6,7-Dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahydroisoquinolines as superior reversal agents for P-glycoprotein-mediated multidrug resistance

Article abstract of DOI:10.1002/cmdc.201402463

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy. Based on our previous study, 17 novel compounds with the 6,7-dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahydroisoquinoline scaffold were designed and synthesized. Among them, 2-[(1-{4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl]phenyl}-1H-1,2,3-triazol-4-yl)methoxy]-N-(p-tolyl)-benzamide (compound 7h) was identified as a potent modulator of P-gp-mediated MDR, with high potency (EC₅₀ = 127.5 ± 9.1 nM), low cytotoxicity (TI > 784.3), and long duration (> 24 h) in reversing doxorubicin (DOX) resistance in K562/A02 cells. Compound 7h also enhanced the effects of other MDR-related cytotoxic agents (paclitaxel, vinblastine, and daunorubicin), increased the accumulation of DOX and blocked P-gp-mediated rhodamine 123 efflux function in K562/A02 MDR cells. Moreover, 7h did not have any effect on cytochrome (CYP3A4) activity. These results indicate that 7h is a relatively safe modulator of P-gp-mediated MDR that has good potential for further development.

Full text of DOI:10.1002/cmdc.201402463

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DOI: 10.1002/cmdc.201402463
6,7-Dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-
1,2,3,4-tetrahydroisoquinolines as Superior Reversal
Agents for P-Glycoprotein-Mediated Multidrug Resistance
Baomin Liu,[a, b] Qianqian Qiu,[a] Tianxiao Zhao,[a] Lei Jiao,[a] Yunman Li,[c] Wenlong Huang,*[a]
and Hai Qian*[a]
P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is
a major obstacle for successful cancer chemotherapy. Based on
our previous study, 17 novel compounds with the 6,7-dime-
thoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahy-
droisoquinoline scaffold were designed and synthesized.
Among them, 2-[(1-{4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl]phenyl}-1H-1,2,3-triazol-4-yl)methoxy]-N-(p-tolyl)-
benzamide (compound 7h) was identified as a potent modula-
tor of P-gp-mediated MDR, with high potency (EC50 =127.5ꢀ
9.1 nm), low cytotoxicity (TI>784.3), and long duration
(>24 h) in reversing doxorubicin (DOX) resistance in K562/A02
cells. Compound 7h also enhanced the effects of other MDR-
related cytotoxic agents (paclitaxel, vinblastine, and daunorubi-
cin), increased the accumulation of DOX and blocked P-gp-
mediated rhodamine 123 efflux function in K562/A02 MDR
cells. Moreover, 7h did not have any effect on cytochrome
(CYP3A4) activity. These results indicate that 7h is a relatively
safe modulator of P-gp-mediated MDR that has good potential
for further development.
Introduction
Multidrug resistance (MDR) is one of the main impediments to
achieving effective treatments for cancer.[1] Overexpression of
P-glycoprotein (P-gp) by cancer cells is the major contributor
for the development of MDR.[2] P-gp is a member of the ATP-
binding cassette (ABC) transporter superfamily, and has broad
substrate specificity. It pumps drugs out of cells by using ATP
hydrolysis as the energy source, leading to the emergence of
MDR.[3] Development of P-gp inhibitors is considered to be
a promising approach in overcoming MDR.[4] Three generations
of P-gp inhibitors have been identified to date.[5] However,
owing to undesired side effects, no P-gp inhibitor has been ap-
proved for clinical use. For example, first-generation P-gp in-
hibitors, including verapamil (VRP) and cyclosporin A (CsA),
produced disappointing results due to high toxicity. Second-
generation P-gp inhibitors still exhibited some unsatisfactory
levels of toxicity and pharmacokinetic complications that im-
peded drug candidate development. Although some third-gen-
eration P-gp inhibitors such as tariquidar have been in clinical
trials, their clinical efficacies are not yet clear.[6] Therefore, there
is still a great need to discover safe and effective MDR modula-
tors to overcome the problem of MDR in cancer chemothera-
py.[7]
Previously, we reported compound a (Figure 1), with a 6,7-
dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tet-
rahydroisoquinoline scaffold, as a potent modulator of MDR
with low cytotoxicity in cancer cells that overexpress P-gp.[8] In
the present study, we designed and synthesized a new series
of compounds based on the scaffold of compound a via iso-
sterism, with the aim to obtain more potent P-gp inhibitors.
All synthesized compounds were evaluated for cytotoxicity
and for the capacity to reverse P-gp-mediated MDR against
doxorubicin (DOX). Among the active compounds, 7h showed
[a] Dr. B. Liu,+ Q. Qiu,+ T. Zhao, L. Jiao, Prof. W. Huang, Prof. H. Qian
Center of Drug Discovery, State Key Laboratory of Natural Medicines
China Pharmaceutical University
24 Tongjiaxiang, Nanjing 210009 (P.R. China)
E-mail: ydhuangwenlong@126.com
qianhai24@163.com
[b] Dr. B. Liu+
Nanjing Research and Development Center
CTTQ Pharmaceutical Research Institute, Building 9, No. 699-8
Xuanwu Dadao, Xuanwu District, Nanjing, Jiangsu (P.R. China)
[c] Prof. Y. Li
Department of Physiology, China Pharmaceutical University
24 Tongjiaxiang, Nanjing 210009 (P.R. China)
[+] These authors contributed equally to this work.
Figure 1. Desirable target compounds derived from scaffold a via isosterism.
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the most potent reversal activity and was selected for further
biological activity evaluations.
sistant subline K562/A02, which overexpress P-gp, were deter-
mined by MTT assays; DOX was used as positive control. As
shown in Table 1, it is clear that K562/A02 cells (DOX IC50
96.42ꢀ3.40 mm) displayed 205.1-fold greater resistance than
non-resistant K562 cells (DOX IC50 0.47ꢀ0.03 mm). Most of the
target compounds showed no intrinsic cytotoxicity (IC50 values
>100 mm) against both K562 and K562/A02 cells. In addition,
both K562 and K562/A02 cells were almost equally insensitive
to most of the target compounds, suggesting that, unlike
DOX, they were not likely to be substrates for P-gp.
Results and Discussion
Chemistry
The synthetic routes used to access target compounds 7a–q
are shown in Schemes 1 and 2. Compounds 1 and 4 were syn-
thesized according to our previous report.[8a] Aromatic sulfonyl
chloride in dichloromethane was added to a solution of com-
pound 1 and triethylamine in dichloromethane to afford the
desired products 2a–f (Scheme 1). Compound 5 and thionyl
chloride were combined with heating to yield compound 6,
which was allowed to react with the appropriately substituted
aniline to afford compounds 3g–q. Sodium ascorbate and cop-
per(II) sulfate were added to a solution of 2a–f and 3g–q and
4 in methanol to furnish the desire products 7a–q in high
purity (Scheme 2). The structures of all target compounds are
presented in Table 1.
MDR reversal activity
The effects of the target compounds on the cytotoxicity of
DOX were evaluated preliminarily in K562/A02 cells at 1.0 mm.
In brief, the cytotoxicity of DOX against K562/A02 cells was
evaluated in the presence or absence of the target compounds
at 1.0 mm by MTT assay, using VRP, WK-X-34, and compound
a as controls. As listed in Table 2, all tested compounds dis-
played reversal potency at 1.0 mm if co-administered with DOX.
Moreover, most of the target compounds exhibited more
potent reversal activity than the classical P-gp inhibitor VRP
under the same conditions. Among the active compounds,
compounds a, 7a, 7h, and 7i showed the most promising re-
versal potency with reversal fold (RF) values of 17.8, 26.8, 18.0,
and 18.3, respectively, at 1.0 mm. In addition, the four active
compounds displayed similar reversal activity with the positive
control WK-X-34 (RF: 19.2).
Biological studies
Cytotoxicity
Before MDR reversal experiments, the cytotoxicities of the
target compounds against the K562 cell line and its drug-re-
To further differentiate reversal
activity of the target compounds
to identify the most promising
compound, we tested the target
compounds at lower concentra-
tion (0.5 mm). Notably, com-
pound 7h showed the highest
activity, with higher RF (12.6)
than compound a (RF: 9.2), sug-
gesting that compound 7h is
the most promising compound.
Scheme 1. Synthesis of target compounds 7a–f: Reagents and conditions: a) arylsulfonyl chloride, Et3N, CH2Cl2,
08C!RT, 24 h; b) 2a–f, sodium ascorbate, CuSO4, 75% CH3OH, RT, 24–48 h.
Dose–response relationship and
therapeutic index
To provide a quantitative mea-
surement of MDR-modulating ef-
ficacy, we determined the EC50
value of 7h in reversing DOX re-
sistance in K562/A02 cells as pre-
viously reported.[9] As shown in
Figure 2, 7h was found to be
very potent in reversing DOX re-
sistance, with an EC50 value in
the nanomolar range (127.5ꢀ
9.1 nm). The cytotoxicity of 7h
against normal human gastric
epithelial cell strain-1 (GES-1)
was determined to evaluate its
Scheme 2. Synthesis of target compounds 7g–q: Reagents and conditions: a) SOCl2, DMF, 508C, 2 h; b) ArNH2, pyri-
dine, DMAP, CH2Cl2, 08C!RT, 12 h; c) 3-bromo-1-propyne, K2CO3, acetone, reflux, 3 h; d) sodium ascorbate, CuSO4,
75% CH3OH, RT, 24–48 h.
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Table 1. Structures and cytotoxicity of the target compounds against
Table 2. DOX-resistance reversal activity of the target compounds.
K562 and K562/A02 cell lines.
Compd[b]
IC50 of DOX on K562/A02 [mm] (RF)[a]
Compd at 1.0 mm Compd at 0.5 mm
a
7a
7b
7c
7d
7e
7 f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
5.4ꢀ0.31
3.6ꢀ0.11
7.1ꢀ0.15
6.7ꢀ0.21
7.9ꢀ0.33
5.9ꢀ0.24
10.5ꢀ0.37
7.2ꢀ0.22
5.4ꢀ0.18
5.3ꢀ0.10
9.4ꢀ0.29
11.2ꢀ0.20
15.1ꢀ0.23
26.3ꢀ1.36
41.9ꢀ1.84
11.2ꢀ0.17
26.1ꢀ1.01
27.4ꢀ0.83
5.0ꢀ0.14
79.8ꢀ5.12
96.4ꢀ3.4
(17.8)
(26.8)
(13.6)
(14.3)
(12.2)
(16.2)
(9.2)
(13.3)
(18.0)
(18.3)
(10.2)
(8.6)
(6.4)
(3.7)
(2.3)
(8.6)
(3.7)
(3.5)
(19.2)
(1.2)
(1.0)
10.5ꢀ0.16
(9.2)
(4.9)
(1.7)
(2.2)
(1.9)
(2.9)
(1.9)
(8.8)
(12.6)
(6.2)
(3.5)
(4.0)
(5.7)
(2.1)
(1.5)
(3.9)
(2.4)
(2.9)
(14.4)
ND
19.5ꢀ0.27
56.0ꢀ4.41
44.6ꢀ3.51
51.2ꢀ3.10
33.2ꢀ2.41
51.1ꢀ4.26
10.9ꢀ0.37
7.6ꢀ0.23
15.6ꢀ0.87
27.9ꢀ2.11
24.4ꢀ3.01
16.9ꢀ1.40
45.5ꢀ3.87
63.4ꢀ4.19
24.8ꢀ2.34
40.9ꢀ2.77
33.3ꢀ1.76
6.7ꢀ0.5
Compd
X
R
IC50 [mm][a]
K562/A02
K562
>100
7a
7b
7c
7d
NHSO2
NHSO2
NHSO2
NHSO2
>100
>100
>100
>100
>100
>100
>100
7e
NHSO2
>100
>100
>100
7q
WK-X-34
VRP
DMSO[c]
7 f
NHSO2
CONH
CONH
27.93ꢀ0.59
ND[d]
ND
ND
7g
7h
18.55ꢀ0.42
>50
[a] IC50 value toward DOX was determined in the presence of modulator
at 1.0 or 0.5 mm (values are the mean ꢀSEM of three independent experi-
ments); reversal fold (RF) values in brackets refer to fold change in drug
sensitivity: RF=(IC50 without modulator)/(IC50 with modulator). [b] All
compounds were dissolved in DMSO for testing. [c] DMSO (0.1%) was
added as solvent control for testing P-gp-modulating activity. [d] ND: not
determined.
>100
>100
>100
>100
7i
7j
7k
CONH
CONH
CONH
>100
>100
10.57ꢀ0.38 >100
66.12ꢀ0.55 >100
7l
CONH
CONH
7m
>100
>100
>100
7n
CONH
>100
>100
7o
7p
CONH
CONH
>100
>100
>100
>100
>100
7q
CONH
–
DOX
–
0.47ꢀ0.03
96.42ꢀ3.40
[a] Determined by MTT assay; values are the mean ꢀSEM of three inde-
Figure 2. Determination of the EC50 value for 7h in lowering DOX resistance
in K562/A02 cells. The percent of IC50 of DOX was calculated as: [(IC50 of
DOX at each modulator concentration)/(IC50 of DOX without modula-
tor)]ꢁ100. EC50 is defined as the concentration of modulator required to de-
crease the %IC50 of DOX by half. Values are the average ꢀSEM of three inde-
pendent experiments.
pendent experiments.
safety. The results indicated that 7h shows no cytotoxic effect
toward GES-1 cells (IC50 >100 mm). Therefore, the therapeutic
index (TI, ratio of IC50 toward GES-1 to EC50 for reversing DOX
resistance) for 7h was very high (>784.3), indicating that 7h
may be very safe for normal human cells if co-administered
with anticancer agents as an MDR modulator.
Effect of 7h on reversing P-gp toward other anticancer drugs
We were interested in determining if 7h could also reverse P-
gp-mediated resistance toward other drugs such as paclitaxel,
vinblastine, daunorubicin, and 5-fluorouracil (5-FU).[10] Figure 3
clearly illustrates that 7h enhanced the cytotoxic effects of var-
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Figure 3. Reversal effect of 7h on ABCB1-mediated drug resistance to anticancer agents. Paclitaxel, vincristine, and daunorubicin are P-gp substrates, and 5-
fluorouracil is not. Values are the average ꢀSEM of three independent experiments.
ious agents (paclitaxel, vinblastine and daunorubicin) associat-
ed with the P-gp-mediated MDR phenotype in K562/A02 cells,
but did not affect the activity of non-MDR cytotoxicity (5-FU)
in resistant cells. These results suggest that reversal of resist-
ance is attributable to the inhibition of P-gp function.
Effect on accumulation of DOX
One property of the MDR phenotype is a defect in the accu-
mulation of MDR cytotoxic compounds relieved by MDR mod-
ulators. Therefore, we investigated whether the reversal effect
of 7h is associated with a concomitant increase in DOX accu-
mulation. DOX is an autofluorescent compound, which allowed
us to establish a straightforward accumulation assay by spec-
trofluorimetry; the results are illustrated in Figure 4. In the ab-
sence of modulator, DOX accumulation in K562 cells was ~4.4-
fold (p<0.001) higher than that in K562/A02 cells. This is prob-
Figure 4. Effect of compound 7h on intracellular DOX accumulation in
K562/A02 cells. DMSO (0.1%) was used as negative control; VRP and WK-X-
34 were chosen as positive controls. Results are presented as the mean
ꢀSEM of n=2 independent experiments: *p<0.05, **p<0.01 relative to
negative control (K562/A02).
ably attributed to P-gp-mediated DOX efflux. Both 7h and WK-
X-34 significantly enhanced the 2.5 h accumulation of DOX
into K562/A02 cells at modulator concentrations down to
0.5 mm. However, verapamil increased DOX accumulation only
if present at 10 mm, the highest level tested. The results indi-
cate that 7h exhibits greater potency than verapamil in in-
creasing the accumulation of DOX into K562/A02 cells, and
that 7h is a potent P-gp inhibitor.
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Inhibition of P-gp-mediated Rh123 efflux
Table 3. Duration of MDR reversal in K562/A02 cells after incubation and
washout of verapamil or 7h.
It is known that the efflux of the fluorescent dye rhoda-
mine 123 (Rh123) is P-gp dependent; this has been used ex-
tensively to evaluate the effect of MDR modulators on P-gp
efflux function. To further determine whether 7h can inhibit P-
gp-mediated drug efflux, intracellular Rh123 was monitored by
flow cytometry. As demonstrated in Figure 5, 7h at 1.0 mm can
Treatment
IC50/DOX [mm] (RF)[a]
10 mm VRP
Control
1.0 mm 7h
no wash
wash, 0 h
wash, 6 h
wash, 12 h ND
wash, 24 h ND
90.71ꢀ3.25 (1.0)
45.03ꢀ2.88 (2.0)
7.78ꢀ0.22 (11.7)
7.89ꢀ0.56 (11.5)
8.87ꢀ0.56 (10.2)
10.49ꢀ0.36 (8.6)
22.90ꢀ0.63 (4.0)
ND[b]
ND
>75
ND
ND
ND
[a] Values are the mean ꢀSEM of 2–3 independent experiments; reversal
fold (RF) values in brackets refer to fold change in drug sensitivity: RF=
(IC50 without modulator)/(IC50 with modulator). [b] ND: not determined.
trol VRP (<6 h) after removal of the agent from the culture
medium.
Effect of 7h on CYP3A4
It is known that P-gp and CYP3A4 have a considerable overlap
in tissue distribution and substrate preference. Several of the
second-generation P-gp modulators are substrates for CYP3A4,
giving rise to unpredictable pharmacokinetic interactions with
chemotherapeutic agents.[11] However, the new generation of
P-gp modulators do not affect CYP3A4 at relevant concentra-
tions.[12] To further investigate the character of 7h as a P-gp
modulator, we determined its effect on rat liver CYP3A4 in
vitro (Figure 6). Ketoconazole, a potent inhibitor of CYP3A4,
significantly inhibited the activity of CYP3A4 in a concentra-
tion-dependent manner. However, 7h did not affect the activi-
ty of CYP3A4 even at 40 mm, which is a concentration signifi-
cantly higher than that required to reverse MDR in vitro. The
results showed that 7h is a safe P-gp modulator when co-ad-
ministered with chemotherapeutic drugs metabolized by
CYP3A4.
Figure 5. Effect of 7h on efflux of Rh123 from K562/A02 cells. DMSO (0.1%)
was used as negative control. VRP was chosen as positive control. Results
are presented as the mean ꢀSEM of n=2 independent experiments.
significantly inhibit the efflux of Rh123 from K562/A02 cells.
The rate constant (K) for the efflux of Rh123 in 7h-treated
K562/A02 cells was 0.0043ꢀ0.00014 minꢁ1, lower than in con-
trol cells (0.0151ꢀ0.00028 minꢁ1) and in verapamil-treated
K562/A02 cells (0.0136ꢀ0.00042 minꢁ1). Moreover, the intracel-
lular Rh123-associated mean fluorescence intensity (MFI) at 5,
10, 25, 30, 60, and 90 min in 1.0 mm 7h-treated cells was
higher than in control and in 10 mm verapamil-treated cells, in-
dicating that the modulatory activity of 7h is more potent
than that of verapamil. The results suggest that 7h can inhibit
P-gp-mediated drug efflux to reverse MDR, and that the activi-
ty is higher than that of verapamil.
Duration of drug effect
We further evaluated the duration of action of 7h as a modula-
tor. The experiment was carried out as previously reported.[8a]
The results listed in Table 3 indicate that the modulating activi-
ty of verapamil at 10 mm nearly disappears right after its re-
moval from the medium, suggesting that the action of verapa-
mil persists no more than 6 h. In contrast, compound 7h at
1.0 mm showed significant reversal effect, even 24 h after its re-
moval. Moreover, in contrast to verapamil, the concentration
of 7h used in this experiment (1.0 mm) was well below the cy-
totoxic IC50 value of 7h against K562/A02 cells (>100 mm,
Table 1). Hence, one conclusion can be drawn from these re-
sults: 7h displayed potent MDR reversal effects, which persist-
ed for a much longer time (>24 h) than with the positive con-
Figure 6. The effect of 7h and ketoconazole on CYP3A4. Ketoconazole, a spe-
cific inhibitor of CYP3A4, was chosen as positive control. Data represent the
mean ꢀSEM of three independent experiments.
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5 mmol), and K2CO3 (1.5 g, 11 mmol) in acetone (20 mL) was
heated at reflux for 3 h to obtain 3g–q. Sodium ascorbate (30 mg)
and CuSO4 (10 mg) were added to a solution of 2a–f and 3g–q
(1 mmol) and 4 (1 mmol) in 75% CH3OH (40 mL). The reaction was
stirred at room temperature for 24–48 h. The mixture was then fil-
tered to give the desires products 7a–q in high purity.
Conclusions
We designed and synthesized a series of novel compounds
with the 6,7-dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]eth-
yl}-1,2,3,4-tetrahydroisoquinoline scaffold as MDR modulators
with low cytotoxicity. This investigation resulted in the identifi-
cation of compound 7h as a potent P-gp modulator. Com-
pound 7h was found to be superior to compound a and VRP
in reversing P-gp-mediated DOX resistance in K562/A02 cells,
with an EC50 value in the nanomolar range. The effect of MDR
reversal by compound 7h persisted more than 24 h. Com-
pound 7h also enhanced the cytotoxicity of other MDR-related
anticancer agents (paclitaxel, vinblastine, and daunorubicin)
toward K562/A02 cells. Moreover, compound 7h did not exhib-
it toxicity up to a concentration of 100 mm in the cell lines
studied. Intracellular accumulation of DOX and efflux of Rh123
in P-gp-overexpressing K562/A02 cells clearly demonstrated
that compound 7h inhibits the transport activity of P-gp.
Unlike several second-generation P-gp inhibitors, compound
7h did not affect the activity of CYP3A4. Future efforts will
focus on the evaluation of compound 7h in vivo.
N-(2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)e-
thyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl) benzenesulfo-
namide (7a): White solid (0.45 g, 71.2%): Rf =0.31 (CH2Cl2/CH3OH
1
20:1); mp: 162–1648C; H NMR (300 MHz, [D6]DMSO): d=9.47 (br,
1H), 8.77 (s, 1H), 7.82 (d, J=7.8 Hz, 2H), 7.67 (d, J=7.5 Hz, 2H),
7.55 (d, J=7.8 Hz, 2H), 7.46 (dd, J=7.4, 7.1 Hz, 1H), 7.39–7.34 (m,
2H), 7.28 (d, J=7.8 Hz, 1H), 7.16–7.11 (m, 2H), 6.94–6.90 (m, 1H),
6.68, 6.66 (s, 2H), 4.98 (s, 2H2), 3.70–3.46 (m, 8H), 2.98–2.78 ppm
(m, 8H); 13C NMR (75 MHZ, [D6]DMSO): d=150.9, 147.3, 147.0,
143.8, 141.0, 140.3, 134.7, 132.3, 130.1, 128.6, 126.6, 126.5, 125.7,
125.5, 125.3, 122.4, 120.9, 120.0, 113.0, 111.7, 109.9, 61.9, 59.7, 58.5,
55.5, 55.4, 54.6, 50.2, 31.9, 27.7 ppm; IR (KBr): n˜ =2869.39, 1611.05,
1596.32, 1518.78, 1466.67, 1369.43, 1255.69, 1158.75, 756.78 cmꢁ1
;
MS (ESI, 40 eV) m/z (%): 626.2 (100) [M+H]+; Anal. calcd for
C34H35N5O5S (625.74): C 65.26, H 5.64, N 11.19%, found: C 65.37, H
5.47, N 11.32%.
N-(2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)e-
thyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-methylben-
zenesulfonamide (7b): White solid (0.42 g, 65.2%): Rf =0.27
(CH2Cl2/CH3OH 20:1); mp: 160–1618C; 1H NMR (300 MHz,
[D6]DMSO): d=9.36 (s, 1H), 8.77 (s, 1H), 7.81 (d, J=7.7 Hz, 2H),
7.54 (d, J=7.7 Hz, 4H), 7.28 (d, J=7.7 Hz, 1H), 7.15–7.11 (m, 4H),
6.94–6.90 (m, 1H), 6.66, 6.63 (s, 2H), 5.00 (s, 2H), 3.69 (s, 6H), 3.57
(s, 2H), 2.94–2.72 (m, 8H), 1.98 ppm (s, 3H); 13C NMR (75 MHZ,
[D6]DMSO): d=150.7, 147.1, 146.9, 143.9, 142.7, 141.5, 137.5, 134.6,
130.1, 129.0, 126.5, 125.8, 125.1, 122.3, 120.9, 199.8, 113.0, 111.8,
109.9, 62.0, 59.0, 55.5, 55.4, 55.0, 50.4, 32.3, 28.3, 20.8 ppm; IR (KBr):
n˜ =2832.65, 1593.37, 1518.07, 1493.19, 1463.72, 1372.38, 1255.87,
1157.04, 763.91 cmꢁ1; MS (ESI, 40 eV) m/z (%): 640.3 (100) [M+H]+;
Anal. calcd for C35H37N5O5S (639.77): C 65.71, H 5.83, N 10.95%,
found: C 65.53, H 5.65, N 11.17%.
Experimental Section
Chemistry
The structures of target compounds were characterized by NMR
spectroscopy, mass spectrometry, and elemental analysis. NMR
spectra were recorded in [D6]DMSO on a Bruker ACF-300Q instru-
1
ment (300 MHz for H, 75 MHz for 13C; Bruker Instruments Inc., Bill-
erica, MA, USA). Chemical shift values (d) are expressed in ppm,
with tetramethylsilane (TMS) as internal standard. Data are report-
ed as follows: chemical shift, multiplicity (s=singlet, d=doublet,
t=triplet, q=quartet, br=broad, m=multiplet), coupling constant
(Hz), integration. ESI-MS data were recorded with Waters ACQUITY
UPLC Systems with Mass (Waters, Milford, MA, USA). Elemental
analyses were carried out on a CHN-O-Rapid instrument. Melting
points were measured with a Mel-TEMP II melting point apparatus
and are uncorrected. Thin-layer chromatography (TLC) was per-
formed on GF/UV 254 plates, and chromatograms were visualized
under UV light at l 254 and 365 nm.
N-(2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)e-
thyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-methoxy-
benzenesulfonamide (7c): White solid (0.39 g, 59.3%): Rf =0.29
(CH2Cl2/CH3OH 20:1); mp: 138–1408C; 1H NMR (300 MHz,
[D6]DMSO): d=9.27 (s, 1H), 8.79 (s, 1H), 7.80 (d, J=8.4 Hz, 2H),
7.55 (dd, J=8.7, 8.4 Hz, 4H), 7.26 (d, J=7.4 Hz, 1H), 7.17–7.10 (m,
2H), 6.93–6.83 (m, 3H), 6.65, 6.63 (s, 2H), 5.00 (s, 2H), 3.69, 3.67
(2 s, 9H), 3.56 (s, 2H), 2.96–2.71 ppm (m, 8H); 13C NMR (75 MHZ,
[D6]DMSO): d=162.08, 150.67, 147.11, 146.85, 143.88, 141.51,
134.64, 131.93, 130.11, 128.74, 126.55, 126.40, 125.86, 125.77,
125.04, 122.36, 120.93, 119.90, 113.74, 113.00, 111.73, 109.91, 61.96,
59.00, 55.44, 55.36, 55.04, 50.44, 32.30, 28.25 ppm; IR (KBr): n˜ =
2837.76, 1596.32, 1517.69, 1498.56, 1463.72, 1330.62, 1255.84,
1172.34, 756.64 cmꢁ1; MS (ESI, 40 eV) m/z (%): 656.3 (100) [M+H]+;
Anal. calcd for C35H37N5O6S (655.77): C 64.11, H 5.69, N 10.68%,
found: C 64.27, H 5.81, N 10.49%.
Compounds 1 and 4 were synthesized according our previous re-
port.[8a] Aromatic sulfonyl chloride (5 mmol) in CH2Cl2 (15 mL) was
added to a solution of compound 1 (0.74 g, 5.0 mmol) and Et3N
(0.61 g, 6 mmol) in CH2Cl2 (20 mL) at 08C. The mixture was then
stirred at room temperature for 24 h. The reaction solution was
washed with 3% HCl (20 mL), saturated aqueous Na2CO3 (20 mL),
and brine (20 mL) in sequence. The organic layer was dried
(Na2SO4), and the solvent was evaporated to afford the desired
products 2a–f. Compound 5 (1.0 g, 7.0 mmol) and SOCl2 (35 mmol)
were heated at 508C for 2 h; the solution was then evaporated to
yield compound 6. Compound 6 (7 mmol) in CH2Cl2 (15 mL) was
added at 08C to a solution of substituted aniline (7.0 mmol) and
pyridine (0.67 g, 8.4 mmol) in CH2Cl2 (20 mL), and the mixture was
stirred at room temperature for 12 h. The solution was washed
(3% HCl), and the solvent was evaporated to obtain a residue,
which was dissolved in 2n NaOH to obtain an aqueous solution;
this was then adjusted to acidic pH 5–6. The solid product was fil-
tered and recrystallized (CH3CN/H2O 9:1) to yield compounds 2g–
q. The mixture of 2g–q (5 mmol), 3-bromoprop-1-yne (0.6 g,
4-Chloro-N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)ben-
zenesulfonamide (7d): White solid (0.38 g, 58.1%): Rf =0.36
(CH2Cl2/CH3OH 20:1); mp: 130–1328C; 1H NMR (300 MHz,
[D6]DMSO): d=9.62 (s, 1H), 8.76 (s, 1H), 7.80 (d, J=8.2 Hz, 2H),
7.64 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.4 Hz,
2H), 7.25 (d, J=7.8 Hz, 1H), 7.17 (d, J=3.9 Hz, 2H), 6.95–6.90 (m,
1H), 6.66 (s, 1H), 6.63 (s, 1H), 5.00 (s, 2H), 3.69 (s, 6H), 3.57 (s, 2H),
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2.93–2.72 ppm (m, 8H); 13C NMR (75 MHZ, [D6]DMSO): d=151.14,
147.12, 146.86, 143.71, 141.49, 139.26, 137.30, 134.63, 130.10,
128.77, 128.47, 127.03, 126.53, 126.03, 125.84, 125.10, 122.35,
120.97, 119.94, 113.03, 111.74, 109.92, 61.80, 58.96, 55.42, 32.29,
28.24 ppm; IR (KBr): n˜ =1560.96, 1517.67, 1499.07, 1465.61,
1333.17, 1159.61, 751.20, 554.50 cmꢁ1; MS (ESI, 40 eV) m/z (%):
661.6 (100) [M+H]+; Anal. calcd for C34H34ClN5O5S (660.19): C
61.86, H 5.19, N 10.61%, found: C 62.06, H 5.27, N 10.43%.
136.44, 134.48, 132.41, 132.25, 130.21, 130.01, 129.06, 126.56,
125.84, 124.61, 122.59, 121.25, 120.01, 119.32, 113.61, 111.73,
109.91, 62.21, 58.97, 55.41, 55.02, 50.41, 32.27, 28.27, 20.39 ppm; IR
(KBr): n˜ =3345.13, 2934.69, 2910.20, 1655.66, 1598.03, 1516.74,
1484.35, 1450.78, 1339.96, 1289.87, 751.08 cmꢁ1; MS (ESI, 40 eV) m/
z (%): 604.2 (100) [M+H]+; Anal. calcd for C36H37N5O4 (603.72): C
71.62, H 6.18, N 11.60%, found: C 71.76, H 6.09, N 11.87%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(2-methoxyphenyl)ben-
zamide (7i): White solid (0.38 g, 62.1%): Rf =0.30 (CH2Cl2/CH3OH
N-(2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)e-
thyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-nitrobenze-
nesulfonamide (7e): White solid (0.36 g, 53.3%): Rf =0.30 (CH2Cl2/
CH3OH 20:1); mp: 116-1188C; 1H NMR (300 MHz, [D6]DMSO): d=
9.75 (s, 1H), 8.76 (s, 1H), 7.93–7.63 (m, 6H), 7.53 (d, J=8.0 Hz, 2H),
7.21–6.93 (m, 4H), 6.66, 6.64 (s, 2H), 5.00 (s, 2H), 3.69 (s, 6H), 3.59
(s, 2H), 2.94–2.73 ppm (m, 8H); 13C NMR (75 MHZ, [D6]DMSO): d=
147.17, 146.86, 141.33, 134.61, 133.87, 132.11, 130.10, 129.67,
125.71, 124.29, 122.58, 121.04, 119.98, 113.12, 111.72, 109.90, 61.69,
58.80, 55.42, 54.87, 50.38, 32.14, 28.07 ppm; IR (KBr): n˜ =2938.05,
1464.92, 1343.02, 1257.58, 1172.34, 546.56 cmꢁ1; MS (ESI, 40 eV) m/
z (%): 671.8 (100) [M+H]+; Anal. calcd for C34H34N6O7S (670.74): C
60.88, H 5.11, N 12.53%, found: C 61.06, H 5.19, N 12.31%.
1
20:1); mp: 118–1198C; H NMR (300 MHz, [D6]DMSO): d=10.34 (s,
1H), 8.98 (s, 1H), 8.42 (d, J=7.7 Hz, 1H), 8.11 (d, J=7.7 Hz, 1H),
7.78 (d, J=8.1, 2H), 7.63–7.45 (m, 4H), 7.18 (t, J=7.4 Hz, 1H), 7.05–
6.91 (m, 3H), 6.65, 6.62 (2 s, 2H), 5.56 (s, 2H), 3.69 (s, 6H), 3.56 (m,
5H), 2.90–2.69 ppm (m, 8H); 13C NMR (75 MHZ, [D6]DMSO): d=
162.1, 155.8, 148.2, 147.1, 146.8, 142.8, 141.5, 134.4, 133.4, 131.5,
130.0, 127.6, 126.5, 125.8, 123.7, 123.1, 121.8, 121.65, 120.4, 119.9,
119.7, 113.9, 111.7, 110.5, 109.9, 62.4, 58.9, 55.5, 55.0, 50.4, 32.2,
28.2 ppm; IR (KBr): n˜ =3351.03, 2832.65, 1649.36, 1559.26, 1530.61,
1518.87, 1484.35, 1460.77, 1295.76, 1248.62, 757.51 cmꢁ1; MS (ESI,
40 eV) m/z (%): 620.3 (100) [M+H]+; Anal. calcd for C36H37N5O5
(619.72): C 69.77, H 6.02, N 11.30%, found: C 69.96, H 6.19, N
11.03%.
N-(2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)e-
thyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-fluoroben-
zenesulfonamide (7 f): White solid (0.39 g, 60.3%): Rf =0.35
(CH2Cl2/CH3OH 20:1); mp: 118–1208C; 1H NMR (300 MHz,
[D6]DMSO): d=9.52 (s, 1H), 8.78 (s, 1H), 7.80 (d, J=8.0 Hz, 2H),
7.70 (dd, J=7.9, 5.1 Hz, 2H), 7.53 (d, J=8.0 Hz, 2H), 7.27 (d, J=
7.7 Hz, 1H), 7.21–7.16 (m, 4H), 6.95–6.93 (m, 1H), 6.66, 6.63 (s, 2H),
4.99 (s, 2H), 3.70 (s, 6H), 3.57 (s, 2H), 2.93–2.72 ppm (m, 8H);
13C NMR (75 MHZ, [D6]DMSO): d=165.65, 162.32, 151.10, 147.11,
146.85, 143.71, 141.50, 136.67, 134.63, 130.10, 129.61, 129.48,
126.96, 126.52, 125.94, 125.84, 125.19, 122.41, 120.95, 119.95,
115.91, 115.61, 112.98, 111.73, 109.91, 61.77, 58.97, 55.41, 55.02,
50.43, 32.29, 28.24 ppm; IR (KBr): n˜ =1328.18, 1155.87, 2825.96,
1255.75, 1593.37, 1517.71, 1497.44, 1460.77, 752.95 cmꢁ1; MS (ESI,
40 eV) m/z (%): 644.7 (100) [M+H]+; Anal. calcd for C34H34FN5O5S
(643.73): C 63.44, H 5.32, N 10.88%, found: C 63.56, H 5.09, N
11.03%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(4-fluorophenyl)benza-
mide (7j): White solid (0.40 g, 65.4%): Rf =0.34 (CH2Cl2/CH3OH
20:1); mp: 119-1218C; 1H NMR (300 MHz, [D6]DMSO): d=10.12 (s,
1H), 8.84 (s, 1H), 7.76–7.73 (m, 3H), 7.56–7.41 (m, 6H), 7.26 (d, J=
7.8 Hz, 2H), 7.13 (t, J=7.0 Hz, 1H), 6.64 (d, J=6.4 Hz, 2H), 5.44 (s,
2H), 3.69 (s, 6H), 3.55 (s, 2H), 2.91–2.71 ppm (m, 8H); 13C NMR
(75 MHZ, [D6]DMSO): d=163.5, 155.4, 147.1, 146.8, 145.7, 143.5,
141.5, 136.3, 134.5, 132.3, 130.2, 130.0, 125.8, 125.2, 124.5, 122.6,
121.2, 120.0, 119.0, 113.6, 111.7, 109.8, 62.2, 58.9, 55.3, 54.9, 50.4,
33.9, 32.2, 31.0, 28.2 ppm; IR (KBr): n˜ =3362.83, 2835.41, 1658.09,
1600.02, 1519.13, 1484.78, 1448.99, 1303.54, 1254.84, 757.20 cmꢁ1
;
MS (ESI, 40 eV) m/z (%): 608.4 (100) [M+H]+; Anal. calcd for
C35H34FN5O4 (607.69): C 69.18, H 5.64, N 11.52%, found: C 70.06, H
5.52, N 11.87%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-phenylbenzamide (7g):
White solid, (0.44 g, 74.3%): Rf =0.38 (CH2Cl2/CH3OH 20:1); mp:
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(3,4-dimethoxyphenyl)-
benzamide (7k): White solid (0.43 g, 67.2%): Rf =0.24 (CH2Cl2/
CH3OH 20:1); mp: 163.0–165.08C; 1H NMR (300 MHz, [D6]DMSO):
d=10.08 (s, 1H), 8.83 (s, 1H), 7.79 (dd, J=7.7, 1.7 Hz, 1H), 7.73 (d,
J=8.5 Hz, 2H), 7.56 (ddd, J=8.5, 8.5, 1.8 Hz, 1H), 7.49–7.41 (m,
3H), 7.32 (d, J=2.4 Hz, 1H), 7.25 (dd, J=8.6, 2.4 Hz, 1H), 7.14 (t,
J=7.6 Hz, 1H), 6.85 (d, J=8.8 Hz, 1H), 6.64 (d, J=7.2 Hz, 2H), 5.44
(s, 2H), 3.70–3.66 (m, 12H), 3.54 (s, 2H), 2.91 (t, J=6.9 Hz, 2H),
2.50 ppm (br, 6H); 13C NMR (75 MHZ, [D6]DMSO): d=163.21, 155.41,
148.57, 146.86, 143.43, 134.49, 132.50, 132.35, 130.32, 130.04,
125.81, 124.27, 122.54, 121.31, 119.98, 113.57, 111.97, 111.74, 111.29,
109.93, 104.49, 62.22, 58.94, 55.67, 55.42, 55.25, 54.97, 50.43,
32.23 ppm; IR (KBr): n˜ =3315.63, 2832.27, 1643.46, 1599.26,
1515.41, 1485.10, 1451.93, 1301.66, 1252.88, 755.06 cmꢁ1; MS (ESI,
40 eV) m/z (%): 650.5 (100) [M+H]+; Anal. calcd for C37H39N5O6
(649.75): C 68.40, H 6.05, N 10.78%, found: C 68.17, H 6.19, N
10.91%.
1
150–1528C; H NMR (300 MHz, [D6]DMSO): d=10.20 (s, 1H), 8.82 (s,
1H), 7.76–7.64 (m, 5H), 7.58–7.42 (m, 4H), 7.28 (t, J=7.6 Hz, 2H),
7.13 (t, J=7.5 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H), 6.65, 6.63 (2 s, 2H),
5.44 (s, 2H), 3.69 (s, 6H), 3.55 (s, 2H), 2.91–2.71 ppm (m, 8H);
13C NMR (75 MHZ, [D6]DMSO): d=163.8, 155.3, 147.0, 146.8, 143.5,
141.6, 138.9, 134.4, 132.2, 130.1, 130.0, 128.6, 126.5, 125.8, 124.6,
123.4, 122.6, 121.2, 120.0, 119.3, 113.6, 111.7, 109.9, 62.1, 58.9, 55.4,
50.3, 32.2, 28.2 ppm; IR (KBr): n˜ =3351.02, 1669.98, 1540.33,
1519.11, 1451.93, 1313.44, 1253.57, 753.01, 593.37 cmꢁ1; MS (ESI,
40 eV) m/z (%): 590.4 (100) [M+H]+; Anal. calcd for C35H35N5O4
(589.70): C 71.29, H 5.98, N 11.88%, found: C 71.06, H 5.79, N
12.07%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(p-tolyl)benzamide
(7h): White solid (0.40 g, 65.7%): Rf =0.33 (CH2Cl2/CH3OH 20:1);
mp: 158–1608C; 1H NMR (300 MHz, [D6]DMSO): d=10.12 (s, 1H),
8.8 (s, 1H), 7.77–7.72 (m, 3H), 7.56–7.40 (m, 6H), 7.15–7.05 (m, 3H),
6.64 (d, J=7.3 Hz, 2H), 5.43 (s, 2H), 3.69 (s, 6H), 3.55 (s, 2H), 2.90–
2.88 (m, 2H), 2.50 (br, 6H), 2.23 ppm (s, 3H); 13C NMR (75 MHZ,
[D6]DMSO): d=163.58, 155.37, 147.11, 146.86, 143.53, 141.59,
N-(3-Chlorophenyl)-2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoqui-
nolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)benza-
mide (7l): White solid (0.42 g, 67.7%): Rf =0.29 (CH2Cl2/CH3OH
1
20:1); mp: 125–1278C; H NMR (300 MHz, [D6]DMSO): d=10.35 (s,
1H), 8.80 (s, 1H), 7.83 (s, 1H), 7.73–7.04 (m, 3H), 7.56 (d, J=7.7 Hz,
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2H), 7.49–7.43 (m, 3H), 7.29 (dd, J=8.1, 8.1 Hz, 1H), 7.15–7.08 (m,
2H), 6.65, 6.63 (2 s, 2H), 5.43 (s, 2H), 3.69 (s, 6H), 3.45 (s, 2H), 3.17–
2.70 ppm (m, 8H); 13C NMR (75 MHZ, [D6]DMSO): d=164.3, 155.3,
147.1, 146.8, 143.5, 141.6, 140.3, 134.4, 133.1, 132.5, 130.3, 130.1,
130.0, 126.5, 125.8, 124.4, 123.1, 122.5, 121.2, 119.9, 118.8, 117.7,
113.6, 111.7, 109.9, 62.2, 58.9, 55.4, 55.0, 50.4, 32.2, 28.2 ppm; IR
(KBr): n=3339.23, 2825.96, 1655.25, 1540.33, 1407.73, 1253.86
678.65 cmꢁ1; MS (ESI, 40 eV) m/z (%): 625.4 (100) [M+H]+; Anal.
calcd for C35H34ClN5O4 (624.14): C 67.35, H 5.49, N 11.22%, found: C
67.46, H 5.31, N 11.47%.
8.85 (s, 1H), 7.78–7.71 (m, 3H), 7.58–7.42 (m, 5H), 7.34 (s, 1H), 7.14
(m, 2H), 6.83 (d, J=7.4 Hz, 2H), 6.65, 6.63 (2 s, 2H), 5.42 (s, 2H),
3.69–3.35 (m, 8H), 2.91–2.71 (m, 8H), 2.15 ppm (s, 3H); 13C NMR
(75 MHZ, [D6]DMSO): d=163.61, 155.44, 147.10, 146.85, 143.51,
141.62, 138.79, 137.92, 134.47, 132.41, 130.29, 130.05, 128.54,
126.57, 125.86, 124.32, 124.14, 122.61, 121.29, 119.89, 119.76,
116.48, 113.58, 111.73, 109.92, 62.25, 58.97, 55.41, 55.04, 50.42,
32.25, 28.27, 20.95 ppm; IR (KBr): n˜ =3351.03, 2926.25, 1652.46,
1288.83, 1254.17, 1611.39, 1599.33, 1516.62, 1488.19, 775.58 cmꢁ1
;
MS (ESI, 40 eV) m/z (%): 604.2 (100)[M+H]+; Anal. calcd for
C36H37N5O4 (603.72): C 71.62, H 6.18, N 11.60%, found: C 71.71, H
6.21, N 11.47%.
N-(4-Chlorophenyl)-2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoqui-
nolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)benza-
mide (7m): White solid (0.39 g, 61.8%): Rf =0.31 (CH2Cl2/CH3OH
N-(4-(tert-Butyl)phenyl)-2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroi-
soquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-
benzamide (7q): White solid (0.48 g, 73.8%): Rf =0.33 (CH2Cl2/
CH3OH 20:1); mp: 117–1198C; 1H NMR (300 MHz, [D6]DMSO): d=
10.10 (s, 1H), 8.83 (s, 1H), 7.74 (d, J=7.6 Hz, 3H), 7.57–7.41 (m,
7H), 7.25 (d, J=8.5 Hz, 1H), 7.14 (d, J=7.3 Hz, 1H), 6.65, 6.63 (2 s,
2H), 5.43 (s, 2H), 3.69 (s, 6H), 3.56 (s, 2H), 2.91–2.71 (m, 8H),
1.22 ppm (s, 9H); 13C NMR (75 MHz, [D6]DMSO): d=163.56, 155.41,
146.86, 145.80, 143.53, 141.59, 136.32, 134.50, 132.29, 130.22,
130.02, 125.84, 125.26, 124.55, 122.71, 121.27, 120.07, 119.07,
113.64, 111.74, 109.93, 62.25, 55.42, 55.01, 50.44, 33.93, 32.28,
31.10, 28.21 ppm; IR (KBr): n˜ =3351.03, 2949.85, 1658.10, 1596.32,
1519.02, 1481.40, 1451.93, 1407.73, 1378.27, 1363.54, 1304.06,
757.12 cmꢁ1; MS (ESI, 40 eV) m/z (%): 646.7 (100) [M+H]+; Anal.
calcd for C39H43N5O4 (645.80): C 72.53, H 6.71, N 10.84%, found: C
72.77, H 6.62, N 10.79%.
1
20:1); mp: 100–1028C; H NMR (300 MHz, [D6]DMSO): d=10.35 (s,
1H), 8.80 (s, 1H), 7.78–7.62 (m, 5H), 7.57–7.41 (m, 4H), 7.34 (d, J=
7.9 Hz, 2H), 7.12 (dd, J=7.2 Hz, 1H), 6.65, 6.62 (2 s, 2H), 5.42 (s,
2H), 3.69 (s, 6H), 3.55 (s, 2H), 2.91–2.70 ppm (m, 8H); 13C NMR (75
MHZ, [D6]DMSO): d=164.21, 155.31, 147.09, 146.84, 143.58, 141.58,
137.91, 134.46, 132.31, 130.01, 128.59, 127.01, 126.53, 125.84,
124.80, 122.51, 121.22, 121.07, 120.89, 120.00, 113.67, 111.71,
109.90, 62.19, 58.98, 55.40, 55.03, 50.43, 32.27, 28.23 ppm; IR (KBr):
n˜ =3339.23, 2837.76, 1652.32, 1399.55, 1292.19, 1256.10,
686.71 cmꢁ1; MS (ESI, 40 eV) m/z (%): 625.5 (100) [M+H]+; Anal.
calcd for C35H34ClN5O4 (624.14): C 67.35, H 5.49, N 11.22%, found: C
67.51, H 5.39, N 11.33%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(o-tolyl)benzamide
(7n): White solid (0.35 g, 57.8%): Rf =0.32 (CH2Cl2/CH3OH 20:1);
mp: 108–1108C; 1H NMR (300 MHz, [D6]DMSO): d=9.67 (s, 1H),
8.91 (s, 1H), 7.94 (d, J=6.9 Hz, 1H), 7.88 (d, J=6.9 Hz, 1H), 7.75 (d,
J=8.1 Hz, 2H), 7.60–7.47 (m, 4H), 7.16–7.00 (m, 5H), 6.65, 6.62 (2 s,
2H), 5.50 (s, 2H), 3.69 (s, 6H), 3.55 (s, 2H), 2.91–2.70 (m, 8H),
1.97 ppm (s, 3H); 13C NMR (75 MHZ, [D6]DMSO): d=163.10, 155.60,
147.10, 146.85, 142.93, 141.61, 136.41, 134.50, 132.80, 131.05,
130.06, 129.48, 126.02, 125.84, 124.70, 124.51, 123.31, 123.13,
121.56, 121.35, 120.06, 113.66, 113.56, 111.72, 109.91, 79.13, 62.05,
58.95, 56.67, 55.41, 55.01, 50.42, 32.25, 28.23, 17.86, 17.14 ppm; IR
(KBr): n˜ =3365.33, 2930.32, 1653.10, 1589.10, 1535.43, 1517.67,
1482.23, 1457.60, 1291.45, 1256.18, 754.13 cmꢁ1; MS (ESI, 40 eV) m/
z (%): 605.3 (100) [M+H]+; Anal. calcd for C36H37N5O4 (603.72): C
71.62, H 6.18, N 11.60%, found: C 71.53, H 6.31, N 11.79%.
Biological studies
Materials: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), doxorubicin (DOX), vinblastine, paclitaxel, daunorubi-
cin, 5-fluorouracil (5-FU), verapamil (VRP), and other chemicals
were purchased from Sigma–Aldrich (St. Louis, MO, USA). All
common chemicals were of analytical grade or higher.
Cell lines and culture: Human leukemia cell line K562 and its doxor-
ubicin-selected P-gp-overexpressing daughter cell line K562/A02
were kindly provided by Professor Bao-An Chen (Department of
Hematology, The Affiliated Zhong-Da Hospital, Southeast Universi-
ty, Nanjing, China). The cell lines were grown in RPMI-1640
medium containing 10% fetal bovine serum (FBS) in a humidified
incubator at 378C with 5% CO2. To confirm drug resistance charac-
teristics, 1 mgmLꢁ1 doxorubicin was added to K562/A02 cultures
and maintained in drug-free medium for 2 weeks before use. All
experiments were performed with cells in exponential growth.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(3-methoxyphenyl)ben-
zamide (7o): White solid (0.42 g, 67.3%): Rf =0.30 (CH2Cl2/CH3OH
1
20:1); mp: 130–1328C; H NMR (300 MHz, [D6]DMSO): d=10.19 (s,
1H), 8.81 (s, 1H), 7.77 (d, J=7.2 Hz, 1H), 7.71 (d, J=8.0 Hz, 2H),
7.58–7.42 (m, 4H), 7.31 (s, 1H), 7.26–7.11 (m, 3H), 6.65–6.63 (m,
3H), 5.43 (s, 2H), 3.69–3.56 (m, 11H), 2.91–2.71 ppm (m, 8H);
13C NMR (75 MHZ, [D6]DMSO): d=163.81, 159.50, 155.39, 147.10,
146.85, 143.49, 141.61, 140.04, 134.47, 132.42, 130.26, 130.02,
129.49, 125.85, 124.39, 122.61, 121.28, 120.00, 113.56, 111.73,
111.62, 109.91, 109.02, 105.06, 62.19, 58.92, 55.41, 54.87, 50.43,
32.26, 28.28 ppm; IR (KBr): n˜ =3345.13, 2843.66, 1677.25, 1599.66,
Cytotoxicity assays: Cell viability was determined using the MTT
method with a minor modification.[13] K562 and K562/A02 cells
were harvested during logarithmic growth phase, and were grown
in 96-well microtiter plates at 1ꢁ104 cells per well. The cells were
cultured for 24 h before the addition of drugs. P-gp inhibitors were
prepared as 5 mm DMSO stocks. In the MTT assay for anticancer
MDR-reversal experiments, cells were incubated in the presence of
anticancer agents (doxorubicin, vinblastine, paclitaxel, daunorubi-
cin, or 5-FU) with or without P-gp inhibitors. When the MTT assay
was used for cytotoxicity evaluation of P-gp inhibitors, cells were
incubated in the presence of various concentrations of P-gp inhibi-
tors alone. After the addition of drugs, cells were incubated for an-
other 48 h followed by the addition of 20 mm freshly prepared MTT.
Then the cells were incubated for another 4 h in a 378C incubator
containing 5% CO2 before the plates were read on a microplate
reader (Thermo, USA) at a test wavelength of 490 nm. The IC50
1548.56, 1518.19, 1492.47, 1287.18, 1255.01, 769.31, 683.99 cmꢁ1
;
MS (ESI, 40 eV) m/z (%): 620.4 (100) [M+H]+; Anal. calcd for
C36H37N5O5 (619.72): C 69.77, H 6.02, N 11.30%, found: C 69.65, H
6.13, N 11.41%.
2-((1-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-N-(m-tolyl)benzamide
(7p): White solid (0.39 g, 64.3%): Rf =0.33 (CH2Cl2/CH3OH 20:1);
mp: 120–1228C; 1H NMR (300 MHz, [D6]DMSO): d=10.11 (s, 1H),
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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values of the compounds for cytotoxicity were calculated by
GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA,
USA) from the dose–response curves.
Diego, CA, USA). Unpaired comparisons were analyzed using the
two-tailed Student t-test, unless otherwise stated.
DOX accumulation: The experiment was carried out according to
the reported procedures with minor modification.[14] Briefly, 4ꢁ105
cells of K562 and K562/A02 were incubated with 20 mm doxorubi-
cin and various concentrations of compound 7h, VRP or WK-X-34
for 150 min at 378C; 0.1% DMSO was used as a negative control.
After incubation, the cells were washed with cold PBS and lysed
with lysis buffer (0.75m HCl, 0.2% Triton-X100 in isopropanol). The
fluorescence level of ADM in the lysate was determined by fluores-
cence spectrophotometer (RF-5301 PC, Shimadzu) using an excita-
tion and emission wavelengths of 460 and 587 nm.
Acknowledgements
We thank Professor Bao-An Chen (Southeast University, Nanjing,
China) for providing cells and Dr. Liqun Jiang and Fang Li (De-
partment of Pharmaceutics, China Pharmaceutical University,
Nanjing, China) for technical assistance. This work was supported
by the National Science and Technology Major Project of the Min-
istry of Science and Technology of China (2009ZX09102-033) and
the National Natural Science Foundation of China (81173088).
Rh123 efflux: As previously reported, K562/A02 cells were incubat-
ed with medium containing 5 mm Rh123 at 378C for 90 min,
washed three times with Rh123-free medium, and then incubated
in the presence or absence of 1.0 mm and 10 mm verapamil at 378C
for 5, 10, 25, 30, 60, or 90 min.[15] The rate constant (K) was ob-
tained from fitting data to Ft =F0eꢁkt, for which Ft is the Rh123-as-
sociated mean fluorescence intensity (MFI) at time t. The MFI was
measured by flow cytometry. Graphs were plotted of cell-associat-
ed MFI against time.
Keywords: accumulation · antitumor agents · modulators ·
multidrug resistance · P-glycoprotein
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per well were incubated for 24 h with or without 1.0 mm com-
pound 7h, 10 mm VRP or PBS before being washed 0 or 3 times
with growth medium. Then, the cells were incubated for 0, 6, 12,
or 24 h before the addition of various concentrations of doxorubi-
cin or vehicle. The incubation was continued for 48 h prior to the
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[6] A. Palmeira, E. Sousa, M. H. Vasconcelos, M. M. Pinto, Curr. Med. Chem.
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CYP3A4 assays: Rat liver microsomes were prepared by differential
centrifugation, and the protein concentration was determined by
the Bradford method.[17] The activity of CYP3A4 was determined
using testosterone (substrate of CYP3A4) and HPLC analysis as pre-
viously described with minor modification.[18] The column used was
an octyldecylsilyl (C18) reversed-phase HPLC column (5 mm,
150 mm, 4.6 mm). Column temperature was set at 358C, and flow
rate was 1.0 mLminꢁ1. Mobile phase A consisted of water. Mobile
phase B consisted of acetonitrile. From 0 to 7.5 min, the percent-
age of mobile phase B was 35%, at which time the percentage of
mobile phase B was immediately increased to 48%, where it re-
mained until 14 min. At 14 min, the percentage of mobile phase B
was returned to 35%, where it remained until the end of the run
at 19 min. Detection was by UV absorbance at 245 nm. The liver
microsomes were incubated with testosterone (50 mm) in the pres-
ence or absence of 7h and ketoconazole, an inhibitor of CYP3A4,
as the positive control.
Statistical analysis: Statistical analyses were performed using specif-
Received: October 25, 2014
ic software (GraphPad InStat version 5.00, GraphPad Software, San
Published online on && &&, 0000
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B. Liu, Q. Qiu, T. Zhao, L. Jiao, Y. Li,
W. Huang,* H. Qian*
Efflux blockers! Seventeen compounds
based on the 6,7-dimethoxy-2-{2-[4-(1H-
1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tet-
rahydroisoquinoline scaffold were syn-
thesized. Among them, compound 7h
(shown) was found to be more potent
than the L-type calcium channel blocker
verapamil in reversing P-glycoprotein-
mediated multidrug resistance; it was
also found to persist for a longer time.
&& – &&
6,7-Dimethoxy-2-{2-[4-(1H-1,2,3-
triazol-1-yl)phenyl]ethyl}-1,2,3,4-
tetrahydroisoquinolines as Superior
Reversal Agents for P-Glycoprotein-
Mediated Multidrug Resistance
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
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These are not the final page numbers!