRP inhibited Rh123 efflux
from K562/A02 cells, and the inhibitory effect of compound 4 was obviously better than VRP over the
90 min interval (Fig. 3). The rate constant (K) of efflux in compound 4-treated K562/A02 cells was
0.0020±0.00053s-1, which is lower than the value for the control group (0.0253±0.0069 s-1) and the
VRP-treated group (0.0085±0.0012 s-1). The calculated data for K were shown as the mean±SD for 3

independent tests. These results suggest that the efflux function of P-gp on anticancer drugs, such as
ADM, could be effectively blocked by compound 4. Moreover, the potency of compound 4 is much
higher than the classical P-gp inhibitor VRP under the same conditions.
The results above verified our presumption that compound 4 was an effective P-gp inhibitor
which possessed the ability to enhance the sensitivity of anticancer drugs to P-gp overexpressing
drug-selected cell lines by effectively blocking the drug efflux function of P-gp and showed higher
potency than the classical P-gp inhibitor VRP under the same conditions.
200
Control
5.0 μM Compound 4
5.0 μM VRP
150
100
50
0
0
15
30
45
60
75
90
Time (min)
Figure 3. Effect of compound 4 and verapamil (VRP) on the efflux of rhodamine 123 (Rh123) in
K562/A02 cells. K562/A02 cells were incubated in 5μM of Rh123 at 37 °C for 60 min, washed 3 times
with phosphate-buffered saline at 4 °C, and then respectively incubated with or without 5μM of
compound 4 or VRP at 37 °C for 0,5, 10, 25, 30, 60 and 90 min. The results were shown as the
mean±SD for 3 independent tests.
3.2. Structure–activity relationships
The analysis of structure–activity relationships (SARs) according to the results in Table 1
indicates that different substituents in R' of compounds 1–25 could affect MDR reversal activities
towards K562/A02 cells: (a) Electron-donating groups in R' might be beneficial for the MDR reversal
activity. For example, compounds 11, 19 and compound 12, 20 which possessed electron-donating
group like –CH(CH3)2 and –C(CH3)3 showed higher MDR reversal activity than VRP. However,
compound 9, 17 and 23, which was substituted by –OCH3, showed lower MDR reversal activity than
VRP. In addition, electron-withdrawing groups in R' represented poor MDR reversal activity , such as
compound 14, 25. (b) The substitution position of electron-donating group in R' has an influence on the
reverse activity. Such as compound 20, which was substituted in para-position, was more potent than
compound 24 in ortho-position. (c) The size of substituent in R' is likely to affect MDR reversal
activity. For example, compound 20 (RF = 20.4) containing 4-tert-butyl in R' was more potent than
compound 16, 18, 19 which were substituted by other alkyls. It suggested that the introduction of
4-tertiary butyl to the molecule of compound 20 could dramatically strengthen its binding affinity to
P-gp, leading to more ADM accumulation in K562/A02 cells. (d) Interestingly, compound 4 and
compound 20 bearing the close molecular weight (compound 4: 470.56, compound 20: 450.57) showed
the best MDR reversal activity among the twenty-five target compounds, whose IC50 were respectively
1.21±0.18μmol/L and 1.86±0.15μmol/L and close to the lead compound LBM-A5
(IC50=1.65±0.22μmol/L). Compounds with triazol-N-phenethyl-tetrahydroisoquinoline (1–12) were

generally more potent than compounds with triazol-N-ethyl-tetrahydroisoquinoline (13–25) with the
same substituent R', and compound 4 showed the strongest activity (RF = 31.4).
4. Conclusions
In summary, twenty-five compounds containing triazol-N-phenethyl-tetrahydroisoquinoline or
triazol-N-ethyl-tetrahydroisoquinoline were synthesized based on the click chemistry and evaluated in
vitro as P-gp-mediated MDR reversal agents. Among them, compound 4 with low cytotoxicity could
significantly reverse MDR in a dose-dependent manner and persist longer chemo-sensitizing effect
than VRP with reversibility. Additionally, compound 4 could remarkably increase the intracellular
accumulation of ADM in K562/A02 cells as well as inhibit Rh123 efflux from the cells. These results
suggest that compound 4 could effectively block the drug efflux function of P-gp and lead to increased
drug accumulation in MDR cells. Therefore, compound 4 might be a promising candidate to develop
P-gp-mediated MDR reversal modulator in cancer chemotherapy.
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 of the target
compounds were analyzed by 1H NMR, 13C NMR (Bruker ACF-300Q, 300 MHz), MS (1100 LC/
MSD spectrometer; Hewlett–Packard) and elemental analyses (CHN-O-Rapid instrument); Thin-layer
chromatography (TLC) was performed on GF/UV 254 plates and the chromato-grams were visualized
under UV light at 254 and 365 nm. Compound e was prepared as previously described.11
5.2. Synthesis of 2-azidoethanol (a)
To a 100mL round bottom flask was added 2-bromoethanol (5.3 g, 42.4mmol) and sodium azide (5.5 g,
84.6 mmol) in water (50mL). The mixture was stirred at 80°C for 24 h, and then, cooled to room
temperature. The solution was extracted with ethyl acetate (4 × 30 mL) and the 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
1
next step directly. H NMR (300 MHz, DMSO-d6): δ= 3.79-3.76 (t, J = 5.1 Hz, 2H, OCH2CH2N3),
3.47-3.44 (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 (10mL, 72.1 mmol) in dry
dichloromethane (25 mL), 4-toluenesulfonylchloride (6.2 g, 32.5 mmol) in dry dichloromethane (25
mL) was added drop-wise under constant stirring at 0°C 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 1 N 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%. 1H NMR (300 MHz, DMSO-d6): δ= 7.81 (d, J
= 8.3 Hz, 2H, ArH), 7.50 (d, J = 8.0 Hz, 2H, ArH), 4.16 (t, J=6.1Hz, 2H, OCH2), 3.56 (t, J = 5.1 Hz, 2H,
CH2), 2.42 (s, 3H, CH3).
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= 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 = 6.1Hz, 2H, ArCH2), 2.80 (dd, 6H,
J = 13.3, 6.1Hz, 3×CH2).
5.5. General procedure for the preparation of 1d–17d
The aromatic phenol (3mmol) and 3-bromo-prop-1-yne (0.25 ml, 3.2 mmol) was dissolved in
acetone (15 mL) and added potassium carbonate (1.0 g, 7.3 mmol), then the mixture was heated to
reflux for 4 h. Then the reaction mixture was cooled to room temperature followed by filtration and
evaporated in vacuo to get the desired product compound 1d–17d.
5.5.1. 1-nitro-2-prop-2-ynyloxy-benzene (1d)
Yield 82.6%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.80 (1H, dd, J = 7.8, 1.2 Hz,
ArH), 7.53 (1H, td, J = 7.8, 1.8 Hz, ArH), 7.23 (1H, d, J = 7.8Hz, ArH), 7.08-7.03 (1H, m), 4.81 (2H, d,
J = 2.4 Hz, OCH2), 2.58 (1H, t, J = 2.4 Hz, C≡CH).
5.5.2. 2-prop-2-ynyloxy-naphthalene (2d)
Yield 87.9%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.61-7.45 (m, 2H, ArH),
7.38-7.23 (m, 5H, ArH), 4.91 (d, J = 2.4 Hz, 2H, OCH2), 2.65 (t, J = 2.4 Hz, 1H, C≡CH).
5.5.3. 1-nitro-4-prop-2-ynyloxy-benzene (3d)
Yield 86.0%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 8.39 (d, J = 8Hz, 2H, ArH),
7.36 (d, J = 8 Hz, 2H, ArH), 4.92 (d, J = 2.4 Hz, 2H, OCH2), 2.69 (t, J = 2.4 Hz, 1H, C≡CH).
5.5.4. prop-2-ynyloxy-benzene (4d)
Yield 85.5%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.30-7.27 (m, 2H, ArH),
7.20-7.15 (m, 3H, ArH), 4.80 (d, J = 2.4 Hz, 2H, OCH2), 2.61 (t, J = 2.4Hz, 1H, C≡CH).
5.5.5. 1-prop-2-ynyloxy-naphthalene (5d)
Yield 86.8%; colorless oil; 1H NMR (300 MHz, CDCl3): δ= 8.29 (m, 1H, ArH), 7.82 (m, 1H,
ArH), 7.55-7.48 (m, 3H, ArH), 7.40 (t, J = 8.0 Hz, 1H, ArH), 4.91 (d, J = 2.5 Hz, 2H, OCH2), 2.57 (q, J
= 2.5 Hz, 1H, C≡CH).
5.5.6. 8-prop-2-ynyloxy-quinoline (6d)
Yield 72.8%; brown solid; 1H NMR (300 MHz, CDCl3): δ= 8.82 (d, 1H, J = 4.1 Hz), 7.97 (d, 1H,
J = 8.3 Hz), 7.37-7.26(m, 3H, ArH), 7.14 (d, 1H, J = 7.2 Hz, ArH), 4.92 (s, 2H, OCH2), 2.44 (t, J = 2.5
Hz, 1H, C≡CH).
5.5.7. 1-methyl-4-prop-2-ynyloxy-benzene (7d)
Yield 88.8%; colorless oil; 1H NMR (300 MHz, CDCl3): δ= 7.10 (d, J = 8.5 Hz, 2H, ArH), 6.86 (d,
J = 8.5 Hz, 2H, ArH), 4.66 (d, J = 2.5 Hz, 2H, OCH2), 2.50 (t, J = 2.5 Hz, 1H, C≡CH), 2.29 (s, 3H,
CH3).
5.5.8. 1-prop-2-ynyloxy-4-trifluoromethoxy-benzene (8d)
Yield 72.6%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.17- 6.94 (m, 4H, ArH), 4.68
(d, J = 2.4 Hz, 2H, OCH2), 2.53(t, J = 2.4 Hz, 1H, C ≡ CH).
5.5.9. 1-methoxy-4-prop-2-ynyloxy-benzene (9d)
Yield 88.8%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 6.93 (d, J = 9.0 Hz, 2H, ArH),
6.85 (d, J = 9.0 Hz, 2H, ArH), 4.64 (d, J = 2.5 Hz, 2H, OCH2), 3.78 (s, 3H, OCH3), 2.51 (t, J = 2.5 Hz,
1H, C≡CH).
5.5.10. 1-ethyl-4-prop-2-ynyloxy-benzene (10d)

Yield 86.3%; colorless oil; 1H NMR (300 MHz, CDCl3): δ= 7.17 (d, J = 8.7 Hz, 2H, ArH), 6.95 (d,
J = 8.7 Hz, 2H, ArH), 4.70(d, J = 2.4 Hz, 2H, OCH2), 2.65 (q, J = 7.6 Hz, 2H, CH2CH3), 2.54 (t, J = 2.4
Hz, 1H, C≡CH), 1.26 (t, J = 7.6 Hz, 3H, CH2CH3).
5.5.11. 1-isopropyl-4-prop-2-ynyloxy-benzene (11d)
Yield 80.3%; colorless oil; 1H NMR (300 MHz, CDCl3): δ= 7.13 (d, J = 8.4 Hz, 2H, ArH), 6.92 (d,
J = 8.5 Hz, 2H, ArH), 4.68(d, J = 2.4 Hz, 2H, OCH2), 2.84 – 2.79 (m, 1H, –CH (CH3)2), 2.52 (t, J = 2.4
Hz, 1H, C≡CH), 1.18 (d, J = 6.9 Hz, 6H, –CH (CH3)2).
5.5.12. 1-(tert-butyl)-4-prop-2-ynyloxy-benzene (12d)
Yield 83.0%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.27 (d, J = 7.4 Hz, 2H, ArH),
6.92 (d, J = 7.5 Hz, 2H, ArH), 4.65 (s, 2H, OCH2), 2.54 (t, J = 2.4 Hz, 1H, C≡CH), 1.23 (s, 9H,
–C(CH3)3).
5.5.13. 1,2-dimethyl-4-prop-2-ynyloxy-benzene (13d)
Yield 87.3%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.05 (d, J = 8.1 Hz, 1H, ArH),
6.77 (d, J = 2.7 Hz, 1H, ArH), 6.72 (dd, J = 8.1, 2.7 Hz, 1H, ArH), 4.65(d, J = 2.4 Hz, 2H, OCH2), 2.49
(t, J = 2.4 Hz, 1H, C≡CH), 2.24 (s, 3H, CH3), 2.19 (s, 3H, CH3).
5.5.14. 2,4-dimethyl-1-prop-2-ynyloxy-benzene (14d)
Yield 77.9%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 6.93 (q, J = 8.0 Hz, 3H, ArH),
4.68 (s, 2H, OCH2), 2.50 (t, J = 2.4 Hz, 1H, C≡CH), 2.18 (s, 3H, –CH3), 2.04 (s, 3H, –CH3).
5.5.15. 1-methoxy-2-prop-2-ynyloxy-benzene (15d)
Yield 72.9%; pale yellow powder; 1H NMR (300 MHz, CDCl3): δ= 7.09 (d, J = 7.6 Hz, 1H, ArH),
6.98 – 6.82 (m, 3H, ArH), 4.68 (s, 2H, OCH2), 3.69 (s, 3H, OCH3), 2.58 (t, J = 2.5 Hz, 1H, C≡CH).
5.5.16. 1-(tert-butyl)-2-prop-2-ynyloxy-benzene (16d)
Yield 73.1 %; white powder; 1H NMR (300 MHz, DMSO-d6): δ= 7.31 – 7.05 (m, 3H, ArH), 6.87
(d, J = 6.1 Hz, 1H, ArH), 4.72 (s, 2H, OCH2), 2.60 (t, J = 2.5 Hz, 1H, C≡CH), 1.21 (s, 9H, –C(CH3)3).
5.5.17. 1-fluoro-4-prop-2-ynyloxy-benzene (17d)
Yield 88.0%; colorless oil; 1H NMR (300 MHz, CDCl3): δ= 7.02-6.94 (m, 4H, ArH), 4.66 (d, J =
2.5 Hz, 2H, OCH2), 2.52 (t, J = 2.5 Hz, 1H, C≡CH).
5.6. General procedure for the preparation of compounds 1–12
To the solution of 1d–12d (1 mmol) and e(1 mmol) in 75% methanol (40 mL), ascorbate sodium
(30 mg) and CuSO4 (10 mg) were added, respectively. The reaction solution was stirred at room
temperature for 24–48 h. Then, the mixture was filtered to give the desire product with high purity.
5.6.1.
6,7-dimethoxy-2-(4-(4-((2-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetrahyd
roisoquinoline (1)
Yield 45.5%; pale yellow powder; mp: 96-98°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.91 (s, 1H,
-CH=C-), 7.88 (d, J = 7.6 Hz, 1 H, ArH), 7.81 (d, J = 8.0 Hz, 2 H, ArH), 7.72-7.61 (m, 2H, ArH), 7.49
(d, J = 7.8 Hz, 2 H, ArH), 7.16 (dd, J =7.5, 7.5 Hz, 1 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 5.44 (s, 2H,
OCH2), 3.69 (s, 6 H, 2×OCH3), 3.55 (s, 2 H, ArCH2N), 2.91-2.70 (m, 8 H, 4 × CH2). 13C NMR (75
MHz, DMSO-d6 , δ ppm): 28.2, 32.3, 50.4, 55.0, 55.4, 59.0, 62.4, 109.9, 111.7, 115.6, 120.0, 121.0,
123.1, 124.9, 125.9, 126.6, 130.0, 134.3, 134.5, 139.8, 141.6, 142.7, 146.9, 147.1, 150.5; ESI-MS m/z:
516.5 ([M + H]+); Anal. calcd. For C28H29N5O5: C, 65.23; H, 5.67; N, 13.58; Found: C, 65.28; H, 5.65;
N, 13.55.
5.6.2.
6,7-dimethoxy-2-(4-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetra

hydroisoquinoline (2)
Yield 41.9%; pale yellow powder; mp: 162−164 °C; 1H NMR (300 MHz, DMSO-d6): δ= 8.94 (s,
1H, -CH=C-), 7.85-7.80 (m, 5 H, ArH), 7.55 (s, 1 H, ArH), 7.49-7.64 (m, 3 H, ArH), 7.36 (t, J = 7.5 Hz,
1 H, ArH), 7.23 (dd, J = 7.5, 1.8 Hz, 1 H, ArH), 6.65, 6.62 (2×s, 2 H, ArH), 5.35 (s, 2 H, OCH2), 3.69
(s, 6 H, 2×OCH3), 3.54 (s, 2 H, ArCH2N), 2.90-2.69 (m, 8 H, 4×CH2); 13C NMR (75 MHz, DMSO-d6 ,
δ ppm): 28.2, 32.2, 50.4, 55.0, 55.4, 58.9, 61.1, 107.2, 109.9, 111.7, 118.6, 120.0, 122.8, 123.7, 125.8,
126.4, 126.5,126.7, 127.4,128.6, 129.3, 130.0, 134.1, 134.6, 141.4, 143.6, 146.8, 147.1, 155.8; ESI-MS
m/z: 521.8 ([M + H]+); Anal. calcd. For C32H32N4O3: C, 73.82; H, 6.20; N, 10.76; Found: C, 73.88; H,
6.18; N, 10.73.
5.6.3.
6,7-dimethoxy-2-(4-(4-((4-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetrahyd
roisoquinoline (3)
Yield 52.4%; pale yellow powder; mp: 132-135°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.94 (s,
1H, -CH=C-), 8.24 (d, J = 9.2 Hz, 2H, ArH), 8.07 (d, J = 9.2 Hz, 2H, ArH), 7.81 (d, J = 8.3 Hz, 2H,
ArH), 7.49 (d, J = 8.3 Hz, 2H, ArH), 7.31(d, J = 9.0 Hz, 2H), 6.66(s, 1H, ArH), 6.63(s, 1H, ArH),
5.42(s, 2H, OCH2), 3.69(s, 6H, 2×OCH3), 3.56(s, 2H, ArCH2N), 2.92-2.71(m, 8H, 4×CH2); 13C NMR
(75 MHz, DMSO-d6 , δ ppm): 28.2, 32.2, 50.4, 55.0, 55.4, 58.9, 61.8, 109.9, 111.7, 115.3, 120.1, 123.1,
125.9, 130.0, 134.5, 141.1, 141.6, 142.8, 146.8, 147.1, 163.2; ESI-MS m/z: 516.8 ([M + H]+); Anal.
calcd. For C28H29N5O5: C, 65.23; H, 5.67; N, 13.58; Found: C, 65.20; H, 5.69; N, 13.56.
5.6.4.
6,7-dimethoxy-2-(4-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetrahydroisoqui
noline (4)
Yield 42.5%; pale yellow powder; mp: 118-120°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.91 (s,
1H, -CH=C-), 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.50 (d, J = 8.4Hz, 2H, ArH), 7.49 (d, J = 8.3 Hz, 2H,
ArH),7.32 (dd, J = 8.1, 8.1Hz, 2H, ArH), 7.07 (d, J = 8.1Hz, 2H, ArH), 6.97 (dd, J = 7.3, 7.3 Hz, 1H),
6.66 (s,1H, ArH, ArH), 6.63 (s, 1H, ArH, ArH), 5.23 (s, 2H, OCH2), 3.70 (s, 6H, 2×OCH3), 3.55 (s, 2H,
ArCH2N), 2.92-2.71 (m, 8H, 4×CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 28.2, 32.3, 50.5, 55.0,
55.4, 59.0, 60.9, 109.9, 111.7, 114.7, 120.0, 120.9, 122.7, 125.8, 126.6, 129.5, 130.0, 134.6, 141.5,
143.8, 146.8, 147.1, 157.9; ESI-MS m/z: 471.2 ([M + H]+); Anal. calcd. For C28H30N4O3: C, 71.47; H,
6.43; N, 11.91; Found: C, 71.42; H, 6.44; N, 11.93.
5.6.5.
6,7-dimethoxy-2-(4-(4-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetra
hydroisoquinoline (5)
Yield 38.5%; pale yellow powder; mp: 130-132°C; 1H NMR (300 MHz, DMSO-d6): δ=
9.02(s, 1H, -CH=C-), 8.20 (d, J = 7.9 Hz, 1H, ArH), 7.89-7.84 (m, 3H, ArH), 7.54-7.43 (m, 6H, ArH),
7.23 (d, J = 7.3 Hz, 1H, ArH), 6.66 (s, 1H, ArH), 6.63 (s, 1H, ArH), 5.43 (s, 2H, OCH2), 3.70 (s, 6H,
2×OCH3), 3.55(s, 2H, ArCH2N), 2.92-2.71(m, 8H, 4×CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm):
28.3, 32.3, 50.4, 55.0, 55.4, 59.0, 61.7, 105.8, 109.9, 111.7, 120.0, 120.4, 121.7, 122.6, 124.9, 125.3,
125.8, 126.1, 126.5, 126.6, 127.4, 130.0, 134.0, 134.6, 141.5, 143.9, 1468.8, 147.1, 153.4; ESI-MS m/z:
522.2 ([M + H]+); Anal. calcd. For C32H32N4O3: C, 73.82; H, 6.20; N, 10.76; Found: C, 73.87; H, 6.19;
N, 10.74.
5.6.6.
8-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)m
ethoxy)quinoline (6)

Yield 38.5%; pale yellow powder; mp: 100-102°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.98
(s, 1 H, ArH), 8.84 (s, 1 H, NCH=C), 8.32 (d, J = 8.1 Hz, 1H, ArH), 7.84 (d, J = 8.2 Hz, 2 H, ArH),
7.55-7.44 (m, 6 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 5.44 (s, 2 H, OCH2), 3.70 (s, 6 H, 2 × OCH3), 3.54
(s, 2 H, ArCH2N), 2.91-2.70 (m, 8 H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 28.2, 32.3,
50.4, 55.0, 55.4, 59.0, 61.8, 109.9, 110.2, 111.7, 120.1, 120.2, 121.8, 123.1, 125.9, 126.6, 126.7, 129.1,
130.0, 134.6, 135.8, 139.7, 141.5, 143.6, 146.9, 147.1, 149.0, 153.8; ESI-MS m/z: 523.0 ([M + H]+);
Anal. calcd. For C31H31N5O3: C, 71.38; H, 5.99; N, 13.43; Found: C, 71.33; H, 6.01; N, 13.45.
5.6.7.
6,7-dimethoxy-2-(4-(4-((p-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetrahydroiso
quinoline (7)
Yield 62.0%; pale yellow powder; mp: 122-124°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.86 (s, 1
H, NCH=C), 7.80 (d, J = 8.0 Hz, 2 H, ArH), 7.47 (d, J = 8.0 Hz, 2 H, ArH), 7.10 (d, J = 8.0 Hz, 2 H,
ArH), 6.96 (d, J = 8.1 Hz, 2 H, ArH), 6.65, 6.62 (2s, 2 H, ArH), 5.17 (s, 2 H, OCH2), 3.69 (s, 6 H, 2 ×
OCH3), 3.54 (s, 2 H, ArCH2N), 2.90-2.70 (m, 8 H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm):
20.0, 28.3, 32.3, 50.4, 55.0, 55.4, 59.0, 61.0, 109.9, 111.7, 114.5, 120.0, 122.6, 125.9, 126.6, 129.6,
129.8, 130.0, 134.6, 141.4, 143.9, 146.9, 147.1, 155.9; ESI-MS m/z: 485.3 ([M + H]+); Anal. calcd. For
C29H32N4O3: C, 71.88; H, 6.66; N, 11.56; Found: C, 71.82; H, 6.68; N, 11.58.
5.6.8.
6,7-dimethoxy-2-(4-(4-((4-(trifluoromethoxy)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,
2,3,4-tetrahydroisoquinoline (8)
Yield 54.2%; pale yellow powder; mp: 90-92°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.90 (s, 1 H,
NCH=C), 7.80 (d, J = 8.0 Hz, 2 H, ArH), 7.47 (d, J = 8.0 Hz, 2 H, ArH), 7.23 (d, J = 8.9 Hz, 2 H, ArH),
7.17 (d, J = 8.9 Hz, 2 H, ArH), 6.64, 6.62 (2s, 2 H, ArH), 5.25 (s, 2 H, OCH2), 3.69 (s, 6 H, 2 × OCH3),
3.55 (s, 2 H, ArCH2N), 2.90-2.70 (m, 8 H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 28.3,32.3,
50.4, 55.0, 55.4, 59.0, 61.5, 109.9, 111.7, 116.0, 116.3, 118.4, 120.0, 121.8, 122.3, 122.5, 122.8, 125.9,
126.5, 130.0, 134.6, 141.5, 142.0, 143.4, 146.9, 147.1,156.8; ESI-MS m/z: 555.2 ([M + H]+); Anal.
calcd. For C29H29F3N4O4: C, 62.81; H, 5.27; F, 10.28; N, 10.10; Found: C, 62.88; H, 5.25; F, 10.31; N,
10.08.
5.6.9.
6,7-dimethoxy-2-(4-(4-((4-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-1,2,3,4-tetra
hydroisoquinoline (9)
Yield 40.2%; pale yellow powder; mp: 136-138°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.88 (s, 1
H, NCH=C), 7.83 (d, J = 7.0 Hz, 2 H, ArH), 7.51 (d, J = 7.0 Hz, 2 H, ArH), 7.04 (d, J = 7.8 Hz, 2 H,
ArH), 6.91 (d, J = 7.7 Hz, 2 H, ArH), 6.68, 6.66 (2s, 2 H, ArH), 5.18 (s, 2 H, OCH2), 3.73 (s, 6 H, 2 ×
OCH3), 3.58 (s, 2 H, ArCH2N), 2.93-2.74 (m, 8 H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm):
28.3, 32.3, 50.4, 55.0, 55.2, 55.4, 59.0, 61.5, 109.9, 111.7, 114.6, 115.7, 120.0, 122.6, 125.9, 126.6,
130.0, 134.6, 141.4, 144.0, 146.9, 147.1, 152.0, 153.6; ESI-MS m/z: 501.4 ([M + H]+); Anal. calcd. For
C29H32N4O4: C, 69.58; H, 6.44; N, 11.19; Found: C, 69.52; H, 6.46; N, 11.21.
5.6.10.
2-(4-(4-((4-ethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-6,7-dimethoxy-1,2,3,4-tetrahyd
roisoquinoline (10)
Yield 50.2%; yellow powder; mp: 114-116°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.88 (s, 1 H,
NCH=C), 7.81 (d, J = 8.1 Hz, 2 H, ArH), 7.47 (d, J = 8.1Hz, 2 H, ArH), 7.13 (d, J = 8.1 Hz, 2 H, ArH),
6.98 (d, J = 8.3 Hz, 2 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 5.18 (s, 2 H, OCH2), 3.69 (s, 6 H, 2 × OCH3),

3.54 (s, 2 H, ArCH2N), 2.90-2.70 (m, 8 H, 4 × CH2), 2.57-2.52 (m, 2 H, CH2CH3), 1.14 (t, J = 7.5 Hz, 3
H, CH2CH3); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 15.8, 28.3, 32.3, 50.4, 55.0, 55.4, 59.0, 61.0,
109.9, 111.7, 114.5, 120.0, 122.6, 125.9, 126.6, 128.6, 130.0, 134.6, 136.1, 141.4, 143.9, 146.9, 147.1,
156.0; ESI-MS m/z: 499.3 ([M + H]+); Anal. calcd. For C30H34N4O3: C, 72.26; H, 6.87; N, 11.24;
Found: C, 72.30; H, 6.89; N, 11.23.
5.6.11.
2-(4-(4-((4-isopropylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-6,7-dimethoxy-1,2,3,4-tetr
ahydroisoquinoline (11)
Yield 19.5%; pale yellow powder; mp: 113-115°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.88 (s, 1
H, NCH=C), 7.81 (d, J = 8.2 Hz, 2 H, ArH), 7.48 (d, J = 8.2 Hz, 2 H, ArH), 7.16 (d, J = 8.4 Hz, 2 H,
ArH), 6.98 (d, J = 8.4 Hz, 2 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 5.18 (s, 2 H, OCH2), 3.69 (s, 6 H, 2 ×
OCH3), 3.54 (s, 2 H, ArCH2N), 2.93-2.70 (m, 9 H, 4 × CH2, CH), 1.1 (d, J = 6.8 Hz, 6 H, 2 × CH3); 13C
NMR (75 MHz, DMSO-d6 , δ ppm): 24.0, 28.3, 32.3, 32.5, 50.4, 55.0, 55.4, 59.0, 61.0, 109.9, 111.7,
114.5, 120.0, 122.6, 125.9, 126.6, 127.1, 130.0, 134.6, 140.8, 141.4, 143.9, 146.9, 147.1, 156.1;
ESI-MS m/z: 513.4([M + H]+); Anal. calcd. For C31H36N4O3: C, 72.63; H, 7.08; N, 10.93; Found: C,
72.68; H, 7.10; N, 10.96.
5.6.12.
2-(4-(4-((4-(tert-butyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-6,7-dimethoxy-1,2,3,4-tet
rahydroisoquinoline (12)
Yield 38.0%; yellow powder; mp: 118-120°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.89 (s, 1 H,
NCH=C), 7.81 (d, J = 8.3 Hz, 2 H, ArH), 7.48 (d, J = 8.3 Hz, 2 H, ArH), 7.31 (d, J = 8.6 Hz, 2 H, ArH),
6.98 (d, J = 8.6 Hz, 2 H, ArH), 6.65, 6.63 (2s, 2 H, ArH), 5.19 (s, 2H, OCH2), 3.69 (s, 6 H, 2 × OCH3),
3.54 (s, 2 H, ArCH2N), 2.93-2.70 (m, 8 H, 4 × CH2), 1.25 (s, 9 H, 3 × CH3); 13C NMR (75 MHz,
DMSO-d6 , δ ppm): 28.3, 31.3, 32.3, 33.7, 55.0, 55.4, 59.0, 61.0, 109.9, 111.7, 114.1, 120.0, 122.6,
125.9, 126.1, 126.6, 130.0, 134.6, 141.4, 143.1, 143.9, 146.9, 147.1, 155.7; ESI-MS m/z: 527.3 ([M +
H]+); Anal. calcd. For C32H38N4O3: C, 72.98; H, 7.27; N, 10.64; Found: C, 72.93; H, 7.28; N, 10.67.
5.7. General procedure for the preparation of compounds 13–25
To the solution of 2d, 3d, 5d, 7d, 9d–17d (1 mmol) and c(1 mmol) in 75% methanol (40 mL),
ascorbate sodium (30 mg) and CuSO4 (10 mg) were added, respectively. The reaction solution was
stirred at room temperature for 24–48 h. Some of the reaction solutions precipitated white solid
(compound 13 and 17), the others none. The solution with precipitate appeared filtered directly and the
solution without precipitate appeared evaporated in vacuo, 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 13–25.
5.7.1.
6,7-dimethoxy-2-(2-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydr
oisoquinoline (13)
Yield 24.6%; white powder; mp: 142–144°C ; 1H NMR (300 MHz, DMSO-d6): δ= 8.28 (s, 1H,
NCH=C), 7.89 – 7.72 (m, 3H, ArH), 7.54 – 7.42 (m, 2H, ArH), 7.39 – 7.31 (m, 1H, ArH), 7.17 (dd, J =
9.0, 2.5 Hz, 1H, ArH), 6.62 (s, 1H, ArH), 6.58 (s, 1H, ArH), 5.25 (s, 2H, OCH2), 4.59 (t, J = 6.1 Hz, 2H,
CHNCH2), 3.69 (s, 6H, 2 × OCH3), 3.54 (s, 2H, ArCH2N), 2.91 (t, J = 6.1 Hz, 2H, NCH2), 2.65 (s, 4H,
2 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 28.3, 32.1, 50.4, 55.1, 55.6, 58.9, 61.1, 107.3, 109.9,
111.7, 118.5, 120.0, 122.8, 123.8, 125.7, 126.4, 127.4, 128.6, 129.3, 130.1, 134.5, 141.4, 143.6, 146.7,

155.7; ESI-MS m/z: 445.1 ([M + H]+); Anal. calcd. For C26H28N4O3: C, 70.25; H, 6.35; N, 12.60;
Found: C, 70.30; H, 6.33; N, 12.58.
5.7.2.
6,7-dimethoxy-2-(2-(4-((4-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroiso
quinoline(14)
Yield 70.6%; white powder; mp: 113–115°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.25 (s,
1H, NCH=C), 7.14 – 7.07 (m, 2H, ArH), 7.04 – 6.98 (m, 2H, ArH), 6.65 (s, 1H, ArH), 6.56 (s, 1H,
ArH), 5.15 (s, 2H, OCH2), 4.58 (t, J = 6.0 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.53 (s, 2H,
ArCH2N), 2. 90 (t, J = 6.0 Hz, 2H, NCH2), 2.66 (s, 4H, 2 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ
ppm): 28.2, 32.2, 50.3, 54.9, 55.4, 58.8, 61.8, 109.9, 111.7, 115.3, 120.0, 123.1, 125.7, 130.0, 134.3,
141.1, 142.7, 146.5, 147.0, 162.0; ESI-MS m/z: 444.0([M + H]+); Anal. calcd. For C22H25N5O5: 60.13;
H, 5.73; N, 15.94; Found: C, 60.10; H, 5.74; N, 15.95.
5.7.3.
6,7-dimethoxy-2-(2-(4-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydr
oisoquinoline (15)
Yield 61.5%; pale yellow powder; mp: 110-112°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.33 (s,
1H, NCH=C), 8.07 (d, J = 7.7 Hz, 1H, ArH), 7.86 (d, J = 7.5 Hz, 1H, ArH), 7.56 – 7.36 (m, 4H, ArH),
7.15 (d, J = 7.4 Hz, 1H, ArH), 6.61 (dd, J = 13.6, 2.8 Hz, 2H, ArH), 5.32 (d, J = 2.7 Hz, 2H, OCH2),
4.59 (s, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.54 (s, 2H, ArCH2N), 2.92 (s, 2H, NCH2), 2.66 (s, 4H,
2 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 28.3, 32.3, 50.3, 55.0, 55.6, 59.1, 61.5, 105.7, 109.7,
111.7, 120.3, 121.6, 122.5, 124.8, 125.1, 125.7, 126.5, 127.3, 130.1, 134.5, 141.3, 143.8, 146.8, 153.2;
ESI-MS m/z: 445.0 ([M + H]+); Anal. calcd. For C26H28N4O3: C, 70.25; H, 6.35; N, 12.60; Found: C,
70.29; H, 6.34; N, 12.58.
5.7.4
6,7-dimethoxy-2-(2-(4-((p-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroisoquino
line (16)
Yield 62.0%; pale yellow powder; mp: 108-110°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.19 (s,
1H, NCH=C), 7.06 (d, J = 8.3 Hz, 2H, ArH), 6.89 (d, J = 8.5 Hz, 2H, ArH), 6.65 (s, 1H, ArH), 6.59 (s,
1H, ArH), 5.07 (s, 2H, OCH2), 4.57 (t, J = 5.9 Hz, 2H, CHNCH2), 3.69 (s, 6H, 2 × OCH3), 3.54 (s, 2H,
ArCH2N), 2.94 – 2.84 (m, 2H, NCH2), 2.67 (s, 4H, 2 × CH2), 2.22 (s, 3H, -CH3); 13C NMR (75 MHz,
DMSO-d6 , δ ppm): 20.1, 28.2, 32.1, 50.4, 55.0, 55.4, 59.0, 60.9, 109.9, 111.7, 114.5, 119.8, 122.5,
125.8, 126.5, 129.8, 134.7, 141.3, 143.8, 146.8, 155.7; ESI-MS m/z: 409.1 ([M + H]+); Anal. calcd. For
C23H28N4O3: C, 67.63; H, 6.91; N, 13.72; Found: C, 67.59; H, 6.90; N, 13.74.
5.7.5
6,7-dimethoxy-2-(2-(4-((4-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydr
oisoquinoline (17)
Yield 23.3%; pale yellow powder; mp: 118-121°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.18 (s,
1H, NCH=C), 6.94 (d, J = 9.1 Hz, 2H, ArH), 6.83 (d, J = 9.1 Hz, 2H, ArH), 6.64 (s, 1H, ArH), 6.59 (s,
1H, ArH), 5.04 (s, 2H, OCH2), 4.57 (t, J = 6.0 Hz, 2H, CHNCH2), 3.68 (s, 9H, 3× OCH3), 3.53 (s, 2H,
ArCH2N), 2.90 (t, J = 5.6 Hz, 2H, NCH2), 2.66 (s, 4H, 2 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ
ppm): 28.2, 32.4, 50.4, 55.0, 55.2, 55.4, 59.1, 61.3, 109.9, 111.8, 114.6, 115.7, 120.0, 122.8, 125.9,
126.7, 130.0, 134.6, 141.5, 144.0, 146.9, 153.8; ESI-MS m/z: 425.1 ([M + H]+); Anal. calcd. For
C23H28N4O4: C, 65.08; H, 6.65; N, 13.20; Found: C, 65.12; H, 6.64; N, 13.18.
5.7.6.

2-(2-(4-((4-ethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroiso
quinoline (18)
Yield 47.3%; pale yellow powder; mp: 106–108°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.20 (s,
1H, NCH=C), 7.10 (d, J = 8.3 Hz, 2H, ArH), 6.92 (d, J = 8.3 Hz, 2H, ArH), 6.62 (d, J = 16.3 Hz, 2H,
ArH), 5.08 (s, 2H, OCH2), 4.64–4.47 (m, 2H, CHNCH2), 3.69 (s, 6H, 2 × OCH3), 3.54 (s, 2H,
ArCH2N), 2.94–2.83 (m, 2H, NCH2), 2.67 (s, 4H, 2 × CH2), 2.56–2.52 (m, 2H,–CH2CH3), 1.14 (t, J =
7.5 Hz, 3H,–CH2CH3); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 15.8, 28.5, 32.4, 50.5, 55.3, 55.9, 57.2,
61.5, 110.3, 112.2, 116.5, 121.1, 124.6, 125.1, 126.7, 128.7, 130.6, 134.7, 140.8, 143.7, 147.9, 156.8;
ESI-MS m/z: 423.1 ([M + H]+); Anal. calcd. For C24H30N4O3: C, 68.22; H, 7.16; N, 13.26; Found: C,
68.18; H, 7.17; N, 13.28.
5.7.7.
2-(2-(4-((4-isopropylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahyd
roisoquinoline (19)
Yield 70.4%; pale yellow powder; mp: 102–105°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.20 (s,
1H, NCH=C), 7.13 (d, J = 8.4 Hz, 2H, ArH), 6.92 (d, J = 8.5 Hz, 2H, ArH), 6.64 (s, 1H, ArH), 6.59 (s,
1H, ArH), 5.08 (s, 2H, OCH2), 4.57 (t, J = 5.7 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.54 (s, 2H,
ArCH2N), 2.89 (dd, J = 12.3, 6.6 Hz, 2H, NCH2), 2.84 – 2.79 (m, 1H, –CH(CH3)2), 2.67 (s, 4H, 2 ×
CH2), 1.16 (d, J = 6.9 Hz, 6H, –CH(CH3)2); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 23.8, 28.2, 32.2,
32.5, 50.4, 55.0, 55.4, 59.0, 60.9, 109.9, 111.7, 114.4, 120.0, 122.5, 125.8, 126.5, 127.2, 130.1, 134.6,
140.7, 143.8, 147.1, 156.2; ESI-MS m/z: 437.1 ([M + H]+); Anal. calcd. For C25H32N4O3: C, 68.78; H,
7.39; N, 12.83; Found: C, 68.82; H, 7.38; N, 12.81.
5.7.8.
2-(2-(4-((4-(tert-butyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahy
droisoquinoline (20)
Yield 74.4%; white powder; mp: 108–111°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.20 (s, 1H,
NCH=C), 7.27 (d, J = 7.4 Hz, 2H, ArH), 6.92 (d, J = 7.5 Hz, 2H, ArH), 6.64 (s, 1H, ArH), 6.59 (s, 1H,
ArH), 5.08 (s, 2H, OCH2), 4.57 (t, J = 5.8 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.54 (s, 2H,
ArCH2N), 2.90 (s, 2H, NCH2), 2.66 (s, 4H, 2 × CH2), 1.23 (s, 9H, –C(CH3)3); 13C NMR (75 MHz,
DMSO-d6 , δ ppm): 28.3. 31.2, 32.3, 33.8, 55.0, 55.4, 59.1, 61.0, 109.9, 111.7, 114.4, 120.0, 122.6,
125.9, 126.0, 126.7, 130.0, 134.6, 141.4, 143.9, 147.0, 155.8; ESI-MS m/z: 451.2 ([M + H]+); Anal.
calcd. For C26H34N4O3: C, 69.31; H, 7.61; N, 12.43; Found: C, 69.26; H, 7.62; N, 12.45.
5.7.9.
2-(2-(4-((3,4-dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahy
droisoquinoline (21)
Yield 68.7%; pale yellow powder; mp: 110–112°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.18 (s,
1H, NCH=C), 7.00 (d, J = 8.3 Hz, 1H, ArH), 6.80 (d, J = 2.4 Hz, 1H, ArH), 6.72 (dd, J = 8.2, 2.6 Hz,
1H, ArH), 6.64 (s, 1H, ArH), 6.59 (s, 1H, ArH), 5.05 (s, 2H, OCH2), 4.57 (t, J = 6.1 Hz, 2H, CHNCH2),
3.68 (s, 6H, 2 × OCH3), 3.53 (s, 2H, ArCH2N), 2.90 (t, J = 6.2 Hz, 2H, NCH2), 2.66 (s, 4H, 2 × CH2),
2.16 (s, 3H, –CH3), 2.12 (s, 3H, –CH3); 13C NMR (75 MHz, DMSO-d6 , δ ppm): 18.9, 20.1, 28.5, 32.4,
50.5, 55.3, 55.9, 57.2, 61.5, 110.3, 112.1, 116.5, 121.1, 124.6, 125.1, 126.2, 128.7, 130.6, 137.7, 140.8,
143.7, 147.9, 156.7; ESI-MS m/z: 423.1 ([M + H]+); Anal. calcd. For C24H30N4O3: C, 68.22; H, 7.16; N,
13.26; Found: C, 68.18; H, 7.17; N, 13.28.
5.7.10.
2-(2-(4-((2,4-dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahy

droisoquinoline (22)
Yield 78.1%; pale yellow powder; mp: 112–114°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.18 (s,
1H, NCH=C), 6.93 (q, J = 8.0 Hz, 3H, ArH), 6.64 (s, 1H, ArH), 6.58 (s, 1H, ArH), 5.07 (s, 2H, OCH2),
4.57 (t, J = 6.1 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.53 (s, 2H, ArCH2N), 2.90 (t, J = 6.1 Hz,
2H, NCH2), 2.66 (s, 4H, 2 × CH2), 2.18 (s, 3H, –CH3), 2.04 (s, 3H, –CH3); 13C NMR (75 MHz,
DMSO-d6 , δ ppm): 16.4, 20.5, 28.6, 32.5, 50.5, 55.3, 55.9, 57.1, 62.1, 110.3, 112.2, 116.4, 121.1, 125.0,
126.2, 126.7, 127.4, 129.6, 131.6, 137.2, 140.7, 143.5, 147.6, 154.4; ESI-MS m/z: 423.1 ([M + H]+).
Anal. calcd. For C24H30N4O3: C, 68.22; H, 7.16; N, 13.26; Found: C, 68.25; H, 7.15; N, 13.28.
5.7.11.
6,7-dimethoxy-2-(2-(4-((2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydr
oisoquinoline (23)
Yield 67.4%; pale yellow powder; mp: 116–119°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.20 (s,
1H, NCH=C), 7.09 (d, J = 7.6 Hz, 1H, ArH), 6.98 – 6.82 (m, 3H, ArH), 6.65 (s, 1H, ArH), 6.59 (s, 1H,
ArH), 5.08 (s, 2H, OCH2), 4.58 (t, J = 5.8 Hz, 2H, CHNCH2), 3.69 (d, J = 5.4 Hz, 9H, 3 × OCH3), 3.54
(s, 2H, ArCH2N), 2.90 (t, J = 5.9 Hz, 2H, NCH2), 2.67 (s, 4H, 2 × CH2); 13C NMR (75 MHz, DMSO-d6 ,
δ ppm): 28.3, 32.4, 50.4, 55.0, 55.3, 55.5, 59.1, 61.4, 109.9, 111.7, 114.6, 115.7, 120.0, 122.8, 125.9,
126.7, 129.9, 134.6, 141.5, 144.0, 146.9, 153.8; ESI-MS m/z: 425.1 ([M + H]+). Anal. calcd. For
C23H28N4O4: C, 65.08; H, 6.65; N, 13.20; Found: C, 65.13; H, 6.64; N, 13.17.
5.7.12.
2-(2-(4-((2-(tert-butyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahy
droisoquinoline (24)
Yield 66.5 %; white powder; mp: 120–122°C; 1H NMR (300 MHz, DMSO-d6): δ= 8.18 (s, 1H,
NCH=C), 7.31 – 7.05 (m, 3H, ArH), 6.87 (d, J = 6.1 Hz, 1H, ArH), 6.63 (s, 1H, ArH), 6.57 (s, 1H,
ArH), 5.13 (s, 2H, OCH2), 4.60 (t, J = 5.8 Hz, 2H,CHNCH2), 3.62 (s, 6H, 2 × OCH3), 3.52 (s, 2H，
ArCH2N), 2.88 (s, 2H, NCH2), 2.65 (s, 4H, 2 × CH2), 1.21 (s, 9H, –C(CH3)3); 13C NMR (75 MHz,
DMSO-d6 , δ ppm): 28.3. 31.2, 32.3, 33.8, 55.0, 55.4, 59.1, 61.0, 109.9, 111.7, 114.4, 120.0, 122.6,
125.9, 126.0, 126.7, 130.0, 134.6, 141.4, 143.9, 147.0, 155.8; ESI-MS m/z: 451.1 ([M + H]+); Anal.
calcd. For C26H34N4O3: C, 69.31; H, 7.61; N, 12.43; Found: C, 69.35; H, 7.59; N, 12.40.
5.7.13.
2-(2-(4-((4-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6,7-dimethoxy-1,2,3,4-tetrahydrois
oquinoline (25)
Yield 38.1%; pale yellow powder; mp: 111–113°C; 1H NMR (300 MHz, DMSO-d6):δ= 8.23 (s,
1H, NCH=C), 7.14 – 7.06 (m, 2H, ArH), 7.04 – 6.98 (m, 2H, ArH), 6.64 (s, 1H, ArH), 6.58 (s, 1H,
ArH), 5.09 (s, 2H, OCH2), 4.57 (t, J = 6.0 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 × OCH3), 3.53 (s, 2H,
ArCH2N), 2.89 (t, J = 6.0 Hz, 2H, NCH2), 2.66 (s, 4H, 2 × CH2); 13C NMR (75 MHz, DMSO-d6 , δ
ppm): 28.2, 32.2, 50.3, 54.9, 55.4, 58.8, 61.8, 109.9, 111.6, 115.2, 120.0, 123.1, 125.7, 130.0, 134.3,
141.1, 142.7, 146.5, 147.0, 158.7; ESI-MS m/z: 413.0 ([M + H]+), 435.1([M + Na]+); Anal. calcd. For
C22H25FN4O3: C, 64.06; H, 6.11; F, 4.61; N, 13.58; Found: C, 64.12; H, 6.09; F, 4.57; N, 13.60.
5.8. Biological assays
5.8.1. Cytotoxicity assay
K562 and K562/A02 cells were grown in 96-well microtiter plates at 1~2×104 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

exponentially growing cancer cells were incubated 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 5.0 software from the
dose–response curves.
5.8.2. MTT assay
K562/A02 cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine
serum at 37°C in a 5% CO2 humidified atmosphere. K562/A02 cells (cells per well) were seeded in
96-well plates. 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 5.0 software from the dose–response
curves. Experiments were conducted in triplicates and repeated three times independently.
5.8.3. Adriamycin intracellular accumulation
K562 and K562/A02 cells were seeded into 24-well plates at 1.5×105 /well. Different
concentrations of compound 4 and VRP were pre-incubated with cells for 60 min. Then 20μM ADM
was added into every 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
intracellular ADM was estimated by BD FACSCalibur flow cytometer.
5.8.4. 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 4 or VRP (0.5, 2.5, 5μM) for another 90 min. Afterwards
the cells were washed twice in ice-cold PBS. The mean fluorescence intensity of
retained intracellular Rh123 was estimated by BD FACSCalibur flow cytometer.
Acknowledgments
The work was supported by the National Science and Technology Major Projects of China (No.
2009ZX09102-033) and the National Natural Science Foundation of China (No. 81173088).
References and notes
1. B.; Homolya, L.; Szakacs, G.; Varadi, A. Physiol. Rev 2006, 86, 1179.
2. Turk, D.; Hall, M. D.; Chu, B. F.; Ludwig, J. A.; Fales, H. M.; Gottesman, M. M.; Szakacs, G. J.
Cancer Res. 2009, 69, 8293.
3. Breier, A.; Gibalova, L.; Seres, M.; Barancik, M.; Sulova, Z. J. Anti-Cancer Agent Me. 2013, 13,
159.
4. Pires, M. M.; Emmert, D.; Hrycyna, C. A.; Chmielewski, J. J. Mol. Pharm. 2009, 75, 92.
5. Mihalyi, A.; Gaspar, R.; Zalan, Z.; Lazar, L.; Fülöp, F.; de Witte, P. A. M. J. Anticancer Res.
2004, 24, 1631.
6. Robert, J.; Jarry, C. J. J. Med. Chem. 2003, 46, 4805–4817.
7. Henderson, M. J.; Haber, M.; Porro, A.; Munoz, M. A.; Iraci, N.; Xue, C. J. J. Natl. Cancer I
2011, 103, 1236.
8. Singh, S.; Prasad, N. R.; Chufan, E. E.; Patel, B. A.; Wang, Y. J.; Chen, Z. S.; Ambudkar, S. V.;

Talele, T. T. J. J. Med. Chem. 2014, 57, 4058.
9. Kwak, J. O.; Lee, S. H.; Lee, G. S.; Kim, M. S.; Ahn, Y. G.; Lee, J. H.; Kim, S. W.; Kim, K. H.;
Lee, M. G. J. Eur. J. Pharmacol. 2010, 627, 92−98.
10. Wijtmans, M.; de Graaf, C.; de Kloe, G.; Istyastono, E. P.; Smit, J.; Lim, H.; Boonnak, R.;
Nijmeijer, S.; Smits, R. A.; Jongejan, A.; Zuiderveld, O.; de Esch, I. J.; Leurs, R. J. J. Med. Chem.
2011, 54, 1693.
11. Liu, B.; Qiu, Q.; Zhao, T.; Jiao, L.; Hou, J.; Li, Y.; Qian, H.; Huang, W. J. Chem. Biol. Drug Des.
2014, 84, 182.
12. Zhao, T.; Song, Y.; Liu, B.; Qiu, Q.; Jiao, L.; Li, Y; Huang, W; Qian, H. J. Can. J. Physiol.
Pharmacol. 2015, 93, 33.
13. Kolb, H. C.; Sharpless, K. B. J. Drug Discovery Today. 2003, 8, 1128.
14. Yu, S. S.; Lau, C. M.; Barham, W. J.; Onishko, H. M.; Nelson, C. E.; Li, H.; Smith, C. A.; Yull, F.
E.; Duvall, C. L.; Giorgio, T. D. J. Mol. Pharm. 2013, 10, 975.
15. Sane, P. S.; Tawade, B. V.; Palaskar, D. V.; Menon, S. K.; Wadgaonkar, P. P. J. React. Funct.
Polym. 2012, 72, 713.
16. Tahtaoui, C.; Parrot, I.; Klotz, P.; Guillier, F.; Galzi, J.-L.; Hibert, M.; Ilien, B. J. J. Med. Chem.
2004, 47, 4300.
17. Gu, X.; Ren, Z.; Tang, X.; Peng, H.; Zhao, Q.; Lai, Y.; Peng, S.; Zhang, Y. J. Eur. J. Med. Chem.
2012, 51, 137.
18. Gu, X.; Ren, Z.; Tang, X.; Peng, H.; Ma, Y.; Lai, Y.; Peng, S.; Zhang, Y. J. Bioorg. Med. Chem.
2012, 20, 2540.
19. Jiao, L.; Qiu, Q.; Liu, B.; Zhao, T.; Huang, W; Qian, H. J. Bioorg. Med. Chem. 2014, 22, 6857.
20. Shi, Y. Q.; Qu, X. J.; Liao, Y. X.; Xie, C. F.; Cheng, Y. N.; Li, S.; Lou, H. X. J. Eur. J.
Pharmacol. 2008, 584, 66.
21. Ji, B.-S.; He, L.; Liu, G.-Q. Life Sci. 2005, 77, 2221.

Graphical abstract